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Biomedical subjects

W L Hoffman

Publications and source records attributed to W L Hoffman.

18 recordsLinked to original sources
PubMed1992

Characterization of immune complex components by dot blot analysis.

A method is described for the characterization of immune complex components by dot blot analysis. After isolation by chromatographic techniques and precipitation with polyethylene glycol, immune complexes were dissociated in 0.1 M phosphate (pH 2) and bound to a nitrocellulose membrane in a dot blot unit. Biotinylated probes were then used to identify the following immune complex components: specific antigens, biologically active antibodies, antibody isotypes, antibody subclasses, antibody idiotypes, and rheumatoid factors. This nonradioactive procedure takes less than 2 h to perform and has been used to analyze immune complexes isolated from sera (rabbit and human) and synovial fluid (human).

Acids↗
PubMed1992

Microanatomic clonality of gamma delta T cells in human leishmaniasis lesions.

T cells bearing gamma delta Ag receptors accumulate in the lesions of patients with localized American cutaneous leishmaniasis (LCL), and are thought to be involved in immunity to the parasite. To obtain clues as to the nature of the Ag recognized by these cells, we analyzed the diversity of the TCR delta-chain in LCL lesions. Using mAb against variable (V) encoded determinants with immunoperoxidase, both V delta 1 and V delta 2 subpopulations were identified in the dermal granulomas. However, within the epidermis of LCL lesions, the majority of the gamma delta T cells were V delta 1 positive. PCR analysis of lesion-derived DNA using oligonucleotide primers for V and junctional (J) gene segments revealed preferential usage of J delta 1 in lesions compared with the peripheral blood of these patients. Nucleotide sequence analysis of the V-J junction indicated limited diversity of gamma delta T cells within specific microanatomic regions. In addition, use of a single diversity (D) gene segment, D delta 3, in V delta 2 cells in lesions was observed, as opposed to multiple D delta gene segment usage in the blood of the same individuals. The distribution, gene segment usage and clonality of gamma delta T cells in lesions of leishmaniasis was remarkably similar to that observed in leprosy. Therefore, gamma delta T cells responding to infection may recognize a limited set of nominal Ag, perhaps common to distinct pathogens and/or those expressed by the host. Our findings are most consistent with a model in which specific gamma delta T cells are clonally selected by these Ag in lesions and undergo oligoclonal expansion within a microanatomic region.

Amino Acid Sequence↗
PubMed1991

Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction for the amount of antibody remaining on the membrane after the milk block, it was found that acid-bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid binding of proteins could increase the sensitivity of nitrocellulose membrane assays using a milk block.

Animals↗
PubMed1991

The role of infections in the rheumatic diseases: molecular mimicry between bacterial and human stress proteins?

Infections can cause or exacerbate the rheumatic diseases in several ways, including immune cross-reactivity between bacterial heat shock proteins and similar proteins in normal human tissues. This may lead to autoimmunity in rheumatoid arthritis and systemic lupus. In addition, increased activation of the gene regulating the synthesis of a heat shock protein has been found in scleroderma fibroblasts. As an infection-induced model for other rheumatic diseases, rheumatic fever (RF), with its well-established link to prior group A streptococcal infection, will be revisited. The lessons learned from RF and other rheumatic diseases directly linked to infection will be applied to ankylosing spondylitis, rheumatoid arthritis, Sjogren's syndrome and polymyositis, for which a mounting body of circumstantial evidence suggests a probable infectious cause. The interplay of genetic susceptibility and infection with particular organisms and the implications of this new information for present and future therapy of the rheumatic diseases will also be presented.

Antigens, Bacterial↗
PubMed1990

Characterization of immune complexes by isoelectric focusing in agarose gels.

A method is described for the characterization of immune complexes on thin-layer agarose isoelectric focusing (IEF) gels. This method involves dissociating immune complexes and then maintaining this dissociation during IEF in agarose gels containing 9 M urea. After IEF, the immune complex components can be quantitatively transferred to nitrocellulose in less than 15 min, and a variety of immunostaining procedures can be used to probe these blotted components. No loss of biological activity was detected in any of the blotted components.

