Hannah Blau

DNAI2 Mutations Cause Primary Ciliary Dyskinesia with Defects in the Outer Dynein Arm

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic destructive airway disease and randomization of left/right body asymmetry. Males often have reduced fertility due to impaired sperm tail function. The complex PCD phenotype results from dysfunction of cilia of the airways and the embryonic node and the structurally related motile sperm flagella. This is associated with underlying ultrastructural defects that frequently involve the outer dynein arm (ODA) complexes that generate cilia and flagella movement. Applying a positional and functional candidate-gene approach, we identified homozygous loss-of-function DNAI2 mutations (IVS11+1G > A) in four individuals from a family with PCD and ODA defects. Further mutational screening of 105 unrelated PCD families detected two distinct homozygous mutations, including a nonsense (c.787C > T) and a splicing mutation (IVS3-3T > G) resulting in out-of-frame transcripts. Analysis of protein expression of the ODA intermediate chain DNAI2 showed sublocalization throughout respiratory cilia. Electron microscopy showed that mutant respiratory cells from these patients lacked DNAI2 protein expression and exhibited ODA defects. High-resolution immunofluorescence imaging demonstrated absence of the ODA heavy chains DNAH5 and DNAH9 from all DNAI2 mutant ciliary axonemes. In addition, we demonstrated complete or distal absence of DNAI2 from ciliary axonemes in respiratory cells of patients with mutations in genes encoding the ODA chains DNAH5 and DNAI1, respectively. Thus, DNAI2 and DNAH5 mutations affect assembly of proximal and distal ODA complexes, whereas DNAI1 mutations mainly disrupt assembly of proximal ODA complexes.

Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis

In a subset of patients with cystic fibrosis (CF), nonsense mutations (premature stop codons) disrupt production of full-length, functional CF transmembrane conductance regulator (CFTR). Ataluren (PTC124) allows ribosomal readthrough of premature stop codons in mRNA. We evaluated drug activity and safety in patients with nonsense mutation CF who took ataluren three times daily (morning, midday and evening) for 12 weeks at either a lower dose (4, 4 and 8 mg·kg−1) or higher dose (10, 10 and 20 mg·kg−1). The study enrolled 19 patients (10 males and nine females aged 19–57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

Clinical and Genetic Risk Factors for Cystic Fibrosis-related Liver Disease

Objective. The aim of this study was to define the role of possible risk factors for the development of cystic fibrosis (CF)-related liver disease and to analyze the association between liver disease and the different genotypes present in the Israeli CF patient population. Patients and Methods. All patients followed at the seven CF centers in Israel were included in this study. Liver disease was determined by persistently elevated serum liver enzymes and/or bilirubin, and/or significant ultrasonographic changes suggestive of chronic liver disease. The following clinical parameters were evaluated: ethnic origin, age at assessment of liver function, sex, history of meconium ileus, pancreatic function, history of distal intestinal obstruction syndrome, pulmonary function, and cystic fibrosis transmembrane conductance regulator mutation analysis. Results. Of the 288 patients screened, 80 (28%) had liver disease. Of the 256 patients with pancreatic insufficiency, 80 (31%) had liver disease compared with none of the 32 patients with pancreatic sufficiency. Genotype-phenotype correlation was performed on 207 patients carrying identified mutations that were previously classified according to phenotype severity. Liver disease was found in 56 (32%) of 173 patients carrying mutations associated with a severe phenotype and in 6 (38%) of 16 patients carrying at least one mutation associated with a variable genotype (G85E and/or 5T allele). None of the 18 patients carrying the 3849+10kb C->T mutation had liver disease. Prevalence of liver disease increased with age. No correlation was found between liver disease and severity of lung disease, nutritional status, history of meconium ileus, or distal intestinal obstruction syndrome. Conclusion. CF patients who have pancreatic insufficiency and carry mutations associated with a severe or a variable genotype are at increased risk to develop liver disease.

Nasal potential difference measurements in patients with atypical cystic fibrosis

The diagnosis of cystic fibrosis (CF) is based on characteristic clinical and laboratory findings. However, a subgroup of patients present with an atypical phenotype that comprises partial CF phenotype, borderline sweat tests and one or even no common cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The aim of this study was to evaluate the role of nasal potential difference (PD) measurements in the diagnosis of CF patients with an atypical presentation and in a population of patients suspected to have CF. Nasal PD was measured in 162 patients from four different groups: patients with classical CF (n = 31), atypical phenotype (n = 11), controls (n = 50), and patients with questionable CF (n = 70). The parameter, or combination of nasal PD parameters was calculated in order to best discriminate all CF patients (including atypical CF) from the non-CF group. The patients with atypical CF disease had intermediate values of PD measurements between the CF and non-CF groups. The best discriminate model that assigned all atypical CF patients as CF used: e(response to chloride-free and isoproterenol/response to amiloride) with a cut-off >0.70 to predict a CF diagnosis. When this model was applied to the group of 70 patients with questionable CF, 24 patients had abnormal PD similar to the atypical CF group. These patients had higher levels of sweat chloride concentration and increased rate of CFTR mutations. Nasal potential difference is useful in diagnosis of patients with atypical cystic fibrosis. Taking into account both the sodium and chloride transport elements of the potential difference allows for better differentiation between atypical cystic fibrosis and noncystic fibrosis patients. This calculation may assist in the diagnostic work-up of patients whose diagnosis is questionable.

