Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy.

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

The aspartate transaminase/alanine transaminase (DeRitis) ratio predicts mid-term mortality and renal and respiratory dysfunction after left ventricular assist device implantation.

OBJECTIVES: Preoperative liver dysfunction is a well-known risk factor for adverse events after major surgery. However, there is only little data regarding the precise role of the Model of End-Stage Liver Disease (MELD) score and the De Ritis ratio (DRR, alanine transaminase/aspartate aminotransferase) as a predictor for outcome after left ventricular assist device (LVAD) implantation. METHODS: A retrospective analysis of all patients undergoing LVAD implantation at our institution between January 2012 and August 2014 was performed. The primary outcome was survival at 180 days after surgery. RESULTS: During the observation period, 63 patients underwent LVAD implantation (mean age 59.9 ± 8.3 years, 50% male). Mean preoperative ejection fraction was 16.3 ± 7.7, 13 patients required preoperative renal replacement therapy and 9 patients were on extracorporeal life support. Mean Interagency Registry for Mechanically Assisted Circulatory Support level was 2.8 ± 1.3, mean preoperative MELD was 12.7 ± 7.2, mean preoperative DRR was 2.01 ± 4.4. Aspartate aminotransferase (102 ± 220.8 vs 57.8 ± 123.4 U/l, P = 0.041), MELD score (16.1 ± 8.8 vs 11.4 ± 6.1, P = 0.017) and DRR (4.2 ± 7.8 vs 1.1 ± 1.1, P = 0.001) were significantly higher in non-survivors than in survivors after 180 days. Using logistic regression analyses, a DRR >1.37 was an independent predictor for 30-day mortality [odds ratio (OR) 4.5] and 180-day mortality (OR 4.1). In addition, the DRR was associated with postoperative acute kidney injury with need for renal replacement therapy (OR 4.2) and prolonged postoperative ventilation time >72 h (OR 3.8). Using receiver operator characteristics analyses, DRR showed a sensitivity of 0.80 and a specificity of 0.81 (area under the curve 0.834, cut-off 1.37) for 180-day mortality. CONCLUSIONS: The DRR is predictive of early and mid-term mortality as well as relevant morbidities in patients undergoing LVAD implantation. Therefore, the DRR should be considered within the preoperative risk stratification and patient selection for LVAD implantation.

Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice.

Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine.

Association of ADAM33 gene polymorphism and arginase activity with susceptibility to ventilatory impairment in wood dust-exposed workers.

ADAM33 represents an important gene of susceptibility for lung function impairment. This work aimed to evaluate the association between genetic polymorphism of ADAM33 at four single nucleotide polymorphisms (T1, T2, S1, and Q1) and arginase activity with respiratory functions impairment in wood workers. The study was done to compare ventilatory functions and arginase activity of 82 wood workers and 81 controls. Genotyping was determined by using the polymerase chain restriction fragment length polymorphism method. Forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and peak expiratory flow rate (PEF) of the workers were significantly reduced compared with the controls. T1 single nucleotide polymorphism (SNP) was associated with obvious decline in the FEV1, FVC, and PEF in wood workers, while T2 SNP was associated with decline in FEV1 and PEF. A significant increase in arginase activity was found in T2 and S1 SNPs of the exposed workers. Increase in duration of exposure was correlated with the decline in ventilatory functions. This inverse correlation was significant for pulmonary function indices in AA and GG genotypes of T1 and T2, respectively. Moreover, significance was detected for FVC and FEV1 in AA and GA genotypes of S1 and Q1. A positive correlation between arginase activity and duration of exposure was found to be significant in GG genotype of S1 SNP. An association between ADAM33 gene polymorphism and impaired lung functions was detected in wood dust-exposed workers. Arginase activity may play an associated important role in increasing this impairment in wood workers.

A case of respiratory depression in a child with ultrarapid CYP2D6 metabolism after tramadol.

