Front Cell Infect Microbiol. 2024 Aug 13;14:1426773. doi: 10.3389/fcimb.2024.1426773. eCollection 2024.

ABSTRACT

INTRODUCTION: The Burkholderia cepacia complex encompasses a group of gram-negative opportunistic pathogens that cause chronic lung infections in people with cystic fibrosis. Distinct from other respiratory pathogens, Burkholderia causes a unique clinical disease in a subset of patients known as 'cepacia syndrome', fulminant pneumonia accompanied by bacteraemia and sepsis with a mortality rate of up to 75%. Due to the bacteraemia associated with this disease, the mechanisms that allow Burkholderia to resist the bactericidal effects of serum complement-depending killing are vital. Antibodies usually promote serum killing; however, we have described 'cloaking antibodies', specific for lipopolysaccharides that paradoxically protect serum-sensitive bacteria from complement-mediated lysis. Cloaking antibodies that protect Pseudomonas aeruginosa have been found in 24%-41% of patients with chronic lung diseases. The presence of these antibodies is also associated with worse clinical outcomes. Here, we sought to determine the relevance of cloaking antibodies in patients with Burkholderia infection.

METHODS: Twelve Burkholderia spp. were isolated from nine pwCF and characterised for susceptibility to healthy control serum. Patient serum was analysed for the titre of the cloaking antibody. The ability of the patient serum to prevent healthy control serum (HCS) killing of its cognate isolates was determined.

RESULTS: We found that several of the Burkholderia strains were shared between patients. Ten of the 12 isolates were highly susceptible to HCS killing. Four of nine (44%) patients had cloaking antibodies that protected their cognate strain from serum killing. Depleting cloaking antibodies from patient serum restored HCS killing of Burkholderia isolates.

DISCUSSION: Cloaking antibodies are prevalent in patients with Burkholderia pulmonary infection and protect these strains from serum killing. Removal of cloaking antibodies via plasmapheresis, as previously described for individuals with life-threatening Pseudomonas infection, may be a useful new strategy for those with serious and life-threatening Burkholderia infection.

PMID:39193503 | PMC:PMC11347948 | DOI:10.3389/fcimb.2024.1426773

ERJ Open Res. 2024 Aug 27;10(4):00256-2024. doi: 10.1183/23120541.00256-2024. eCollection 2024 Jul.

ABSTRACT

OBJECTIVES: Lung disease progression in people with cystic fibrosis (pwCF) varies from one individual to another. Different immunological characteristics have been suggested to explain this variation, and we hypothesised that lung capacity may be associated with the innate immune response in pwCF. In an exploratory study, we aimed to investigate potential links between the innate immune response and lung function in pwCF using the standardised immune function assay TruCulture.

METHODS: In a single-centre study with combined cross-sectional and longitudinal data before and after intravenous antibiotics, blood was sampled from Pseudomonas aeruginosa-infected pwCF. Whole blood was analysed by TruCulture to reveal the unstimulated and stimulated cytokine release. Tobit regressions and Spearman's correlations were used to estimate the associations between lung function and cytokine release.

RESULTS: We included 52 pwCF in the cross-sectional study and 24 in the longitudinal study. In the cross-sectional study, we found that compared to a healthy population, the release of toll-like receptor (TLR)3, TLR4- and TLR7/8-stimulated interferon-γ, and interleukin (IL)-12p40 was reduced. Although TLR3-stimulated IL-1β and IL-6 release increased with lung function, overall, cytokine release did not correlate well with lung function. In the longitudinal study, the cytokine release was modified by antibiotic treatment, but the cytokine release before antibiotic treatment did not associate with changes in lung function after treatment.

CONCLUSION: The stimulated cytokine release could not predict lung function levels or changes in pwCF, but our data indicate that pwCF experience exhaustion in the innate immune response after years of chronic bacterial infection.

PMID:39193378 | PMC:PMC11347999 | DOI:10.1183/23120541.00256-2024

Sci Rep. 2024 Aug 27;14(1):19822. doi: 10.1038/s41598-024-70778-9.

