rectal-organoid-morphology-analysis-roma-diagnostic-assay-cystic

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

KU Leuven

University Hospitals Leuven

In children, post-obstructive pulmonary edema is a rare condition, caused by a sudden change in upper airway patency. It causes dyspnea, tachypnea, hypoxemia, and at times hemoptysis and respiratory insufficiency. It occurs as a complication in the immediate post-operative period. Pediatricians should be aware of this clinical entity.

Hemoptysis after orthopedic surgery in an adolescent boy.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease, caused by specific primary structural and/or functional abnormalities of the motile cilia, in contrast with the transitory abnormalities seen in secondary ciliary dyskinesia. Disease-causing mutations in at least 16 genes have already been identified. The true incidence of PCD may be higher than currently reported, because the diagnosis is challenging and often missed. For the confirmation of PCD, both ciliary motility as well as ciliary ultrastructure must be evaluated. An early and adequate diagnosis and therapy can theoretically prevent bronchiectasis. Measurement of nasal nitric oxide has some value as a screening test but cannot be performed in young children. In the respiratory tract epithelium, impaired mucociliary clearance leads to chronic and/or recurrent upper and lower respiratory tract infections. In up to 75 % of the patients, respiratory manifestations start in the newborn period, although the diagnosis is often missed at that time. During embryogenesis, nodal cilia, which are motile cilia, determine the correct lateralization of the organs. Dysfunction of these cilia leads to random lateralization and thus situs inversus in approximately 50 % of the patients with PCD. The tail of a spermatozoon has a structure similar to that of a motile cilium. Consequently, male infertility due to immotile spermatozoa is often part of the characteristics of PCD. Given the heterogeneity and the rarity of the disorder, therapy is not evidence-based. Many treatment schedules are proposed in analogy with the treatment for cystic fibrosis.

Primary ciliary dyskinesia, an orphan disease.

Primary ciliary dyskinesia (PCD) is a rare disorder with variable disease progression. To date, mutations in more than 20 different genes have been found. At present, PCD subtypes are described according to the ultrastructural defect on transmission electron microscopy (TEM) of the motile cilia. PCD with normal ultrastructure (NU) is rarely reported because it requires additional testing. Biallelic mutations in DNAH11 have been described as one cause of PCD with NU.The aim of our study was to describe the clinical characteristics of a large population of patients with PCD, in relation to the ultrastructural defect. Additionally, we aimed to demonstrate the need for biopsy and cell culture to reliably diagnose PCD, especially the NU subtype.

Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure.

Primary ciliary dyskinesia (PCD) and humoral mmunodeficiency (HID) are both rare disorders which cause recurrent upper and lower respiratory tract infections.

Primary ciliary dyskinesia and humoral immunodeficiency--is there a missing link?

After repeated cycles of lung infection and inflammation, patients with cystic fibrosis (CF) evolve to respiratory insufficiency. Although histology and imaging have provided descriptive information, a thorough morphometric analysis of end-stage CF lung disease is lacking.

Morphometric Analysis of Explant Lungs in Cystic Fibrosis.

Nebulized drugs are used in the treatment of cystic fibrosis (CF) lung disease, asthma, and COPD, and increasingly also in other chronic lung diseases. Their use in CF is reasonably evidence based, but this is not so for use in other orphan diseases. Potential side effects often have not been studied. Therefore, we evaluated the influence of nebulized drugs on ciliary activity in an in vitro model.

The Influence of Nebulized Drugs on Nasal Ciliary Activity.

Serotype-specific antibody responses to unconjugated pneumococcal polysaccharide vaccine (PPV) evaluated by a World Health Organization (WHO)-standardized enzyme-linked immunosorbent assay (ELISA) are the gold standard for diagnosis of specific polysaccharide antibody deficiency (SAD). The American Academy of Allergy, Asthma and Immunology (AAAAI) has proposed guidelines to interpret the PPV response measured by ELISA, but these are based on limited evidence. Additionally, ELISA is costly and labor-intensive. Measurement of antibody response to  () Vi vaccine and serum allohemagglutinins (AHA) have been suggested as alternatives. However, there are no large cohort studies and cutoff values are lacking.

