Adeno-associated virus (AAV) in a small replication deficient DNA virus belonging to the Parvovirinae family. It has a single-stranded approximately 4.7kb genome. Recombinant AAV (rAAV) is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. The short viral inverted terminal repeats must remain intact for replication and packaging in production as well as vector genome processing and persistence in the transduction process. The AAV capsid (serotype) determines the tissue tropism of the rAAV vector. In this unit we will discuss serotype selection for lung targeting along with the factors effecting efficient delivery of rAAV vectors to the murine lung. Detailed procedures for lung delivery (intranasal, orotracheal, and surgical tracheal injection), sample collection and post-mortem tissue processing will be described.
Adeno-associated virus; vector delivery; lung; gene therapy
The purpose of this study was to report on the incidence, diagnosis and clinical manifestation of VAI following cervical spine injuries observed in a prospective observational study with a standardized clinical and radiographical protocol.
During a 16-year period, 69 (mean age: 43 ± 20.7 years; 25 female, 44 male) of 599 patients had cervical spine injury suspicious for VAI due to facet luxation and/or fractures extending into the transverse foramen. Diagnosis and management of these patients followed a previously published protocol (Kral in Zentralbl Neurochir 63:153–158, 2002). Digital subtraction angiography (DSA) was performed in all 69 patients. Injury grading of VAI was done according to Biffl et al. (Ann Surg 231:672–681, 2000). All patients with VAI were treated with anticoagulation (heparin followed by ASS) for 6 months.
In cases suspicious for VAI, the incidence of VAI detected by DSA was 27.5% (n = 19 of 69 patients). VAI Grade I occurred in 15.8%, Grade II in 26.3%, Grade IV in 52.6% and Grade V in 5.2%. Of 19 patients, 4 (21%) had clinical signs of vertebrobasilar ischemia. Two patients died in hospital after 4 and 21 days respectively. Of 69 patients, 33 (47.8%) with suspected VAI had unstable spine injuries and were treated surgically.
In patients with cervical spine fractures or dislocations crossing the course of the vertebral artery, VAI are relatively frequent and may be associated with significant morbidity and mortality. VAI were identified by DSA in 27.5%. Despite anticoagulation therapy, 5.8% became clinically symptomatic and 2.9% died due to cerebrovascular ischemia.
Vertebral artery injury; Arteriography; Cervical spine trauma; CT-angiography; MR-angiography
In hemodialysis patients, elevated plasma troponin concentrations are a common finding that has even increased with the advent of newly developed sensitive assays. However, the interpretation and relevance of this is still under debate.
In this cross-sectional study, we analyzed plasma concentrations of sensitive troponin I (TnI) and troponin T (TnT) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality.
In all of the enrolled patients, plasma TnI or TnT was detectable at a median concentration of 14 pg/ml (interquartile range: 7–29) using the Siemens TnI ultra assay and 49 pg/ml (31–74) using the Roche Elecsys high sensitive TnT assay. Markedly more patients exceeded the 99th percentile for TnT than for TnI (95% vs. 14%, p<0.0001). In a multivariate linear regression model, TnT was independently associated with age, gender, systolic dysfunction, time on dialysis, residual diuresis and systolic blood pressure, whereas TnI was independently associated with age, systolic dysfunction, pulse pressure, time on dialysis and duration of a HD session. During a follow-up period of nearly two years, TnT concentration above 38 pg/mL was associated with a 5-fold risk of death, whereas elevation of TnI had a gradual association to mortality.
In hemodialysis patients, elevations of plasma troponin concentrations are explained by cardiac function and dialysis-related parameters, which contribute to cardiac strain. Both are highly predictive of increased risk of death.
Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α1-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×1011, 1.9×1012, and 6.0×1012 vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8+ subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.
Flotte and colleagues report on a phase 2 trial in which the same α1-antitrypsin (AAT) AAV vector as in phase 1 is administered intramuscularly to nine AAT-deficient individuals at one of three doses. Vector-derived expression of normal (M-type) AAT in serum is shown to be dose dependent, peaks on day 30, and persists for at least 90 days, although AAT levels were sub-therapeutic.
AIM: To investigate the actual injury situation of seniors in traffic accidents and to evaluate the different injury patterns.
METHODS: Injury data, environmental circumstances and crash circumstances of accidents were collected shortly after the accident event at the scene. With these data, a technical and medical analysis was performed, including Injury Severity Score, Abbreviated Injury Scale and Maximum Abbreviated Injury Scale. The method of data collection is named the German In-Depth Accident Study and can be seen as representative.
