Proteolysis in homogenates of perfused rat liver: responses to insulin, glucagon and amino acids.
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Amino Acids↗
Biomedical subjects
A N Neely
Publications and source records attributed to A N Neely.
EGTA and proteinase reversal of cellular aggregation of activated lymphocytes.
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Calcium↗
Proteolytic activity in human burn wounds.
Wound healing is the result of a dynamic balance between synthetic and degradative processes. After a burn, proteolytic activity increases at the wound site. Excised burn wounds and donor skin were examined from 20 pediatric burn patients, to determine which of two classes of neutral proteinases, serine or metalloproteinases, accounts for the majority of this proteolytic activity in these tissues; to examine messenger RNA expression of three of the principal enzymes and inhibitors of this class; and to measure enzymatic activity of two of these metalloproteinases. The majority of the increased proteolysis was due to metalloproteinases. By polymerase chain reaction assays, messenger RNAs for matrix metalloproteinase-1, -3, and -9 were strongly expressed in burn tissue and absent or weakly expressed in unburned skin. Messenger RNA for tissue inhibitor of metalloproteinase-1 and -2 was consistently present in burned and unburned skin. By zymography, there was a significant increase in matrix metalloproteinase-2 (twofold to threefold) and matrix metalloproteinase-9 (20- to 30-fold) activity in burned versus unburned skin. We suggest that postburn there is an upregulation of some matrix metalloproteinases that exceeds the level of inhibitors with the net result of an increase in proteolysis in burned tissue. This increased proteolysis may play a role in wound repair and scar formation.
Journal Article↗
Gelatinase activity in keloids and hypertrophic scars.
Keloids and hypertrophic scars are characterized by excessive deposition of collagen, which may result from insufficient protein degradation. Little is known about the levels of two gelatinases, matrix metalloproteinase-2 (72 kD type IV collagenase) and matrix metalloproteinase-9 (matrix metalloproteinase-9; 92 kD type IV collagenase) in these abnormal scars. The purpose of this study was to determine levels of these proteinases in tissue from hypertrophic scars, keloids, and donor skin. Ten hypertrophic scar samples, 9 keloid samples, and 10 donor skin samples were frozen, pulverized, homogenized, clarified by centrifugation, and analyzed for matrix metalloproteinases by quantitative zymography. Identity of matrix metalloproteinases was determined using a conditioned media reference standard, molecular weight ladders, and Western blotting. Levels of matrix metalloproteinase-9 activity were very low or undetectable in all samples. However, matrix metalloproteinase-2 activity was significantly elevated in keloids and hypertrophic scars vs. donor samples: 2.6 and 3.9-fold increases for latent matrix metalloproteinase-2, 7.8 and 6.9-fold increases for active matrix metalloproteinase-2, respectively. We conclude that little matrix metalloproteinase-9 activity (the gelatinase involved in early tissue repair) is present in keloids and hypertrophic scars, while matrix metalloproteinase-2 activity (the gelatinase involved in prolonged tissue remodeling) is present in donor skin and is significantly increased in hypertrophic scars and keloids.
Blotting, Western↗
Combined host and specific anti-Pseudomonas-directed therapy for Pseudomonas aeruginosa infections in burned mice: experimental results and theoretic considerations.
Four strains of Pseudomonas aeruginosa showed significant variation in the elaboration of the virulence-associated exoproducts, exotoxin A and proteases. These variations appeared to affect the mortality observed when these strains were used to infect burned mice. Further, these variations seemed to influence the efficacy of individual after sepsis treatments designed to (1) reduce total microbial load in the host (HIg treatment), (2) reduce total circulating protease load in the host (alpha 1-PI treatment), or (3) neutralize the specific effects of exotoxin A (antitoxin treatment). Combined treatment, using all of these approaches, was significantly better than individual treatment.
ADP Ribose Transferases↗
Hageman factor-dependent kinin activation in burns and its theoretical relationship to postburn immunosuppression syndrome and infection.
