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Biomedical subjects

M K Robinson

Publications and source records attributed to M K Robinson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Skeletal muscle phosphocreatine depletion depresses myocellular energy status during sepsis.

OBJECTIVE: To determine the effects of phosphocreatine (PCr) depletion on myocellular energetics. DESIGN: Randomized controlled study. SETTING: University laboratory. MATERIALS: Thirty-eight adult male Wistar rats (110-121 g). METHODS: The poorly metabolized creatine analogue beta-guanidinopropionic acid, (beta-GPA, 2% of a gel diet) was fed to the rats for 14 days to replace (75%) endogenous PCr stores before cecal ligation and puncture (CLP). Rats were randomized to receive sham operation and gel diet (sham-gel group [n=10]), sham operation and beta-GPA diet (sham-beta-GPA group [n=9]), CLP and gel diet (CLP-gel group [n=10]), and CLP and beta-GPA diet (CLP-beta-GPA group [n=9]). On day 14, all animals underwent operation. Twenty-four hours later, in vivo phosphorus 31-labeled magnetic resonance spectroscopy (31P-MRS) of the gastrocnemius muscle was performed. Muscle samples were collected to determine enzyme activities of beta-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, citrate synthase, and the metabolites adenosine triphosphate (ATP), PCr, inorganic phosphate, and creatine. Free adenosine diphosphate levels, the phosphorylation potential, and free energy change of ATP hydrolysis were then calculated. RESULTS: All animals undergoing CLP but no controls had positive results of blood cultures. Although sham-beta-GPA animals had altered bioenergetics, CLP-beta-GPA rats experienced a greater deterioration of energy state compared with CLP-gel controls. Glycolytic and oxidative enzyme activities were not significantly different between groups and therefore could not explain the observed differences. CONCLUSIONS: There is an overall decrease in energy availability during sepsis, which is worsened by PCr depletion. These changes support the contention that PCr plays an important role as an ATP buffer during systemic infection.

Animals↗
PubMed1998

Oxidant inhibition of alphaLbeta2 integrin adhesion: evidence for coordinate effects on conformation and cytoskeleton linkage.

Dithiothreitol (DTT) and other dithiol antioxidants with closely spaced thiol pairs strongly activate leukocyte function antigen-1 (LFA-1, alphaLbeta2 integrin) to bind intercellular adhesion molecule-1 (ICAM-1). Because direct biochemical modification of LFA-1 by DTT is not apparently involved, we investigated the possible role of a reduction-oxidation (redox)-sensitive adhesion-regulatory pathway. Phenylarsine oxide (PAO), an oxidant selectively reactive with closely spaced pairs of thiol groups, inhibited LFA-1-dependent adhesion of human natural killer and HSB2 T leukemia cells to murine cells expressing human ICAM-1. PAO also induced disappearance of a conformation-sensitive LFA-1 epitope recognized by KIM127 antibodies and promoted an increase in total apparent cytoskeleton-linked LFA-1 in which a novel cytochalasin D-resistant linkage was involved. Exposure of PAO-pretreated cells to DTT caused a decline in LFA-1/cytoskeleton linkages in conjunction with rapid restoration of KIM127 epitope expression and LFA-1 adhesive function. Implicating an intracellular site of action were findings that (1) an epitope-tagged PAO probe bound predominantly to intracellular proteins but not detectably to immunoprecipitation-purified LFA-1 chains, and (2) membrane permeant but not impermeant dithiol antioxidants reversed PAO adhesion-inhibitory effects. These results support the concept of a reversible redox-sensitive linkage between LFA-1 and cytoskeleton by which oxidants and antioxidants may exert profound opposing effects on LFA-1 conformation and adhesive function.

Adenosine Triphosphate↗
PubMed1998

Use of the mouse intranasal test (MINT) to determine the allergenic potency of detergent enzymes: comparison to the guinea pig intratracheal (GPIT) test.