RESULTS: A total of 4430 injured seniors in traffic accidents were evaluated. The incidence of sustaining severe injuries to extremities, head and maxillofacial region was significantly higher in the group of elderly people compared to a younger age (P < 0.05). The number of accident-related injuries was higher in the group of seniors compared to other groups.
CONCLUSION: Seniors are more likely to be involved in traffic injuries and to sustain serious to severe injuries compared to other groups.
Traffic accidents; Seniors; Head injury; Injury severity score; Abbreviated injury scale
Local renal ischemia is regarded as an important factor in the development of contrast-induced nephropathy (CIN). Mannose-binding lectin (MBL) is involved in the tissue damage during experimental ischemia/reperfusion injury of the kidneys. The aim of the present study was to investigate the association of MBL deficiency with radiocontrast-induced renal dysfunction in a large prospective cohort.
246 patients with advanced non–dialysis-dependent renal dysfunction who underwent radiographic contrast procedures were included in the study. Baseline serum MBL levels were analyzed according to the occurrence of a creatinine-based (increase of ≥0.5 mg/dL or ≥25% within 48 hours) or cystatin C-based (increase of ≥10% within 24 hours) CIN.
The incidence of creatinine-based and cystatin C-based CIN was 6.5% and 24%, respectively. MBL levels were not associated with the occurrence of creatinine-based CIN. However, patients that experienced a cystatin C increase of ≥10% showed significantly higher MBL levels than patients with a rise of <10% (median 2885 (IQR 1193–4471) vs. 1997 (IQR 439–3504)ng/mL, p = 0.01). In logistic regression analysis MBL deficiency (MBL levels≤500 ng/ml) was identified as an inverse predictor of a cystatin C increase ≥10% (OR 0.34, 95% CI 0.15-0.8, p = 0.01).
MBL deficiency was associated with a reduced radiocontrast-induced renal dysfunction as reflected by the course of cystatin C. Our findings support a possible role of MBL in the pathogenesis of CIN.
Complement; Mannose-binding lectin; Contrast-induced nephropathy; Ischemia/reperfusion injury; Acute kidney injury
The aim of this review is to examine the causes, pathophysiology and experimental models of non-infectious pharyngitis (sore throat).
The causes of sore throat can be infectious (viruses, bacteria, and fungi) or non-infectious, although the relative proportion of each is not well documented.
A PubMed database search was performed for studies of non-infectious sore throat.
Results and conclusions
Non-infectious causes of sore throat include: physico-chemical factors, such as smoking, snoring, shouting, tracheal intubation, medications, or concomitant illness; and environmental factors including indoor and outdoor air pollutants, temperature and humidity, and hazardous or occupational irritants. The pathophysiology underlying non-infectious sore throat is largely uncharacterised, although neurogenic inflammation looks to be a promising candidate. It is likely that there will be individual disposition factors or the coincidence of more than one irritant with possible—up to now unknown—interactions between them. Therefore, experimental models with defined conditions and objective endpoints are needed. A new model using cold dry air to directly induce pharyngeal irritation in humans, with pharyngeal lavage to measure biomarkers, may provide a useful tool for the study of mechanisms and treatment of non-infectious sore throat.
Air pollution; Cold dry air; Experimental model; Inflammation; Occupational exposure; Therapy; Pain
Chronic lung colonization with Pseudomonas aeruginosa is anticipated in cystic fibrosis (CF). Abnormal terminal glycosylation has been implicated as a candidate for this condition. We previously reported a down-regulation of mannose-6-phosphate isomerase (MPI) for core N-glycan production in the CFTR-defective human cell line (IB3). We found a 40% decrease in N-glycosylation of IB3 cells compared with CFTR-corrected human cell line (S9), along with a threefold-lower surface attachment of P. aeruginosa strain, PAO1. There was a twofold increase in intracellular bacteria in S9 cells compared with IB3 cells. After a 4-hour clearance period, intracellular bacteria in IB3 cells increased twofold. Comparatively, a twofold decrease in intracellular bacteria occurred in S9 cells. Gene augmentation in IB3 cells with hMPI or hCFTR reversed these IB3 deficiencies. Mannose-6-phosphate can be produced from external mannose independent of MPI, and correction in the IB3 clearance deficiencies was observed when cultured in mannose-rich medium. An in vivo model for P. aeruginosa colonization in the upper airways revealed an increased bacterial burden in the trachea and oropharynx of nontherapeutic CF mice compared with mice treated either with an intratracheal delivery adeno-associated viral vector 5 expressing murine MPI, or a hypermannose water diet. Finally, a modest lung inflammatory response was observed in CF mice, and was partially corrected by both treatments. Augmenting N-glycosylation to attenuate colonization of P. aeruginosa in CF airways reveals a new therapeutic avenue for a hallmark disease condition in CF.