Nontuberculous mycobacteria in cystic fibrosis associated with allergic bronchopulmonary aspergillosis and steroid therapy

Nontuberculous mycobacterial (NTM) infection, particularly due to Mycobacterium abscessus, is an emerging disease that can be relentlessly progressive, particularly in cystic fibrosis (CF) patients. The risk factors that were associated with this increasingly symptomatic infection in a group of CF patients were investigated. A total of 139 CF patients aged 2–52 yrs were reviewed. Sputum was cultured for NTM annually or whenever clinical deterioration was unexplained. In total, 12 patients (8.6%) had positive cultures and six (4.3%) met the criteria for NTM pulmonary disease (five with M. abscessus). Five had allergic bronchopulmonary aspergillosis (ABPA) compared with one out of 133 patients without NTM disease. Five had received systemic steroids (four as a treatment for ABPA) compared with only one out of 133 without NTM lung disease. All six NTM patients deteriorated markedly following mycobacterial infection, and forced expiratory volume in one second dropped 18–46%. Despite prolonged triple antibiotic therapy, M. abscessus was not eradicated, and four out of six did not return to baseline clinically. In conclusion, severe nontuberculous mycobacterial lung disease, particularly with Mycobacterium abscessus, is becoming a perplexing challenge in cystic fibrosis patients. Allergic bronchopulmonary aspergillosis and systemic steroids appear to be risk factors, although small patient numbers limit this to a descriptive observation. When pulmonary condition deteriorates, increased surveillance for mycobacteria would enable prompt diagnosis and treatment.

Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-kappaB and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines

ABSTRACT We previously showed that moxifloxacin (MXF) exerts protective anti-inflammatory effects in immunosuppressed mice infected with Candida albicans by inhibiting interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) production in the lung. Immunohistochemistry demonstrated inhibition of nuclear factor (NF)-κB translocation in lung epithelium and macrophages in MXF-treated mice. In the present study we investigated the effects of MXF on the production of proinflammatory cytokines (i.e., IL-8, TNF-α, and IL-1β) by activated human peripheral blood monocytes and THP-1 cells and analyzed the effects of the drug on the major signal transduction pathways associated with inflammation: NF-κB and the mitogen-activated protein kinases ERK and c-Jun N-terminal kinase (JNK). The levels of IL-8, TNF-α, and IL-1β secretion rose 20- and 6.7-fold in lipopolysaccharide (LPS)-activated monocytes and THP-1 cells, respectively. MXF (5 to 20 μg/ml) significantly inhibited cytokine production by 14 to 80% and 15 to 73% in monocytes and THP-1 cells, respectively. In THP-1 cells, the level of NF-κB nuclear translocation increased fourfold following stimulation with LPS-phorbol myristate acetate (PMA), and this was inhibited (38%) by 10 μg of MXF per ml. We then assayed the degradation of inhibitor (I)-κB by Western blotting. LPS-PMA induced degradation of I-κB by 73%, while addition of MXF (5 μg/ml) inhibited I-κB degradation by 49%. Activation of ERK1/2 and the 46-kDa p-JNK protein was enhanced by LPS and LPS-PMA and was significantly inhibited by MXF (54 and 42%, respectively, with MXF at 10 μg/ml). We conclude that MXF suppresses the secretion of proinflammatory cytokines in human monocytes and THP-1 cells and that it exerts its anti-inflammatory effects in THP-1 cells by inhibiting NF-κB, ERK, and JNK activation. Its anti-inflammatory properties should be further assessed in clinical settings.

Immunomodulatory and Protective Effects of Moxifloxacin against Candida albicans-Induced Bronchopneumonia in Mice Injected with Cyclophosphamide

