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Biomedical subjects

B H Schofield

Publications and source records attributed to B H Schofield.

At least 37 records · Page 2Linked to original sources
PubMed1994

Histochemical demonstration of hydrogen peroxide production by leukocytes in fixed-frozen tissue sections of inflammatory lesions.

The production of H2O2 by cells in cold paraformaldehyde-fixed frozen sections of inflammatory lesions was histochemically demonstrated by incubating them with diaminobenzidine (DAB) for 2 to 6 h. Catalase (150 micrograms/ml, about 1400 U/ml) inhibited the reaction, indicating that H2O2 was required to produce the chromogenic DAB product. Granulocytes (PMNs and eosinophils) were the main types of cells stained by the DAB reaction. Positive staining of macrophages was less frequent. The H2O2 was produced by metabolic enzymes that were still active after cell death and mild fixation. An atmosphere of 95 to 100% oxygen enhanced the specific DAB reaction, and an atmosphere of 100% nitrogen eliminated it. The DAB histochemical reaction to detect H2O2 requires the presence of peroxidases to produce the colored reaction product. Within our tissue sections, such peroxidases were evidently present in excess, because addition of low concentrations of H2O2 significantly increased the reaction product. Although some of the H2O2 produced by the granulocytes may have been derived from the dismutation of superoxide (O2-), the NADPH oxidase pathway for O2- formation did not seem to be involved: NADPH oxidase, a rather labile enzyme, should not be active after mild fixation, and diphenyleneiodonium (100 microM), an inhibitor of flavine-requiring NADPH oxidase, did not inhibit the reaction. Reactive nitrogen intermediates were also not involved, because NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthetase, did not appreciably inhibit the reaction. We conclude that stable, non-flavine-requiring oxidases, possibly cyclooxygenases or lipoxygenases, produced the H2O2 measured histochemically by our DAB reaction. These studies were made on tissue sections of acute dermal inflammatory lesions produced in rabbits by the topical application of 1% sulfur mustard [bis(2-chloroethyl) sulfide] in methylene chloride. Both intact PMNs and disintegrating PMNs in the base of the crust produced H2O2. Despite the production of H2O2 and the presence of peroxidase activity, no tissue damage was seen microscopically near the H2O2-producing cells, which indicates that the tissues are well protected by the antioxidants present in this self-limiting inflammatory reaction.

Amitrole↗
PubMed1994

Physiologic modulation of bronchial epithelial cell barrier function by polycationic exposure.

Bronchial epithelial cells provide a functional barrier to the movement of water and solutes between the luminal and interstitial compartments of the lung. Barrier integrity can be compromised by a variety of factors, including polycationic proteins released by inflammatory cells. We investigated the characteristics of epithelial barrier function and its modulation by cationic stimuli in canine bronchial epithelial (CBE) cells grown in culture. Morphologic characteristics were examined, and barrier function was assessed by measurements of transepithelial mannitol flux (flux) and electrical resistance (RT) during a stable, 3- to 14-day culture period. CBE cultures exhibited progressive mucociliary differentiation and contained nonciliated, ciliated, and neutral and acidic mucin-secretory cells. The synthetic polycation, poly-L-lysine (PLL), from 2.5 to 10 micrograms/ml, caused dose-related increases in flux and decreases in RT that were not accompanied by detectable release of lactate dehydrogenase (LDH) or changes in histochemical appearance. The effect on RT spontaneously reversed over a 15-h recovery period. The action of PLL on flux was not attenuated by treatment of the cells to stabilize cytoskeletal contractile elements but was immediately attenuated by the addition of heparin to the challenged cells. These results indicate that modulation of the barrier integrity of bronchial epithelial cells by cationic proteins, such as those released by inflammatory cells, represents a physiologic process that may be regulated by endogenous anionic factors.

Animals↗
PubMed1994

Dry air-induced mucosal cell injury and bronchovascular leakage in canine peripheral airways.

The purpose of this study was to examine the relationship between hyperpnea-induced mucosal injury, bronchovascular hyperpermeability, and airway reactivity. Hyperpnea-induced bronchoconstriction was assessed by measuring peripheral airway resistance (Rp) in anesthetized mechanically ventilated male mongrel dogs. Either colloidal carbon or monastral blue was used to localize bronchovascular leakage after a 5-min exposure to either a 1000 ml/min dry, 2000 ml/min wet, or 2000 ml/min dry air challenge. Morphometric analyses of cross-sectioned bronchi revealed that hyperpnea with dry air stimulated goblet cell degranulation, damaged the bronchial mucosa, and increased bronchovascular permeability. Exposure to only a 2000 ml/min dry air challenge produced marked mucosal injury when compared with control. Regardless of treatment, bronchial vessels lying below normal mucosa characterized by goblet/ciliated cell (G/C) ratios > or = 0.3 did not leak. A G/C transition zone between 0 and 0.3 separated normal from damaged mucosa. Within this zone, vascular permeability was inversely correlated with G/C ratio. In addition, airflow-induced changes in Rp were inversely related to G/C ratio and positively correlated with bronchovascular leakage. Although these correlations are consistent with the speculation that bronchovascular leakage and edema formation are responsible for the dry air-induced changes in Rp, it is equally plausible that bronchovascular leakage is not the cause of but occurs in concert with airway narrowing to protect cells in the bronchial mucosa from excessive losses of heat and water.

