Neuropathological and molecular comparison between clinical and asymptomatic bovine spongiform encephalopathy cases

Interest in the proper neuropathological and molecular characterization of bovine spongiform encephalopathy (BSE) has increased since asymptomatic and atypical cases were detected in the cattle population by active disease surveillance. In this respect we investigated a total of 95 confirmed BSE cases originating from different active and passive surveillance categories (clinical suspects, emergency-slaughter, fallen stock and routinely slaughter) in Switzerland for their neuropathological and molecular phenotype. We looked for measurable differences between these categories in lesion profile, severity of spongiform change, degree of astrocytosis as well as immunohistochemical and molecular patterns of the disease-associated isoform of the prion protein (PrPd) in the caudal brainstem. Our results indicate significantly higher intensities of spongiform change in clinically affected compared to asymptomatic BSE cases. Similar effects were in trend observed for the intensities of PrPd deposition and astrocytosis, whereas the frequencies of morphological PrPd types and the molecular patterns in Western immunoblot were not different. Importantly, none of the animals included in this study revealed features of atypical BSE. Taken together, this study suggests that both clinically affected as well as asymptomatic Swiss BSE cases in cattle share the neuropathological and molecular phenotype of classical BSE and that asymptomatic classical BSE cases are at a pre-clinical stage of the disease rather than representing a true sub-clinical form of BSE.

Atypical scrapie in a Swiss goat and implications for transmissible spongiform encephalopathy surveillance

Different types of transmissible spongiform encephalopathies (TSEs) affect sheep and goats. In addition to the classical form of scrapie, both species are susceptible to experimental infections with the bovine spongiform encephalopathy (BSE) agent, and in recent years atypical scrapie cases have been reported in sheep from different European countries. Atypical scrapie in sheep is characterized by distinct histopathologic lesions and molecular characteristics of the abnormal scrapie prion protein (PrP(sc)). Characteristics of atypical scrapie have not yet been described in detail in goats. A goat presenting features of atypical scrapie was identified in Switzerland. Although there was no difference between the molecular characteristics of PrP(sc) in this animal and those of atypical scrapie in sheep, differences in the distribution of histopathologic lesions and PrP(sc) deposition were observed. In particular the cerebellar cortex, a major site of PrP(sc) deposition in atypical scrapie in sheep, was found to be virtually unaffected in this goat. In contrast, severe lesions and PrP(sc) deposition were detected in more rostral brain structures, such as thalamus and midbrain. Two TSE screening tests and PrP(sc) immunohistochemistry were either negative or barely positive when applied to cerebellum and obex tissues, the target samples for TSE surveillance in sheep and goats. These findings suggest that such cases may have been missed in the past and could be overlooked in the future if sampling and testing procedures are not adapted. The epidemiological and veterinary public health implications of these atypical cases, however, are not yet known.

Continuous monitoring of bovine spongiform encephalopathy rapid test performance by weak positive tissue controls and quality control charts

Bovine spongiform encephalopathy (BSE) rapid tests and routine BSE-testing laboratories underlie strict regulations for approval. Due to the lack of BSE-positive control samples, however, full assay validation at the level of individual test runs and continuous monitoring of test performance on-site is difficult. Most rapid tests use synthetic prion protein peptides, but it is not known to which extend they reflect the assay performance on field samples, and whether they are sufficient to indicate on-site assay quality problems. To address this question we compared the test scores of the provided kit peptide controls to those of standardized weak BSE-positive tissue samples in individual test runs as well as continuously over time by quality control charts in two widely used BSE rapid tests. Our results reveal only a weak correlation between the weak positive tissue control and the peptide control scores. We identified kit-lot related shifts in the assay performances that were not reflected by the peptide control scores. Vice versa, not all shifts indicated by the peptide control scores indeed reflected a shift in the assay performance. In conclusion these data highlight that the use of the kit peptide controls for continuous quality control purposes may result in unjustified rejection or acceptance of test runs. However, standardized weak positive tissue controls in combination with Shewhart-CUSUM control charts appear to be reliable in continuously monitoring assay performance on-site to identify undesired deviations.

