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1.  Recovery of Olfactory Function Induces Neuroplasticity Effects in Patients with Smell Loss 
Neural Plasticity  2014;2014:140419.
The plasticity of brain function, especially reorganization after stroke or sensory loss, has been investigated extensively. Based upon its special characteristics, the olfactory system allows the investigation of functional networks in patients with smell loss, as it holds the unique ability to be activated by the sensorimotor act of sniffing, without the presentation of an odor. In the present study, subjects with chronic peripheral smell loss and healthy controls were investigated using functional magnetic resonance imaging (fMRI) to compare functional networks in one of the major olfactory areas before and after an olfactory training program. Data analysis revealed that olfactory training induced alterations in functional connectivity networks. Thus, olfactory training is capable of inducing neural reorganization processes. Furthermore, these findings provide evidence for the underlying neural mechanisms of olfactory training.
PMCID: PMC4269319  PMID: 25544900
2.  Carrageenan nasal spray in virus confirmed common cold: individual patient data analysis of two randomized controlled trials 
Clinical trials applying iota-carrageenan nasal spray have previously shown to reduce duration of virus-confirmed common cold. The present study pooled data of two similar clinical trials to provide further evidence for the antiviral effectiveness of carrageenan.
Individual patient data were analyzed from two randomized double blind placebo controlled trials assessing the therapeutic effectiveness of carrageenan nasal spray in acute common cold. Patients with virus-confirmed common cold (n = 254, verum 126, placebo 128) were included and the following parameters were appraised: duration of disease, number of patients with relapses, number of respiratory viruses and viral titers at inclusion (visit 1) compared to days 3–5 (visit 2).
Carrageenan treated patients showed a significant reduction in duration of disease of almost 2 days (p < 0.05) as well as significantly fewer relapses during 21 days of observation period (p < 0.05). The virus clearance between visit 1 and visit 2 was significantly more pronounced in the carrageenan group (p < 0.05). In both studies, virus-confirmed common cold was caused by three main virus subtypes: human rhinovirus (46%), human coronavirus (25%) and influenza A (14%) virus. Carrageenan nasal spray showed significant antiviral efficacy in all three virus subgroups, the highest effectiveness was observed in human corona virus-infected patients. The reduced duration of disease was 3 days (p < 0.01) and the number of relapses was three times less (p < 0.01) in carrageenan treated corona-virus-infected patients compared to control patients.
Administration of carrageenan nasal spray in children as well as in adults suffering from virus-confirmed common cold reduced duration of disease, increased viral clearance and reduced relapses of symptoms. Carrageenan nasal spray appeared as an effective treatment of common cold in children and adults.
Trial registration
Pooled data from ISRCTN52519535 and ISRCTN80148028
PMCID: PMC4236476  PMID: 25411637
Carrageenan; Common cold; Coronavirus; Respiratory disease; Rhinovirus; Influenza; Virus
3.  BNP but Not s-cTnln Is Associated with Cardioembolic Aetiology and Predicts Short and Long Term Prognosis after Cerebrovascular Events 
PLoS ONE  2014;9(7):e102704.
We analyzed the prognostic value of b-type natriuretic peptide (BNP) and sensitive cardiac Troponin (s-cTnI) in patients with ischemic stroke or transient ischemic attack (TIA) and their significance in predicting stroke aetiology.
In a prospectively enrolled cohort we measured BNP and s-cTnI levels upon admission. Primary endpoints were mortality, unfavorable functional outcome and stroke recurrence after 90 days and after 12 months. Secondary endpoint was cardioembolic aetiology.
In 441 patients BNP but not s-cTnI remained an independent predictor for death with an adjusted HR of 1.2 (95% CI 1.1–1.4) after 90 days and 1.2 (95% CI 1.0–1.3) after one year. The comparison of the Area under Receiver Operating Characteristic (AUROC) of model A (age, NIHSS) and model B (age, NIHSS, BNP) showed an improvement in the prediction of mortality (0.85 (95% CI 0.79–0.90) vs. 0.86 (95% CI 0.81–0.92), Log Rank p = 0.004). Furthermore the category free net reclassification improvement (cfNRI) when adding BNP to the multivariate model was 57.5%, p<0.0001. For the prediction of functional outcome or stroke recurrence both markers provided no incremental value. Adding BNP to a model including age, atrial fibrillation and heart failure lead to a higher discriminatory accuracy for identification of cardioembolic stroke than the model without BNP (AUC 0.75 (95% CI 0.70–0.80) vs. AUC 0.79, (95% CI 0.75–0.84), p = 0.008).
