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PubMed1978

Heterogeneous staining of rat hepatocyte nuclei by HBFP technique.

The non-enzymatic histochemical technique Haematoxylin--Basic Fuchsin--picric acid (HBFP) was studied in fresh-frozen and Carnoy-fixed, paraffin-embedded rat liver sections. The hepatocyte nuclei fell into two populations and showed either a crimson red or purple staining in frozen as well as paraffin sections. The heterogeneous staining of the rat heptocyte nuclei was also present when the tissue sections were stained by Methyl Green--Pyronin stain. The differing nuclear staining was present in the isolated nuclei also. The HBFP technique, therefore, appears potentially useful when applied to liver and other tissues as well.

Animals↗

PubMed1991

Atypical large plasma cells in lymph node granulomas in cat-scratch disease.

A histological variant of plasma cells found in the granulomas of cat-scratch disease (CSD) lymphadenitis is reported. Though the lesion shows the typical features of suppurative granulomatous lymphadenitis, many atypical giant cells which have abundant basophilic cytoplasm and bizarre nuclei with occasional multinucleated forms are noted among epithelioid histiocytes. The diagnosis of CSD lymphadenitis was confirmed by comparing clinical; histopathological, and histochemical (Warthin-Starry silver impregnation stain) studies on lymph node sections from five cases with features typical of the disease. Histochemical (methyl green-pyronine stain) and immunohistochemical examination provided several lines of evidence indicating that the atypical giant cells in our case were plasmacytic and confirmed that its proliferation was reactive, not neoplastic. Multinucleated giant cells were also occasionally present in the other five cases, but they had histological and immunohistochemical features of Langhans' type giant cells. We stress the importance of distinguishing such atypical large plasma cells from neoplastic cells.

Adolescent↗

PubMed1986

Theileria infectivity of Hyalomma ticks in Haryana, India.

Theileria infection of Hyalomma ticks collected from three districts of Haryana was assessed in whole salivary glands by the methyl-green pyronin staining method. Of 1,662 ticks screened, 546 (32.8%) were found Theileria positive. Infection rate in 935 female ticks (36.9%) was more than that of 727 male ticks (27.6%). Density of Theileria infection (number of infected acini per infected tick) was also higher in female ticks. Theileria infection rate of ticks varied greatly in the three districts viz. only 12.0% in Rohtak, 25.8% in Hisar and 48.3% in Karnal. Per cent infection rate was high (63.7%) in ticks from indigenous cattle and low (18.6%) in those collected from buffaloes. However, the intensity of infection in infected ticks from cross-bred cattle was comparatively much higher. Frequency distribution of Theileria positive acini in infected ticks revealed a low density of infection per infected tick. This points to the largely stable endemic situation prevailing in Haryana. Only a single salivary acinus was found infected in 16.6% of the infected ticks, about 70% had up to 10 infected acini while only about 10% had over 25 infected acini per tick. The masses in acini presumed to be Theileria were confirmed by demonstrating parasitic masses on staining one of a pair of salivary glands and inoculating the suspension of the other half of the gland into two cross-bred cow calves which developed clinical signs and lesions typical of theileriosis.

Animals↗

PubMed1990

Sites of action of soltriol (vitamin D) in hamster spleen, thymus, and lymph node, studied by autoradiography.