Burn injury and intradermal injection of bradykinin or histamine cause permeability changes visualized as dye-release lesions in the skin of guinea pigs injected intravenously with Evans blue dye. Antihistamine pretreatment ablates the histamine but not the effect of thermal injury or bradykinin. Bradykinin is generated via activation of Hageman factor in a two-step reaction. Steps 1 and 2 can be inhibited by corn trypsin inhibitor and soy bean trypsin inhibitors, respectively. Dye-release lesions were reduced from thermal injury and bradykinin injections when these substances were injected into the skin first. Angiotensin-converting enzyme deactivates bradykinin by degrading it. Angiotensin-converting enzyme inhibitor neutralizes angiotensin-converting enzyme. Dye-release lesions from both thermal injury and bradykinin injection were enhanced because of continued bradykinin build-up when these treatments were preceded by subcutaneous injections of angiotensin-converting enzyme inhibitor. Thus bradykinin is generated in thermal injury via the Hageman factor-dependent pathway. Hageman factor sits at the apex of a series of interrelated cascade systems, all of which impinge on the animal's immune status. Uncontrolled Hagemen factor activation in thermal injury may be the link among all the events collectively known as the "post thermal injury immunosuppression syndrome."
Animals↗
Causes of colonization of autografted burn wounds.
The progression of autograft colonization to infection presents significant problems in the care of grafted burn wounds. To determine whether graft colonization arises from self-contamination or from some exogenous source, we characterized pregraft and graft Candida albicans isolates through serotyping and biotyping (Leicester system) and Pseudomonas aeruginosa isolates through serotyping (Japan Pseudomonas aeruginosa Society system). In 21 of 24 patients, the serotype/biotype of the graft isolate was the same as the serotype/biotype of a C. albicans strain previously isolated from the same patient. Fifteen of 16 P. aeruginosa graft contaminants also showed the same serotype as that in a pregraft isolate. Therefore, whereas it has long been surmised that the grafts of patients with burns are self-colonized, this study provides definite data showing that most C. albicans and P. aeruginosa autograft isolates are indeed self-contaminants.
Bacteria↗
Candida albicans growth studies: a hypothesis for the pathogenesis of Candida infections in burns.
The proteolytic environment in which Candida albicans exists strongly affects its virulence. To determine whether virulence might be related to C. albicans growth in different proteolytic environments, we measured renal fungal load in burned mice and found significantly greater Candida census in kidneys from mice that were challenged with a high proteinase-generating parent C. albicans (MY 1044) versus those that were challenged with its low proteinase-generating mutant (MY 1049). In vitro, MY 1044 cells grew faster than MY 1049 cells in media that contained sera from burned mice as the only nitrogen source. Augmentation of media with proteinase or a mixture of amino acids increased growth of MY 1049 cells, whereas augmentation with proteinase inhibitor decreased MY 1044 growth. In conclusion, in vitro growth of both the mutant and its parent strain was affected by the proteolytic environment in which they existed; thus, virulence differences for MY 1044 and MY 1049 could be due in part to growth differences between these two strains in different proteolytic environments. These results were combined with existing observations, and we proposed a theory for the pathogenesis of C. albicans in burns.
Amino Acids↗
Biologic response modifier OK-432 pretreatment reduces mortality in burned mice infected with gram-negative bacteria.
When compared with a control group, mice pretreated with the biologic response modifier OK-432 showed increased survival when burned and infected with several strains of Pseudomonas aeruginosa and a Klebsiella pneumoniae isolate. Quantitative microbial counts in the skin, liver, and kidneys of burned mice infected with Pseudomonas aeruginosa showed that although skin counts were not different between control groups pretreated or untreated with OK-432, liver and kidney counts were reduced significantly in the group pretreated with OK-432. We speculate from these results that OK-432 pretreatment may enhance survival by enhancing host clearance of microorganisms entering the circulation from the burn wound. Making mice granulocytopenic by cyclophosphamide treatment or macrophage-deficient/dysfunctional by carrageenan treatment nullified any survival advantage given by OK-432 pretreatment. These results supported our speculation and suggested that both of these cell types were important in the mechanisms of enhanced survival afforded by OK-432 pretreatment.