BNP is an independent prognostic maker for overall mortality in patients with ischemic stroke or TIA and may improve the diagnostic accuracy to identify cardioembolic aetiology.
Trial Registration NCT00390962
PMCID: PMC4114527  PMID: 25072816
Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2–15% of patients. We hypothesized that sensitization/challenge of CFTR−/− mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF.
FABP-hCFTR+/−-CFTR−/− mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines.
Pla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR−/− mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells.
The distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF.
PMCID: PMC4086715  PMID: 22985691
Allergy; Lung; Lipid Mediators; Inflammation
5.  Prognostic Value and Link to Atrial Fibrillation of Soluble Klotho and FGF23 in Hemodialysis Patients 
PLoS ONE  2014;9(7):e100688.
Deranged calcium-phosphate metabolism contributes to the burden of morbidity and mortality in dialysis patients. This study aimed to assess the association of the phosphaturic hormone fibroblast growth factor 23 (FGF23) and soluble Klotho with all-cause mortality. We measured soluble Klotho and FGF23 levels at enrolment and two weeks later in 239 prevalent hemodialysis patients. The primary hypothesis was that low Klotho and high FGF23 are associated with increased mortality. The association between Klotho and atrial fibrillation (AF) at baseline was explored as secondary outcome. AF was defined as presence of paroxysmal, persistent or permanent AF. During a median follow-up of 924 days, 59 (25%) patients died from any cause. Lower Klotho levels were not associated with mortality in a multivariable adjusted analysis when examined either on a continuous scale (HR 1.25 per SD increase, 95% CI 0.84–1.86) or in tertiles, with tertile 1 as the reference category (HR for tertile two 0.65, 95% CI 0.26–1.64; HR for tertile three 2.18, 95% CI 0.91–2.23). Higher Klotho levels were associated with the absence of AF in a muItivariable logistic regression analysis (OR 0.66 per SD increase, 95% CI 0.41–1.00). Higher FGF23 levels were associated with mortality risk in a multivariable adjusted analysis when examined either on a continuous scale (HR 1.45 per SD increase, 95% CI 1.05–1.99) or in tertiles, with the tertile 1 as the reference category (HR for tertile two 1.63, 95% CI 0.64–4.14; HR for tertile three 3.91, 95% CI 1.28–12.20). FGF23 but not Klotho levels are associated with mortality in hemodialysis patients. Klotho may be protective against AF.
PMCID: PMC4084634  PMID: 24991914
6.  Association of Adrenal Function and Disease Severity in Community-Acquired Pneumonia 
PLoS ONE  2014;9(6):e99518.
Rapid and accurate risk stratification in patients with community-acquired pneumonia (CAP) is an unmet clinical need. Cortisol to dehydroepiandrosterone (DHEA) ratio was put forward as a prognostic marker in sepsis. We herein validated the prognostic value of the adrenal hormones DHEA, DHEA-Sulfate (DHEAS), cortisol/DHEA-, cortisol/DHEAS- and DHEA/DHEAS – ratios in patients with CAP.
We assessed severity of illness using the pneumonia severity index (PSI) and measured adrenal hormone concentrations in 179 serum samples of prospectively recruited patients hospitalized with CAP. We calculated spearman rank correlation, logistic regression analysis and Kaplan Meier curves to study associations of adrenal hormones and outcomes.
There was a significant correlation between PSI score and total cortisol (r = 0.24, p = 0.001), DHEAS (r = −0.23, p = 0.002), cortisol/DHEA (r = 0.23, p = 0.003), cortisol/DHEAS (r = 0.32, p = <0.0001) and DHEA/DHEAS (r = 0.20, p = 0.009). In age and gender adjusted logistic regression analysis, cortisol (OR: 2.8, 95% CI: 1.48–5.28) and DHEA (OR: 2.62, 95% CI: 1.28–5.34), but not DHEAS and the different ratios were associated with all-cause mortality. The discriminatory accuracy of cortisol and DHEA in ROC analysis (area under the curve) was 0.74 and 0.61. In Kaplan Meier analysis, patients in the highest deciles of cortisol and DHEA (p = 0.005 and p = 0.015), and to a lesser extent of cortisol/DHEAS ratio (p = 0.081) had a higher risk of death.
