Glioma infiltration and extracellular matrix: key players and modulators.

An outstanding characteristic of gliomas is their infiltration into brain parenchyma, a property that impairs complete surgical resection; consequently, these tumors might recur, resulting in a high mortality rate. Gliomas invade along preferential routes, such as those along white matter tracts and in the perineuronal and perivascular spaces. Brain extracellular components and their partners and modulators play a crucial role in glioma cell invasion. This review presents an extensive survey of the literature, showing how the brain extracellular matrix (ECM) is modulated during the glioma infiltration process. We explore aspects of ECM interaction with glioma cells, reviewing the main glycosaminoglycans, glycoproteins and proteoglycans. We discuss the roles of ECM-binding proteins, including CD44, RHAMM, integrins and axonal guidance molecules, and highlight the role of proteases and glycosidases in glioma infiltration; in binding and release chemokines, cytokines and growth factors; and in generating new bioactive ECM fragments. We also consider the roles of cytoskeletal signaling, angiogenesis, miRNAs and the glial-to-mesenchymal transition linked to glioma invasion. We closely discuss therapeutic approaches based on the modulation of the extracellular matrix, targeting the control of glioma infiltration, its relative failure in clinical trials, and potential means to overcome this difficulty.

The Chemokine Receptor CXCR6 Evokes Reverse Signaling via the Transmembrane Chemokine CXCL16.

Reverse signaling is a signaling mechanism where transmembrane or membrane-bound ligands transduce signals and exert biological effects upon binding of their specific receptors, enabling a bidirectional signaling between ligand and receptor-expressing cells. In this study, we address the question of whether the transmembrane chemokine (C-X-C motif) ligand 16, CXCL16 is able to transduce reverse signaling and investigate the biological consequences. For this, we used human glioblastoma cell lines and a melanoma cell line as in vitro models to show that stimulation with recombinant C-X-C chemokine receptor 6 (CXCR6) or CXCR6-containing membrane preparations induces intracellular (reverse) signaling. Specificity was verified by RNAi experiments and by transfection with expression vectors for the intact CXCL16 and an intracellularly-truncated form of CXCL16. We showed that reverse signaling via CXCL16 promotes migration in CXCL16-expressing melanoma and glioblastoma cells, but does not affect proliferation or protection from chemically-induced apoptosis. Additionally, fast migrating cells isolated from freshly surgically-resected gliomas show a differential expression pattern for CXCL16 in comparison to slowly-migrating cells, enabling a possible functional role of the reverse signaling of the CXCL16/CXCR6 pair in human brain tumor progression in vivo.

The role of Fc-receptors in the uptake and transport of therapeutic antibodies in the retinal pigment epithelium.

In the ophthalmological clinic, intravitreally applied antibodies or Fc-containing fusion proteins are frequently used, but the biology and pharmacokinetics of these therapeutics in the retina are not well understood. We have previously shown intracellular uptake of Fc-containing molecules in RPE cells. In this study, we investigated the involvement of Fc-receptors, both Fcγ-receptors and the neonatal Fc-receptor (FcRn) in the uptake and intracellular trafficking of the VEGF-antagonists bevacizumab, aflibercept and the anti-CD20 antibody rituximab in three different model systems, primary porcine RPE cells, ARPE-19 cells and porcine RPE/choroid explants. The expression of Fcγ-receptors was tested in primary porcine RPE cells, and the expression of Fcγ-receptors I and II could be shown in RT-PCR and qRT-PCR, while the expression of FcRn was additionally confirmed in Western blot and immunocytochemistry. All three compounds, bevacizumab, rituximab and aflibercept, were taken up into the cells and displayed a characteristic time-dependent pattern, as shown in Western blot and immunohistochemistry. The uptake was not altered by the inhibition of Fcγ-receptors using different inhibitors (TruStain FcX, genistein, R406). However, the inhibition of FcRn with an antagonistic antibody reduced intracellular IgG in porcine RPE cells (rituximab) and ARPE-19 cells (bevacizumab, rituximab). Colocalisations between the tested compounds and myosin7a could be found. In addition, limited colocalization with FcRn and the tested compounds, as well as triple localization between compound, FcRn and myosin7a could be detected, indicating a role of myosin7a in FcRn mediated transport. However, the colocalizations are restricted to small fractions of the Fc-containing compounds. Furthermore, the FcRn is mainly found in the membrane section, where only minute amounts of the Fc-containing compounds are seen, suggesting a limited interaction. An apical to choroidal transport of IgG through the RPE/choroid can be found in RPE/choroid explants. Inhibition of FcRn increases the amount of bevacizumab found on the choroidal side, suggesting a role of FcRn in the recycling of bevacizumab. In conclusion, our data indicate a role for FcRn, but not Fcγ-receptors, in the uptake and transport of Fc-containing molecules in the RPE and indicate a recycling function of FcRn in the retina.

