Wikky Tigchelaar

Endogenous activated protein C limits cancer cell extravasation through sphingosine-1-phosphate receptor 1–mediated vascular endothelial barrier enhancement

Activated protein C (APC) has both anticoagulant activity and direct cell-signaling properties. APC has been reported to promote cancer cell migration/invasion and to inhibit apoptosis and therefore may exacerbate metastasis. Opposing these activities, APC signaling protects the vascular endothelial barrier through sphingosine-1-phosphate receptor-1 (S(1)P(1))activation, which may counteract cancer cell extravasation. Here, we provide evidence that endogenous APC limits cancer cell extravasation, with in vivo use of monoclonal antibodies against APC. The protective effect of endogenous APC depends on its signaling properties. The MAPC1591 antibody that only blocks anticoagulant activity of APC does not affect cancer cell extravasation as opposed to MPC1609 that blocks anticoagulant and signaling properties of APC. Combined administration of anti-APC antibodies and S(1)P(1) agonist (SEW2871) resulted in a similar number of pulmonary foci in mice in presence and absence of APC, indicating that the protective effect of APC depends on the S(1)P(1) pathway. Moreover, endogenous APC prevents cancer cell-induced vascular leakage as assessed by the Evans Blue Dye assay, and SEW2871 treatment reversed MPC1609-dependent vascular leakage. Finally, we show that cancer cells combined with MPC1609 treatment diminished endothelial VE-cadherin expression. In conclusion, endogenous APC limits cancer cell extravasation because of S(1)P(1)-mediated VE-cadherin-dependent vascular barrier enhancement.

Effects of Mechanical Compression of a Fibrous Tissue Interface on Bone with or without High-Density Polyethylene Particles in a Rabbit Model of Prosthetic Loosening

BACKGROUND The mechanisms leading to aseptic loosening of a total hip replacement are not fully understood. A fibrous tissue interface can be present around the implant. Hypothetically, component micromovements can compress this interface and cause increased fluid pressure according to biphasic models. We tested the hypothesis that compression of a fibrous membrane with or without the presence of high-density polyethylene particles leads to bone degradation. METHODS A titanium implant was inserted in forty-five rabbit tibiae, and, after osseous integration was achieved, a fibrous tissue interface was generated. The animals were randomized to undergo a sham operation, treatment with compression of the fibrous membrane, treatment with high-density polyethylene particles, or treatment with both compression and particles. Morphometric analysis of the surrounding bone was performed on cryostat sections after Giemsa staining and staining of tartrate-resistant acid phosphatase activity. RESULTS Forty specimens were available for analysis; five tibiae with an infection were excluded. After nine weeks, the controls showed vital bone, whereas the specimens treated with compression showed necrosis of bone and replacement of bone by cartilage in a discontinuous layer (p < 0.05 for both) but not fibrous tissue. Treatment with high-density polyethylene particles caused replacement of bone by fibrous tissue (p < 0.05) but not necrosis or cartilage formation. Compression combined with the presence of high-density polyethylene particles caused bone necrosis and loss of bone with replacement by cartilage and fibrous tissue (p < 0.05). CONCLUSIONS In this in vivo study in rabbits, fibrous membrane compression led to bone necrosis and cartilage formation, possibly because of fluid pressure or fluid flow, whereas the presence of high-density polyethylene particles led to the loss of bone with replacement of bone by fibrous tissue. Cartilage formation may be a protective response to fluid pressure and/or fluid flow. Fibrous membrane compression may play an important role in the early stages of loosening of a total hip replacement.

