Net Proteolytic Activity Research Articles

Atypical response to bacterial coinfection and persistent neutrophilic bronchoalveolar inflammation distinguish critical COVID-19 from influenza.

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non–COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1β, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.

Bifunctional inhibitors of urokinase and metalloproteinase-9 for cancer treatment - in silico evaluation

Matrix metalloproteinase-9 (MMP-9), and urokinase plasminogen activator (uPA) overexpression or/and increased activity are considered causative elements for cancer invasion and metastasis. These enzymes are degrading the extracellular matrix (ECM) providing space for cancer progression and cancer cell mobility. Process of angiogenesis, in which microvascular endothelial cells form blood vessels, requires local degradation of the underlying basal lamina to invade into the stroma proximal to cancer, and it strongly depends on the activity of MMP-9 and uPA as well. Malignant tumor invasion, cancer metastasis and angiogenesis have been documented as a fundamental factors in the morbidity and mortality among cancer patients, thus their inhibition can be exploit therapeutically. Numerous in vivo and in vitro studies have demonstrated that inhibition of proteolytic activity can reduce caner invasion, tumor size and limit angiogenesis. Consequently human clinical studies were designed inhibiting urokinase or MMPs, but these target specific inhibitors produce mix results. One of the possible explanation could be that cancers are overexpressing more than one enzyme simultaneously – for instance urokinase and MMPs. Thus upregulated net proteolytic activity should be normalized rather than trying to inhibit single proteolytic enzyme. Therefore, starting from specific inhibitors we have created - in silico - several hybrid molecules that could inhibit both uPA and MMP-9. The best hybrid (UI1xAGB) had theoretical affinities of Ki = 1.61-9 mol for MMP-9 and Ki = 1.36-9 mol for uPA. In the future each individual hybrid would need to be successfully synthesized and checked in the in vitro and in vivo analyses.

Cerebrospinal fluid matrix metalloproteinase 9 levels, blood-brain barrier permeability, and treatment outcome in tuberculous meningitis

ObjectivesTuberculous meningitis is characterized by elevated levels of matrix metalloproteinase 9 (MMP9) in the cerebrospinal fluid (CSF). However, it is unclear whether elevated MMP9 levels are associated with poor treatment outcome. We tested the hypothesis that pretreatment MMP9 levels in the CSF would be higher in tuberculous meningitis patients experiencing a poor treatment outcome.MethodsWe prospectively assessed the treatment outcome in a consecutive sample of human immunodeficiency virus-negative patients with tuberculous meningitis. We defined good outcome as survival without severe neurological disability (modified Rankin scale scores 0–2). We estimated levels of MMP9 and its tissue inhibitor (TIMP1) on pretreatment CSF samples. We used albumin index to assess blood-brain barrier permeability.ResultsWe studied 40 patients (23 males [58%]) with tuberculous meningitis. Sixteen patients (40%) had stage 3 disease. On follow-up, 18 (45%) patients had a poor treatment outcome—15 patients died and 3 had severe neurological disability. Pretreatment MMP9 levels were not associated with treatment outcome (median [interquartile range], 254 [115–389] vs. 192 [60–383] ng/mL in good vs. poor outcome groups; P = 0.693). MMP9 levels did not correlate with the albumin index (Spearman’s rho = 0.142; P = 0.381). However, MMP9 levels significantly correlated with CSF glucose levels (rho = −0.419; P = 0.007) and admission Glasgow coma scale score (rho = 0.324; P = 0.032). Likewise, TIMP1 levels also did not differ by treatment outcome (1239 [889–1511] vs. 1522 [934–1949] ng/mL; P = 0.201). MMP9/TIMP1 ratio that reflects net proteolytic activity was also not different between the two groups (0.191 [0.107–0.250] vs. 0.163 [0.067–0.34]; P = 0.625).ConclusionOur findings do not support the hypothesis that pretreatment levels of MMP9 would be higher in tuberculous meningitis patients experiencing a poor treatment outcome. Further, MMP9 levels in the CSF did not correlate with blood-brain barrier permeability in patients with tuberculous meningitis.