Fifth Percentile Cutoff Values for Antipneumococcal Polysaccharide and Anti- Vi IgG Describe a Normal Polysaccharide Response.

We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development.

Differential antiviral activities of respiratory syncytial virus (RSV) inhibitors in human airway epithelium.

The correlation between different methods for the detection of pneumococcal polysaccharide vaccine (PPV) responses to diagnose specific polysaccharide antibody deficiency (SAD) is poor and the criteria for defining a normal response lack consensus. We previously proposed fifth percentile (p5) values of PPV responses as a new cutoff for SAD.

Defining Polysaccharide Antibody Deficiency: Measurement of Anti-Pneumococcal Antibodies and Anti-Salmonella typhi Antibodies in a Cohort of Patients with Recurrent Infections.

Most patients with cystic fibrosis (CF) suffer from pancreatic insufficiency (PI), leading to fat malabsorption, malnutrition, abdominal discomfort and impaired growth. Pancreatic enzyme replacement therapy (PERT) is effective, but evidence based guidelines for dose adjustment are lacking. A mobile app for self-management of PERT was developed in the context of the HORIZON 2020 project MyCyFAPP. It contains an algorithm to calculate individual PERT-doses for optimal fat digestion, based on in vitro and in vivo studies carried out in the same project. In addition, the app includes a symptoms diary, educational material, and it is linked to a web tool allowing health care professionals to evaluate patient's data and provide feedback.

Use of a mobile application for self-management of pancreatic enzyme replacement therapy is associated with improved gastro-intestinal related quality of life in children with Cystic Fibrosis.

Given the vast number of cystic fibrosis transmembrane conductance regulator () mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF).

Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis.

Matrix metalloproteinases (MMPs) are enzymes with critical roles in biology and pathology. Glycosylation, nitrosylation and proteolysis are known posttranslational modifications (PTMs) regulating intrinsically the activities of MMPs. We discovered MMP citrullination by peptidyl arginine deiminases (PADs) as a new PTM. Upon hypercitrullination, MMP-9 acquired a higher affinity for gelatin than control MMP-9. Furthermore, hypercitrullinated proMMP-9 was more efficiently activated by MMP-3 compared to control MMP-9. JNJ0966, a specific therapeutic inhibitor of MMP-9 activation, inhibited the activation of hypercitrullinated proMMP-9 by MMP-3 significantly less in comparison with control proMMP-9. The presence of citrullinated/homocitrullinated MMP-9 was detected in vivo in neutrophil-rich sputum samples of cystic fibrosis patients. In addition to citrullination of MMP-9, we report efficient citrullination of MMP-1 and lower citrullination levels of MMP-3 and MMP-13 by PAD2 in vitro. In conclusion, citrullination of MMPs is a new PTM worthy of additional biochemical and biological studies.

Citrullination as a novel posttranslational modification of matrix metalloproteinases.

Diagnosing cystic fibrosis (CF) when sweat chloride is not in the CF range and less than 2 disease-causing  mutations are found requires physiological CFTR assays, which are not always feasible or available. We developed a new physiological CFTR assay based on the morphological differences between rectal organoids from subjects with and without CF. In organoids from 167 subjects with and 22 without CF, two parameters derived from a semi-automated image analysis protocol (rectal organoid morphology analysis, ROMA) fully discriminated CF subjects with two disease-causing mutations from non-CF subjects (p<0.001). ROMA, feasible at all ages, can be centralised to improve standardisation.

Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis.

Cystic fibrosis, a multi-organ genetic disease, is characterized by abnormal function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel at the apical membrane of several epithelia. In recent years, therapeutic strategies have been developed to correct the CFTR defect. To evaluate CFTR function at baseline for diagnosis, or the efficacy of CFTR-restoring therapy, reliable tests are needed to measure CFTR function, in vitro, ex vivo and in vivo. In vitro techniques either directly or indirectly measure ion fluxes; direct measurement of ion fluxes and quenching of fluorescence in cell-based assays, change in transmembrane voltage or current in patch clamp or Ussing chamber, swelling of CFTR-containing organoids by secondary water influx upon CFTR activation. Several cell or tissue types can be used. Ex vivo and in vivo assays similarly evaluate current (intestinal current measurement) and membrane potential differences (nasal potential difference), on tissues from individual patients. In the sweat test, the most frequently used in vivo evaluation of CFTR function, chloride concentration or stimulated sweat rate can be directly measured. Here, we will describe the currently available bio-assays for quantitative evaluation of CFTR function, their indications, advantages and disadvantages, and correlation with clinical outcome measures.

