Asparaginase inhibits the lectin pathway of complement activation.

Oncological treatment has been associated with an increased risk of infection, most often related to therapy-induced pancytopenia. However, limited research has been conducted on the effect of oncological therapy on the complement system, being part of the non-cellular innate immune system. This became the rationale for an observational clinical study (C2012) in which we have investigated the prevalence of transient complement defects. Once we had observed such defects, a correlation of the complement defects to specific clinical parameters or to specific therapeutic regimens was investigated. A prominent defect observed in C2012 was the inhibition of the lectin pathway (LP) of complement activation during the treatment of acute lymphoblastic leukemia (ALL), which we could directly associate to the use of asparaginase (ASNase). Ex-vivo experiments confirmed a direct dose-dependent inhibitory effect of ASNase on the LP functionality.

Production of monoclonal antibodies against inactivated alpha 1-antitrypsin. Cross-reactivity with complexed alpha 1-antitrypsin and application in an assay to determine inactivated and complexed alpha 1-antitrypsin in biological fluids.

15 different monoclonal antibodies (mcAbs) have been raised against the cleaved (inactive) form of the serpin alpha 1-antitrypsin (AT). In initial experiments these mcAbs were analysed for their ability to bind the native and the cleaved form of this inhibitor: eight of the 15 mcAbs appeared to react predominantly with cleaved AT. Additional experiments with mixtures of purified native AT, AT complexed to neutrophilic elastase and inactivated AT revealed that all mAbs that preferentially reacted with inactivated AT also bound to complexed AT. Using two of the mcAbs against inactivated AT a quantitative and sensitive sandwich-type radioimmunoassay was developed to determine levels of proteolytically inactivated AT in biological fluids. With this assay increased levels of inactivated AT were found in synovial fluid from patients with rheumatoid arthritis corresponding to about 2.4% (range 0.3-11%) of total AT. Approximately 10% of this inactivated AT appeared to consist of AT complexed to neutrophil elastase. The mcAbs described here further illustrate the structural resemblance between the complexed and cleaved forms of AT. In addition, these mcAbs appear to be useful tools for the study of AT in human disease.

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins.

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

Assessment of the relative contribution of different protease inhibitors to the inhibition of plasmin in vivo.

It has been shown that the most important inhibitor of plasmin is alpha 2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed. To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin. It was confirmed that alpha 2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than alpha 2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-alpha 1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with alpha 2-macroglobulin and with antithrombin III were significantly elevated. In conclusion, we confirmed the important role of alpha 2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Conidium production by insect pathogenic fungi on commercially available agars.

AIMS: Conidium production by three species of insect pathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, was assessed on various depths and types of commercially available agars. METHODS: Conidium production was assessed after 14 d of growth on commercially available media as well as at three different agar depths. RESULTS: Metarhizium anisopliae and B. bassiana isolates showed greatest conidium production on potato dextrose agar (PDA) at a depth of 2 mm, whereas V. lecanii showed greatest conidium production on yeast extract-peptone-dextrose agar (YPDA) regardless of agar depth. Optimum conidium production for M. anisopliae and B. bassiana was not only dependent upon the isolate used but also on the medium type and agar depth. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia are the infective structures for insect pathogenic fungi and this study suggests a rationale basis for consistent conidium production for laboratory and commercial practices.

Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors.

Granzyme A (GrA) and B (GrB) together with perforin are the main constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytotoxic proteins are released to deliver a lethal hit during contact between the CTL or NK cell and target cell. With the use of an enzyme-linked immunosorbent assay for antigenic levels, we showed in a recent study that plasma of patients with activated CTLs and NK cells contain elevated levels of extracellular GrA. In this study, we determined the form and proteolytic capacity of this extracellular GrA detected in plasma. With the use of various assays, we show that part of the extracellular GrA circulates in the mature conformation and is bound to proteoglycans that protect it against inactivation by protease inhibitors, such as antithrombin III and alpha-2-macroglobulin, whereas another part of GrA circulates as a complex with antithrombin III. Finally, with the use of a novel assay for active GrA, we demonstrate that some plasma samples with high levels of extracellular GrA contain active GrA. These results suggest that various forms of extracellular GrA occur in vivo and that the regulation of GrA activity may be modified by proteoglycans. These data support the notion that granzymes may exert extracellular functions distant from the site of CTL or NK cell interaction with their target cells. (Blood. 2000;95:1465-1472)

Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.