Protean proteases: at the cutting edgeoflung diseases.

Proteases were traditionally viewed as mere protein-degrading enzymes with a very restricted spectrum of substrates. A major expansion in protease research has uncovered a variety of novel substrates, and it is now evident that proteases are critical pleiotropic actors orchestrating pathophysiological processes. Recent findings evidenced that the net proteolytic activity also relies upon interconnections between different protease and protease inhibitor families in the protease web.In this review, we provide an overview of these novel concepts with a particular focus on pulmonary pathophysiology. We describe the emerging roles of several protease families including cysteine and serine proteases.The complexity of the protease web is exemplified in the light of multidimensional regulation of serine protease activity by matrix metalloproteases through cognate serine protease inhibitor processing. Finally, we will highlight how deregulated protease activity during pulmonary pathogenesis may be exploited for diagnosis/prognosis purposes, and utilised as a therapeutic tool using nanotechnologies.Considering proteases as part of an integrative biology perspective may pave the way for the development of new therapeutic targets to treat pulmonary diseases related to intrinsic protease deregulation.

Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE−/− mice prevent collateral host tissue damage.

Plasminogen activator inhibitor-1 in kidney pathology

Plasminogen activator inhibitor type-1 (PAI-1) inhibits tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which convert plasminogen to plasmin, a strong proteolytic enzyme. Thus, PAI-1 is a primary and negative regulator of plasmin-driven proteolysis. In addition to its main role as an inhibitor of fibrinolysis, PAI‑1 has been implicated as a mediator in other processes, including fibrosis, rheumatoid arthritis, atherosclerosis, tumor angiogenesis and bacterial infections. It also significantly modulates cellular adhesion or migration, wound healing, angiogenesis and tumor cell metastasis. However, in the present study, we have reviewed the literature in relation to different kidney diseases where PAI-1 regulates fibrinolysis and acts independently of proteolysis. PAI-1 is normally produced in trace amounts in healthy kidneys but is synthesized in a wide variety of both acute and chronic diseased kidneys. We reviewed the role of PAI-1 in diabetic kidney nephropathy, chronic kidney disease, hemodialysis, peritoneal dialysis and in kidney transplantation. Increased PAI-1 expression results in accumulation of extracellular matrix (ECM) leading to numerous kidney diseases. Predisposition to some diseases is due to the genetic role of PAI-1 in their development. A number of studies demonstrated that the inhibition of PAI-1 activity or therapy with a mutant PAI-1 increases matrix turnover and reduces glomerulosclerosis by competing with endogenous PAI-1. This strongly suggests that PAI-1 is a valid target in the treatment of fibrotic renal disease. However, net proteolytic activity depends on the delicate balance between its negative regulation by PAI-1 and activation by uPA and tPA. Also, plasmin activated by its inhibitors upregulates activity of other enzymes. Thus, assessment of prognosis for the diseased kidney should include a variety of proteolysis regulators and enzymes.

Challenging delivery of VLHL NS plasminogen activator inhibitor-1 by osmotic pumps in diabetic mouse: A case report

