Julia Hentschel

Sinonasal persistence of Pseudomonas aeruginosa after lung transplantation

UNLABELLED We report on two CF patients who received double lung transplantation (LTX) due to Pseudomonas aeruginosa related pulmonary destruction. Prior to LTX we detected P. aeruginosa in nasal lavages (NL) and sputum cultures from both patients. Donor lungs of patient 1 became colonized within four weeks with P. aeruginosa identical in genotype with isolates from his pre-transplant sputum cultures and pre- and post-transplant NL. In contrast, patient 2 remained P. aeruginosa free in lower airway samples (bronchial lavage/sputum) for now up to 30 months, despite persistent detection of P. aeruginosa that was identical in genotype with pre-transplant NL and sputum isolates in NL and even in throat swabs. For prevention of pulmonary re-colonization patient 2 has continuously inhaled Colomycin 1 MIU once daily during the preceding more than 36 months with the novel Pari Sinus™ nebulizer, which in scintigraphic studies was shown to deliver vibrating aerosols into paranasal sinuses, additional to bronchial antibiotic inhalation. DISCUSSION Pulmonary colonization of transplanted donor lungs with identical clones previously colonizing the explanted lungs has been described previously and the upper airways were postulated as reservoir for descending colonization. However, this remained speculative, as upper airway sampling which does not belong to current standards, was not performed in these studies. Our report demonstrates persistence of identical P. aeruginosa genotypes in CF upper airways prior to and after LTX underlining risks of descending colonization of transplanted lungs with P. aeruginosa, which increases risks of graft dysfunction. Therefore, we recommend regular assessment of sinonasal colonization prior to and after LTX. Sinonasal inhalation with antimicrobials should be investigated in prospective trials.

Delineating the GRIN1 phenotypic spectrum A distinct genetic NMDA receptor encephalopathy

Objective: To determine the phenotypic spectrum caused by mutations in GRIN1 encoding the NMDA receptor subunit GluN1 and to investigate their underlying functional pathophysiology. Methods: We collected molecular and clinical data from several diagnostic and research cohorts. Functional consequences of GRIN1 mutations were investigated in Xenopus laevis oocytes. Results: We identified heterozygous de novo GRIN1 mutations in 14 individuals and reviewed the phenotypes of all 9 previously reported patients. These 23 individuals presented with a distinct phenotype of profound developmental delay, severe intellectual disability with absent speech, muscular hypotonia, hyperkinetic movement disorder, oculogyric crises, cortical blindness, generalized cerebral atrophy, and epilepsy. Mutations cluster within transmembrane segments and result in loss of channel function of varying severity with a dominant-negative effect. In addition, we describe 2 homozygous GRIN1 mutations (1 missense, 1 truncation), each segregating with severe neurodevelopmental phenotypes in consanguineous families. Conclusions: De novo GRIN1 mutations are associated with severe intellectual disability with cortical visual impairment as well as oculomotor and movement disorders being discriminating phenotypic features. Loss of NMDA receptor function appears to be the underlying disease mechanism. The identification of both heterozygous and homozygous mutations blurs the borders of dominant and recessive inheritance of GRIN1-associated disorders.

Influences of nasal lavage collection-, processing- and storage methods on inflammatory markers--evaluation of a method for non-invasive sampling of epithelial lining fluid in cystic fibrosis and other respiratory diseases.

