In-vivo-Studien der Gene Atm und Ccnd1 in der Pathogenese lymphatischer Neoplasien

Lymphatische Neoplasien stellen eine heterogene Gruppe proliferativer Erkrankungen der Lymphozyten dar. Zwar konnten die zugrundeliegenden Pathomechanismen in einigen wenigen Fällen bereits entschlüsselt werden, doch sind sie bei der Mehrzahl der lymphatischen Leukämien und Lymphome unbekannt. So besteht auch für das bisher unheilbare Mantelzell-Lymphom (MCL) großer Bedarf an der Entschlüsselung der zugrundeliegenden molekularen Vorgänge, die letztlich zur Entstehung des Lymphoms führen. Erste Anhaltspunkte lieferte bereits die Aufdeckung der chromosomalen Translokation t(11;14)(q13;q32), welche letztendlich zu der für das MCL charakteristischen Überexpression von Cyclin D1 führt. Ferner konnte in 75 % der MCL-Fälle eine Inaktivierung der Proteinkinase ATM nachgewiesen werden. Ziel dieser Arbeit war es, durch Kombination der genannten Aberrationen im murinen System ein Modell zu schaffen, an dem die Pathomechanismen von B-Zell-Lymphomen und insbesondere von MCL untersucht werden können. Zu diesem Zweck wurden zwei unterschiedliche Ansätze gewählt. Zum einen wurden die genetischen Aberrationen durch Kreuzung eines Atm-knock-out und eines Ccnd1 transgenen Mausstammes zusammengeführt, zum anderen wurden mit Ccnd1-rekombinanten Retroviren infizierte, Atm-defiziente hämatopoetische Stammzellen zur Rekonstitution des hämatopoetischen Systems letal bestrahlter Mäuse genutzt. In keinem der Ansätze konnte nach langfristiger Beobachtung und eingehender Untersuchung die Entstehung eines B-Zell-Lymphoms festgestellt werden. Hingegen wurden Hinweise gefunden, wonach die molekularen Folgen des Atm-Verlusts dem Effekt der Ccnd1-Überexpression entgegenwirken. Darüber hinaus entwickelten alle Atm-defizienten Mäuse unabhängig von Ccnd1-Status thymische T-Zell-Lymphome. Anhand immunphänotypischer und molekulargenetischer Untersuchungen wurde die weitestgehend unverstandene Genese dieser durch Atm-Verlust verursachten Tumoren untersucht. Die neoplastischen Zellen der T-Zell-Lymphome wurden umfassend charakterisiert und konnten als nicht aktivierte, CD4 CD8 doppelt positive T-Lymphozyten identifiziert werden, die eine ausgeprägte Deregulation der entwicklungsspezifischen Oberflächenmoleküle CD25 und T-Zell-Rezeptor β (TCRβ) zeigen. Erstmalig konnten in dieser Arbeit sowohl der oligoklonale Ursprung dieser Tumorzellen als auch deren inkorrekte Rekombination des TCR β Gens nachgewiesen werden. Die Untersuchung der Atm-/- T-Zell-Lymphome mit hochauflösenden aCGH-microarrays ließ charakteristische genomische Imbalancen (Chromosom 12, 14 und 15) erkennen, von welchen der TCRα-Lokus auf Chromosom 14 grundsätzlich in sämtlichen untersuchten Proben betroffen war. Basierend auf der Analyse von Atm-/- Thymi ohne Lymphom konnten genomische Tumorvorstufen als Stadien beschrieben werden, deren mittels microarray-basierter Expressionsanalyse erstellte Transkriptionsprofile grundlegend von den Profilen genomisch unveränderter Thymi abweichen. Die weitere Auswertung der gewonnen Expressionsdaten ergab neue Erkenntnisse über die transkriptionelle Deregulation tumorassoziierter Gene, wie z. B. Tuba1 und Aurka. Die Mehrzahl der hier untersuchten T-Zell-Lymphome wies darüber hinaus einen genomischen Notch1-Zugewinn auf, der sich in der Deregulation Notch1-regulierter Gene, insbesondere c-Myc, Dtx1 und Hes1, auswirkte. Durch bioinformatorische Auswertung der gewonnenen Expressionsdaten konnte ein Netzwerk deregulierter Gene postuliert werden, welches auf eine Dysregulation der CD95 vermittelten Apoptose in der betroffenen T-Zellpopulation der Lymphome hinweist