ALZET® osmotic pumps are implantable devices used in animals for the continuous infusion of drugs or proteins at controlled rates from 1 day to 4 weeks. Pumps have been used successfully in a number of studies on the effects of controlled delivery of a wide range of experimental agents, independent of their properties. In the present study, use of these pumps was made in mice with diabetic nephropathy. Plasminogen activator inhibitor-1 (PAI-1) mediates diabetic nephropathy, which is characterized by the excessive accumulation of extracellular matrix (ECM) in the kidney. Disproportionate PAI-1 inactivates tissue plasminogen activator, which is one of the proteolytic enzymes in a cascade responsible for ECM remodeling in the kidney. The decrease of PAI-1 in the kidney has been shown to arrest the progression of nephropathy in experimental animals. This was achieved using inactive PAI-1R which increased the clearance of wild-type PAI-1 in order to protect net proteolytic activity and ECM clearance. However, this protein has a brief half-life in vivo, therefore, high and frequent doses are required. Thus, VLHL NS PAI-1 protein with a long half-life of over 700 h (Gln197Cys, Gly355Cys) inactivated by single point mutation (Arg369Ala) was used. Following the sacrifice of animals the tips of the flow moderators of the osmotic pumps in the treated animals were found to be clogged. In addition, from each pump from the treatment group, but not controls, we collected 50–150 μl of clear liquid containing VLHL NS PAI-1, cellular and serum proteins suggesting early pump sealing by cellular material. In conclusion, despite encouraging results obtained for the PAI-1R protein, the method of VLHL PAI-1 delivery should be ameliorated.

The Cystic Fibrosis Transmembrane Conductance Regulator Impedes Proteolytic Stimulation of the Epithelial Na+ Channel

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl(-) secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na(+) absorption. The mechanistic link between missing CFTR and increased Na(+) absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na(+) channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na(+) absorption.

A novel approach to measure the contribution of matrix metalloproteinase in the overall net proteolytic activity present in synovial fluids of patients with arthritis

Despite decades of research, only a very limited number of matrix metalloproteinase (MMP) inhibitors have been successful in clinical trials of arthritis. One of the central problems associated with this failure may be our inability to monitor the local activity of proteases in the joints since the integrity of the extracellular matrix results from an equilibrium between noncovalent, 1:1 stoichiometric binding of protease inhibitors to the catalytic site of the activated forms of the enzymes. In the present work, we have measured by flow cytometry the net proteolytic activity in synovial fluids (SF) collected from 95 patients with osteoarthritis and various forms of inflammatory arthritis, including rheumatoid arthritis, spondyloarthropathies, and chronic juvenile arthritis. We found that SF of patients with inflammatory arthritis had significantly higher levels of proteolytic activity than those of osteoarthritis patients. Moreover, the overall activity in inflammatory arthritis patients correlated positively with the number of infiltrated leukocytes and the serum level of C-reactive protein. No such correlations were found in osteoarthritis patients. Members of the MMP family contributed significantly to the proteolytic activity found in SF. Small-molecular-weight MMP inhibitors were indeed effective for inhibiting proteolytic activity in SF, but their effectiveness varied greatly among patients. Interestingly, the contribution of MMPs decreased in patients with very high proteolytic activity, and this was due both to a molar excess of tissue inhibitor of MMP-1 and to an increased contribution of other proteolytic enzymes. These results emphasize the diversity of the MMPs involved in arthritis and, from a clinical perspective, suggest an interesting alternative for testing the potential of new protease inhibitors for the treatment of arthritis.

Effect of doxycycline on sulfur mustard-induced respiratory lesions in guinea pigs

Respiratory tract lesions induced by the chemical warfare agent sulfur mustard (SM) are characterized by epithelial damages associated with inflammatory cell infiltration. Here we evaluated the imbalance between gelatinase and tissue inhibitors of metalloproteinases (TIMPs), and we tested pretreatment with the protease inhibitor doxycycline. Guinea pigs were intoxicated intratracheally with SM and evaluated 24 h after exposure. Matrix metalloproteinase (MMP) gelatinase activity of bronchial lavage (BL) fluid from SM-exposed guinea pigs was high compared with controls, as shown by both zymography and biotinylated substrate degradation, whereas TIMP-1 and -2 levels by immunoblotting were similar. Extensive areas of lysis were evidenced by in situ zymography, indicating imbalance between gelatinases and inhibitors towards net proteolytic activity. Doxycycline pretreatment resulted in 1) decreased gelatinase activity (zymography, free gelatinase activity assay, and in situ zymography); 2) decreased inflammation (BL fluid cellularity and protein level); and 3) dramatic decrease in histological epithelial lesions. Our results suggest inadequate levels of TIMP to counteract increased gelatinase activity and further support a role for MMP gelatinases in SM-induced respiratory lesions. They also suggest that doxycycline may hold promise as a therapeutic tool.