BACKGROUND Non-invasive sampling of airway epithelial-lining-fluid by nasal lavage (NL) is an emerging method to monitor allergy, infection and inflammation in patients with respiratory diseases. However, the influences of collection-, processing- and storage-methods have not been sufficiently evaluated and standardized. METHODS Influences of repeated NL, centrifugation setups, repeated freezing and thawing, and protease inhibitors on mediator concentration were evaluated in healthy controls and CF patients, which serve as a model for chronic bacterial infection and inflammation. Polymorphonuclear leukocyte elastase (NE)/myeloperoxidase (MPO)/interleukin (IL)-1/IL-6/IL-8 and tumour necrosis factor alpha (TNF) concentrations were measured using ELISA and Multiplex Bead-Arrays. RESULTS NL-repetition within 0.5-4h markedly decreased NE, IL-8 and MPO-concentrations for up to 70%. NL centrifugation up to 250×g for cellular differentiation did not significantly influence mediator concentration in native and processed NL fluid. NL freezing and thawing markedly decreased IL-8 and MPO concentrations by up to 50% while NE remained stable. In contrast to preceding reports, storing at -70°C for ≥5 years led to significantly reduced mediator concentrations in NL compared to contemporary analyses, being most pronounced for IL-1β, IL-6 and TNFa. Storing of samples in the presence of protease inhibitors led to an increase in marker concentration for IL-8 (+27%) and MPO (+15%) even after one year of storage. CONCLUSIONS NL is an easy and robust technique for inflammation monitoring of the upper airways. For the first time we have shown that diagnostic NL should be performed only once daily to get comparable results. Whereas NL-fluid can be stored unprocessed at -70°C for cytokine analysis over 1-2 years with protease inhibitors supporting stability, ≥5 years storage as well as repeated freezing and thawing should be avoided.

Non-invasive assessment of upper and lower airway infection and inflammation in CF patients

The upper (UAW) and lower (LAW) airways of patients with cystic fibrosis (CF) have the same ion‐channel defects, but little is known about similarities and differences in host immunological responses at the two levels.

Reduced Nasal Nitric Oxide Production in Cystic Fibrosis Patients with Elevated Systemic Inflammation Markers

Background Nitric oxide (NO) is produced within the respiratory tract and can be detected in exhaled bronchial and nasal air. The concentration varies in specific diseases, being elevated in patients with asthma and bronchiectasis, but decreased in primary ciliary dyskinesia. In cystic fibrosis (CF), conflicting data exist on NO levels, which are reported unexplained as either decreased or normal. Functionally, NO production in the paranasal sinuses is considered as a location-specific first-line defence mechanism. The aim of this study was to investigate the correlation between upper and lower airway NO levels and blood inflammatory parameters, CF-pathogen colonisation, and clinical data. Methods and Findings Nasal and bronchial NO concentrations from 57 CF patients were determined using an electrochemical analyser and correlated to pathogen colonisation of the upper and lower airways which were microbiologically assessed from nasal lavage and sputum samples. Statistical analyses were performed with respect to clinical parameters (lung function, BMI), laboratory findings (CRP, leucocytes, total-IgG, fibrinogen), and anti-inflammatory and antibiotic therapy. There were significant correlations between nasal and bronchial NO levels (rho = 0.48, p<0.001), but no correlation between NO levels and specific pathogen colonisation. In patients receiving azithromycin, significantly reduced bronchial NO and a tendency to reduced nasal NO could be found. Interestingly, a significant inverse correlation of nasal NO to CRP (rho = −0.28, p = 0.04) and to leucocytes (rho = −0.41, p = 0.003) was observed. In contrast, bronchial NO levels showed no correlation to clinical or inflammatory parameters. Conclusion Given that NO in the paranasal sinuses is part of the first-line defence mechanism against pathogens, our finding of reduced nasal NO in CF patients with elevated systemic inflammatory markers indicates impaired upper airway defence. This may facilitate further pathogen acquisition in the sinonasal area, with consequences for lung colonisation and the overall outcome in CF.

BCR-ABL- and Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML

Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drugs action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

Soluble inflammation markers in nasal lavage from CF patients and healthy controls

BACKGROUND CF sinonasal and bronchial mucosa reveal identical ion channel defects. Nasal Lavage (NL) allows non-invasive repeated sampling of airway surface liquid. We compared inflammatory mediators in NL from CF-patients and healthy controls, and in CF in relation to sinonasal pathogen colonization. METHODS From 40 CF-patients (mean age 21.8yrs, SD 11.8yrs.) and 52 healthy controls (mean age 31.9yrs., SD 13.7yrs.) NL-fluid (10ml/nostril) concentrations of MPO, IL-8, IL-17A, sICAM-1, IL-1β, IL-6, TNF-α, IL-10 and IL-5 were determined using cytometric bead arrays for flow cytometry. RESULTS CF-patients showed significantly higher MPO-concentrations in NL-fluid and higher IL-8-levels (n.s.) than controls. MPO, IL-8, IL-17A, sICAM-1, IL-1β and IL-6 were significantly more often detectable in CF-patients than in controls. CF-patients with S. aureus colonization in both upper and lower airways had significantly elevated MPO and IL-8 levels in NL-fluid compared to S. aureus negatives. CONCLUSION NL-fluid differed substantially between CF-patients and healthy controls with most promising results for IL-8 and MPO, a primarily in CF-NL assessed mediator. Further studies are required to assess effects of sample collection and processing on concentrations of inflammatory markers and to evaluate potentials of NL analysis in research and clinical routine.

