Claire Jackson

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

Background and Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor β (TGFβ) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFβ signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn’s disease (CD). Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFβ blocking antibody or TGFβ1. TGFβ transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. Results: TGFβ transcripts, phosphorylated Smad2–Smad3 (pSmad2–3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFβ transcripts, a greater pSmad2–3 response to TGFβ, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFβ blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. Conclusions: Changes in TGF-β signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed “radial spoke defect.” We sequenced CCDC39 and CCDC40 in 54 “radial spoke defect” families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by “null” alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as “IDA and microtubular disorganisation defect,” rather than “radial spoke defect.”

Nitric oxide in primary ciliary dyskinesia

Nitric oxide is continually synthesised in the respiratory epithelium and is upregulated in response to infection or inflammation. Primary ciliary dyskinesia (PCD) is characterised by recurrent sinopulmonary infections due to impaired mucociliary clearance. Despite chronic infections, nasal nitric oxide in such patients is markedly reduced and is used as a screening test for this condition. These low levels were first described >15 yrs ago but the underlying mechanisms have yet to be fully elucidated. We review epithelial nitric oxide synthesis, release and measurement in the upper airways with particular reference to PCD. The key hypotheses that have been proposed to explain the low nitric oxide levels in this condition are explored and the potential benefits of augmenting airway nitric oxide levels are considered. Further work in these patients clarifying both whether the respiratory epithelium is able to biosynthesise normal levels of nitric oxide and the role played by abnormalities in the anatomy of the paranasal sinuses is essential. While nitric oxide augmentation is unlikely to be beneficial in common PCD phenotypes, it has potential in the treatment of secondary dyskinesias and may also improve treatment of bacterial infections, particularly where biofilms are implicated.

α-Melanocyte-Stimulating Hormone Suppresses Antigen-Induced Lymphocyte Proliferation in Humans Independently of Melanocortin 1 Receptor Gene Status

Studies in mice indicate that α-melanocyte-stimulating hormone (αMSH) is immunosuppressive, but it is not known whether αMSH suppresses human immune responses to exogenous Ags. Human PBMCs, including monocytes, express the melanocortin 1 receptor (MC1R), and it is thought that the ability of αMSH to alter monocyte-costimulatory molecule expression and IL-10 release is mediated by this receptor. However, the MC1R gene is polymorphic, and certain MC1R variants compromise receptor signaling via cAMP, resulting in red hair and fair skin. Here, we have investigated whether αMSH can suppress Ag-induced lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. αMSH suppressed streptokinase-streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10−13–10−11 M αMSH. Anti-IL-10 Abs failed to prevent suppression by αMSH, indicating that it was not due to MC1R-mediated IL-10 release by monocytes. Despite variability in the degree of suppression between subjects, similar degrees of αMSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R alleles. RT-PCR of streptokinase-streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macrophages, MC1R and MC3R expression by B lymphocytes, but no melanocortin receptor expression by T lymphocytes. In addition, αMSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas αMSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that αMSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lymphocytes, has immunosuppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by αMSH is independent of MC1R gene status.

Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

Background The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. Methods We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. Results Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. Conclusions The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

Static respiratory cilia associated with mutations in Dnahc11/DNAH11: a mouse model of PCD

Primary ciliary dyskinesia (PCD) is an inherited disorder causing significant upper and lower respiratory tract morbidity and impaired fertility. Half of PCD patients show abnormal situs. Human disease loci have been identified but a mouse model without additional deleterious defects is elusive. The inversus viscerum mouse, mutated at the outer arm dynein heavy chain 11 locus (Dnahc11) is a known model of heterotaxy. We demonstrated immotile tracheal cilia with normal ultrastructure and reduced sperm motility in the Dnahc11iv mouse. This is accompanied by gross rhinitis, sinusitis, and otitis media, all indicators of human PCD. Strikingly, age‐related progression of the disease is evident. The Dnahc11iv mouse is robust, lacks secondary defects, and requires no intervention to precipitate the phenotype. Together these findings show the Dnahc11iv mouse to be an excellent model of many aspects of human PCD. Mutation of the homologous human locus has previously been associated with hyperkinetic tracheal cilia in PCD. Two PCD patients with normal ciliary ultrastructure, one with immotile and one with hyperkinetic cilia were found to carry DNAH11 mutations. Three novel DNAH11 mutations were detected indicating that this gene should be investigated in patients with normal ciliary ultrastructure and static, as well as hyperkinetic cilia. Hum Mutat 33:495–503, 2012.