We discuss a case of severe respiratory depression in a child, with ultrarapid CYP2D6 genotype and obstructive sleep apnea syndrome, after taking tramadol for pain relief related to a day-case tonsillectomy.

Critical illness in energy metabolism genetic disorder: rhabdomyolysis, acute kidney injury, respiratory arrest / Enfermedad crítica por trastorno genético en el metabolismo de la energía: rabdomiolisis, lesión renal aguda, paro respiratorio

In very long-chain acylCoA dehydrogenase deficiency (VLCAD), the activity of this enzyme is either reduced or absent with the inability to use long-chain fatty acids as energy substrates. A 25-year old male with VLCAD was admitted to the Emergency Department of Policlinico Teaching Hospital (Modena, Italy) for generalized weakness and oliguria, after a period of physical and mental stress and inadequate compliance to a long-chain fatty acid free diet. Laboratory tests were compatible with acute kidney injury. Seventy-two hours after admission, the subject had an episode of chest pain with elevated markers of myocardial necrosis. The rapid deterioration of muscular strength and the subsequent worsening respiratory failure necessitated ventilator support within the local Medical Intensive Care Unit. There, the patient showed a prompt normalization of respiratory parameters and a steady improvement of renal function. An inadequate compliance to lifestyle and dietary restriction in VLCAD may trigger severe and potentially lethal crisis. The in-hospital management of these patients calls for early intensive care admission as their conditions may deteriorate without warning.

En el caso de deficiencia de acylCoA deshidrogenasa de cadena muy larga (VLCAD), o bien se reduce la actividad de esta enzima, o la misma se halla ausente con la consiguiente incapacidad para utilizar los ácidos grasos de cadena larga como sustratos de energía. Un hombre de 25 años fue ingresado con VLCAD fue admitido en el Departamento de emergencia del Hospital Policlínico Docente Hospital de Modena, en Italia, a causa de presentar debilidad generalizada y oliguria, después de un período de estrés físico y mental, y por no cumplir adecuadamente con una dieta libre de ácidos grasos de cadena larga. Las pruebas de laboratorio eran compatibles con una lesión renal aguda. Setenta y dos horas después de su ingreso, el sujeto tuvo un episodio de dolor en el pecho con marcadores elevados de necrosis miocárdica. El rápido deterioro de la fuerza muscular, y el posterior empeoramiento de la insuficiencia respiratoria requirieron el apoyo de un ventilador en la Unidad de Cuidados Médicos Intensivos. Una vez allí, el paciente mostró una pronta normalización de los parámetros respiratorios, y una constante mejoría de la función renal. Un inadecuado cumplimiento con las restricciones dietéticas y el estilo de vida en los casos de VLCAD, pueden desatar una crisis grave y potencialmente fatal. El tratamiento intrahospitalario de estos pacientes requiere un ingreso temprano en cuidados intensivos, ya que sus condiciones pueden deteriorarse sin previo aviso.

Activity of neutrophil elastase reflects the progression of acute pancreatitis.

OBJECTIVE: Neutrophil elastase (NE) concentration is associated with progression of acute pancreatitis (AP), but measuring total NE concentration includes biologically inactive NE. This study aims to investigate the relationship between NE activity and the aetiology and severity of AP and associated organ failure. METHODS: Seventy-five patients admitted to our surgery department with a first episode of AP during 2004-2005 were age- and sex-matched to 20 healthy volunteers (controls). NE activity was assessed using venous blood samples obtained on patient admission and after 1, 2 and 14 days. One sample was also taken from each control. ANOVA was used for statistical comparison between groups. RESULTS: Baseline NE activity (geometric mean; 95% confidence intervals) differed between patients (58.6 nM of substrate 7-amino-4-methylcoumarin [AMC]/hour; 48.52-70.72) and controls (31.5 nM AMC/hour; 25.5-39.0) (p = 0.0003), and did not correlate with time between symptom onset and admission. Patients with alcohol-induced AP demonstrated higher mean activity (59.1 nM AMC/h; 44.7-78.2) than those with gallstone-induced AP (41.7 nM AMC/h; 33.9-51.4) (p = 0.0496). NE activity was higher overall in patients with predicted severe AP (60.9 nM AMC/h; 48.0-77.2) than in those with predicted mild AP (42.1 nM AMC/h; 34.9-50.8) (p = 0.027). Patients with respiratory failure had higher NE activity (82.5 nM AMC/h; 57.5-118.4) than those without (43.9 nM AMC/h; 37.6-51.3) (p = 0.0024). CONCLUSIONS: NE activity was associated with predicted severity of AP and AP-associated respiratory failure. Specific NE inhibitors may have therapeutic potential in acute pancreatitis.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Activation of protein kinase C (PKC)α or PKCε as an approach to increase morphine tolerance in respiratory depression and lethal overdose.

