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Biomedical subjects

R A Clark

Publications and source records attributed to R A Clark.

At least 307 records · Page 17Linked to original sources
PubMed1986

Basal cell carcinomas grown in nude mice produce and deposit fibronectin in the extracellular matrix.

Epidermal cells in vitro produce and deposit fibronectin (FN) in the pericellular matrix. Such FN production by epidermal cells may be involved in vivo in wound reepithelialization, tissue morphogenesis, and growth of epithelial tumors. The purpose of this study was to examine whether the FN, previously shown to be within and surrounding human basal cell carcinoma (BCC) lobules, was in part the product of epidermal-derived tumor cells. To examine this question we took advantage of our ability to grow human BCC in nude mice. Since we could demonstrate that all stromal cells surrounding the BCC were of mouse origin, antibodies specific for human FN would distinguish epithelial-derived FN from mesenchymal-derived FN. Five solid BCCs were implanted subcutaneously in nude mice. Growing tumors were removed after 60 days, snap-frozen, sectioned on a cryostat, and verified microscopically as BCC. The Hoescht DNA stain, which can distinguish mouse and human nuclei, demonstrated that mouse, not human, fibroblasts occupied the stroma surrounding each tumor lobule. Sections of all 5 BCCs were stained by immunofluorescence and immunoperoxidase techniques with antibodies to bullous pemphigoid (BP) antigen, laminin (LM), and FN. BP antigen and LM were present at the basement membrane zone (BMZ) of all tumor lobules as previously described for in situ BCC. FN staining was present along the BMZ, within the tumor lobules, and in the surrounding stroma. Antibodies to human FN were passed over a mouse FN affinity column to absorb antibodies which cross-reacted with mouse FN. The resultant antibody preparation, which was specific for human FN in this system, continued to demonstrate FN along the BMZ and within the tumors, but failed to stain FN in more distant stroma. Epidermal-derived cells, therefore, can synthesize and deposit FN in vivo in adjacent extracellular matrix. We speculate that this FN matrix may facilitate growth of BCC in this model.

Animals↗
PubMed1986

Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.

Actinobacillus↗
PubMed1986

Fibronectin mediates adherence of rat alveolar type II epithelial cells via the fibroblastic cell-attachment domain.

The lung alveolar surface is composed of types I and II epithelial cells. Extremely attenuated type I cells cover 90% of the surface and are prone to necrosis during acute lung injury. After denudation of type I cells, the alveolar epithelium is restored by proliferation of type II cells. During reepithelialization in vivo the type II cells have been observed to reorganize on an extracellular matrix that contains fibronectin. We thus sought to determine whether type II cells would adhere to purified fibronectin. Adherence assays of primary rat type II cells were performed in protein-coated bacteriologic microtiter wells for 24 h at 37 degrees C. Concentrations of fibronectin from 1 to 300 micrograms/ml mediated type II cell adherence, 10 micrograms/ml gave maximal adherence, and 4 micrograms/ml gave 50% maximal adherence. Adherence progressively increased from 1 to 72 h. Adherence on fibronectin was at least 50% greater than adherence on laminin, types I and III collagen, or IV collagen. Little or no adherence was observed on bacteriologic plastic or albumin. Spreading on these various substrata paralleled adherence. Adherence to fibronectin, laminin, and fibrinogen was specifically blocked by their respective polyclonal antibodies. Monoclonal antibodies (MoAb) to the fibronectin cell-attachment domain blocked adherence to fibronectin, whereas MoAb to other domains did not. From the data reported here and the previously mentioned in vivo study we propose that fibronectin is an important functional component of the extracellular matrix that supports type II cells during alveolar reepithelialization.

Animals↗
PubMed1986

Either exogenous or endogenous fibronectin can promote adherence of human endothelial cells.

