Discussion on the relationship between gut microbiota and glioma through Mendelian randomization test based on the brain gut axis.

BACKGROUND: In the realm of Gut-Brain axis research, existing evidence points to a complex bidirectional regulatory mechanism between gut microbiota and the brain. However, the question of whether a causal relationship exists between gut microbiota and specific types of brain tumors, such as gliomas, remains unresolved. To address this gap, we employed publicly available Genome-Wide Association Study (GWAS) and MIOBEN databases, conducting an in-depth analysis using Two-Sample Mendelian Randomization (MR). METHOD: We carried out two sets of MR analyses. The preliminary analysis included fewer instrumental variables due to a high genome-wide statistical significance threshold (5×10-8). To enable a more comprehensive and detailed analysis, we adjusted the significance threshold to 1×10-5. We performed linkage disequilibrium analysis (R2 <0.001, clumping distance = 10,000kb) and detailed screening of palindromic SNPs, followed by MR analysis and validation through sensitivity analysis. RESULTS: Our findings reveal a causal relationship between gut microbiota and gliomas. Further confirmation via Inverse Variance Weighting (IVW) identified eight specific microbial communities related to gliomas. Notably, the Peptostreptococcaceae and Olsenella communities appear to have a protective effect, reducing glioma risk. CONCLUSION: This study not only confirms the causal link between gut microbiota and gliomas but also suggests a new avenue for future glioma treatment.

Glioma and temozolomide induced alterations in gut microbiome.

The gut microbiome is fundamental in neurogenesis processes. Alterations in microbial constituents promote inflammation and immunosuppression. Recently, in immune-oncology, specific microbial taxa have been described to enhance the effects of therapeutic modalities. However, the effects of microbial dysbiosis on glioma are still unknown. The aim of this study was to explore the effects of glioma development and Temozolomide (TMZ) on fecal microbiome in mice and humans. C57BL/6 mice were implanted with GL261/Sham and given TMZ/Saline. Fecal samples were collected longitudinally and analyzed by 16S rRNA sequencing. Fecal samples were collected from healthy controls as well as glioma patients at diagnosis, before and after chemoradiation. Compared to healthy controls, mice and glioma patients demonstrated significant differences in beta diversity, Firmicutes/Bacteroides (F/B) ratio, and increase of Verrucomicrobia phylum and Akkermansia genus. These changes were not observed following TMZ in mice. TMZ treatment in the non-tumor bearing mouse-model diminished the F/B ratio, increase Muribaculaceae family and decrease Ruminococcaceae family. Nevertheless, there were no changes in Verrucomicrobia/Akkermansia. Glioma development leads to gut dysbiosis in a mouse-model, which was not observed in the setting of TMZ. These findings seem translational to humans and warrant further study.

Gut microbiota alterations affect glioma growth and innate immune cells involved in tumor immunosurveillance in mice.

Glioma is a CNS tumor with few therapeutic options. Recently, host microbiota has been involved in the immune modulation of different tumors, but no data are available on the possible effects of the gut-immune axis on brain tumors. Here, we investigated the effect of gut microbiota alteration in a syngeneic (GL261) mouse model of glioma, treating mice with two antibiotics (ABX) and evaluating the effects on tumor growth, microbe composition, natural killer (NK) cells and microglia phenotype. We report that ABX treatment (i) altered the intestinal microbiota at family level, (ii) reduced cytotoxic NK cell subsets, and (iii) altered the expression of inflammatory and homeostatic proteins in microglia. All these findings could contribute to the increased growth of intracranial glioma that was observed after ABX treatment. These results demonstrate that chronic ABX administration alters microbiota composition and contributes to modulate brain immune state paving the way to glioma growth.

MicroRNA-133b expression associates with clinicopathological features and prognosis in glioma.

BACKGROUND: Recently, microRNA-133b (miR-133b) dysregulation has been shown to play a key role in several human cancers, as well as glioma. In this study, we aimed to investigate the clinical significance and prognostic value of miR-133b in glioma. METHODS: Real-time quantitative PCR was employed to measure the expression level of miR-133b in tissues. Survival analysis was carried out by using the log-rank test and Kaplan-Meier method. Prognostic factors for overall survival were identified by univariate and multivariate analyses using the Cox proportional hazards regression model. RESULTS: The expression level of miR-133b was significantly lower in glioma tissues compared with matched non-cancerous brain tissues (p < .05). Its level was strongly correlated with Karnofsky Performance Scale score (p < .001) and WHO grade (p < .001). Kaplan-Meier survival and log-rank analysis indicated that the decreased expression of miR-133b was strongly correlated with shorter overall survival of patients with glioma (log-rank test, p = .03). CONCLUSIONS: The current investigation demonstrated that miR-133b level is useful for predicting the prognosis of patients with glioma.