A mouse intranasal test (MINT) was developed to determine the relative allergenicity of detergent enzymes. In this simple method, various doses of the enzymes are administered in a detergent matrix, via intranasal instillation, on days 1, 3, and 10, with serum samples collected on day 15. The sera are then analyzed for enzyme specific IgG1 antibody by an antigen specific enzyme immunoassay. The protease enzyme Alcalase (protease Subtilisin Carlsberg) has been used as a benchmark enzyme for development and characterization of the model. The objective of the current studies was to obtain potency comparisons with other protease and nonprotease enzymes and to begin to assess the validity of the model by comparison with potency determinations obtained with the guinea pig intratracheal (GPIT) test. The range of potencies detected among several enzymes of different classes was approximately 60-fold (compared with Alcalase). Modification of the dosing regimen to permit slightly more extended dosing did not change the relative potency determination. Comparison of data from the MINT and GPIT methods indicate that both the mouse and the guinea pig recognize the bacterial amylase Termamyl and a fungal exocellulase as more potent than Alcalase, a serine protease (Subtilisin B) and a fungal alpha-amylase (Fungamyl) as less potent than Alcalase, and the serine protease, Savinase, as equivalent to Alcalase. Also, these data are in alignment with clinical data on the prevalence of occupational enzyme sensitization. Given the simplicity and low cost of the MINT method compared with the GPIT test, these results support continued development of the method as an alternative approach for assessing the allergenicity of enzymes.

Administration, Intranasal↗
PubMed1998

Application of a 4-h human patch test method for comparative and investigative assessment of skin irritation.

A human 4-h patch test has recently been developed for testing the irritation hazard potential of chemicals. The original method was developed for comparative irritation assessments relative to benchmark irritants using simple statistical tests. In this context, the method has been shown to be robust in intralaboratory testing over time. Recent interlaboratory testing has also established the consistency of the method in assessment of the relative irritation potential of selected chemicals. These data help to position the method as a suitable replacement for animal test methods in assessment of skin irritation hazard. In addition, the method has great utility for investigating different parameters of clinical skin irritation. Using kinetic response patterns and curve fitting analysis, we have compared the relative irritation potential of chemicals in greater detail, using as a basis the time required for test subjects to respond as well as the incidence of positive responses. Also, using the response to 20% sodium dodecyl sulfate (SDS) as a benchmark, we've been able to examine the intersubject variation in clinical skin irritation responses. In general, subjects most reactive to 20% SDS, in terms of the exposure time required to produce a positive response, were relatively more sensitive to a 2nd irritant chemical as well. However, this was not an absolute correlation in that some test subjects showed divergent patterns of response. The method was also used to compare directly the relative skin reactivity of different populations, based on race (Caucasian versus Asian) or on neurosensory skin sensitivity. Our results using this acute exposure test method indicate little difference in visually assessed skin irritation among these diverse human subpopulations.

Acetic Acid↗
PubMed1998

CD43 (sialophorin, leukosialin) shedding is an initial event during neutrophil migration, which could be closely related to the spreading of adherent cells.

Leukosialin is a negatively-charged mucin-like membrane protein of leukocytes. This anti-adhesive molecule prevents uncontrolled cellular interactions and is proteolytically cleaved during neutrophil activation. CD43 is shed in vivo during neutrophil migration to the inflammatory site. We have analysed the decrease in CD43 expression during in vitro adherence of TNF-alpha activated PMN. CD43 was quantitated by flow cytometry on TNF-alpha-activated PMN either maintained in suspension or allowed to adhere then detached with EDTA. Although TNF did not induce significant modification of CD43 expression on suspended cells, we showed that 40% of membrane CD43 is released during neutrophil TNF-induced adhesion to serum-coated plates or endothelial cells, and that migration through the endothelial monolayer did not result in further shedding. Adhesion-blocking anti-beta 2 integrin mAbs prevented CD43 shedding. beta 2 integrin "activation" by anti-CD 18 mAbs or Mn ions did not decrease CD43 expression if adhesion was prevented by stirring. Inhibitors of signal transduction or of cytoskeleton association, which allowed cells to adhere but not to spread, inhibited the shedding of CD43 during adhesion. We conclude that CD43 shedding is not promoted by beta 2 integrins engagement or adhesion but is concomitant with spreading of adherent cells.

1-Methyl-3-isobutylxanthine↗
PubMed1997

RTX toxins recognize a beta2 integrin on the surface of human target cells.

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coli alpha-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the beta2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins.

Amino Acid Sequence↗
PubMed1997

Assessment of postpartum depression: a review of the research.

