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PubMed1979

Correlated expression of a B-lymphocyte-specific glycoprotein (gp27/35) and the EBV receptor/C3 receptor complex in sublines from the same Burkitt lymphoma.

A series of virus-producer and non-producer sublines, derived from the Burkitt lymphoma line Jijoye and its P3HR-1 clone, were previously found to differ in the expression of EBV receptors and, in parallel, C3 receptors. The differences could be related to an "internal selection" caused by the cytopathic P3HR-1 virus variant, favouring the growth of receptor-negative cells. We have now analyzed the same lines for the expression of a B-lymphocyte-specific glycoprotein (gp27/35). A close parallellism was found between the expression of the EBV receptor-complement receptor complex and the glycoprotein, measured by quantitative absorption. The results favour a relationship between gp27/35 and EBV/C3 receptor expression.

Antigen-Antibody Complex↗
PubMed1987

Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation.

The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.

Antibodies, Bacterial↗
PubMed1975

The defined antigen substrate spheres (DASS) system and some of its applications.

A quantitative immunofluorescent technique based on the covalent coupling of protein to Sepharose beads, the so-called defined antigen substrate spheres (DASS) system is described, and the main technical aspects are discussed. The method proved to be reproducible and has a lower detection limit equivalent to the amount of antigen, present per bead, that can bind 1 pg fluorescent labeled antibody. The DASS system has many applications, and some of them are discussed, such as the quantitation of antibodies and antigens with Sepharose-coupled antigens or antibodies. Not only antigen-antibody interactions but also any reaction that results in a stable complex can be studied, provided antisera against one of the constituents are available. An example is given by the complement fixation to Sepharose-coupled IgG. For the quantitation of hydrophobic antigens, hydrophobic Sepharise can be used.

Antibodies↗
PubMed1984

Radioimmunoassay for anaphylatoxins: a sensitive method for determining complement activation products in biological fluids.

Activation of the blood complement system generates bioactive fragments called anaphylatoxins. The three anaphylatoxins C3a, C4a, and C5a are released during "classical pathway" activation while only C3a and C5a are released when the "alternative pathway" of complement is activated. Radioimmunoassays were designed to individually detect and quantitate the activation fragments C3a, C4a, and C5a in biological fluids without interference from the precursor molecules C3, C4, and C5. Kinetics of complement activation in fresh human serum exposed to the activators zymosan, heat-aggregated immunoglobulin, or cobra venom factor were monitored using the radioimmunoassay technique. For the first time, activation of components C3, C4, and C5 was followed simultaneously in a single serum sample. Analysis of the patterns and extent of anaphylatoxin formation during activation in serum may be used to screen for deficiencies or defects in the complement cascade. Levels of the anaphylatoxins in freshly drawn serum were much higher than levels detected in EDTA-plasma. Detection limits of anaphylatoxins in plasma are governed by background levels of 152 +/- 69, 155 +/- 33, and 5.4 +/- 6.6 ng/ml for C3a, C4a, and C5a, respectively. Detection of low-level complement activation in patient's blood, urine, or synovial fluid, using anaphylatoxin formation as an indicator, may prove useful in signaling numerous forms of inflammatory reactions. The demonstration of anaphylatoxins in clinical samples is being recognized as a valuable diagnostic tool in monitoring the onset of immune disease.

Anaphylatoxins↗
PubMed1998

Escherichia coli bound to the primate erythrocyte complement receptor via bispecific monoclonal antibodies are transferred to and phagocytosed by human monocytes in an in vitro model.

We have prepared cross-linked, bispecific mAb complexes (heteropolymers) that facilitate rapid and quantitative binding of a prototype pathogen, Escherichia coli, to the complement receptor (CR1) on primate erythrocytes. Incubation of the erythrocyte-heteropolymer-E. coli complexes with freshly isolated human mononuclear cells leads to rapid removal of the E. coli from the erythrocytes, and phagocytosis and killing of the bacteria. The erythrocytes are not lysed or phagocytosed during this transfer reaction, but both heteropolymer and CR1 are removed from the erythrocytes along with the E. coli. These findings parallel observations made in previous in vivo experiments in which heteropolymers were used to facilitate clearance of innocuous prototype pathogens in a monkey model. It should now be possible to extend the heteropolymer paradigm to a live pathogen in a primate model.

Animals↗
PubMed1995

Dimensional growth and extracellular matrix accumulation by neonatal rat mandibular condyles in long-term culture.

