Entwicklung einer PCR zum Nachweis der EAV-Infektion

The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Elevated levels of endocannabinoids in chronic hepatitis C may modulate cellular immune response and hepatic stellate cell activation.

The endocannabinoid (EC) system is implicated in many chronic liver diseases, including hepatitis C viral (HCV) infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC), however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH) and monoaclyglycerol lipase (MAGL) activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC), ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC) co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects.

Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

Echinococcus multilocularis Detection in Live Eurasian Beavers (Castor fiber) Using a Combination of Laparoscopy and Abdominal Ultrasound under Field Conditions.

Echinococcus multilocularis is an important pathogenic zoonotic parasite of health concern, though absent in the United Kingdom. Eurasian beavers (Castor fiber) may act as a rare intermediate host, and so unscreened wild caught individuals may pose a potential risk of introducing this parasite to disease-free countries through translocation programs. There is currently no single definitive ante-mortem diagnostic test in intermediate hosts. An effective non-lethal diagnostic, feasible under field condition would be helpful to minimise parasite establishment risk, where indiscriminate culling is to be avoided. This study screened live beavers (captive, n = 18 or wild-trapped in Scotland, n = 12) and beaver cadavers (wild Scotland, n = 4 or Bavaria, n = 11), for the presence of E. multilocularis. Ultrasonography in combination with minimally invasive surgical examination of the abdomen by laparoscopy was viable under field conditions for real-time evaluation in beavers. Laparoscopy alone does not allow the operator to visualize the parenchyma of organs such as the liver, or inside the lumen of the gastrointestinal tract, hence the advantage of its combination with abdominal ultrasonography. All live beavers and Scottish cadavers were largely unremarkable in their haematology and serum biochemistry with no values suspicious for liver pathology or potentially indicative of E. multilocularis infection. This correlated well with ultrasound, laparoscopy, and immunoblotting, which were unremarkable in these individuals. Two wild Bavarian individuals were suspected E. multilocularis positive at post-mortem, through the presence of hepatic cysts. Sensitivity and specificity of a combination of laparoscopy and abdominal ultrasonography in the detection of parasitic liver cyst lesions was 100% in the subset of cadavers (95%Confidence Intervals 34.24-100%, and 86.7-100% respectively). For abdominal ultrasonography alone sensitivity was only 50% (95%CI 9.5-90.6%), with specificity being 100% (95%CI 79.2-100%). For laparoscopy alone sensitivity was 100% (95% CI 34.2-100%), with specificity also being 100% (95% CI 77.2-100%). Further immunoblotting, PCR and histopathological examination revealed one individual positive for E. multilocularis, whilst the other individual was positive for Taenia martis.

Inverse fusion PCR cloning.

Inverse fusion PCR cloning (IFPC) is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

Shifts in microbial communities do not explain the response of grassland ecosystem function to plant functional composition and rainfall change

Ecosystem functioning in grasslands is regulated by a range of biotic and abiotic factors, and the role of microbial communities in regulating ecosystem function has been the subject of much recent scrutiny. However, there are still knowledge gaps regarding the impacts of rainfall and vegetation change upon microbial communities and the implications of these changes for ecosystem functioning. We investigated this issue using data from an experimental mesotrophic grassland study in south-east England, which had been subjected to four years of rainfall and plant functional composition manipulations. Soil respiration, nitrogen and phosphorus stocks were measured, and the abundance and community structure of soil microbes were characterised using quantitative PCR and multiplex-TRFLP analysis, respectively. Bacterial community structure was strongly related to the plant functional composition treatments, but not the rainfall treatment. However, there was a strong effect of both rainfall change and plant functional group upon bacterial abundance. There was also a weak interactive effect of the two treatments upon fungal community structure, although fungal abundance was not affected by either treatment. Next, we used a statistical approach to assess whether treatment effects on ecosystem function were regulated by the microbial community. Our results revealed that ecosystem function was influenced by the experimental treatments, but was not related to associated changes to the microbial community. Overall, these results indicate that changes in fungal and bacterial community structure and abundance play a relatively minor role in determining grassland ecosystem function responses to precipitation and plant functional composition change, and that direct effects on soil physical and chemical properties and upon plant and microbial physiology may play a more important role.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

RASSF7 expression and its regulatory roles on apoptosis in human intervertebral disc degeneration