Indirect Comparison of Topiramate and Monoclonal Antibodies Against CGRP or Its Receptor for the Prophylaxis of Episodic Migraine: A Systematic Review with Meta-Analysis

Background: Head-to-head comparator trials between first-line oral migraine preventatives and the new monoclonal antibodies (mAbs) blocking the calcitonin gene-related peptide (CGRP) pathway have not been published to date. Objectives: This study aimed to indirectly compare the clinical efficacy and safety of mAbs against CGRP or its receptor (CGRPR) and topiramate in episodic migraine prophylaxis using meta-analysis. Methods: We included controlled trials testing efficacy and safety of erenumab, galcanezumab, fremanezumab, eptinezumab, and topiramate in adults diagnosed with episodic migraine. We searched PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), and ClinicalTrials.gov from January 2000 to November 2020. We used the Risk of Bias 2 (RoB2) tool to assess the risk of bias and report pooled mean effects (mean difference and risk ratio) as estimated in a random effect model. For efficacy analysis, we determined the reduction of monthly migraine days (MMDs), reduction of days with acute medication (AMDs), and 50% responder rates (50% RR). For safety, we determined adverse events (AEs) occurring in >= 2% of study participants and the number of patients who discontinue treatment due to AEs (DAEs). The number needed to treat (NNT) and to harm (NNH) were estimated as well as the likelihood to help or harm (LLH). Results: We included 13 trials involving 7557 patients: three trials with erenumab, two trials with galcanezumab, two trials with fremanezumab, one trial with eptinezumab, and five trials with topiramate, for the prophylaxis of episodic migraine in adults. The placebo-subtracted reduction (pooled mean difference) of MMDs were - 1.55 (95% CI - 1.86 to - 1.24; active drug n = 3326 vs placebo n = 2219, 8 studies) for the CGRP(R) mAb and - 1.11 (95% CI - 1.62 to - 0.59; active drug n = 1032 vs placebo n = 543, 4 studies) for topiramate (p for subgroup difference = 0.15). 'Cognitive' and 'sensory & pain'-related adverse events occurred more often in patients treated with topiramate compared with those treated with a CGRP(R) mAb (p for subgroup difference 0.03 and < 0.001, respectively). Based on the 50% RR and DAE, the NNT, NNH, and LHH for the CGRP(R) mAbs were 6, 130, and 24.3:1, respectively. For topiramate, these values were 7, 9, and 1.8:1, respectively. Conclusion: The efficacy of CGRP(R) mAbs to reduce migraine days does not differ from topiramate. However, the safety profile is in favor of the CGRP(R) mAbs, with a higher likelihood to help than to harm compared with topiramate. The diversity of endpoint determination and the heterogeneity between studies for some endpoints cause some limitations for this study

Deterioration of headache impact and health-related quality of life in migraine patients after cessation of preventive treatment with CGRP(−receptor) antibodies