Pseudomonas aeruginosa Acquisition in Cystic Fibrosis Patients in Context of Otorhinolaryngological Surgery or Dentist Attendance: Case Series and Discussion of Preventive Concepts

Introduction. P. aeruginosa is the primary cause for pulmonary destruction and premature death in cystic fibrosis (CF). Therefore, prevention of airway colonization with the pathogen, ubiquitously present in water, is essential. Infection of CF patients with P. aeruginosa after dentist treatment was proven and dental unit waterlines were identified as source, suggesting prophylactic measures. For their almost regular sinonasal involvement, CF patients often require otorhinolaryngological (ORL) attendance. Despite some fields around ORL-procedures with comparable risk for acquisition of P. aeruginosa, such CF cases have not yet been reported. We present four CF patients, who primarily acquired P. aeruginosa around ORL surgery, and one around dentist treatment. Additionally, we discuss risks and preventive strategies for CF patients undergoing ORL-treatment. Perils include contact to pathogen-carriers in waiting rooms, instrumentation, suction, drilling, and flushing fluid, when droplets containing pathogens can be nebulized. Postsurgery mucosal damage and debridement impair sinonasal mucociliary clearance, facilitating pathogen proliferation and infestation. Therefore, sinonasal surgery and dentist treatment of CF patients without chronic P. aeruginosa colonization must be linked to repeated microbiological assessment. Further studies must elaborate whether all CF patients undergoing ORL-surgery require antipseudomonal prophylaxis, including nasal lavages containing antibiotics. Altogether, this underestimated risk requires structured prevention protocols.

A recurrent mutation in KCNA2 as a novel cause of hereditary spastic paraplegia and ataxia.

The hereditary spastic paraplegias (HSPs) are heterogeneous neurodegenerative disorders with over 50 known causative genes. We identified a recurrent mutation in KCNA2 (c.881G>A, p.R294H), encoding the voltage‐gated K+‐channel, KV1.2, in two unrelated families with HSP, intellectual disability (ID), and ataxia. Follow‐up analysis of > 2,000 patients with various neurological phenotypes identified a de novo p.R294H mutation in a proband with ataxia and ID. Two‐electrode voltage‐clamp recordings of Xenopus laevis oocytes expressing mutant KV1.2 channels showed loss of function with a dominant‐negative effect. Our findings highlight the phenotypic spectrum of a recurrent KCNA2 mutation, implicating ion channel dysfunction as a novel HSP disease mechanism. Ann Neurol 2016

A Novel Homozygous WDR72 Mutation in Two Siblings with Amelogenesis Imperfecta and Mild Short Stature

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous group of inherited defects of enamel formation. In isolated AI (no additional segregating features), mutations in at least 7 genes are known so far, causing dominant, recessive or X-linked AI and allowing the identification of the molecular etiology in 40–50% of affected families. We report on 2 siblings (an 11-year-old female and a 7-year-old male) born to consanguineous Turkish parents, with AI and mild, proportionate short stature. Both parents have normal teeth, but mother, maternal grandmother and great-grandfather are/were also of short stature. A spine X-ray performed in the girl excluded brachyolmia. Affymetrix GenomeWide SNP6.0 Array analysis identified no pathogenic copy number changes, but showed sharing of large homozygous regions, including chromosome band 15q21.3 containing the WDR72 gene. WDR72 sequence analysis in both siblings revealed homozygosity for a novel stop mutation in exon 10 (c.997A>T, p.Lys333X) explaining the AI phenotype. Mutations in WDR72 are a very rare cause of autosomal-recessive hypomaturation type of isolated AI. The mutation described in our patients specifies the diagnosis AI IIA3 and represents only the sixth WDR72 mutation reported so far. The WDR72 protein is critical for dental enamel formation, but its exact function is still unknown.