"Inverse signaling" of the transmembrane chemokine CXCL16 contributes to proliferative and anti-apoptotic effects in cultured human meningioma cells.

BACKGROUND: Chemokines and their receptors play a decisive role in tumor progression and metastasis. We recently found a new signaling mechanism in malignant glioma cells mediated by transmembrane chemokines that we termed "inverse signaling". According to this hypothesis, soluble (s)-CXCL16 binds to the surface-expressed transmembrane (tm) -CXCL16, and induces signaling and different biological effects in the stimulated cells, so that the transmembrane ligand itself acts as a receptor for its soluble counterpart. Now, we hypothesized that "inverse signaling" via tm-CXCL16 might also take place in meningiomas, a completely different, benign tumor entity. METHODS: We used quantitative reverse-transcription polymerase chain reaction, immunocytochemistry and western blot to detect CXCL16 and CXCR6 in human meningioma cells isolated from 28 human meningiomas. Subsequently, we stimulated cultured human tm-CXCL16-positive, CXCR6-negative meningioma cells with recombinant s-CXCL16 and analyzed binding, signaling and biological effects using RNAi silencing to verify specificity. RESULTS: In fact, cultured human meningioma cells considerably express CXCL16, but substantially lack CXCR6, the only known CXCL16 receptor. These receptor-negative cells could bind s-CXCL16, and responded to s-CXCL16 application with activation of the intracellular kinases ERK1/2 und Akt. As a consequence, we observed increased proliferation and rescue of apoptosis of cultured meningioma cells. Since binding and signaling were abolished by siRNA silencing, we concluded that tm-CXCL16 specifically acts as a receptor for s-CXCL16 also in human meningioma cells. CONCLUSION: These findings underline our recent report on the mechanism of inverse signaling as a broad biological process also observable in more benign tumor cells and contributing to tumor progression.

Effects of the chemokine CXCL12 and combined internalization of its receptors CXCR4 and CXCR7 in human MCF-7 breast cancer cells.

The chemokine CXCL12 (stromal cell-derived factor-1, SDF-1) and its receptor CXCR4 play a major role in tumor initiation, promotion, progression and metastasis, especially for breast cancer cells. Recently, CXCR7 has been identified as a second receptor for CXCL12; nevertheless, it also binds CXCL11 (interferon-inducible T cell α chemoattractant, I-TAC). However, little is known about the co-expression of the two receptors and their interactions. Quantitative reverse transcription plus the polymerase chain reaction has demonstrated that both receptors are frequently co-expressed in breast cancer cell lines, whereas other tumor cell lines often express only one of them. For interaction studies, we chose MCF-7 breast cancer cells, since they highly express CXCR4 and CXCR7 at the protein level but not CXCR3 (another target for CXCL11). Immunofluorescence and gold-labeling by light and electron microscopy, respectively, revealed that both receptors were localized at the cell surface in non-stimulated cells. After exposure to CXCL12 or CXCL11, the receptors were rapidly internalized alone or in close proximity. Stimulation with the CXCR4- or CXCR7-selective non-peptide antagonists AMD3100 and CCX733 resulted not only in single internalization but partly also in co-internalization of the two receptors. Furthermore, both chemokine ligands reduced staurosporine-induced apoptosis and caspase-3/7 activation; however, the selective inhibitors merely had partial inhibitory effects on these biological responses. Our findings suggest that CXCR4 and CXCR7 closely interact in breast cancer cells. Both are co-internalized, transduce signals and induce further biological effects partly independently of a selective stimulus or antagonist.

The transcription factor Forkhead box P3 (FoxP3) is expressed in glioma cells and associated with increased apoptosis.

The forkhead transcription factor FoxP3 is critically involved in the development and function of regulatory T cells (Tregs) that populate tumors and are considered as powerful parts of their immune evasion. However, also tumor cells are reported to express FoxP3. Since gliomas are particularly immunosuppressive tumors, we investigated the occurrence and possible functions of FoxP3 in these malignant cells. By quantitative RT-PCR, immunohistochemistry and FACS analysis, we detected FoxP3 in glioma cells in situ and in vitro. After exposure of glioma cell lines to chemotherapeutics, expression of FoxP3 was significantly enhanced, and it was dislocated from more nuclear to perinuclear localization. Overexpression of FoxP3 in glioma cell lines considerably favored apoptotic damage of nuclei, DNA fragmentation, increased cleavage of the pro-apoptotic enzyme poly(ADP-ribose) polymerase (PARP) and basal activities of effector caspases-3/7. In FoxP3-transfected cells, apoptotic stimuli like Camptothecin, Temozolomide or tumor necrosis factor-α synergistically enhanced caspases-3/7-activities over controls. Taking together, FoxP3 occurs in glioma cells, is induced by chemotherapeutics, and its expression is correlated with increased apoptosis of glioma cells, especially when propagated by apoptotic stimuli. Thus, FoxP3 is a novel pro-apoptotic transcription factor in gliomas that is critically involved in the action of apoptotic agents.