Background Migraine preventive treatment with CGRP(-receptor) monoclonal antibodies (mAbs) has a positive effect on patients' health-related quality of life (HRQoL). The German treatment guidelines recommend discontinuing successful treatment with CGRP(-receptor) mAbs after 6-12 months. We aimed to evaluate headache-specific and generic HRQoL for three months after discontinuation of CGRP(-receptor) mAb treatment. Methods We conducted a prospective, longitudinal cohort study, including patients with migraine after 8-12 months of therapy with a CGRP(-R) mAb and before a planned discontinuation attempt. HRQoL was assessed at the time of the last mAbs injection (V1), eight weeks later (V2), and sixteen weeks later (V3). For headache-specific HRQoL, we used the Headache Impact Test-6 (HIT-6). Generic HRQoL was determined with the EuroQol-5-Dimension-5-Level (ED-5D-5L) form, and the Short-Form 12 (SF-12), which comprises a Physical Component Summary (PCS-12) and a Mental Component Summary (MCS-12). Questionnaires' total scores were compared across the three observation points using nonparametric procedures. Results The study cohort consisted of n = 61 patients (n = 29 treated with the CGRP-receptor mAb erenumab and n = 32 with the CGRP mAbs galcanezumab or fremanezumab). The HIT-6 sum score was 59.69 +/- 6.90 at V1 and increased by 3.69 +/- 6.21 at V3 (p < 0.001), indicating a greater headache impact on patients' lives. The mean total EQ-D5-L5 score declined from 0.85 +/- 0.17 at V1 by - 0.07 +/- 0.18 at V3 (p = 0.013). Both Mental and Physical Component Scores of the SF-12 worsened significantly during treatment discontinuation: The PCS-12 total score decreased by - 4.04 +/- 7.90 from V1 to V3 (p = 0.013) and the MCS-12 score by - 2.73 +/- 9.04 (p = 0.003). Changes in all questionnaires' scores but the MCS-12 were already significant in the first month of the drug holiday (V2). Conclusions Our results show a significant decline in headache impact and generic HRQoL of migraine patients after treatment discontinuation of a CGRP(-R) mAb. The observed deterioration is above the established minimally clinically important differences for each of the questionnaires and can therefore be considered clinically meaningful. Monitoring HRQoL during a discontinuation attempt could facilitate the decision whether or not to resume preventive treatment with CGRP(-R) mAbs

Patients’ and Health Care Workers’ Perception of Migraine Images on the Internet: Cross-sectional Survey Study

Background: The representation of migraine in the media is stereotypical. Standard images of migraine attacks display stylish young women holding their head in a pain pose. This representation may contribute to the social stigmatization of patients with migraine. Objective: We aimed to analyze how patients with migraine and health care workers perceive online images of migraine. Methods: The study consisted of an anonymous web-based survey of patients with migraine at the Headache Center of Charite - Universitatsmedizin Berlin (migraine group) and employees and students at our university (health care group). A total of 10 frequently used Adobe Stock photos of migraine attacks were presented to the participants. Each photo was rated on a scale of 0% to 100% based on how closely it resembled a realistic migraine attack (realism score). Patients with migraine also indicated how much each photo corresponded to their own experience of migraine as a percentage (representation score). We calculated the mean realism and representation scores for all photos and conducted further analyses using the categories male or female models, younger or older models, and unilateral or bilateral pain pose. Results: A total of 367 patients with migraine and 331 health care employees and students completed the survey. In both groups, the mean realism score was <50% (migraine group: 47.8%, SD 18.3%; health care group: 46.0%, SD 16.2%). Patients with migraine identified their own migraine experience in these photos to a lesser degree (mean representation score 44.4%, SD 19.8%; P<.001 when compared to the realism score). Patients and health care workers considered photos with male models to be more realistic than photos with females (P<.001) and photos with older models to be more realistic than those with younger people (P<.001). In the health care group only, a bilateral pain posture was deemed more realistic than a unilateral pose (P<.001). Conclusions: Standard images of migraine attacks are considered only slightly or moderately realistic by patients and health care workers. Some characteristics perceived as more realistic such as male sex or older age are in contrast with migraine epidemiology. A more accurate representation of migraine in the media could help to raise awareness for migraine and reduce the associated stigma

Primary headaches during the COVID-19 lockdown in Germany: analysis of data from 2325 patients using an electronic headache diary

