A single-centre cohort of patients with systemic light chain AL-amyloidosis treated with conventional chemotherapy or with high-dose chemotherapy and autologous stem cell transplantation.

BACKGROUND High-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) has been reported to confer better prognosis in systemic light chain AL-amyloidosis as compared with conventional chemotherapy. However, only limited data are available so far on treatment and outcome of AL-amyloidosis patients in Switzerland. METHODS Within a single-centre cohort of patients with biopsy confirmed AL-amyloidosis diagnosed between January 1995 and December 2012, we aimed to investigate treatment effects in patients treated with conventional chemotherapy versus HDCT with ASCT. RESULTS We identified 50 patients with AL-amyloidosis treated with conventional chemotherapy and 13 patients who received HDCT with ASCT. Clinical characteristics differed between the groups for the age of the patients (59 years for patients with ASCT/HDCT vs 69 years; p= 0.0006) and the troponin-T value (0.015 μg/l vs 0.08 μg/l; p = 0.0279). Patients with ASCT showed a trend towards better overall survival, with median survival not yet reached compared with 53 months in patients on conventional chemotherapy (p = 0.0651). CONCLUSION Our results suggest that light chain AL-amyloidosis patients considered fit to undergo HDCT and ASCT may have a better outcome than patients treated exclusively with conventional chemotherapy regimens; however, the better performance status of patients receiving HDCT may have added to this treatment effect.

Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

Ultrasound-assisted versus conventional catheter-directed thrombolysis for acute iliofemoral deep vein thrombosis

BACKGROUND For patients with acute iliofemoral deep vein thrombosis, it remains unclear whether the addition of intravascular high-frequency, low-power ultrasound energy facilitates the resolution of thrombosis during catheter-directed thrombolysis. METHODS AND RESULTS In a controlled clinical trial, 48 patients (mean age 50±21 years, 52% women) with acute iliofemoral deep vein thrombosis were randomized to receive ultrasound-assisted catheter-directed thrombolysis (N=24) or conventional catheter-directed thrombolysis (N=24). Thrombolysis regimen (20 mg r-tPA over 15 hours) was identical in all patients. The primary efficacy end point was the percentage of thrombus load reduction from baseline to 15 hours according to the length-adjusted thrombus score, obtained from standardized venograms and evaluated by a core laboratory blinded to group assignment. The percentage of thrombus load reduction was 55%±27% in the ultrasound-assisted catheter-directed thrombolysis group and 54%±27% in the conventional catheter-directed thrombolysis group (P=0.91). Adjunctive angioplasty and stenting was performed in 19 (80%) patients and in 20 (83%) patients, respectively (P>0.99). Treatment-related complications occurred in 3 (12%) and 2 (8%) patients, respectively (P>0.99). At 3-month follow-up, primary venous patency was 100% in the ultrasound-assisted catheter-directed thrombolysis group and 96% in the conventional catheter-directed thrombolysis group (P=0.33), and there was no difference in the severity of the post-thrombotic syndrome (mean Villalta score: 3.0±3.9 [range 0-15] versus 1.9±1.9 [range 0-7]; P=0.21), respectively. CONCLUSIONS In this randomized controlled clinical trial of patients with acute iliofemoral deep vein thrombosis treated with a fixed-dose catheter thrombolysis regimen, the addition of intravascular ultrasound did not facilitate thrombus resolution. CLINICAL TRIAL REGISTRATION URL http://www.clinicaltrials.gov. Unique identifier: NCT01482273.

Infection rate of emergency bolt-kit vs. non-emergency conventional implanted silver bearing external ventricular drainage catheters.