ABSTRACT

Our study probed the differences in ion channel gene expression in the endometrium of women with Recurrent Implantation Failure (RIF) compared to fertile women. We analyzed the relative expression of genes coding for T-type Ca2+, ENaC, CFTR, and KCNQ1 channels in endometrial samples from 20 RIF-affected and 10 control women, aged 22-35, via microarray analysis and quantitative real-time PCR. Additionally, we examined DNA methylation in the regulatory region of KCNQ1 using ChIP real-time PCR. The bioinformatics component of our research included Gene Ontology analysis, protein-protein interaction networks, and signaling pathway mapping to identify key biological processes and pathways implicated in RIF. This led to the discovery of significant alterations in the expression of ion channel genes in RIF women's endometrium, most notably an overexpression of CFTR and reduced expression of SCNN1A, SCNN1B, SCNN1G, CACNA1H, and KCNQ1. A higher DNA methylation level of KCNQ1's regulatory region was also observed in RIF patients. Gene-set enrichment analysis highlighted a significant presence of genes involved with ion transport and membrane potential regulation, particularly in sodium and calcium channel complexes, which are vital for cation movement across cell membranes. Genes were also enriched in broader ion channel and transmembrane transporter complexes, underscoring their potential extensive role in cellular ion homeostasis and signaling. These findings suggest a potential involvement of ion channels in the pathology of implantation failure, offering new insights into the mechanisms behind RIF and possible therapeutic targets.

PMID:39192025 | DOI:10.1038/s41598-024-70778-9

J Cyst Fibros. 2024 Aug 26:S1569-1993(24)00804-X. doi: 10.1016/j.jcf.2024.07.019. Online ahead of print.

ABSTRACT

BACKGROUND: Factors associated with severe COVID-19 infection have been identified; however, the impact of infection on longer-term outcomes is unclear. The objective of this study was to examine the impact of COVID-19 infection on the trajectory of lung function and nutritional status in people with cystic fibrosis (pwCF).

METHODS: This is a retrospective global cohort study of pwCF who had confirmed COVID-19 infection diagnosed between January 1, 2020 and December 31, 2021. Forced expiratory volume in one second percent predicted (ppFEV1) and body mass index (BMI) twelve months prior to and following a diagnosis of COVID-19 were recorded. Change in mean ppFEV1 and BMI were compared using a t-test. A linear mixed-effects model was used to estimate change over time and to compare the rate of change before and after infection.

RESULTS: A total of 6,500 cases of COVID-19 in pwCF from 33 countries were included for analysis. The mean difference in ppFEV1 pre- and post-infection was 1.4 %, (95 % CI 1.1, 1.7). In those not on modulators, the difference in rate of change pre- and post-infection was 1.34 %, (95 % CI -0.88, 3.56) per year (p = 0.24) and -0.74 % (-1.89, 0.41) per year (p = 0.21) for those on elexacaftor/tezacaftor/ivacaftor. No clinically significant change was noted in BMI or BMI percentile before and after COVID-19 infection.

CONCLUSIONS: No clinically meaningful impact on lung function and BMI trajectory in the year following infection with COVID-19 was identified. This work highlights the ability of the global CF community to unify and address critical issues facing pwCF.

PMID:39191560 | DOI:10.1016/j.jcf.2024.07.019

Am J Respir Crit Care Med. 2024 Aug 27. doi: 10.1164/rccm.202406-1128RL. Online ahead of print.

NO ABSTRACT

PMID:39189854 | DOI:10.1164/rccm.202406-1128RL

J Infect Dis. 2024 Aug 27:jiae407. doi: 10.1093/infdis/jiae407. Online ahead of print.

ABSTRACT

BACKGROUND: Mycobacterium abscessus complex (MABC), an opportunistic nontuberculous mycobacteria (NTM), can lead to poor clinical outcomes in pulmonary infections. Conflicting data exist on person-to-person transmission of MABC within and across healthcare facilities. To investigate further, a comprehensive retrospective study across five healthcare institutions on the Island of Montréal was undertaken.

METHODS: We analyzed the genomes of 221 MABC isolates obtained from 115 individuals (2010-2018) to identify possible links. Genetic similarity, defined as ≤25 single-nucleotide polymorphisms (SNPs), was investigated through a blinded epidemiological inquiry.

RESULTS: Bioinformatics analyses identified 28 sequence types (STs), including globally observed dominant circulating clones (DCCs). Further analysis revealed 210 isolate pairs within the SNP threshold. Among these pairs, there was one possible lab contamination where isolates from different patients processed in the same lab differed by only 2 SNPs. There were 37 isolate pairs from patients who had provided specimens from the same hospital; however, epidemiological analysis found no evidence of healthcare-associated person-to-person transmission between these patients. Additionally, pan-genome analysis showed higher discriminatory power than core genome analysis for examining genomic similarity.