Hepsin and Maspin are Inversely Expressed in Laser Capture Microdissectioned Prostate Cancer

Recent studies have shown that hepsin, a serine protease, is over expressed in prostate cancers, implicating hepsin activity in tumor invasion. Using microarray technology we have previously identified 22 genes that were up-regulated in high grade prostate cancers compared with benign prostatic hyperplasia. Of them hepsin was the most differentially over expressed. In the current report we compare hepsin to maspin (BD Transduction Laboratories, San Diego, California), a serine protease inhibitor (serpin), to measure the balance between levels of serine proteases and serpins, which are considered to be a critical determinant of net proteolytic activity. We combined the technique of laser capture microdissection with gene expression monitoring by micro-array analysis to investigate the gene expression profiles of prostate cells of different histological types. We also studied maspin immunohistochemically. We observed that hepsin as well as 7 of 22 previously reported up-regulated genes demonstrated a pattern of increasing expression with increasing malignant phenotype. In contrast, the expression of maspin (a serpin) decreased with increasing malignancy of prostate cancers. Using immunohistochemistry we observed that maspin protein is expressed strongly in benign prostatic tissues and slightly in grade 3 prostate cancers, and is absent in grade 4/5 cancers. We conclude that the increased ratio of hepsin-to-maspin may have an important role in prostate cancer progression and invasion.

Matrix metalloproteinases: regulation and dysregulation in the failing heart.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible for myocardial extracellular protein degradation. Several MMP species identified within the human myocardium may be dysregulated in congestive heart failure (CHF). For example, MMPs that are expressed at very low levels in normal myocardium, such as collagenase-3 (MMP-13) and the membrane-type-1 MMPs, are substantially upregulated in CHF. However, MMP species are not uniformly increased in patients with end-stage CHF, suggesting that a specific portfolio of MMPs are expressed in the failing myocardium. With the use of animal models of CHF, a mechanistic relationship has been demonstrated with respect to myocardial MMP expression and the left ventricular (LV) remodeling process. The tissue inhibitors of the MMPs (TIMPs) are locally synthesized proteins that bind to active MMPs and thereby regulate net proteolytic activity. However, there does not appear to be a concomitant increase in myocardial TIMPs during the LV remodeling process and progression to CHF. This disparity between MMP and TIMP levels favors a persistent MMP activation state within the myocardium and likely contributes to the LV remodeling process in the setting of developing CHF. The elucidation of upstream signaling mechanisms that contribute to the selective induction of MMP species within the myocardium as well as strategies to normalize the balance between MMPs and TIMPs may yield some therapeutic strategies by which to control myocardial extracellular remodeling and thereby slow the progression of the CHF process.

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571–572 and Leu578–579) and tyrosine573 residues are important for the internalization, and the μ2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus.

The local increase in the secretion of extracellular proteases, allowing cleavage of the extracellular matrix and thereby facilitating the infiltration of T cells, monocytes and neutrophils, is a hallmark of chronic inflammation and autoimmunity. In pulmonary genetic diseases, such as emphysema and cystic fibrosis, proteases can also favour the development of local immunodeficiency by degrading key regulators of the immune response, such as CD4, CD8, IgG, ICAM-1 and C3b receptors. Since several infectious agents can give rise to severe pulmonary disorders associated with opportunistic infections, we sought to determine whether an increase in proteolytic activity occurred during infection with porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of a new disease in swine characterized by severe respiratory problems in young pigs. Piglets were infected with the virus and bronchoalveolar lavages were collected at various times post-infection to measure the net proteolytic activity. It was shown that PRRSV infection leads to a significant increase in proteolytic activity in pulmonary fluids. Maximal activity was found at 7 and 14 days post-infection, with a return towards normal levels at day 42. Zymographic analyses showed a significant increase in the secretion of matrix metalloproteases (MMPs) 2 and 9, two enzymes involved in tissue remodelling. Histological analyses showed a correlation between the increase in proteolytic activity and the appearance of lesions that were characterized by massive lymphomononuclear cell infiltration. These results suggest that virus infection of the lungs can lead to a transient increase in proteolytic activity that could favour opportunistic infection.