Siberian hamsters (Photopus sungorus) were injected with 3H dihydroxycholecalciferol (vitamin D, soltriol). Autoradiograms of spleen, thymus, and lymph nodes revealed nuclear concentration of the hormone in a select population of cells in all of these organs. In the spleen, labeled cells were abundant in the red pulp, but sparse in the white pulp. In the periarterial lymphatic sheath (PALS) labeled cells were found predominantly at the outer rim, with a few scattered labeled cells in the inner PALS and in the marginal zone. Lymphocytes, including pyronin-positive plasma cells, did not display nuclear labeling. In the red pulp, some of the labeled cells contained pigmented inclusions in the cytoplasm, while most of the labeled cells did not appear phagocytic under the conditions of the experiment. In the thymus, labeled cells were most numerous in the medulla, but sparse in the cortex. Many of the thymic target cells were larger than the unlabeled lymphocytes, with a large and pale nucleus, sometimes containing a distinct nucleous, and with large and dendritic cytoplasm, having the appearance and distribution of epithelio-reticular cells. In lymph nodes, scattered labeled cells were conspicuous in or near the subcapsular sinus, while other cells did not concentrate radioactivity in their nuclei. The results indicate that nuclear receptors and direct genomic actions for soltriol exist in certain cell populations of lymphatic tissues that probably include reticular cells and a subpopulation of macrophages. These target cells may mediate effects of the steroid on lymphocytes that appear to have no or only very low numbers of nuclear receptors.

Animals↗

PubMed1979

EDTA soluble protein of human mature normal enamel.

Pure human mature enamel was prepared using a careful microdissection technique. After EDTA dissolution, the soluble proteins were recovered representing a concentration of 0.035% in the initial enamel. When the samples were analyzed with polyacrylamide gel electrophoresis, Coomassie Brilliant Blue staining revealed only one sharp fast migrating band, whereas o-toluidine blue, methylene blue, Amido Black 10B, and pyronine red G showed a thin double band at the same migration distance. Ultracentrifugation studies suggested that the proteins were of low molecular weight or of weak density. Absorption spectra showed a strong absorbance at 260 nm. After hydrolysis, amino acid analyses yielded a composition of 25% Gly, 13.5% Glu, 11% Ser, and 11% Pro. Cysteine measured as cysteic acid was present at 2%, and 2% hydroxyproline was found. A carbohydrate content of 15% was estimated by the anthrone method. Glucose, galactose, mannose, and fucose, identified through gas chromatography, were in a molar ratio of 9:4:3:1. Thus the organic matrix of adult human enamel consists of one or possibly two acidic glycoproteins.

Amino Acids↗

PubMed1976

Further observations on the chemistry of pararosaniline-Feulgen staining.

Pararosaniline-Feulgen staining of cells in suspension produces nucleus- and chromatin-specific fluorescence as well as color. Experiments were designed to test postulated reaction mechanisms responsible for the fluorescent staining with the nonfluorescent pararosaniline. The reduction in fluorescent-staining intensity by pretreatment of cells with 2.2 x 10-2M K2S2O5 tends to rule out the alkysulfonic acid pathway; conditions favoring the formation of this intermediate reduce staining intensity. The fluorescence enhancement, observed when cells stained in pararosaniline without K2S2O5 are post-treated with K2S2O5, suggests that there is an initial Schiff-base linkage between pararosaniline and an aldehyde of hydrolyzed DNA, and that this linkage is stabilized in the presence of K2S2O5. Microspectrofluorometer measurements of cells stained at various pararosaniline concentrations in 2.2x10-2M K2S2O5, show that the fluorescence emission maximum ranges from about 627 nm at 3.1x10-3 M pararosaniline to about 604 nm at 3.1x10-5M. All of the employed staining protocols appear to produce the same fluorescent product, perhaps a heterocyclic pyronin analog formed from pararosaniline. Flow microfluorometric analysis of cells stained in suspension verified that the relative fluorescence intensity represents relative DNA content. Staining at reduced pararosaniline concentration (3.1x10-4M) reduces the coefficient of variation of the flow microfluorometric histograms, showing that maximum quantitation does not necessarily correlate with maximum staining intensity.

Animals↗

PubMed1999

Bunina bodies in amyotrophic lateral sclerosis on Guam: a histochemical, immunohistochemical and ultrastructural investigation.