Modification of redox processes in astroglial cells induced by microbial and viral infections.

BACKGROUND: The authors hypothesized that the cell redox state might be modified during microbial and viral infections. To detect and evaluate changes in astroglial cell redox state, rat C6 glioma cells after exposure to lipopolysaccharide (LPS) or after herpes simplex virus type 1 (HSV-1) inoculation were used. Redox state modification of glioma cells was determined by the change in menadione-induced superoxide yield. MATERIAL/METHODS: Menadione-induced superoxide formation was registered by the lucigenin-enhanced chemiluminescence (CL) method. RESULTS: The results demonstrate that exposure of C6 glioma cells to LPS for 24 hours resulted in a dose-dependent increase in the mitotic index and integral intensity of menadione-induced lucigenin-enhanced CL. Menadione-induced ROS generation in C6 cells during HSV-1 infection changed depending on the time after HSV-1 inoculation. CONCLUSIONS: The redox state of astroglial cells is modified during microbial and viral infections. The use of redox-active quinones is an informative model for determining cell redox state change and analyzing cells' functional state.

Space-time clustering of glioma cannot be attributed to specific histological subgroups.

We previously showed that infectious exposures may be involved in the aetiology of adult glioma, by analysing for space-time clustering using population-based data from the South of the Netherlands. Here we extended these analyses and describe in detail the space-time clustering patterns in glioma subgroups, gender and age-categories. Knox tests for space-time interactions between cases were applied with fixed thresholds of close in space, <5 km, and close in time, <1 year apart. We used the spatial coordinates of the addresses at diagnosis in the analyses. Tests were repeated replacing geographical distance with distance to the Nth nearest neighbour. N was chosen such that the mean distance was 5 km. Data were also analysed by a second order procedure based on K-functions. There was only statistically significant space-time clustering for oligodendroglioma. Clustering was present for adults aged 30-54 years and was more pronounced among males. Given the low prior probability of an infectious aetiology for this specific subgroup, these results should probably be interpreted as false-positive. We conclude that space-time clustering of glioma cannot be attributed to a specific glioma subgroup. The observed clustering in our previous study is therefore probably an overall effect within and between glioma subgroups.

[Effect of HCMV on p38MAPK, apoptosis and cell cycle of human glioma U251 cells].

OBJECTIVE: To study the changes of p38MAPK expressions, the frequency of apoptosis and the distribution of cell cycle of hunan Glioma U251 cells after HCMV infection. METHODS: The expression of total p38 (both phosphorylated and nonphosphorylated p38) and phosphorylated p38 in U251 cells were detected by Western blotting at 15 min, 30 min, 1 h, 6 h, 10 h, 16 h, 24 h, 36 h and 48 h after HCMV infection. The apoptosis percentage and the cell cycle distribution of U251 cells at 2 d, 5 d and 7 d after HCMV infection were detected by flow cytometry (FCM). RESULTS: The results of Western blotting demonstrated that a strong increase in phosphorylated p38 was detected from 6 h to 10 h after HCMV infection, with mean gray scales 186.33 +/- 7.51 (t = 5.37, P < 0.01) and 188.00 +/- 7.02 (t = 5.26, P < 0.01 for all) at 6 h and 10 h, respectively, and p38 phosphorylation decreased to the basic level at 16 h after HCMV infection. But the overall levels of p38 protein were not significantly altered during the course of infection. FCM analysis showed that HCMV could significantly increase the apoptotic rates of U251 cells compared with controls (t = 10.84, P < 0.01), and the apoptotic percentages of the cells reached to peak [(10.18 +/- 1.24)%] at 5 d after HCMV infection. The data of FCM showed that HCMV could decrease the number of U251 cells in G1 phase and arrest the cells in S and G2 phase. The numbers of G1 phase U251 cells were significantly lowered to (56.50 +/- 2.57)% (t = 26.45, P < 0.01), (62.33 +/- 2.64)% (t = 21.20, P < 0.01) and (67.45 +/- 4.44)% (t = 10.61, P < 0.01), respectively at 2 d, 5 d and 7 d after infection. CONCLUSION: HCMV could activate p38MAPK pathway and trigger apoptosis and interfere cell cycle in U251 cells.

Space-time clustering patterns of gliomas in The Netherlands suggest an infectious aetiology.