Airway Resistance↗
PubMed1991

Immunologic and ultrastructural changes during early rejection of vascularized bone allografts.

This investigation evaluated ultrastructural changes during the earliest phase of immunologic rejection of vascularized bone allografts in a genetically defined rat model. These results were correlated with the cell-mediated and humoral immunologic responses during this time period. Employing a model for heterotopic allograft transplantation, 33 rats divided into four categories were evaluated. Group I consisted of ungrafted (naive) Lewis and Brown Norway rats; group II consisted of Lewis-to-Lewis vascularized bone isografts; group III consisted of Lewis-to-Brown Norway vascularized bone allografts; and group IV consisted of Lewis-to-Brown Norway vascularized bone allografts in rats receiving cyclosporine (10 mg/kg/day). Experimental animals were sacrificed at 3, 5, and 7 days. Immunologic analysis was performed using a cell-mediated lymphocytotoxicity assay and a complement-dependent cytotoxic antibody assay. The results of this study show that rejection of vascularized bone allografts appears as early as 3 days postoperatively, with osteocytes and vascular endothelium being the first elements affected. This early rejection is probably a manifestation of the humoral response. All changes secondary to rejection were arrested by cyclosporine.

Animals↗
PubMed1990

Full-thickness human skin explants for testing the toxicity of topically applied chemicals.

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

Administration, Topical↗
PubMed1987

A histologic comparison of aseptic loosening of cemented, press-fit, and biologic ingrowth prostheses.

The histology of interface membranes from aseptic loosened prostheses of various types including cemented, press-fit, and biologic ingrowth varieties was compared. Pseudosynovial implant-facing surfaces were present in specimens from all types. The remaining portions of these membranes showed distinct characteristics as well. Cemented implant membranes contained many macrophages and giant cells and evidenced frequent granuloma formation. Press-fit membranes consisted of poorly vascularized, dense fibrous tissue within the loosened press-fit membrane. Macrophages and giant cells were rare, except in one specimen containing ceramic debris particles. Biologic ingrowth membranes were the most vascular and contained loosely organized connective tissue and islands of woven bone. Macrophages were common. One out of six specimens from patients with rheumatoid arthritis contained massive numbers of lymphocytes and plasma cells but not mast cells. The greatest numbers of mast cells were present in membranes from patients with osteoarthritis and in all cases were associated with the presence of stainless steel and/or chrome cobalt particles.

Arthritis, Rheumatoid↗
PubMed1986

Histochemical localization of cathepsin B, dipeptidyl peptidase I, and dipeptidyl peptidase II in rat bone.

The histochemical distribution of the thiol proteases cathepsin B and dipeptidyl peptidase I and the serine protease dipeptidyl peptidase II was examined in rat bone and joint using amino acid derivatives of 4-methoxy-2-naphthylamine (MNA). The liberated MNA was then visualized by simultaneous coupling with fast blue B. Cathepsin B was examined with CBZ-Arg-Arg-MNA, dipeptidyl peptidase I (DPP I) with Gly-Arg- or Pro-Arg-MNA, and dipeptidyl peptidase II (DPP II) with Lys-ALA- or Lys-Pro-MNA. Bright red reaction product indicative of proteolytic activity was observed in most cell types associated with bone and its surrounding connective tissues, including osteocytes, osteoblasts, chondrocytes, chondroblasts, fibroblasts, and macrophages. Surprisingly, protease activity in osteoclasts could not be established with certainty, and it was concluded that these enzymes are either absent, present in very low amounts, or secreted as soon as they are synthesized rather than stored within the cell. The cells of the resting zone of the growth plate were intensely reactive for DPP II but were only moderately reactive for cathepsin B and DPP I. The reverse was true of the proliferating and hypertrophic layers. The protease activity observed in bone, cartilage, tendon, ligament, and synovium would be expected to contribute significantly to normal protein metabolism as well as to pathological destruction in these tissues.

Animals↗
PubMed1984

Pathogenesis of skin lesions caused by sulfur mustard.