The retinal pigment epithelium (RPE) induces FasL and reduces iNOS and Cox2 in primary monocytes.

PURPOSE: Retinal pigment epithelium (RPE) cells may alter the phenotype of monocytes by soluble factors that may be influenced by stimulation of the RPE. Since RPE cells carry the toll-like receptor-3 (TLR3) that detects and reacts to viral infection through binding of dsRNA we investigated the effects of RPE cells with or without TLR3 stimulation on blood-derived monocytes with respect to regulation of pro-/anti-inflammatory cytokines, anti-angiogenic factors and migratory properties. METHODS: Primary RPE cells were prepared from porcine eyes; monocytes were prepared from porcine blood. TLR3 activation was induced by polyinosinic:polycytidylic acid (Poly I:C). RPE cells were stimulated with Poly I:C in different concentrations for 24 hours and a cell culture supernatant was applied to the monocytes. Expression of CD14 and Fas ligand (FasL) was determined via flow cytometry. The expression of IL-6, IL-1ß, TNFα, Cox2, iNOS and IL-10 was determined via quantitative RT-PCR. Migration was determined using Boyden chamber experiments. RESULTS: The supernatant of RPE cells, irrespective of TLR3 activation, induced FasL expression in the monocytes. Expression of iNOS and Cox2 was reduced by RPE cells and the reduction of Cox2 but not if iNOS was lost under TLR3 activation. No induction of IL-6, IL-1ß, IL-10 or TNFα by the RPE was seen. TLR3-activated RPE cells induced monocyte migration. CONCLUSION: RPE cells induce an upregulation of FasL and a downregulation of iNOS and Cox2 without upregulating inflammatory cytokines, possibly inducing an anti-angiogenic phenotype in the monocytes. This phenotype is still upheld after challenging RPE cells with dsRNA, mimicking a viral infection.

The antimicrobial peptide lysozyme is induced after multiple trauma.

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction.

Lost in disruption: role of proteases in glioma invasion and progression.

A characteristic feature of malignant glial tumors (gliomas) is their tendency to diffusely infiltrate the nervous system preventing their complete surgical resection. Proteases play a decisive role in this malignant process, either by degradation of brain extracellular matrix (ECM) components, adhesion molecules, or by regulating the activity of growth and chemotactic factors. Secreted matrix metalloproteinases (MMPs) and ADAMTS proteases (ADAMs with thrombospondin motifs) cleave different ECM components like the proteoglycans (lecticans) aggrecan, versican, neurocan and brevican with selective preferences; they are further regulated by endogenous inhibitors and activating metallo- and serine proteases. Cell surface proteases of the ADAM family (A Disintegrin And Metalloproteinase), but also serine proteases regulate the activity of growth factors and chemokines that act as autocrine / paracrine stimulators within gliomas. Thus, proteases play a decisive role for the spread and growth of gliomas and are prominent targets for their therapy.

The presumed atypical chemokine receptor CXCR7 signals through G(i/o) proteins in primary rodent astrocytes and human glioma cells.

SDF-1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF-1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin-sensitive G(i/o) proteins. SDF-1-dependent activation of G(i/o) proteins and subsequent increases in intracellular Ca(2+) concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7-mediated effects of SDF-1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF-1-induced activation of Erk and Akt in CXCR7-only expressing human glioma cell lines. Finally, consistent with a ligand-biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I-TAC/CXCL11, activated Erk and Akt through ß-arrestin. The demonstration that SDF-1-bound CXCR7 activates G(i/o) proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.