Animals↗
Patients undergoing reconstructive surgery versus unburned children as "controls" in studies of pediatric patients with burns.
A valid control group is an essential part of any patient study. We asked whether burned children returning for reconstructive surgery could be used as "controls" in a study of seven proteolytic elements in the circulation. Functional levels of elastase, plasminogen, prekallikrein, antithrombin, alpha 2-antiplasmin, alpha 2-macroglobulin, and total proteolytic activity in 30 healthy unburned children were compared with levels in 29 patients admitted for reconstructive surgery an average of 7.9 years after burn. The two groups were not statistically different in distributions of sex and race but differed in mean age. Levels of six of the seven parameters were equal in the two groups. However, even when correction for the age difference was done between the groups, alpha 2-macroglobulin in the patients undergoing reconstructive surgery still was significantly less (p < 0.021) than in the unburned group. Therefore caution may be needed when readily available patients undergoing reconstructive surgery are used as "controls" in studies of patients with acute burns.
Adolescent↗
Efficacy of a rise in C-reactive protein serum levels as an early indicator of sepsis in burned children.
C-Reactive protein serum levels were measured in 57 pediatric patients with 3% to 92% total body surface area burns to determine whether a defined rise in C-reactive protein serum levels could indicate sepsis earlier in burn patients. A rise in C-reactive protein serum levels was defined as an increase of at least 3 mg/dL for 2 days or 10 mg for 1 day. Increases the first 2 days after the burn or the day after surgery were excluded, since these injuries increase C-reactive protein serum levels. Patients were defined as septic when they were on systemic antibiotics and exhibited at least two of 16 specific clinical parameters. C-Reactive protein serum levels correctly predicted sepsis 82% of the time (efficiency=82%). Nonseptic patients generally did not show increased C-reactive protein serum levels (specificity=69%). When sepsis did occur, it always was preceded by increased C-reactive protein (sensitivity=100%), and the increased C-reactive protein occurred 2.3+/-0.5 days before the patient was deemed septic clinically. Hence, a defined rise in C-reactive protein serum levels can predict sepsis sooner in burned children.
Adolescent↗
The 1999 Lindberg award. 3% hydrogen peroxide for the gram-positive disinfection of fabrics.
Because of growing concern about the spread of antibiotic-resistant gram-positive bacteria in burn and trauma units, an inexpensive, safe, effective means of spot-disinfecting fabrics (such as privacy curtains) that remain in clinic or patient rooms as various patients use the rooms was sought. From comparisons of cost and safety data, 3% hydrogen peroxide was chosen to be tested for its efficacy in the control of these bacteria. Systematic laboratory testing used 30 antibiotic-resistant and sensitive staphylococci and enterococci and 4 common hospital fabrics: cotton (clothing), terry cloth (towels), cotton-polyester blend (scrub suits), and polyester (curtains). Without disinfection, bacteria survived for many hours to several days. After a single spraying with 3% hydrogen peroxide, all bacteria on all fabrics were dead within 5 to 120 minutes. On-site testing targeted privacy curtains in patients' rooms. Curtain edges that tended to be grabbed when moving the curtain showed a mixture of gram-positive and gram-negative bacteria (median, 22 bacteria/24 cm2). After these areas were sprayed with 3% hydrogen peroxide, no bacteria were found. It was concluded that spraying with 3% hydrogen peroxide is a safe, inexpensive, effective means of spot-disinfecting fabrics in patients' rooms; this simple procedure may limit the spread of potentially pathogenic antibiotic-resistant bacteria.