An investigation of Bunina bodies is important when studying the pathoetiology and pathomechanisms involved in amyotrophic lateral sclerosis (ALS). It may serve as a clue essential for the study of the pathogenesis of Guamanian amyotrophic lateral sclerosis (ALS-G), and it may provide a means of answering the question of whether ALS-G is the same disease as classical ALS or a different entity. In ALS-G, however, no precise histochemical, immunohistochemical, or detailed ultrastructural examination has been published to date. To elucidate the pathological differences/similarities of Bunina bodies between classical ALS and ALS-G, we performed histochemical, immunohistochemical, topographic and ultrastructural examinations. Histochemically, hematoxylin and eosin, Masson's trichrome, methylgreen-pyronin, phosphotungstic acid-hematoxylin, Klüver-Barrera, Bodian and periodic acid-Schiff staining were utilized. Immunohistochemical examination was performed using antibodies for cystatin C, ubiquitin, Tau-2, Cu/Zn superoxide dismutase, phosphorylated neurofilament and glial fibrillary acidic protein. Histochemical findings were consistent with those previously described for classical ALS. The immunohistochemical study showed that in ALS-G Bunina bodies were intensely labeled by an anti-cystatin C antibody. Topographic examination demonstrated that Bunina bodies were distributed in the spinal anterior horns and Clarke's column in the spinal cord. Ultrastructurally, Bunina bodies were composed of electron-dense amorphous/ granular material accompanied by vesicular structures and neurofilaments. The results of the present study have revealed that the pathological features of Bunina bodies in ALS-G are identical to those seen in classical ALS. These findings strongly suggest that a similar degenerative process occurs in the spinal anterior horn cells in both ALS-G and classical ALS.

Adult↗

PubMed1997

Use of the polymerase chain reaction for identification and quantification of Theileria parva protozoa in Rhipicephalus appendiculatus ticks.

The polymerase chain reaction (PCR) was adapted for detection of Theileria parva sporoblasts in Rhipicephalus appendiculatus ticks by comparison with staining of histological preparations of ticks with methyl green and pyronin (MGP). Two 32mer primers (IL174 and IL179) were used to amplify Theileria parva (Muguga isolate) DNA from the TPR 1 region of the genome by the PCR. Detection of T. parva was carried out with dissected salivary glands and whole ticks preserved in ethanol. Adult ticks which fed as nymphs on a T. parva infected calf were used in three experiments. Firstly, 70 whole ticks divided into 7 batches representing the rising and falling parasitaemia of the calf were used to show that detection of infection by the PCR was significantly correlated with MGP staining. Secondly, 120 dissected ticks were used from 4 different batches representative of the overall infection profile within the ticks to show a high correlation between PCR quantification within tick salivary glands and MGP count data of the paired gland. Thirdly, 120 ticks were used in batches selected for high and low infections. Bloodmeal contaminants from partially fed adult ticks, present in 60 out of the 120 ticks used, did not inhibit the PCR amplification of T. parva DNA. This experiment also showed a great increase in infection detection in partially fed batches of ticks compared to the untreated batches.

Animals↗

PubMed2003

Apoptosis in canine distemper.

Canine distemper is a systemic viral disease characterized by immunosuppression followed by secondary infections. Apoptosis is observed in several immunosuppressive diseases and its occurrence on canine distemper in vivo has not been published. In this study, the occurrence of apoptosis was determined in lymphoid tissues of thirteen naturally infected dogs and nine experimentally inoculated puppies. Healthy dogs were used as negative controls. Samples of lymph nodes, thymus, spleen and brain were collected for histopathological purposes. Sections, 5 microm thick, of retropharingeal lymph nodes were stained by HE, Shorr, Methyl Green-Pyronin and TUNEL reaction. Shorr stained sections were further evaluated by morphometry. Canine distemper virus nucleoprotein was detected by immunohistochemistry. Retropharingeal lymph nodes of naturally and experimentally infected dogs had more apoptotic cells per field than controls. In addition, DNA from thymus of infected dogs were more fragmented than controls. Therefore, apoptosis is increased in lymphoid depletion induced by canine distemper virus and consequently play a role in the immunosuppression seen in this disease.

