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Aim: Ventilation with pure oxygen (hyperoxic ventilation: HV) is thought to decrease whole body oxygen consumption (VO(2)). However, the validity and impact of this phenomenon remain ambiguous; until now, under hyperoxic conditions, VO(2) has only been determined by the reverse Fick principle, a method with inherent methodological problems. The goal of this study was to determine changes of VO(2), carbon dioxide production (VCO(2)), and the respiratory quotient (RQ) during normoxic and hyperoxic ventilation, using a metabolic monitor.
Methods: After providing signed informed consent and institutional acceptance, 14 healthy volunteers were asked to sequentially breathe room air, pure oxygen, and room air again. VO(2), VCO(2), RQ, and energy expenditure (EE) were determined by indirect calorimetry using a modified metabolic monitor during HV.
Results: HV reduced VO(2) from 3.4 (3.0/4.0) mL/kg/min to 2.8 (2.5/3.6) mL/kg/min (P < 0.05), whereas VCO(2) remained constant (3.0 [2.6/3.6] mL/kg/min versus 3.0 [2.6/3.5] mL/kg/min, n.s.). After onset of HV, RQ increased from 0.9 (0.8/0.9) to 1.1 (1.0/1.1). Most changes during HV were immediately reversed during subsequent normoxic ventilation.
Conclusion: HV not only reduces VO(2), but also increases the respiratory quotient. This might be interpreted as an indicator of the substantial metabolic changes induced by HV. However, the impact of this phenomenon requires further study.
Hemorrhagic shock leads to hepatic hypoperfusion and activation of mitogen-activated stress kinases (MAPK) like c-Jun N-terminal kinase (JNK) 1 and 2. Our aim was to determine whether mitochondrial dysfunction leading to hepatic necrosis and apoptosis after hemorrhage/resuscitation (H/R) was dependent on JNK2. Under pentobarbital anesthesia, wildtype (WT) and JNK2 deficient (KO) mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R, ALT in WT-mice increased from 130 U/L to 4800 U/L. In KO-mice, ALT after H/R was blunted to 1800 U/l (P < 0.05). Necrosis, caspase-3 activity and ROS were all substantially decreased in KO compared to WT mice after H/R. After sham operation, intravital microscopy revealed punctate mitochondrial staining by rhodamine 123 (Rh123), indicating normal mitochondrial polarization. At 4 h after H/R, Rh123 staining became dim and diffuse in 58% of hepatocytes, indicating depolarization and onset of the mitochondrial permeability transition (MPT). By contrast, KO mice displayed less depolarization after H/R (23%, P < 0.05). In conclusion, JNK2 contributes to MPT-mediated liver injury after H/R.
Orthotopic liver transplantation in human-immunodeficiency-virus-positive patients in Germany
(2012)
Objectives: This summary evaluates the outcomes of orthotopic liver transplantation (OLT) of HIV-positive patients in Germany.
Methods: Retrospective chart analysis of HIV-positive patients, who had been liver-transplanted in Germany between July 1997 and July 2011.
Results: 38 transplantations were performed in 32 patients at 9 German transplant centres. The reasons for OLT were end-stage liver disease (ESLD) and/or liver failure due to hepatitis C (HCV) (n = 19), hepatitis B (HBV) (n = 10), multiple viral infections of the liver (n = 2) and Budd-Chiari-Syndrome. In July 2011 19/32 (60%) of the transplanted patients were still alive with a median survival of 61 months (IQR (interquartile range): 41-86 months). 6 patients had died in the early post-transplantation period from septicaemia (n = 4), primary graft dysfunction (n = 1), and intrathoracal hemorrhage (n = 1). Later on 7 patients had died from septicaemia (n = 2), delayed graft failure (n = 2), recurrent HCC (n = 2), and renal failure (n = 1). Recurrent HBV infection was efficiently prevented in 11/12 patients; HCV reinfection occurred in all patients and contributed considerably to the overall mortality.
Conclusions: Overall OLT is a feasible approach in HIV-infected patients with acceptable survival rates in Germany. Reinfection with HCV still remains a major clinical challenge in HIV/HCV coinfection after OLT.