Evidence for altered balance between matrix metalloproteinases and their inhibitors in human aortic diseases.

Although abdominal aortic aneurysms (AAAs) exhibit increased expression of matrix metalloproteinases (MMPs), the functional balance between MMPs and their tissue inhibitors (TIMPs) remains uncertain. This report compares the proteolytic activity in normal aorta, aorto-occlusive disease (AOD), and AAA by use of a novel in situ zymographic technique. Infrarenal aortic specimens were obtained from 25 patients undergoing surgery for AOD or AAA and were compared with normal aortic tissue (n = 7) obtained from cadavers. Immunohistochemical staining was performed for collagenase (MMP-1), gelatinase A (MMP-2), stromelysin (MMP-3), TIMP-1, and TIMP-2. Net proteolytic activity was determined with in situ zymography whereby aortic sections were incubated on fluorescently labeled substrate. Proteolytic activity was detected under epifluorescent examination. Compared with normal aortic tissue, AOD and AAA tissue demonstrated marked increases in MMP-1 and MMP-3 immunoreactivity, predominantly in the neointima, and modest increases in TIMP-1. MMP-2 was increased in the diseased aortas, and TIMP-2 was abundant in normal, AOD, and AAA samples. Zymography revealed proteolytic activity in AOD and AAA tissues with active digestion of casein and gelatin substrate, particularly on the luminal portion of the specimens. Normal specimens exhibited no lytic activity. Comparison of AOD and AAA specimens revealed no difference in MMP/TIMP immunoreactivity or net proteolytic activity. MMP expression is markedly increased in AOD and AAA samples, and an imbalance between MMPs and their inhibitors results in similar proteolytic activity. The eventual formation of aneurysmal or occlusive lesions appears not to result from an ongoing difference in the proteolytic pattern.

Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue

Many physiological and pathological processes involve tissue remodeling due in part to degradation of extracellular matrix. Several factors limit current approaches used for detection of matrix-degrading enzymes in tissues. Matrix metalloproteinases (MMPs), enzymes specialized in catabolism of extracellular matrix constituents, require processing from inactive zymogen precursors to gain enzymatic function. Presently available antibodies do not distinguish between precursor and proteolytically processed forms of MMPs. Also, ubiquitous tissue inhibitors of metalloproteinases (TIMPs) could prevent matrix degradation by MMPs even if the enzymes were in an active form. For these reasons immunocytochemistry does not provide information regarding the functional state of these enzymes. Biochemical studies of tissue extracts preclude localization and entail the possibility of artifactual activation of the enzymes consequent to tissue disruption. To obviate these problems, we have adapted substrate zymography to frozen tissue sections to assess net proteolytic activity in situ. We report here the details and the validation of this methodology. Initial experiments defined casein fluorescently labeled with resorufin as a useful substrate for detecting stromelysin, and fluoresceinated gelatin or autoradiographic emulsion as suitable for detecting gelatinolytic activity by this approach. Either TIMP-1 or the Zn chelator 1,10-phenanthroline reduced the zymographic activity in cryosections of atheroma from humans or rabbits. Inhibitors of serine proteases did not reduce the extent of substrate lysis substantially. In situ zymography preserves the fine morphological details of the tissue and can complement the study of enzyme expression by other microscopic techniques, such as immunocytochemistry. This approach may prove generally applicable for the detection of protease activity in tissue sections permitting exploration of the roles of these enzymes in pathobiology.

