Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG

Background: Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level. Results: LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis. Conclusions: The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations

Determination of phosphate-activated glutaminase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

Phosphate-activated glutaminase (PAG) converts glutamine to glutamate as part of the glutaminolysis pathway in mitochondria. Two genes, GLS1 and GLS2, which encode for kidney-type PAG and liver-type PAG, respectively, differ in their tissue-specific activities and kinetics. Tissue-specific PAG activity and its kinetics were determined by metabolic mapping using a tetrazolium salt and glutamate dehydrogenase as an auxiliary enzyme in the presence of various glutamine concentrations. In kidney and brain, PAG showed Michaelis-Menten kinetics with a K(m) of 0.6 mM glutamine and a V(max) of 1.1 µmol/mL/min when using 5 mM glutamine. PAG activity was high in the kidney cortex and inner medulla but low in the outer medulla, papillary tip, glomeruli and the lis of Henle. In brain tissue sections, PAG was active in the grey matter and inactive in myelin-rich regions. Liver PAG showed allosteric regulation with a K(m) of 11.6 mM glutamine and a V(max) of 0.5 µmol/mL/min when using 20 mM glutamine. The specificity of the method was shown after incubation of brain tissue sections with the PAG inhibitor 6-diazo-5-oxo-L-norleucine. PAG activity was decreased to 22% in the presence of 2 mM 6-diazo-5-oxo-L-norleucine. At low glutamine concentrations, PAG activity was higher in periportal regions, indicating a lower K(m) for periportal PAG. When compared with liver, kidney and brain, other tissues showed 3-fold to 6-fold less PAG activity. In conclusion, PAG is mainly active in mouse kidney, brain and liver, and shows different kinetics depending on which type of PAG is expresse

Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 m

Structure of the periodontium in cathepsin C-deficient mice

Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency leads to changes in the structure of the periodontal tissues as a result of which its barrier function to pathogens is compromised. We studied the structure of the periodontium in 9-month-old cathepsin C-deficient mice (cathepsin C(-/-)) and compared this with age-matched wild-type mice. Our observations showed that the overall structure of the gingiva, periodontal ligament, alveolar process, and cementum layer are normal in cathepsin C(-/-) mice, with one exception, namely that epithelial rests of Malassez in the periodontal ligament of the cathepsin C(-/-) mice are slightly enlarged. In both experimental and control animals, we noted cyst formation in rests of Malassez. No signs of periodontal infection were observed. It is concluded that cathepsin C deficiency does not lead to major changes in the structure of the periodontiu

Functional changes in articular cartilage after meniscal allograft transplantation: A quantitative histochemical evaluation in rabbits

Purpose: To evaluate quantitatively functional changes in articular cartilage after immediate and delayed meniscus transplantation in rabbits. Type of Study: Experimental study. Methods: Thirty rabbits were divided into 5 groups: groups A and C were subjected to meniscectomy only, groups B and D underwent meniscal transplantation immediately after meniscectomy, and group E had delayed transplantation 6 weeks after meniscectomy. Six nonoperated knees served as controls. Functional changes in articular cartilage were examined at 6 weeks (groups A, B) and I year (groups C, D, E, controls) after surgery by measuring proteoglycan content of the extracellular matrix as a measure of its quality and lactate dehydrogenase (LDH) activity in chondrocytes as a measure of their vitality. Results: At 6-week and I-year follow-up, no significant differences were found between the immediate transplant group and postmeniscectomy group. The delayed transplant group showed a significantly decreased proteoglycan content compared with the postmeniscectomy group. No significant differences in cellular LDH activity were found between the immediate transplant group and postmeniscectomy group at 6 weeks and I year. However, the delayed transplant group showed a significant decrease in LDH activity compared with the postmeniscectomy group. Conclusions: Immediate meniscal transplantation in rabbits did not significantly reduce degenerative changes of articular cartilage in comparison with meniscectomy on a short-term and long-term basis, whereas delayed transplantation led to more degenerative changes than meniscectomy. Clinical Relevance: Before meniscus transplantation can be considered as an alternative to meniscectomy in clinical practice, it has to be determined whether this procedure has any protective effect on articular cartilage on the long ter

Plasminogen activators are involved in the degradation of bone by osteoclasts

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and Urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were Cultured for 6 or 24 11 ill the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed ill uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA(-/-) bones. nolocalization of the integrin alpha(v)beta(3) of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA(-/-) mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings Suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone, (c) 2008 Elsevier Inc. All rights reserved.status: publishe

Polyethylene wear particles do not induce inflammation or gelatinase (MMP-2 and MMP-9) activity in fibrous tissue interfaces of loosening total hip arthroplasties

In vitro and in vivo studies have suggested that polyethylene wear particles are the main cause for osteolysis in prosthetic loosening. Elevated amounts of proteases including gelatinases (or matrix metalloproteinases MMP-2 and MMP-9) have been found in fibrous tissue interfaces of loosened total hip arthroplasties suggesting that proteolysis plays a role in osteolysis. The presence of proteases does not mean that they are active, because activity of proteases is highly regulated at the post-translational level. We investigated whether the activity of two major proteases that are active extracellularly and have been associated with loosening, MMP-2 and MMP-9, is involved in loosening of non-cemented hip implants with polyethylene acetabular components. Eight interface tissues retrieved during revision were studied with light and electron microscopy and by in situ zymography to localize MMP-2 and MMP-9 activity in combination with immunohistochemistry to localize MMP-2 and MMP-9 proteins. All interface tissues contained large amounts of polyethylene wear particles, either in large accumulations or dispersed in the extracellular matrix or intracellularly in fibroblasts. Particles were not encountered in association with MMP-2 or MMP-9 activity or leukocytes. Inflammation was never found. MMP-9 activity was restricted to macrophages and MMP-2 activity was restricted to microvascular endothelial cells mainly outside areas where particles were present. Our data indicate that wear particles do not induce activation of leukocytes or MMP-2 or MMP-9 activity. Therefore, aseptic loosening may not be particle induced but initiated by other mechanisms such as mechanical stress. (C) 2010 Elsevier GmbH. All rights reserve

The Thyroid Hormone Inactivating Enzyme Type 3 Deiodinase is Present in Bactericidal Granules and the Cytoplasm of Human Neutrophils

Neutrophils are important effector cells of the innate immune system. Thyroid hormone (TH) is thought to play an important role in their function. Intracellular TH levels are regulated by the deiodinating enzymes. The TH-inactivating type 3 deiodinase (D3) is expressed in infiltrating murine neutrophils, and D3 knockout mice show impaired bacterial killing upon infection. This suggests that D3 plays an important role in the bacterial killing capacity of neutrophils. The mechanism behind this effect is unknown. We aimed to assess the presence of D3 in human neutrophils, and determine its subcellular localization using confocal and electron microscopy, because this could give important clues about its function in these cells. D3 appeared to be present in the cytoplasm and in myeloperoxidase containing azurophilic granules and as well as lactoferrin containing specific granules within human neutrophils. This subcellular localization did not change upon activation of the cells. D3 is observed intracellularly during neutrophil extracellular trap formation, followed by a reduction of D3 staining after release of the neutrophil extracellular traps into the extracellular space. At the transcriptional level, human neutrophils expressed additional essential elements of TH metabolism, including TH transporters and TH receptors. Here, we demonstrate the presence and subcellular location of D3 in human neutrophils for the first time and propose a model, in which D3 plays a role in the bacterial killing capacity of neutrophils either through generation of iodide for the myeloperoxidase system or through modulation of intracellular TH bioavailability