Objective. To examine the effects of clinical hypnosis versus NLP intervention on the success rate of ECV procedures in comparison to a control group.
Methods. A prospective off-centre randomised trial of a clinical hypnosis intervention against NLP of women with a singleton breech fetus at or after 370/7 (259 days) weeks of gestation and normal amniotic fluid index. All 80 participants heard a 20-minute recorded intervention via head phones. Main outcome assessed was success rate of ECV. The intervention groups were compared with a control group with standard medical care alone (n=122).
Results. A total of 42 women, who received a hypnosis intervention prior to ECV, had a 40.5% (n=17), successful ECV, whereas 38 women, who received NLP, had a 44.7% (n=17) successful ECV (P > 0.05). The control group had similar patient characteristics compared to the intervention groups (P > 0.05). In the control group (n = 122) 27.3% (n = 33) had a statistically significant lower successful ECV procedure than NLP (P = 0.05) and hypnosis and NLP (P = 0.03).
Conclusions. These findings suggest that prior clinical hypnosis and NLP have similar success rates of ECV procedures and are both superior to standard medical care alone.
To compare the development of the auditory system in hearing and completely acoustically deprived animals, naive congenitally deaf white cats (CDCs) and hearing controls (HCs) were investigated at different developmental stages from birth till adulthood. The CDCs had no hearing experience before the acute experiment. In both groups of animals, responses to cochlear implant stimulation were acutely assessed. Electrically evoked auditory brainstem responses (E-ABRs) were recorded with monopolar stimulation at different current levels. CDCs demonstrated extensive development of E-ABRs, from first signs of responses at postnatal (p.n.) day 3 through appearance of all waves of brainstem response at day 8 p.n. to mature responses around day 90 p.n.. Wave I of E-ABRs could not be distinguished from the artifact in majority of CDCs, whereas in HCs, it was clearly separated from the stimulus artifact. Waves II, III, and IV demonstrated higher thresholds in CDCs, whereas this difference was not found for wave V. Amplitudes of wave III were significantly higher in HCs, whereas wave V amplitudes were significantly higher in CDCs. No differences in latencies were observed between the animal groups. These data demonstrate significant postnatal subcortical development in absence of hearing, and also divergent effects of deafness on early waves II–IV and wave V of the E-ABR.
Measles virus glycoprotein-based lentiviral targeting vectors that avoid neutralizing antibodies
(2012)
Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of α-MV antibody-positive human plasma. At plasma dilution 1:160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1:80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans.
In our previous work we showed that NGAL, a protein involved in the regulation of proliferation and differentiation, is overexpressed in human breast cancer (BC) and predicts poor prognosis. In neoadjuvant chemotherapy (NACT) pathological complete response (pCR) is a predictor for outcome. The aim of this study was to evaluate NGAL as a predictor of response to NACT and to validate NGAL as a prognostic factor for clinical outcome in patients with primary BC. Immunohistochemistry was performed on tissue microarrays from 652 core biopsies from BC patients, who underwent NACT in the GeparTrio trial. NGAL expression and intensity was evaluated separately. NGAL was detected in 42.2% of the breast carcinomas in the cytoplasm. NGAL expression correlated with negative hormone receptor (HR) status, but not with other baseline parameters. NGAL expression did not correlate with pCR in the full population, however, NGAL expression and staining intensity were significantly associated with higher pCR rates in patients with positive HR status. In addition, strong NGAL expression correlated with higher pCR rates in node negative patients, patients with histological grade 1 or 2 tumors and a tumor size <40 mm. In univariate survival analysis, positive NGAL expression and strong staining intensity correlated with decreased disease-free survival (DFS) in the entire cohort and different subgroups, including HR positive patients. Similar correlations were found for intense staining and decreased overall survival (OS). In multivariate analysis, NGAL expression remained an independent prognostic factor for DFS. The results show that in low-risk subgroups, NGAL was found to be a predictive marker for pCR after NACT. Furthermore, NGAL could be validated as an independent prognostic factor for decreased DFS in primary human BC.