Human granzyme A is one of the serine proteinases present in the granules of cytotoxic T lymphocytes and natural killer cells. Granzymes are synthesized as inactive proenzymes with an amino-terminal prodipeptide, which is processed during transport of granzymes to the cytotoxic granules, where they are stored as active proteinases. In this study, we explored the possibility of producing recombinant granzymes. Recombinant human granzyme A zymogen was expressed in several eukaryotic cell lines (HepG2, Jurkat, and COS-1) after infection with a recombinant vaccinia virus containing full-length granzyme A cDNA. Immunoblot analysis of cell lysates showed that all infected cells produced a disulfide-linked homodimer of identical molecular weight as natural granzyme A. Infected HepG2 cells produced the largest amount of this protease (approximately 160 times more than lymphokine activated killer (LAK) cells). The recombinant protein only had high mannose type oligosaccharides as did the natural protein. Although infected HepG2 and COS cells contained high granzyme A antigen levels, lysates from these cells did not show any granzyme A proteolytic activity. However, the inactive proenzyme could be converted into active granzyme A by incubation with the thiol proteinase cathepsin C (dipeptidyl peptidase I). This study is the first to demonstrate expression of an active recombinant human cytotoxic lymphocyte proteinase and conversion of inactive progranzyme A into an active enzyme by cathepsin C. We suggest that a similar approach can be used for the production of other granzymes and related proteinases.

Relative contribution of contact and complement activation to inflammatory reactions in arthritic joints.

Although both the complement and contact system are thought to contribute to the inflammatory reaction in arthritic joints, only activation of complement has so far been well established, whereas contact activation and its contribution to arthritis has not been systematically explored. Complement and contact activation were assessed in 71 patients with inflammatory arthropathies and 11 with osteoarthritis using sensitive assays for C3a, and C1-inhibitor (C1INH)-kallikrein and C1INH-factor XIIa complexes respectively. Increased plasma concentrations of kallikrein-and factor XIIa-C1INH complexes were found in two and seven of the 71 patients with inflammatory arthropathies, respectively, and in none of the patients with osteoarthritis. Increased synovial fluid concentrations of kallikrein and factor XIIa complexes occurred in 13 and 15 patients with inflammatory joint diseases respectively, and in two patients with osteoarthritis. Contact system parameters did not correlate with clinical symptoms, local activity, or neutrophil activation. In contrast, synovial fluid concentrations of C3a and C1INH-C1 complexes were increased in all patients and in 20 patients with inflammatory arthropathies respectively, and were higher in patients with a higher local activity score. Synovial fluid C3a correlated with parameters of neutrophil activation such as lactoferrin. Increased plasma concentrations of C3a and C1INH-C1 complexes occurred in 13 and 11 patients with inflammatory joint diseases, and in one and two patients with osteoarthritis respectively. Plasma concentrations of C3a correlated with the number of painful joints. Thus contact activation occurs only sporadically in patients with arthritis and contributes little if anything to the local inflammatory reaction and neutrophil activation. These latter events are significantly related to the extent of complement activation.

Predominant role of neutrophils in the inactivation of alpha 2-macroglobulin in arthritic joints.