Recently, we have presented evidence that proliferating blood vessels produce and deposit fibronectin in situ during the angiogenesis of wound repair. This report extends these observations by demonstrating that human endothelial cells from both large and small vessels depend on fibronectin for their adherence in vitro. Endothelial cells were grown from human umbilical veins (HUVEC) by the method of Gimbrone and from the microvasculature of human omentum by the method of Kern, Knedler and Eckel. Second-passage cells were plated into microtitre wells that had been coated with 100 micrograms ml-1 of fibronectin, types I and III collagen, type IV collagen or laminin. After a 3-h incubation, adherent cells were solubilized with Zap-Isoton and quantified on a Coulter Counter. Under normal culture conditions HUVEC showed no preference for fibronectin substrates while microvascular cells always demonstrated a striking preference for fibronectin substrates. However, when HUVEC were exposed to 2.5 or 25 micrograms ml-1 of cycloheximide for 4 h before and during the adherence assays, the adherence to fibronectin was 50-200% greater than to types I and III collagen. Immunofluorescence studies showed that while HUVEC expressed a large quantity of surface fibronectin, microvascular cells expressed very little. Metabolic labelling studies confirmed that HUVEC cultures had substantial quantities of fibronectin in their cell layer while microvascular cells did not. In antibody blocking experiments, preincubation of fibronectin-coated surfaces with anti-fibronectin antibodies totally blocked microvascular cell adhesion but only abrogated HUVEC adherence by 50%, presumably since these latter cells were able to deposit additional fibronectin onto the surface during the 3 h assay period. In the presence of cycloheximide anti-fibronectin antibodies totally blocked HUVEC adherence. These results demonstrate that both endothelial cell types rely, at least in part, on fibronectin for adherence in vitro. HUVEC can synthesize, secrete and deposit enough fibronectin for their adherence in vitro, while microvascular cells rely on an exogenous source of fibronectin under these culture conditions. Thus, the increased blood vessel fibronectin observed during angiogenesis in vivo may mediate adherence of the proliferating and migrating endothelial cells.

Cell Adhesion↗
PubMed1986

Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing.

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.

Bone Marrow↗
PubMed1986

Juvenile dermatomyositis: histological findings and pathogenetic hypothesis for the associated skin changes.

Skin biopsies from patients with scleroderma and juvenile dermatomyositis (DM) share many histologic features. Characteristics common to both diseases are particularly evident in the dermal microvasculature and include endothelial swelling and concentric thickening of the vascular basement membrane. Biopsies performed on 3 patients with the severe vasculitic form of juvenile DM showed these changes as well as dropout of vessels and linear deposition of collagen. The latter findings, seen late in the course of the disease, are indistinguishable from those of advanced scleroderma. A hypothesis is presented which attempts to relate these histological findings to a common underlying pathophysiologic mechanism.

Basement Membrane↗
PubMed1985

Relationships between the rate of synthesis of ATP and the concentrations of reactants and products of ATP hydrolysis in maize root tips, determined by 31P nuclear magnetic resonance.

Using 31P NMR spectroscopy, we have measured the rate of ATP synthesis, and the free concentrations of ATP, ADP, cytoplasmic Pi, and H+ in maize root tips under a wide range of conditions. We show that the ratio [ATP]/[ADP] in normoxic root tips is greater than 25. We found no simple relationship between the concentration of ATP and the rate of ATP synthesis: when the rate of ATP synthesis decreases in response to different treatments, the concentration of ATP can increase, decrease, or remain unchanged. Clear relationships were obtained, however, when the rate of synthesis of ATP was plotted against the logarithm of the ratio psi, defined as [ATP]/[ADP][Pi][H+]. Two curves were obtained, depending on which of two situations pertained. First, if mitochondrial ATP synthesis was inhibited, e.g., by KCN or hypoxia, ln psi decreased monotonically as rates of ATP synthesis decreased. The decrease in ln psi may account for decreases in the rates of biosynthetic reactions dependent on ATP, such as protein synthesis, as they approach equilibrium. Second, if consumption of ATP for biosynthetic reactions was inhibited, by treatment with succinate, ln psi increased as rates of ATP synthesis decreased. The increase in ln psi may account for decreases in the rate of ATP synthesis, as oxidative phosphorylation approaches equilibrium.

Adenosine Diphosphate↗
PubMed1985

Improved control of cisplatin-induced emesis with high-dose metoclopramide and with combinations of metoclopramide, dexamethasone, and diphenhydramine. Results of consecutive trials in 255 patients.

A series of consecutive trials were undertaken to determine whether higher doses of intravenous metoclopramide and combinations of metoclopramide, dexamethasone, and diphenhydramine would improve antiemetic control or decrease treatment-related side effects in patients receiving cisplatin at 120 mg/m2. Metoclopramide and dexamethasone were studied because of their proven efficacy as single agents and their differing mechanisms of action and side effects. Diphenhydramine was used because of its possible antiemetic properties and its ability to control acute dystonic reactions. Two hundred fifty-five patients who had never received chemotherapy or antiemetics were observed in the hospital for the 24 hours following cisplatin administration. The addition of dexamethasone or dexamethasone plus diphenhydramine to intravenous metoclopramide 2 mg/kg produced both improved antiemetic control and a decrease in treatment-associated diarrhea (P = 0.002). The use of metoclopramide alone at a dose of 3 mg/kg for only two doses appeared as effective as 2 mg/kg for five doses. When dexamethasone and diphenhydramine were given with metoclopramide 3 mg/kg for two intravenous dosages, 81% of patients experienced no emesis and 93% had two or fewer vomiting episodes. The antiemetic results of this 2-hour "short-course" regimen were superior to metoclopramide 2 mg/kg, with (P = 0.002) or without (P = 0.0001) dexamethasone and diphenhydramine. It was concluded that combinations of metoclopramide plus dexamethasone plus diphenhydramine improve antiemetic control, facilitate the usage of higher doses of metoclopramide, and decrease the incidence of treatment-related side effects.

