Potential role of ticks as vectors of bluetongue virus

When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not to survive the northern European winter, and transovarial transmission in Culicoides is not recorded, we examined the potential vector role of ixodid and argasid ticks for bluetongue virus. Four species of ixodid ticks (Ixodes ricinus, Ixodes hexagonus, Dermacentor reticulatus and Rhipicephalus bursa) and one soft tick species, Ornithodoros savignyi, ingested BTV8-containing blood either through capillary feeding or by feeding on artificial membranes. The virus was taken up by the ticks and was found to pass through the gut barrier and spread via the haemolymph into the salivary glands, ovaries and testes, as demonstrated by real-time reverse transcriptase PCR (PCR-test). BTV8 was detected in various tissues of ixodid ticks for up to 21 days post feeding and in Ornithodoros ticks for up to 26 days. It was found after moulting in adult Ixodes hexagonus and was also able to pass through the ovaries into the eggs of an Ornithodoros savignyi tick. This study demonstrates that ticks can become infected with bluetongue virus serotype 8. The transstadial passage in hard ticks and transovarial passage in soft ticks suggest that ticks have potential vectorial capacity for bluetongue virus. Further studies are required to investigate transmission from infected ticks to domestic livestock. This route of transmission could provide an additional clue in the unresolved mystery of the epidemiology of Bluetongue in Europe by considering ticks as a potential overwintering mechanism for bluetongue virus

Characterization of long-term functional outcome in a murine model of mild brain ischemia

Zweck der Arbeit war es, das Wissen über die Verhaltenstestung in der Maus nach Schlaganfall zu erweitern und zu konsolidieren. Die folgenden Ziele waren dabei vorrangig: 1. Charak- terisierung des sensomotorischen Defizites und seiner Erholung im Langzeitverlauf nach 30-minütigem MCA-Verschluss, 2. Etablierung neuer bzw. verbesserter Testverfahren mit besserer Langzeitsensitivität oder klinischer Relevanz, 3. Standardisierung und Optimisierung der Testmethoden. In der Summe sollte uns unsere Arbeit ermöglichen, begründete Aussagen darüber zu treffen, welches der richtige Verhaltenstest für den jeweiligen Zeitpunk und das zu untersuchende Defizit ist, um damit die Entscheidung darüber, welche Verhaltenstests nach Schlaganfall durchgeführt werden, auf eine experimentell validierte Basis zu stellen. Wir führten daher eine umfassende Charakterisierung des sensomotorischen Defizi- tes im Langzeitverlauf nach 30-minütigem proximalen MCA-Verschluss (MCAo) bei der Maus durch. Es ist dies das Modell eines milden ischämischen Schadens und für die Evaluation von langfristigen Reperaturmechanismen gut geeignet. Aufgrund des nur sehr geringfügigen längerfristigen funktionellen Defizites werden für die Verhaltenstestung je- doch besonders sensitive Testverfahren benötigt. Zwei Kohorten von C57Bl/6N Mäusen wurden in zwei Batterien von Verhaltenstests in definierten Zeitabständen über bis zu 28 Tage untersucht. Im Kurzzeitintervall von 1-2 Wochen erwiesen sich “Pole Test” und, mit einigen Einschränkungen, der “Rotarod” als gut geeignete Tests von motorischer Stärke und Koordination. Die bereits etablierten Langzeittests “Corner Test” und “Adhesive Removal Test” zeigten auch im 30-minütigen MCAo-Modell eine gute Sensitivität über bis zu vier Wochen. Da der “Corner Test” unter einigen methodischen Problemen wie der subjektiven Versuchsauswertung und der geringen Anzahl von Testversuchen pro Tier leidet, entwickelten wir das “Corner Rotation” – Paradigma. Dieser Test bewährte sich als ein einfacher, objektiver und langfristig sensitiver Test speziell der posturalen Asymmetrie. Ferner führten wir mit dem “Bowl Test” ein alternatives automatisiertes Testverfahren für die langfristige Evaluation von posturalen Asymmetrien nach Schlaganfall ein. Mit dem “Paw Preference Test” und dem “Catwalk” - System konnten wir des weiteren zwei Testverfahren für die Evaluation von klinisch besonders relevanten Defiziten, nämlich Feinmotorik- respektive Gangstörung, etablieren. Insbesondere die Analyse des Gangbildes nach MCAo mithilfe des “Catwalk”-Systems stellt die erste umfassende Beschreibung dieses Testverfahrens im Langzeitverlauf nach MCAo in der Maus dar und zeigt sowohl die Schwierigkeiten wie auch das Potential dieses neuartigen Systems. In Zusammenschau unserer Ergebnisse empfehlen wir im 30-minütigen MCAo-Modell für die Routineanwendung den “Pole Test” als primären Test für kurzfristige motorische Defizite, ergänzt durch “Corner” oder “Corner Rotation” Test im Langzeitverlauf. Für die Evaluation sensorischer Defizite im Langzeitverlauf ist der “Adhesive Removal Test” ge- eignet. Weiter vervollständigt werden kann die sensomotorische Testbatterie durch “Paw Preference Test” “Catwalk” für die Evaluation von Feinmotorik und Gang.Evaluation of functional outcome over the course of several weeks after ischemia is a key component in improving the clinical relevance of experimental stroke studies. Using a battery of behavioral tests, we characterized functional outcome in mice over 4 weeks following 30min of proximal middle cerebral artery occlusion (MCAo). We evaluated rotarod, chimney, pole and cylinder tests to assess short term functional deficits in a transient stroke model which induces infarcts mainly in the striatum. The corner test, adhesive removal test, cylinder test, catwalk, paw preference test and novel tests of rotation were evaluated for long-term functional outcome. Rotarod detected deficits within the first week and pole test was reliable up to intermediate time points after MCAo. Corner test, adhesive removal test, catwalk and paw preference test detected deficits for up to 4 weeks, as did the novel corner rotation and bowl tests. Chimney and cylinder test did not prove useful in our model of mild stroke. In summary, we established the pole test and rotarod as useful tools to evaluate sensory motor deficits early after mild stroke, and corner test and adhesive removal test at later time-points. Alternatively, corner rotation may be a suitable test of long-term function. Test batteries may be further complemented by catwalk and paw preference test for clinically relevant deficits. There was no correlation of behavioral outcome with lesion size at 28 days, and determining whether these tests are useful for detecting a potential benefit of neuroprotective or regenerative therapies requires further testing