Accuracy of diagnostic testing in primary ciliary dyskinesia

Diagnosis of primary ciliary dyskinesia (PCD) lacks a “gold standard” test and is therefore based on combinations of tests including nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA), genotyping and transmission electron microscopy (TEM). There are few published data on the accuracy of this approach. Using prospectively collected data from 654 consecutive patients referred for PCD diagnostics we calculated sensitivity and specificity for individual and combination testing strategies. Not all patients underwent all tests. HSVMA had excellent sensitivity and specificity (100% and 93%, respectively). TEM was 100% specific, but 21% of PCD patients had normal ultrastructure. nNO (30 nL·min−1 cut-off) had good sensitivity and specificity (91% and 96%, respectively). Simultaneous testing using HSVMA and TEM was 100% sensitive and 92% specific. In conclusion, combination testing was found to be a highly accurate approach for diagnosing PCD. HSVMA alone has excellent accuracy, but requires significant expertise, and repeated sampling or cell culture is often needed. TEM alone is specific but misses 21% of cases. nNO (≤30 nL·min−1) contributes well to the diagnostic process. In isolation nNO screening at this cut-off would miss ∼10% of cases, but in combination with HSVMA could reduce unnecessary further testing. Standardisation of testing between centres is a future priority. Combination testing in PCD diagnosis remains the most accurate approach, but standardisation is needed http://ow.ly/TLEDu

Ciliary Beat Pattern Analysis Below 37°C May Increase Risk of Primary Ciliary Dyskinesia Misdiagnosis

We thank Dr Mao and colleagues for their comments on our article in CHEST . 1 Investigations on lung transplantation are often single-center studies with limited external validity; thus, including data from two study sites strengthened our investigation. An analysis by study site showed comparable results for allograft function, and introducing study site as a variable into the Cox proportional hazard models for the occurrence of bronchiolitis obliterans syndrome (BOS) showed no signifi cant interaction between study site and BOS ( P 5 .748 for univariate and P 5 .774 for multivariate model). Information on transplant protocols and recipient characteristics by study site for the same cohorts as in this investigation has been published. 2 The concern was raised that oversizing the allograft could be associated with an increase in early postoperative complications. We reported in a subsequent study that oversized allografts, as estimated by a predicted total lung capacity ratio . 1.0, were not associated with an increase in complications after bilateral lung transplant. 3 The latter investigation was limited to the post-lung-allocation-score era, and the undersized cohort had a signifi cantly higher lung allocation score, was more likely to be in the ICU prior to transplant, and had a higher need for cardiopulmonary bypass during transplant. 3 Thus, differences in the acuity and complexity of undersized compared with oversized patients might account for some of the observed differences. Lung trimming because of an oversized allograft was required in only one patient in the study reporting on the effects of lung size mismatch on complications 3 ; however, we do not have comprehensive data on this for the entire cohort for this study, which we expressed in the discussion of study limitations. 1

Neuronal NOS localises to human airway cilia

BACKGROUND Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. METHODS AND RESULTS Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. CONCLUSIONS We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function.

Ciliated Cultures From Patients With Primary Ciliary Dyskinesia Produce Nitric Oxide in Response to Haemophilus influenzae Infection and Proinflammatory Cytokines

1article. The study was approved by the Southampton and South West Hampshire Research Ethics Committee (06/Q1702/109). Airway epithelium from patients with PCD and control patients without PCD was obtained by nasal brushing and was cultured at the air-liquid interface until differentiated and ciliated. We quantifi ed the presence of NO using a total NO detection assay (Enzo Life Sciences, Inc) within phosphate-buffered saline washes applied to the apical surface of air-liquid interface cultures. NO levels were measured before and after epithelial cells were apically cocultured for 72 h with NTHi at a multiplicity of infection of 100 to evaluate biofi lm infection. Cell viability was demonstrated by daily stable transepithelial electrical resistance (PCD and non-PCD) and ciliary beat frequency measurements (control subjects without PCD). We also measured levels following an 18-h incubation with a cocktail of proinflammatory cytokines (10 ng/mL each of IL-1 b /interferon- g /tumor necrosis factor- a ) applied basolaterally to the cells (n 5 14 for each experiment). Our baseline data were consistent with that of Smith et al, 1