Cortisol, DHEAS and their ratios correlate with CAP severity, and cortisol and DHEA predict mortality. Adrenal function in severe pneumonia may be an important factor for CAP outcomes.
PMCID: PMC4049821  PMID: 24910975
7.  Diagnostic accuracy of combined cardiac troponin and copeptin assessment for early rule-out of myocardial infarction: a systematic review and meta-analysis 
This systematic review aimed to investigate the diagnostic accuracy of combined cardiac troponin (cTn) and copeptin assessment in comparison to cTn alone for early rule-out of acute myocardial infarction (AMI).
Primary studies were eligible if they evaluated diagnostic accuracy for cTn with and without copeptin in patients with symptoms suggestive of AMI. AMI was defined according to the universal definition, using detection of cTn as a marker for myocardial necrosis. Eligible studies were identified by searching electronic databases (Medline, EMBASE, Science Citation Index Expanded, CINAHL, Pascal, and Cochrane) from inception to March 2013, reviewing conference proceedings and contacting field experts and the copeptin manufacturer.
In 15 studies totalling 8740 patients (prevalence of AMI 16%), adding copeptin improved the sensitivity of cTn assays (from 0.87 to 0.96, p=0.003) at the expense of lower specificity (from 0.84 to 0.56, p<0.001). In 12 studies providing data for 6988 patients without ST-segment elevation, the summary sensitivity and specificity estimates were 0.95 (95% CI 0.89 to 0.98) and 0.57 (95% CI 0.49 to 0.65) for the combined assessment of cTn and copeptin. When a high-sensitivity cTnT assay was used in combination with copeptin, the summary sensitivity and specificity estimates were 0.98 (95% CI 0.96 to 1.00) and 0.50 (95% CI 0.42 to 0.58).
Despite substantial between-study heterogeneity, this meta-analysis demonstrates that copeptin significantly improves baseline cTn sensitivity. Management studies are needed to establish the effectiveness and safety of measuring copeptin in combination with high-sensitivity cTnT for early rule-out of AMI without serial testing.
PMCID: PMC3932778  PMID: 24562800
copeptin; diagnostic accuracy; myocardial infarction; troponin
8.  Gene Transfer in Skeletal and Cardiac Muscle Using Recombinant Adeno-Associated Virus 
Current protocols in microbiology  2013;0 14:Unit-14D.3.
Adeno-associated virus (AAV) is a DNA virus with a small (~4.7kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer vector, is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. Only the viral inverted terminal repeats (ITRs) are required in cis for replication and packaging during production. The ITRs are also necessary and sufficient for vector genome processing and persistence during transduction. The tissue tropism of the rAAV vector is determined by the AAV capsid. In this unit we will discuss several methods to deliver rAAV in order to transduce cardiac and/or skeletal muscle, including: intravenous delivery, intramuscular delivery, isolated limb infusion, intrapericardial injection in neonatal mice, and left ventricular wall injection in adult rats.
PMCID: PMC3641885  PMID: 23408131
Adeno-associated virus; gene therapy; heart; skeletal muscle; vector delivery
9.  Plasma Concentrations of the Vasoactive Peptide Fragments Mid-Regional Pro-Adrenomedullin, C-Terminal Pro-Endothelin 1 and Copeptin in Hemodialysis Patients: Associated Factors and Prediction of Mortality 
PLoS ONE  2014;9(1):e86148.