Animals↗
PubMed1990

Synthesis of specific IgG idiotypes by rheumatoid synovium.

Synovial tissue samples from 6 patients with rheumatoid arthritis were cultured, and the IgG antibodies isolated from the synovial culture supernatants were used to immunize rabbits to make 6 antiidiotypic (anti-Id) antibody preparations. After extensive adsorption, the rabbit anti-Id were tested in a solid-phase enzyme-linked immunosorbent assay (ELISA). Each anti-Id reacted predominantly with the immunizing synovial IgG and showed almost no reactivity with either pooled normal human serum IgG or with IgG from 50 normal donors. When identical amounts of matched rheumatoid arthritis serum IgG and synovial culture supernatant IgG were probed simultaneously with the corresponding rabbit anti-Id in an ELISA, 3 of 6 pairs demonstrated an increased concentration of specific idiotypes in the synovial culture supernatant IgG. Furthermore, when these 6 matched samples were subsequently analyzed by isoelectric focusing, individual IgG antibodies in 5 of 6 synovial IgG samples revealed enhanced reactivity with the corresponding rabbit anti-Id preparations, when compared with matched serum IgG. This increased synovial concentration of specific idiotypes detected by both the ELISA and isoelectric focusing was compatible with enhanced synovial tissue synthesis of the antibodies involved. These specific Id/anti-Id reactivities were not blocked by excess normal human Fc, Fab, or F(ab')2 fragments, indicating a lack of association of the stimulating synovial antibodies with rheumatoid factors or antibodies against other IgG fragments (pepsin agglutinators).

Arthritis, Rheumatoid↗
PubMed1989

Rehydratable agarose gels: application to isoelectric focusing in 9 molar urea.

A method is described for the preparation of rehydratable agarose gels, with specific application to the direct incorporation of 9 M urea and carrier ampholytes into rehydratable agarose gels for use in isoelectric focusing. After drying the agarose gel containing an uncharged linear polyacrylamide, one gel volume of a 9 M urea-carrier ampholyte solution is absorbed directly into the gel in 60 min, eliminating equilibration or dialysis of the gel in larger volumes of the 9 M urea-carrier ampholyte solution. Proteins with a molecular mass of 970,000 Da can be separated by isoelectric focusing in these rehydratable gels. The focused proteins can then be quantitatively transferred to nitrocellulose in less than 10 min, and any immunostaining procedure can be used to probe the blotted proteins. These agarose gels are easy to make, they rehydrate rapidly and they can be used in applications other than isoelectric focusing.

Acrylic Resins↗
PubMed1989

Inhibition of the streptavidin-biotin interaction by milk.

Nonfat dried milk is routinely used as a blocking agent and diluent in immunoassays. However, the data presented in this paper demonstrate that milk contains an inhibitor of the biotin-streptavidin interaction. It is recommended that milk be dialyzed or used at lower concentrations when it is employed as a diluent of streptavidin.

Animals↗
PubMed1988

Site-specific immobilization of antibodies by their oligosaccharide moieties to new hydrazide derivatized solid supports.

This report describes a new method for immobilization of antibodies to solid supports. Antibodies are bound to the solid supports by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibody and hydrazide groups on the solid support. The hydrazone bonds that are formed are stable at least from pH 2-10, permitting the acid elution of antigens from the affinity column. Over 25 mg of affinity-purified rabbit IgG binds per ml of solid support, with most of the bound antibodies retaining biological activity. Advantages of this new affinity support over existing technology are discussed along with procedures for the preparation and use of affinity columns containing monoclonal or polyclonal antibodies.

Animals↗
PubMed1987

Site-directed immobilization of glycoproteins on hydrazide-containing solid supports.

