Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers

Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders1, 2. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin3, 4 and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure5 and neurocognitive impairment6. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Electromyographic activity of back muscles during stochastic whole body vibration

OBJECTIVES: Stochastic resonance whole body vibrations (SR-WBV) may reduce and prevent musculoskeletal problems (MSP). The aim of this study was to evaluate how activities of the lumbar erector spinae (ES) and of the ascending and descending trapezius (TA, TD) change in upright standing position during SR-WBV. METHODS: Nineteen female subjects completed 12 series of 10 seconds of SR-WBV at six different frequencies (2, 4, 6, 8, 10, 12Hz) and two types of "noise"-applications. An assessment at rest had been executed beforehand. Muscle activities were measured with EMG and normalized to the maximum voluntary contraction (MVC%). For statistical testing a three-factorial analysis of variation (ANOVA) was applied. RESULTS: The maximum activity of the respective muscles was 14.5 MVC% for the ES, 4.6 MVC% for the TA (12Hz with "noise" both), and 7.4 MVC% for the TD (10Hz without "noise"). Furthermore, all muscles varied significantly at 6Hz and above (p⋜0.047) compared to the situation at rest. No significant differences were found at SR-WBV with or without "noise". CONCLUSIONS: In general, muscle activity during SR-WBV is reasonably low and comparable to core strength stability exercises, sensorimotor training and "abdominal hollowing" in water. SR-WBV may be a therapeutic option for the relief of MSP.

Determination of respiratory gas flow by electrical impedance tomography in an animal model of mechanical ventilation

Background A recent method determines regional gas flow of the lung by electrical impedance tomography (EIT). The aim of this study is to show the applicability of this method in a porcine model of mechanical ventilation in healthy and diseased lungs. Our primary hypothesis is that global gas flow measured by EIT can be correlated with spirometry. Our secondary hypothesis is that regional analysis of respiratory gas flow delivers physiologically meaningful results. Methods In two sets of experiments n = 7 healthy pigs and n = 6 pigs before and after induction of lavage lung injury were investigated. EIT of the lung and spirometry were registered synchronously during ongoing mechanical ventilation. In-vivo aeration of the lung was analysed in four regions-of-interest (ROI) by EIT: 1) global, 2) ventral (non-dependent), 3) middle and 4) dorsal (dependent) ROI. Respiratory gas flow was calculated by the first derivative of the regional aeration curve. Four phases of the respiratory cycle were discriminated. They delivered peak and late inspiratory and expiratory gas flow (PIF, LIF, PEF, LEF) characterizing early or late inspiration or expiration. Results Linear regression analysis of EIT and spirometry in healthy pigs revealed a very good correlation measuring peak flow and a good correlation detecting late flow. PIFEIT = 0.702 · PIFspiro + 117.4, r2 = 0.809; PEFEIT = 0.690 · PEFspiro-124.2, r2 = 0.760; LIFEIT = 0.909 · LIFspiro + 27.32, r2 = 0.572 and LEFEIT = 0.858 · LEFspiro-10.94, r2 = 0.647. EIT derived absolute gas flow was generally smaller than data from spirometry. Regional gas flow was distributed heterogeneously during different phases of the respiratory cycle. But, the regional distribution of gas flow stayed stable during different ventilator settings. Moderate lung injury changed the regional pattern of gas flow. Conclusions We conclude that the presented method is able to determine global respiratory gas flow of the lung in different phases of the respiratory cycle. Additionally, it delivers meaningful insight into regional pulmonary characteristics, i.e. the regional ability of the lung to take up and to release air.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses.

The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-β activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-β. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-β activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-β-treated MΦ (P<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-β stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information important not only to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

The role of cold stress in predicting extra cardiovascular and respiratory admissions

Although several studies have examined effects of air temperature and/or other meteorological variables separately on disease rates, the relationship of meteorological variables and human disease is, in fact, rather complex in the “real-world” [1,2] including the number of potential variables to be considered and their weighting. In other words, 1 °C of air temperature difference in a warm climate may not necessarily mean the same in a cold climate across regions on Earth [3,4]. Why some seasonality was observed in certain regions at certain times only is likely due in part to the imprecise weather estimation from mean, maximum, or minimum air temperature or the definition of study catchments or time period to be included.

Enhanced-depth optical coherence tomography for imaging horizontal rectus muscles in Graves' orbitopathy.

PURPOSE Graves' orbitopathy (GO) is an extraocular eye disease with symptoms ranging from minor discomfort from dry eyes to strabismus and visual loss. One of the hallmarks of active GO is visible hyperemia at the insertion of the extraocular muscles. The aim of the present study was to evaluate the use of enhanced-depth imaging spectral domain anterior segment optical coherence tomography (EDI SD AS-OCT) for detecting pathological changes in horizontal recti muscles of patients with GO. METHODS Prospective cross sectional study of 27 eyes. Only women were included. EDI AS-OCT was used to measure the thickness of the tendons of the horizontal recti muscles in a predefined area in patients with GO and healthy controls. RESULTS EDI AS-OCT was able to image the tendons of the horizontal recti muscles in both healthy controls and patients suffering from GO. The mean thickness of the medial rectus muscle (MR) tendon was 256.4 μm [±17.13 μm standard deviation (SD)] in the GO group and, therefore, significantly thicker (p = 0.046) than in the healthy group which had a mean thickness of 214.7 μm (±5.516 μm SD). There was no significant difference in the mean thickness of the tendon of the lateral recti muscles (LRs) between these groups. CONCLUSION This is the first report showing that EDI AS-OCT is suitable to detect swelling at the insertion site of the MR muscle in GO. MR tendon thickness may be a useful parameter to monitor activity in these patients.

CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.

Ectodysplasin-1 deficiency in a German Holstein bull associated with loss of respiratory mucous glands and chronic rhinotracheitis

A 2-year-old German Holstein bull was identified as a carrier of a mutation within the X-chromosomal ED1 gene, which encodes a TNF-related signalling molecule mainly involved in ectodermal development. The clinicopathological appearance was associated with hypotrichosis, hypodontia, and a reduced number of eccrine glands, in addition to chronic rhinotracheitis and partial squamous metaplasia. Furthermore, for the first time in an ED1-deficient animal, a complete lack of respiratory mucous glands was observed. This suggests that the ED1 gene plays a role in the development of mucous glands, the absence of which resembles a feature of X-linked anhidrotic ectodermal dysplasia (ED1) in human patients.