Multimodal Targeted Nanoparticle-Based Delivery System for Pancreatic Tumor Imaging in Cellular and Animal Models.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), which ranks forth on the cancer-related death statistics still is both a diagnostic and a therapeutic challenge. Adenocarcinoma of the exocrine human pancreas originates in most instances from malignant transformation of ductal epithelial cells, alternatively by Acinar-Ductal Metaplasia (ADM). RA-96 antibody targets to a mucin M1, according to the more recent nomenclature MUC5AC, an extracellular matrix component excreted by PDAC cells. In this study, we tested the usability of multimodal nanoparticle carrying covalently coupled RA-96 Fab fragments for pancreatic tumor imaging. METHODS: In order to make and evaluate a novel, better targeting, theranostic nanoparticle, iron nanoparticles and the optical dye indocyanin green (ICG) were encapsulated into the cationic sphingomyelin (SM) consisting liposomes. RA-96 Fab fragment was conjugated to the liposomal surface of the nanoparticle to increase tumor homing ability. ICG and iron nanoparticle-encapsulated liposomes were studied in vitro with cells and (i) their visibility in magnetic resonance imaging (MRI), (ii) optical, (iii) Magnetic particle spectroscopy (MPS) and (iv) photoacoustic settings was tested in vitro and also in in vivo models. The targeting ability and MRI and photoacoustic visibility of the RA-96-nanoparticles were first tested in vitro cell models where cell binding and internalization were studied. In in vivo experiments liposomal nanoparticles were injected into the tail vain using an orthotopic pancreatic tumor xenograft model and subcutaneous pancreatic cancer cell xenografts bearing mice to determine in vivo targeting abilities of RA-96-conjugated liposomes Results: Multimodal liposomes could be detected by MRI, MPS and by photoacoustic imaging in addition to optical imaging showing a wide range of imaging utility. The fluorescent imaging of ICG in pancreatic tumor cells Panc89 and Capan-2 revealed an increased association of ICG-encapsulated liposomes carrying RA-96 Fab fragments in vitro compared to the control liposomes without covalently linked RA-96. Fluorescent molecular tomography (FMT) studies showed increased accumulation of the RA96-targeted nanoparticles in the tumor area compared to non-targeted controls in vivo. Similar accumulation in the tumor sites could be seen with liposomal ferric particles in MRI. Fluorescent tumor signal was confirmed by using an intraoperative fluorescent imaging system, which showed fluorescent labeling of pancreatic tumors. CONCLUSION: These results suggest that RA-96-targeted liposomes encapsulating ICG and iron nanoparticles can be used to image pancreatic tumors with a variety of optical and magnetic imaging techniques. Additionally, they might be a suitable drug delivery tool to improve treatment of PDAC patients.

Expression and role of the cell surface protease seprase/fibroblast activation protein-α (FAP-α) in astroglial tumors.

Seprase or fibroblast activation protein-α (FAP-α) is a cell-surface serine protease that was previously described nearly exclusively on reactive and tumor stromal fibroblasts and thought to be involved in tissue remodeling. We investigated the expression and significance of FAP-α in astrocytomas/glioblastomas. As shown by quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, FAP-α was elevated in whole glioblastoma tissues and in particular in most glioma cells in situ and in vitro. In glioma stem-like cells (gliospheres), FAP-α was detected at low levels; however, FAP-α was considerably induced upon differentiation with 10% fetal calf serum. To explore its functional role, FAP-α was silenced by siRNA transfection. In Boyden chamber assays, FAP-α silenced cells migrated similar as control cells through non-coated or Matrigel (basal lamina)-coated porous membranes, but significantly slower through membranes coated with gelatin or brevican, a major component of brain extracellular matrix. Furthermore, FAP-α-silenced glioma cells migrated through murine brain slices much slower under the conditions tested than differentially fluorescent-labeled control cells. Thus, FAP-α is highly expressed on the surface of glioma cells and contributes to diffuse glioma invasion through extracellular matrix components.

CX3CR1 promotes recruitment of human glioma-infiltrating microglia/macrophages (GIMs).

The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets.

Interleukin-1alpha treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2.

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.

Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging.

Superparamagnetic iron-oxide particles (SPIO) are used in different ways as contrast agents for magnetic resonance imaging (MRI): Particles with high nonspecific uptake are required for unspecific labeling of phagocytic cells whereas those that target specific molecules need to have very low unspecific cellular uptake. We compared iron-oxide particles with different core materials (magnetite, maghemite), different coatings (none, dextran, carboxydextran, polystyrene) and different hydrodynamic diameters (20-850 nm) for internalization kinetics, release of internalized particles, toxicity, localization of particles and ability to generate contrast in MRI. Particle uptake was investigated with U118 glioma cells und human umbilical vein endothelial cells (HUVEC), which exhibit different phagocytic properties. In both cell types, the contrast agents Resovist, B102, non-coated Fe(3)O(4) particles and microspheres were better internalized than dextran-coated Nanomag particles. SPIO uptake into the cells increased with particle/iron concentrations. Maximum intracellular accumulation of iron particles was observed between 24 h to 36 h of exposure. Most particles were retained in the cells for at least two weeks, were deeply internalized, and only few remained adsorbed at the cell surface. Internalized particles clustered in the cytosol of the cells. Furthermore, all particles showed a low toxicity. By MRI, monolayers consisting of 5000 Resovist-labeled cells could easily be visualized. Thus, for unspecific cell labeling, Resovist and microspheres show the highest potential, whereas Nanomag particles are promising contrast agents for target-specific labeling.