Pre-publication peer review of scientific literature in its present state suffers from a lack of evaluation validity and transparency to the community. Inspired by social networks, we propose a framework for the open exchange of post-publication evaluation to complement the current system. We first formulate a number of necessary conditions that should be met by any design dedicated to perform open scientific evaluation. To introduce our framework, we provide a basic data standard and communication protocol. We argue for the superiority of a provider-independent framework, over a few isolated implementations, which allows the collection and analysis of open evaluation content across a wide range of diverse providers like scientific journals, research institutions, social networks, publishers websites, and more. Furthermore, we describe how its technical implementation can be achieved by using existing web standards and technology. Finally, we illustrate this with a set of examples and discuss further potential.
The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or –inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1′s) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540–580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.
Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.
The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C−/− mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C−/− mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C−/− C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C−/− mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3rd ventricle in JAM-C−/− C57BL/6 mice. Taken together, our study suggests that JAM-C−/− C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.
Background: Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRs) circulating in the blood are an emerging new class of biomarkers. In particular, the serum level of the liver-specific miR-122 might be a clinically useful new parameter in patients with acute or chronic liver disease.
Aim: Here we investigated if the serum level of miR-122 might be a prognostic parameter in patients with liver cirrhosis.
Methods: 107 patients with liver cirrhosis in the test cohort and 143 patients in the validation cohort were prospectively enrolled into the present study. RNA was extracted from the sera obtained at the time of study enrollment and the level of miR-122 was assessed. Serum miR-122 levels were assessed by quantitative reverse-transcription PCR (RT-PCR) and were compared to overall survival time and to different complications of liver cirrhosis.
Results: Serum miR-122 levels were reduced in patients with hepatic decompensation in comparison to patients with compensated liver disease. Patients with ascites, spontaneous bacterial peritonitis and hepatorenal syndrome had significantly lower miR-122 levels than patients without these complications. Multivariate Cox regression analysis revealed that the miR-122 serum levels were associated with survival independently from the MELD score, sex and age.
Conclusions: Serum miR-122 is a new independent marker for prediction of survival of patients with liver cirrhosis.
Background: The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol.
Results: Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose.
Conclusion: A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes and engineering of co-factor imbalances should lead to even higher isobutanol production.
Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de.
Cytokines play an important role in ischemic injury and repair. However, little is known about their prognostic value in cardiovascular disease. The aim of this study was to investigate the prognostic importance of chemokines CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC for the risk of future cardiovascular events in patients with acute coronary syndromes (ACS). Baseline levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined in ACS patients from the Bad Nauheim ACS II registry (n = 609). During the following 200 days, patients were monitored for the occurrence of fatal and non-fatal cardiovascular events. Patients with CCL3/MIP1α, CCL5/RANTES and CCL18/PARC concentrations in the highest tertile were associated with an increased risk of a fatal event during follow-up (HR: 2.19, 95%CI: 1.04–4.61 for CCL3/MIP1α, HR: 3.45, 95%CI: 1.54–7.72 for CCL5/RANTES and HR: 3.14, 95%CI: 1.33–7.46 for CCL18/PARC). This risk was highest for patients with all three biomarkers concentrations in the upper tertile (HR: 2.52, 95%CI: 1.11–5.65). Together with known risk predictors of cardiovascular events, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC combined improved the c-statistics from 0.74 to 0.81 (p = 0.007). In conclusion, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC are independently associated with the risk of short-term mortality in ACS patients. Combining all three biomarkers further increased their prognostic value.
In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca2+-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ1 subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ1−/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ1−/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β1 subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β1 subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.