We studied the state of alpha 2-macroglobulin (alpha 2M), an important inhibitor of cartilage-degrading proteinases, in relation to activation of neutrophils in 82 patients with several types of arthritis, including 52 with rheumatoid arthritis and 11 with osteoarthritis. Levels of total inactive alpha 2M (i alpha 2M), which comprises alpha 2M complexed to proteinases and alpha 2M inactivated by oxidation or hydrolysis, were measured with a monoclonal antibody specific for i alpha 2M. In addition, levels of alpha 2M complexed to proteinases were quantitated with specific assays. Neutrophil activation was assessed by measuring elastase-alpha 1-antitrypsin complexes and lactoferrin. In 83% of the 82 patients tested, the synovial fluid (SF) to plasma ratio of i alpha 2M exceeded 1, indicating an intraarticular generation. Levels of i alpha 2M significantly correlated with neutrophil numbers (P less than 0.0005) and with levels of elastase-alpha 1-antitrypsin complexes and of lactoferrin (P less than 0.00001 for both). Moreover, part of i alpha 2M consisted of alpha 2M complexed to elastase-like and chymotrypsin-like proteinases, presumably, neutrophil elastase and cathepsin G, respectively. However, the amount of i alpha 2M was approximately 10-fold larger than the amount complexed to these proteinases. In vitro inactivation of alpha 2M by activated neutrophils was only partly inhibitable by eglin C, a specific inhibitor of both elastase and cathepsin G. Release of reactive oxygen species was presumably responsible for the additional inactivation of alpha 2M, because eglin C completely abolished the inactivation of alpha 2M by cell-free supernatant of activated neutrophils. Thus, our results suggest a predominant role of neutrophils in the inactivation of alpha 2M in the SF of patients with inflammatory joint diseases. However, this inactivation could be explained only in part by the release of neutrophilic proteinases. We propose that the inactivation of alpha 2M in SF was due to the concerted action of both reactive oxygen species and lysosomal proteinases.

Distribution pattern and functional state of alpha 1-antichymotrypsin in plaques and vascular amyloid in Alzheimer's disease. A immunohistochemical study with monoclonal antibodies against native and inactivated alpha 1-antichymotrypsin.

Monoclonal antibodies (mAbs) were raised against inactivated alpha 1-antichymotrypsin (ACT) to study the presence and functional state of the serine protease inhibitor alpha 1-antichymotrypsin in cerebral amyloid deposits in Alzheimer's disease. A panel of seven different mAbs was obtained; six of them were directed against neoepitopes that are expressed on ACT after interaction with proteases (inactivated ACT) and one mAb was directed against an epitope that is exposed both on native and inactivated ACT. The mAbs against neoepitopes could discriminate native ACT from complexed and inactivated ACT in vitro as shown in binding experiments in the presence of either native or inactivated ACT. With the mAbs against ACT we found that: (a) besides classical congophilic plaques, amorphous noncongophilic beta/A4-positive plaques were stained; (b) amorphous and classical plaques reacted with both types of mAbs against ACT indicating that this ACT was either complexed to a protease or proteolytically inactivated; (c) vascular amyloid was not stained for ACT. The presence of ACT in amorphous and classical plaques and its absence in vascular amyloid may indicate differences in the proteolytic degradation of preamyloid into amyloid fibrils. Our study strongly suggests that ACT is biologically active in amyloid plaques from an early stage.

Habitat association in two genetic groups of the insect-pathogenic fungus metarhizium anisopliae: uncovering cryptic species?

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungus Metarhizium anisopliae from forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8 degrees C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37 degrees C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide, M. anisopliae may be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.

Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood.

Cytoplasmic granules from activated natural killer (NK) and cytotoxic T lymphocytes (CTL) contain a pore-forming protein, perforin, and several homologous serine proteinases called granzymes. Expression of these proteins correlates with the cytolytic potential of cytotoxic lymphocytes. Using a panel of MoAbs specific for human granzyme A and B, respectively, expression of these proteinases in non-pathological lymphoid tissue and peripheral blood lymphocyte (PBL) subpopulations was investigated. Using immunohistochemistry and double stainings, the phenotype of granzyme-expressing cells in lymphoid tissue was investigated. Granzyme-positive cells were detected in all lymphoid tissues tested. No large differences in the number and distribution between granzyme A- and granzyme B-positive cells were observed. The highest number of positive cells was located in the red pulp of the spleen. Significant numbers were detected in tonsil, lymph nodes, liver and thymus. Low numbers were present in the lamina propria of non-inflamed stomach, small intestine and colon. Phenotypic analysis and cell sorting showed that most of the granzyme-positive cells in lymphoid tissue and PBL consisted of CD3-CD16+CD56+ lymphocytes. Hardly any granzyme-positive CD3+CD8+ CTL were present in peripheral blood. The synthesis of granzyme A as well as B by both CD3+CD16+CD56+ and CD3+CD8+ cells in peripheral blood was increased upon IL-2 stimulation. These results indicate that in normal lymphoid tissue the predominant cytolytic cell population is formed by the NK cells, and activated CTL are rare.

