Upper and lower airway cultures in children with cystic fibrosis: Do not neglect the upper airways

AbstractBackgroundAirways of cystic fibrosis (CF) patients are colonised with bacteria early in life. We aimed to analyse differences between results of simultaneously taken upper airway (UAW) and lower airway (LAW) cultures, to describe clinical characteristics of patients with positive versus negative cultures and to follow up the patients with P. aeruginosa positive UAW cultures.MethodsBacteriological and clinical data from 157 children were collected during annual check up. The number of positive UAW and LAW cultures and correspondence between these results and clinical characteristics were analysed.ResultsPositive LAW and UAW cultures were found in 79.6% and 43.9% of patients respectively (p<0.001). Patients with positive LAW cultures were significantly older (11.9 vs. 9.8years, p<0.05) and had more LAW symptoms (73.6% vs. 46.7%, p<0.05), especially when P. aeruginosa was found. Patients with positive UAW cultures (especially S. aureus) had more nasal discharge (50.7% vs. 25.0%, p<0.001). In 65% of patients with positive UAW and negative LAW culture for P. aeruginosa the next LAW became P. aeruginosa positive.ConclusionUAW cultures and LAW cultures differ in children with CF and there are differences in clinical characteristics between patients with positive versus negative culture results. P. aeruginosa positive UAW cultures appeared to precede positive LAW cultures in a substantial part of patients, suggesting some kind of cross-infection between the UAW and LAW

Forskolin-induced Organoid Swelling is Associated with Long-term CF Disease Progression

RATIONALE: Cystic fibrosis (CF) is a monogenic life-shortening disease associated with highly variable individual disease progression which is difficult to predict. Here we assessed the association of forskolin-induced swelling (FIS) of patient-derived organoids (PDO) with long-term CF disease progression in multiple organs and compared FIS with the golden standard biomarker sweat chloride concentration (SCC). METHODS: We retrieved 9-year longitudinal clinical data from the Dutch CF Registry of 173 people with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individual CFTR function was defined by FIS, measured as the relative size increase of intestinal organoids after stimulation with 0.8 µM forskolin, quantified as area under the curve (AUC). We used linear mixed effect models and multivariable logistic regression to estimate the association of FIS with long-term FEV1pp decline and development of pancreatic insufficiency, CF-related liver disease and diabetes. Within these models, FIS was compared with SCC. RESULTS: FIS was strongly associated with longitudinal changes of lung function, with an estimated difference in annual FEV1pp decline of 0.32% (95%CI: 0.11%-0.54%; p=0.004) per 1000-points change in AUC. Moreover, increasing FIS levels were associated with lower odds of developing pancreatic insufficiency (adjusted OR: 0.18, 95%CI: 0.07-0.46, p<0.001), CF-related liver disease (adjusted OR: 0.18, 95%CI: 0.06-0.54, p=0.002) and diabetes (adjusted OR: 0.34, 95%CI: 0.12-0.97, p=0.044). These associations were absent for SCC. CONCLUSION: This study exemplifies the prognostic value of a PDO-based biomarker within a clinical setting, which is especially important for people carrying rare CFTR mutations with unclear clinical consequences

Updated guidance on the management of children with cystic fibrosis transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID)

Over the past two decades there has been considerable progress with the evaluation and management of infants with an inconclusive diagnosis following Newborn Screening (NBS) for cystic Fibrosis (CF). In addition, we have an increasing amount of evidence on which to base guidance on the management of these infants and, importantly, we have a consistent designation being used across the globe of CRMS/CFSPID. There is still work to be undertaken and research questions to answer, but these infants now receive more consistent and appropriate care pathways than previously. It is clear that the majority of these infants remain healthy, do not convert to a diagnosis of CF in childhood, and advice on management should reflect this. However, it is also clear that some will convert to a CF diagnosis and monitoring of these infants should facilitate their early recognition. Those infants that do not convert to a CF diagnosis have some potential of developing a CFTR-RD later in life. At present, it is not possible to quantify this risk, but families need to be provided with clear information of what to look out for. This paper contains a number of changes from previous guidance in light of developing evidence, but the major change is the recommendation of a detailed assessment of the child with CRMS/CFSPID in the sixth year of age, including respiratory function assessment and imaging. With these data, the CF team can discuss future care arrangements with the family and come to a shared decision on the best way forward, which may include discharge to primary care with appropriate information. Information is key for these families, and we recommend consideration of a further appointment when the individual is a young adult to directly communicate the implications of the CRMS/CFSPID designation

Measuring cystic fibrosis drug responses in organoids derived from 2D differentiated nasal epithelia

Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1&lt;i&gt;β&lt;/i&gt; and interleukin-1&lt;i&gt;β&lt;/i&gt;, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids

Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu

Mini-guts in a dish : Perspectives of adult Cystic Fibrosis (CF) patients and parents of young CF patients on organoid technology

Background: Organoid technology enables the cultivation of human tissues in a dish. Its precision medicine potential could revolutionize the Cystic Fibrosis (CF) field. We provide a first thematic exploration of the patient perspective on organoid technology to set the further research agenda, which is necessary for responsible development of this ethically challenging technology. Methods: 23 semi-structured qualitative interviews with 14 Dutch adult CF patients and 12 parents of young CF patients to examine their experiences, opinions, and attitudes regarding organoid technology. Results: Four themes emerged: (1) Respondents express a close as well as a distant relationship to organoids; (2) the open-endedness of organoid technology sparks hopes and concerns, (3) commercial use evokes cautiousness. (4) Respondents mention the importance of sound consent procedures, long-term patient engagement, responsible stewardship, and stringent conditions for commercial use. Conclusions: The precision medicine potential of organoid technology can only be realized if the patient perspective is taken adequately into account

Optimal correction of distinct CFTR folding mutants in rectal cystic fibrosis organoids

Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu

Rectal Organoids Enable Personalized Treatment of Cystic Fibrosis

Contains fulltext : 201349.pdf (publisher's version ) (Open Access

Potentiator synergy in rectal organoids carrying S1251N, G551D, or F508del CFTR mutations

The potentiator VX-770 (ivacaftor/KALYDECO™) targets defective gating of CFTR and has been approved for treatment of cystic fibrosis (CF) subjects carrying G551D, S1251N or one of 8 other mutations. Still, the current potentiator treatment does not normalize CFTR-dependent biomarkers, indicating the need for development of more effective potentiator strategies. We have recently pioneered a functional CFTR assay in primary rectal organoids and used this model to characterize interactions between VX-770, genistein and curcumin, the latter 2 being natural food components with established CFTR potentiation capacities. Results indicated that all possible combinations of VX-770, genistein and curcumin synergistically repaired CFTR-dependent forskolin-induced swelling of organoids with CFTR-S1251N or CFTR-G551D, even under suboptimal CFTR activation and compounds concentrations, conditions that may predominate in vivo. Genistein and curcumin also enhanced forskolin-induced swelling of F508del homozygous organoids that were treated with VX-770 and the prototypical CFTR corrector VX-809. These results indicate that VX-770, genistein and curcumin in double or triple combinations can synergize in restoring CFTR-dependent fluid secretion in primary CF cells and support the use of multiple potentiators for treatment of CF