Hrvoje Miletic

Both TLR2 and TLR4 are required for the effective immune response in Staphylococcus aureus-induced experimental murine brain abscess.

Toll-like receptors (TLRs) play central roles in the innate reaction to bacterial products and transmit specific immune responses against these pathogens. TLRs are expressed on numerous cell types, including innate immune cells, and on astrocytes, neurons, and microglial cells of the central nervous system (CNS). Lipoproteins and lipopolysaccharides are specifically recognized by TLR2 and TLR4, respectively. We examined the in vivo role of TLR2 and TLR4 in Staphylococcus aureus-induced brain abscess. Phenotypically, 87% of TLR2(-/-) mice and 43% of TLR4(-/-) mice died whereas all wild-type (WT) mice recovered. Clearance of bacteria from the CNS was significantly delayed in TLR2(-/-) mice compared with TLR4(-/-) and WT animals. Recruitment of granulocytes and macrophages to the CNS, as well as microglial activation and expansion, was up-regulated in TLR2(-/-) mice. Although inflammation persisted especially in the CNS of TLR2(-/-) mice, but also of TLR4(-/-) mice, WT mice terminated the infection more effectively. Collectively, these data show that the immune response to experimental S. aureus-induced brain abscess depends crucially on the recognition of S. aureus by TLR2 but that TLR4 is also required for an optimal intracerebral immune response in this disorder.

Protective Immunosurveillance of the Central Nervous System by Listeria-Specific CD4 and CD8 T Cells in Systemic Listeriosis in the Absence of Intracerebral Listeria

The invasion of the CNS by pathogens poses a major risk for damage of the highly vulnerable brain. The aim of the present study was to analyze immunological mechanisms that may prevent spread of infections to the CNS. Intraperitoneal application of Listeria monocytogenes to mice induced infection of the spleen, whereas pathogens remained absent from the brain. Interestingly, Listeria-specific CD4 and CD8 T cells homed to the brain and persisted intracerebrally for at least 50 days after both primary and secondary infection. CD4 and CD8 T cells resided in the leptomeninges, in the choroid plexus, and, in low numbers, in the brain parenchyma. CD4 and CD8 T cells isolated from the brain early after infection (day 7) were characterized by an activated phenotype with spontaneous IFN-γ production, whereas at a later stage of infection (day 28) restimulation with Listeria-specific peptides was required for the induction of IFN-γ production by CD4 and CD8 T cells. In contrast to splenic T cells, T cells in the brain did not exhibit cytotoxic activity. Adoptively transferred T cells isolated from the brains of Listeria-infected mice reduced the bacterial load in cerebral listeriosis. The frequency of intracerebral Listeria-specific T cells was partially regulated by the time of exposure to Listeria and cross-regulated by CD4 and CD8 T cells. Collectively, these data reveal a novel T cell-mediated pathway of active immunosurveillance of the CNS during bacterial infections.

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells

Early clinical trials for gene therapy of human gliomas with retroviral packaging cells (PC) have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor. In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus (LCMV) based on tumor‐infiltrating progenitor cells.

Regulation of the inflammatory response to Staphylococcus aureus-induced brain abscess by interleukin-10

A characteristic of brain abscess is a localized suppurative infection leading to substantial damage of the adjacent central nervous system tissue. The orchestrated interplay of pro- and antiinflammatory cytokines released by leukocytes as well as resident cells of the central nervous system is crucial for both an effective host defense and for limiting tissue damage in brain abscess. To study the regulatory role of interleukin (IL)-10 in brain abscess in vivo, IL-10-deficient (IL-100/0) mice were stereotaxically infected with Staphylococcus aureus-laden agarose beads. Increased numbers of intracerebral (IC) granulocytes, macrophages, CD4+ and CD8+ T cells, and higher levels of TNF, IL-1β, and iNOS were observed in IL-100/0 mice than in wild-type mice, whereas chemokines were induced earlier and more pronounced in wild-type mice. Together with prominent microvascular hemorrhage, necrotic vasculitis, severe brain edema, and markedly increased abscess size, these alterations led to an increased morbidity of IL-100/0 mice. Nevertheless, the hyperinflammatory response of IL-100/0 mice did not improve bacterial elimination. Collectively, these data outline the important role of IL-10 in vivo for the regulation of the IC host immune response in experimental S. aureus-induced brain abscess.