Proteolytic inactivation of alpha 1-antitrypsin and alpha 1-antichymotrypsin by neutrophils in arthritic joints.

OBJECTIVE: In vitro, activated neutrophils create a microenvironment in which proteinase inhibitors are inactivated through the coordinate action of reactive oxygen species and released elastase. We investigated whether such a mechanism may contribute to the destruction of the joint tissues in arthritis. METHODS: We analyzed the state of alpha 1-antitrypsin (alpha 1AT) and alpha 1-antichymotrypsin (alpha 1ACT), the two major inhibitors of the neutrophilic serine proteinases, in synovial fluid (SF) from patients with inflammatory arthropathies (n = 71) and osteoarthritis (OA) (n = 11), and related the results to neutrophil activation in SF. RESULTS: The ratio of functional to antigenic levels of alpha 1AT in SF of patients with inflammatory joint diseases was similar to that of alpha 1AT in normal plasma, whereas that of alpha 1ACT was significantly decreased. Patients with inflammatory arthropathies had significantly higher levels of inactivated alpha 1AT (i alpha 1AT) and inactivated alpha 1ACT (i alpha 1ACT) in SF (as determined with monoclonal antibodies specific for the inactivated [i.e., proteolytically inactivated and/or complexed] forms of these inhibitors) than patients with OA (P < 0.005). Inactivated alpha 1AT and i alpha 1ACT levels corresponded to 0.3-11% and 3-99%, respectively, of the total amount of these inhibitors in SF. Most of the i alpha 1AT in SF had a lower M(r) than that of native alpha 1AT. Inactivated alpha 1ACT in SF had an M(r) identical to that of nonfunctional alpha 1ACT in plasma treated with chymotrypsin. Levels of both i alpha 1AT and i alpha 1ACT correlated significantly with lactoferrin and elastase levels. CONCLUSION: These results suggest that alpha 1AT and alpha 1ACT in arthritic joints are inactivated in part by activated neutrophils, suggesting a role for these cells in impairment of the local balance between proteinases and their inhibitors in arthritis.

Analysis of intraarticular fibrinolytic pathways in patients with inflammatory and noninflammatory joint diseases.

OBJECTIVE: Intraarticular activation of the fibrinolytic system has been suspected to occur in patients with arthritis. We undertook the present study to investigate the relation of this activation to clinical symptoms, and the molecular pathways involved. METHODS: We quantitatively assessed levels of plasmin-alpha 2-antiplasmin (PAP) complexes in synovial fluid (SF) from 25 patients with rheumatoid arthritis (RA), 7 with seronegative spondylarthropathy (SSA), and 10 with osteoarthritis (OA), and conducted an analysis to determine the plasminogen-activating pathway via which these complexes were generated. In addition, we studied the relationship of intraarticular fibrinolysis to clinical and biochemical parameters. RESULTS: All patients studied had increased SF levels of PAP complexes. Levels in patients with RA and SSA were slightly higher than those in patients with OA. These complexes were probably formed by activation of urokinase-type plasminogen activator (u-PA), and not tissue-type plasminogen activator (t-PA), since SF levels of both u-PA antigen and u-PA-plasminogen activator inhibitor (PAI) complexes were increased in 27 of the 42 patients. Conversely, SF levels of t-PA were below normal in all but 1 patient. In some patients, activation of factor XII presumably also contributed to plasminogen activation in SF, since levels of factor XIIa-C1 inhibitor in SF were increased in 8 of the 42 patients and correlated, as did u-PA-PAI levels, with levels of PAP complexes. Several of the parameters of fibrinolysis in SF, particularly u-PA antigen and u-PA-PAI-1 complexes, were found to correlate with clinical and biochemical parameters. CONCLUSION: Our results suggest that plasminogen is frequently activated in the joints of patients with inflammatory or noninflammatory arthropathy and that this activation mainly occurs via a u-PA-, and in some cases also via a factor XII-, dependent pathway. The possible relation of this activation process to stimulation of synovial cells by cytokines is discussed.

Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection.