Adult↗
PubMed1985

Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix.

Fibronectin and laminin production by human keratinocytes cultured in serum-free, low-calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7-day keratinocyte cultures was determined by 18 hr 35S-methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.

Autoradiography↗
PubMed1985

Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis.

We explored the pathologic changes in the skin of mice undergoing a chronic graft-versus-host (GVH) reaction. In rodents and in man, chronic GVH includes the deposition of excess collagen in the skin-a reaction which resembles idiopathic scleroderma. GVH disease across minor histocompatibility barriers was produced by injecting B10.D2 cells into irradiated BALB/c mice. These strains are identical at the H-2 and Mls loci but differ in minor histocompatibility antigens. Control BALB/c mice received irradiation and BALB/c cells. Serial skin biopsies were taken and studied for histological changes characteristic of chronic GVHD, for mast cell density, and for the deposition of immunoreactants. GVHD was produced in B10.D2----BALB/c mice as measured by body weight loss and the production of skin changes including dermal fibrosis, loss of fat and appendages, and a mononuclear cell infiltrate. Dermal mast cells, assessed by toluidine blue staining, were normal at Day 11, but had disappeared by Days 21-63 and returned to normal by Day 104. Immunoglobulins IgG, IgA, and IgM appeared at the dermo-epidermal junction and along the basement membrane zone of hair follicles. This deposition was maximal at Day 42 and waned thereafter. Thus the appearance of immunoglobulins in the skin was maximal when mast cell staining was minimal. The changes in this GVHD model leading to a scleroderma-like picture in the skin are compatible with an immune etiology for the fibrosis. Vasodilation following liberation of mast cell mediators would facilitate the deposition of immunoglobulins. The disappearance of mast cell staining may be caused by extensive degranulation. We postulate an interaction between GVHD-activated T cells, mast cell stimulation, fibroblast activation, and fibrosis.

Animals↗
PubMed1985

Computed tomography of abdominal and pelvic trauma.

Computed tomography (CT) is now considered an accepted modality in the initial and follow-up evaluations of traumatized patients. The purpose of this article is to reaffirm the important role of CT in the assessment of trauma. The subject matter covered includes trauma involving the abdomen and pelvis, including the spine and hips.

Abdominal Injuries↗
PubMed1985

Cutaneous tissue repair: basic biologic considerations. I.

Wound repair of the integument is reviewed in the context of new developments in cell biology and biochemistry. Injury of the skin and concomitant blood vessel disruption lead to extravasation of blood constituents, followed by platelet aggregation and blood clotting. These events initiate inflammation and set the stage for repair processes. The macrophage plays a pivotal role in the transition between wound inflammation and repair (granulation tissue formation), since this cell both scavenges tissue debris and releases a plethora of biologically active substances that include growth factors. Although concrete evidence is lacking, growth factors are probably at least partially responsible for the angiogenesis and fibroplasia (granulation tissue) that gradually fill the wound void. If the epidermal barrier is disrupted during injury, reepithelialization begins within 24 hours and proceeds first over the margin of residual dermis and subsequently over granulation tissue. The signals for angiogenesis, fibroplasia, neomatrix formation, and reepithelialization in wound repair are not known, but a number of possibilities are discussed. Matrix remodeling is the last stage of wound repair and gradually increases the scar tensile strength to 70% to 80% of normal skin.

Animals↗
PubMed1985

Cutaneous tissue repair: practical implications of current knowledge. II.

This article reviews the scientific basis for the certain factors that delay wound repair in the clinical setting. A brief history of wound healing is given, followed by a discussion of endogenous local factors (bacterial infection, hypoxia, foreign body, and desiccation) and endogenous systemic factors (nutritional deficiencies, aging, coagulation disorders, and the Ehlers-Danlos syndromes) associated with poor wound repair. Also reviewed are the mechanisms by which exogenously administered agents (glucocorticoids, antineoplastic agents, and anticoagulants) may delay healing. Commonly used topical antimicrobials, their spectrum of activity, and evidence of effects on wound healing are examined. Finally, properties of commercially available wound coverings and wound care in the future are discussed.

Aging↗
PubMed1985

Phase 1 trial of levonantradol in chemotherapy-induced emesis.