Mandibular condyles in organ culture commonly have been used as a model system for examination of the factors that influence skeletal growth and development. The work reported here complements previously published histological studies by providing quantitative temporal information on growth and matrix accumulation. Condyles maintained for as long as 5 weeks in serum-free and 1% serum-supplemented culture media were found to remain viable and metabolically active as demonstrated by continued dimensional growth as well as cell and matrix accumulation. Growth occurred by a combination of cell proliferation, matrix synthesis and accumulation, and cell hypertrophy (with the latter two mechanisms dominating). Increases in tissue volume correlated directly with increased glycosaminoglycan content; both increased 7-fold over 5 weeks. In comparison with serum-free culture, after 35 days in medium containing 1% serum, glycosaminoglycan content was 24% lower and collagen content was 36% higher, whereas dry weight, condyle length, and DNA content were not significantly different; in addition, histological observation suggested that, for samples cultured with serum, chondrogenic phenotype had been lost from some regions. The temporal behavior for all growth parameters exhibited a transient phase 1-2 weeks in duration followed by a steady-state period in which dimensions and tissue constituents or content increased at a constant or near constant rate. Comparison of the rates of incorporation of [35S]sulfate with glycosaminoglycan content in serum-free cultures suggests that the loss of glycosaminoglycan occurs only initially or is negligible; therefore, under these baseline conditions, cartilage glycosaminoglycan content reflects the biosynthetic rate. The high degree of reproducibility seen during steady-state growth suggests that these data provide reliable baseline information and further supports the notion that this model system is useful for investigation of the effects of specific physical factors on in vitro growth and development.

Animals↗
PubMed1987

Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd1) among denitrifying pseudomonads.

The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques. Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization. The assignment of P. perfectomarina to the stutzerigroup (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase. Crossreactivity of nitrite reductase (cytochrome cd1) with a respective P. perfectomarina rabbit antiserum was limited to strain DSM 50227 of P. stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species.

Complement Fixation Tests↗
PubMed1976

[Immunological investigations in lichen planus patients (author's transl)].

30 patients suffering from generalized lichen planus (= L.p.) have been examined by the following methods in order to more precise information regarding their immune-status: 1. Quantitative determination of immuno-globulins including complement fraction (beta 1C/A) 2. Intracutaneous tests with recall-antigens 3. Investigation of cutaneous sensitivity to DNCB 4. Lymphocytemigrationinhibition- and Lymphocytetransformationstests. The results obtained were not statistically significant when comparing the results of this investigation in patients suffering from L.p. with the control group. Therefore, the theory that immunological processes are the basis of these occurences is still under discussion. Further studies applying more specific methods should be conducted in order to clarify this problem.

Adult↗
PubMedDate not supplied

A model of adaptive visual processes of primary image processing.

A computer model of adaptive segmentation of two-dimensional visual objects was developed, based on neurophysiological and psychophysiological principles. The model imitates several stages of visual information processing. At the first stage, a preliminary assessment of the image is performed using a brightness pattern analyzer. At the second stage, control parameters are formed on the bases of the initial assessment. A defined control vector is synthesized for each type of starting image, which allows adaptive processing; this initiates two parallel mechanisms of primary image description: one contains the outlines of images and the sharp boundaries between their fragments, and the second contains areas of uniform intensity. The control parameter vector is applied to these two descriptions to analyze their brightnesses and spatial characteristics, and this is used as the basis for forming the regime for subsequent processing, which includes a set of processing operators whose parameters are tuned for each fragment of the image. The primary phasic and tonic descriptions are then used to extract individual fragments of the image (i.e., discrimination of figures from the background) and to form the final presentation. The resulting descriptions complement each other, creating a basis for quantitative measurements of image characteristics and allowing various signs needed for image classification to be formed. The computer program for adaptive image segmentation was tested using a large number of different two-dimensional half-tone objects, the purposes of these exercises including segmentation and measurement of objects in morphometric and cytometric studies.

Adaptation, Physiological↗
PubMedDate not supplied

Images from Waves--photoelastic modelling of bones. 8th Samuel Haughton Lecture, Bioengineering Section of Royal Academy of Medicine in Ireland. January 2002.

BACKGROUND: This paper cites the development of the principles of photoelastic stress analysis, contemporary to the life of Samuel Haughton. Subsequent studies of bone and joint replacements are discussed, with reference to hypotheses regarding bone, including the coincidence of trabecular structure with principal stresses. Issues regarding assumptions of homogeneous and isotropic properties in photoelastic modelling are acknowledged. AIM: Awareness of photoelastic methods is often through the visual appeal of the coloured fringe patterns. The aim of this paper is to complement this awareness by demonstration of the quantitative analyses that may be conducted through biomechanical examples. METHODS: Examples of new pseudo three-dimensional model analyses are presented together with a method for photoelastic study of cancellous bone, which entails novel procedures for preparation of replicate models and for optical evaluation of fringes. CONCLUSION: Photoelastic analysis offers novel solutions to studies in biomechanics, which are facilitated by contemporary modelling materials.