bacterial clearance; cystic fibrosis; gene therapy; N-glycosylation
Cystic fibrosis (CF), the most common fatal monogenic disease in the United States, results from mutations in CF transmembrane conductance regulator (CFTR), a chloride channel. The mechanisms by which CFTR mutations cause lung disease in CF are not fully defined but may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr−/− mice mount an exaggerated IgE response toward Aspergillus fumigatus, with higher levels of IL-13 and IL-4, mimicking both the T helper cell type 2–biased immune responses seen in patients with CF. Herein, we demonstrate that these aberrations are primarily due to Cftr deficiency in lymphocytes rather than in the epithelium. Adoptive transfer experiments with CF splenocytes confer a higher IgE response to Aspergillus fumigatus compared with hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount T helper cell type 2 responses with high IL-13 and IL-4 was confirmed by in vitro antigen recall experiments. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T cell lineages developed higher IgE than their wild-type control littermates. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca2+ flux in response to T cell receptor activation. This was accompanied by an increase in nuclear localization of the calcium-sensitive transcription factor, nuclear factor of activated T cell, which could drive the IL-13 response. In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. These findings implicate the lymphocyte population as a potentially important target for CF therapeutics.
cystic fibrosis transmembrane conductance regulator; lymphocytes; allergy; intracellular calcium; nuclear factor of activated T cell
Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.
Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia-regulated Hypoxia-inducible factor 1-α, (HIF1α) may regulate liporegulatory genes but the relationship of HIF1 to steatosis remains unknown. We investigated HIF1α in alcohol-induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels and serum ALT suggesting liver injury in WT mice. There was increased hepatic HIF1α mRNA, protein and DNA-binding activity in alcohol-fed mice compared to controls. Mice engineered with hepatocyte-specific HIF1 activation (HIF1dPA) had increased HIF1α mRNA, protein, and DNA-binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared to WT. In contrast, hepatocyte-specific deletion of HIF1α (HIF-1α(Hep-/-), protected mice from alcohol- and LPS-induced liver damage, serum ALT elevation, hepatomegaly and lipid accumulation. HIF-1α(Hep-/-), WT, and HIF1dPA mice had equally suppressed levels of PPARα mRNA after chronic ethanol, while the HIF target, ADRP, was upregulated in WT, but not in HIF-1α(Hep-/-) ethanol fed/LPS challenged mice. The chemokine, MCP-1, was cooperatively induced by alcohol feeding and LPS in WT but not in HIF-1α(Hep-/-) mice. Using Huh7 hepatoma cells in vitro, we found that MCP-1 treatment induced lipid accumulation and increased HIF1α protein expression as well as DNA-binding activity. SiRNA inhibition of HIF1α prevented MCP-1-induced lipid accumulation suggesting a mechanistic role for HIF1α in hepatocyte lipid accumulation.
Alcohol feeding results in lipid accumulation in hepatocytes involving HIF1α activation. The alcohol-induced chemokine, MCP-1, triggers lipid accumulation in hepatocytes via HIF1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease.
Hypoxia-Inducible Factor-1 alpha; Monocyte-Chemoattractant Protein-1; Steatosis; Alcoholic Liver Disease
Alpha-1 antitrypsin (AAT) deficiency is a common single-gene disorder among Northern Europeans and North Americans. The carrier frequency for the common missense mutation (Z-AAT) ranges from 4% in the US to nearly 25% in the Republic of Ireland. Severe AAT deficiency (plasma levels below 11 μm) is most commonly associated with an adult-onset lung disease, with pan-acinar emphysema and airway inflammation, which is thought to be primarily owing to the loss of function of AAT in neutralizing neutrophil elastase and other pro-inflammatory enzymes. In 5–10% of patients, severe liver disease may develop. This may occur at any time from infancy to adulthood, and is thought to be owing to toxicity from the Z-AAT mutant protein that folds poorly and forms insoluble polymers within the hepatocyte, which is the primary site for AAT production. Thus, gene therapy for AAT lung disease is conceived of as augmentation of serum levels (a prolonged form of protein replacement, which is currently in use), while gene therapy for liver disease presents the problem of also having to downregulate the production of Z-AAT protein. Over the years, numerous strategies have been employed for the gene therapy of both AAT-deficient lung disease and liver disease. These will be reviewed with an emphasis on modalities that have reached clinical trials recently.