Assays of CFTR Function In Vitro, Ex Vivo and In Vivo.

To date, investigations of the microbiota in the lungs of people with Cystic Fibrosis (PWCF) have primarily focused on microbial community composition in luminal mucus, with fewer studies observing the microbiota in tissue samples from explanted lung tissue. Here, we analysed both tissue and airway luminal mucus samples extracted from whole explanted lungs of PWCF and unused donor lungs. We determined if the lung microbiota in end-stage CF varied within and between patients, was spatially heterogeneous and related to localized structural damage. Microbial community composition was determined by Illumina MiSeq sequencing and related to the CF-Computed Tomography (CT) score and features of end-stage lung disease on micro-CT. Ninety-eight CF tissue (n=11 patients), 20 CF luminal mucus (n=8 patients) and 33 donor tissue (n=4 patients) samples were analysed. Additionally, we compared 20 paired CF tissue and luminal mucus samples that enabled a direct "geographical" comparison of the microbiota in these two niches. Significant differences in microbial communities were apparent between the 3 groups. However, overlap between the three groups, particularly between CF and donor tissue and CF tissue and CF luminal mucus was also observed. Microbial diversity was lower in CF luminal mucus compared to CF tissue, with dominance higher in luminal mucus. For both CF and donor tissue, intra- and inter-patient variability in ecological parameters was observed. No relationships were observed between ecological parameters and CF-CT score, or features of end-stage lung disease. The end-stage CF lung is characterised by a low diversity microbiota, differing within and between individuals. No clear relationship was observed between regional microbiota variation and structural lung damage.

Article	Year
Hemoptysis after orthopedic surgery in an adolescent boy. Pediatric pulmonology\| PubMed ID: 22038784	2012
Primary ciliary dyskinesia, an orphan disease. European journal of pediatrics\| PubMed ID: 22777640	2013
Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure. Orphanet journal of rare diseases\| PubMed ID: 24450482	2014
Primary ciliary dyskinesia and humoral immunodeficiency--is there a missing link? Respiratory medicine\| PubMed ID: 24768622	2014
Morphometric Analysis of Explant Lungs in Cystic Fibrosis. American journal of respiratory and critical care medicine\| PubMed ID: 26551917	2016
The Influence of Nebulized Drugs on Nasal Ciliary Activity. Journal of aerosol medicine and pulmonary drug delivery\| PubMed ID: 26741301	2016
Fifth Percentile Cutoff Values for Antipneumococcal Polysaccharide and Anti- Vi IgG Describe a Normal Polysaccharide Response. Frontiers in immunology\| PubMed ID: 28553290	2017
Differential antiviral activities of respiratory syncytial virus (RSV) inhibitors in human airway epithelium. The Journal of antimicrobial chemotherapy\| PubMed ID: 29596680	2018
Defining Polysaccharide Antibody Deficiency: Measurement of Anti-Pneumococcal Antibodies and Anti-Salmonella typhi Antibodies in a Cohort of Patients with Recurrent Infections. Journal of clinical immunology\| PubMed ID: 31705452	2020
Use of a mobile application for self-management of pancreatic enzyme replacement therapy is associated with improved gastro-intestinal related quality of life in children with Cystic Fibrosi... Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 32335023	2020
Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis. The European respiratory journal\| PubMed ID: 32747394	2021
Citrullination as a novel posttranslational modification of matrix metalloproteinases. Matrix biology : journal of the International Society for Matrix Biology\| PubMed ID: 33157227	2021
Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis. Thorax\| PubMed ID: 33859053	2021
Assays of CFTR Function In Vitro, Ex Vivo and In Vivo. International journal of molecular sciences\| PubMed ID: 35163362	2022
Microbial Community Composition in Explanted Cystic Fibrosis and Control Donor Lungs. Frontiers in cellular and infection microbiology\| PubMed ID: 35368453	2021

Mieke Boon

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