Tumour tissue is infiltrated by myeloid cells that are reprogrammed into alternatively activated/regenerative (M2) macrophages. The contribution of major signalling pathways and their ...modulators/targets involved in the macrophage reprogramming is poorly known. Glioblastoma (malignant brain tumour) attracts and reprograms brain-resident microglia and peripheral macrophages into cells that increase invasion, angiogenesis and suppress antitumour immunity. Using a 'function-first' approach and glioma secretome proteomics we identified osteopontin and lactadherin as proteins that cooperatively activate amoeboid transformation, phagocytosis and motility of primary microglia cultures via integrins and FAK-Akt (focal adhesion kinase-Akt) signalling. A synthetic peptide interfering with integrin ligands blocks glioma-microglia communication, functional activation and M2 gene expression. We found that osteopontin/secreted phosphoprotein 1 (Spp1) produced by non-transformed cells acts as a proinflammatory factor inducing inflammatory signalling and M1 genes, and counteracts the action of lactadherin. Using constructs encoding functional mutants of osteopontin, we demonstrated sequential processing of Spp1 by thrombin and matrix metalloproteinase-3 and/or -7 (MMP-3 and/or -7) in glioma cells, which generates a microglia-activating form devoid of the inflammatory activity, while retaining the M2 reprogramming potential. A similar form of osteopontin is secreted by human glioma cells but not normal human astrocytes. Knockdown of osteopontin or lactadherin in glioma cells reduces intracranial glioma growth, blocks amoeboid transformation of myeloid cells and affects M2 reprogramming of microglia/macrophages. Our findings demonstrate how glioma cells misuse macrophage-activating signals and redesign primarily proinflammatory signals towards their advantage to induce M2 reprogramming of tumour-infiltrating brain macrophages. Oncogene (2016) 35, 6366-6377; doi: 10.1038/onc.2016.55; published online 4 April 2016
Trapped-ion systems are a promising route toward the realization of both near-term and universal quantum computers. However, one of the pressing challenges is improving the fidelity of two-qubit ...entangling gates. These operations are often implemented by addressing individual ions with laser pulses using the Mølmer-Sørensen (MS) protocol. Amplitude modulation (AM) is a well-studied extension of this protocol, where the amplitude of the laser pulses is controlled as a function of time. We present an analytical study of AM using a Fourier series expansion so that the laser amplitude may be represented as a general continuous function. Varying the Fourier coefficients used to generate the pulse produces trade-offs between the laser power, gate time, and fidelity. We specifically study gate-timing errors, and we have shown that the sensitivity of the fidelity to these errors can be improved without a significant increase in the average laser power or the gate time. We plot atomic population vs. time for both the traditional MS protocol and the protocol with AM, highlighting the increased robustness of the AM gates. Our central result is that we improve the leading order dependence on gate timing errors from O(∆t2 ) to O(∆t6 ), and the protocol allows for arbitrarily high orders of scaling to be achieved in principle.
Malignant gliomas are highly resistant to current therapeutic approaches due to genetic alterations rendering them resistant to cell death. CK2, a ubiquitous and constitutively active ...serine/threonine kinase, frequently elevated in tumors, contributes to enhanced cell proliferation and resistance to apoptosis. Inhibition of CK2 expression or treatment with inhibitors of CK2 affected survival or induced apoptosis in various cancer cells. Here we compared cytotoxic effects of well-known and new CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), the related 3-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)propan-1-ol (MB001), 3-(4,5,6,7-tetrabromo-1H-1,2,3-benzotriazol-1-yl) propan-1-ol (MB002), 3-(4,5,6,7-tetrabromo-2H-1,2,3-benzotriazol-2-yl)propan-1-ol (MB003) and also structurally similar to above compounds pentabromobenzylisothiourea (ZKK1) and its derivatives (ZKK2-8) on cultured malignant glioma cells. TBI, ZKK1 and MB001-3 were more effective than TBB in inducing growth arrest and cell death in glioma cells. TBI and ZKK1 strongly induced apoptotic death involving caspase 3 and 7 activation followed by PARP cleavage. DMAT strongly upregulated the expression of cytotoxic ligand and its receptor Fas. Structural modifications of ZKK1 largely affected its efficacy: exchange of Br- to Cl- or F-substituents on the pentabromophenyl ring and inclusion of the bulky N-phenyl substituent in thiourea fragment of ZKK1 diminished cytotoxic activity, while N-substitution with short alkyl groups or an allyl group had opposite effects. Interestingly, TBI at moderate dose did not affect viability of non-transformed astrocytes, suggesting some specificity toward tumor cells in cytotoxic action. TBI, DMAT and ZKK1-induced apoptosis associated with caspase cascade activation in human malignant glioblastoma cells with mutated PT53 and PTEN genes. The reported data demonstrate that suitably modified polybromobenzene molecules exhibit a significant cytotoxic potential towards malignant glioblastoma cells.
A fed‐batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. ...GOX is a deaminating enzyme that shares substrate specificity with d‐amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed‐batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g·L−1). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U·g−1 cell dry weight and 1154 U·L−1 with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26‐fold in the cost of the culture medium.