Biomarkers complement other clinical information by proving quantitative data regarding a pathophysiological mechanism that can be used for the early diagnosis of a specific disease, to monitor and guide treatment, and to predict the risk of death or other adverse events. The stronger the link between the information provided by the biomarker and the immediate clinical course of action that we physicians take in response, the higher the clinical utility of the biomarker. This link is weakest for prognostic biomarkers applied in patients with a wide variety of diseases, such as in unselected intensive care unit (ICU) patients. Although the added value on top of current ICU mortality scores seems to be too low to justify clinical use, the observation that hemodynamic cardiac stress and inflammation are present in multiple conditions provides important insights into the pathophysiology of common disorders in the ICU.
α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.
Biomarkers have proven their ability in the evaluation of cardiopulmonary diseases. We investigated the utility of concentrations of the biomarker procalcitonin (PCT) alone and with clinical variables for the diagnosis of pneumonia in patients presenting to emergency departments (EDs) with a chief complaint of shortness of breath.
Methods and results
The BACH trial was a prospective, international, study of 1641 patients presenting to EDs with dyspnoea. Blood samples were analysed for PCT and other biomarkers. Relevant clinical data were also captured. Patient outcomes were assessed at 90 days. The diagnosis of pneumonia was made using strictly validated guidelines. A model using PCT was more accurate [area under the curve (AUC) 72.3%] than any other individual clinical variable for the diagnosis of pneumonia in all patients, in those with obstructive lung disease, and in those with acute heart failure (AHF). Combining physician estimates of the probability of pneumonia with PCT values increased the accuracy to >86% for the diagnosis of pneumonia in all patients. Patients with a diagnosis of AHF and an elevated PCT concentration (>0.21 ng/mL) had a worse outcome if not treated with antibiotics (P = 0.046), while patients with low PCT values (<0.05 ng/mL) had a better outcome if they did not receive antibiotic therapy (P = 0.049).
Procalcitonin may aid in the diagnosis of pneumonia, particularly in cases with high diagnostic uncertainty. Importantly, PCT may aid in the decision to administer antibiotic therapy to patients presenting with AHF in which clinical uncertainty exists regarding a superimposed bacterial infection.
Trial registration: NCT00537628
Acute heart failure; Procalcitonin; Pneumonia; Survival; Diagnosis
α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke–induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL–deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.
The accurate prediction of acute kidney injury (AKI) in patients with acute heart failure (AHF) is an unmet clinical need. Neutrophil gelatinase-associated lipocalin (NGAL) is a novel sensitive and specific marker of AKI.
A total of 207 consecutive patients presenting to the emergency department with AHF were enrolled. Plasma NGAL was measured in a blinded fashion at presentation and serially thereafter. The potential of plasma NGAL levels to predict AKI was assessed as the primary endpoint. We defined AKI according to the AKI Network classification.
Overall 60 patients (29%) experienced AKI. These patients were more likely to suffer from pre-existing chronic cardiac or kidney disease. At presentation, creatinine (median 140 (interquartile range (IQR), 91 to 203) umol/L versus 97 (76 to 132) umol/L, P < 0.01) and NGAL (114.5 (IQR, 67.1 to 201.5) ng/ml versus 74.5 (60 to 113.9) ng/ml, P < 0.01) levels were significantly higher in AKI compared to non-AKI patients. The prognostic accuracy for measurements obtained at presentation, as quantified by the area under the receiver operating characteristic curve was mediocre and comparable for the two markers (creatinine 0.69; 95%CI 0.59 to 0.79 versus NGAL 0.67; 95%CI 0.57 to 0.77). Serial measurements of NGAL did not further increase the prognostic accuracy for AKI. Creatinine, but not NGAL, remained an independent predictor of AKI (hazard ratio (HR) 1.12; 95%CI 1.00 to 1.25; P = 0.04) in multivariable regression analysis.
Plasma NGAL levels do not adequately predict AKI in patients with AHF.