We study how species richness of arthropods relates to theories concerning net primary productivity, ambient energy, water-energy dynamics and spatial environmental heterogeneity. We use two datasets of arthropod richness with similar spatial extents (Scandinavia to Mediterranean), but contrasting spatial grain (local habitat and country). Samples of ground-dwelling spiders, beetles, bugs and ants were collected from 32 paired habitats at 16 locations across Europe. Species richness of these taxonomic groups was also determined for 25 European countries based on the Fauna Europaea database. We tested effects of net primary productivity (NPP), annual mean temperature (T), annual rainfall (R) and potential evapotranspiration of the coldest month (PETmin) on species richness and turnover. Spatial environmental heterogeneity within countries was considered by including the ranges of NPP, T, R and PETmin. At the local habitat grain, relationships between species richness and environmental variables differed strongly between taxa and trophic groups. However, species turnover across locations was strongly correlated with differences in T. At the country grain, species richness was significantly correlated with environmental variables from all four theories. In particular, species richness within countries increased strongly with spatial heterogeneity in T. The importance of spatial heterogeneity in T for both species turnover across locations and for species richness within countries suggests that the temperature niche is an important determinant of arthropod diversity. We suggest that, unless climatic heterogeneity is constant across sampling units, coarse-grained studies should always account for environmental heterogeneity as a predictor of arthropod species richness, just as studies with variable area of sampling units routinely consider area.
Background: Vesicular stomatitis virus (VSV) is a potent candidate vaccine vector for various viral diseases (e.g. HIV, HCV, RSV). The biggest limitation of VSV, however, is its neurotoxicity, which limits application in humans. The second drawback is that VSV induces neutralizing antibodies rapidly and is thus ineffective as a vaccine vector upon repeated applications. Our group has recently shown that VSV pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), VSV-GP, is not neurotoxic. The aim of this project was to evaluate the potential of VSV-GP as a vaccine vector.
Methods: For this purpose, we used Ovalbumin (OVA) as a model antigen and analyzed immunogenicity of GP-pseudotyped and wildtype VSV containing OVA (VSV-GP-OVA and VSV-OVA) in vitro and in vivo in mouse models.
Results: We showed that both vectors infected murine bone marrow-derived dendritic cells (bmDCs) in vitro. These bmDCs were able to activate OVA specific CD8+ and CD4+ T cells. Immunization experiments in mice revealed that both VSV-OVA and VSV-GP-OVA induced functional OVA-specific cytotoxic T cells (CTLs) after a single immunization. In addition, with both viruses, mice generated antibodies against OVA. However, boosting with the same virus was only possible for the GP-pseudotyped virus but not for wild type VSV. The efficacy of repeated immunization with VSV-OVA was most likely limited by high levels of neutralizing antibodies, which we detected after the first immunization. In contrast, no neutralizing antibodies against VSV-GP were induced even after boosting.
Conclusion: Taken together, we showed that the non-neurotoxic VSV-GP is able to induce specific T cell and B cell responses against the model antigen OVA to the same level as the wild type VSV vector. However, in contrast to wild type VSV, VSV-GP-OVA boosted the immune response upon repeated applications. Thus, VSV-GP is a promising novel vaccine vector.
Background: Both standard and low-dose allergen provocations are an established tool in asthma research to improve our understanding of the pathophysiological mechanism of allergic asthma. However, clinical symptoms are less likely to be induced. Therefore, we designed a protocol for repetitive high-dose bronchial allergen challenges to generate clinical symptoms and airway inflammation.
Methods: A total of 27 patients aged 18 to 40 years with positive skin-prick tests and mild asthma underwent repetitive high-dose allergen challenges with household dust mites for four consecutive days. Pulmonary function and exhaled NO were measured at every visit. Induced sputum was analysed before and after the allergen challenges for cell counts, ECP, IL-5, INF-γ, IL-8, and the transcription factor Foxp3.
Results: We found a significant decrease in pulmonary function, an increased use of salbutamol and the development of a late asthmatic response and bronchial hyperresponsiveness, as well as a significant induction of eNO, eosinophils, and Th-2 cytokines. Repeated provocation was feasible in the majority of patients. Two subjects had severe adverse events requiring prednisolone to cope with nocturnal asthma symptoms.
Conclusions: Repeated high-dose bronchial allergen challenges resulted in severe asthma symptoms and marked Th-2-mediated allergic airway inflammation. The high-dose challenge model is suitable only in an attenuated form in diseased volunteers for proof-of-concept studies and in clinical settings to reduce the risk of severe asthma exacerbations.
Trial registration: ClinicalTrials.govNCT00677209
Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal) and Homo sapiens (epithelial) tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties.