for highly malignant gliomas (World Health Organization grade III and IV) there is no successful treatment; patients have an average survival time of approximately 1 y after diagnosis. Glioma cells are highly invasive and infiltrate normal brain tissue, and as a result, surgical resection is always incomplete. Degradation of ECM by membrane-bound and secreted metalloproteases facilitates glioma invasion. In particular, the membrane-bound metalloproteases are pivotal for tumor invasion as they very efficiently digest extracellular matrix proteins and also activate secreted metalloproteases (1) like matrix metalloproteinase-2 (MMP-2, also known as gelatinase A), which is one of the major proteases involved in glioma invasion in mouse models (2) and probably also in humans (3). Hence, membrane-inserted metalloproteases like membrane type 1 matrix metalloproteinase (MT1-MMP) can enable gliomas to invade the brain parenchyma as single cells (4).Microglia are the intrinsic immune cells of the brain; they control the innate and the adaptive immune response in the CNS and are activated by inflammatory or other pathological stimuli (5). Activation of microglial toll-like receptors (TLRs) triggers the innate immune response and can initiate host-defense and tissue repair mechanisms, but also CNS inflammation, neurodegeneration, and trauma (5, 6). As microglial cells are attracted toward glioma in large numbers-glioma tissue consists of as much as 30% microglial cells-and because microglia density in gliomas positively correlates with malignancy, invasiveness, and grading of the tumors (7-9), we investigated if microglia may actively contribute to glioma expansion. Here, we show that soluble factors released from glioma stimulate microglial TLRs, resulting in microglial MT1-MMP expression via the TLR downstream signaling molecules MyD88 and p38 MAPK. In turn, MT1-MMP expression and activity in these immune cells promotes glioma cell invasion and tumor expansion. ResultsGlioma Associated Microglia Over-Express MT1-MMP. We analyzed the expression pattern of the matrix protease MT1-MMP in mouse and human gliomas and found the enzyme to be expressed predominantly in microglial cells closely associated with the tumors. Whereas tumor-free human brain samples showed virtually no MT1-MMP expression, we detected intense MT1-MMP labeling, especially in higher-grade gliomas. Importantly, in human samples, immunolabeling for the microglial marker Iba1 and for MT1-MMP largely overlapped [supporting information (SI) Fig. S1 A-D and Table S1]. Likewise, after injection of a human glioma cell line (U373 cells) into immunodeficient mice, we detected that microglia represent the predominant cell type contributing intratumoral MT1-MMP expression (see Fig. S1E).In our in vivo mouse model, the glioma cells were identified by stable expression of EGFP and microglial cells by immunolabeling for Iba1. In sections obtained from mice 2 weeks after intracerebral injection with isogenic glioma cells (GL261 cells), we found an increased density of mic...

Primary astrocytomas of World Health Organization grade 3 and grade 4 (HG-astrocytomas) are preponderant among adults and are almost invariably fatal despite multimodal therapy. Here, we show that the juvenile brain has an endogenous defense mechanism against HG-astrocytomas. Neural precursor cells (NPCs) migrate to HG-astrocytomas, reduce glioma expansion and prolong survival by releasing a group of fatty acid ethanolamides that have agonistic activity on the vanilloid receptor (transient receptor potential vanilloid subfamily member-1; TRPV1). TRPV1 expression is higher in HG-astrocytomas than in tumor-free brain and TRPV1 stimulation triggers tumor cell death via the activating transcription factor-3 (ATF3) controlled branch of the ER stress pathway. The anti-tumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid Arvanil, suggesting that TRPV1 agonists hold potential as new HG-astrocytoma therapeutics.

Glioblastoma cells with stem-like properties control brain tumour growth and recurrence. Here, we show that endogenous neural precursor cells perform an anti-tumour response by specifically targeting stem-like brain tumour cells. In vitro, neural precursor cells predominantly express bone morphogenetic protein-7; bone morphogenetic protein-7 is constitutively released from neurospheres and induces canonical bone morphogenetic protein signalling in stem-like glioblastoma cells. Exposure of human and murine stem-like brain tumour cells to neurosphere-derived bone morphogenetic protein-7 induces tumour stem cell differentiation, attenuates stem-like marker expression and reduces self-renewal and the ability for tumour initiation. Neurosphere-derived or recombinant bone morphogenetic protein-7 reduces glioblastoma expansion from stem-like cells by down-regulating the transcription factor Olig2. In vivo, large numbers of bone morphogenetic protein-7-expressing neural precursors encircle brain tumours in young mice, induce canonical bone morphogenetic protein signalling in stem-like glioblastoma cells and can thereby attenuate tumour formation. This anti-tumour response is strongly reduced in older mice. Our results indicate that endogenous neural precursor cells protect the young brain from glioblastoma by releasing bone morphogenetic protein-7, which acts as a paracrine tumour suppressor that represses proliferation, self-renewal and tumour-initiation of stem-like glioblastoma cells.