Hyperbaric oxygen (HBO) and normobaric hyperoxia (NBO) protect the brain parenchyma and the cerebral microcirculation against ischemia. We studied their effect on secondary hemorrhage after thrombolysis in two thromboembolic middle cerebral artery occlusion (MCAO) (tMCAO) models. Beginning 60 minutes after tMCAO with either thrombin-induced thromboemboli (TT) or calcium-induced thromboemboli (CT), spontaneously hypertensive rats (n=96) breathed either air, 100% O2 (NBO), or 100% O2 at 3 bar (HBO) for 1 hour. Immediately thereafter, recombinant tissue plasminogen activator (rt-PA, 9 mg/kg) was injected. Although significant reperfusion was observed after thrombolysis in TT-tMCAO, vascular occlusion persisted in CT-tMCAO. In TT-tMCAO, NBO and HBO significantly reduced diffusion-weighted imaging–magnetic resonance imaging (MRI) lesion volume and postischemic blood–brain barrier (BBB) permeability on postcontrast T1-weighted images. NBO and, significantly more potently, HBO reduced macroscopic hemorrhage on T2* MRI and on corresponding postmortem cryosections. Oxygen therapy lowered hemoglobin content and attenuated activation of matrix metalloproteinases in the ischemic hemisphere. In contrast, NBO and HBO failed to reduce infarct size in CT but both decreased BBB damage and microscopic hemorrhagic transformation. Only HBO reduced hemoglobin extravasation in the ischemic hemisphere. In conclusion, NBO and HBO decrease infarct size after thromboembolic ischemia only if recanalization is successful. As NBO and HBO also reduce postthrombolytic intracerebral hemorrhage, combining the two with thrombolysis seems promising.
cerebral ischemia; hemorrhage; hyperbaric oxygen; normobaric hyperoxia; thrombolysis
Recombinant adeno-associated viruses (rAAVs) that can cross the blood-brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5′ and 3′ mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
rAAV; intravascular delivery; CNS transduction; miRNA-binding site; miRNA regulation
Neutrophil gelatinase-associated lipocalin (NGAL) is a measure of acute kidney injury. Renal dysfunction portends significant risk after discharge from acute heart failure (AHF). Thus, a sensitive marker of renal injury might also help to risk stratify HF patients.
Methods and results
GALLANT [NGAL EvaLuation Along with B-type NaTriuretic Peptide (BNP) in acutely Decompensated Heart Failure] was a multicentre, prospective study to assess the utility of plasma NGAL, alone and in combination with BNP, as an early risk marker of adverse outcomes. We studied 186 patients (61% male). There were 29 events (AHF readmissions and all-cause mortality) at 30 days (16%). Patients with events had higher levels of NGAL than those without (134 vs. 84 ng/mL, P < 0.001). The area under the receiver operating characteristic curve was higher for NGAL (0.72) than BNP (0.65), serum creatinine (0.57), or estimated glomerular filtration rate (eGFR; 0.55). In multivariable analyses, NGAL predicted events (P= 0.001), BNP approached significance (P= 0.052 and 0.070 without creatinine and GFR, respectively) while neither serum creatinine nor eGFR were significant. The addition of discharge NGAL over BNP alone improved classification by a net 10.3% in those with events and 19.5% in those without events, for a net reclassification improvement of 29.8% (P= 0.010). Subjects with both BNP and NGAL elevated were at significant risk [hazard ratio (HR) = 16.85, P= 0.006], as were subjects with low BNP and high NGAL (HR = 9.95, P= 0.036).
Plasma NGAL is a measure of kidney injury that at the time of discharge is a strong prognostic indicator of 30 days outcomes in patients admitted for AHF.
Clinical trial registration number: NCT 00693745
Renal injury; Acute heart failure; Biomarkers; BNP
The diagnostic and prognostic value of arterial blood gas analysis (ABGA) parameters in unselected patients presenting with acute dyspnea to the Emergency Department (ED) is largely unknown.
We performed a post-hoc analysis of two different prospective studies to investigate the diagnostic and prognostic value of ABGA parameters in patients presenting to the ED with acute dyspnea.
We enrolled 530 patients (median age 74 years). ABGA parameters were neither useful to distinguish between patients with pulmonary disorders and other causes of dyspnea nor to identify specific disorders responsible for dyspnea. Only in patients with hyperventilation from anxiety disorder, the diagnostic accuracy of pH and hypoxemia rendered valuable with an area under the receiver operating characteristics curve (AUC) of 0.86. Patients in the lowest pH tertile more often required admission to intensive care unit (28% vs 12% in the first tertile, P < 0.001) and had higher in-hospital (14% vs 5%, P = 0.003) and 30-day mortality (17% vs 7%, P = 0.002). Cumulative mortality rate was higher in the first (37%), than in the second (28%), and the third tertile (23%, P = 0.005) during 12 months follow-up. pH at presentation was an independent predictor of 12-month mortality in multivariable Cox proportional hazard analysis both for patients with pulmonary (P = 0.043) and non-pulmonary disorders (P = 0.038).
