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PubMed1975

Microbial adjuvant and autoimmunity. I. Induction of antibody responses to syngeneic tissue extracts in mice treated with capsular polysaccharide of Klebsiella pneumoniae.

With the use of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a powerful adjuvant, high precipitin responses could be induced in mice to syngeneic eyeball extracts and thyroid gland extracts which were normally nonimmunogenic. Only very weak responses were induced to eyeball extracts by Freund's complete adjuvant. Repeated administrations of the antigens mixed with CPS-K at time intervals of 30 days (more than twice for the eyeballs or more than three times for the thyroid glands) were required for induction of high precipitin responses. Antibody responses detectable by the immunofluorescent technique could be induced to syngeneic lymphoid tissue extracts by injecting the mixture of antigen and CPS-K more than five times at time intervals of 30 days. These findings suggest that repeated stimulation by autoantigens together with such a strong adjuvant as CPS-K can terminate natural tolerance against autoantigens.

Adjuvants, Immunologic↗
PubMed1983

Selenium-dependent and non-selenium-dependent glutathione peroxidases in human tissue extracts.

A method for the assessment of both selenium-dependent and non-selenium-dependent glutathione peroxidases on crude tissue extracts in human is described. The enzyme activity is measured by the coupled assay system in which oxidation of reduced glutathione (GSH) is coupled to NADPH oxidation catalyzed by glutathione reductase. Total glutathione peroxidase activity is measured with cumene hydroperoxide as substrate. Selenium-dependent glutathione peroxidase is measured with tert-butyl hydroperoxide. This substrate is preferable to H2O2 which gives too high blank values compared to the assay values. The difference between total glutathione peroxidase and selenium-dependent glutathione peroxidase activities represents the non-selenium-dependent glutathione peroxidase activity. Studies of substrate specificity of the two glutathione peroxidases separated by gel filtration as well as linearity and recovery studies are presented. For a given tissue, the relative amounts of the two glutathione peroxidases given by our assay are identical to those estimated by quantifying the elution peaks after gel filtration. Based on the percentages of the two glutathione peroxidases, human tissues can be classified in four groups: (1) the non-selenium-dependent glutathione peroxidase is predominant in liver, in renal cortex and skeletal muscle; (2) non-selenium-dependent and selenium-dependent glutathione peroxidases are in equal amounts in renal medulla; (3) the selenium-dependent glutathione peroxidase is predominant in adrenal glands and platelets; (4) the selenium-dependent glutathione peroxidase represents 100% of the glutathione peroxidase activity in the other organs. The heart and the brain are of special interest in this group because of the physiological role and the regulation of the selenoenzyme.

Chromatography, Gel↗
PubMed1994

Cell death of spinal motoneurons in the chick embryo following deafferentation: rescue effects of tissue extracts, soluble proteins, and neurotrophic agents.

In the absence of descending spinal and supraspinal afferent inputs, neurons in the developing lumbar spinal cord of the chick embryo undergo regressive changes including cellular atrophy and degeneration between embryonic days 10 and 16. There are significant decreases in the number of motoneurons, interneurons, and sensory (dorsal root ganglion) neurons. Although there are several possible explanations for how afferents might regulate the maintenance of neuronal viability, we have focused attention on the putative role of neurotrophic agents in these events. Previous studies have shown that specific tissue extracts (e.g., muscle, brain), soluble proteins, growth factors, and trophic agents can promote the in vitro and in vivo survival of avian motoneurons during the period of natural cell death (embryonic days 6-10). Several of these agents were also effective following deafferentation. These included brain extract (BEX), muscle extract (MEX), conditioned medium from astrocyte cultures (ACM), as well as the following neurotrophic agents: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), S-100, insulin-like growth factor-I (IGF-I), ciliary neurotrophic factor (CNTF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (CDF/LIF). Both transforming growth factor-beta (TGF-beta) and acidic fibroblast growth factor (aFGF) were ineffective. Although considerable more work is needed to determine which (and how) specific CNS-derived trophic agents regulate motoneuron survival, the present results are consistent with the notion that neurotrophic agents released from or modulated by synaptic inputs to target neurons promote neuronal differentiation and survival in the CNS.

Afferent Pathways↗
PubMed1975

Carcinoembryonic antigen (CEA) of human tissue extracts: partial characterization of two variants separated by affinity chromatography on concanavalin A.