CONCLUSIONS: Genomics alone is insufficient to establish MABC transmission, particularly considering the genetic similarity and wide distribution of DCCs, although pan-genome analysis has the potential to add further insight. Our findings indicate that MABC infections in Montréal are unlikely attributable to healthcare-associated person-to-person transmission.

PMID:39189818 | DOI:10.1093/infdis/jiae407

Microbiol Spectr. 2024 Aug 27:e0036224. doi: 10.1128/spectrum.00362-24. Online ahead of print.

ABSTRACT

Mycobacterium abscessus (Mab) is an emerging pathogen that poses a severe health threat, especially in people with cystic fibrosis and other chronic lung diseases. Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used in vitro for identifying hits in a high-throughput manner and in vivo to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to in vivo infection (using the Galleria mellonella model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way.

IMPORTANCE: Mycobacterium abscessus (Mab) is currently considered an "incurable nightmare." Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between in vitro susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as in vitro drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success.

PMID:39189762 | DOI:10.1128/spectrum.00362-24

Tracheostomy (Warrenville). 2024;1(2):16-26. Epub 2024 Jun 30.

ABSTRACT

OBJECTIVE: To evaluate an educational intervention to promote confidence, knowledge, and skills in tracheostomy tube change among nursing students.

METHODS: The study, conducted at the at the Johns Hopkins Center for Immersive Learning and Digital Innovation, enrolled nursing students without prior experience in tracheostomy tube change. The intervention included a pre-recorded presentation, faculty demonstrations with a Tracheostomy Care Training Simulation Model, and participant practice demonstrating skills. Primary outcomes included confidence, knowledge, and competency with tracheostomy tube changes. Secondary outcomes included number of attempts required to achieve competency and time required per attempt. The study followed STROBE guidelines with repeated measure design.

RESULTS: Participants in the study (n=50) had a mean age of 30 years, were predominantly female (83%) with a bachelor's degree (76%), most often in the third semester of nursing school (45%). Participants showed a mean improvement of 3.58 points out of five (SD: 0.56, P<0.001) across 11 pre- and post-test items. Every confidence assessment improved, with the largest increase in assessing tube placement. Knowledge assessments improved across all eight test items in the first test-retest interval, showing an improvement of 1.14 points out of five (SD: 0.89, P<0.001). Competency assessment improved in the first test-retest interval of 1.01 points out of five (SD: 0.65, P<0.001). On serial assessments, time to complete tracheostomy tube change decreased from 2.39 to 0.60 minutes. Faculty deemed 95% of participants competent after only one skill testing iteration.

CONCLUSION: An educational intervention, combining digital presentations with interactive faculty-led simulations and practical skill assessments, successfully elevated nursing students' confidence, knowledge, and competency in tracheostomy tube changes.

PMID:39188760 | PMC:PMC11345849

J Clin Transl Endocrinol. 2024 Jul 24;37:100362. doi: 10.1016/j.jcte.2024.100362. eCollection 2024 Sep.

ABSTRACT

BACKGROUND: Cystic fibrosis (CF) is a multi-organ disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Individuals with CF often have gastrointestinal (GI) dysbiosis due to chronic inflammation and antibiotic use. Previous studies suggested a role for vitamin D in reversing the GI dysbiosis found in CF.

OBJECTIVE: To explore the potential role of a combination of high-dose oral cholecalciferol (vitamin D3) and fermentable dietary fiber, inulin, to impact bacterial composition, richness, and diversity of intestinal and airway microbiota in adults with CF.

METHODS: This was a 2 × 2 factorial, double-blinded, placebo-controlled, randomized, pilot clinical trial in which adults with CF received oral cholecalciferol (vitamin D3) (50,000 IU/week) and/or inulin (12 g/day) for 12 weeks. Thus, there were 4 study groups (n = 10 subjects per group); 1) placebo 2) vitamin D3 3) inulin 4) vitamin D3 plus inulin. Stool and sputum samples were collected at baseline (just before) and after the intervention and were analysed using 16S ribosomal RNA gene sequencing for gut and airway microbiota composition. Statistical analyses assessed alpha and beta diversity to evaluate microbial community changes.