Levonantradol is a synthetic cannabinoid with demonstrated preclinical antiemetic activity. The current phase I trial was undertaken to determine: 1) the maximally tolerated dose; 2) the side effects at the different dosage levels; and 3) to evaluate the antiemetic efficacy of levonantradol in patients receiving emesis-producing chemotherapy. Thirty-four patients received 52 courses of levonantradol. Concurrent chemotherapy most frequently consisted of high dose cisplatin (120 mg/m2), either alone or in combination with other agents. Levonantradol dosage was escalated through seven treatment levels (0.5-4.0 mg per dose) and was given intramuscularly every 4 hours. Toxicity was similar to that observed with other cannabinoids and primarily consisted of dizziness (65%), burning and erythema at the injection site (48%), mild sedation (44%), orthostatic hypotension (37%), dysphoria (29%), and urinary retention (10%). Marked urinary retention occurred in three of seven patients at the 4.0 mg per dose level, and two of 24 patients at either the 2.5 mg and 3.0 mg levels. Major or minor antiemetic responses (0-2 or 3-5 emetic episodes, respectively) occurred in 23% of patients receiving cisplatin and in 53% of patients receiving non-cisplatin containing chemotherapy. Intramuscular levonantradol can be given safely at doses up to 3.0 mg/kg, with toxicity and antiemetic efficacy similar to that observed with other cannabinoids.

Adult↗
PubMed1985

Fibronectin, as well as other extracellular matrix proteins, mediate human keratinocyte adherence.

During the reepithelialization of cutaneous wounds, the migratory epidermis transits over a provisional matrix of fibronectin and fibrin in the absence of laminin and type IV collagen as well as ultrastructurally identifiable basement membrane. Since significant quantities of fibronectin occur at these sites of reepithelialization, we surmised that fibronectin is a suitable substrate for keratinocyte adherence and therefore undertook the in vitro investigations reported here. Purified human plasma fibronectin precoated on bacteriologic microtiter wells was demonstrated to mediate human keratinocyte adherence when concentrations greater than 10 micrograms/ml fibronectin were used. Maximal keratinocyte adherence was obtained in wells precoated with 100 micrograms/ml fibronectin and when cells were incubated with substrate for 60 min or longer at 37 degrees C. Both primary and second-passaged human keratinocytes adhered as well or better to fibronectin than to types I and III collagen, laminin, or type IV collagen under both high- and low-Ca++ culture conditions. However maximal adherence to all substrates occurred when second-passaged keratinocytes were assayed in low-Ca++ medium. Under these latter culture conditions, keratinocyte phenotype resembles the phenotype of cells in the migrating epidermis. To determine specificity of these adherence reactions, antifibronectin antibodies were shown to block keratinocyte adherence to fibronectin but not to laminin substrates. Conversely, antilaminin antibodies blocked adherence to laminin but not fibronectin substrates. Thus, human keratinocytes demonstrate specific adherence to fibronectin in a time- and dose-dependent fashion and this adherence relies on de novo protein synthesis. We take these observations to support our hypothesis that the provisional fibronectin matrix observed beneath the migrating epithelium during tissue repair plays a functional role in the reepithelialization process.

Binding Sites, Antibody↗
PubMed1985

Expression of fibronectin, laminin, and factor VIII-related antigen during development of the human cutaneous microvasculature.

The regulation of angiogenesis during human skin development is poorly understood. Since fibronectin is involved in cell movement and organization during embryogenesis and morphogenesis in a variety of species, we investigated the expression of fibronectin and factor VIII-related antigen, a marker for endothelial cells, at various stages in the development of the human cutaneous microvasculature. Skin specimens were obtained from 4 human fetuses during the second trimester (14-18 weeks), from newborn foreskins, and from consenting normal adults. Cryostat sections were stained with both fluorescein-conjugated rabbit antihuman fibronectin and rhodamine-conjugated goat antihuman factor VIII-related antigen. Expression of fibronectin in the microvasculature was striking in fetal skin but became progressively less prominent with maturation. Fibronectin appeared in fetal blood vessels as a bright continuous linear array, in neonatal blood vessels as a bright interrupted linear and speckled array, and in adult blood vessels as a sparse interrupted linear and speckled array. In contrast, expression of factor VIII-related antigen by the endothelium became more prominent with the degree of maturation of the microvasculature. Granular factor VIII-related antigen staining was scant in the newly forming blood vessels of fetal skin, bright but focal in the microvasculature of newborn skin, and intense and almost confluent in the blood vessels of adult skin. Although expression of fibronectin and factor VIII-related antigen changed, expression of laminin was consistent throughout development. Staining of the same skin specimens with fluorescein-conjugated sheep antihuman laminin produced a bright continuous linear pattern in all blood vessels. The reciprocal relationship manifested by intense fibronectin staining during human blood vessel development and prominent factor VIII-related antigen staining in mature blood vessels supports the hypotheses that fibronectin plays a role in human blood vessel modulation and morphogenesis, and that factor VIII-related antigen is a marker for endothelial cell differentiation.

Adult↗