Malignant gliomas attract resident microglia and macrophages and re-program these cells into pro-invasive, immunosuppressive cells. It results in formation of tumor supportive microenvironment and ...evasion of antitumor responses. The analysis of marker genes in microglia and macrophages infiltrating experimental gliomas indicates different kinetics and functions of various myeloid subpopulations during glioma progression. Signals responsible for recruitment and polarization of tumor infiltrating myeloid cells are poorly known. Proteomic analysis of glioma secretome revealed a presence of osteopontin (SPP1) and lactadherin (MGF-E8) in microglia-activating fractions. Both proteins stimulated microglia via alpha v beta 3 integrin signaling that results in activation of PI-3K/Akt and FAK, enhancement of microglial migration, phagocytosis and transcriptional responses. Inhibition of ligand-integrin interactions with short interfering peptides abolished pro-invasive polarization in glioma-microglia co-cultures. We found that SPP1, highly overexpressed in glioma cells, was specifically processed by sequential thrombin and metalloproteinase-dependent cleavage. This type of processing results in losing SPP1 pro-inflammatory activity leaving intact its pro-tumorigenic activity and does not occur in non-transformed cells. Knockdown of SPP1 in glioma cells strongly reduced growth of intracranial gliomas. The number of infiltrating microglia/macrophages (Iba1+) was not affected, but amoeboid transformation, expression of the alternative phenotype markers and angiogenesis were reduced in SPP1-depleted gliomas. Interestingly, infiltrating Iba1+ cells did not undergo pro-invasive polarization. Moreover, SPP1-depleted tumors were infiltrated with T cytotoxic lymphocytes, while accumulation of T regulatory cells was significantly reduced. The expression of SPP1 is up-regulated in human glioblastoma and inversely correlated with patient's survival. Our findings demonstrate that glioma-derived and specifically processed osteopontin/SPP1 polarizes glioma-infiltrating microglia and contributes to transformation of tumor microenvironment. This defines osteopontin/SPP1 a new biomarker and target for glioma therapy, and shows that targeting glioma-microglia interactions within tumor microenvironment is a promising strategy to fight these tumors. Studies were supported by a grant 2012/04/A/NZ3/00630 from the National Science Center.
The old urinary calculi of the votive offerings in the pilgrimage church at Grafrath offer the possibility of analysing the components by infrared spectroscopy to give insights into factors that ...might influence their formation. A total of 166 specimens were taken from 139 objects (134 stones, 5 bones), in some stones from different layers.
Spectral analysis showed typical components for urinary calculi in 127 stones. These were compared with a control group of 98 urinary stones from carriers (77 male, 21 female) during 2007/2008 in Bavaria.
The percentage of occurrence of ammonium acid urate (NH(4)U) was high in the old stones (68.0%) versus the 2007/2008 group (1.0%). In uric acid (HS) there was no relevant difference between the two groups, whereas the occurrence of the oxalates whewellite (Whe) and weddellite (Wed) was much less in the old stones (Whe 18.1-69.4%, Wed 7.9-26.5 %). The phosphates differ in the components in favour of brushite in the old stones. The high occurrence of NH(4) in the old stones is comparable with (a) the old pre-1900 collection of Norwich (England), especially with the pre-1800 juvenile bladder stones, and (b) urinary stones in endemic areas of stone disease in children such as in North Thailand. Data about the Grafrath stone carriers (name, age, hometown) are not available but can indirectly be derived from the miracle books (1444-1728) of Grafrath with 12,131 reports; 1,165 had urologic disease of which 70% were children with urinary calculi coming from areas of Upper Bavaria and Swabia.
The finding of a high NH(4)U content indicates that this area might have been a stone belt for bladder stones in children. Under- or malnutrition with low protein and low fluid intake may be the aetiologic factor.
Malignant gliomas are fast-growing, heterogeneous and invasive brain tumors strongly infiltrated by non-tumor cells. Glioma attracts variety of immune cells, in particular microglia/macrophages and ...re-program these cells into immunosuppressive, tumor-supporting cells. Factors responsible for pro-invasive macrophage polarization and shaping tumor microenvironment in tumor-supporting manner are poorly known. We analyzed glioma secretome using proteomical approach and identified lactadherin (Mfge8) and osteopontin (Spp1) in microglia-activating fractions. Both osteopontin and lactadherin are αvβ3/αvβ5 integrin ligands able to interact with receptors present on microglia and macrophages and thus could be involved in pro-invasive polarization of microglia/macrophages. Moreover, both Spp1 and Mfge8 are overexpressed in glioma cells, but not in non-transformed astrocytes. C6 glioma cells stably expressing shRNA specific to lactadherin (shMfge8), osteopontin (shSpp1) and negative shRNA (shNeg) were implanted into striatum of Wistar rats. There was no difference in proliferation and viability of C6 glioma cells, cells stably expressing shRNA specific to lactadherin, ostopontin and negative shRNA in vitro, that demonstrates the negligible effect of autocrine production of both protein on tumor cell growth. Knockdown of Spp1 and Mfge8 resulted in significant reduction of tumor volume in rat model of glioma. Immunochemical analysis of brain sections revealed similar numbers of infiltrating microglia/macrophages (Iba1 staining), but the reduced number of ameboid, arginase 1 expressing cells in Mfge8 – depleted tumor. Treatment of endothelial cells with rhMFGE8 revealed significant effect of that protein on angiogenesis in vitro, however lactadherin-depleted tumors do not exhibit reduced blood vessel density in rat glioma model. FACS analysis showed that silencing of Spp1 does not affect total number of CD11b-positive cells, but strongly modulates microenvironment by leading to significant changes in percentage of Tc and Treg cells infiltrating tumor-bearing hemisphere. Our results suggest that glioma-derived integrin ligands are important factor in polarization of glioma infiltrating microglia/macrophages into the pro-invasive phenotype and its targeting could be a new therapeutic strategy.
Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the ...present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate
fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.
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