Apoptosis plays an important role in intervertebral disc degeneration (IDD). Overwhelming evidence indicates that RASSF7 is essential for cell growth and apoptosis. Recently, it has been noted that the JNK signaling can be negatively regulated by suppressing phosphorylated-MKK7 activation during pro-apoptosis. We aimed to investigate the RASSF7 expression level in human degenerative nucleus pulposus (NP) cells and non-degenerative NP cells and the link between RASSF7-JNK with NP cells apoptosis. We harvested NP tissues from 20 IDD patients as disease group and 8 cadaveric donors as normal controls. We detected RASSF7 expression by Real-time-PCR and western blotting. Consequently, we found that the expression of RASSF7 was higher in non-degenerative group than in degenerative group (P<0.05). Overexpression of RASSF7 in degenerative NP cells led to decreased apoptosis rate than that in scramble group (P<0.05). Collectively, our findings suggest that RASSF7 plays an important role in human IDD and RASSF7 might be potentially developed as a curative agent.

Investigating the role of free-ranging wild boar (Sus scrofa) in the re-emergence of enzootic pneumonia in domestic pig herds: a pathological, prevalence and risk-factor study

Enzootic pneumonia (EP) caused by Mycoplasma hyopneumoniae has a significant economic impact on domestic pig production. A control program carried out from 1999 to 2003 successfully reduced disease occurrence in domestic pigs in Switzerland, but recurrent outbreaks suggested a potential role of free-ranging wild boar (Sus scrofa) as a source of re-infection. Since little is known on the epidemiology of EP in wild boar populations, our aims were: (1) to estimate the prevalence of M. hyopneumoniae infections in wild boar in Switzerland; (2) to identify risk factors for infection in wild boar; and (3) to assess whether infection in wild boar is associated with the same gross and microscopic lesions typical of EP in domestic pigs. Nasal swabs, bronchial swabs and lung samples were collected from 978 wild boar from five study areas in Switzerland between October 2011 and May 2013. Swabs were analyzed by qualitative real time PCR and a histopathological study was conducted on lung tissues. Risk factor analysis was performed using multivariable logistic regression modeling. Overall prevalence in nasal swabs was 26.2% (95% CI 23.3-29.3%) but significant geographical differences were observed. Wild boar density, occurrence of EP outbreaks in domestic pigs and young age were identified as risk factors for infection. There was a significant association between infection and lesions consistent with EP in domestic pigs. We have concluded that M. hyopneumoniae is widespread in the Swiss wild boar population, that the same risk factors for infection of domestic pigs also act as risk factors for infection of wild boar, and that infected wild boar develop lesions similar to those found in domestic pigs. However, based on our data and the outbreak pattern in domestic pigs, we propose that spillover from domestic pigs to wild boar is more likely than transmission from wild boar to pigs.

Detection of Chlamydia pneumoniae in a collection of captive snakes and response to treatment with marbofloxacin.

In a collection of 58 snakes comprising predominantly Eurasian vipers in Switzerland, five snakes died unexpectedly during hibernation from 2009 to 2012. In one snake, organisms resembling chlamydiae were detected by immunohistochemistry in multiple histiocytic granulomas. Real-time quantitative PCR and microarray analysis were used to determine the presence of Chlamydia pneumoniae in tissue samples and cloacal/choanal swabs from snakes in the collection; 8/53 (15.1%) of the remaining snakes were positive. Although one infected snake had suppurative periglossitis, infection with C. pneumoniae did not appear to be associated with specific clinical signs in snakes. Of seven snakes treated with 5 mg/kg marbofloxacin IM once daily, five became PCR negative for C. pneumoniae following treatment, whereas one animal remained positive and one snake was lost to follow-up.

Hepatitis B Infection, Viral Load and Resistance in HIV-Infected Patients in Mozambique and Zambia.