Sulfur mustard (SM) (di-2-chlorethyl sulfide), used for chemical warfare in World War I, is a highly reactive radiomimetic alkylating agent. When applied to the skin of rabbits and guinea pigs, it produced vascular leakage, leukocyte infiltration, and slow death of basal epidermal cells. Thirty to sixty minutes after exposure to SM, injury to the superficial microvasculature (beneath the SM application site) was detected by measuring vascular leakage with Evans blue dye and also with horseradish peroxidase. At this same time, injury to the superficial fibroblasts was observed ultrastructurally; and an unexpectedly high percentage of basophils was found among the early infiltrating granulocytes. At 2 to 4 hr, the vascular leakage ceased, and had resumed by 8 hr in a more diffuse form. At this time, the basal epidermal cells showed pyknotic nuclei, an increase in their lysosomal enzymes (observed histochemically), and autophagic vacuoles (observed ultrastructurally). Leukocyte infiltration was marked, consisting mostly of heterophils (PMN) with a reduced percentage of basophils. During the next 24 to 72 hr, the entire inflammatory reaction reached its peak; and a superficial, crust-covered ulcer developed. Then, over the next 10 days, the lesion gradually subsided with concomitant repair and healing. Glucocorticosteroids decreased the early edematous phase, but did not affect the rate of healing. These findings suggest that the skin response to sulfur mustard has an immediate and a delayed phase. The immediate phase, i.e., within the first hour, was characterized by injury to the superficial fibroblasts and to the endothelium of superficial capillaries and venules, possibly because of direct damage to their cell membranes. At this time, a restricted vascular leakage and a selective granulocyte infiltration containing many basophils occurred. The delayed phase, which became evident after 8 hr, was characterized by the death of basal epidermal cells, probably because of DNA damage. This phase was accompanied by generalized vascular leakage, by massive heterophil immigration, and eventually by ulceration.

Acid Phosphatase↗
PubMed1983

Direct bone resorbing activity of murine myeloma cells.

The cellular mechanism(s) responsible for tumor-associated bone resorption in multiple myeloma remain uncertain. Both in vivo and in vitro evidence is presented for the direct resorption of bone by mouse plasmacytomas. Morphological examination of autopsy specimens from tumor-bearing mice revealed in vivo erosion of bony surfaces at sites of tumor cell-bone matrix apposition. No osteoclastic bone resorptive activity was evident. Using a 45Ca-labelled, devitalized bone explant assay system, mouse myeloma cells caused the release of isotope at levels from 200-300% above control values. Control cells such as normal spleen lymphocytes and liver cells did not resorb bone. Demonstration of the ability of myeloma cells to independently destroy bone is important to the understanding of the causes of and development of chemotherapeutic approaches to myelomatous bone resorption.

Animals↗
PubMed1982

Two clonal cell populations (mosaicism) in a 46,XY male with mucolipidosis II (I-cell disease)--an autosomal recessive disorder.

Cultured fibroblasts from a 46,XY male with an atypical form of mucolipidosis II (I-cell disease) had two distinct phenotypes. One population of these fibroblasts had the morphological and biochemical features characteristic of I-cell disease, while the remaining cells were indistinguishable from normal fibroblasts. Direct evidence that the patient was a mosaic, having two cell populations, was provided by the establishment of pure, stable clones of both wild type and I-cell fibroblasts from each of two biopsies obtained several months apart. Additionally, it was shown that the I-cell fibroblasts lacked UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosaminylphosphotransferase while the morphologically normal cells contained levels of this enzyme just below or at the lower end of the normal range.

Cells, Cultured↗
PubMed1980

Selective localization of a Golgi apparatus acid phosphatase isoenzyme in bone using pyridoxal-5'-phosphate.

Substrates commonly used for localizing bone Golgi apparatus (GA) acid phosphatase (AcPase), e.g., beta-glycerophosphate, p-nitrophenylphosphate, cytidine-5'-monophosphate, and di(dicyclohexylammonium)-2-napthylthiolphosphate, give strong staining not only of GA but also of lysosomes. Thiamine pyrophosphate and inosine-5'-monophosphate--substrates that give good GA staining in some soft tissues--give only lysosomal staining in bone. No previously used substrate or substrate-effector combination has selectively localized the GA acid phosphatase in bone. This article describes results using a new AcPase medium having pyridoxal-5'-phosphate (PLP) as substrate. In bone this medium produced strong staining of the osteoblast GA, but relatively little staining of lysosomes, including lysosomes in osteoclasts. The weak lysosomal staining was almost totally eliminated, without affecting the GA reaction, by pretreating the tissue in 0.3% NH3 solution. Conversely, elevated ionic strength of the substrate medium eliminated the GA reaction, while having little effect on lysosomal staining. The GA enzyme was very sensitive to 1 mM tartrate whereas the lysosomal enzyme was not. These differences suggest the presence of distinct isoenzymes in the two locations. The distribution of osteoblasts with stained GA coincided with the distribution of strongest alkaline phosphatase activity and rapid bone mineralization, supporting previous suggestions that osteoblast GA AcPase is involved in the processing of one or more newly synthesized bone matrix components.

