A large animal model of spinal muscular atrophy and correction of phenotype.

OBJECTIVES Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein, which results in motoneuron loss. Therapeutic strategies to increase SMN levels including drug compounds, antisense oligonucleotides, and scAAV9 gene therapy have proved effective in mice. We wished to determine whether reduction of SMN in postnatal motoneurons resulted in SMA in a large animal model, whether SMA could be corrected after development of muscle weakness, and the response of clinically relevant biomarkers. METHODS Using intrathecal delivery of scAAV9 expressing an shRNA targeting pig SMN1, SMN was knocked down in motoneurons postnatally to SMA levels. This resulted in an SMA phenotype representing the first large animal model of SMA. Restoration of SMN was performed at different time points with scAAV9 expressing human SMN (scAAV9-SMN), and electrophysiology measurements and pathology were performed. RESULTS Knockdown of SMN in postnatal motoneurons results in overt proximal weakness, fibrillations on electromyography indicating active denervation, and reduced compound muscle action potential (CMAP) and motor unit number estimation (MUNE), as in human SMA. Neuropathology showed loss of motoneurons and motor axons. Presymptomatic delivery of scAAV9-SMN prevented SMA symptoms, indicating that all changes are SMN dependent. Delivery of scAAV9-SMN after symptom onset had a marked impact on phenotype, electrophysiological measures, and pathology. INTERPRETATION High SMN levels are critical in postnatal motoneurons, and reduction of SMN results in an SMA phenotype that is SMN dependent. Importantly, clinically relevant biomarkers including CMAP and MUNE are responsive to SMN restoration, and abrogation of phenotype can be achieved even after symptom onset.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Adaptive, convergent origins of the pygmy phenotype in African rainforest hunter-gatherers

Tropical rainforest hunter-gatherer populations worldwide share the pygmy phenotype, or small human body size. The evolutionary history of this phenotype is largely unknown. Here we studied DNA from the Batwa, a rainforest hunter-gatherer population from east central Africa, to identify regions of the Batwa genome that underlie the pygmy phenotype. We then performed population genomic analyses to study the evolution of these regions, including comparisons with the Baka, a west central African rainforest hunter-gatherer population. We conclude that the pygmy phenotype likely arose due to positive natural selection and that it arose possibly multiple times within Africa. These results support longstanding anthropological hypotheses that small body size confers an important selective advantage for human rainforest hunter-gatherers.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application.

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

From quantum to classical dynamics: The relativistic O ( N ) model in the framework of the real-time functional renormalization group

We investigate the transition from unitary to dissipative dynamics in the relativistic O(N) vector model with the λ(φ2)2 interaction using the nonperturbative functional renormalization group in the real-time formalism. In thermal equilibrium, the theory is characterized by two scales, the interaction range for coherent scattering of particles and the mean free path determined by the rate of incoherent collisions with excitations in the thermal medium. Their competition determines the renormalization group flow and the effective dynamics of the model. Here we quantify the dynamic properties of the model in terms of the scale-dependent dynamic critical exponent z in the limit of large temperatures and in 2≤d≤4 spatial dimensions. We contrast our results to the behavior expected at vanishing temperature and address the question of the appropriate dynamic universality class for the given microscopic theory.

Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells.

The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

The viral protein Npro is unique to the genus Pestivirus within the family Flaviviridae. After autocatalytic cleavage from the nascent polyprotein, Npro suppresses type I IFN (IFN-α/β) induction by mediating proteasomal degradation of IFN regulatory factor 3 (IRF-3). Previous studies found that the Npro-mediated IRF-3 degradation was dependent of a TRASH domain in the C-terminal half of Npro coordinating zinc by means of the amino acid residues C112, C134, D136 and C138. Interestingly, four classical swine fever virus (CSFV) isolates obtained from diseased pigs in Thailand in 1993 and 1998 did not suppress IFN-α/β induction despite the presence of an intact TRASH domain. Through systematic analyses, it was found that an amino acid mutation at position 40 or mutations at positions 17 and 61 in the N-terminal half of Npro of these four isolates were related to the lack of IRF-3-degrading activity. Restoring a histidine at position 40 or both a proline at position 17 and a lysine at position 61 based on the sequence of a functional Npro contributed to higher stability of the reconstructed Npro compared with the Npro from the Thai isolate. This led to enhanced interaction of Npro with IRF-3 along with its degradation by the proteasome. The results of the present study revealed that amino acid residues in the N-terminal domain of Npro are involved in the stability of Npro, in interaction of Npro with IRF-3 and subsequent degradation of IRF-3, leading to downregulation of IFN-α/β production.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

Postnatal persistent infection with classical Swine Fever virus and its immunological implications.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.

Comparative dendritic cell biology of veterinary mammals.

Dendritic cells (DC) have a main function in innate immunity in that they sense infections and environmental antigens at the skin and mucosal surfaces and thereby critically influence decisions about immune activation or tolerance. As professional antigen-presenting cells, they are essential for induction of adaptive immune responses. Consequently, knowledge on this cell type is required to understand the immune systems of veterinary mammals, including cattle, sheep, pigs, dogs, cats, and horses. Recent ontogenic studies define bona fide DC as an independent lineage of hematopoietic cells originating from a common precursor. Distinct transcription factors control the development into the two subsets of classical DC and plasmacytoid DC. These DC subsets express a distinguishable transcriptome, which differs from that of monocyte-derived DC. Using a comparative approach based on phenotype and function, this review attempts to classify DC of veterinary mammals and to describe important knowledge gaps.