Axel Pagenstecher

A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells

Cavernous vascular malformations occur with a frequency of 1:200 and can cause recurrent headaches, seizures and hemorrhagic stroke if located in the brain. Familial cerebral cavernous malformations (CCMs) have been associated with germline mutations in CCM1/KRIT1, CCM2 or CCM3/PDCD10. For each of the three CCM genes, we here show complete localized loss of either CCM1, CCM2 or CCM3 protein expression depending on the inherited mutation. Cavernous but not adjacent normal or reactive endothelial cells of known germline mutation carriers displayed immunohistochemical negativity only for the corresponding CCM protein but not for the two others. In addition to proving loss of function at the protein level, our data are the first to demonstrate endothelial cell mosaicism within cavernous tissues and provide clear pathogenetic evidence that the endothelial cell is the cell of disease origin.

IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis

IL-17-producing CD8+ T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8+ T cells and subsequent immunization for EAE induction in these mice, the CD8+ T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4+ T cells alone did not evoke EAE, but when transferred together with CD8+ T cells, IL-17-producing CD4+ (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8+ T cells, suggesting that Tc17 cells are required to promote CD4+ T cell-mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.

Late-Onset Chronic Inflammatory Encephalopathy in Immune-Competent and Severe Combined Immune-Deficient (SCID) Mice with Astrocyte-Targeted Expression of Tumor Necrosis Factor

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.

The TIMPs tango with MMPs and more in the central nervous system

The matrix metalloproteinases (MMPs) are a family of zinc‐dependent extracellular proteases that have been implicated in CNS development and disease. Crucial homeostatic regulation of MMPs is mediated through the expression and actions of the tissue inhibitors of metalloproteinases (TIMPs). Although the TIMPs are recognized inhibitors of the MMPs, recent studies have revealed that these proteins also can exhibit biological activities that are distinct from their interactions with or inhibition of the MMPs. With our understanding of the roles of the TIMPs in the CNS continuously emerging, this review examines the current state of knowledge regarding the multifarious and novel functions of this family of proteins, with particular attention to their increasing potential in the development, plasticity, and pathology of the CNS.

Malignant pineal parenchymal tumors in adult patients : Patterns of care and prognostic factors

OBJECTIVE The aim of our study was to analyze patterns of care and to identify prognostic factors in patients at least 18 years of age who received radiotherapy for malignant pineal parenchymal tumors. METHODS In a multicenter, retrospective study, we analyzed data for 37 previously published cases and 64 patients treated at the participating institutions. RESULTS A total of 56 patients received postoperative radiotherapy, and 45 patients received primary radiotherapy. Chemotherapy was administered to 34 patients. The median follow-up period was 38 months, and median overall survival was 100 months. The variables that significantly influenced overall survival were the extent of disease (localized versus disseminated;P = 0.0002), differentiation (pineal parenchymal tumor of intermediate differentiation versus pineoblastoma;P = 0.001), and residual disease (≥50% versus <50% reduction in size;P < 0.0001). In a multivariate analysis, the parameters turned out to be independent risk factors. The median survival in patients with local or spinal failure was 15 months. Local control was better in older patients (≥32 yr versus <32 yr;P = 0.02). Spinal control was more successful in patients with pineal parenchymal tumors of intermediate differentiation than it was in patients with pineoblastomas (P = 0.03). Nine of 45 treatment failures occurred later than 5 years after treatment. CONCLUSION Stage, histological characteristics, and response are independent risk factors in adults with malignant pineal parenchymal tumors. Late relapses are common.

Metalloproteinase Disintegrins ADAM8 and ADAM19 Are Highly Regulated in Human Primary Brain Tumors and their Expression Levels and Activities Are Associated with Invasiveness

Patients with primary brain tumors have bleak prognoses and there is an urgent desire to identify new markers for sensitive diagnosis and new therapeutic targets for effective treatment. A family of proteins, the disintegrin and metalloproteinases (ADAMs or adamalysins), are cell surface and extracellular multidomain proteins implicated in cell-cell signaling, cell adhesion, and cell migration. Their putative biological and pathological roles make them candidates for promoting tumor growth and malignancy. We investigated the expression levels of 12 cerebrally expressed ADAM genes in human primary brain tumors (astrocytoma WHO grade I-III, glioblastoma WHO grade IV, oligoastrocytoma WHO grade II and III, oligodendroglioma WHO grade II and III, ependymoma WHO grade II and III, and primitive neuroectodermal tumor WHO grade IV) using real-time PCR. The mRNAs of the five ADAMs 8, 12, 15, 17, and 19 were significantly upregulated. The ADAM8 and ADAM19 proteins were mainly located in tumor cells and in some tumors in endothelia of blood vessels. In brain tumor tissue, ADAM8 and ADAM19 undergo activation by prodomain removal resulting in active proteases. By using specific peptide substrates for ADAM8 and ADAM19, respectively, we demonstrated that the proteases exert enhanced proteolytic activity in those tumor specimens with the highest expression levels. In addition, expression levels and the protease activities of ADAM8 and ADAM19 correlated with invasive activity of glioma cells, indicating that ADAM8 and ADAM19 may play a significant role in tumor invasion that may be detrimental to patients survival.

Nuclear Factor I X Deficiency Causes Brain Malformation and Severe Skeletal Defects

ABSTRACT The transcription factor family of nuclear factor I (NFI) proteins is encoded by four closely related genes: Nfia, Nfib, Nfic, and Nfix. A potential role for NFI proteins in regulating developmental processes has been implicated by their specific expression pattern during embryonic development and by analysis of NFI-deficient mice. It was shown that loss of NFIA results in hydrocephalus and agenesis of the corpus callosum and that NFIB deficiency leads to neurological defects and to severe lung hypoplasia, whereas Nfic knockout mice exhibit specific tooth defects. Here we report the knockout analysis of the fourth and last member of this gene family, Nfix. Loss of NFIX is postnatally lethal and leads to hydrocephalus and to a partial agenesis of the corpus callosum. Furthermore, NFIX-deficient mice develop a deformation of the spine, which is due to a delay in ossification of vertebral bodies and a progressive degeneration of intervertebral disks. Impaired endochondral ossification and decreased mineralization were also observed in femoral sections of Nfix−/− mice. Consistent with the defects in bone ossification we could show that the expression level of tetranectin, a plasminogen-binding protein involved in mineralization, is specifically downregulated in bones of NFIX-deficient mice.

C10 Is a Novel Chemokine Expressed in Experimental Inflammatory Demyelinating Disorders that Promotes Recruitment of Macrophages to the Central Nervous System

Chemokines may be important in the control of leukocytosis in inflammatory disorders of the central nervous system. We studied cerebral chemokine expression during the evolution of diverse neuroinflammatory disorders in transgenic mice with astrocyte glial fibrillary acidic protein-targeted expression of the cytokines IL-3, IL-6, or IFN-alpha and in mice with experimental autoimmune encephalomyelitis. Distinct chemokine gene expression patterns were observed in the different central nervous system inflammatory models that may determine the phenotype and perhaps the functions of the leukocytes that traffic into the brain. Notably, high expression of C10 and C10-related genes was found in the cerebellum and spinal cord of GFAP-IL3 mice with inflammatory demyelinating disease and in mice with experimental autoimmune encephalomyelitis. In both these neuroinflammatory models, C10 RNA and protein expressing cells were predominantly macrophage/microglia and foamy macrophages present within demyelinating lesions as well as in perivascular infiltrates and meninges. Intracerebroventricular injection of recombinant C10 protein promoted the recruitment of large numbers of Mac-1(+) cells and, to a much lesser extent, CD4(+) lymphocytes into the meninges, choroid plexus, ventricles, and parenchyma of the brain. Thus, C10 is a prominent chemokine expressed in the central nervous system in experimental inflammatory demyelinating disease that, we show, also acts as a potent chemotactic factor for the migration of these leukocytes to the brain.

Stereotactic management of lesions of the pineal region.

OBJECTIVE The relevance of the computed tomography-guided stereotactic approach for the management of lesions of the pineal region is analyzed. METHODS In a retrospective analysis conducted between 1985 and 1993, the risk profile, the diagnostic accuracy, and the therapeutic relevance of the stereotactic approach in 106 patients was studied. Survival analysis was used to assess the reliability of the stereotactically obtained diagnosis in terms of follow-up observation. RESULTS A histological diagnosis was obtained in 103 of the 106 patients. In three patients, a conclusive diagnosis could not be established because of intraoperative complications. One lesion was misdiagnosed as a pineocytoma instead of a pineoblastoma. Two of the 106 patients died; 9 patients experienced perioperative morbidity. In 38 patients, the stereotactic approach was also useful for therapy. Cyst aspiration and/or internal drainage was performed in 18 patients with symptomatic cystic lesions, and radiosurgical treatment with use of interstitial 125iodine was performed in 16 patients with low-grade tumors and in 4 patients with solitary metastases. In 12 patients, the obtained tissue diagnosis was the basis for deferring additional therapy. In 43 patients with germ-cell tumors, pineoblastomas, or malignant gliomas, a stereotactic biopsy was the starting point for additional radiotherapy/chemotherapy. Open tumor resection played a minor role (five patients). CONCLUSION The stereotactic approach to the pineal region is a relatively safe procedure in experienced hands. The diagnosis obtained by computed tomography-guided stereotactic biopsy is a valid basis for treatment decisions. Long-term follow-up observation of the benign lesions is necessary for a definite confirmation of diagnostic accuracy.

Astrocyte-Targeted Expression of IL-12 Induces Active Cellular Immune Responses in the Central Nervous System and Modulates Experimental Allergic Encephalomyelitis

The role of IL-12 in the evolution of immunoinflammatory responses at a localized tissue level was investigated. Transgenic mice were developed with expression of either both the IL-12 subunits (p35 and p40) or only the IL-12 p40 subunit genes targeted to astrocytes in the mouse CNS. Glial fibrillary acidic protein (GF)-IL-12 mice, bigenic for the p35 and p40 genes, developed neurologic disease which correlated with the levels and sites of transgene-encoded IL-12 expression. In these mice, the brain contained numerous perivascular and parenchymal inflammatory lesions consisting of predominantly CD4+ and CD8+ T cells as well as NK cells. The majority of the infiltrating T cells had an activated phenotype (CD44high, CD45Rblow, CD62Llow, CD69high, VLA-4 high, and CD25+). Functional activation of the cellular immune response was also evident with marked cerebral expression of the IFN-γ, TNF, and IL-1αβ genes. Concomitant with leukocyte infiltration, the CNS expression of immune accessory molecules was induced or up-regulated, including ICAM-1, VCAM-1, and MHC class II and B7-2. Glial fibrillary acidic protein-p40 mice with expression of IL-12 p40 alone remained asymptomatic, with no inflammation evident at any age studied. The effect of local CNS production of IL-12 in the development of experimental autoimmune encephalomyelitis was studied. After immunization with myelin oligodendrocyte glycoprotein-peptides, GF-IL-12 mice had an earlier onset and higher incidence but not more severe disease. We conclude that localized expression of IL-12 by astrocytes can 1) promote the spontaneous development of activated type 1 T cell and NK cellular immunity and cytokine responses in the CNS, and 2) promote more effective Ag-specific T cell dynamics but not activity in experimental autoimmune encephalomyelitis.