[Show abstract][Hide abstract] ABSTRACT: In mature skeletal muscle, the intracellular Ca2+ concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca2+ release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1’s potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca2+ signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca2+ signaling during muscle organ development.
Full-text · Article · Feb 2016 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GBM) is treated by surgical resection followed by radiochemotherapy. Bevacizumab is commonly deployed for anti-angiogenic therapy of recurrent GBM; however, innate immune cells have been identified as instigators of resistance to bevacizumab treatment. We identified angiopoietin-2 (Ang-2) as a potential target in both naive and bevacizumab-treated glioblastoma. Ang-2 expression was absent in normal human brain endothelium, while the highest Ang-2 levels were observed in bevacizumab-treated GBM. In a murine GBM model, VEGF blockade resulted in endothelial upregulation of Ang-2, whereas the combined inhibition of VEGF and Ang-2 leads to extended survival, decreased vascular permeability, depletion of tumor-associated macrophages, improved pericyte coverage, and increased numbers of intratumoral T lymphocytes. CD206(+) (M2-like) macrophages were identified as potential novel targets following anti-angiogenic therapy. Our findings imply a novel role for endothelial cells in therapy resistance and identify endothelial cell/myeloid cell crosstalk mediated by Ang-2 as a potential resistance mechanism. Therefore, combining VEGF blockade with inhibition of Ang-2 may potentially overcome resistance to bevacizumab therapy.
Full-text · Article · Dec 2015 · EMBO Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: The immunoregulatory cytokine IL-10 suppresses T-cell immunity. The complementary question, whether IL-10 is also involved in limiting the collateral damage of vigorous T cell responses, has not been addressed in detail. Here, we report that the particularly strong virus-specific immune response during acute primary infection with the lymphocytic choriomeningitis virus (LCMV) in mice is significantly further increased in Il10-deficient mice, particularly regarding frequencies and cytotoxic activity of CD8(+) T cells. This increase results in exacerbating immunopathology in select organs, ranging from transient local swelling to an increased risk for mortality. Remarkably, LCMV-induced, T cell-mediated hepatitis is not affected by endogenous Il10. The alleviating effect of Il10 on LCMV-induced immunopathology was found to be operative in delayed-type hypersensitivity footpad-swelling reaction and in debilitating meningitis in mice of both the C57BL/6 and BALB/c strains. These strains are prototypic counterpoles for genetically imprinted type 1-biased versus type 2-biased T cell-mediated immune responses against various infectious pathogens. However, during acute LCMV infection, neither systemic cytokine patterns nor the impact of Il10 on LCMV-induced immunopathology differed conspicuously between these two strains of mice. This study documents a physiological role of Il10 in the regulation of a balanced T-cell response limiting immunopathological damage.
Preview · Article · Nov 2015 · American Journal Of Pathology
[Show abstract][Hide abstract] ABSTRACT: We evaluated the treatment of oligodendroglial brain tumors with interstitial brachytherapy (IBT) using (125)iodine seeds ((125)I) and analyzed prognostic factors.
Between January 1991 and December 2010, 63 patients (median age 43.3 years, range 20.8-63.4 years) suffering from oligodendroglial brain tumors were treated with (125)I IBT either as primary, adjuvantly after incomplete resection, or as salvage therapy after tumor recurrence. Possible prognostic factors influencing disease progression and survival were retrospectively investigated.
The actuarial 2-, 5-, and 10-year overall and progression-free survival rates after IBT for WHO II tumors were 96.9, 96.9, 89.8 % and 96.9, 93.8, 47.3 %; for WHO III tumors 90.3, 77, 54.9 % and 80.6, 58.4, 45.9 %, respectively. Magnetic resonance imaging demonstrated complete remission in 2 patients, partial remission in 13 patients, stable disease in 17 patients and tumor progression in 31 patients. Median time to progression for WHO II tumors was 87.6 months and for WHO III tumors 27.8 months. Neurological status improved in 10 patients and remained stable in 20 patients, while 9 patients deteriorated. There was no treatment-related mortality. Treatment-related morbidity was transient in 11 patients. WHO II, KPS ≥ 90 %, frontal location, and tumor surface dose > 50 Gy were associated with increased overall survival (p ≤ 0.05). Oligodendroglioma and frontal location were associated with a prolonged progression-free survival (p ≤ 0.05).
Our study indicates that IBT achieves local control rates comparable to surgery and radio-/chemotherapy treatment, is minimally invasive, and safe. Due to the low rate of side effects, IBT may represent an attractive option as part of a multimodal treatment schedule, being supplementary to microsurgery or as a salvage therapy after chemotherapy and conventional irradiation.
No preview · Article · Aug 2015 · Strahlentherapie und Onkologie
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) contribute to immune homeostasis under physiological conditions and regulate the immune activation during infection. The deubiquitinase A20 inhibits the activation of NF-κB-dependent immune reactions, and prevents the hyperactivation of DCs under steady-state conditions. However, the role of DC-specific A20 under pathological conditions is unknown. Here, we demonstrate that upon injection of low-dose LPS, mice with DC-specific A20 deletion (CD11c-Cre A20(fl/fl) ) died within 6 hours, whereas A20(fl/fl) controls survived. LPS-induced mortality in CD11c-Cre A20(fl/fl) mice was characterised by increased serum levels of IL-2, IL-10, IL-12, IFN-γ, and TNF. Upon LPS stimulation, the activation of NF-κB and ERK-NFATc3 pathways were enhanced in A20-deficient DCs, resulting in an increased production of IL-2, IL-12, and TNF both in vitro and in vivo. Targeted inhibition of ERK in A20-deficient DCs abolished the increased production of IL-2. A20-deficient DCs failed to induce LPS tolerance, which was independent of T cells and the intestinal flora, since T-cell depletion and decolonisation of CD11c-Cre A20(fl/fl) mice could not prevent death of LPS-challenged CD11c-Cre A20(fl/fl) mice. In conclusion, these findings show that DC-specific A20 preserves immune homeostasis in steady-state conditions and is also required for LPS tolerance. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Full-text · Article · Mar 2015 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Primary lymphoma of the central nervous system (PCNSL) is a diffuse large B-cell lymphoma confined to the CNS. It has been hypothesized that antigen(s) in the CNS may trigger tumor cell proliferation. Because efforts to identify potential antigens have been unsuccessful to date, we studied the B-cell receptor in detail in a comprehensive series of 50 PCNSLs to obtain indirect information on potential antigens. Potentially functional V-D-J rearrangements were identified in all PCNSLs analyzed. Immunoglobulin heavy-chain variable gene segment (IGHV), IGHV4, was the predominant family used by 66% (33 of 50) of PCNSLs with a preferential rearrangement of the IGHV4-34 gene segment (18 [55%] of 33). The IGHV genes showed mutation frequencies from 0% to 29%, with a high average mutation frequency of 10%. In addition to 48% (24 of 50) of PCNSLs being highly mutated, 22% (11 of 50) defined a low-level mutated group. Antigen selection of the tumor cells or their precursors was indicated by replacement/silent mutation ratios and ongoing somatic hypermutation. Complementarity determining region 3 length and composition as well as the lack of stereotyped B-cell receptors suggest involvement of several antigens instead of a unique antigen recognized by the tumor cells.
No preview · Article · Nov 2014 · Journal of Neuropathology and Experimental Neurology
[Show abstract][Hide abstract] ABSTRACT: To decipher the mutational pattern of primary CNS lymphoma (PCNSL) we performed whole exome-sequencing to a median coverage of 103x followed by mutation verification in 9 PCNSL and validation using Sanger sequencing in 22 PCNSL. We identified a median of 202 (range: 139-251) potentially somatic single nucleotide variants (SNV) and 14 small indels (range: 7-22) with potentially protein-changing features per PCNSL. Mutations affected the B cell receptor-, TLR-, and NF-κB-and genes involved in chromatin structure and modifications, cell cycle regulation or immune recognition. A median of 22.2% (range: 20.0%-24.7%) of somatic SNVs in the 9 PCNSL overlap with the RGYW motif targeted by somatic hypermutation (SHM); a median of 7.9% (range: 6.2%-12.6%) affect its hotspot position suggesting a major impact of SHM on PCNSL pathogenesis. In addition to well-known targets of aberrant SHM (PIM1) our data suggest new targets of aberrant SHM (KLHL14, OSBPL10, SUSD2). Among the four most frequently mutated genes was ODZ4 showing protein-changing mutations in 4/9 PCNSL. Together with mutations affecting CSMD2, CSMD3, and PTPRD these findings may suggest that alterations in genes playing a role in CNS development may facilitate DLBCL manifestation in the CNS. This may point to intriguing mechanisms of CNS tropism in PCNSL.Leukemia accepted article preview online, 05 September 2014. doi:10.1038/leu.2014.264.
[Show abstract][Hide abstract] ABSTRACT: The role of the type 2 helper T cell (Th2)-polarizing cytokines IL-4 and IL-10 has not yet been studied in P0106-125-induced murine experimental autoimmune neuritis (EAN). We, therefore, addressed the functional relevance of these cytokines and signaling via the IL-4-associated transcription factor STAT6. The clinical course of P0106-125-induced EAN in mice deficient for IL-10(0/0), IL-4(0/0), or STAT6(0/0) was significantly aggravated compared with that of wild-type control mice. In addition, treatment of P0106-125-immunized C57BL/6 mice at the onset of clinical symptoms with a monoclonal IL-10 neutralizing antibody aggravated symptoms and prolonged disease to a similar degree as in IL-10(0/0) mice. This exacerbated course was attributed to a more prominent Th1 immune response associated with a persistent M1 milieu in the sciatic nerve and in the regional and systemic lymphatic system. These data suggest a Th2-polarized milieu being required to prevent axonal damage of the sciatic nerve and to terminate the P0106-125-specific immune response in EAN. Beyond the already known role of macrophages as pathogenic effector cells in EAN, these data suggest that M2-differentiated macrophages do not damage and may even protect neural tissues in EAN. Thus, these data highlight the pathogenetic relevance of the macrophage polarization status in EAN. Therapeutic modulation of immune responses from an M1 toward an M2 milieu may be a promising novel strategy in peripheral nervous system neuritis.
No preview · Article · Aug 2014 · American Journal Of Pathology
[Show abstract][Hide abstract] ABSTRACT: Myelin protein 0 peptide 106-125-induced murine experimental autoimmune neuritis (EAN) is a CD4-positive T cell-mediated monophasic axonal inflammatory neuropathy; interferon-γ is the key proinflammatory mediator. Experimental autoimmune neuritis is well suited for elucidating pathogenetic mechanisms underlying human acute axonal Guillain-Barré syndrome. Here, the functional role of the costimulatory molecule CD40 was defined by characterization of EAN in CD40-deficient mice. In contrast to immunized C57BL/6 mice, CD40-deficient mice were resistant to EAN owing to impaired priming of CD4-positive T-effector cells. To determine whether CD40 is a suitable candidate for the treatment of EAN, we administered monoclonal anti-CD40 antibody either before immunization or upon onset of neurologic signs. Prophylactic anti-CD40 treatment completely abolished CD4-positive T-cell priming. Therapeutic application of anti-CD40 prevented full activation of CD4-positive T cells that were in the process of priming and suppressed production of interferon-γ in peripheral lymph nodes, spleen, and serum, and of interleukin-6, interleukin-12p40, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, which are associated with activation of the nuclear factor-κB signaling pathway. This resulted in enhanced recovery by early generation of CD25-positive, Foxp3-positive, CD4-positive regulatory T cells. Thus, these experiments highlight the crucial role of CD40 as an important costimulatory molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis.
[Show abstract][Hide abstract] ABSTRACT: Primary lymphoma of the central nervous system (CNS, PCNSL) is a specific diffuse large B cell lymphoma entity arising in and confined to the CNS. Despite extensive research since many decades, the pathogenetic mechanisms underlying the remarkable tropism of this peculiar malignant hematopoietic tumor remain still to be elucidated. In the present review, we summarize the present knowledge on the genotypic and phenotypic characteristics of the tumor cells of PCNSL, give an overview over deregulated molecular pathways in PCNSL and present recent progress in the field of preclinical modeling of PCNSL in mice. With regard to the phenotype, PCNSL cells resemble late germinal center exit IgM+IgD+ B cells with blocked terminal B cell differentiation. They show continued BCL6 activity in line with ongoing activity of the germinal center program. This together with the pathways deregulated by genetic alterations may foster B cell activation and brisk proliferation, which correlated with the simultaneous MYC and BCL2 overexpression characteristic for PCNSL. On the genetic level, PCNSL are characterized by ongoing aberrant somatic hypermutation that, besides the IG locus, targets the PAX5, TTF, MYC, and PIM1 genes. Moreover, PCNSL cells show impaired IG class switch due to sμ region deletions, and PRDM1 mutations. Several important pathways, i.e., the B cell receptor (BCR), the toll-like receptor, and the nuclear factor-κB pathway, are activated frequently due to genetic changes affecting genes like CD79B, SHIP, CBL, BLNK, CARD11, MALT1, BCL2, and MYD88. These changes likely foster tumor cell survival. Nevertheless, many of these features are also present in subsets of systemic DLBLC and might not be the only reasons for the peculiar tropism of PCNSL. Here, preclinical animal models that closely mimic the clinical course and neuropathology of human PCNSL may provide further insight and we discuss recent advances in this field. Such models enable us to understand the pathogenetic interaction between the malignant B cells, resident cell populations of the CNS, and the associated inflammatory infiltrate. Indeed, the immunophenotype of the CNS as well as tumor cell characteristics and intracerebral interactions may create a micromilieu particularly conducive to PCNSL that may foster aggressiveness of tumor cells and accelerate the fatal course of disease. Suitable animal models may also serve as a well-defined preclinical system and may provide a useful tool for developing new specific therapeutic strategies.
No preview · Article · Nov 2013 · Acta Neuropathologica
[Show abstract][Hide abstract] ABSTRACT: Single-nucleotide polymorphisms in the tumor necrosis factor, alpha-induced protein 3 gene, which encodes the ubiquitin-modifying protein A20, are linked to susceptibility to multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS). Since it is unresolved how A20 regulates MS pathogenesis, we examined its function in a murine model of MS, namely experimental autoimmune encephalomyelitis (EAE). Deletion of A20 in neuroectodermal cells (astrocytes, neurons, and oligodendrocytes; Nestin-Cre A20(fl/fl) mice) or selectively in astrocytes (GFAP-Cre A20(fl/fl) mice) resulted in more severe EAE as compared to control animals. In Nestin-Cre A20(fl/fl) and GFAP-Cre A20(fl/fl) mice demyelination and recruitment of inflammatory leukocytes were increased as compared to A20(fl/fl) control mice. Importantly, numbers of encephalitogenic CD4(+) T cells producing interferon (IFN)-γ, interleukin (IL)-17, and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively, as well as mRNA production of IFN-γ, IL-17, tumor necrosis factor (TNF), GM-CSF, IL-6, CXCL1, CCL2, and CXCL10 were significantly increased in spinal cords of Nestin-Cre A20(fl/fl) and GFAP-Cre A20(fl/fl) mice, respectively. Compared to A20-sufficient astrocytes, A20-deficient astrocytes displayed stronger activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in response to TNF, IL-17, and GM-CSF, and of signal transducer and activator of transcription 1 (STAT1) upon IFN-γ stimulation. Due to NF-κB and STAT1 hyperactivation, A20-deficient astrocytes produced significantly more chemokines in response to these key encephalitogenic cytokines of autoimmune CD4(+) T cells resulting in an amplification of CD4(+) T cell recruitment to the CNS. Thus, astrocytic A20 is an important inhibitor of autoimmune-mediated demyelination in the CNS.
No preview · Article · Sep 2013 · Acta Neuropathologica
[Show abstract][Hide abstract] ABSTRACT: The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld(-/-) mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld(-/-) mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.
[Show abstract][Hide abstract] ABSTRACT: Primary lymphoma of the central nervous system (PCNSL) is defined as lymphoma of the diffuse large B-cell type confined to the CNS. To understand the effects of the CNS microenvironment on the malignant B cells and their interactions with the cells of the target organ, we analyzed a syngeneic mouse model. Transplantation of BAL17 cells into the frontal white matter of syngeneic BALB/c mice induced lymphomas with major clinical and neuropathologic features that parallel those of human PCNSL, including an angiocentric growth pattern in the brain parenchyma and tropism for the inner and outer ventricular system. Seven cycles of repeated isolation of lymphoma cells from the CNS and their intracerebral reimplantation induced genotypic and phenotypic alterations in resulting BAL17VII cells; the affected genes regulate apoptosis and are of the JAK/STAT pathway. Because lymphoma growth of BAL17VII cells was significantly accelerated, that is, shortening the time to death of the mice, these data indicate that prolonged stay of the lymphoma cells in the CNS was associated with worse outcome. These findings suggest that the CNS microenvironment fosters aggressiveness of lymphoma cells, thereby accelerating the lethal course of PCNSL.
[Show abstract][Hide abstract] ABSTRACT: The differential diagnosis of lymphoid lesions in the central nervous system covers a broad spectrum of neoplastic and inflammatory disorders. Complex cases benefit from the combined expertise in the fields of hematopoietic and neuroepithelial tumors as well as neuroimmunology. The Network Lymphomas and Lymphomatoid Lesions in the Nervous System (NLLLN) recommends performing a biopsy prior to any therapeutic intervention as a precise diagnosis was impossible in approximately 50 % of patients pretreated with corticosteroids. This is based on the analysis of approximately 1,000 cases in the past 4 years. In addition to total NLLLN experiences the characteristics, pathogenesis and differential diagnosis of primary lymphoma of the central nervous system are discussed.
[Show abstract][Hide abstract] ABSTRACT: Aim:
The role of chemokines and their receptors, which regulate trafficking and homing of leucocytes to inflamed organs in human or murine autoimmune neuritis, has not yet been elucidated in detail, Therefore, the role of the chemokine receptors CXCR4 and CXCR7 and their ligand CXCL12 was studied in autoimmune-mediated inflammation of the peripheral nervous system.
CXCL12/CXCR4 and/or CXCL12/CXCR7 interactions were specifically inhibited by the compounds AMD3100 or CCX771, respectively, in experimental autoimmune neuritis (EAN) of C57BL/6J mice immunized with P0106-125 peptide.
Disease activity was significantly suppressed by blocking CXCR7 while antagonization of CXCR4 enhanced disease activity. Enhanced disease activity was accompanied by significantly increased transcription of IFN-γ, IL-12 and TNF-α mRNA in regional lymph nodes and spleen as well as by increased serum levels of IFN-γ. Furthermore, by blocking CXCR4, expression of the cell adhesion molecules ICAM-1 and VCAM-1 was upregulated on vascular endothelial cells of the sciatic nerve, which coincided with significantly increased infiltration of the sciatic nerve by CD4+ T cells and macrophages. Remarkably, combined antagonization of both CXCR4 and CXCR7 significantly suppressed disease activity. This was accompanied by increased frequencies of activated and highly IFN-γ-expressing, P0106-125 -specific T cells in regional lymph nodes and spleen; however, these cells were unable to infiltrate the sciatic nerve.
These data suggest differential and hierarchically ordered roles for CXCR4/CXCL12- vs. CXCR7/CXCL12-dependent effects during EAN: CXCR7/CXCL12 interaction is a gatekeeper for pathogenic cells, regardless of their CXCR4/CXCL12-dependent state of activation.
No preview · Article · Mar 2013 · Neuropathology and Applied Neurobiology