The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6.

Chemokines are implicated in developmental and inflammatory processes in the brain. The transmembrane chemokine CXCL16 is produced in brain endothelial and reactive astroglial cells and released by shedding. Its receptor CXCR6 is detected during brain development highest at postnatal day 6, found in glial precursor cells differentiated from neural stem cells and in an A2B5-positive glial precursor cell line. Their stimulation by soluble CXCL16 induces the PI3-kinase/Akt and Erk pathways resulting in the activation of the transcription factor AP-1. As biological responses, soluble CXCL16 upregulates its own receptor, increases cell proliferation, stimulates cell migration in wound-healing and in spheroid confrontation assays. Invasion of CXCR6-positive glial cells into CXCL16-expressing spheroids can be blocked by sheddase inhibitors and CXCL16-antibody. Since CXCL16 is induced by cytokines at sites of inflammation, neurodegeneration, ischemia and malignant transformation, it should attract CXCR6-positive glial precursor cells, enhance their invasion and proliferation and thus favor astrogliosis.

Iron oxide-loaded liposomes for MR imaging.

In this study a liposome cell labeling system was developed for non-target-specific labeling of glioma cells with superparamagnetic iron oxide nanoparticles for magnetic resonance imaging (MRI). A high non-target-specific uptake is ideal for in vitro labeling of cells and subsequently for cell tracking and visualization of phagocytic cells in vivo. The preparation of iron oxide-loaded liposomes was optimized and the biological properties of the liposomes were investigated. Cytotoxicity and cell viability were examined and showed limited cytotoxic effects. Non-target-specific labeling of glioma cells in vitro for subsequent specific labeling of molecules for MR imaging was tested by T2*-weighted MRI at 3T. The glioma cells showed a strong initial uptake of the iron oxide liposomes and the uptake was not saturable within 24 h exposure. The uptake of liposomes was superior to non-coated magnetite nanoparticles. Using PEG-ylated liposomes, the non-specific uptake could be decreased fundamentally (86% lower) in comparison to conventional liposomes. Furthermore, the ability of liposomes as contrast agents for MR imaging was investigated. Cells labeled with iron oxide nanoparticles by treatment with liposomes showed a negative contrast in MRI and consequently successful cellular labeling. Thus, iron oxide-loaded liposomes are well suited for non-target-specific cell labeling for MR imaging.

Glial cross-talk by transmembrane chemokines CX3CL1 and CXCL16.

The transmembrane chemokines CX3CL1 and CXCL16 mediate chemotaxis and cell to cell adhesion by interaction with their receptors CXCR6 and CX3CR1, respectively. We here summarize reports on the expression, regulation and proteolytic processing of transmembrane chemokines by glial cells, their activity on glial cells/glial precursor cells and their role in glial cell cross-talk under physiological and pathophysiological conditions.

A methylation-specific and SYBR-green-based quantitative polymerase chain reaction technique for O6-methylguanine DNA methyltransferase promoter methylation analysis.

The O(6)-methylguanine DNA methyltransferase (MGMT) gene encodes a DNA repair enzyme whose activity is a major mechanism of resistance to alkylating drugs in glioblastoma treatment. Hypermethylation of the MGMT promoter is associated with chemosensitivity because it reduces MGMT activity. Here we present a method combining methylation-specific and SYBR-green-based quantitative PCR (MSQP) for MGMT promoter methylation analysis. This highly specific, sensitive, and reproducible method allows the quantification of fully methylated and fully unmethylated MGMT DNA species in terms of percentage. Values are related to standard curves, corrected for DNA input by an internal standard, and calculated in relation to methylated and unmethylated control DNAs as a percentage share. Finally, values are defined relative to the sum of fully methylated and unmethylated MGMT DNA sample amount to obtain percentage of methylated reference and percentage of unmethylated reference results. We have used this technique to investigate MGMT promoter methylation in relation to MGMT mRNA expression in nine tumor cell lines and 15 primary glioblastoma patients. Presented data confirm that this assay is suitable for detection of low amounts of methylated and unmethylated MGMT promoter DNA. Carefully validated quantitative MSQP assays will be useful in both research and clinical molecular diagnosis.