The carcinoembryonic antigen (CEA) of normal and pathological tissue extracts was separated into two variants, Concanavalin A reactive (CEAr) and non-reactive CEA (CEAn) by affinity chromatography on Con A Sepharose columns. CEAr was the quantitatively predominant variant. CEAn varied in concentration between 0.2 and 6 percent of the total CEA activity. The affinity of CEAn for anti-CEA antibodies was significantly lower than that of CEAr. Pooled extracts of primary adenocarcinomas of the colon contained CEAn in the lowest concentration and with the least affinity for antibodies. It is suggested that a deficiency and/or steric blocking of alpha-D-mannopyranosyl residues in CEAn reduce the affinities for both antibodies and Con A.

Adenocarcinoma↗
PubMed1971

Helper activity of human leukemic tissue extracts for leukemia virus expression in mice.

Extracts of cultured human leukemic tissues increased the spleen focus-forming activity of Friend leukemia virus preparations in BALB/c and other partially resistant mice. Such mice carry the Fv-1(b) gene, which inhibits the expression of helper virus indigenous to the Friend virus complex, and allows co-infecting leukemia viruses of mice, cats, or chickens to substitute for the inhibited helper virus and increase the expression of the spleen focus-forming virus. The helper activity of extracts from human leukemic tissues shared several important properties with that associated with known leukemogenic viruses of animals.

Animals↗
PubMed1985

Radioimmunoassay for neuropeptide Y (NPY): chromatographic characterization of immunoreactivity in plasma and tissue extracts.

A sensitive and specific radioimmunoassay was developed to determine the occurrence and concentration of neuropeptide Y (NPY) in plasma and tissue extracts. Furthermore, NPY-like immunoreactivity (NPY-Li) was characterized by means of three different chromatographic systems. The NPY antiserum used (NI) did not cross-react with related peptides of the pancreatic polypeptide family except avian pancreatic polypeptide (1% cross-reactivity). Unextracted plasma contained high molecular weight proteins which interfered in the assay. Acid ethanol extraction removed this protein interference allowing a 90% recovery of NPY-Li. The content of NPY-Li in human plasma from healthy subjects was close to or below the detection limit (less than 22 pmol/l). Sympathetic nerve stimulation in the cat increased the output of NPY-Li from the splenic vein suggesting the release of this peptide upon sympathetic activation. The major peak of NPY-Li in spleen extracts and splenic vein plasma co-eluted with synthetic porcine NPY and a minor peak with larger Stokes radius was also present. The present radioimmunoassay enables further studies on the physiological and pathophysiological role of NPY.

Animals↗
PubMed1975

[The diangostic value of skin tests with tissue-extracts, especially in lepra and sarcoidosis (author's transl)].

The immunological aspects of lepra (India, Germany) and Sarcoidosis are presented under consideration of cutaneous reaction to lepromin and Kveim-extracts. Patients with lepra, erythema nodosum, sarcoidosis and different dermatoses have been tested with lepromin, Kveim-Antigen and tuberculin. The specifity of the results has been discussed. Patients with tuberculoid lepra did not react on Kveim-test, but patients with sarcoidosis can give positive reaction on lepromin. Patients with erythema nodosum without clinical signs of sarcoidosis are Kveim negativ and a positive lepromin-test is an exception. The positive Kveim-test is bound to a histological examination. The positive lepromin-test in non lepraendemic countries should be interpreted also by histological examination. Granulomatous dermatoses can show positive lepromin-tests, but the histological picture is different from the lepromin reaction in tuberculoid lepra. The positive reactions on tissue extracts are not combined with positive immunfluorescence results. Intracutaneous injection of lepra-bacilli in lepromin extract can be presented days to weeks after intracutaneous application in biopsies.

Antigens↗
PubMed2003

Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signaling lipids in cell and tissue extracts.

We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotinylated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay procedure was validated using recombinant PI 3-kinase gamma with diC8PtdIns(4,5)P(2) as substrate and general receptor for phosphoinositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P(3)-specific probe. This PI 3-kinase assay was robust, was suitable for high-throughput screening platforms, and delivered expected IC(50) values for reference compounds. The approach is adaptable to a wide range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was measured using the same reagents but with diC8PtdIns(3,4,5)P(3) as substrate. PtdIns(3,4,5)P(3) present in lipid extracts of Swiss 3T3 and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity. The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1 (which binds PtdIns(3,4)P(2) and not PtdIns(3,4,5)P(3)). This system was used to monitor the accumulation of PtdIns(3,4)P(2) in Swiss 3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-characterized protein domains with appropriate phosphoinositide-binding specificity.