Prevalence of deleterious germline variants in risk genes including BRCA1/2 in consecutive ovarian cancer patients (AGO-TR-1)

Background: Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated. Methods: Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history. Results: In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16–93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients <60 years was 30.2% (33.2%) versus 10.6% (18.9%) in patients ≥60 years. Family history was positive in 43% of all patients. Patients with a positive family history had a prevalence of deleterious variants of 31.6% (36.0%) versus 11.4% (17.6%) and histologic subtype of high grade serous ovarian cancer versus other showed a prevalence of deleterious variants of 23.2% (29.1%) and 10.2% (14.8%), respectively. Testing only for BRCA1/2 would miss in our series more than 5% of the patients with a deleterious variant in established risk genes. Conclusions: 26.4% of all patients harbor at least one deleterious variant in established risk genes. The threshold of 10% mutation rate which is accepted for reimbursement by health care providers in Germany was observed in all subgroups analyzed and neither age at primary diagnosis nor histo-type or family history sufficiently enough could identify a subgroup not eligible for genetic counselling and testing. Genetic testing should therefore be offered to every patient with invasive epithelial ovarian cancer and limiting testing to BRCA1/2 seems to be not sufficient

Essential role of interleukin-6 in post-stroke angiogenesis

Ambivalent effects of interleukin-6 on the pathogenesis of ischaemic stroke have been reported. However, to date, the long-term actions of interleukin-6 after stroke have not been investigated. Here, we subjected interleukin-6 knockout (IL-6(−/−)) and wild-type control mice to mild brain ischaemia by 30-min filamentous middle cerebral artery occlusion/reperfusion. While ischaemic tissue damage was comparable at early time points, IL-6(−/−) mice showed significantly increased chronic lesion volumes as well as worse long-term functional outcome. In particular, IL-6(−/−) mice displayed an impaired angiogenic response to brain ischaemia with reduced numbers of newly generated endothelial cells and decreased density of perfused microvessels along with lower absolute regional cerebral blood flow and reduced vessel responsivity in ischaemic striatum at 4 weeks. Similarly, the early genomic activation of angiogenesis-related gene networks was strongly reduced and the ischaemia-induced signal transducer and activator of transcription 3 activation observed in wild-type mice was almost absent in IL-6(−/−) mice. In addition, systemic neoangiogenesis was impaired in IL-6(−/−) mice. Transplantation of interleukin-6 competent bone marrow into IL-6(−/−) mice (IL-6(chi)) did not rescue interleukin-6 messenger RNA expression or the early transcriptional activation of angiogenesis after stroke. Accordingly, chronic stroke outcome in IL-6(chi) mice recapitulated the major effects of interleukin-6 deficiency on post-stroke regeneration with significantly enhanced lesion volumes and reduced vessel densities. Additional in vitro experiments yielded complementary evidence, which showed that after stroke resident brain cells serve as the major source of interleukin-6 in a self-amplifying network. Treatment of primary cortical neurons, mixed glial cultures or immortalized brain endothelia with interleukin 6-induced robust interleukin-6 messenger RNA transcription in each case, whereas oxygen–glucose deprivation did not. However, oxygen–glucose deprivation of organotypic brain slices resulted in strong upregulation of interleukin-6 messenger RNA along with increased transcription of key angiogenesis-associated genes. In conclusion, interleukin-6 produced locally by resident brain cells promotes post-stroke angiogenesis and thereby affords long-term histological and functional protection