An acute phase protein α1-acid glycoprotein mitigates AKI and its progression to CKD through its anti-inflammatory action.

The molecular mechanism for acute kidney injury (AKI) and its progression to chronic kidney disease (CKD) continues to be unclear. In this study, we investigated the pathophysiological role of the acute phase protein α1-acid glycoprotein (AGP) in AKI and its progression to CKD using AGP KO mice. Plasma AGP levels in WT mice were increased by about 3.5-fold on day 1-2 after renal ischemia-reperfusion (IR), and these values then gradually decreased to the level before renal IR on day 7-14. On day 1 after renal IR, the AGP KO showed higher renal dysfunction, tubular injury and renal inflammation as compared with WT. On day 14, renal function, tubular injury and renal inflammation in WT had recovered, but the recovery was delayed, and renal fibrosis continued to progress in AGP KO. These results obtained from AGP KO were rescued by the administration of human-derived AGP (hAGP) simultaneously with renal IR. In vitro experiments using RAW264.7 cells showed hAGP treatment suppressed the LPS-induced macrophage inflammatory response. These data suggest that endogenously induced AGP in early renal IR functions as a renoprotective molecule via its anti-inflammatory action. Thus, AGP represents a potential target molecule for therapeutic development in AKI and its progression CKD.

α2-Macroglobulin: Autologous Protease Inhibition Technology.

α2-Macroglobulin (A2M) is a plasma glycoprotein best known for its ability to inhibit a broad spectrum of serine, threonine, and metalloproteases as well as inflammatory cytokines by a unique bait-and-trap method. A2M has emerged as a unique potential treatment of cartilage-based pathology and inflammatory arthritides. This article describes the unique method by which A2M not only inhibits the associated inflammatory cascade but also disrupts the catabolic process of cartilage degeneration. Autologous concentrated A2M from plasma is currently in use to successfully treat various painful arthritides. Future directions will focus on recombinant variants that enhance its anti-inflammatory and disease-modifying potential.

Cardiovascular hemodynamics after opsonic alpha-2-surface binding glycoprotein therapy in injured patients.

Depression of reticuloendothelial (RE) phagocytic function has been clearly documented following trauma and operation. This phagocytic failure is mediated in part by depletion of an opsonic glycoprotein. Depletion of this opsonic protein may result in prolonged blood retention of potentially harmful particulates that may interfere with the microcirculation and may possibly result in altered organ function. Isolation and identification of this opsonic protein has led to the finding of the identity between opsonic glycoprotein and cold insoluble globulin (CIg) or so-called plasma fibronectin. Since CIg is concentrated in cryoprecipitate, this blood component was used as a readily available source of opsonic protein for replacement studies. Nine patients were studied following a 1-hour infusion of cryoprecipitate obtained from 10 units of plasma and suspended in a volume of 250 ml. Both the pulmonary shunt fraction and the fraction of dead space ventilation decreased significantly (P = 0.02) after cryoprecipitate administration. Limb blood flow (P = 0.001), limb oxygen consumption (P = 0.001), and reactive hyperemia of the limb (P = 0.05) increased significantly following cryoprecipitate infusion. Cardiac output, total oxygen consumption did not change consistently. The data demonstrate that the infusion of cryoprecipitate resulted in improved pulmonary and microcirculatory function--possibly due to opsonic glycoprotein replacement.

Collagenase inhibitors: rationale for their use in treating corneal ulceration.

Tissue collagenases have been implicated in corneal ulceration in human corneal disease and in ulceration of the rabbit cornea that has served as a model system. Such enzymes from the rabbit and human cornea are inhibited by metal-binding agents of the EDTA type, by thiols, and by the human serum antiprotease alpha2-macroglobulin. Determination of the relative efficacies of collagenase inhibitors indicates that EDTA and Ca-EDTA are about one hundred times more effective on a molar basis than L-cysteine and its derivatives, N-acetyl-L-cysteine and D-penicillamine. The alpha2-macroglobulin on a molar basis, is superior as an inhibitor to the metal-binding agents and thiols. Although Ca may be a necessary cofactor of the corneal collagenases, such a requirement has not been established unequivocally. Inhibition and isotope studies do indicate a requirement for Zn. Thiols are thought to inhibit corneal collagenases by binding to or removing an intrinsic metal cofactor (Zn), and/or possibly by reducing one or more disulfide bonds. Inhibition by both EDTA-type agents and thiols is largely reversible by dialysis. The human alpha2-macroglobulin appears to inhibit corneal colleagenases irreversibly by forming tight complexes with them. Ca-EDTA, cysteine, and acetylcysteine, given as eyedrops, are able to prevent or retard ulceration in the alkali-burned rabbit cornea. They appear to have some efficacy in the prevention of corneal ulceration in humans. EDTA-type compounds are quite stable under routine storage, while acetylcysteine is more stable than cysteine. EDTA is quite toxic and should not be used as eye medication. Ca-EDTA has a low toxicity, and cysteine and acetylcysteine have even lower toxicity. It is not yet certain which inhibitor has the most favorable therapeutic index for clinical use, or is the optimal mode of drug delivery known. However, the collagenase inhibitors seem to have therapeutic promise in the prevention of corneal ulceration.

