Chemical and physical properties of a hydrogel wound dressing.

Geliperm hydrogel provides optimal physiological conditions for wound healing. The material is composed of two interlaced networks, one of polyacrylamide and one of agar, and contains about 96% firmly bound water. It is supplied in smooth, elastic, transparent sheets which are impermeable to bacteria but permeable to gases, salts, metabolites and proteins. Geliperm is nontoxic and has no irritative properties. Mechanical properties, water retention and diffusion of dyes and proteins are reported. Bacterial size should preclude penetration of the gel. The hydrogel in granular form represents a coherent material which could be used in deep fissured wounds and for the treatment of injuries with a large amount of exudation and contamination.

Skin reactivity of unsensitized monkeys upon challenge with staphylococcal enterotoxin B: a new approach for investigating the site of toxin action.

The correlation between skin tests and emetic responses in unsensitized monkeys was used to elucidate the cellular site of action of staphylococcal enterotoxin B (SEB). Evidence is presented that SEB administered intradermally provoked immediate-type skin reactions associated with mild degranulation of cutaneous mast cells. The cytoplasma showed signs of synthetic and metabolic activity, with formation of vesicles and increased prominence of mitochondria. Carboxymethylation of histidine residues of SEB altered the molecule (cSEB) from more alkaline components to more acidic species with increased microheterogeneity. This modification caused a loss in toxicity and completely abrogated the skin-sensitizing activity without changing the immunological specificity. cSEB, however, could compete with SEB for binding sites on the target cell surface. Previously, compound 48/80-treated skin sites behaved refractively to challenge with SEB, indicating that mediators from cutaneous mast cells are required for SEB-induced skin reactions. Skin reactions as well as emetic responses challenged with SEB were completely inhibited by H2 receptor antagonists and calcium channel blockers but not by H1 antihistamine or competitive antagonists of serotonin. This new approach provides a model for investigating the mechanisms of SEB action.

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.

Partial purification and chemical characterization of macrophage cytotoxicity factor (MCF, MAF) and its separation from migration inhibitory factor (MIF).

Macrophage cytotoxicity factor (MCF) was purified in 3 consecutive steps including adsorption chromatography on Matrex Gel Red A, hydrophobic chromatography on phenylalanine-Sepharose, and isoelectric focusing. MCF was characterized as a protein with a m.w. of approximately 30,000 by gel filtration on Sephadex G-100 with 2 isoelectric points at 7.4 and 8.4 in the presence of urea. The unpurified supernatant was fairly stable provided that manipulations favoring adsorption to membrane materials used for dialysis or ultrafiltration were omitted. The partially purified preparation was highly unstable. Trypsin treatment did not affect MCF activity, whereas chymotrypsin destroyed it. Treatment with glycosidases and neuraminidase or cultivation of cells in the presence of 2-deoxy-D-glucose or tunicamycin did not impair the MCF activity. MCF was separated from migration inhibitory factor (MIF) by 2 methods: first, isoelectric focusing in the presence of urea, and second by gel filtration on Ultrogel. MCF could be separated from interferon by chromatography on poly(I)-Sepharose.

Chemical characterization of macrophage cytotoxicity factor, macrophage migration inhibitory factor, T-helper cell-replacing factor and colony-stimulating factor from culture supernatants of concanavalin A-stimulated murine spleen cells.

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatograhy of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.

Characterization of macrophage cytotoxicity factor (MCF).

Allogenetically sensitized T lymphocytes secrete in the presence of the specific antigen a factor which renders macrophages cytotoxic to target cells in vitro, macrophage cytotoxicity factor. Cytotoxicity is expressed as 51Cr release from labeled target cells. Experiments are described to characterize chemically nonspecific MCF activity. After purification on a DEAE-cellulose column, active material elutes from Sephadex G-100 in a single peak. Assaying individual fractions of this peak shows that nonspecific MCF activity is found at a mol. wt. of 28,000+/-5,000. This material is stable at -70 degrees C several months.

[Complex formation between monoclonal IgM and albumin in a patient with macroglobulinaemia (author's transl)]. / Stark ausgeprägte IgM-albumin-Komplexbildung bei monoklonaler IgM-gammopathie

A remarkable interaction between a monoclonal IgM(x) protein and autologous albumin was found in the serum of a 76 year old male patient (Ke.E) without morphological characteristics of Waldenströms disease. The components were bound noncovalently. Immunoelectrophoretic analysis of 50 additional monoclonal IgM and 10 IgA sera showed a complex formation with albumin in 62% (IgM) and in 90% (IgA). The degree of interaction was, however, less pronounced in comparison with that observed in the serum of Ke.E.