Kupffer cells and pit cells are not effective in the defense against experimentally induced colon carcinoma metastasis in rat liver

The present study was performed to investigate processes involved in circumvention of the immune system by advanced stages of tumor growth in the liver. The efficacy of Kupffer cells and pit cells against cancer cells was tested in vivo in an experimental model of colon carcinoma metastasis in rat liver. Liver tumors were induced by administration of CC531 colon cancer cells into the vena portae. After 3 weeks, livers were obtained and partly fixed for electron microscopic procedures or frozen in liquid nitrogen for enzyme and immunohistochemistry at the light microscope level. The activation status of Kupffer cells was studied by expression of la-antigen (MHC class II) and by measurement of glucose-6-phosphate dehydrogenase (G6PDH) activity in the cells in situ as a measure of production of reactive oxygen species. Large numbers of Kupffer cells were found in liver parenchyma surrounding colon carcinomas when compared with levels in control livers, but these cells were not activated. Large numbers of activated monocytes and macrophages, cytotoxic T cells but only a few pit cells were found to be recruited to the boundary between liver parenchyma and tumors or their stroma. In those areas where cancer cells invaded liver parenchyma, only newly recruited macrophages and some Kupffer cells were present but few cytotoxic T cells or pit cells were found. The low activation status of Kupffer cells both in terms of production of reactive oxygen species and Ia-antigen expression and the absence of significant numbers of pit cells at tumor sites suggest that Kupffer cells and pit cells do not play a significant role in advanced stages of tumor growth. High levels of prostaglandin E2 were detected in the parenchyma of livers containing tumors and transforming growth factor β was detected in the stroma of the tumors, therefore suggest that cytotoxicity of newly recruited monocytes, macrophages and cytotoxic T cells may be limited in these stages because of local production of these immunosuppressive factors.

BENIGN CELLULAR RESPONSES IN RATS TO DIFFERENT WEAR PARTICLES IN INTRA-ARTICULAR AND INTRAMEDULLARY ENVIRONMENTS

We examined the cellular responses to various particles injected into the knees and the intramedullary femoral cavities of rats in the presence of polymethylmethacrylate (PMMA) plugs. The intra-articular particles were mainly ingested by synovial fibroblasts. Increased numbers of macrophages were not detected and there was only a slight increase in synovial thickness. Cellular responses in the intramedullary space were similarly mild and bone resorption around the PMMA plug did not occur. Bone formation was inhibited only by polyethylene particles. In contrast to current views, our study shows that wear particles per se do not initiate bone resorption.

Receptor usage dictates HIV-1 restriction by human TRIM5α in dendritic cell subsets

The most prevalent route of HIV-1 infection is across mucosal tissues after sexual contact. Langerhans cells (LCs) belong to the subset of dendritic cells (DCs) that line the mucosal epithelia of vagina and foreskin and have the ability to sense and induce immunity to invading pathogens. Anatomical and functional characteristics make LCs one of the primary targets of HIV-1 infection. Notably, LCs form a protective barrier against HIV-1 infection and transmission. LCs restrict HIV-1 infection through the capture of HIV-1 by the C-type lectin receptor Langerin and subsequent internalization into Birbeck granules. However, the underlying molecular mechanism of HIV-1 restriction in LCs remains unknown. Here we show that human E3-ubiquitin ligase tri-partite-containing motif 5α (TRIM5α) potently restricts HIV-1 infection of LCs but not of subepithelial DC-SIGN+ DCs. HIV-1 restriction by TRIM5α was thus far considered to be reserved to non-human primate TRIM5α orthologues, but our data strongly suggest that human TRIM5α is a cell-specific restriction factor dependent on C-type lectin receptor function. Our findings highlight the importance of HIV-1 binding to Langerin for the routeing of HIV-1 into the human TRIM5α-mediated restriction pathway. TRIM5α mediates the assembly of an autophagy-activating scaffold to Langerin, which targets HIV-1 for autophagic degradation and prevents infection of LCs. By contrast, HIV-1 binding to DC-SIGN+ DCs leads to disassociation of TRIM5α from DC-SIGN, which abrogates TRIM5α restriction. Thus, our data strongly suggest that restriction by human TRIM5α is controlled by C-type-lectin-receptor-dependent uptake of HIV-1, dictating protection or infection of human DC subsets. Therapeutic interventions that incorporate C-type lectin receptors and autophagy-targeting strategies could thus provide cell-mediated resistance to HIV-1 in humans.

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

BackgroundPoint 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.ResultsLC-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.ConclusionsThe 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.