ABSTRACT In a previous study, moxifloxacin was shown to ameliorate immunosuppression and enhance cytokine production in several tissues, including the lungs of cyclophosphamide-injected mice. We examined here the effects of moxifloxacin on Candida albicans lung infection in cyclophosphamide-injected mice. Mice were injected on day 0 with 250 mg of cyclophosphamide/kg, and on days 1 to 4 they were given moxifloxacin at 22.5 mg/kg/day compared to controls given ceftazidime at 75 mg/kg/day or saline. On day 6, C. albicans (10 7 CFU/mouse) was inoculated intratracheally, and animals were observed for the development of bronchopneumonia, weight loss, mortality, the presence of C. albicans, and lung cytokine production. Histopathology on day 10 postinoculation revealed bronchopneumonia in 50, 67, and 0% of saline-, ceftazidime-, and moxifloxacin-treated mice, respectively (P < 0.05). The mortality rates were 28, 17, and 5%, respectively (P < 0.05), and weight loss occurred at 20, 32, and 0%, respectively (P < 0.05). By day 15, C. albicans was eliminated from all moxifloxacin-treated mice but was still isolated from lung homogenates of 50 to 60% of the saline- and ceftazidime-treated groups. Among the cytokines tested on days 0 to 15, we found an increased production of tumor necrosis factor alpha, KC (functional interleukin-8), and gamma interferon in the lungs of ceftazidime- and saline-treated controls compared to the moxifloxacin pretreatment that abolished their secretion. In conclusion, moxifloxacin protected cyclophosphamide-injected mice from C. albicans-induced lung infection and significantly reduced pneumonia, weight loss, and mortality despite the lack of direct antifungal activity. This is most likely due to an immunomodulating activity conferred by moxifloxacin, as shown in this model and in our previous studies. Its potential protective role should be studied in patients undergoing chemotherapy and immune suppression.

Rapid induction of clinical response with a short-term high-dose starting schedule of budesonide nebulizing suspension in young children with recurrent wheezing episodes

BACKGROUND There are no data currently available on the correct schedule for the initiation of treatment with nebulized suspension of budesonide in children with recurrent wheezing episodes. We compared the efficacy and safety of starting with a high dose followed by a stepwise decrease to a continuous low dose. METHODS In a double-blind design, 42 children aged 6 months to 3 years were randomly allocated to receive either a high starting dose of 1 mg budesonide twice daily followed by a stepwise decrease of 25% every second day for 1 week (group A) or a low dose of 0.25 mg twice daily for 1 week (group B). Efficacy was assessed with daily symptom scores and the systemic effect of the corticosteroids with the adrenocorticotropic hormone test. RESULTS The two groups were comparable for all parameters evaluated. During the first week of treatment, there was a significant decrease in asthmatic symptomatology only in group A: a 59% decrease for wheezing (p = 0.0001), 39% for diurnal cough (p = 0.036), and 39% for nocturnal cough (p = 0.04). Mean time to clinical response was 3.0 days in group A and 5.7 days in group B (p = 0.02). This early improvement was sustained for the rest of the follow-up period. The high dose starting schedule was not associated with any change in serum cortisol level. CONCLUSIONS The administration of nebulized suspension of budesonide at a high starting dose schedule followed by a rapid (1 week) stepwise decrease yields a significant early improvement in asthma symptoms and causes no change in serum cortisol levels.

Long term nutritional rehabilitation by gastrostomy in Israeli patients with cystic fibrosis: clinical outcome in advanced pulmonary disease.

Objectives: Several studies have shown a linear correlation between nutritional status and pulmonary function in patients with cystic fibrosis. Our study aims were: 1) To evaluate the effect of nutritional supplementation via gastrostomy on nutritional, clinical, and pulmonary parameters, and 2) To identify predicting factors for success of long-term nutritional rehabilitation. Methods: Twenty-one Israeli patients, aged 8 months to 20 years, underwent gastrostomy insertion from 1992 to 2001. All patients were pancreatic insufficient, and all carried severe mutations (W1282X in 62% of the patients). Anthropometric and clinical data were obtained for each patient: 0−12 months before and 6−12 months and 18−24 months after gastrostomy placement. Standard deviation scores (SDS) for height, weight, and body mass index as well as percent of height-appropriate body weight were calculated. Results: The mean percent-of-predicted forced expiratory volume in 1 second (FEV1) decreased significantly during the first year of gastrostomy feeding (n = 16), from 44.2% ± 13.9 to 41% ± 13.3 (P = 0.05). However, during the second year of therapy (n = 10), a trend toward improvement was observed (from 39.4 ± 12.1 to 41.4 ± 16.1). Weight, and BMI z-scores as well as weight percent-of ideal body weight increased significantly. Height z-score for age decreased during the first year (from −1.9 ± 1.3 to −2.1 ± 1.4), However, a trend toward improvement was observed during the second year. A significant correlation was found between the change in weight z-score and height z-score during the first (r = 0.488, P = 0.016) and the second (r = 0.825, P < 0.001) years. There was no difference between compliers and noncompliers regarding height, weight, and BMI either before or after gastrostomy placement. A significant correlation between age at insertion of gastrostomy and improvement in height z-score (r = 0.52, P = 0.016) was observed. Cystic fibrosis related diabetes (n = 8) did not affect the response to supplemental feeding. Conclusions: We observed a trend toward improvement of pulmonary disease during the second year, and a significant improvement in weight, height, and BMI z-scores. Compliance, diabetes, and young age prior to tube insertion did not predict success of nutritional rehabilitation.