A rapid method for dry weight determination of proteins on a micro scale with an electrobalance.

A method to determine the dry weight (0.25-2 mg) of aqueous protein solutions within 1 h, using an electrobalance, is described. The drying of 50-200 mul solution pipetted onto a glass fiber disc is carried out in vacuo at 70 degrees C until the recorded dry weight becomes constant (within 25-40 min). It has been shown that the dried residue can subsequently be used for other purposes, such as quantitative amino acid analyses. The method is also suitable for the determination of moisture content in lyophilized protein samples.

In vitro studies on the selective binding of IgG from different species to tissue sections of the bovine mammary gland.

Tissue sections of the mammary gland of cattle provide a sensitive and reproducible model for studying the initial events of the selective transport of bovine IgGs by the acinar epithelium (AE). Mammary tissue was reacted with purified antibody to horseradish peroxidase (HRP) and exposed to HRP. The sites of peroxidase activity were revealed cytochemically. The highly selective binding of IgGs by AE of the colostrum-forming gland but never of the lactating gland was most notable, whereas IgG1 and IgG2 subclasses and IgM and IgA lacked any binding capacity. An inhibition assay showed that the inhibitory capacity of IgGs was abolished by either alanylation or acetylation, whereas binding of IgGs was blocked by both Fab/c and isolated H-chains, but not by F(ab')2, Fab and Fc. The inhibitory pattern suggests that the region on the IgGs molecule involved in binding to the AE receptor may be located within the CH2 domain. In addition, IgG preparations of human, sheep and rabbit origin were found to be equally efficient in inhibiting the binding of IgGs to AE, whereas no inhibition was obtained with nonmammalian (turtle and chicken) IgG. Similarly, human myeloma proteins of the subclasses IgG1 and IgG3 caused complete inhibition, while IgG2 and IgG4 were inefficient. It is suggested that molecular evolution of IgG within mammalian species is accompanied by the conservation of structures fitting to receptors on the cell surface of different species.

Experimental studies on the bridging hypothesis of anaphylaxis: haptenic determinants required to elicit immediate-type reactions in calf skin by separate or concurrent sensitization with reagins of different specificity.

The present study has established, that cows suitably immunized with either DNP-edestin (DNP-Ed), di-DNP-gramicidin-J [(DNP)(2)-Gram], respectively, or p-azobenzenearsonate-Ed (ABA-Ed) synthesized and secreted reaginic antibodies (IgE) into colostrum. Whereas ABA-Ed failed to elicit more than a low response, there was however a persistent and increased antibody synthesis between 10 and 56 days after priming with DNP-Ed. Bivalent and multivalent DNP haptens differing in molecular size, degree of substitution, and rigidity were compared for their effectiveness in eliciting Prausnitz-Küstner (P-K) reactions in either newborn colostrum-deprived calves or in those 4 wk of age. The sensitization with reaginic anti-DNP antibody has been accomplished either by feeding colostrum of the immunized dam or by intradermal injection of reaginic serum or colostral whey. It could be demonstrated that equimolar doses of the bivalent alpha,N-(epsilon,N-DNP-aminocaproyl-)-epsilon,N-DNP-L-lysine and the multivalent dinitrophenylated bovine serum albumin were equally effective in eliciting reactions in skin sites provided that a high affinity antibody was used for sensitization. By contrast, the comparatively rigid, bivalent hapten, (DNP)(2)-Gram consistently failed to induce comparable reactions. Furthermore, it was clearly shown that optimal distances of determinant groups on the haptenic molecule are a prerequisite for positive P-K reactions, since alpha,epsilon,N-bis-DNP-lysine failed to induce comparable reactions. Concurrent sensitization of skin sites with reaginic anti-DNP and anti-ABA antibodies provides the final proof that cross-linking of two adjacent reaginic molecules on the mast cell surface by a bivalent hapten is required for effective elicitation of immediate-type reactions. This has been accomplished by utilizing the bivalent epsilon,N-DNP-alpha,N-[(4-hydroxy-3-azobenzenearsonic acid)-phenacetyl]-L-lysine (DNP-ABA) carrying noncross-reactive haptenic groups, which was consistently effective in eliciting P-K reactions in doubly but never in singly sensitized skin sites. It is apparent from the results that equimolar doses of monovalent haptens could completely inhibit the response to DNP-ABA. The present studies finally establish that mast cells of newborn colostrum-deprived calves lack IgE molecules on their surface. Thus, mast cells of newborn calves may be unique, to investigate the molecular mechanisms involved in immediate-type reactions more precisely.