A Targeted Survey for Scrapie in Jos Plateau State, Nigeria

Scrapie, a disease of sheep and goats with a progressive course and fatal outcome, has not been identified in Nigeria. Anecdotal scrapie reports by livestock workers abound. Livestock diseases like scrapie form huddles in livestock economics of countries. For 8 months we surveyed for scrapie targeting emergency/casualty slaughter sheep and goats in Jos, Nigeria. We clinically examined 510 sheep and 608 goats of local breeds, aged from 12 months to 5 years. In total 31 (5.10%) goats and no sheep were clinically suspicious for scrapie. Caudal brainstem tissues of suspect animals collected postmortem were analyzed for the disease specific form of the prion protein, PrPSc, using Bio-Rad’s TeSeE ELISA rapid test kit. No sample was positive for scrapie. Fluorescent antibody test for rabies and H&E staining on samples were carried out for differential diagnosis. These showed no pathological lesions indicative for neurological disease. While our findings do not exclude the presence of scrapie in Jos, we demonstrate that targeted sampling of small ruminants for neuroinfectious disease is feasible in developing countries, pointing to the possibility of implementing such a monitoring scheme in Nigeria to prevent economic losses in small ruminant livestock as scrapie caveats from endemic countries have shown.

A pilot study for targeted surveillance of bovine spongiform encephalopathy in Nigeria.

Bovine spongiform encephalopathy (BSE), popularly known as 'mad cow disease', led to an epidemic in Europe that peaked in the mid-1990s. Its impact on developing countries, such as Nigeria, has not been fully established as information on livestock and surveillance has eluded those in charge of this task. The BSE risk to Nigeria's cattle population currently remains undetermined, which has resulted in international trade restrictions on commodities from the cattle population. This is mainly because of a lack of updated BSE risk assessments and disease surveillance data. To evaluate the feasibility of BSE surveillance in Nigeria, we carried out a pilot study targeting cattle that were presented for emergency or casualty slaughter. In total, 1551 cattle of local breeds, aged 24 months and above were clinically examined. Ataxia, recumbency and other neurological signs were topmost on our list of criteria. A total of 96 cattle, which correspond to 6.2%, presented clinical signs that supported a suspect of BSE. The caudal brainstem tissues of these animals were collected post-mortem and analysed for the disease-specific form of the prion protein using a rapid test approved by the International Animal Health Organization (OIE). None of the samples were positive for BSE. Although our findings do not exclude the presence of BSE in Nigeria, they do demonstrate that targeted sampling of clinically suspected cases of BSE is feasible in developing countries. In addition, these findings point to the possibility of implementing clinical monitoring schemes for BSE and potentially other diseases with grave economic and public health consequences.

Quantitative analysis of the native presynaptic cytomatrix by cryoelectron tomography

The presynaptic terminal contains a complex network of filaments whose precise organization and functions are not yet understood. The cryoelectron tomography experiments reported in this study indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations are consistent with an exocytosis model in which vesicles are first anchored by long (>5 nm) tethers that give way to multiple short tethers once vesicles enter the readily releasable pool. The formation of short tethers was inhibited by tetanus toxin, indicating that it depends on soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor complex assembly. Vesicles were extensively interlinked via a set of connectors that underwent profound rearrangements upon synaptic stimulation and okadaic acid treatment, suggesting a role of these connectors in synaptic vesicle mobilization and neurotransmitter release.