BACKGROUND Bolt-kit systems are increasingly used as an alternative to conventional external cerebrospinal fluid (CSF) drainage systems. Since 2009 we regularly utilize bolt-kit external ventricular drainage (EVD) systems with silver-bearing catheters inserted manually with a hand drill and skull screws for emergency ventriculostomy. For non-emergency situations, we use conventional ventriculostomy with subcutaneous tunneled silver-bearing catheters, performed in the operating room with a pneumatic drill. This retrospective analysis compared the two techniques in terms of infection rates. METHODS 152 patients (aged 17-85 years, mean=55.4 years) were included in the final analysis; 95 received bolt-kit silver-bearing catheters and 57 received conventionally implanted silver-bearing catheters. The primary endpoint combined infection parameters: occurrence of positive CSF culture, colonization of catheter tips, or elevated CSF white blood cell counts (>4/μl). Secondary outcome parameters were presence of microorganisms in CSF or on catheter tips. Incidence of increased CSF cell counts and number of patients with catheter malposition were also compared. RESULTS The primary outcome, defined as analysis of combined infection parameters (occurrence of either positive CSF culture, colonization of the catheter tips or raised CSF white blood cell counts >4/μl)was not significantly different between the groups (58.9% bolt-kit group vs. 63.2% conventionally implanted group, p=0.61, chi-square-test). The bolt-kit group was non-inferior and not superior to the conventional group (relative risk reduction of 6.7%; 90% confidence interval: -19.9% to 25.6%). Secondary outcomes showed no statistically significant difference in the incidence of microorganisms in CSF (2.1% bolt-kit vs. 5.3% conventionally implanted; p=0.30; chi-square-test). CONCLUSIONS This analysis indicates that silver-bearing EVD catheters implanted with a bolt-kit system outside the operating room do not significantly elevate the risk of CSF infection as compared to conventional implant methods.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.

Seroprevalence of hepatitis E virus in domestic pigs and wild boars in Switzerland.

Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6-21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV-specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.

CT coronary angiography versus conventional invasive coronary angiography - the view of the referring physician

PURPOSE Assessment of experience gained by local referring physicians with the procedure of coronary computed tomographic angiography (CCTA) in the everyday clinical routine. MATERIALS AND METHODS A 25-item questionnaire was sent to 179 physicians, who together had referred a total of 1986 patients for CCTA. They were asked about their experience to date with CCTA, the indications for coronary imaging, and their practice in referring patients for noninvasive CCTA or invasive catheter angiography. RESULTS 53 questionnaires (30 %) were assessable, corresponding to more than 72 % of the patients referred. Of the referring physicians who responded, 94 % saw a concrete advantage of CCTA in the treatment of patients, whereby 87 % were 'satisfied' or 'very satisfied' with the reporting. For excluding coronary heart disease (CHD) where there was a low pre-test probability of disease, the physicians considered CCTA to be superior to conventional coronary diagnosis (4.2 on a scale of 1 - 5) and vice versa for acute coronary syndrome (1.6 of 5). The main reasons for unsuitability of CCTA for CT diagnosis were claustrophobia and the absence of a sinus rhythm. The level of exposure to radiation in CCTA was estimated correctly by only 42 % of the referring physicians. 90 % of the physicians reported that their patients evaluated their coronary CT overall as 'positive' or 'neutral', while 87 % of the physicians whose patients had undergone both procedures reported that the patients had experienced CCTA as the less disagreeable of the two. CONCLUSION CCTA is accepted by the referring physicians as an alternative imaging procedure for the exclusion of CHD and received a predominantly positive assessment from both the referring physicians and the patients.

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

Comparison of morphological and rheological conditions between conventional and eversion carotid endarterectomy using computational fluid dynamics - a pilot study

PURPOSE To compare postoperative morphological and rheological conditions after eversion carotid endarterectomy versus conventional carotid endarterectomy using computational fluid dynamics. BASIC METHODS Hemodynamic metrics (velocity, wall shear stress, time-averaged wall shear stress and temporal gradient wall shear stress) in the carotid arteries were simulated in one patient after conventional carotid endarterectomy and one patient after eversion carotid endarterectomy by computational fluid dynamics analysis based on patient specific data. PRINCIPAL FINDINGS Systolic peak of the eversion carotid endarterectomy model showed a gradually decreased pressure along the stream path, the conventional carotid endarterectomy model revealed high pressure (about 180 Pa) at the carotid bulb. Regions of low wall shear stress in the conventional carotid endarterectomy model were much larger than that in the eversion carotid endarterectomy model and with lower time-averaged wall shear stress values (conventional carotid endarterectomy: 0.03-5.46 Pa vs. eversion carotid endarterectomy: 0.12-5.22 Pa). CONCLUSIONS Computational fluid dynamics after conventional carotid endarterectomy and eversion carotid endarterectomy disclosed differences in hemodynamic patterns. Larger studies are necessary to assess whether these differences are consistent and might explain different rates of restenosis in both techniques.

