Proteometabolomics of initial and recurrent glioblastoma highlights an increased immune cell signature with altered lipid metabolism.

BACKGROUND: There is an urgent need to better understand the mechanisms associated with the development, progression, and onset of recurrence after initial surgery in glioblastoma (GBM). The use of integrative phenotype-focused -omics technologies such as proteomics and lipidomics provides an unbiased approach to explore the molecular evolution of the tumor and its associated environment. METHODS: We assembled a cohort of patient-matched initial (iGBM) and recurrent (rGBM) specimens of resected GBM. Proteome and metabolome composition were determined by mass spectrometry-based techniques. We performed neutrophil-GBM cell coculture experiments to evaluate the behavior of rGBM-enriched proteins in the tumor microenvironment. ELISA-based quantitation of candidate proteins was performed to test the association of their plasma concentrations in iGBM with the onset of recurrence. RESULTS: Proteomic profiles reflect increased immune cell infiltration and extracellular matrix reorganization in rGBM. ASAH1, SYMN, and GPNMB were highly enriched proteins in rGBM. Lipidomics indicates the downregulation of ceramides in rGBM. Cell analyses suggest a role for ASAH1 in neutrophils and its localization in extracellular traps. Plasma concentrations of ASAH1 and SYNM show an association with time to recurrence. CONCLUSIONS: We describe the potential importance of ASAH1 in tumor progression and development of rGBM via metabolic rearrangement and showcase the feedback from the tumor microenvironment to plasma proteome profiles. We report the potential of ASAH1 and SYNM as plasma markers of rGBM progression. The published datasets can be considered as a resource for further functional and biomarker studies involving additional -omics technologies.

Identification of Dysregulated microRNAs in Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

Alternative splicing of the TNFSF13B (BAFF) pre-mRNA and expression of the BAFFX1 isoform in human immune cells.

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.

Levetiracetam but not valproate inhibits function of CD8+ T lymphocytes.

PURPOSE: To further elucidate possible immune-modulatory effects of valproate (VPA) or levetiracetam (LEV), we investigated their influence on apoptosis and cytotoxic function of CD8+ T lymphocytes in humans. METHODS: In 15 healthy subjects (9 female (60%), 35.7±12.1 years), apoptosis and cytotoxic function of CD8+ T lymphocytes were measured using flow cytometry following in vitro exposure to LEV (5 mg/L and 50 mg/L) and VPA (10mg/L and 100 mg/L). Apoptosis rates were determined after incubation with LEV or VPA for 1 h or 24 h. Cytotoxic function was assessed following 2h stimulation with mixed virus peptides, using perforin release, CD107a/b expression and proliferation. The presence of synaptic vesicle protein 2A (SV2A) was investigated in human CD8+ T lymphocytes by flow cytometry analysis, Western blot and real time polymerase chain reaction (rtPCR). RESULTS: High concentration of LEV decreased perforin release of CD8+ T lymphocytes (LEV 50 mg/L vs. CEF only: 21.4% (interquartile range (IQR) 16.5-35.9%) vs. 16.6% (IQR 12-24.9%), p=0.002). LEV had no influence on apoptosis and proliferation (p>0.05). VPA (100 mg/L) slowed apoptosis of CD8() T lymphocytes after 24h (VPA 100mg/L vs. control: 7.3% (IQR 5.4-9.5%) vs. 11.3% (IQR 8.2-15.1%), p<0.001), but had no effects on perforin release (p>0.05). SV2A protein was detected in CD8+ T lymphocytes. CONCLUSION: LEV decreased degranulation of CD8+ T lymphocytes which may contribute to the increased incidence of upper respiratory tract infections in LEV treated patients. Inhibition of SV2A may be responsible for this effect.

Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis.