The postpartum period is a time when many women are vulnerable for the development of a variety of emotional syndromes. One of the problems with treating the emotional problems is the difficulty in differentiating them. For example, there are at least three distinct postpartum depression disorders: postpartum blues, postpartum psychosis, and postpartum depression. Each of these syndromes is discussed. A review of the instruments used to test for risk factors for the development of postpartum affective disorders indicates that conceptual muddiness still exists in differentiating the three disorders. The notion of whether postpartum depression is a reactive depression to stress or a syndrome is examined. A suggestion is made to more closely examine the role of culture in the development of affective disorders in the puerperium.

Journal Article↗
PubMed1997

Expression of vascular cell adhesion molecule-1 by vascular endothelial cells in immune and nonimmune inflammatory reactions in the skin.

Expression of VCAM-1 was compared with that of E-selectin in cytokine-induced lesions and in delayed-type hypersensitivity reactions to tuberculin purified protein derivative (PPD) in pig skin. Lumenally expressed Ags were quantified by measuring localization in skin of i.v. injected (111)In-mAb 10.2C7 (anti-vascular cell adhesion molecule-1 (anti-VCAM-1), (125)I-mAb 1.2B6 (anti-E-selectin), and (99m)Tc-MOPC21 (control IgG1). Anti-VCAM-1 mAb uptake was greater following intradermal (i.d.) injection of TNF-alpha than following injection of IL-1, while the two cytokines induced similar uptake of anti-E-selectin. In immunologically naive pigs there was no detectable increase in anti-VCAM-1 after i.d. injection of PPD, although anti-E-selectin uptake was increased at 3 and 6 h. In contrast, i.d. injection of PPD in sensitized pigs led to increased uptake of both anti-VCAM-1 and anti-E-selectin at 6, 8, 24, and 48 h, each of which was significantly greater than the uptake of control IgG1 into the same lesions (each p < 0.01). Anti-TNF-alpha mAb abolished the increased uptake of anti-VCAM-1 3 and 8 h following i.d. injection of PPD in sensitized pigs and significantly inhibited uptake at 24 h (p = 0.0025), but did not significantly reduce uptake of anti-E-selectin. We conclude that in this delayed-type hypersensitivity model 1) E-selectin expression by endothelial cells follows sequential Ag nonspecific and immune-specific phases, 2) increased VCAM-1 expression by endothelial cells is only seen in sensitized animals, and 3) expression of VCAM-1 appears to be relatively more dependent on TNF-alpha than E-selectin. Differential expression of E-selectin and VCAM-1 may influence the leukocytic infiltrate during the course of nonspecific and immune-specific inflammatory reactions.

Animals↗
PubMed1997

Comparison of E-selectin-binding glycoprotein ligands on human lymphocytes, neutrophils, and bovine gamma delta T cells.

We compared E-selectin-binding cell surface ligands on bovine gamma delta T cells and human leukocytes using an E-selectin/Ig chimera. The chimera worked well in flow cytometric studies and showed that bovine gamma delta T cells were the only lymphocyte population in newborn animals that bound E-selectin chimera. Furthermore, the chimera blocked gamma delta T cell rolling on E-selectin. Chimera reacted with four potential glycoprotein ligands of 180, 200, 250, and 300 kDa in Western blot analysis of gamma delta T cell detergent lysates, and it specifically precipitated at least two of these E-selectin ligands (200 and 250 kDa) from lysates of cell surface biotinylated gamma delta T cells. Preclearing bovine gamma delta lysates of GD3.5 Ag and workshop cluster 1 did not abrogate E-selectin ligand precipitation, suggesting that these surface markers do not represent E-selectin ligands. Human neutrophils possessed three E-selectin-binding ligands of approximately 80 to 90, 130, and 230 kDa, while human lymphocytes variably possessed three ligands of 120, approximately 220 to 240, and 260 kDa. Cross-precipitation experiments confirmed the results of others that neutrophil L-selectin serves as the 80 to 90-kDa E-selectin ligand. The human lymphocyte approximately 220 to 240-kDa and 260-kDa ligands may be analogous to the bovine gamma delta T cell molecules, whereas the 120-kDa is unique to human cells. The identities of the human and bovine lymphocyte E-selectin ligands are unknown. Finally, E-selectin ligand-1 apparently may have a minimal role, if any, in lymphocyte/E-selectin interactions, since a polyclonal anti-E-selectin ligand-1 serum stained a minimal number of cutaneous lymphocyte-associated Ag-positive human lymphocytes and bovine gamma delta T cells.