Distinct reactivities of interleukin-4-specific antibodies with recombinant and native canine interleukin-4 in various assays

Interleukin 4 (IL-4) plays a central role in immune responses to parasites and allergens. IL-4 drives the differentiation of naive T cells into Th2 cells and regulates immunoglobulin class switching to IgE.Little is known about the role of IL-4 in canine allergies and parasite infections. Most of the information derives from measurement of IL-4 mRNA expression in dog tissues, but detection of IL-4 protein has been difficult so far, probably due to low sensitivity of available methods. Antibodies (Ab) specific for canine IL-4 are available from various sources, but these Ab have been produced against recombinant Escherichia coli-expressed canine IL-4 and there is only limited information on their reactivities with native canine IL-4. Therefore, in the present study, we tested six available canine IL-4-specific Ab for their reactivities with recombinant canine IL-4 expressed in E. coli (rec.IL-4) or in mammalian cells (mam.IL-4), and with supernatants from stimulated canine peripheral blood mononuclear cells (PBMCs) using several detection methods, including Western blotting, ELISA, cytokine bead assay, and intracellular IL-4 staining. Additionally, we tested a bovine IL-4-specific antibody that has been previously shown to cross-react with canine IL-4. All tested Ab except anti-bovine IL-4 reacted with rec.IL-4, and most of them reacted with mam.IL-4. However, only the cytokine bead assay was sensitive enough to allow the detection of IL-4 in supernatants of canine PBMCs.

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca(2+) release and store-operated Ca(2+) entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1microM) and the Src inhibitor PP2 (10microM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca(2+) transients were reduced by imatinib and/or PP2. Ca(2+) transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca(2+) transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCalpha catalytic activity and PKCalpha co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca(2+) influx was reduced by complexing extracellular Ca(2+) with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca(2+) transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

C-reactive protein production in term human placental tissue

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Vaccination with microneme protein NcMIC4 increases mortality in mice inoculated with Neospora caninum

NcMIC4 is a Neospora caninum microneme protein that has been isolated and purified on the basis of its unique lactose-binding properties. We have shown that this protein binds to galactosyl residues of lactose; antibodies directed against NcMIC4 inhibit host cell interactions in vitro, thus making it a vaccine candidate. Because of this feature, NcMIC4 was first purified on a larger scale in its native, functionally active form using lactose-agarose affinity chromatography. Second, NcMIC4 was expressed in Escherichia coli as a histidine-tagged recombinant protein (recNcMIC4) and purified through Ni-affinity chromatography. Third, NcMIC4 cDNA was cloned into the mammalian pcDNA3.1 DNA vector and expression was confirmed upon transfection of Vero cells in vitro. For vaccination studies, we employed the murine cerebral infection model based on C57Bl/6 mice, employing experimental groups of 10 mice each. Two groups were injected intraperitoneally with purified native NcMIC4 and recNcMIC4, respectively, employing RIBI adjuvant. The third group was vaccinated intramuscularly with pcDNA-NcMIC4. Control groups included an infection control, an adjuvant control, and a pcDNA3.1 control group. Following 3 injections at 4-wk intervals, mice were challenged by i.p. inoculation of 2 x 10(6) N. caninum tachyzoites (Nc-1 isolate). During the course of parasite challenge (3 wk), mice from the 3 different test groups showed varying degrees of symptoms bearing a semblance to neosporosis, i.e., walking disorder, rounded back, apathy, and paralysis of the hind limbs. Control groups showed no symptoms at all. Most notably, vaccination with pcDNA-MIC4 proved antiprotective, with 60% of mice succumbing to infection within 3 wk, and all mice lacking a measurable anti-NcMIC4 IgG response. NcMIC4 in its native form elicited a substantial humoral IgG1 immune response and a reduction in cerebral parasite load compared to the controls, but 20% of mice succumbed to infection. Vaccination with recNcMIC4 also resulted in 20% of mice dying; however, in this group, cerebral parasite load was similar to the controls, and recNcMIC4 vaccination elicited a mixed IgG1/IgG2 response. In conclusion, vaccines based on NcMIC4, especially pcDNA-NcMIC4, render mice more susceptible to cerebral disease upon challenge with N. caninum tachyzoites.

Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Probing the function of ionotropic and G protein-coupled receptors in surface-confined membranes

This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.