Lactate formation in highly proliferative tumors such as malignant gliomas is associated with poor survival and contributes to the suppression of local immunity. Here, we report that diclofenac used at nontoxic concentrations significantly decreased lactate production in murine glioma cells and inhibited the expression of lactate dehydrogenase-A in vitro. Lactate reduction was accompanied by a dose-dependent inhibition of cell growth and a cell cycle arrest at the G2/M checkpoint. In the presence of diclofenac, murine bone marrow-derived dendritic cells (DCs) showed enhanced IL-12, but decreased IL-10 secretion on Toll-like receptor stimulation with R848 that correlated with reduced lactate levels in the glioma cell coculture and a blockade of signal transducers and activators of transcription 3 phosphorylation. In vivo, diclofenac treatment diminished intratumoral lactate levels and resulted in a significant delay of glioma growth. Ex vivo analyses revealed that tumor-infiltrating DCs regained their capacity to produce IL-12 on R848 stimulation. Moreover, diclofenac reduced the number of tumor-infiltrating regulatory T cells and impaired the upregulation of the Treg activation marker CD25. Nevertheless, a single intratumoral injection of R848 combined with diclofenac failed to induce an additional survival advantage in glioma-bearing mice. Further analyses illustrated that the presence of diclofenac during T-cell activation compromised INF-c production and T-cell proliferation, indicating that immunotherapeutic approaches have to be carefully timed when combined with diclofenac. In summary, diclofenac appears as an attractive agent for targeting lactate production and counteracting local immune suppression in malignant gliomas.One of the most important adaptive responses of quickly proliferating tumors is the metabolic switch from the oxidative to the glycolytic pathway. The driving force, among others, is hypoxia, which induces the upregulation of glycolytic enzymes and glucose transporters via HIF-1 activation. 1,2 Alternatively, glycolysis can be stimulated by oncogenic transformation mediated by c-myc. 3 As a consequence of elevated glycolysis, tumors upregulate lactate dehydrogenase (LDH), leading to an increased production of lactate, which is released into the tumor stroma. 4 LDH is a tetrameric enzyme composed of 4 muscle (M) and/or heart (H) subunits, encoded by two distinct genes, LDH-A and LDH-B. An increase of LDH-A over LDH-B activation favors the production of a specific LDH isoenzyme (e.g., LDH5) that is more efficient in catalyzing the conversion of pyruvate to lactate. The LDH-A gene is under the direct transcriptional regulation of HIF-1 and c-myc. 5,6 Increased LDH5 expression and elevated lactate levels correlate with poorer prognosis, poor disease-free or metastasis-free survival and poor overall survival in several tumor entities, including malignant gliomas. [7][8][9][10] Accumulating data suggest that dendritic cells (DCs) play a central role in the initiation of immune responses in the ...

Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia in humans. Adherence of M. pneumoniae to host cells requires several adhesin proteins, such as P1, P30, and P116. A major limitation in developing a specific diagnostic test for M. pneumoniae is the inability to express adhesin proteins in heterologous expression systems due to unusual usage of the UGA stop codon, leading to premature termination of these proteins in Escherichia coli. In the present study, we successfully expressed the C-terminal (P1-C1) and N-terminal (P1-N1) regions of the P1 protein in E. coli. On screening these recombinant proteins with sera from M. pneumoniae-infected patients, only the P1-C1 protein was found to be immunogenic. This protein can be used as an antigen for immunodiagnosis of M. pneumoniae infection, as well as in adherence inhibition studies to understand the pathophysiology of the disease.Mycoplasma pneumoniae is the causative agent of atypical pneumonia and is also responsible for other respiratory tract infections such as tracheobronchitis, bronchiolitis, croup, and less severe upper respiratory tract infections in older children and young adults (3,14). It has been estimated that between 10 and 20% of X-ray-proven pneumonia cases that occur in the endemic period and that up to 50% of all cases that occur in the epidemic period are caused by M. pneumoniae (7,23).Adherence of M. pneumoniae to the human host respiratory epithelium is a prerequisite for colonization of the respiratory epithelium and subsequent induction of disease (12,14). Cytadherence is mediated by a specialized tip-like attachment organelle found in M. pneumoniae (8,21). It requires a complex interaction of several M. pneumoniae proteins present on the attachment organelle, including the major surface adhesins P1 (170 kDa), P30 (30 kDa), and P116 and proteins HMW1 to HMW5, as well as proteins A, B and C (1, 10, 14, 24, 29, 30). These proteins cooperate structurally and functionally so that M. pneumoniae major surface adhesins display polar clustering at the organelle tip assisted by HMW proteins. The major proteins P1 and P30 appear to be directly involved in receptor binding (5,13,21). The HMW proteins and proteins A, B, and C are accessory proteins since they are not adhesins but are required for proper functioning. The native adhesins P1 and P30 are also known to be strongly immunogenic in humans and experimental animals infected with M. pneumoniae.The true incidence of M. pneumoniae associated infection is not clear because of the nonavailability of rapid and specific diagnostic tests. Being a fastidious organism, M. pneumoniae grows slowly and poorly. The standard methods for the diagnosis of M. pneumoniae are culture, serology, and PCR. Since M. pneumoniae can be difficult to isolate (11), laboratory diagnosis is based on serological tests, such as complement fixation and enzyme-linked immunosorbent assays (ELISAs) (31). PCR has also been used for its detection (6, 34). The complement fixation test has limited value and produ...