Long-term use of opioids is hindered by respiratory depression and the possibility for fatal overdose in drug abusers. This is attributed to higher levels of tolerance that develops against antinociception than to respiratory depression. Identifying important mechanisms that would increase morphine respiratory depression and overdose tolerance could lead to the safer use of opioids. Because protein kinase C (PKC) activity mediates the development and maintenance of morphine antinociceptive tolerance, we hypothesized that activating PKCα or PKCε at the pre-Bötzinger complex (preBötC) can increase morphine tolerance in respiration and overdose. Laser microdissection and quantitative reverse transcriptase-polymerase chain reaction were used to compare the relative mRNA abundances of PKCα, γ, and ε between ventrolateral periaqueductal gray (vlPAG) and preBötC. To test whether PKCα or ε could enhance morphine tolerance in respiratory depression and overdose, lentivirus carrying the wild type, constitutively activated mutants, and small interference RNA against PKCα or ε was stereotaxically injected into the preBötC. Expression of constitutively active PKC (CAPKC) α or ε, but not wild-type PKC (WTPKC) α or ε, at the preBötC allowed rats to develop tolerance to morphine respiratory depression. In terms of lethality, expression of WTPKCε, CAPKCα, or CAPKCε at preBötC increased morphine tolerance to lethal overdose. CAPKCε-expressing rats developed the highest level of respiratory depression tolerance. Furthermore, when CAPKCε lentivirus was injected into the vlPAG, rats were able to develop significant antinociceptive tolerance at low doses of morphine that normally do not cause tolerance. The approach of increasing morphine respiratory depression and lethality tolerance by increasing PKCα or ε activity at preBötC could be used to make opioids safer for long-term use.

Effects of enzyme replacement therapy on five patients with advanced late-onset glycogen storage disease type II: a 2-year follow-up study.

We examined the efficacy of 2-year enzyme replacement therapy (ERT) using recombinant human α-glucosidase (GAA; Myozyme®) in five long-term ventilator-dependent adults and aged patients with advanced, late-onset glycogen storage disease type II (GSDII, also known as Pompe disease). Although all patients had advanced respiratory failure and were ventilator-dependent for more than 6 years, four showed obvious improvements in muscle strength, pulmonary function, and activities of daily living after ERT. Improvement in each parameter was more prominent in the first year than in the second year. Values in the second year were still significantly better than those at study entry and indicate stabilization in the clinical status of all patients. These results suggest that ERT continues to be effective in the second year of treatment even in patients suffering from advanced late-onset GSDII disease with severe respiratory failure.

[Clinical manifestations, course and outcome of enzyme replacement therapy in Hungarian patients with Pompe's disease]. / Pompe-kór fenotípusvariációi, kórlefolyása és az enzimpótló kezelés eredményei: hazai tapasztalatok.

