Reliability and validity of the modified shuttle test-paeds to measure cardiorespiratory fitness in children.

BACKGROUND: The Modified Shuttle Test-Paeds (Paeds), a recently developed 10-meter Shuttle run test for aerobic capacity in children. This study aims to investigate the construct validity (known-group and convergent validity) and test-retest reliability of the recently developed test for cardiorespiratory fitness, the Modified Shuttle Test-Paeds (Paeds). METHODS: A total of 144 participants (6-12 y) were tested on the Paeds test, and 84 children were tested on the 20-meter Shuttle Run test (20 m-SRT) to assess construct validity. To evaluate test-retest reliability, 46 children were tested twice on the Paeds. RESULTS: No sex differences were found, but there was an age effect. A strong correlation was found between Paeds and the 20 m-SRT (rs=0.78, p < 0.001). The test-retest reliability was good (ICC 0.84; 95% CI 0.74-0.91). CONCLUSION: Paeds appears to be a reliable and valid tool for estimating cardiorespiratory fitness in typically developing children aged 6-12 years and has the advantages of being shorter, needing less space, not requiring pacing and being self-motivational. More studies are needed to assess whether children reach an aerobic steady state in three minutes and how much of the results of the Paeds test can be explained by the agility component of the task (turning and grasping or aiming a bean bag). For clinical use, psychometric properties should be studied in various patient groups (e.g., ADHD, DCD, and children with intellectual disabilities).

Climate footprint assessment of plastic waste pyrolysis and impacts on the Danish waste management system.

Increased plastic recycling is necessary to reduce environmental impacts related to manufacturing and end-of-life of plastic products, however, mechanical recycling (MR) - currently the most widespread recycling option for plastic waste - is limited by quality requirements for inputs and reduced quality of outputs. In this study, pyrolysis of plastic waste is assessed against MR, municipal solid waste incineration (MSWI) and fuel substitution through climate footprint assessment (CFA) based on primary data from pyrolysis of plastic waste sourced from Danish waste producers. Results of the CFA are scaled to the Danish plastic waste resource in an impact assessment of current Danish plastic waste management, and scenarios are constructed to assess reductions through utilization of pyrolysis. Results of the CFA show highest benefits utilizing pyrolysis for monomer recovery (-1400 and -4800 kg CO2e per ton polystyrene (PS) and polymethyl methacrylate (PMMA), respectively) and MR for single polymer polyolefins (-1000 kg CO2e per ton PE). The two management options perform similarly with mixed plastic waste (200 kg CO2e per ton plastic waste). MSWI has the highest impact (1600-2200 kg CO2e per ton plastic waste) and should be avoided when alternatives are available. Scaling the results of the CFA to the full Danish plastic waste resource reveals an impact of 0.79 Mt CO2e in year 2020 of current plastic waste management. Utilizing pyrolysis to manage MR residues reduces the system impact by 15%. Greater reductions are possible through increased separation of plastic from residual waste. The best performance is achieved through a combination of MR and pyrolysis.

Serum neurofilament light chain, contactin-1 and complement activation in anti-MAG IgM paraprotein-related peripheral neuropathy.

INTRODUCTION: In anti-myelin-associated glycoprotein IgM paraprotein-related peripheral neuropathy (anti-MAG PN), there is a lack of reliable biomarkers to select patients eligible for therapy and for evaluating treatment effects, both in routine practice and in clinical trials. Neurofilament light chain (NfL) and contactin-1 (CNTN1) can serve as markers of axonal and paranodal damage. Complement activation is involved in the pathogenesis in anti-MAG PN. We, therefore, hypothesized that serum NfL, CNTN1, C3b/c and C4b/c may function as biomarkers of disease activity in anti-MAG PN. METHODS: In this prospective cohort study, we included 24 treatment-naïve patients with anti-MAG PN (mean age 69 years, 57% male) that had IgM paraproteinemia, a high IgM MAG-antibody, and clinical diagnosis of anti-MAG PN by a neurologist specialized in peripheral nerve disorders. We measured serum NfL, CNTN1, C3b/c and C4b/c, reference values were based on healthy controls. As controls, 10 treatment-naïve patients with IgM Monoclonal gammopathy of undetermined significance (MGUS) or Waldenström's Macroglobulinemia (mean age 69 years, 60% male) without signs of neuropathy were included (non-PN). RESULTS: NfL, CNTN1 levels in serum were mostly normal in anti-MAG PN patients and comparable to non-PN patients. C3b/c and C4b/c levels were normal in anti-MAG PN patients. CONCLUSION: Our results do not support serum NfL, CNTN1, and C3b/c and C4b/c as potential biomarkers in anti-MAG PN, although we cannot exclude that subgroups or subtle abnormalities could be found in a much larger cohort with longitudinal follow-up.