RESULTS: Of a total of 254 screened participants, 40 eligible participants were randomized to one of the 4 treatment arms. Participants receiving vitamin D3 plus inulin exhibited greater changes in microbiome indexes in both intestinal and airway relative to those in the other study groups. Specific taxonomic changes supported the potential beneficial influence of this combination to mitigate both intestinal and airway dysbiosis in adults with CF.

CONCLUSION: This pilot study established that the combination of oral vitamin D3 and the prebiotic inulin was well tolerated over 12 weeks in adults with CF and altered gut and airway bacterial communities. Future research appear warranted to define clinical outcomes and the role of microbiota changes therein with this approach.

PMID:39188269 | PMC:PMC11345930 | DOI:10.1016/j.jcte.2024.100362

In Vivo. 2024 Sep-Oct;38(5):2294-2299. doi: 10.21873/invivo.13694.

ABSTRACT

BACKGROUND/AIM: Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle.

MATERIALS AND METHODS: Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations.

RESULTS: The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 μA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 μA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 μA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells.

CONCLUSION: Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.

PMID:39187341 | DOI:10.21873/invivo.13694

Diabetes Res Clin Pract. 2024 Aug 24:111839. doi: 10.1016/j.diabres.2024.111839. Online ahead of print.

ABSTRACT

AIMS: To evaluate the impact of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on Cystic Fibrosis Related Diabetes (CFRD) glycemic control and insulin treatment in patients with CFRD during clinical practice METHODS: We carried out a retrospective observational study of 23 adult patients with CFRD who started treatment with ETI. They had, at least, one F508del mutation. Data were collected before ETI initiation and 3, 6, and 12 months after.

RESULTS: Glycemic control measured by HbA1c significantly improved by 0.3 % (0.1-0.5) after 3 months of ETI therapy (p = 0.004) and kept this improvement during follow-up (p < 0.001). The proportion of patients needing multiple daily injections of insulin was reduced by 16 % (p = 0.023). Total daily insulin dose dropped by 0.12 (0.05-0.18) UI/kg/day (p < 0.001). Data derived from Flash Continuous Glucose Monitoring (CGM) for patients treated with insulin stayed unchanged after insulin reduction, except for a significant 8 % (0.3-15.6) increase in the Time In Tight Range (TITR) between 70 and 140 mg/dL (p = 0.043).

CONCLUSION: ETI therapy impacted CFRD in clinical practice reducing insulin needs and improving glycemic control measured by HbA1c and CGM. The improvements can be observed from the first 3 months of treatment.

PMID:39187175 | DOI:10.1016/j.diabres.2024.111839

Clin Nutr ESPEN. 2024 Aug 24:S2405-4577(24)01287-7. doi: 10.1016/j.clnesp.2024.08.016. Online ahead of print.

ABSTRACT

BACKGROUND & AIMS: Cystic fibrosis (CF)-related diabetes (CFRD), a common comorbidity in CF, is often preceded and characterized with elevated postprandial glycaemia (PPG). In the general population, the consumption of a pre-meal protein snack and/or physical activity (PA) hinder the elevation of PPG levels. Our objective is to evaluate the effect of a pre-meal snack and/or post-meal PA on PPG excursions in CF.

METHODS: This is a double-blinded randomized controlled crossover interventional study in 14 adults with CF, with 4 interventions: placebo pre-meal snack + no PA (control: CTL), pre-meal soy snack + no PA (SK), placebo pre-meal snack + PA (PA), and pre-meal soy snack + PA (SK+PA). The pre-meal soy snack or placebo beverage (vanilla flavoured water) is served at 8 am, followed by a standardized breakfast at 9 am and, postprandially, 5 repeated bouts of 3-min walk every 30 mins or sedentary activity. Blood glucose and insulin were measured every 15 to 30 minutes during the interventions.

RESULTS: Plasma glucose (PG) was higher 30 mins after snack consumption compared to placebo beverage. One-hour post-breakfast, PG levels were lower during both PA interventions than with sedentary behavior. However, the overall 3h post-breakfast glucose area under the curve (AUC) was similar between interventions. Post-breakfast 3h insulin AUC was significantly lower during the SK+PA intervention compared to the sedentary behavior interventions.