Animals↗
PubMed2005

Comparison of rapid liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of glycoalkaloids in transgenic field-grown potatoes.

Two rapid methods for highly selective detection and quantification of the two major glycoalkaloids in potatoes, alpha-chaconine and alpha-solanine, were compared for robustness in high-throughput operations for over 1000 analytical runs using potato tuber samples from field trials. Glycoalkaloids were analyzed using liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode. An electrospray interface was used in the detection of glycoalkaloids in positive ion mode. Classical reversed phase (RP) and hydrophilic interaction (HILIC) columns were investigated for chromatographic separation, ruggedness, recovery, precision, and accuracy. During the validation procedure both methods proved to be precise and accurate enough in relation to the high degree of endogenous biological variability found for field-grown potato tubers. However, the RP method was found to be more precise, more accurate, and, more importantly, more rugged than the HILIC method for maintaining the analytes' peak shape symmetry in high-throughput operation. When applied to the comparison of six classically bred potato cultivars to six genetically modified (GM) lines engineered to synthesize health beneficial inulins, the glycoalkaloid content in potato peels of all GM lines was found within the range of the six cultivars. We suggest complementing current unbiased metabolomic strategies by validating quantitative analytical methods for important target analytes such as the toxic glycoalkaloids in potato plants.

Agriculture↗
PubMed2005

SAGE and the quantitative analysis of gene expression in parasites.

The nature of an organism is defined by the genes that it expresses. Genome- and expressed-sequence-tag (EST) sequencing projects are underway for many of the major parasites of humans and animals. These provide essential datasets that delineate the genes present in an organism and, in the case of ESTs, some quantitative information on gene expression. The temporal and quantitative analysis of gene expression is essential to fully exploit these datasets and define the biology of the parasite at the molecular level. Here, we discuss the application of serial analysis of gene expression (SAGE) for this purpose. SAGE is a technique that allows the rapid, quantitative analysis of thousands of transcripts. It complements microarray analysis with the advantage that it is affordable for standard laboratories. It provides a platform to define complete metabolic pathways and has been applied to study responses to drug treatment and the molecular events that are associated with arrested larval development.

Animals↗
PubMed2000

Yeast surface display for directed evolution of protein expression, affinity, and stability.

The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks. Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.

Amino Acid Sequence↗
PubMedDate not supplied

Determination of atrazine metabolites in human urine: development of a biomarker of exposure.

Enzyme-linked immunosorbent assays (ELISAs) are reported for the detection of atrazine and its principle metabolite in human urine. The ELISAs can be used with crude urine or following extraction and partial purification by methods described in this report. GC, MS, and HPLC techniques were used to confirm and complement the ELISA methods for qualitative and quantitative detection of urinary metabolites. A series of samples from workers applying this herbicide confirmed a mercapturic acid conjugate of atrazine as a major urinary metabolite. The mercapturate was found in concentrations at least 10 times that of any of the N-dealkylated products or the parent compound. Atrazine mercapturic acid was isolated from urine using affinity extraction based upon a polyclonal antibody for hydroxy-s-triazines and yielded products sufficiently pure for structure confirmation by MS/MS. In a pilot study monitoring applicators, a relationship between cumulative dermal and inhalation exposure and total amount of atrazine equivalents excreted over a 10-day period was observed. On the basis of these data, we propose that an ELISA for the mercapturate of atrazine could be developed as a useful marker of exposure.

Acetylcysteine↗
PubMed1994

Alloimmune neonatal neutropenia resulting from immunization to a high-frequency antigen on the granulocyte Fc gamma receptor III.

BACKGROUND: Alloimmune neonatal neutropenia is mainly caused by NA- or NB1-specific alloantibodies. An antibody in the serum of a Turkish mother who had given birth to an infant with alloimmune neonatal neutropenia showed no NA or NB specificity and was therefore investigated further. STUDY DESIGN AND METHODS: The number of antibody-binding sites was calculated by determination of elutable IgG from granulocytes using a quantitative sandwich enzyme-linked immunosorbent assay. Complement activation was tested by immunofluorescence (C3d) and cytotoxicity tests. The antigen was identified using the antigen-capture assay, monoclonal antibody-specific immobilization of granulocyte antigens, and a modified immunoprecipitation method based upon biotinylation of proteins and visualization by luminescence (luminoimmunoprecipitation). Family study and determination of antigen frequency were done by immunofluorescence and agglutination tests. RESULTS: A noncytotoxic, granulocyte-specific alloantibody that recognized the Fc gamma receptor III, independent of the NA phenotype, was detected, and 242,000 binding sites per cell were calculated. Of granulocytes from 150 randomly selected German blood donors, the alloantibody bound to all. The maternal cells were typed NA1/NA2- and NB1-positive. CONCLUSION: These data reveal the presence of a previously unrecognized, high-frequency epitope on the granulocyte Fc gamma receptor III. Luminoimmunoprecipitation proved to be a simple, nonradioactive technique that was useful in identifying the molecule involved.