BACKGROUND Few data on the virological determinants of hepatitis B virus (HBV) infection are available from southern Africa. METHODS We enrolled consecutive HIV-infected adult patients initiating antiretroviral therapy (ART) at two urban clinics in Zambia and four rural clinics in Northern Mozambique between May 2013 and August 2014. HBsAg screening was performed using the Determine® rapid test. Quantitative real-time PCR and HBV sequencing were performed in HBsAg-positive patients. Risk factors for HBV infection were evaluated using Chi-square and Mann-Whitney tests and associations between baseline characteristics and high level HBV replication explored in multivariable logistic regression. RESULTS Seventy-eight of 1,032 participants in Mozambique (7.6%, 95% confidence interval [CI]: 6.1-9.3) and 90 of 797 in Zambia (11.3%, 95% CI: 9.3-13.4) were HBsAg-positive. HBsAg-positive individuals were less likely to be female compared to HBsAg-negative ones (52.3% vs. 66.1%, p<0.001). Among 156 (92.9%) HBsAg-positive patients with an available measurement, median HBV viral load was 13,645 IU/mL (interquartile range: 192-8,617,488 IU/mL) and 77 (49.4%) had high values (>20,000 UI/mL). HBsAg-positive individuals had higher levels of ALT and AST compared to HBsAg-negative ones (both p<0.001). In multivariable analyses, male sex (adjusted odds ratio: 2.59, 95% CI: 1.22-5.53) and CD4 cell count below 200/μl (2.58, 1.20-5.54) were associated with high HBV DNA. HBV genotypes A1 (58.8%) and E (38.2%) were most prevalent. Four patients had probable resistance to lamivudine and/or entecavir. CONCLUSION One half of HBsAg-positive patients demonstrated high HBV viremia, supporting the early initiation of tenofovir-containing ART in HIV/HBV-coinfected adults.

Lichtheimia Infection in a Lymphoma Patient: Case Report and a Brief Review of the Available Diagnostic Tools

We describe the case of a patient with a T-lymphoblastic lymphoma whose disseminated mucormycosis was diagnosed with delay, and we address the diagnostic and therapeutic decision-making process and review the diagnostic workup of patients with potential IFD. The diagnosis was delayed despite a suggestive radiological presentation of the patient's pulmonary lesion. The uncommon risk profile (T-lymphoblastic lymphoma, short neutropenic phases) wrongly led to a low level of suspicion. The diagnosis was also hampered by the lack of indirect markers for infections caused by Mucorales, the low sensitivity of both fungal culture and panfungal PCR, and the limited availability of species-specific PCR. A high level of suspicion of IFD is needed, and aggressive diagnostic procedures should be promptly initiated even in apparently low-risk patients with uncommon presentations. The extent of the analytical workup should be decided on a case-by-case base. Diagnostic tests such as the galactomannan and β-D-glucan test and/or PCR on biological material followed by sequencing should be chosen according to their availability and after evaluation of their specificity and sensitivity. In high-risk patients, preemptive therapy with a broad-spectrum mould-active antifungal agent should be started before definitive diagnostic findings become available.

Dynamics of the Developing Chick Chorioallantoic Membrane Assessed by Stereology, Allometry, Immunohistochemistry and Molecular Analysis.

The chick chorioallantoic membrane (CAM) is a widely used model for the study of angiogenesis, tumour growth, as well as drug efficacy. In spite of this, little is known about the developmental alteration from its appearance to the time of hatching. In the current study the CAM has been studied by classical stereology and allometry. Expression levels of selected angiogenesis-related molecules were estimated by RT-PCR and cell dynamics assessed by proliferation and apoptosis assays. Absolute CAM volume increased from a low of 0.47 ± 0.11 cm3 at embryonic day 8 (E8) to a high of 2.05 ± 0.27 cm3 at E18, and then decreased to 1.6 ± 0.47 cm3 at E20. On allometric analysis, three growth phases were identifiable. Between E8-13 (phase I), the CAM grew fastest; moderately in phase II (E13-18) but was regressing in phase III (E18-20). The chorion, the mesenchyme and the allantoic layers grew fastest in phase I, but moderately in phase II. The mesenchyme grew slowly in phase III while the chorion and allantois were regressing. Chorionic cell volume increased fastest in phase I and was regressing in phase III. Chorionic capillaries grew steadily in phase I and II but regressed in phase III. Both the chorion and the allantois grew by intrinsic cell proliferation as well as recruitment of cells from the mesenchyme. Cell proliferation was prominent in the allantois and chorion early during development, declined after E17 and apoptosis started mainly in the chorion from E14. VEGFR2 expression peaked at E11 and declined steadily towards E20, VEGF peaked at E13 and E20 while HIF 1α had a peak at E11 and E20. Studies targeting CAM growth and angiogenesis need to take these growth phases into consideration.