UNLABELLED: Pompe's disease is an autosomal recessive disease caused by deficiency of acid-alpha-glucosidase. AIMS AND METHODS: Authors analyzed the phenotype of 11 Hungarian patients with Pompe's disease and evaluated clinical parameters and response to enzyme replacement therapy during a long-term follow-up in 8 patients. RESULTS: One patient with atypical infantile form presented with cardiomyopathy and a very slow progression of motor deficits; after 2 years of enzyme replacement therapy no disability was present at the age 6 years. Another patient was asymptomatic at the age of 2.5 years. The adult onset form was characterized by slight to prominent limb-girdle myopathy with an age of onset between 20 and 50 years. In 3 of such cases respiratory insufficiency was also present. CONCLUSIONS: Hungarian patients with Pompe's disease presented with a wide phenotypic variability ranging from atypical early childhood form with slowly progressive course to late-onset limb-girdle myopathy with variable courses. Enzyme replacement therapy resulted in significant improvement in motor and respiratory functions in most of the patients.

Inhibiting lung elastase activity enables lung growth in mechanically ventilated newborn mice.

RATIONALE: Mechanical ventilation with O2-rich gas (MV-O2) offers life-saving treatment for respiratory failure, but also promotes lung injury. We previously reported that MV-O2 of newborn mice increased lung elastase activity, causing elastin degradation and redistribution of elastic fibers from septal tips to alveolar walls. These changes were associated with transforming growth factor (TGF)-ß activation and increased apoptosis leading to defective alveolarization and lung growth arrest, as seen in neonatal chronic lung disease. OBJECTIVES: To determine if intratracheal treatment of newborn mice with the serine elastase inhibitor elafin would prevent MV-O2-induced lung elastin degradation and the ensuing cascade of events causing lung growth arrest. METHODS: Five-day-old mice were treated via tracheotomy with recombinant human elafin or vehicle (lactated-Ringer solution), followed by MV with 40% O2 for 8-24 hours; control animals breathed 40% O2 without MV. At study's end, lungs were harvested to assess key variables noted below. MEASUREMENTS AND MAIN RESULTS: MV-O2 of vehicle-treated pups increased lung elastase and matrix metalloproteinase-9 activity when compared with unventilated control animals, causing elastin degradation (urine desmosine doubled), TGF-ß activation (pSmad-2 tripled), and apoptosis (cleaved-caspase-3 increased 10-fold). Quantitative lung histology showed larger and fewer alveoli, greater inflammation, and scattered elastic fibers. Elafin blocked these MV-O2-induced changes. CONCLUSIONS: Intratracheal elafin, by blocking lung protease activity, prevented MV-O2-induced elastin degradation, TGF-ß activation, apoptosis, and dispersion of matrix elastin, and attenuated lung structural abnormalities noted in vehicle-treated mice after 24 hours of MV-O2. These findings suggest that elastin breakdown contributes to defective lung growth in response to MV-O2 and might be targeted therapeutically to prevent MV-O2-induced lung injury.

Recombinant human acid alpha-glucosidase (rhGAA) in adult patients with severe respiratory failure due to Pompe disease.

Pompe disease is a rare metabolic myopathy caused by lysosomal α-glucosidase deficiency. Pompe disease ranges from a rapidly progressive course when symptoms present in infancy to a more slowly progressive rate when symptoms present in childhood or adulthood. This open-label prospective exploratory study investigated the effect of 12 months of recombinant enzyme replacement therapy in 5 adult patients who had already advanced to a very severe stage of Pompe disease. Muscular and respiratory function, quantitative muscle testing and spirometry were assessed. Four patients were tracheostomized. Respiratory parameters did not deteriorate. A moderate improvement in sitting/supine slow vital capacity in 2 patients (from 7% to 11% and 28% to 32% of predicted) and reductions of ventilation support in 2 patients was observed. Three patients, wheelchair bound at baseline, improved sitting and proximal motor function; 2 patients improved in their ability to stand and transfer. The treatment was well tolerated. Alglucosidase alfa may stabilize or even slightly improve muscle strength and respiratory function among patients with severe Pompe disease.