To test the hypothesis that infectious exposures may be involved in glioma aetiology, we have analysed space-time clustering and seasonal variation using population-based data from the South of The Netherlands between 1983 and 2001. Knox tests for space-time interactions between cases were applied, with spatial coordinates of the addresses at time of diagnosis, and with distance to the Nth nearest neighbour. Data were also analysed by a second order procedure based on K-functions. Tests for heterogeneity and Edwards' test for sinusoidal variation were applied to examine seasonal variation of incidence. There was statistically significant space-time clustering in the Eastern, but not in the Western part of the region. Clustering was only present in adults, particularly in less densely populated areas. There was no evidence for seasonal variation. The results support a role for infectious exposures in glioma aetiology that may act preferentially in certain geographical areas.

Falsification of tetrazolium dye (MTT) based cytotoxicity assay results due to mycoplasma contamination of cell cultures.

Mycoplasma contamination of cell cultures is a frequently observed problem. Due to the inconspicuous growth in cell cultures, periodical screening procedures represent the only protection. Many influences of mycoplasma on cell culture parameters have been described. We addressed the question of whether mycoplasma contamination affects the most frequently used cytotoxicity assay, the tetrazolium based MTT assay. We contaminated C6 glioma cells with mycoplasma and performed MTT assays with doxorubicin, vincristine, etoposide and cisplatinum under various conditions. Contaminated cells demonstrated significant different results when tested with the MTT assay than mycoplasma free controls. Differences were not detectable when cells were counted as toxicity assay. Due to an additional reduction of tetrazolium by mycoplasmas, contaminated cells appeared up to 15 fold resistant to doxorubicin, vincristine and etoposide, but not to cisplatinum. Differences decreased with decreasing drug doses and decreasing plated cell count. Our findings confirm the compelling need for periodical mycoplasma screening, especially when tetrazolium based cytotoxicity assay (MTT) are used.

Infection of human brain cells by HIV-1: restricted virus production in chronically infected human glial cell lines.

OBJECTIVE: To study expression of HIV-1 in human glial cell lines. DESIGN: Chronically HIV-1-infected glial cell lines were established to evade potential artefacts resulting from unphysiological viral entry (i.e., transfection). These cell lines were used to study viral expression and regulation. METHODS: Chronically infected glial cell lines were established by terminal dilution cloning of human glioma cells exposed to HIV-1. Virus production and expression were assayed by measuring reverse transcriptase activity, p24-antigen levels and syncytia-inducing capacity in C8166 target cells (extracellular), or by indirect immunoperoxidase staining, immunoblot analysis, and p24- and Nef-antigen-capture enzyme-linked immunosorbent assays (intracellular). HIV-long terminal repeat (LTR)-dependent expression of the chloramphenicol acetyltransferase reporter gene was determined in transient transfection assays. RESULTS: Culture supernatant from chronically HIV-1-infected glial cells contained only low levels of virus compared with chronically HIV-infected fibroblasts and T-lymphoma cells. Detailed study of HIV-antigen expression in representative glial cell line TH4-7-5 indicated the presence of all major structural proteins, albeit at low levels, and of Vif, Tat, Rev and Nef. Intracellular levels of Nef exceeded p24-antigen levels by approximately 10-fold. Virus was recovered from TH4-7-5 cells by cocultivation with blood-derived target cells, indicating that low-level virus production is not due to defective provirus. Prominent negative regulatory element (NRE)-mediated suppression of exogenous HIV-LTR activity was observed in TH4-7-5 cells and was unequalled by chronically HIV-producing fibroblast cells or by uninfected fibroblast and glial cells. CONCLUSIONS: Our results suggest that restricted virus production by chronically infected glial cells involves LTR-mediated regulation of virus expression.

Differential susceptibility of a rat glioma cell line and its clones to herpes simplex virus types 1 and 2.

A rat glioma cell line, C6-BU-1, showed differential susceptibility to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), namely, all the HSV-1 strains tested so far persisted in this cell line but the HSV-2 strains did not. Two clones were derived from C6-BU-1 cells and designated C-17 and C-72. The C-72 as well as the parental C6-BU-1 cells supported the replication of HSV-1, but not that of HSV-2. In contrast, C-17 was highly resistant to both HSV-1 and HSV-2. Hydrocortisone treatment converted C-17 to being susceptible to HSV-1, but not to HSV-2. Therefore, C6-BU-1 cells consist of subpopulations heterogeneous in susceptibility to HSV-1 which may be possibly interchangeable.

Target cells for HIV in the central nervous system: macrophages or glial cells?