Background Lockdown measures due to the COVID-19 pandemic have led to lifestyle changes, which in turn may have an impact on the course of headache disorders. We aimed to assess changes in primary headache characteristics and lifestyle factors during the COVID-19 lockdown in Germany using digital documentation in the mobile application (app) M-sense. Main body We analyzed data of smartphone users, who entered daily data in the app in the 28-day period before lockdown (baseline) and in the first 28 days of lockdown (observation period). This analysis included the change of monthly headache days (MHD) in the observation period compared to baseline. We also assessed changes in monthly migraine days (MMD), the use of acute medication, and pain intensity. In addition, we looked into the changes in sleep duration, sleep quality, energy level, mood, stress, and activity level. Outcomes were compared using paired t-tests. The analysis included data from 2325 app users. They reported 7.01 +/- SD 5.64 MHD during baseline and 6.89 +/- 5.47 MHD during lockdown without significant changes (p > 0.999). MMD, headache and migraine intensity neither showed any significant changes. Days with acute medication use were reduced from 4.50 +/- 3.88 in the baseline to 4.27 +/- 3.81 in the observation period (p < 0.001). The app users reported reduced stress levels, longer sleep duration, reduced activity levels, along with a better mood, and an improved energy level during the first lockdown month (p <= 0.001). In an extension analysis of users who continued to use M-sense every day for 3 months after initiation of lockdown, we compared the baseline and the subsequent months using repeated-measures ANOVA. In these 539 users, headache frequency did not change significantly neither (6.11 +/- 5.10 MHD before lockdown vs. 6.07 +/- 5.17 MHD in the third lockdown month, p = 0.688 in the ANOVA). Migraine frequency, headache and migraine intensity, and acute medication use were also not different during the entire observation period. Conclusion Despite slight changes in factors that contribute to the generation of headache, COVID-19-related lockdown measures did not seem to be associated with primary headache frequency and intensity over the course of 3 months

A Novel In Vitro Method for the Detection and Characterization of Photosensitizers

Photoactivation and binding of photoactive chemicals to proteins is a known prerequisite for the formation of immunogenic photoantigens and the induction of photoallergy. The intensive use of products and the availability of new chemicals, along with an increasing exposure to sun light contribute to the risk of photosensitizing adverse reactions. Dendritic cells (DC) play a pivotal role in the induction of allergic contact dermatitis. Human peripheral blood monocyte derived dendritic cells (PBMDC) were thus perceived as an obvious choice for the development of a novel in vitro photosensitization assay using the modulation of cell surface protein expression in response to photosensitizing agents. In this new protocol, known chemicals with photosensitizing, allergenic or non-allergenic potential were pre-incubated with PBMDCs prior to UVA irradiation (1 J/cm2). Following a 48 h incubation, the expression of the cell surface molecules CD86, HLA-DR and CD83 was measured by flow cytometry. All tested photosensitizers induced a significant and dose-dependent increase of CD86 expression after irradiation compared to non-irradiated controls. Moreover, the phototoxicity of the chemicals could also be determined. In contrast, (i) CD86 expression was not affected by the chosen irradiation conditions, (ii) increased CD86 expression induced by allergens was independent of irradiation and (iii) no PBMDC activation was observed with the non-allergenic control. The assay proposed here for the evaluation of the photoallergenic potential of chemicals includes the assessment of their allergenic, phototoxic and toxic potential in a single and robust test system and is filling a gap in the in vitro photoallergenicity test battery

Treatment with Helicobacter pylori-derived VacA attenuates allergic airway disease

BACKGROUND: Asthma is an incurable heterogeneous disease with variations in clinical and underlying immunological phenotype. New approaches could help to support existing therapy concepts. Neonatal infection of mice with Helicobacter pylori or administration of H. pylori-derived extracts or molecules after birth have been shown to prevent the development of allergic airway disease later in life. This study evaluated the potential therapeutic efficacy of H. pylori vacuolating cytotoxin A (VacA) in allergic airway inflammation and investigated the underlying immunological mechanisms for its actions. METHODS: Murine models of allergic airway diseases, and murine and human in vitro models were used. RESULTS: In both an acute model and a therapeutic house dust mite model of allergic airway disease, treatment with H. pylori-derived VacA reduced several asthma hallmarks, including airway hyperresponsiveness, inflammation and goblet cell metaplasia. Flow cytometry and ELISA analyses revealed induction of tolerogenic dendritic cells (DC) and FoxP3 positive regulatory T cells (Tregs), and a shift in the composition of allergen-specific immunoglobulins. Depletion of Tregs during treatment with VacA reversed treatment-mediated suppression of allergic airway disease. Human monocyte derived DCs (moDC) that were exposed to VacA induced Tregs in co-cultured naïve autologous T cells, replicating key observations made in vivo. CONCLUSION: H. pylori-derived VacA suppressed allergic airway inflammation via induction of Tregs in both allergic airway disease models. These data suggest that the immunomodulatory activity of VacA could potentially be exploited for the prevention and treatment of allergic airway disease

JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis