Paul M. Bronson

Opsonic properties of human serum alpha-2 hs glycoprotein.

Human serum α2HS glycoprotein (α2HS) has been isolated to a purity of more than 95%. At a concentration of 0.1%= α2HS significantly enhances the phagocytosis of Escherichia coli and of Staphylococcus aureus by human neutrophils in vitro. At the same concentration, α2HS causes an increase in the hydrophobicity of these microorganisms, as measured by the sessile saline drop contact angle method. The level of α2HS is much decreased in the sera of trauma patients. Alpha-2 HS may be the human homologue of the recently described rat α2 opsonic protein (1).

The valency of IgM and IgG rabbit anti-dextran antibody as a function of the size of the dextran molecule

Abstract The valency of IgM and IgG anti-dextran antibody was studied as a function of the size of the ligand. Scatchard plots were made utilizing 3H-labeled NRRL B-512 dextran fractions of molecular weights varying from 342 to 1,870,000. The size and shape of the dextran molecules were found from weight average molecular weight (Mw), number average molecular weight, sedimentation, diffusion, partial specific volume and viscosity data. Effective valency was found to be a function of the molecular size of the dextran used in its determination. The valency of IgM varied between 10 (for Mw 342) through 5 (for Mw 7100 to 237,000) down to 2·3 (for Mw 1,870,000) and the valency of IgG varied between 2 (from Mw 342 to 492,000) and 1·1 (for Mw 1,870,000). Since all dextran molecules, regardless of molecular weight are otherwise of the same physical-chemical configuration, our results substantiate the theory that steric hindrance plays a dominant role in determining the valency of IgM as well as of IgG antibody with respect to a ligand.

Studies on thyroid proteins: II. Purification of human thyroid proteins by gel filtration and the isolation of 19-S, 7-S, and 4-S components

Abstract A procedure is described by which the major soluble proteins of normal human thyroid tissue can be separated. Gel filtration on Sephadex G-200 yields three fractions. The first eluted fraction (A) contains samples of well-purified thyroglobulin. Each of these samples also contains varying proportions of the 34-S, 27-S and 12-S thyroid proteins. Some of the samples from Fraction A contain only the 27-S and heavier proteins. The second fraction (B) contains nearly pure 7-S thyroid protein, as judged by ultracentrifugal analysis. By this same criterion, the third fraction (C) has essentially pure 4-S material, designated thyralbumin. The components in Fraction A cannot be further separated by paper curtain electrophoresis. The 7-S protein in Fraction B is, however, separated into nearly equal amounts of two subfractions by curtain electrophoresis. Likewise, the 4-S protein in Fraction C is separated into two nearly equal amounts. These procedures have separated eight major protein fractions in human thyroid extract. Furthermore, one of the 7-S protein subfractions and both of the 4-S subfractions have been shown to contain thyroid-specific antigenic activity.

Cryo-Immunology A Method of Immunization to Autologous Tissue.

Summary By means of a blind test, in which serum samples from a group of rabbits were relabeled with code designations, it was shown clearly that definite antibody production had occurred as a result of freezing of tissue in the animal. Freezing was done by means of a liquid nitrogen probe and the target tissue was the coagulating gland and seminal vesicle of the urogenital tract. Antibodies were detected by tanned cell hemag-glutination and were found to be present within 7 days after cryosurgery. No antibody was detected in any of 15 sera from 5 control animals, whereas 6 out of 7 rabbits exposed to freezing showed positive results. Titers as high as 1:4096 were obtained, and there was a progressive increase with time.

Phagocytosis-Inhibiting Properties of Human Serum Alpha-1 Acid Glycoprotein

Human serum α1 acid glycoprotein (α1A) or orosomucoid has been obtained in a virtually pure state. At a concentration of 0.1% α1A significantly depressed the phagocytosis of Escherichia coli and of Staphylococcus aureus by human neutrophils in vitro. At the same concentration α1A causes a decrease in the hydrophobicity of these microorganisms, as measured by the sessile saline drop contact angle method. Alpha-1 A, purified from Cohns Fraction V (1), though in most respects identical to our purified product, nevertheless appears somewhat denatured in that its phagocytosis inhibiting properties and its capacity to increase the hydrophobicity of microorganisms are less pronounced. Alpha-1 A is one of the constituents of Mowbrays rat serum fraction which induces a prolongation of skin homografts (2,3).

Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase.

Matrix metalloprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (—660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

Characteristics of a Protein Concentrating Anisotropic Cellulose Acetate Membrane

Abstract A high flux anisotropic (“skinned”) protein concentrating membrane made of cellulose acetate is described. Its method of preparation is given and the influence of the thickness of the membrane on its flux determined. The membranes characteristics under different pressures and when ultrafiltering protein solutions of varying concentration are studied. A slightly denser variant of the same membrane, useful for the concentration of some of the smaller pathological serum proteins, is also described. The characteristics of both new membranes are compared with those of some of the commercially available membranes.

The allergic response to stinging insects. VII. Fractionation of whole body and venom sac extracts of yellow jacket

Abstract Extracts of whole body and of venom sac of yellow jackets (Vespula pennsylvanicus) were fractionated by DEAE-cellulose chromatography. Ten fractions were separated from whole body, and seven fractions from venom sac preparations. Only fraction 1 of venom sac was active in direct skin testing. While several fractions of whole body were active by skin testing, only one of them, fraction 1, was significantly active in a patient who was sensitive to yellow jacket only. Other fractions, therefore, reflected the cross-reaction between the different insects. The antigenic heterogeneity of each fraction was analyzed by appropriate rabbit antisera to yellow jacket extracts. Eight of the ten body fractions gave precipitation, as did three of the seven venom sac fractions. They variously gave one or two lines of precipitation. In addition, fractions 6, 7, and 8 of whole body showed, by testing with rabbit antisera, common reactions with wasp and bee. An analysis of the studies comparing the findings of bee, wasp, and yellow jacket extracts and fractions with each other is presented at the end of this paper.

Preparative cell electrophoresis with D2O as a stabilizing agent.

The method of ascending preparative cell electrophoresis in a nonstabilized vertical column can be greatly improved by layering the cells on a starting cushion of buffer prepared in D2O and by stabilizing the liquid column above it with a D2O gradient. D2O/H2O mixtures have no apparent biochemical effects on the cells, and their physiochemical effects are short-lived and reversible. It was possible, by this method, to separate a mixture of glutaraldehyde-fixed erythrocytes of three species (rabbit, horse and chicken) into three distinct zones after 15 minutes of electrophoresis. It was also possible to obtain an enriched human lymphocyte fraction containing 96% T-cells, after 1 hour of electrophoresis.

Preparative Electrophoresis of Human Lymphocytes I. Purification of Non-Immunoglobulin-Bearing Lymphocytes by Electrophoretic Levitation

Abstract When 10–30 × 106 human peripheral lymphocytes are electrophoresed in an upward direction in a vertical column for 1 to 1–1/2 hour at 12 V/cm at pH 7.1, the fastest migrating fraction of 3–10 × 10 lymphocytes consists of 98–100%. non-immunoglobulin-bearing lymphocytes, as determined by immunofluorescence with anti-human immunoglobulin conjugate. The method can be applied to fresh human lymphocytes as well as to lymphocytes that have been frozen and thawed and, if glycerol is added to the buffer as a cryoprotectant, the fast “T” cell fraction can be frozen immediately, to be stored for later use. Similar separations can be obtained with lymphocytes from human tonsils.