A 43-YEAR-OLD WOMAN WITH A TEMPORAL MASS

clinicAl history A 43-year-old woman was admitted with headache over a 3-week period. The clinical examination was completely unremarkable. The past medical history included breast cancer 3 years before presentation with a contralateral relapse another 2 years later. Treatment consisted of surgical resection, radiotherapy and post-operative chemotherapy with 4 cycles of epirubicine and cyclophosphamide. The relapse was treated by resection, radiotherapy and anti-estrogen therapy. Two years before current admission, acute myeloid leukemia FAB M4 Eo was diagnosed and treated with induction and consolidation chemotherapy according to the German AMLCG protocol with TAD/HAM double-induction and TAD consolidation chemotherapy followed by 4 weekly alternating maintenance chemotherapy. T-1 weighted MRI scans showed a hypointense mass in the left temporal lobe with little surrounding edema and compression of the left cerebral peduncle (Figure 1A). After intravenous Gadolinium application, the tumor exhibited homogenous contrast enhancement (Figure 1B). Metastasis and meningeoma were considered as differential diagnoses.

Diagnostic impact of ornithine decarboxylase in meningiomas

The objective of this study was to investigate whether activity and protein expression of ornithine decarboxylase (ODC) the metabolism-rate limiting enzyme of the polyamine biosynthesis, which is involved in the regulation of cellular growth and differentiation, reflects the distinct histopathologic characteristics indicative of atypia and anaplasia in meningiomas as well as their impact in recurrences. The authors previously evaluated its value as a critical factor of tumor development in various brain tumors. Among 152 meningiomas, World Health Organisations (WHO) grade I meningiomas (n = 121) exhibited a low ODC activity, while meningiomas WHO grade II (n = 22) and WHO grade III (n = 9), respectively, showed a significantly increased ODC activity. Recurrent WHO grade I meningiomas exhibited the same low enzyme activity and immunohistochemical staining index for ODC positive tumor cells as their primary tumors, whereas raising ODC activity and protein expression in recurrent meningiomas paralleled malignant transformation. These results indicate that ODC reflects aggressive growth and malignization in meningiomas. Especially in recurrent meningiomas, the determination of its activity and protein expression by immunohistochemistry may provide a useful diagnostic tool to recognize malignant progression.

977. Selective Transduction of Malignant Glioma by Lentiviral Vectors Pseudotyped with LCMV Glycoproteins

Malignant glioma are the most frequent primary brain tumors and have a dismal prognosis due to their infiltrative growth. Gene therapy using viral vectors represents an attractive alternative to conventional cancer therapies. In a previous study, we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus (LCMV) glycoproteins (GP) and demonstrated transduction of human malignant glioma cells in culture. In the current approach, we compared the transduction efficacy of LCMV GP- and vesicular stomatitis virus glycoprotein (VSV G)-pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo. LCMV GP pseudotypes transduced predominantly astrocytes in vivo and in vitro, whereas VSV G pseudotypes infected neurons as well as astrocytes. LCMV pseudotypes showed an efficient transduction of solid glioma parts and very specific transduction of infiltrating tumor cells. In contrast, VSV G-pseudotyped lentiviral vectors transduced only a few tumor cells in solid tumor parts and infected mostly normal brain cells in infiltrating tumor areas. In conclusion, lentiviral vectors pseudotyped with LCMV glycoproteins represent an attractive option for gene therapy of malignant glioma.

277. Variability in Infectivity of Primary Cell Cultures of Human Brain Tumors with HSV-1 Amplicon Vectors

Background and Purpose. The discrepancy between successful experimental gene therapy protocols and limited clinical translation might – among other reasons – be due to the variability in infectivity and transgene expression in tumors from different patients using vectors. We investigated the variability in infectivity of cells derived from primary brain tumor samples from different patients using HSV-1 amplicons.