Granzymes A and B are serine-proteinases stored in the granules of activated cytotoxic T-lymphocytes and natural killer (NK) cells. Expression of granzymes in tissues can be used as an activation marker for cytotoxic cells. Using mAbs specific for human granzyme A or B in immunohistochemical staining techniques we investigated expression of granzyme A and B by lymphocytes infiltrating acutely rejected renal allografts. Twelve core needle biopsies were taken from ten different patients during an episode of acute rejection. Eleven biopsies contained high numbers of granzyme A and B positive lymphocytes infiltrating tubular epithelium, and vascular and glomerular structures. In one patient infiltrating lymphocytes did not express granzyme A and only low amounts of granzyme B. No correlation was found between the number of granzyme positive cells and the severity of the rejection as classified by conventional histological criteria. In one tissue specimen from a patient with a renal allograft without signs of rejection, the number of granzyme positive cells was much lower compared to that of the transplant group. In spite of the presence of a marked inflammatory infiltrate, no granzyme positive cells were detected in renal biopsies from patients with various inflammatory, not transplant-related, renal diseases. Phenotypic analysis showed that granzymes A and B were expressed by CD56+ NK cells and CD3+ cells, representing cytotoxic T-lymphocytes. Thus, this study demonstrates that granzyme A and B protein-expressing lymphocytes infiltrate the kidney allografts during an acute cellular rejection but not in several other inflammatory renal diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of granzymes A and B in synovial tissue from patients with rheumatoid arthritis and osteoarthritis.

Granzymes A and B are serine proteinases which are stored in the granules of activated cytotoxic T cells and NK cells. Expression of these granzymes by cytotoxic cells in tissues can be used as an activation marker for these cells. To investigate a possible role of cytotoxic lymphocytes in rheumatoid arthritis (RA) and osteoarthritis (OA), we assessed the expression of granzymes A and B by cytotoxic lymphocytes in synovial biopsies from five RA and five OA patients using mAb specific for these serine proteinases. In three of the five RA patients but also in two of the five OA patients granzyme A- and B-expressing lymphocytes were observed in the synovium. Double-labeling immunohistochemical techniques revealed that up to 75% of the granzyme-positive synovial lymphocytes had the CD16+ or CD56+ natural killer cell phenotype. Less than 5% were CD3+, CD8+ cytotoxic T cells, whereas in some patients the phenotype of up to 50% of these cells could not be identified. The presence of granzymes A and B in the synovium of both RA as well as OA patients was confirmed on the molecular level in a second group of 11 RA and 5 OA patients using the polymerase chain reaction. Thus, expression of granzymes A and B occurs in the synovium in patients with RA as well as those with OA. These proteins are mainly expressed by NK cells that may therefore play a role in the pathogenesis of these diseases.

OKT3-induced nephrotoxicity is associated with release of group II secretory phospholipase A2.

Administration of the murine IgG2a CD3 monoclonal antibody OKT3 exerts a transient nephrotoxic effect. Increased levels of group II secretory phospholipase A2 (sPLA2-II) might account for this nephrotoxicity as sPLA2-II induces the biosynthesis of prostaglandins, vasoactive lipid mediators that influence glomerular haemodynamics and renal function. Furthermore, extracellular phospholipases seem to be involved in proximal tubular cell injury. We studied plasma sPLA2-II levels in relation to circulating creatinine, tumour necrosis factor alpha, interleukin 6 and C-reactive protein levels in 15 renal allograft recipients receiving rejection treatment with OKT3. As a control group, we studied 15 renal allograft recipients receiving rejection treatment with methylprednisolone. A maximal fourfold increase in sPLA2-II levels was observed 48 h after the first OKT3 administration, preceded by increased tumour necrosis factor alpha and interleukin 6 levels and accompanied by increased C-reactive protein levels. Creatinine levels reached a maximal increase 72 h after initiation of treatment. During methylprednisolone treatment no increase in any of the studied parameters was observed. Thus, administration of OKT3 induces increased sPLA2-II levels, presumably via generation of cytokines. We hypothesize that sPLA2-II may contribute to the nephrotoxic effect of OKT3 by inducing vasoconstrictive prostaglandins and renal tubular cell injury.