Vasopressin, endothelin and adrenomedullin are vasoactive peptides that regulate vascular tone and might play a role in hypertensive diseases. Recently, laboratory assays have been developed to measure stable fragments of vasopressin, endothelin and adrenomedullin. Little is known about their diagnostic and prognostic value in hemodialysis patients. In this study, we measured the plasma concentration of copeptin, mid-regional-pro-adrenomedullin (MR-pro-ADM) and C-terminal pro-endothelin 1 (CT-pro-ET1) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality. In all patients enrolled, the plasma concentrations of copeptin, MR-pro-ADM and CT-pro-ET1 were largely elevated with a median concentration of 132 pmol/L (interquartile range [IQR] 78–192) for copeptin, 1.26 nmol/L (IQR 1.02–1.80) for MR-pro-ADM and 149 pmol/L (IQR 121–181) for CT-pro-ET1. The plasma concentrations of all vasoactive peptide fragments correlated with time on dialysis and plasma β2-microglobulin concentration and were negatively correlated to residual diuresis. The plasma concentration of MR-pro-ADM was a strong predictor of all-cause (univariate hazard ratio for a 10-fold increase 9.94 [3.14;32], p<0.0001) and cardiovascular mortality (hazard ratio 34.87 [5.58;217], p = 0.0001) within a 3.8-year follow-up. The associations remained stable in models adjusted for dialysis specific factors and were attenuated in a full model adjusted for all prognostic factors. Plasma copeptin concentration was weakly associated with cardiovascular mortality (only in univariate analysis) and CT-pro-ET1 was not associated with mortality at all. In conclusion, vasoactive peptide fragments are elevated in hemodialysis patients because of accumulation and, most likely, increased release. Increased concentrations of MR-pro-ADM are predictive of mortality.
PMCID: PMC3899212  PMID: 24465926
10.  Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice 
Gene therapies for neurological diseases with autonomic or gastrointestinal involvement may require global gene expression. Gastrointestinal complications are often associated with Parkinson's disease and autism. Lewy bodies, a pathological hallmark of Parkinson's brains, are routinely identified in the neurons of the enteric nervous system (ENS) following colon biopsies from patients. The ENS is the intrinsic nervous system of the gut, and is responsible for coordinating the secretory and motor functions of the gastrointestinal tract. ENS dysfunction can cause severe patient discomfort, malnourishment, or even death as in intestinal pseudo-obstruction (Ogilvie syndrome). Importantly, ENS transduction following systemic vector administration has not been thoroughly evaluated. Here we show that systemic injection of AAV9 into neonate or juvenile mice results in transduction of 25–57% of ENS myenteric neurons. Transgene expression was prominent in choline acetyltransferase positive cells, but not within vasoactive intestinal peptide or neuronal nitric oxide synthase cells, suggesting a bias for cells involved in excitatory signaling. AAV9 transduction in enteric glia is very low compared to CNS astrocytes. Enteric glial transduction was enhanced by using a glial specific promoter. Furthermore, we show that AAV8 results in comparable transduction in neonatal mice to AAV9 though AAV1, 5, and 6 are less efficient. These data demonstrate that systemic AAV9 has high affinity for peripheral neural tissue and is useful for future therapeutic development and basic studies of the ENS.
PMCID: PMC4197761  PMID: 25360081
AAV9; adeno-associated virus; enteric nervous system; myenteric plexus; functional gastrointestinal motility disorders; enteric glia; enteric neuropathy; gene therapy
11.  Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression 
The Journal of Clinical Investigation  2013;123(12):5310-5318.
Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.
PMCID: PMC3859421  PMID: 24231351
12.  Efficacy of a Carrageenan nasal spray in patients with common cold: a randomized controlled trial 
Respiratory Research  2013;14(1):124.
The common cold is the most widespread viral infection in humans. Iota-carrageenan has previously shown antiviral effectiveness against cold viruses in clinical trials. This study investigated the efficacy of a carrageenan-containing nasal spray on the duration of the common cold and nasal fluid viral load in adult patients.
In a randomized, double-blind, placebo-controlled trial, 211 patients suffering from early symptoms of the common cold were treated for seven days. Application was performed three times daily with either a carrageenan-supplemented nasal spray or saline solution as placebo with an overall observation period of 21 days. The primary endpoint was the duration of disease defined as the time until the last day with symptoms followed by all other days in the study period without symptoms. During the study, but prior unblinding, the definition of disease duration was adapted from the original protocol that defines disease duration as the time period of symptoms followed by 48 hours without symptoms.