CONCLUSION: Repeated short bouts of post-meal physical activity may positively impact PPG control in adults with CF, with or without the addition of a pre-meal soy snack. A pre-meal snack alone does not improve PPG.

PMID:39187012 | DOI:10.1016/j.clnesp.2024.08.016

J Vis Exp. 2024 Aug 9;(210). doi: 10.3791/66380.

ABSTRACT

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time series of each voxel is analyzed with regard to the cardiac and breathing frequency components. Using a standard 2D spoiled gradient-echo sequence with a temporal resolution of ~300 ms, an image-sorting algorithm was developed to produce phase-resolved functional lung imaging (PREFUL) with an increased temporal resolution. Thus, it is feasible to evaluate regional flow volume loops (FVL) during tidal volume breathing and depict the propagation of the pulse wave during the cardiac cycle. This method can be applied at 1.5T or 3T with standard MR hardware without the necessity for sequence programming, as the described protocol can be implemented with the default SPGRE sequence on most systems. PREFUL ventilation MRI has been validated using 129Xe and 19F gas imaging with good regional agreement. Perfusion-weighted PREFUL MRI has been validated using SPECT as well as dynamic contrast enhanced (DCE) MRI. PREFUL has been tested in a dual center dual vendor setting and is currently applied in several ongoing multicenter trials. Furthermore, it is feasible across a range of field strengths (0.55T-3T) and different age groups, including newborns. Quantitative V/Q PREFUL MRI has been used in patients with cystic fibrosis, chronic obstructive pulmonary disease, chronic thromboembolic pulmonary hypertension, and corona virus disease-2019 to quantify disease and monitor treatment change after therapy. Furthermore, PREFUL V/Q imaging has been shown to predict transplant loss due to chronic lung allograft dysfunction in patients after lung transplantation. In summary, PREFUL MRI is a validated technique for quantitative ventilation and pulmonary pulse wave/perfusion imaging for regional pulmonary disease detection, quantification, and treatment monitoring with potential added value to the current clinical routine.

PMID:39185874 | DOI:10.3791/66380

ACS Nano. 2024 Aug 26. doi: 10.1021/acsnano.4c10344. Online ahead of print.

ABSTRACT

Sweat analysis has advanced from diagnosing cystic fibrosis and testing for illicit drugs to noninvasive monitoring of health biomarkers. This article introduces the rapid development of wearable and flexible sweat sensors, highlighting key milestones and various sensing strategies for real-time monitoring of analytes. We discuss challenges such as developing high-performance nanomaterial-based biosensors, ensuring continuous sweat production and sampling, achieving high sweat/blood correlation, and biocompatibility. The potential of machine learning to enhance these sensors for personalized healthcare is presented, enabling real-time tracking and prediction of physiological changes and disease onset. Leveraging advancements in flexible electronics, nanomaterials, biosensing, and data analytics, wearable sweat biosensors promise to revolutionize disease management, prevention, and prediction, promoting healthier lifestyles and transforming medical practices globally.

PMID:39185844 | DOI:10.1021/acsnano.4c10344

An Sist Sanit Navar. 2024 Aug 26;47(2):e1084. doi: 10.23938/ASSN.1084.

ABSTRACT

BACKGROUND: To analyze the knowledge, abilities, and emotional state of cystic fibrosis patients during a specific follow-up period and compare this with the recall they had of the transition (planned and gradual shift from the pediatric unit) / transfer (direct change skipping the steps recommended by the guidelines) to a specialized cystic fibrosis adult unit.

METHODS: Prospective cross-sectional study with cystic fibrosis adult patients under follow-up in a specialist consultation. Group 1 were patients who transitioned and Group 2 were transferred patients. The following information was collected: sociodemographic variables, degree of knowledge, skills, and emotional state using a survey designed for this purpose (as part of the internal consistency validation process). Participants also completed the emotional subscale of Cystic Fibrosis Questionnaire-Revised. Inter-group comparisons were made for the transition/transfer, at the follow-up, and during the evolution.

RESULTS: Thirty-five patients were analyzed; 65.8% male; mean age 31.9 years (SD =10.1). At the transition, Group 1 (n=19; 54.3%) had greater knowledge about their medication and reduced ability to manage appointments and making decisions in comparison to Group 2 at transfer. At follow-up, Group 1 made a better report on their emotional state and significantly improved their ability to manage appointments, communication, and decision-making.