Animals↗
PubMed1997

Starvation enhances hepatic free radical release following endotoxemia.

Although it is well known that malnourished patients who become septic have an increased risk of organ failure and death compared to normally nourished individuals, the pathological processe(s) underlying this observation are unknown. To evaluate one possible explanation for this finding, we tested the hypothesis that malnutrition depresses hepatic antioxidant stores and accelerates hepatic release of oxygen free radicals in an animal model of sepsis. Male rats were either fasted (n = 14) or fed (n = 14) for 3 days prior to receiving lipopolysaccharide (LPS, 17 mg/kg intraperitoneally). Animals were weighed daily and then sacrificed 6 and 24 hr after LPS administration to determine hepatic superoxide anion (an oxygen free radical) release and liver glutathione (GSH, an antioxidant) content. Fasted rats were severely malnourished as indicated by a 23% decrease in body weight compared to fed rats, which gained 11% (P < 0.05). Liver GSH was depressed by 30% (P < 0.05) and 20% (P = 0.066) in the fasted compared to fed animals 6 and 24 hr after LPS administration. In addition, hepatic superoxide anion release was 210 and 75% higher in the fasted animals 6 and 24 hr after LPS injection (P < 0.05 at both time points). Liver superoxide anion release and GSH content were negatively correlated (P < 0.001, R = - 0.73) indicating that superoxide anion release increased as GSH content fell. Malnutrition leads to depletion of liver antioxidant stores with accelerated release of hepatic oxygen free radicals. Oxidant-mediated organ damage may be one cause of increased morbidity and mortality in malnourished, systemically infected patients.

Animals↗
PubMed1997

Interlaboratory evaluation of a human patch test for the identification of skin irritation potential/hazard.

The human 4 hr patch test provides an opportunity to identify substances with significant skin irritation potential without recourse to the use of animals. To demonstrate the validity of the method it must be relevant and reliable. It is self-evident that the method is relevant to the identification of skin irritation hazards to humans. However, it is essential that the results be reproducible. This paper presents data on a number of substances tested by different laboratories. Eight substances were tested by two or more laboratories and the data compared with a standard positive control, 20% sodium dodecyl sulfate. In almost all cases, the outcome of this comparison was identical. Thus, despite the fact that there is known variability among human subjects in terms of skin reactivity to irritants, this simple method showed good reproducibility for the classification of acute skin irritation potential. Therefore, it is argued that this human 4-hr patch test is a valid alternative to the equivalent rabbit test for the assessment of skin irritation hazard to humans.

Adult↗
PubMed1997

The in vivo and in vitro characterisation of an engineered human antibody to E-selectin.

BACKGROUND: E-selectin is an endothelial cell specific adhesion molecule that is believed to play an important role in the early stages of leukocyte extravasation. OBJECTIVES: Here we describe the construction and evaluation of an engineered human monoclonal antibody that blocks E-selectin function. RESULTS: SPLAT-1 is an engineered human monoclonal antibody that has a very similar affinity for E-selectin as its murine parent antibody. In vitro SPLAT-1 blocks the binding of human leukocytes to E-selectin and does not mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated lysis of endothelial cells. In vivo, SPLAT-1 inhibits the recruitment of leukocytes to cytokine-inflamed human skin grafted on to SCID mice and has a long circulating half-life in primates. It does not appear to provoke an immune response in primates even on repeat administration. CONCLUSIONS: SPLAT-1 has the characteristics of a antibody suitable for human therapy studies.

Animals↗
PubMed1997

Superiority of blood over saline resuscitation from hemorrhagic shock: a 31P magnetic resonance spectroscopy study.

OBJECTIVE: To study the relation between blood and saline administration, postresuscitation hematocrit (Hct) level, and metabolic recovery after hemorrhagic shock. SUMMARY BACKGROUND DATA: It is generally believed that crystalloid can be substituted, in whole or in part, for blood during resuscitation of hemorrhagic shock. This is based on the belief that Hct can be safely reduced but should not fall below a critical level. METHODS: Male rats weighing 200 g were subjected to an isobaric hemorrhagic shock at a mean arterial pressure of 30 mmHg for 14 minutes, after which they were randomized to one of three resuscitation regimens. Control group (n = 36) were resuscitated by return of all shed blood. Mid-Hct (n = 39) and low-Hct (n = 60) groups were depleted of one third and one half of their circulating blood volumes, respectively, and were resuscitated with three times that volume of normal saline. Skeletal muscle intracellular energetics and pH were measured serially using 31P magnetic resonance spectroscopy at baseline, during shock, and after resuscitation. Arterial blood was sampled at the same time points. The number of surviving animals in each group at 24 hours was recorded. RESULTS: After resuscitation, surviving rats in the low-Hct group demonstrated a greater consumption of high-energy phosphocreatine stores than did the other groups (control = 0.479 +/- 0.003, mid-Hct = 0.465 +/- 0.004, low-Hct = 0.457 +/- 0.007, mean +/- standard error of the mean; p < 0.01 low-Hct vs. other groups by analysis of variance). The rats that received saline resuscitation developed a relative intracellular acidosis (control = 7.29 +/- 0.02, mid-Hct = 7.25 +/- 0.02, low-Hct = 7.23 +/- 0.02; p < 0.05 controls vs. other groups by analysis of variance). At 24 hours, the death rates were significantly different among the groups: control = 1 of 36 rats (2.8%), mid-Hct = 6 of 39 (15.4%), and low-Hct = 14 of 60 (23.3%) (p < 0.05 by chi square analysis). CONCLUSION: The oxygen-carrying capacity of resuscitation fluid has an important impact on intracellular metabolism and outcome.

Animals↗
PubMed1997

Competition and its implications for managing health care organizations.

Reports that the US health care system has been shifting since the 1960s to a competitive environment with many new and innovative organizational forms. Describes the nature of the competitive environment for health care and some of the basic forms of the new organizational arrangements. Explores some of the implications for managing successful health care organizations by the understanding of interorganizational linkages in the health care industry and a partnership approach to marketing and management.

Capitation Fee↗
PubMed1996

The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs.

We have studied the role of E-selectin in leukocyte accumulation into Ag-specific cutaneous delayed-type hypersensitivity reactions in pigs sensitized to the topical application of 2,4-dinitro-1-fluorobenzene or to the intradermal injection of bacillus Calmette-Guérin. The delayed-type hypersensitivity reactions were shown to be specific for the sensitizing Ag and characterized by the up-regulation of E-selectin, as demonstrated by the uptake of tracer 99mTc-labeled monoclonal anti-E-selectin mAb and entry of 51Cr-labeled PBL and (111)In-labeled polymorphonuclear cells (PMN). Intravenous injection of 5 mg/kg of a F(ab')2 preparation of a monoclonal anti-E-selectin Ab at peak times of leukocyte entry resulted in a significant inhibition of entry of both PMN and lymphocytes. The anti-E-selectin Ab inhibited PMN recruitment by 70 to 90% and lymphocyte recruitment by 50 to 60%. In comparison, an anti-CD18 treatment reduced PMN recruitment by 70 to 90% and lymphocyte recruitment by 60 to 70% in this model. These data confirm an important role for E-selectin in the recruitment of both PMN and lymphocytes to sites of immune-based dermal inflammation.

Age Factors↗
PubMed1996

Workshop overview. Identification of respiratory allergens.

A variety of chemicals and proteins can sensitize the respiratory tract. Among these are materials of industrial importance, including certain diisocyanates, acid anhydrides, reactive dyes, and enzymes. Currently, no widely accepted or well-validated methods for the prospective identification of respiratory allergens exist. Most progress has been made with guinea pig methods where sensitizing potential is measured usually by assessment of changes in pulmonary function induced following sensitization and challenge. However, these methods are often prohibitively expensive, particularly for screening purposes. A number of alternative approaches are under consideration and are described here. The nature of the health problems associated with occupational respiratory sensitization, chemical structure-activity analyses as a tool for detecting pulmonary allergens, approaches used to test for respiratory allergens in guinea pigs, and alternative approaches using mice are all discussed. Finally, regulatory issues and needs with respect to respiratory sensitization are outlined.

Allergens↗
PubMed1996

Specific antibody responses to subtilisin Carlsberg (Alcalase) in mice: development of an intranasal exposure model.

An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 x DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-microliters aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.

Administration, Intranasal↗