3T3 Cells↗
PubMed1998

Monoamines, amino acids and acetylcholine in the preoptic area and anterior hypothalamus of rats: measurements of tissue extracts and in vivo microdialysates.

A microbore column high-performance liquid chromatography (HPLC) system was used to measure neurotransmitters in tissue extracts and in vivo microdialysates obtained from the preoptic area (PO) and anterior hypothalamus (AH) of rats. The extracts contained norepinephrine, epinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine, 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), aspartate, glutamate, GABA, acetylcholine (ACh) and choline. The microdialysates obtained from the PO and AH of freely moving rats contained all of these substances except for norepinephrine, epinephrine, dopamine, and 5-HT. During collection of microdialysate from the PO and AH, core body temperature and locomotor activity were simultaneously measured by means of telemetry. The locomotor activity and body temperature increased during the night. This was accompanied by increased levels of 5-HIAA. The results suggest that serotonergic neuronal mechanisms in the PO and AH may be involved in hypothalamic regulation of spontaneous behaviors and body temperature.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1998

External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunosorbent assay) of urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) in human breast cancer tissue extracts.

High levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in breast cancer tissue extracts have been associated with rapid disease progression. In these studies, different enzyme-linked immunosorbent assay (ELISA) kits have been applied for the quantification, and consequently the ranges of uPA and PAI-1 levels reported differ considerably. Therefore, the Receptor and Biomarker Study Group (RBSG) of the European Organization for Research and Treatment of Cancer (EORTC) and a consortium of the BIOMED-1 project 'Clinical Relevance of Proteases in Tumor Invasion and Metastasis' initiated three collaborative between-laboratory assessment trials aimed at controlling uPA and PAI-1 antigen analyses. For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-101094). The lyophilized preparations were stable for prolonged times (at least 3 and 27 months respectively) at 4 degrees C. Furthermore, a good parallelism following dilution was found for uPA and PAI-1. The data from QC trial no. 1 clearly indicated that acceptable between-laboratory coefficients of variation (CVs) for uPA (<8.2%) and PAI-1 (<16.6%) in QC-240893 could be achieved when the same type of ELISA kit (American Diagnostica) was used. From the second trial, in which ten EORTC laboratories each received five identical lyophilized QC-101094 samples, it appeared that the within-laboratory variations for uPA and PAI-1 determinations obtained by 'experienced' laboratories were lower (<12.9%) than those from non-experienced laboratories (<36.4%). In a third QC trial, five BIOMED-1 laboratories, all of which employed ELISA procedures for uPA and PAI-1, participated in six subsequent quality assessment rounds receiving five samples of QC-101094. Although for each laboratory the within-run CVs for uPA as well as for PAI-1 were low (<7.8%), the between-run CVs were found to be considerably higher (up to 56.2% for uPA and to 27.6% for PAI-1). Consequently, because of the different ELISA formats used, the absolute analyte values measured in the different laboratories varied substantially. The use of 'common external standards' in the different ELISAs resulted in a significant reduction of the between-laboratory CVs from 61.3% to 15.7% (uPA) and from 42.1% to 19.1% (PAI-1). The present data demonstrate that in multicentre studies the same ELISA kit should be used, and that external quality assurance (QA) is mandatory. Furthermore, it appears from the present study that standardization of the protein assay as a tissular parameter is imperative.

Animals↗
PubMed1990

Towards large-scale purification of natural CSF-1 from human placenta tissue extracts.

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.

Blotting, Western↗
PubMed1984

Use of high-performance liquid chromatography in the analysis of retinyl and 3,4-didehydroretinyl compounds in tissue extracts of bullfrog tadpoles and goldfish.

HPLC (high-performance liquid chromatography) was used to analyse retinyl and 3,4-didehydroretinyl compounds in tissue extracts from goldfish and bullfrog tadpoles. Using silica columns (packed with 10-micron mu Porasil or 5-micron Ultrasphere particles) eluted with n-hexane (containing a small amount of dioxane or diethyl ether), the authentic all-trans retinyl and 3,4-didehydroretinyl palmitates, retinal and 3,4-didehydroretinal, retinol and 3,4-didehydroretinol were completely separated. Liver and eye extracts of the goldfish and bullfrog tadpoles had mainly esterified all-trans retinol and all-trans 3,4-didehydroretinol. In the liver, these vitamin A were conjugated to a number of fatty acids whereas in the eye, principally one fatty acid was used. Moreover, the relative proportions of all-trans retinol and all-trans 3,4-didehydroretinol (obtained by analysing the saponified esters) were significantly different between some of these body compartments.

Animals↗