CONCLUSIONS: Patients who were moved to an adult cystic fibrosis unit through transition were more knowledgeable about their medications. However, those who were transferred managed their appointments and decision-making better, but felt sadder.

PMID:39185776 | DOI:10.23938/ASSN.1084

bioRxiv [Preprint]. 2024 Aug 12:2024.08.12.607571. doi: 10.1101/2024.08.12.607571.

ABSTRACT

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which the CFTR cDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of the CFTR cDNA and an enrichment cassette into the CFTR locus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improve CFTR cDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.

PMID:39185207 | PMC:PMC11343149 | DOI:10.1101/2024.08.12.607571

bioRxiv [Preprint]. 2024 Aug 16:2024.08.14.607921. doi: 10.1101/2024.08.14.607921.

ABSTRACT

In our era of rising antibiotic resistance, Stenotrophomonas maltophilia (STM) is an understudied, gram-negative, aerobic bacterium widespread in the environment and increasingly causing opportunistic infections. Treating STM infections remains difficult, leading to an increase in disease severity and higher hospitalization rates in people with Cystic Fibrosis (pwCF), cancer, and other immunocompromised health conditions. The lack of effective antibiotics has led to renewed interest in phage therapy; however, there is a need for well-characterized phages. In response to an oncology patient with a respiratory infection, we collected 18 phages from Southern California wastewater influent that exhibit different plaque morphology against STM host strain B28B, cultivated from a blood sample. Here, we characterize the genomes and life cycle kinetics of our STM phage collection. We hypothesize that genetically distinct phages give rise to unique lytic life cycles that can enhance bacterial killing when combined into a phage cocktail compared to the individual phages alone. We identified three genetically distinct clusters of phages, and a representative from each group was screened for potential therapeutic use and investigated for infection kinetics. The results demonstrated that the three-phage cocktail significantly suppressed bacterial growth compared to individual phages when observed for 48 hours. We also assessed the lytic impacts of our three-phage cocktail against a collection of 46 STM strains to determine if a multi-phage cocktail can expand the host range of individual phages. Our phages remained strain-specific and infect >50% of tested strains. The multi-phage cocktail maintains bacterial growth suppression and prevents the emergence of phage-resistant strains throughout our 40-hour assay. These findings suggest specialized phage cocktails may be an effective avenue of treatment for recalcitrant STM infections resistant to current antibiotics.

IMPORTANCE: Phage therapy could provide a vital strategy in the fight against antimicrobial resistance (AMR) bacterial infections; however, significant knowledge gaps remain. This study investigates phage cocktail development for the opportunistic pathogen Stenotrophomonas maltophilia (STM). Our findings contribute novel phages, their lytic characteristics, and limitations when exposed to an array of clinically relevant STM strains. Eighteen bacteriophages were isolated from wastewater influent from Escondido, California, and subjected to genomic analysis. We investigated genetically distinct phages to establish their infection kinetics and developed them into a phage cocktail. Our findings suggest that a genetically distinct STM phage cocktail provides an effective strategy for bacterial suppression of host strain B28B and five other clinically relevant STM strains. Phage therapy against STM remains poorly understood, as only 39 phages have been previously isolated. Future research into the underlying mechanism of how phage cocktails overwhelm the host bacteria will provide essential information that could aid in optimizing phage applications and impact alternative treatment options.

PMID:39185190 | PMC:PMC11343209 | DOI:10.1101/2024.08.14.607921

F1000Res. 2022 Nov 22;11:379. doi: 10.12688/f1000research.110472.3. eCollection 2022.