Transcriptomic profiling of cells treated with Golgi-disrupting compounds reveals that some target genes including several spliceosome components are controlled by ELK1, GABPA, and ETS1, the activity of which is regulated by MEK/ERK signaling. Furthermore, brefeldin A and golgicide A cause increased splicing of the proapoptotic MCL1-S isoform.

Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, we show that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and increases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines, as shown by the following experiments: (1) downregulating TfR expression reduced proliferation in vitro and in vivo; (2) forced TfR expression in low-grade glioma accelerated proliferation to the level of high-grade glioma; (3) iron and oxidant chelators attenuated tumor proliferation in vitro and tumor size in vivo. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused N-methyl-D-aspartate-receptor-mediated reduction of neuron cell mass. Our results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass.

Members of the transforming growth factor ␤ (TGF-␤) family of proteins modulate the proliferation, differentiation, and survival of many different cell types. Neural stem and progenitor cells (NPCs) in the adult brain are inhibited in their proliferation by TGF-␤ and by bone morphogenetic proteins (BMPs). Here, we investigated neurogenesis in a hypomorphic mouse model for the TGF-␤ and BMP inhibitor Smad7, with the hypothesis that NPC proliferation might be reduced due to increased TGF-␤ and BMP signaling. Unexpectedly, we found enhanced NPC proliferation as well as an increased number of label-retaining cells in vivo. The enhanced proliferation potential of mutant cells was retained in vitro in neurosphere cultures. We observed a higher sphere-forming capacity as well as faster growth and cell cycle progression. Use of specific inhibitors revealed that these effects were independent of TGF-␤ and BMP signaling. The enhanced proliferation might be at least partially mediated by elevated signaling via epidermal growth factor (EGF) receptor, as mutant cells showed higher expression and activation levels of the EGF receptor. Conversely, an EGF receptor inhibitor reduced the proliferation of these cells. Our data indicate that endogenous Smad7 regulates neural stem/ progenitor cell proliferation in a TGF-␤-and BMP-independent manner.The transforming growth factor ␤ (TGF-␤) superfamily of cytokines, consisting of TGF-␤s, activins, and bone morphogenetic proteins (BMPs), participates in the regulation of a multitude of cellular processes, such as proliferation, apoptosis, differentiation, and extracellular matrix production. In consequence, TGF-␤ has important functions in embryonic development as well as in the adult organism (reviewed in references 22 and 26).Recently, it was shown that TGF-␤ is involved in the regulation of adult neural stem cells located in two neurogenic niches, the dentate gyrus of the hippocampus (HC) and the subventricular zone (SVZ) of the lateral ventricles (reviewed in references 1 and 2). Treating adult neural stem/progenitor cells in culture, we have been able to demonstrate that TGF-␤ strongly suppresses proliferation of these cells. Moreover, intracerebroventricular infusion of TGF-␤ reduced the proliferation of neural progenitor cells in vivo (33). Similar results were reported in a transgenic approach involving overexpression of TGF-␤ in astrocytes (5).The cellular functions of TGF-␤ are mediated via ligandinduced hetero-oligomerization of type I (T␤RI) and type II (T␤RII) serine/threonine kinase receptors and subsequent phosphorylation of T␤RI by the constitutively active T␤RII. Receptor-regulated Smad proteins (R-Smads), that is, Smad2 and -3 for TGF-␤ and activin and Smad1, -5, and -8 for BMPs, are phosphorylated by T␤RI and heterotrimerize with the coSmad, Smad4. This complex then translocates to the nucleus, where it regulates gene transcription in association with coactivators or repressors (reviewed in references 27, 30, and 31). Negative feedback regulation of TGF-␤ signaling i...