Animal production, animal health and food safety: Gaps and challenges in the chilean industry.

This paper summarizes the gaps and challenges related to animal production, health, and food safety as discussed by a panel at the 1st International Symposium of Food Safety (ISFS) in Santiago, Chile, in December 2016. Participating representatives of academia, industry, and government and statements from the audience confirmed that food safety is essential for increasing food security. First, panelists identified the need for a science-based regulatory framework to implement effective regulations. Second, they highlighted the importance of a risk analysis framework to quantify the risk of the potential for antimicrobial resistance associated with the use of antimicrobials, and the need of studies to evaluate foodborne prevention/control strategies. Third, the challenges of filling the gaps between industry and academia were addressed, including examples of successful collaboration, opportunities, and weakness identified by industry. Finally, challenges in animal food production included issues related to changing consumer preferences, animal welfare, the use of antimicrobials, and sustainable animal production. The symposium provided a regional platform to share experiences from the implementation of methods and approaches for food safety. The roundtable successfully explored the future science and technology challenges that are of strategic importance for Chile and the region in animal health and food safety.

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.

Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants - from the field to the test tube and back.

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.

Soil feedback of exotic savanna grass relates to pathogen absence and mycorrhizal selectivity.

Enemy release of exotic plants from soil pathogens has been tested by examining plant-soil feedback effects in repetitive growth cycles. However, positive soil feedback may also be due to enhanced benefit from the local arbuscular mycorrhizal fungi (AMF). Few studies actually have tested pathogen effects, and none of them did so in arid savannas. In the Kalahari savanna in Botswana, we compared the soil feedback of the exotic grass Cenchrus biflorus with that of two dominant native grasses, Eragrostis lehmanniana and Aristida meridionalis. The exotic grass had neutral to positive soil feedback, whereas both native grasses showed neutral to negative feedback effects. Isolation and testing of root-inhabiting fungi of E. lehmanniana yielded two host-specific pathogens that did not influence the exotic C. biflorus or the other native grass, A. meridionalis. None of the grasses was affected by the fungi that were isolated from the roots of the exotic C. biflorus. We isolated and compared the AMF community of the native and exotic grasses by polymerase chain reaction-denaturing gradient gel elecrophoresis (PCR-DGGE), targeting AMF 18S rRNA. We used roots from monospecific field stands and from plants grown in pots with mixtures of soils from the monospecific field stands. Three-quarters of the root samples of the exotic grass had two nearly identical sequences, showing 99% similarity with Glomus versiforme. The two native grasses were also associated with distinct bands, but each of these bands occurred in only a fraction of the root samples. The native grasses contained a higher diversity of AMF bands than the exotic grass. Canonical correspondence analyses of the AMF band patterns revealed almost as much difference between the native and exotic grasses as between the native grasses. In conclusion, our results support the hypothesis that release from soil-borne enemies may facilitate local abundance of exotic plants, and we provide the first evidence that these processes may occur in arid savanna ecosystems. Pathogenicity tests implicated the involvement of soil pathogens in the soil feedback responses, and further studies should reveal the functional consequences of the observed high infection with a low diversity of AMF in the roots of exotic plants.

Algorithmic approaches for computing elementary modes in large biochemical reaction networks.

The concept of elementary (flux) modes provides a rigorous description of pathways in metabolic networks and proved to be valuable in a number of applications. However, the computation of elementary modes is a hard computational task that gave rise to several variants of algorithms during the last years. This work brings substantial progresses to this issue. The authors start with a brief review of results obtained from previous work regarding (a) a unified framework for elementary-mode computation, (b) network compression and redundancy removal and (c) the binary approach by which elementary modes are determined as binary patterns reducing the memory demand drastically without loss of speed. Then the authors will address herein further issues. First, a new way to perform the elementarity tests required during the computation of elementary modes which empirically improves significantly the computation time in large networks is proposed. Second, a method to compute only those elementary modes where certain reactions are involved is derived. Relying on this method, a promising approach for computing EMs in a completely distributed manner by decomposing the full problem in arbitrarity many sub-tasks is presented. The new methods have been implemented in the freely available software tools FluxAnalyzer and Metatool and benchmark tests in realistic networks emphasise the potential of our proposed algorithms.

Economic and environmental effects of the manure policy in The Netherlands: synthesis of integrated ex-post and ex-ante evaluation.