Serum folate, cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute, chronic or subclinical Lawsonia intracellularis infection.

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B(9)) and cobalamin (vitamin B(12)) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

Comparison of the diagnostic significance of clinical, radiographic and ultrasonographic results after an experimental aerosol infection of pigs with Actinobacillus pleuropneumoniae

Scoring schemes for clinical, ultrasonographic and radiographic findings in pigs were developed based upon a standardized animal model for Actinobacillus pleuropneumoniae infection.The results of these methods were compared to each other as well as with the corresponding pathomorphological findings during necropsy. Altogether 69 pigs of different breeding lines (Hampshire, Pietrain and German Landrace were examined. Positive correlations were found between the results of all three methods as well as with the necropsy scores (p <0.0001). Different pathomorphological findings were detected either by radiographic or by ultrasonographic examination dependent upon the type of lung tissue alterations: Alterations of the pleura as well as sequestration of lung tissue on the lung surface could be clearly identified during the ultrasonographic examination while deep tissue alterations with no contact to the lung surface could be detected reliably by radiographic examination. Both methods complement each other, and the application of a combined ultrasonographic and radiographic examination of the thorax allows a comprehensive inspection of the lung condition. Particularly during the acute phase of the disease the extent of lung tissue damage can be estimated more precisely than by clinical examination alone.

Comparative proteomic analysis of lung tissue from guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a particularly severe form of leptospirosis. LPHS is increasingly recognized in both humans and animals and is characterized by rapidly progressive intra-alveolar haemorrhage leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood which hampers the application of effective treatment regimes. In this study a 2-D guinea pig proteome lung map was created and used to investigate the pathogenic mechanisms of LPHS. Comparison of lung proteomes from infected and non-infected guinea pigs via differential in-gel electrophoresis revealed highly significant differences in abundance of proteins contained in 130 spots. Acute phase proteins were the largest functional group amongst proteins with increased abundance in LPHS lung tissue, and likely reflect a local and/or systemic host response to infection. The observed decrease in abundance of proteins involved in cytoskeletal and cellular organization in LPHS lung tissue further suggests that infection with pathogenic Leptospira induces changes in the abundance of host proteins involved in cellular architecture and adhesion contributing to the dramatically increased alveolar septal wall permeability seen in LPHS. BIOLOGICAL SIGNIFICANCE The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, the comparative proteomic analysis of lung tissue from experimentally infected guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) revealed a decrease in abundance of proteins involved in cellular architecture and adhesion, suggesting that loss or down-regulation of cytoskeletal and adhesion molecules plays an important role in the pathogenesis of LPHS. A publically available guinea pig lung proteome map was constructed to facilitate future pulmonary proteomics in this species.

In vivo degradation of magnesium plate/screw osteosynthesis implant systems: Soft and hard tissue response in a calvarial model in miniature pigs.

Biodegradable magnesium plate/screw osteosynthesis systems were implanted on the frontal bone of adult miniature pigs. The chosen implant geometries were based on existing titanium systems used for the treatment of facial fractures. The aim of this study was to evaluate the in vivo degradation and tissue response of the magnesium alloy WE43 with and without a plasma electrolytic surface coating. Of 14 animals, 6 received magnesium implants with surface modification (coated), 6 without surface modification (uncoated), and 2 titanium implants. Radiological examination of the skull was performed at 1, 4, and 8 weeks post-implantation. After euthanasia at 12 and 24 weeks, X-ray, computed tomography, and microfocus computed tomography analyses and histological and histomorphological examinations of the bone/implant blocks were performed. The results showed a good tolerance of the plate/screw system without wound healing disturbance. In the radiological examination, gas pocket formation was found mainly around the uncoated plates 4 weeks after surgery. The micro-CT and histological analyses showed significantly lower corrosion rates and increased bone density and bone implant contact area around the coated screws compared to the uncoated screws at both endpoints. This study shows promising results for the further development of coated magnesium implants for the osteosynthesis of the facial skeleton.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.