The sequence polymorphism of MnSOD gene in subjects with respiratory insufficiency in COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with the development of respiratory failure, which, in turn, exposes the tissues to oxidative stress, which is both a cause and a result of respiratory insufficiency. The first-line defense against oxidative stress is provided by the mitochondrial enzyme manganese superoxide dismutase (MnSOD), which is a superoxide anion scavenger. It is unknown whether genetic variability of the enzymes protecting against reactive oxygen species (ROS) can influence the development of respiratory failure in COPD patients. The aim of the study was to determine the correlation between polymorphism of MnSOD signal peptide and the occurrence of respiratory failure in the course of COPD. MATERIAL/METHODS: The study group consisted of 162 COPD patients (113 men and 49 women). The control group consisted of 63 subjects. Respiratory failure was diagnosed in 42 COPD patients. In all the examined subjects, the polymorphism resulting in alanine at residue 9 being replaced by valine and the expression of MnSOD in blood cells were determined. RESULTS: The Val/Val phenotype was demonstrated to occur in COPD patients more frequently than in the control group, as well as being associated with a lower expression level of MnSOD mRNA. Respiratory failure in the course of COPD also correlates with lower expression of MnSOD mRNA. CONCLUSIONS: The presence of valine at position 9 of the MnSOD signal peptide encoded by exon 2 is a risk factor for the occurrence of respiratory failure in the course of COPD in the Polish population.

Severe congenital myasthenia gravis of the presynaptic type with choline acetyltransferase mutation in a Chinese infant with respiratory failure.

We report a severe case of congenital myasthenia gravis in a Chinese newborn who presented with complete ptosis, severe hypotonia, dysphagia and respiratory insufficiency with recurrent apnea that required mechanical ventilatory support since birth. Routine neurophysiologic studies, including the 3-Hz repetitive stimulation test and electromyogram were normal. Neostigmine and edrophonium tests were also negative. However, decremental response to 3-Hz stimulation became apparent after depleting the muscles with trains of 10-Hz stimuli for 10 min. The infant was subsequently confirmed to have heterozygous mutations in the choline acetyltransferase genes, p.T553N and p.S704P. Both missense mutations are novel mutations. The child remained on positive pressure ventilation at 3 years of age despite treatment with high-dose anticholinesterase. This case highlights the difficulty of making an early diagnosis based on clinical presentation and routine electrophysiologic tests, especially when neonatologists are not familiar with this condition. Further, as there are different genetic defects causing different types of congenital myasthenia gravis, anticholinesterase therapy may be beneficial to some but detrimental to others. Therefore, the exact molecular diagnosis is an important guide to therapy. A high index of suspicion coupled with extended electrodiagnostic tests in clinically suspected patients will ensure the selection of appropriate genetic molecular study for confirming the diagnosis.

Rhabdomyolysis and respiratory failure: rare presentation of carnitine palmityl-transferase II deficiency.

Carnitine palmityl-transferase (CPT) II deficiency is a rare disorder of the fatty acid beta-oxidation cycle. CPT II deficiency can be associated with rhabdomyolysis in particular conditions that increase the requirement for fatty acid oxidation, such as low-carbohydrate and high-fat diet, fasting, exposure to excessive cold, lack of sleep and prolonged exercise. The best known CPT II deficiency is the muscular form with episodic muscle necrosis and paroxysmal myoglobinuria after prolonged exercise. We report a case of a four-year-old male child, who, after one day of hyperthermia and fasting, developed a massive rhabdomyolysis beginning with acute respiratory failure and later complicated by acute renal failure. Appropriate management in Pediatric Intensive Care Unit (PICU) (mechanical ventilatory support, fluid supply combined with mannitol and bicarbonate infusions, administration of acetaminophen and antibiotics, and continuous venovenous haemofiltration) brought about complete resolution with an excellent outcome. Biochemical investigation of muscle biopsy and genetic analysis showed a deficiency of CPT II. The onset of CPT II deficiency with respiratory failure is extremely rare, but a correct and early diagnosis of rhabdomyolysis is the key to successful treatment. A metabolic myopathy such as CPT II deficiency should be suspected in children affected by rhabdomyolysis if trauma, crash, infections, drugs or extreme exertion can be excluded.