MS is a chronic inflammatory and demyelinating disease of the CNS with as yet unknown etiology. A hallmark of this disease is the occurrence of oligoclonal IgG antibodies in the cerebrospinal fluid (CSF). To assess the specificity of these antibodies, we screened protein expression arrays containing 37,000 tagged proteins. The 2 most frequent MS-specific reactivities were further mapped to identify the underlying high-affinity epitopes. In both cases, we identified peptide sequences derived from EBV proteins expressed in latently infected cells. Immunoreactivities to these EBV proteins, BRRF2 and EBNA-1, were significantly higher in the serum and CSF of MS patients than in those of control donors. Oligoclonal CSF IgG from MS patients specifically bound both EBV proteins. Also, CD8(+) T cell responses to latent EBV proteins were higher in MS patients than in controls. In summary, these findings demonstrate an increased immune response to EBV in MS patients, which suggests that the virus plays an important role in the pathogenesis of disease.

The role of the polio virus receptor and the herpesvirus entry mediator B genes for the development of MS.

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS). Although the cause of MS is still uncertain, it is well accepted that both genetic and environmental factors are important for the development of disease. In this study, we focused on the Polio Virus Receptor (PVR) and Herpesvirus entry mediator B (HVEB) receptor genes, which are located on chromosome 19q13, a region previously linked to MS. Both receptors are expressed in the brain and immune system and play an important role for inter-cellular adhesion and entry of neurotropic viruses to the brain. We identified four new polymorphisms in the PVR gene, which were located in the promoter region and three different exons. All exonic polymorphisms altered the amino acid sequence of the receptor. No new polymorphisms were found in the HVEB gene, but we confirmed a previously identified intronic polymorphism. We analyzed the frequency of the polymorphisms by RFLP analysis in sporadic MS patients, MS families, and healthy controls and determined the surface expression of HVEB and PVR on peripheral blood monocytes. We did not find differences in the frequency of the polymorphisms or surface expression between MS patients and controls. Overall, our findings do not support a role of HVEB and PVR genes in the development of MS.

Clonal accumulation of activated CD8+ T cells in the central nervous system during the early phase of neuroborreliosis.

Borrelia burgdorferi may cause an acute infection of the central nervous system (CNS) that rarely leads to chronic disease. To characterize host immunity to B. burgdorferi in humans, we performed serial T cell receptor (TCR) variable beta (TCRBV) chain analyses in blood and cerebrospinal fluid (CSF) samples from 10 patients with acute neuroborreliosis. In most patients, we found significant differences in TCRBV expression between CSF and peripheral blood T cells, predominantly involving CD8(+) T cells. T cells that accumulated in the CSF had a memory phenotype and expressed high levels of C-C chemokine receptor 5 and CD69. Serial studies demonstrated that CD8(+) T cell accumulation decreased continuously after resolution of the infection. In 2 patients, serial analysis of the TCR-alpha and -beta chain sequences revealed that overexpression of TCRBV in CSF was caused by extensive clonal expansion of CD8(+) T cells. Our findings support the role of CD8(+) T cells during the early host defense against spirochete infection of the CNS.

A novel mutation in PTPRC interferes with splicing and alters the structure of the human CD45 molecule.

CD45, encoded by the protein tyrosine phosphatase receptor type C ( PTPRC) gene, is essentially involved in maturation, activation, and migration of immune cells. Lack of CD45 results in severe immunodeficiency, and alterations of the receptor may result in autoimmunity. Here, we describe a novel mutation in PTPRCas a cause of variant CD45 expression in humans. Several members of a multiple sclerosis multiplex family showed expression of CD45RA on memory T cells and monocytes. The variant expression pattern was linked to the PTPRCgene by DNA microsatellite studies. DNA analysis identified a novel point mutation in exon 4 (position 59 C-->A) in all family members with variant CD45 expression, but not in donors with normal CD45 expression. The mutation interferes with alternative splicing and alters amino acid sequence (H-->Q), interfering with antibody binding to the CD45RA domain. Overall, we describe the first mutation in PTPRCthat interferes with splicing and results in surface expression of a structurally altered CD45 molecule in humans.