Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Neuropathological survey of fallen stock: Active surveillance reveals high prevalence of encephalitic listeriosis in small ruminants

This paper describes the prevalence of brain lesions in the Swiss fallen stock population of small ruminants. 3075 whole brains (75% sheep, 25% goats) were collected as part of a year-long active survey of transmissible spongiform encephalopathies (TSEs) in small ruminants conducted by the Swiss authorities between July 2004 and July 2005. All fallen stock brains were systematically examined by histopathology to obtain reliable data on histologically identifiable brain lesions. Lesions were found in an unexpectedly high number of animals (8.1% of all examined brains). A wide spectrum of diseases was detected showing that this approach provides an excellent opportunity to screen for the prevalence of neurological diseases. Encephalitic listeriosis was by far the most frequent cause of CNS lesions in both species and its prevalence was unexpectedly high when compared to notified confirmed cases. In conclusion, the prevalence of listeriosis as estimated by passive surveillance based on the notification of clinical suspects has been underestimated in the past.

Noninvasive three-dimensional assessment of femoroacetabular impingement

A CT-based method ("HipMotion") for the noninvasive three-dimensional assessment of femoroacetabular impingement (FAI) was developed, validated, and applied in a clinical pilot study. The method allows for the anatomically based calculation of hip range of motion (ROM), the exact location of the impingement zone, and the simulation of quantified surgical maneuvers for FAI. The accuracy of HipMotion was 0.7 +/- 3.1 degrees in a plastic bone setup and -5.0 +/- 5.6 degrees in a cadaver setup. Reliability and reproducibility were excellent [intraclass correlation coefficient (ICC) > 0.87] for all measures except external rotation (ICC = 0.48). The normal ROM was determined from a cohort of 150 patients and was compared to 31 consecutive hips with FAI. Patients with FAI had a significantly decreased flexion, internal rotation, and abduction in comparison to normal hips (p < 0.001). Normal hip flexion and internal rotation are generally overestimated in a number of orthopedic textbooks. HipMotion is a useful tool for further assessment of impinging hips and for appropriate planning of the necessary amount of surgical intervention, which represents the basis for future computer-assisted treatment of FAI with less invasive surgical approaches, such as hip arthroscopy.

Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway

Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.

Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukaryotic elongation factor 1A

In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.

Reactivation of rheumatoid arthritis after pregnancy: increased phagocyte and recurring lymphocyte gene activity

OBJECTIVE: Pregnancy is associated with reduced disease activity in rheumatoid arthritis (RA) and frequently with disease exacerbation after delivery. This study was undertaken to generate a systematic overview of the molecular mechanisms related to disease remission and postpartum reactivation. METHODS: Transcriptomes of peripheral blood mononuclear cells (PBMCs) were generated from RA patients and healthy women by transcription profiling during the third trimester and 24 weeks after delivery. For functional interpretation, signatures of highly purified immune cells as well as Kyoto Encyclopedia of Genes and Genomes pathway annotations were used as a reference. RESULTS: Only minor differences in gene expression in PBMCs during pregnancy were found between RA patients and controls. In contrast, RA postpartum profiles presented the most dominant changes. Systematic comparison with expression signatures of monocytes, T cells, and B cells in healthy donors revealed reduced lymphocyte and elevated monocyte gene activity during pregnancy in patients with RA and in controls. Monocyte activity decreased after delivery in controls but persisted in RA patients. Furthermore, analysis of 32 immunologically relevant cellular pathways demonstrated a significant additional activation of genes related to adhesion, migration, defense of pathogens, and cell activation, including Notch, phosphatidylinositol, mTOR, Wnt, and MAPK signaling, in RA patients postpartum. CONCLUSION: Our findings indicate that innate immune functions play an important role in postpartum reactivation of arthritis. However, this may depend not only on the monocyte itself, but also on the recurrence of lymphocyte functions postpartum and thus on a critical interaction between both arms of the immune system.

Eicosanoids in exhaled breath condensates in the assessment of childhood asthma

The value of measurements of eicosanoids in exhaled breath condensate (EBC) for the evaluation of childhood asthma is still inconclusive most likely because of the limited value of the methods used. In this case-control study in 48 asthmatic and 20 healthy children, we aimed to characterize the baseline profile of the inflammatory mediators cysteinyl leukotrienes (cysLTs), 9(alpha)11(beta)PGF(2), PGE(2), PGF(2alpha), 8-isoprostane (8-iso-PGF(2alpha)) within EBC in asthmatic compared with healthy children using new methods. In addition, we investigated their relation to other inflammatory markers. The assessment included collection of EBC, measurement of fractional exhaled nitric oxide (FE(NO)) and evaluation of urinary excretion of leukotriene E(4.) cysLTs were measured directly in EBC by radioimmunoassay and prostanoids were measured using gas chromatography negative-ion chemical ionization mass spectrometry. Only cysLT levels were significantly higher in asthmatic compared with healthy children (p = 0.002). No significant differences in cysLTs were found between steroid naïve and patients receiving inhaled corticosteroids. In contrast, FE(NO) was significantly higher in steroid naïve compared with steroid-treated asthmatic and healthy children (p = 0.04 and 0.024, respectively). The diagnostic accuracy of cysLTs in EBC for asthma was 73.6% for the whole group and 78.2% for steroid-naïve asthmatic children. The accuracy to classify asthmatic for FE(NO) was poor (62.9%) for the whole group, but improved to 79.9% when only steroid-naïve asthmatic children were taken into consideration. cysLTs in EBC is an inflammatory marker which distinguishes asthmatics, as a whole group, from healthy children.