Infection of foetal or embryonic brain cells and cell lines from human astrocytomas and gliomas with HIV1 derived from T-lymphoma cultures leads to the expression of HIV in about 1 to 2% of the cells in culture. Single-cell cloning of astrocytoma cells shortly after infection resulted in the establishment of persistently HIV1-infected cell lines. These cultures were characterized by low production of virus and moderate intra- and extracellular expression of structural proteins. However, high expression of the nef regulatory protein was found. The virus could be rescued by cocultivation with T cells and primary macrophages giving rise to typical syncytia formation. In contrast to infection with HIV-infected T-lymphoma lines, cocultivation with HIV1-infected primary macrophages or monocytic cell lines induced a reduction in the growth of astrocytes and failed to induce productive infection. These in vitro observations support the hypothesis that astrocytes and glial cells may be a reservoir for HIV in the central nervous system and that macrophages may not carry the virus to the brain, but rather may be infected in the brain after having penetrated the blood-brain barrier.

Fulminant blastomycosis with blastomycotic infection of a cerebral glioma. Light microscopic and ultrastructural observations.

Except for isolated case reports, blastomycosis has not been identified as a significant problem in immunosuppressed patients. We describe an unusual case with blastomycotic infection of a cerebral glioma in a 56-year-old man who underwent radiotherapy for his tumor and died of fulminant blastomycotic pneumonia. This is believed to be the first reported case of Blastomyces dermatitidis infection of a cerebral glioma. The light microscopic and ultrastructural features of B. dermatitidis, the giant forms of which were encountered in our patient, are described, and thr role of immunosuppression due to steroid therapy in the pathogenesis of this fulminant infection are reviewed.

Effect of human T lymphotropic retrovirus-I exposure on cultured human glioma cell lines.

Four different human tumor cell lines of glial origin have been exposed to a human T lymphotropic retrovirus (HTLV-I). All these cell lines were positive for the glial marker glial fibrillary acidic protein (GFAP). The presence of virus RNA was demonstrated by in situ hybridization using an HTLV-I, SStI-SStI viral insert as probe. Virus expression has been monitored through an indirect immunofluorescence assay using a monoclonal antibody against virus core protein p19. All the four glioma cell lines tested became positive for p19 after 2 weeks of co-cultivation and showed a clear alteration of GFAP expression.

Characterization of BK virus variants rescued from human tumours and tumour cell lines.

Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi's sarcoma and in cell lines from brain tumours. Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable elements in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.

[Reproduction of Japanese encephalitis virus in human glioma cells, 118MGC: inhibitory effect of host factor(s)].

Japanese encephalitis viruses (JEV) were well propagated in human glioma cells, 118MGC until the first 24 hrs after virus infection. However, after 24 hrs, virus growth rate was quickly reduced. This unusual pattern of virus growth was different from the cases in others cells, e.g. IMR-32, Vero and C6/36 cells. The fact that actinomycin-D retained the high yields of JEV in 118MGC cells suggests that some suppressing factors against JEV replication are produced in MGC cells. Interestingly, culture fluids of 118MGC cells indicated inhibitory effect to JEV reproduction, but other culture fluids from several cell lines had no effect. This inhibitory effect of the MGC-culture fluids was lost by heat-treatment at 60 C. In addition, the infectivity of JEV was rapidly decreased by the incubation with MGC-culture fluids. These findings suggest that 118MGC cells produce and secret some inhibitory factors against JEV replication.

Morphogenesis of measles virus on C6 rat glioma cells.

Rat glioma cells (C6) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.

Extinction of JC virus tumor-antigen expression in glial cell--fibroblast hybrids.

JC virus (JCV) is a ubiquitous human papovavirus that shares sequence and structural homology with simian virus 40 (SV40). In contrast to SV40, expression of JCV is restricted to a small number of cell types, including human fetal glial cells, uroepithelial cells, amnion cells, and some endothelial cells. To study the control of JCV early region expression, we made heterokaryons and stable hybrids between JCV-transformed hamster glial cells and mouse fibroblasts. Binucleate heterokaryons exhibited extinction of large tumor antigen expression in the hamster nuclei as assayed by indirect immunofluorescence. This extinction was both time and dose dependent: extinction reached maximal levels at 24-36 hr after fusion and was dependent on the ratio of glial cell to fibroblast nuclei in multinucleated heterokaryons. Extinction also was observed in stable hybrids between the glial cells and mouse Ltk- cells. Southern blot analysis showed that the extinguished hybrids contained viral sequences. Reexpression of large tumor antigen was observed in several subclones, suggesting that extinction was correlated with the loss of murine fibroblast chromosomes from these hybrids. The cis-acting region that mediates extinction resides within the viral regulatory region, which contains two 98-base-pair repeats that have enhancer activity. These data demonstrate that cellular factors that negatively regulate viral gene expression contribute to the restricted cell-type specificity of this virus.

Interaction of herpes simplex virus type 2 with a rat glioma cell line.

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.