Animals↗

PubMed1991

A novel approach to nonradioactive hybridization assay of nucleic acids using stained latex particles.

The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods.

Acrolein↗

PubMed1975

Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response.

An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.

Animals↗

PubMed1985

Immunogold staining: an alternative method for lymphocyte subset enumeration. Comparison with immunofluorescence microscopy and flow cytometry.

An immunogold staining procedure for light microscopic enumeration of peripheral blood lymphocyte subsets defined by monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1, Leu 1, Leu 4, Leu 2a, Leu 3a, Leu 10, Leu 12, B1) is described. It uses colloidal gold-labeled goat anti-mouse Ig (GAM G40 and GAM G30) as second layer and a methyl-green pyronin counterstain. Performed on small volumes of blood without sophisticated laboratory equipment, this method allows accurate cell type recognition and permanent records, essential for longitudinal observations. By enumerating the gold particles on positively labeled cells, it was shown that the staining reactivity depended on the monoclonal antibody used. Lymphocytes reacting with OKT8 or OKIa1 or B1 exhibited the strongest labeling whereas OKT4+ cells were weakly labeled. When compared with flow cytometry analysis in healthy subjects, the accuracy, precision and sensitivity of both methods were very similar. Similarly, a close correlation (97%) was found between immunogold staining and immunofluorescence microscopy in 35 patients with various diseases suggesting that immunogold staining may be useful in a clinical context.

Antibodies, Monoclonal↗

PubMed1978

Atypical progressive multifocal leukoencephalopathy and primary cerebral malignant lymphoma.

Atypical progressive multifocal leukoencephalopathy occurred in an 82-year-old man with long-standing chronic lymphocytic leukemia. The multifocal white matter lesions revealed demyelination and gliosis, relative preservation of axons and a prominent plasma cell infiltrate. Bizarre astrocytes were uncommon. There were no changes in the oligodendroglial nuclei and no inclusion bodies. Electron and immunohistofluorescence microscopy for papova virus were negative. In addition to this leukoencephalopathy a small primary cerebral lymphoma was present in the right occipital lobe. The tumor cells were plasmacytoid in appearance and their cytoplasm stained well with pyronin. They contained IgG by immunohistofluorescence microscopy. The neoplasm was considered an immunoblastic sarcoma (reticulum cell sarcoma) and thought to arise directly within foci of demyelination. Both disease processes, leukoencephalopathy and lymphoma, may have occurred on a background of immunosuppression.

Aged↗

PubMed1986

T cell reactivity to penicillin: phenotypic analysis of in vitro activated cell subsets.

Patients with penicillin allergy demonstrate a T cell proliferative response after in vitro stimulation with penicillin G (Pen G) and other beta-lactam antibiotics. To understand better penicillin-allergic reactions, T cell subset stimulation with Pen G was studied and compared with other soluble (tetanus toxoid and purified protein derivative [PPD]) and membrane-bound viral (influenza A and Epstein-Barr viruses) antigens. A double fluorescence method for flow cytometry was used to evaluate the activated cells simultaneously by pyronin Y staining of RNA and by indirect immunofluorescence of cell surface T4, T8, or Leu 8 antigens. The antigens used stimulated mainly the T4+ subset (greater than 90%), whereas the number of activated T8 cells was slightly increased only in Pen G- and influenza A-triggered cultures (5% to 15%). Leu 8 antigen was used to analyze more precisely the activated T4+ cells. Pen G and influenza A and Epstein-Barr viruses stimulated both T4+, Leu 8+ (greater than 50% of activated cells, inducers for suppressor cells), and T4+, Leu 8- (helpers for B cells) subsets, whereas PPD activated mainly T4+, Leu 8- subpopulations. These results indicate that penicillin-allergic patients with skin symptoms demonstrate a T cell subset stimulation that resembles more the reaction versus viral antigens (membrane incorporated) than to soluble antigens like PPD. These results suggest that Pen G is presented to T cells like viral proteins and might thus cause allergic reactions resembling skin symptoms observed in viral diseases.