ABSTRACT

Background: The main aim of this study was to evaluate whether selected polymorphic variants in genes from the inflammatory pathway can be predictors of pulmonary or digestive manifestation of cystic fibrosis, as well as of severity of lung disease. Materials and methods: Using pyrosequencing and sequencing we have genotyped 12 variants in TNF (rs361525, rs1800629), CXCL8 (rs4073, rs2227306, rs2227307, rs188378669), IL1B (rs16944, rs1143634, rs1142639, rs1143627), IL6 (rs1800795) and IL10 (rs1800896) genes in a cohort of 55 Polish patients with diagnosed cystic fibrosis and controls. In our study group, a pulmonary manifestation of disease revealed 44 of subjects (80%), and digestive symptoms dominated in 11 (20%) of analyzed individuals. Severe lung dysfunction has occurred in 20 (36.4%) of patients. Results: We proved, that two promoter variants of IL1B, rs1143627 (c.-118G > A) and rs16944 (c.-598T > C) are presented significantly more often in patients with severe character of lung disease compared to mild (82.5% vs. 62.8%, p-value 0.030, and 87.5% vs. 64.3%, p-value 0.008, respectively) in cystic fibrosis course. Haplotype AC formed by both changes had also a higher frequency (80%) in patients with severe course compared to the mild character (61.4%) of disease. However, the frequency of promoter variant TNF c.-308C > T (rs1800629) was presented at a significantly lower level in the patient's group compared to healthy controls (2.7% vs. 15%, p-value 0.001). Furthermore, the presence of methicillin-resistant Staphylococcus aureus significantly correlated with the lower FEV1% in patients (p-value 0.01). Conclusions: Genetic variants, rs1143627 and rs16944, of IL1B are promising candidates as predictors of the severe character of lung disease in Polish patients with cystic fibrosis.

PMID:39185143 | PMC:PMC11344199 | DOI:10.12688/f1000research.110472.3

Gene Ther. 2024 Aug 25. doi: 10.1038/s41434-024-00480-y. Online ahead of print.

ABSTRACT

Mutation-agnostic treatments such as airway gene therapy have the potential to treat any individual with cystic fibrosis (CF), irrespective of their CF transmembrane conductance regulator (CFTR) gene variants. The aim of this study was to employ two CF rat models, Phe508del and CFTR knockout (KO), to assess the comparative effectiveness of CFTR modulators and lentiviral (LV) vector-mediated gene therapy. Cells were isolated from the tracheas of rats and used to establish air-liquid interface (ALI) cultures. Phe508del rat ALIs were treated with the modulator combination, elexacaftor-tezacaftor-ivacaftor (ETI), and separate groups of Phe508del and KO tracheal epithelial cells were treated with LV-CFTR followed by differentiation at ALI. Ussing chamber measurements were performed to assess CFTR function. ETI-treated Phe508del ALI cultures demonstrated CFTR function that was 59% of wild-type level, while gene-addition therapy restored Phe508del to 68% and KO to 47% of wild-type level, respectively. Our findings show that rat Phe508del-CFTR protein can be successfully rescued with ETI treatment, and that CFTR gene-addition therapy provides significant CFTR correction in Phe508del and KO ALI cultures to levels that were comparable to ETI. These findings highlight the potential of an LV vector-based gene therapy for the treatment of CF lung disease.

PMID:39183346 | DOI:10.1038/s41434-024-00480-y

J Cyst Fibros. 2024 Aug 24:S1569-1993(24)00828-2. doi: 10.1016/j.jcf.2024.08.002. Online ahead of print.

ABSTRACT

BACKGROUND: The objective of this study was to assess the differential times of submission and approval of CFTR modulators in the United States (US) and the European Union (EU).

METHODS: By collecting publicly available data from the websites of the Food and Drug Administration and the European Medicines Agency, we quantified differential times in submission, review duration, and approvals of initial marketing authorization and variation of indications of CFTR modulators in the US and the EU by December 31, 2023.

RESULTS: Applications regarding marketing of 4 CFTR modulators were submitted 103 (SD ±143) days later in the EU than in the US: 31 (SD ±39) days later for initial approval, and 124 (SD ±155) days for supplemental indications. The regulatory review process was completed in 181 days [IQR, 179 - 182] in the US and 325 days [IQR, 276 - 382] in the EU: 167 days [IQR, 102 - 232] in the US and 346 days [IQR, 302 - 400] in the EU for first approvals, 181 days [IQR, 181 - 182] in the US and 324 days [IQR, 264 - 382] in the EU for supplemental indication approvals. CFTR modulators were approved 267 (SD 143) days later in the EU than in the US: 220 (SD ±76) days for initial approval and 280 (SD ±157) days for supplemental indications.

CONCLUSION: We found significant differences in times of submission and for approval of CFTR modulators between the US and EU, whereby initial approvals and subsequent indication approvals were always first granted in the US.

PMID:39183127 | DOI:10.1016/j.jcf.2024.08.002