scholarly journals Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

10.3791/55159 ◽  
2017 ◽  
Author(s):  
Sylvia F. Boj ◽  
Annelotte M. Vonk ◽  
Marvin Statia ◽  
Jinyi Su ◽  
Robert R. G. Vries ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Drug Response ◽  
Intestinal Organoids

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

2020 ◽  
Vol 57 (1) ◽  
pp. 1902426 ◽  
Author(s):  
Anabela S. Ramalho ◽  
Eva Fürstová ◽  
Annelotte M. Vonk ◽  
Marc Ferrante ◽  
Catherine Verfaillie ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Data ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Vast Number ◽  
Sweat Chloride ◽  
Intestinal Organoids ◽  
Rectal Biopsies ◽  
Cftr Modulators

RationaleGiven the vast number of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF).ObjectivesTo study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data.MethodsIntestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature.ResultsAcross 28 genotypes, residual CFTR function correlated (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit.ConclusionsMeasurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.

scholarly journals Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aditya Mithal ◽  
Amalia Capilla ◽  
Dar Heinze ◽  
Andrew Berical ◽  
Carlos Villacorta-Martin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
In Vitro Models ◽  
Directed Differentiation ◽  
Intestinal Organoids ◽  
Differentiation Protocol ◽  
Before And After ◽  
Induced Pluripotent

AbstractEfficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.

scholarly journals Forskolin-induced swelling of intestinal organoids predicts long-term cystic fibrosis disease progression

2021 ◽  
Author(s):  
D. Muilwijk ◽  
E. de Poel ◽  
P. van Mourik ◽  
S.W.F. Suen ◽  
A.M. Vonk ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Disease Progression ◽  
Pancreatic Insufficiency ◽  
Chloride Concentration ◽  
Predictive Biomarker ◽  
Sweat Chloride ◽  
Intestinal Organoids ◽  
Clinical Consequences ◽  
Related Liver Disease

ABSTRACTPatient-derived organoids hold great potential as predictive biomarker for disease expression or therapeutic response. Here, we used intestinal organoids to estimate individual cystic fibrosis transmembrane conductance regulator (CFTR) function of people with cystic fibrosis, a monogenic life-shortening disease associated with more than 2000 CFTR mutations and highly variable disease progression. In vitro CFTR function in CF intestinal organoids of 176 individuals with diverse CFTR mutations was quantified by forskolin induced swelling and was strongly associated with longitudinal changes of lung function and development of pancreatic insufficiency, CF-related liver disease and diabetes. This association was not observed when the commonly used biomarker of CFTR function sweat chloride concentration was used. The data strongly exemplifies the value of an organoid-based biomarker in a clinical disease setting and supports the prognostic value of forskolin induced swelling of intestinal organoids, especially for people with CF who have rare CFTR genotypes with unclear clinical consequences.

Preliminary Report of In vitro and In vivo Effectiveness of Dornase Alfa on SARS-CoV-2 Infection (Preprint)

2020 ◽  
Author(s):  
Hacer Kuzu Okur ◽  
Koray Yalcin ◽  
Cihan Tastan ◽  
Sevda Demir ◽  
Bulut Yurtsever ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Respiratory Tract ◽  
Mononuclear Cells ◽  
Multiple Organ ◽  
Peripheral Blood Mononuclear ◽  
Dornase Alfa ◽  
Tract Infections ◽  
Adult Peripheral Blood

UNSTRUCTURED Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). Coronavirus disease 2019 (COVID-19) pandemic affected more than two million people over the world, resulting in unprecedented health, social and economic crises. The COVID-19, viral pneumonia that progresses to ARDS and even multiple organ failure, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High blood neutrophil levels are an early indicator of SARS-CoV-2 infection and predict severe respiratory diseases. A similar mucus structure is detected in COVID-19 patients due to the accumulation of excessive NET in the lungs. Here, we show our preliminary results with dornase alfa that may have an in-vitro anti-viral effect against SARS-CoV-2 infection in a bovine kidney cell line, MDBK without drug toxicity on healthy adult peripheral blood mononuclear cells. In this preliminary study, we also showed that dornase alfa can promote clearance of NET formation in both an in-vitro and three COVID-19 cases who showed clinical improvement in radiological analysis (2-of-3 cases), oxygen saturation (SpO2), respiratory rate, disappearing of dyspnea and coughing.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

scholarly journals Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2530
Author(s):  
Bijean D. Ford ◽  
Diego Moncada Giraldo ◽  
Camilla Margaroli ◽  
Vincent D. Giacalone ◽  
Milton R. Brown ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bactericidal Activity ◽  
Scavenger Receptor ◽  
Receptor Expression ◽  
Blood Monocytes ◽  
Bacterial Killing ◽  
Blood Neutrophils ◽  
Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

scholarly journals In Vitro Newly Isolated Environmental Phage Activity against Biofilms Preformed by Pseudomonas aeruginosa from Patients with Cystic Fibrosis

2021 ◽  
Vol 9 (3) ◽  
pp. 478
Author(s):  
Ersilia Vita Fiscarelli ◽  
Martina Rossitto ◽  
Paola Rosati ◽  
Nour Essa ◽  
Valentina Crocetta ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
In Vitro Test ◽  
Chronic Infections ◽  
Hospital Environments ◽  
Vitro Test ◽  
General Reliability ◽  
Phage Activity

As disease worsens in patients with cystic fibrosis (CF), Pseudomonas aeruginosa (PA) colonizes the lungs, causing pulmonary failure and mortality. Progressively, PA forms typical biofilms, and antibiotic treatments determine multidrug-resistant (MDR) PA strains. To advance new therapies against MDR PA, research has reappraised bacteriophages (phages), viruses naturally infecting bacteria. Because few in vitro studies have tested phages on CF PA biofilms, general reliability remains unclear. This study aimed to test in vitro newly isolated environmental phage activity against PA isolates from patients with CF at Bambino Gesù Children’s Hospital (OBG), Rome, Italy. After testing in vitro phage activities, we combined phages with amikacin, meropenem, and tobramycin against CF PA pre-formed biofilms. We also investigated new emerging morphotypes and bacterial regrowth. We obtained 22 newly isolated phages from various environments, including OBG. In about 94% of 32 CF PA isolates tested, these phages showed in vitro PA lysis. Despite poor efficacy against chronic CF PA, five selected-lytic-phages (Φ4_ZP1, Φ9_ZP2, Φ14_OBG, Φ17_OBG, and Φ19_OBG) showed wide host activity. The Φ4_ZP1-meropenem and Φ14_OBG-tobramycin combinations significantly reduced CF PA biofilms (p < 0.001). To advance potential combined phage-antibiotic therapy, we envisage further in vitro test combinations with newly isolated phages, including those from hospital environments, against CF PA biofilms from early and chronic infections.

scholarly journals In vitro co-culture of human intestinal organoids and lamina propria-derived CD4+ T cells

2021 ◽  
Vol 2 (2) ◽  
pp. 100519
Author(s):  
Renée R.C.E. Schreurs ◽  
Martin E. Baumdick ◽  
Agata Drewniak ◽  
Madeleine J. Bunders
Keyword(s):  
T Cells ◽  
Lamina Propria ◽  
Cd4 T Cells ◽  
Intestinal Organoids

scholarly journals Kenyan Plasmodium falciparum Field Isolates: Correlation between Pyrimethamine and Chlorcycloguanil Activity In Vitro and Point Mutations in the Dihydrofolate Reductase Domain

1998 ◽  
Vol 42 (1) ◽  
pp. 164-169 ◽  
Author(s):  
A. Nzila-Mounda ◽  
E. K. Mberu ◽  
C. H. Sibley ◽  
C. V. Plowe ◽  
P. A. Winstanley ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Dihydrofolate Reductase ◽  
Drug Response ◽  
Point Mutations ◽  
Distinct Group ◽  
Wild Type ◽  
Field Isolates ◽  
Vitro Data ◽  
In Vitro Data

ABSTRACT Sixty-nine Kenyan Plasmodium falciparum field isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P< 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P < 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

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