Antigens↗
PubMed2006

Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana.

Vitamin E is an essential nutrient for humans and is obtained primarily from food, especially oil, derived from the seed of plants. Genes encoding the committed steps in vitamin E synthesis in plants (VTE, loci 1-5) have been isolated and used for tocopherol pathway engineering with various degrees of success. As a complement to such approaches we have used quantitative trait loci analysis with two sets of Arabidopsis thaliana recombinant inbred lines and have identified 14 QVE (quantitative vitamin E) loci affecting tocopherol content and composition in seeds. Five QVE intervals contain VTE loci that are likely QVE gene candidates. Nine QVE intervals do not contain VTE loci and therefore identify novel loci affecting seed tocopherol content and composition. Several near-isogenic lines containing introgressions of the accession with increased vitamin E levels were shown to confer significantly elevated tocopherol levels compared with the recurrent parent. Fine-mapping has narrowed QVE7 (a gamma-tocopherol quantitative trait loci) to an 8.5-kb interval encompassing two genes. Understanding the basis of the QVE loci in Arabidopsis promises to provide insight into the regulation and/or metabolism of vitamin E in plants and has clear ramifications for improving the nutritional content of crops through marker-assisted selection and metabolic engineering.

Arabidopsis↗
PubMed1979

Immunohistologic evaluation of the lymphoreticular infiltrate of human central nervous system tumors.

Forty-five nervous system tumors (9 glioblastomas, 9 meningiomas, 15 assorted primary neural tumors including 3 medulloblastomas, and 12 brain tumors metastatic to the brain were analyzed for their content of lymphocytes, granulocytes, and macrophages. Cell suspensions were prepared by enzymatic digestion; lymphocytes and granulocytes were quantitated by morphology following cytocentrifugation, and macrophages were quantitated by IgG EAC (erythrocyte-antibody-complement) rosette formation. EA (erythrocyte-antibody) adsorption to sections of tumor was employed to determine the distribution of the IgG Fc receptor-positive cells within the tumors and to serve as quality control for selective release of Fc receptor-positive cells by enzyme digestion. The 9 glioblastomas had a mean macrophage content of 41% (range: 5-78%); the 9 meningiomas, 42% (range: 5-80%); the 3 medulloblastomas, 6% (range: 2-15%); and the metastatic tumors, 21% (range: 2-50%). Lymphocyte contents were variable but generally less than 10%. Most tumors contained less than 10% granulocytes. EA adsorption demonstrated that Fc receptor-positive cells were distributed throughout the tumor mass, although different types of patterns were observed. There was an excellent correlation between the percent EAC rosette-positive cells in suspensions and the extent of EA adsorption to the tumor sections. The significance of the study primarily rests in the demonstration that most nervous system tumors contain high numbers of infiltrating host cells, primarily macrophages.

Brain Neoplasms↗
PubMed1983

Visual field and retinal nerve fibre layer in early glaucoma after optic disc haemorrhage.

Computerized visual field testing with the Competer automatic perimeter including automatic profile perimetry revealed early glaucomatous field loss in 9 of 10 eyes with photographically demonstrable retinal nerve fibre layer (RNFL) defects after optic disc haemorrhages despite the fact than conventional routine perimetry had failed to do so. In 7 of these cases the Competer central pattern test was abnormal; in 2 cases field defects could be shown only when automatic high-resolution static profile perimetry was performed and directed according to the information present in wide-angle RNFL photographs. A field defect was also found in one of five eyes with no photographic RNFL changes. Visual field defects seem to occur in a higher proportion of cases with RNFL defects than was previously thought, but they can be very subtle and hard to find. There are, however, cases where in spite of the presence of an indisputable RNFL defect no visual field defects can be shown even with painstaking technique and conversely field defects may be present without RNFL defects. Automatic perimetry and RNFL photography complement each other in early detection and quantitation of glaucomatous damage.

Aged↗