[Alpha 2-macroglobulin: structure, properties and physiological role]. / Alpha 2-makroglobulin: struktura, svoistva i fiziologicheskaia rol'.

The paper is concerned with the results of recent researches devoted to studies of the structure, properties and physiological role of alpha 2-macroglobulin, one of main inhibitors of blood proteolytic enzymes. Data are presented on its primary and quaternary structure, mechanisms of interaction with proteinases. The role of alpha 2-macroglobulin in regulation of the activity of proteinases participating in blood coagulation, fibrinolysis, kininogenesis, immune reactions is shown. Possibilities of its application in medicine are discussed.

Effect of the treatment with alpha-macroglobulin on the development of experimental pancreatitis.

UNLABELLED: Alpha-macroglobulins are proteinase inhibitors. Monofluorophosphate increases alpha-macroglobulin levels in plasma, inducing a higher survival rate and lower pancreatic damage in rats with pancreatitis. The aim of this study was to evaluate the effect of alpha-macroglobulin on the development of pancreatitis. Pancreatitis was surgically induced in Sprague-Dawley rats divided into groups of 16 rats each and -subjected to the following intravenous treatments for 3 days: CONTROLS: pancreatitis without treatment, Enriched plasma: pancreatitis+-alpha-macroglobulin-enriched plasma, Normal plasma: pancreatitis+plasma with normal levels of alpha-macroglobulin, Saline Solution: pancreatitis+saline solution, Purified alpha-macroglobulin: pancreatitis+purified alpha-macroglobulin. After 14 days pancreatic damage was assessed using a score that measures: edema, fibrin, neutrophils, mononuclear leukocytes, necrosis, vascular congestion, thrombosis, hemorrhage and fibrosis. Pancreatic damage decreased and the percentage of animals with pancreatitis was lower in enriched-plasma and purified alpha-macroglobulin groups. We conclude that the intravenous administration of alpha-macroglobulins causes a reduction in the histological damage produced by pancreatitis.

Polymyxin B-conjugated alpha 2-macroglobulin as an adjunctive therapy to sepsis: Modes of action and impact on lethality.

A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-alpha (TNF-alpha)] should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein alpha2-macroglobulin (A2M). The conjugate binds TNF-alpha as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-alpha from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 microg/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.

[Efficacy of the use of a stabilized alpha2-macroglobulin concentrate in experimental burns]. / Effektivnost' primeneniia stabilizirovannoi kompozitsii kontsentrata alpha2-makroglobulina pri ozhogovoi bolezni v éksperimente.

The effectiveness of the use of a stabilized composition of alpha 2-macroglobulin concentrate obtained from fraction III of plasma proteins according to Cohn in burn disease in the experiment on guinea pigs was studied. Under the influence of intraabdominal administration for 4 days of alpha 2-macroglobulin concentrate with a stabilizer, healing of the burn wounds, normalization of a state of the animals occurred 2 times more rapidly than in control ones, activity of acid proteinases restored by day 5 (in control--by day 14) due to increase in activity of proteolytic enzyme inhibitors.

[Immunosuppression (author's transl)]. / Immunosuppression.

Among different procedures of immunosuppression in man medical treatment with cytotoxic drugs has become the most important. Biological methods are still under development but promise to gain in importance since they are more specific and have fewer side effects. The clinical effect of immunosuppressive therapy in organ transplantation is undisputed. Its effects has been proven in some but not all immuno-inflammatory diseases. Before immunosuppressive therapy is started it must be known whether it is effective in the particular disease involved. The severity of the disease has to be critically balanced against the possible side effects of cytotoxic drugs.

Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: extracellular protease and elastase as in vivo virulence factors.

The effects on mortality of supplemental injections of protease and elastase were determined in burned mice infected with non-lethal inocula of a toxin-producing but non-proteolytic-enzyme-producing strain of Pseudomonas aeruginosa. When a variety of solutions containing proteolytic enzyme were injected under these conditions, the mortality increased significantly. This did not occur when organisms other than P. aeruginosa were used. Injections of the enzyme solutions alone were non-lethal. Injection of a solution of alpha 2-macroglobulin, which was shown to inhibit proteolytic activity, together with a proteolytic enzyme--toxin producing strain of P. aeruginosa caused a significant delay in mortality when compared with controls. It was concluded that protease, elastase, and toxin production were necessary for P. aeruginosa to express full virulence in the burned mouse model.

Regulation of collagenase. Therapeutic considerations.

Why the cornea ulcerates, in the sense of what goes awry, may be related to the trapping of wound healing in a phase of proteolytic debridement related to a persistent epithelial defect. The initial avascularity of the cornea makes it particularly vulnerable to proteolytic damage. Studies on the biochemistry and cell biology of corneal ulceration have indicated that sequential interactions occur which result in the generation of collagenase activity and the development of ulceration. It is likely that the interactions are susceptible to intervention; and it is thought that eventual, successful treatment of corneal ulceration will require a complex therapy, with interventions at multiple sites of regulation.

Suppression of experimental allergic encephalomyelitis by alpha 2-macroglobulin.

Lewis rats sensitized with guinea-pig spinal cord in Freund's complete adjuvant developed an acute-phase protein response. This was characterized by a marked increase in plasma alpha 2-macroglobulin (alpha 2 M) levels which, however, declined towards normal values before the onset of clinical signs of experimental allergic encephalomyelitis (EAE). In contrast, levels of two other acute-phase proteins, fibrinogen and caeruloplasmin, remained variably elevated over the entire study period. Recovery from EAE coincided with an increase in alpha 2 M levels. Infusion of purified alpha 2 M effectively protected the rats against clinical EAE and this was associated with a restimulation of the acute-phase response. The protected rats were shown to be sensitized to myelin basic protein and to have comparable mononuclear infiltration of the central nervous system with the diseased animals. It is postulated that the infusion of alpha 2 M leads to the inhibition of the effector pathways of the delayed type hypersensitivity response.

Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: effect of treatment with protease inhibitors.

Data are presented showing that treatment of burned, Pseudomonas aeruginosa-infected mice with the protease inhibitor alpha 2-macroglobulin but not treatment with phosphoramidon enhanced survival. Treatment with alpha 2-macroglobulin caused reductions in bacterial counts in the skin and livers of infected mice and also protected liver elongation factor 2. Similar results were observed when human IgG was used for treatment. The protective effect of the IgG treatment was probably due to the presence of opsonizing antibodies in the preparation. The protective capacity of the IgG could be removed by its adsorption with heat-killed cells of the strain used for infecting the mice. The protection afforded by alpha 2-macroglobulin was not due to the presence of opsonizing antibodies in the preparation used. It appeared to be due to inhibition of the proteolytic, not the elastolytic, activities of alkaline protease and elastase elaborated by the organisms growing in the burned skin tissue. Proteolytic activities of these enzymes appeared to serve as virulence factors in P. aeruginosa by decreasing the generation time in vivo of the microorganisms growing in the burned skin tissue and by allowing the organisms to spread from this local site into the systemic circulation. Treatment of pseudomonas infections of burn wounds with protease inhibitors may serve as an alternative to antibiotic treatment and/or immunotherapy.

Effective intraperitoneal antiprotease therapy for taurocholate-induced pancreatitis in rats.

In canine pancreatitis, irreversible hypotension and death follow saturation of the antiprotease molecules in peritoneal exudate by activated proteolytic enzymes which are released from the pancreas. This study has examined, in rats with taurocholate-induced pancreatitis, the efficacy of removal of the peritoneal exudate by aspiration and a single lavage, followed by instillation of an exogenous antiprotease solution. Instillation of human fresh frozen plasma, containing alpha 2-macroglobulin and alpha 1-antiprotease, was associated with the longest median survival. Aprotinin, although possessing a much greater trypsin inhibitory capacity, just failed to significantly improve the median survival time compared with the control group. Intraperitoneal antiprotease therapy is simple to perform, has a beneficial effect on survival time in this model and merits investigation in man.