Anti-Bacterial Agents↗

PubMed1980

Immune response to first mating in the female rat.

The iliac and popliteal lymph nodes were removed from 110 Sprague Dawley female rats taken from a closed colony. All were killed at age 12-13 wk in groups of 10 animals; one group of virgin controls, the others at 2-day intervals during the first 10 days of either inbred or allogeneic poregnancy. Every tenth section of the lymph nodes was stained with methyl green pyronin and counts were made of the large pyroninophilic cells in the thymus-dependent area of the cortex for evidence of T cell proliferation and the plasma cells in the medulla for evidence of B cell proliferation. After allogeneic mating significant T cell proliferation was evident in both the iliac and popliteal lymph nodes by the second post-coital day and this proliferative response was sustained until the sixth postcoital day and recurred on the tenth post-coital day. After inbred mating transient T cell proliferation was observed on the fourth post-coital day in the iliac lymph nodes. Significant B cell proliferation occurred in the iliac lymph nodes on the second day of allogeneic pregnancy and on the fourth day of inbred pregnancy, being sustained in both until the end of the observation period. These results suggest that allogeneic mating and possibly semi-allogeneic implantation induce an immune response in the female rat.

Analysis of Variance↗

PubMed1990

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo.

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.

Animals↗

PubMed1989

Localization of a nonintercalative DNA binding antitumour drug in mitochondria: relationship to multidrug resistance.

The bis-(n-butyl) quaternary salt of N,N'-bis-(6-quinolyl)terephthalamide (QBQ), a fluorescent antitumour compound in the phthalanilide series which is thought to bind to the minor groove of the DNA double helix, has been investigated with respect to its in vitro activity and subcellular localization. Cultured MCF-7 human breast carcinoma cells concentrated QBQ in mitochondria by a time-dependent process which was inhibited by the ionophore valinomycin, suggesting a possible mode of antitumour action of QBQ through mitochondrial poisoning. Growth of cultured P388 murine leukaemia cells was inhibited 50% in the presence of 0.52 microM QBQ and multidrug-resistant P388 sublines developed for resistance to actinomycin D, vincristine, Adriamycin and the phthalanilide NSC 38280 were cross-resistant to the drug. Cross-resistance was reduced in all lines by the presence of 11 microM verapamil, suggesting that a transport resistance mechanism operates on QBQ. The actinomycin D-resistant P388 cell line was found to be cross-resistant to the aromatic cations rhodamine 123, which binds to proteins, and ethidium and pyronin Y, which bind intercalatively to DNA. Thus mitochondrion-specific drugs with different macromolecular binding properties all appear to be excluded by multidrug-resistant cells.

Animals↗

PubMed1989

Histopathological study of human TMJ perforated discs with emphasis on synovial membrane response.

Fifteen perforated TMJ discs from human cadavers were studied histologically to examine the synovial membranes and to compare the findings with previous experimental results in monkeys. There were four with perforations in the bilaminar zone (these four discs were displaced anteriorly), three in the medial third of the disc, and eight in the lateral third of the disc. Histopathologically, there was an increase in vascularity and strong methyl pyronine-positive cellularity around the margins of the perforations. A young, loose, collagenous tissue lined the lateral margins of the perforated discs. Increased fibrous tissue content of the synovial subintimal territorial matrix and osteochondroid metaplasia were also seen. Severe synovial hyperplasia was visible in all joint recesses, but was greatest within those associated with displaced discs. There was patchy distribution of acidic glycoproteins, especially in the lateral parts of the perforated discs. As in the animal studies, human TMJ disc perforation was associated with a vigorous synovial reaction that was seen to form lateral bridges along the margins.

Aged↗