Caveolin-1 mediated uptake via langerin restricts HIV-1 infection in human Langerhans cells

BackgroundHuman Langerhans cells (LCs) reside in foreskin and vaginal mucosa and are the first immune cells to interact with HIV-1 during sexual transmission. LCs capture HIV-1 through the C-type lectin receptor langerin, which routes the virus into Birbeck granules (BGs), thereby preventing HIV-1 infection. BGs are langerin-positive organelles exclusively present in LCs, however, their origin and function are unknown.ResultsHere, we not only show that langerin and caveolin-1 co-localize at the cell membrane and in vesicles but also that BGs are langerin/caveolin-1-positive vesicles are linked to the lysosomal degradation pathway in LCs. Moreover, inhibition of caveolar endocytosis in primary LCs abrogated HIV-1 sequestering into langerin+ caveolar structures. Notably, both inhibition of caveolar uptake and silencing of caveolar structure protein caveolin-1 resulted in increased HIV-1 integration and subsequent infection. In contrast, inhibition of clathrin-mediated endocytosis did not affect HIV-1 integration, even though HIV-1 uptake was decreased, suggesting that clathrin-mediated endocytosis is not involved in HIV-1 restriction in LCs.ConclusionsThus, our data strongly indicate that BGs belong to the caveolar endocytosis pathway and that caveolin-1 mediated HIV-1 uptake is an intrinsic restriction mechanism present in human LCs that prevents HIV-1 infection. Harnessing this particular internalization pathway has the potential to facilitate strategies to combat HIV-1 transmission.

Differential Activity of NADPH-Producing Dehydrogenases Renders Rodents Unsuitable Models to Study IDH1R132 Mutation Effects in Human Glioblastoma

The somatic IDH1R132 mutation in the isocitrate dehydrogenase 1 gene occurs in high frequency in glioma and in lower frequency in acute myeloid leukemia and thyroid cancer but not in other types of cancer. The mutation causes reduced NADPH production capacity in glioblastoma by 40% and is associated with prolonged patient survival. NADPH is a major reducing compound in cells that is essential for detoxification and may be involved in resistance of glioblastoma to treatment. IDH has never been considered important in NADPH production. Therefore, the authors investigated NADPH-producing dehydrogenases using in silico analysis of human cancer gene expression microarray data sets and metabolic mapping of human and rodent tissues to determine the role of IDH in total NADPH production. Expression of most NADPH-producing dehydrogenase genes was not elevated in 34 cancer data sets except for IDH1 in glioma and thyroid cancer, indicating an association with the IDH1 mutation. IDH activity was the main provider of NADPH in human normal brain and glioblastoma, but its role was modest in NADPH production in rodent brain and other tissues. It is concluded that rodents are a poor model to study consequences of the IDH1R132 mutation in glioblastoma.

In vivo treatment of rats with unfractionated heparin (UFH) or low molecular weight heparin (LMWH) does not affect experimentally induced colon carcinoma metastasis

Recent randomized trials have suggested that treatment with low molecular weight heparin (LMWH) improves survival of cancer patients with venous thromboembolism, as compared to treatment with unfractionated heparin (UFH). Experimental studies have shown that UFH has activities besides its anticoagulant function which may affect progression of malignancy, including stimulation of new blood vessel formation. In contrast, LMWH has been suggested to inhibit angiogenesis. In the present study, we compared quantitatively the effects of treatment with UFH, LMWH or placebo on the development of experimentally induced colon carcinoma metastases in rat liver and on tumor-associated angiogenesis. It is shown that UFH and LMWH in therapeutic dosages neither affect development of metastases nor tumor blood vessel formation in this animal model. These results indicate that heparins do not affect colon cancer metastasis in liver. Further studies in other animal models are required to establish the mechanisms by which heparins potentially affect cancer.

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 Vmax was 2.5-fold higher than NADP+-dependent Vmax, whereas the Km was similar, 1.92 mM versus 1.66 mM, when NAD+ or NADP+ was used, respectively. With either coenzyme, Vmax was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a Ki 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 mM.