[Effect of alpha 2-macroglobulin on indices of the proteolysis system and organ blood flow in tourniquet shock]. / Vliianie al'fa 2-makroglobulina na pokazateli sistemy proteoliza i organnogo krovotoka pri turniketnom shoke.

It was shown that alpha 2-macroglobulin (50 mg/kg, 30 min before revascularization of previously ischemized extremities) significantly inhibits the blood serum proteolytic activity and exerts similarly to contrykal (10,000 U/kg) the correcting effect on the level of arterial blood pressure and parameters of the blood flow in the kidneys, intestine and previously ischemized extremities.

Plasma proteinase inhibitor activity and hemostasis tests in children with nephrotic syndrome. Effect of prednisone alone and prednisone plus epsilon-aminocaproic acid treatment regimens: a preliminary report.

Neutrophil-derived proteinases cause glomerular injury by proteolysis of the glomerular basement membrane and alterations in glomerular metabolism. Recently, a marked elevation of the plasma elastase complex with alpha1-proteinase inhibitor (alpha 1-PI) both in the acute phase and during remission of nephrotic syndrome (NS) compared with age-matched controls was reported. In experimental immune-mediated glomerulonephritis epsilon-aminocaproic acid (EACA) significantly reduced albuminuria, and it was suggested that this may be linked with the antiproteolytic activity of the drug. We studied plasma antithrombin III (AT-III), alpha 1-PI, alpha 2-antiplasmin (alpha 2-A), alpha 2-macroglobulin (alpha 2-M) activity, and some blood coagulation and fibrinolysis tests in children with frequently relapsing prednisone-responsive NS. Also, the effect of prednisone alone (Group I, n = 9) and prednisone plus EACA (Group II, n = 10) treatment regimens on the studied parameters was estimated. All investigations were performed on admission to the hospital and after approximately 13 days of prednisone alone therapy (Group I), as well as before the administration of prednisone plus EACA and 24 hours after the last dose of EACA, ie, after approximately 5 days of treatment (Group II). Prednisone was administered at the usual dose of approximately 2 mg/kg/d and EACA was given orally at the doses of 72 to 230 mg/kg of body weight per day for 3 to 10 days. In the acute phase of disease, NS patients (n = 19) were shown to have a statistically significant decrease of plasma AT-III (16.4 +/- 4.7 vs. 21.9 +/- 2.5 IU/mL) and alpha 1-PI (1.28 +/- 0.6 vs. 1.97 +/- 0.34 IU/mL) activity, as well as a marked increase in plasma alpha 2-M activity (14.96 +/- 5.81 vs. 9.6 +/- 1.6 IU/mL), and fibrinogen concentration (5.51 +/- 1.78 vs. 2.96 +/- 0.34 g/L) compared to the age-matched controls; no significant changes in plasma alpha 2-A activity, plasminogen concentration, euglobulin clot lysis time, activated partial thromboplastin time (APTT), or thromboplastin time were noted. In children treated with prednisone alone, a marked increase in plasma AT-III (by 76%, P < 0.001) and alpha 2-A (36%, P < 0.019) activity, and a significant decrease of the plasma fibrinogen concentration (6.07 +/- 1.66 vs. 3.17 +/- 1.64 g/L, P < 0.001), and APTT (45.1 +/- 7.6 vs. 33.8 +/- 4.4 s, P < 0.001) were found. Prednisone plus EACA therapy resulted in a significant increase in plasma AT-III activity (by 53%, P < 0.003), whereas plasma fibrinogen concentration and APTT remained unchanged. However, statistically significant differences between the pre- and posttreatment plasma AT-III, alpha 1-PI, and alpha 2-A activities in these patients were observed. There was also a relationship between EACA dose and the percentage change in plasma alpha 2-A activity. In a few patients receiving prednisone plus EACA regimen, side effects that included purulent rhinitis, pharyngitis, increases in body temperature, loose stools, and an approximately 20% to 30% decrease in systolic and diastolic arterial blood pressure were observed. Thus, although the prednisone plus EACA treatment regimen seems to offer new therapeutic possibilities in some patients with NS, it should not be used in acute phase of the disease.