Development of a convenient and sensitive assay for matrix metalloproteinase enzyme activity in synovial fluid samples using fluorigenic peptides

Matrix metalloproteinases (MMPs) are considered to play a major role in the proteolytic degradation of extracellular matrix in joint and skeletal diseases. Inactive forms of these enzymes are secreted by cells of the joint, together with their tissue inhibitors (TIMPs). Extracellular activation results in active MMPs which can degrade most matrix components of articular cartilage such as proteoglycans and collagen proteins. Matrix degradation is inhibited by interaction of TIMPs with active MMPs. Under physiological conditions, active MMPs are efficiently neutralized, predominantly by TIMPs (MMP/TIMP ratio ≤ 1), so that net proteolytic activity is absent or very low. Under pathological conditions like in rheumatoid arthritis and osteoarthritis, levels of active MMPs as well as TIMPs are increased. More importantly, active enzymes are present in excess over the amount of tissue inhibitor (MMP/TIMP ratio>1).

Effect of proteolytic activity on virulence of Candida albicans in burned mice

Total circulating proteolytic activity (PA) was determined by measuring the acid-soluble 125I-protein fragments generated per 100 microliters of serum incubated with 125I-protein at 37 degrees C for 15 min. Normal mice had low circulating PA (1.3 +/- 0.2 micrograms/100 microliters), and burned mice had a higher average PA; the actual value depended on the time of measurement postburn. We measured the effect on mortality and on circulating PA of challenging normal and burned mice with high-virulence strain Candida albicans MY 1044 and its less virulent mutant MY 1049. Burned and normal mice challenged with a high dose (10(5)) of MY 1044 had high mortality (greater than 90%) and high circulating PA (greater than 33 micrograms generated per 100 microliters). Burned mice challenged with a lower dose (10(4] of MY 1044 had moderate mortality (63%) and lower PA (27.2 +/- 4.2 micrograms/100 microliters). All other groups of mice, including burned mice challenged with 10(5) MY 1049, had low mortality (less than 10%), and PAs were less than 22 micrograms/100 microliters. Augmentation of burned mice challenged with 10(5) MY 1049 with proteinase significantly increased mortality; with treatment of burned mice challenged with 10(5) MY 1044 with proteinase inhibitor significantly decreased mortality. We conclude that mortality correlated with total circulating PA; that the contribution to this net PA was the background PA level in the normal mice, the PA associated with the burn, and the PA caused by infection with a C. albicans strain with a particular virulence; that most deaths caused by C. albicans occurred past a PA threshold of 25 micrograms/100 microliters in the host; and that the number of burned and infected mice that died of candidiasis could be modulated by the addition of proteinases or proteinase inhibitors to the host. This last finding may lead to some novel treatments for candidiasis in burned hosts.

Plasma proteolytic activity following burns.

Because a number of metabolic events which are triggered by proteolysis in the bloodstream are activated following trauma, net proteolytic activity (P.A.) in the plasma of 37 pediatric burned patients was measured. The P.A. assay involved incubating plasma with a radioiodinated protein substrate and counting the isotopic activity of the hydrolyzed fragments in the acid-soluble fraction. In patients, plasma P.A. increased in direct proportion to the extent of burn injury. To examine additional trends in plasma P.A. suggested by the patient P.A. data, we measured plasma P.A. in a burned rat model: circulating P.A. was significantly elevated at 6 hours and until at least 2 weeks postburn; infection with Pseudomonas and use of the proteolytic debriding agent, Travase, each further elevated this activity; the plasma P.A. was not directly derived from the burn site. We postulate that this elevated circulating P.A. triggers some of the pathologic as well as some physiologic sequelae which follow burn trauma.