Aerosols↗
Gelatinase activities in wounds of healing-impaired mice versus wounds of non-healing-impaired mice.
The gelatinases, matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), have been implicated in different aspects of wound repair. However, little is known about MMP-2 and MMP-9 activity in animal models of impaired wound healing. We sought to compare serial gelatinase activities for 25 days after full-thickness excisional wounds in genetically diabetic healing-impaired mice and their nondiabetic non-healing-impaired littermates. Wound samples were frozen, homogenized, clarified by centrifugation, and analyzed on zymography gels, and MMP bands were quantitated relative to a conditioned media standard from HT-1080 cells. Gelatinase activity in both diabetic mice and nondiabetic mice increased after the mice were wounded. However, levels of latent gelatinases peaked earlier in the diabetic wounds, and there was more active MMP-2 and MMP-9 in the wounds of the diabetic mice than in the wounds of the nondiabetic mice. Because the higher gelatinase activity in the wounds of the diabetic mice was similar to the higher levels of gelatinase reported in difficult-to-heal wounds such as ulcers and burns, this diabetic mouse model may be useful for studies of these proteinases and their inhibitors in impaired wound healing.
Animals↗
A survey of gram-negative bacteria survival on hospital fabrics and plastics.
One critical factor for the transmission of microorganisms from person to person or from the environment to a person (patient or health care worker) is the ability of the microbe to survive on an environmental surface. The purpose of this study was to determine the length of survival of various gram-negative bacteria on fabrics and plastics commonly used in hospitals. Seven materials were tested: smooth cotton (clothing), cotton terry (towels), 60% cotton-40% polyester blend (scrub suits and lab coats), polyester (drapes), 75% nylon-25% spandex (pressure garments), polyvinyl (splash aprons), and polyurethane (keyboard covers). The following bacteria were tested: Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Acinetobacter species, and Enterobacter species. Swatches of the materials were inoculated with defined amounts of bacteria and assayed at regular intervals. Survival was dependent on the bacterium, its inoculum size, and the material tested. At 102 microorganisms per swatch, bacteria survived from less than 1 hour to 8 days. At 10(4) to 10(5) bacteria per swatch, survival ranged from 2 hours to more than 60 days. These findings emphasize the need for careful disinfection and conscientious contact control procedures in areas that serve immunosuppressed individuals, such as patients with burn injuries.
Bedding and Linens↗
Does the addition of nystatin to 5% mafenide acetate and genitourinary irrigant solutions interfere with their antimicrobial activity? Assessment by two topical antimicrobial test assay systems.
Results previously reported using the Wet Disc Topical Antimicrobial Assay (WDA) suggested that adding nystatin (NY) to a 0.5% mafenide acetate (MA) suspension or genitourinary irrigant (double antibiotic [DAB]) to expand their antimicrobial activity to include Candida sp. antagonized the antibacterial effect of MA but not DAB. We use DAB solution as described by the authors of the previous study, also, but we use a 5% commercially available mafenide acetate solution instead of the in-house prepared 0.5% mafenide acetate suspension that they used. Further, we use both the WDA and the Agar Well Diffusion Topical Antimicrobial Assay (AWDA) to test topical antimicrobials at this institution. In light of the previously reported results, this study 1) examined whether adding nystatin to DAB or the 5% mafenide acetate solution used at this institution caused any interference in the ability of these substances to migrate through the agar matrices and cause zones of growth inhibition in the two test assay systems and 2) compared the assessment of microbial susceptibility (by very precise definition) between the two systems. The addition of nystatin did not interfere with the ability of either DAB or mafenide acetate to migrate through the agar matrices and cause clear zones. However, on the assessment of susceptibility a significantly larger number of organisms were judged susceptible using the AWDA than the WDA. We believe that the disparity is caused by a large difference in agar diffusion kinetics between the two assays. Therefore, we recommend the AWDA rather than the WDA for susceptibility studies.
Administration, Topical↗