Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain

Loss-of-function mutations of Na_V1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na_V1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na_V1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na_V1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na_V1.7 inhibitors and significantly reduced in Na_V1.7^−/− mice. To validate the use of the model for profiling Na_V1.7 inhibitors, we determined the Na_V selectivity and tested the efficacy of the reported Na_V1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na_V1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na_V1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na_V channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na_V1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na_V1.7 inhibitors.     

H3K27me3 loss indicates an increased risk of recurrence in the Tübingen meningioma cohort

BackgroundA loss of the trimethylation of lysine 27 of histone H3 (H3K27me3) in meningioma has been recently suggested as an adjunct to identify subsets of higher risk of recurrence. The aim of the present study was to assess the prognostic value of H3K27 histone trimethylation and its potential clinical utility in the “Tübingen meningioma cohort.”MethodsPatients who underwent meningioma resection between October 2003 and December 2015 at the University Hospital Tübingen were included. Immunohistochemical stainings for H3K27me3 and the proliferation marker MIB1 were assessed and correlated with clinical parameters using univariate and multivariate Cox regressions as well as Pearson''s chi-squared and log-rank test.ResultsOverall, 1268 meningiomas were analyzed with a female to male ratio of 2.6 and a mean age of 58.7 years (range 8.3–91.0). With 163 cases lost to follow up, 1103 cases were available for further analysis with a mean follow-up of 40.3 months (range 1.1–186.3). Male gender, younger age, intracranial tumor localization, progressive tumor, subtotal resection, higher WHO grade, increased MIB1 rate, and loss of H3K27me3 were significant negative prognostic factors in the univariate analysis. H3K27me3 status and all other prognostic factors, except age and tumor location, remained significant in the multivariate model. Furthermore, adjuvant radiotherapy was an independent positive prognostic factor.ConclusionsLoss of H3K27me3 combined with MIB1 labeling index are independent prognostic factors in meningioma. These data from the Tübingen meningioma cohort support the clinical utility of H3K27me3 immunohistochemical staining in meningioma and its integration into the routine histopathological workup.     

Sensitivity of the FPG protein towards alkylation damage in the comet assay

The comet assay (single cell gel electrophoresis) is widely used for the evaluation of DNA-damaging effects in genotoxicity testing and population monitoring. In its standard version at pH >13, DNA double strand breaks (DSB), DNA single strand breaks (SSB) and alkali-labile sites (ALS) lead to increased DNA migration. At reduced pH (12.5-12.1) the expression of ALS as SSB can be eliminated and the effect of SSB only can be identified. Specific endonucleases have been used to characterize specific classes of DNA damage. The formamido pyrimidine glycosylase (FPG) protein has been used to assess oxidative DNA base damage because it detects 8-OH guanine and other oxidatively damaged purines. Here, we show that the FPG protein also detects alkylation damage with high sensitivity in the comet assay. Human whole blood, isolated lymphocytes and V79 cells were treated with alkylating agents and post-incubated with FPG. FPG strongly enhanced MMS- and EMS-induced DNA damage but had no significant effect on ENU-induced DNA damage, indicating that the amount of N-7 guanine alkylation is responsible for the observed effect. Reducing the pH during alkali unwinding and electrophoresis to 12.5 to avoid the contribution of ALS to the comet assay effects, strongly decreased the sensitivity of the comet assay with and without FPG treatment and prevented DNA migration. We conclude that enhanced DNA effects in the comet assay by FPG after exposure to genotoxins with unknown mode of action should not directly be regarded as evidence for the presence of oxidative damage. Furthermore, reducing the pH leads to a considerable loss in sensitivity and should not be used in biomonitoring and other applications which require a sensitive protocol.     

Cancer mortality rates among first and second generation migrants in the Netherlands: Convergence toward the rates of the native Dutch population

This study investigates the difference in cancer mortality rates between migrant groups and the native Dutch population, and determines the extent of convergence of cancer mortality rates according to migrants' generation, age at migration and duration of residence. Data were obtained from the national cause of death and population registries in the period 1995-2000. We used Poisson regression to compare the cancer mortality rates of migrants originating from Turkey, Morocco, Surinam, Netherlands Antilles and Aruba to the rates for the native Dutch. All-cancer mortality among all migrant groups combined was significantly lower when compared to that of the native Dutch population (RR = 0.55, CI: 0.52-0.58). For a large number of cancers, migrants had more than 50% lower risk of death, while elevated risks were found for stomach and liver cancers. Mortality rates for all cancers combined were higher among second generation migrants, among those with younger age at migration, and those with longer duration of residence. This effect was particularly pronounced in lung cancer and colorectal cancer. For most cancers, mortality among second generation migrants remained lower compared to the native Dutch population. Surinamese migrants showed the most consistent pattern of convergence of cancer mortality. The generally low cancer mortality rates among migrants showed some degree of convergence but did not yet reach the levels of the native Dutch population. This convergence implies that current levels of cancer mortality among migrants will gradually increase in future years if no specific preventive measurements are taken.     

Resistance of Sézary cells to TNF-α-induced apoptosis is mediated in part by a loss of TNFR1 and a high level of the IER3 expression

Failure to execute an apoptotic programme is one of the critical steps and a common mechanism promoting tumorogenesis. Immediate early responsive gene 3 (IER3) has been shown to be upregulated in several cancers. IER3 is a stress-induced gene, which upregulation leads to reduction in production of reactive oxygen species (ROS) protecting malignant cells from apoptosis. We observed that malignant lymphocytes from patients with Sézary syndrome (SzS) were resistant to pro-apoptotic dose of tumour necrosis factor-α (TNF-α). The aim of this study was to investigate the role of IER3 in the mechanism of such resistance. CD4+ CD26- lymphocytes from the peripheral blood of patients with SzS and healthy controls were negatively selected using CD4 and CD26 magnetic beads and analysed for expression of TNFR1, TNFR2, IER3 expression, and ROS production in response to TNF-α at an apoptotic dose. Sézary cells with a higher level of IER3 expression retained their viability to TNF-α. IER3 upregulation correlated with a decrease level of intracellular ROS and low TNFR1 expression on malignant cells. Targeting IER3 could be of interest for the development of future therapeutic strategies for patients with SzS.     

An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology

α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.