Methods are described for the preparation and use of solid supports containing hydrazide functions for the immobilization of glycoproteins specifically through the oligosaccharide moieties. The solid supports are prepared from commercial "active ester" agarose by reaction with hydrazine hydrate. Glycoproteins are oxidized with sodium periodate, resulting in the production of aldehydes on the oligosaccharide moieties. Oxidized glycoprotein is then reacted with the hydrazide-derivatized solid support to produce stable hydrazone linkages. Data are presented for the optimization of binding of oxidized glycoprotein to hydrazide-derivatized agarose. Agarose hydrazide/glycoprotein gels were shown to be stable from pH 3 to 10 and activity studies using immobilized avidin show that this method of immobilization results in an increased "specific activity" of bound protein when compared with standard methods of immobilization.

Chromatography, Affinity↗
PubMed1979

Tissue-specific ribosomal protein composition.

Membrane-bound and free polysomes from murine liver and kidney were isolated under identical conditions and their ribosomal proteins were compared by two-dimensional gel electrophoresis. The results demonstrate that these ribosome subpopulations are quantitatively and qualitatively similar except for the presence of one additional protein in the kidney-bound polysomal fraction.

Animals↗
PubMed1977

Analysis by two-dimensional polyacrylamide gel electrophoresis of liver ribosomal subunnit proteins obtained from free and membrane-bound polysomes of unfasted animals.

Ribosomal proteins were analyzed by means of two-dimensional gel electrophoresis. To insure that the analysis included only that fraction of the ribosome actively participating in protein synthesis, only polysomal-bound ribosomes were used. This differs from previously reported analyses of liver ribosomal proteins. The ribosomal proteins were prepared from ribosomes of polysomal origin from membrane-bound and free polysomes. Membrane-bound and free liver polysomes were isolated from unfasted mice. The polysomes were purified on hydroxyapatite under conditions known to result in polysomes and ribosomes that are active in both endogenous and synthetic mRNA translation. Moreover, this is the first time that liver ribosomal protein was obtained and analyzed from animals that have not been starved prior to sacrifice. The puromycin-released ribosomes were dissociated into subunits and ribosomal proteins were analyzed by means of two-dimensional polyacrylamide gel electrophoresis. When 100-200 mug samples of the ribosomal subunit proteins were analyzed by two-dimensional electrophoresis, approximately 32 major proteins were detected for the 60 S ribosomal subunit and 25 major proteins for the 40 S ribosomal subunit. A total of 13 "fractional" ribosomal proteins was also detected in the ribosomal subunit profiles. No differences in number or mobility of the ribosomal proteins were found between the membrane-bound and free ribosome populations. We describe a system in which all ribosomal proteins are completely solubilized and quantitatively move from the first to the second dimension gel. Thus the total sample is separated and fractionated. This procedure elimates artifacts due to incomplete solubilization of ribosomal proteins, which is common for the transfer from the first- to second-dimension gel. Therefore, a more detailed and accurate analysis is achieved.

Animals↗
PubMed1977

The role of the canine spleen in cardiovascular homeostasis during halothane anesthesia.

Barbiturate anesthesia is known to increase canine splenic sequestration of red blood cells. In our laboratory, high halothane concentrations have produced a decreased arterial hematocrit in the dog. In order to assess the role of the spleen in this phenomenon, central hematocrit, plasma volume, and ventricular hemodynamics were studied at low and high halothane concentrations before and after splenectomy in the same group of dogs. Although the hematocrit difference was less after splenectomy, it was not abolished. In addition, there was more cardiovascular depression by equivalent or lower halothane doses after splenectomy. It appears that the dog has other areas of red cell sequestration than the spleen accounting for the persistently lower hematocrit with high halothane concentrations after splenectomy. The modifying effect of the canine spleen on the circulatory depression produced by halothane should be taken into account when the drug is used in the dog.

Anesthesia, Inhalation↗
PubMed1975

Analysis by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes.

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.

Animals↗