Acid Phosphatase↗
PubMed1980

A comparative study of new substrates for the histochemical demonstration of acid phosphomonoesterase activity in tissues which secrete acid phosphatase.

The histochemical demonstration of acid phosphatase activities against phosphoethanolamine (PEA), phosphorylcholine (PC), and D-ephedrine phosphate (DEP) are reported for a variety of rat tissues and are compared to acid beta-glycerophosphatase (beta GPase) activity. Intense acid beta GPase activity was demonstrated in all tissues examined. However, liver, kidney, intestine, spleen and bone marrow cells failed to exhibit any enzyme activity against PEA, PC, or DEP. In addition, significant differences in the hydrolysis of these substrates were noted among the tissues that did demonstrate activity (bone, tooth, oral mucosa, sebaceous gland, and prostate gland). These observations suggest that PEA, PC, and DEP are more specific substrates for acid phosphatase than beta GP and permit the differential localization of several distinct acid phosphatase isoenzymes.

Acid Phosphatase↗
PubMed1979

Histochemical demonstration of enzyme activities in plastic and paraffin embedded tissue sections.

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, beta-galactosidase, and cytochrome oxidase in plastic embedded and ruotine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 micrometers, were far superior to frozen sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.

Acid Phosphatase↗
PubMed1978

Acid D-ephedrine-O-phosphatase activity in rate ventral prostate lysosomes.

Hydrolysis of D-ephedrine-O-phosphate (DEP) by rat ventral prostrate was investigated by electron microscopic hystochemistry. In contrast to previous reports that DEP is exclusitely hydrolyzed by a secreted nonlysosomal acid phosphatase, enzyme reaction product was present in supranuclear and basal lysosomes.

Acid Phosphatase↗
PubMed1976

Enzyme histochemistry of bone and cartilage cells.

Initial studies indicated that bone and cartilage, when treated with a hypertonic glutaraldehyde fixative for a short period, retained significant enzyme activity for histochemistry and also maintained excellent fine structure. We used 6% glutaraldehyde in 0.1 M cacodylate buffer, pH = 7.2, 4 degrees C to fix small pieces of bone or cartilage for three hours while the tissues were being constantly agitated. These samples were demineralized in 10% ethylene diamine tetraacetic acid, buffered to pH = 7.2 with 0.1 M Tris HC1, at 4 degrees C. The demineralized tissue was frozen and cryostat sections 32 microns thick were taken for incubation at 37 degrees C in various media for histochemistry. For electron microscopic localization of enzymes a heavy metal capturing method had to be used. For light microscopy, the azo dye methods were frequently used, but these were not usable for electron microscopy. Alkaline phosphatase was found on the outer surface of osteoblast and hypertrophic cartilage cell membranes. The only intracellular enzyme activity was found on the mitochondrial membranes of the osteoclast and only when the pH of the media was lowered from the optimum 9.5 to 8.5. Alkaline phosphatase was not found along the osteocyte or young cartilage cell membranes...

Acid Phosphatase↗
PubMed1975

Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentation principle.

The critical electrolyte concentration principle was applied to the Alcian Blue staining of rat epiphyseal cartilage proteoglycans for electron microscopy. The distribution and structure of material in glutaraldehyde-fixed cartilage stained at pH 5.8 without MgCl2 and in the presence of 0.05, 0.4, 0.5, 0.9 and 1.0 M MgCl2 was compared with that produced by simultaneous staining and fixation at neutral pH. Both methods resulted in staining of intracellular material within vacuoles as well as staining of non-collagenous matrix material. The structure and distribution of Alcian Blue-positive matrix material consisted of rounded or polygonal granules which accumulated around cells in the proliferative and hypertrophied zones. A similar pattern of distribution was observed in samples stained in the presence of 0.4 or 0.5 M MgCl2. In these cases, however, the stained material exhibited a ribbon-like configuration and granules were few in number. Increasing the MgCl2 concentration to 1.0 M resulted in a marked reduction of Alcian Blue stained material. No ribbon-like structures were observed, and matrix granules were reduced in both number and size. The decreased staining associated with increased electrolyte concentration lends support to the concept that epiphyseal cartilage matrix granules are composed primarily of chondroitin sulphate, and suggest that this same material is present in vacuoles associated with the Golgi apparatus in chondrocytes of the proliferative and hypertrophying zones.

Alcian Blue↗