In patients showing a laboratory-confirmed cold virus infection and adherence to the protocol, alleviation of symptoms was 2.1 days faster in the carrageenan group in comparison to placebo (p = 0.037). The primary endpoint that had been prespecified but was changed before unblinding was not met. Viral titers in nasal fluids showed a significantly greater decrease in carrageenan patients in the intention-to-treat population (p = 0.024) and in the per protocol population (p = 0.018) between days 1 and 3/4.
In adults with common cold virus infections, direct local administration of carrageenan with nasal sprays reduced the duration of cold symptoms. A significant reduction of viral load in the nasal wash fluids of patients confirmed similar findings from earlier trials in children and adults.
Trial registration
Current Controlled Trials ISRCTN80148028
PMCID: PMC3840586  PMID: 24219370
Common cold; Virus; Respiratory; Disease; Carrageenan
13.  Gene Transfer in the Lung Using Recombinant Adeno-Associated Virus 
Current protocols in microbiology  2012;CHAPTER:Unit14D.2.
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.
PMCID: PMC3486738  PMID: 22875566
Adeno-associated virus; vector delivery; lung; gene therapy
14.  Vertebral artery injuries following cervical spine trauma: a prospective observational study 
European Spine Journal  2011;20(12):2202-2209.
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.
PMCID: PMC3229739  PMID: 21717238
Vertebral artery injury; Arteriography; Cervical spine trauma; CT-angiography; MR-angiography
15.  Sensitive Troponins – Which Suits Better for Hemodialysis Patients? Associated Factors and Prediction of Mortality 
PLoS ONE  2012;7(10):e47610.
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.
PMCID: PMC3471860  PMID: 23077650
16.  Phase 2 Clinical Trial of a Recombinant Adeno-Associated Viral Vector Expressing α1-Antitrypsin: Interim Results 
Human Gene Therapy  2011;22(10):1239-1247.
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.
PMCID: PMC3205788  PMID: 21609134
17.  Injury patterns of seniors in traffic accidents: A technical and medical analysis 
World Journal of Orthopedics  2012;3(9):151-155.
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.
PMCID: PMC3502611  PMID: 23173111
Traffic accidents; Seniors; Head injury; Injury severity score; Abbreviated injury scale
18.  Impact of mannose-binding lectin deficiency on radiocontrast-induced renal dysfunction: a post-hoc analysis of a multicenter randomized controlled trial 
BMC Nephrology  2012;13:99.
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.
PMCID: PMC3471006  PMID: 22938690
Complement; Mannose-binding lectin; Contrast-induced nephropathy; Ischemia/reperfusion injury; Acute kidney injury
19.  Environmental and non-infectious factors in the aetiology of pharyngitis (sore throat) 
Inflammation Research  2012;61(10):1041-1052.
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.
PMCID: PMC3439613  PMID: 22890476
Air pollution; Cold dry air; Experimental model; Inflammation; Occupational exposure; Therapy; Pain
20.  N-Glycosylation Augmentation of the Cystic Fibrosis Epithelium Improves Pseudomonas aeruginosa Clearance 
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.
PMCID: PMC3135844  PMID: 20693405
bacterial clearance; cystic fibrosis; gene therapy; N-glycosylation
21.  Lack of Cystic Fibrosis Transmembrane Conductance Regulator in CD3+ Lymphocytes Leads to Aberrant Cytokine Secretion and Hyperinflammatory Adaptive Immune Responses 
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.
PMCID: PMC3135852  PMID: 20724552
cystic fibrosis transmembrane conductance regulator; lymphocytes; allergy; intracellular calcium; nuclear factor of activated T cell
22.  Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy 
Molecular Therapy  2012;20(6):1131-1138.
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.
PMCID: PMC3370259  PMID: 22395529
23.  Happy birthday BNP 
PMCID: PMC3760530  PMID: 24062896
24.  Hepatocyte-specific Hypoxia Inducible Factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice 
Hepatology (Baltimore, Md.)  2011;53(5):1526-1537.
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.
PMCID: PMC3104403  PMID: 21520168
Hypoxia-Inducible Factor-1 alpha; Monocyte-Chemoattractant Protein-1; Steatosis; Alcoholic Liver Disease
25.  Gene therapy for alpha-1 antitrypsin deficiency 
Human Molecular Genetics  2011;20(R1):R87-R92.
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.
PMCID: PMC3095063  PMID: 21498872

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