This paper summarises the results of both an ex-post evaluation of the Dutch Mineral Accounting System (MINAS) and an ex-ante evaluation of the effect of different levy-free surplus values. The MINAS system has been introduced in 1998 in order to reduce nitrate and phosphate leaching from agricultural soils. MINAS resulted in a reduction of the N surplus on dairy farms of approximately 50 kg ha(-1) to limited or no costs to the farms involved. MINAS resulted in higher costs for manure removal for intensive livestock farmers. Though emissions of N and P have decreased significantly during the last five years effects of this reduction in environmental quality cannot be observed, except for a small decrease in nitrate concentration of the upper groundwater. The ex-ante evaluation of different variants for possible future levy-free surplus levels indicated that under the lowest variant, the nitrate concentration in groundwater will exceed 50 mg per litre on 14% of the area. The environmental effect of the different variants for P were not distinguished. The lowering of the levy-free surplus for P will have a drastic effect on the intensive livestock farms. The incorporation of fertiliser P under the MINAS system would be a cheap option to reduce the P surplus.

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.

Irradiated versus nonirradiated endothelial cells: effect on proliferation of vascular smooth muscle cells.

PURPOSE: Endovascular radiation therapy is a promising strategy for the prevention of restenosis. Radiation prevents proliferation of vascular smooth muscle cells, thereby reducing the incidence of restenosis, but may also affect the remaining endothelial cells. For this reason, a comparison was made between irradiated and nonirradiated endothelial cells and their effects on the proliferation of vascular smooth muscle cells in a coculture system was evaluated. MATERIALS AND METHODS: A coculture system was used, in which both endothelial cells and vascular smooth muscle cells were grown on opposite sides of a semipermeable membrane. After a period of growth arrest, the proliferation of vascular smooth muscle cells was measured during four subsequent days. RESULTS: The presence of endothelial cells stimulated the proliferation of vascular smooth muscle cells during the first days of analysis but had an inhibitory effect during the subsequent days (P <.5). gamma-irradiation of endothelial cells resulted in a complete blockage of the proliferation of these cells. However, irradiated endothelial cells affected the proliferation of vascular smooth muscle cells in coculture in a fashion comparable to nonirradiated endothelial cells (P >.5). CONCLUSION: The results suggest that, in endovascular radiation therapy, irradiation of endothelial cells does not change their effects on the proliferative behavior of vascular smooth muscle cells.

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.

Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice.

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37 degrees C reveals a 2- to 3-fold increase in the formation of pre-beta-HDL compared with plasma from wild-type mice. Although pre-beta-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-beta-HDL.

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)

A prospective, randomized clinical trial comparing 150 IU recombinant follicle stimulating hormone (Puregon((R))) and 225 IU highly purified urinary follicle stimulating hormone (Metrodin-HP((R))) in a fixed-dose regimen in women undergoing ovarian stimulation.

A prospective, randomized, open, multicentre (n = 3) study was conducted to compare the efficacy and efficiency of a fixed daily dose of 150 IU (3x50 IU) recombinant follicle stimulating hormone (recFSH, Puregon((R))) and 225 IU (3x75 IU) highly purified urinary FSH (uFSH-HP, Metrodin-HP((R))) in women undergoing ovarian stimulation prior to in-vitro fertilization treatment. A total of 165 women were treated with FSH, 83 subjects with recFSH and 82 subjects with uFSH-HP. In the recFSH group a mean number of 8.8 oocytes were retrieved, compared with 9.8 in the uFSH-HP group (not statistically significant). In the recFSH group, a significantly lower total dose was required compared to the uFSH-HP group, 1479 versus 2139 IU, respectively (P < 0.0001; 95% confidence interval -747 to -572). Treatment with recFSH resulted in a significantly higher embryo development rate (69.6 versus 56.2%; P = 0.003) and more embryos accessible for the embryo freezing programme (3.3 versus 2.0; P = 0.02) compared to uFSH-HP. The vital pregnancy rate per cycle started was 30.2 versus 28.3% in the recombinant and urinary FSH group, respectively. It is concluded that treatment outcome of a fixed daily dose of 150 IU recFSH is comparable to a fixed daily dose of 225 IU uFSH-HP. However, a significantly lower total dose was needed in the recFSH group (nearly 700 IU less).

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver.

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.

Seeding of intravascular stents by the xenotransplantation of genetically modified endothelial cells.

A novel approach of cell seeding of stents using xenotransplanted endothelium is proposed. The advantages of this approach are that these doubly transgenic animals will provide a limitless supply of endothelial cells producing controllable levels of active compound. These foreign cells will act as Trojan horses, graciously accepted at face value by the host organism, but capable of modifying the pathophysiological response to vessel damage, typified by the process of restenosis. Once implanted, the production of the bioactive compound is under exogenous control by means of 'designer' genes coding for modified cell surface receptors, which are introduced with the transgene to provide controllable levels of compound. Interaction of an orally administered compound with the modified cell receptor will switch on the transgene, while in its absence the transgene remains dormant. We have been able to show the feasibility this type of approach has for other animal species, and it shows great potential for application to humans.

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.

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.

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.