ABGA parameters provide limited diagnostic value in patients with acute dyspnea, but pH is an independent predictor of 12 months mortality.
The objective of the study was to describe the technique, accuracy of placement and complications of transpedicular C2 screw fixation without spinal navigation. Patients treated by C2 pedicle screw fixations were identified from the surgical log book of the department. Clinical data were extracted retrospectively from the patients’ charts. Pedicle screw placement accuracy was assessed on postoperative CT scans according to Gertzbein and Robbins (GRGr). A total of 27 patients were included in the study. The mean age of the patients was 56 ± 22.0 years; 51.9% of them were female. As much as 17 patients suffered from trauma, 5 of degenerative disease, 3 of inflammations and 2 of metastatic disease. A total of 47 C2 transpedicular screw fixations were performed. The canulated screws were inserted under visual control following the preparation of the superior surface of the isthmus and of the medial surface of the pedicles of the C2. Intraoperative fluoroscopy was additionally used. The postoperative CT findings showed in 55.3% GRGr 1, in 27.7% GRGr 2, in 10.6% GRGr 3, and in 6.3% GRGr 4 pedicle screw insertion accuracy. Screw insertions GRGr 5 were not observed. Screw malpositioning (i.e., GRGr 3 and 4) was significantly associated with thin (<5 mm) pedicle diameters and with surgery for C2 fractures. In the three patients with screw insertions GRGr 4, postoperative angiographies were performed to exclude vertebral artery affections. In one of these three cases, the screw caused a clinically asymptomatic vertebral artery compression. Hardware failures did not occur. In one patient, postoperative pneumonia resulted in the death of the patient. Careful patient selection and surgical technique is necessary to avoid vertebral artery injury in C2 pedicle screw fixation without spinal navigation. A slight opening of the vertebral artery canal (Gertzbein and Robbins grade ≤3) does not seem to put the artery at risk. However, the high rate of misplaced screws when inserted without spinal navigation, despite the fact that no neurovascular injury occurred, supports the use of spinal navigation in C2 pedicle screw insertions.
C2 pedicle screw; Placement accuracy; Vertebral artery; Spinal navigation
Recombinant adeno-associated viruses (rAAVs) that can cross the blood–brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5′ and 3′ mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
Monitoring treatment efficacy and assessing outcome by serial measurements of natriuretic peptides in acute decompensated heart failure (ADHF) patients may help to improve outcome.
This was a prospective multi-center study of 171 consecutive patients (mean age 80 73-85 years) presenting to the emergency department with ADHF. Measurement of BNP and NT-proBNP was performed at presentation, 24 hours, 48 hours and at discharge. The primary endpoint was one-year all-cause mortality; secondary endpoints were 30-days all-cause mortality and one-year heart failure (HF) readmission.
During one-year follow-up, a total of 60 (35%) patients died. BNP and NT-proBNP levels were higher in non-survivors at all time points (all P < 0.001). In survivors, treatment reduced BNP and NT-proBNP levels by more than 50% (P < 0.001), while in non-survivors treatment did not lower BNP and NT-proBNP levels. The area under the ROC curve (AUC) for the prediction of one-year mortality increased during the course of hospitalization for BNP (AUC presentation: 0.67; AUC 24 h: 0.77; AUC 48 h: 0.78; AUC discharge: 0.78) and NT-proBNP (AUC presentation: 0.67; AUC 24 h: 0.73; AUC 48 h: 0.75; AUC discharge: 0.77). In multivariate analysis, BNP at 24 h (1.02 [1.01-1.04], P = 0.003), 48 h (1.04 [1.02-1.06], P < 0.001) and discharge (1.02 [1.01-1.03], P < 0.001) independently predicted one-year mortality, while only pre-discharge NT-proBNP was predictive (1.07 [1.01-1.13], P = 0.016). Comparable results could be obtained for the secondary endpoint 30-days mortality but not for one-year HF readmissions.
BNP and NT-proBNP reliably predict one-year mortality in patients with ADHF. Prognostic accuracy of both biomarker increases during the course of hospitalization. In survivors BNP levels decline more rapidly than NT-proBNP levels and thus seem to allow earlier assessment of treatment efficacy. Ability to predict one-year HF readmission was poor for BNP and NT-proBNP.
ClinicalTrials.gov identifier: NCT00514384.