Literatura académica sobre el tema "Nasal Potential Difference Measurament"

The gold standard for diagnosing cystic fibrosis (CF) is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD) test.CFTRgene sequencing can identifyCFTRmutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-IIICFTRmutations (10 patients) (G1), CF patients with classes IV-VICFTRmutations (five patients) (G2), and 21 healthy subjects (G3). The CF patients and healthy subjects also underwent the NPD test. A statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis,χ2, and Fisher’s exact tests,α=0.05. No differences were observed between the CF patients and healthy controls for the PDMax, Δamiloride, and Δchloride + free + amiloride markers from the NPD test. For the finger value, a difference between G2 and G3 was described. The Wilschanski index values were different between G1 and G3. In conclusion, our data showed that NPD is useful for CF diagnosis when classes I-IIICFTRmutations are screened. However, if classes IV-VI are considered, the NPD test showed an overlap in values with healthy subjects.

Background Human airway epithelium maintains homeostasis of the fluid and salt composition at the airway surface by a regulated transport of sodium and chloride ions. The volume and composition of airway surface liquid have been shown to be important in the pathogenesis of cystic fibrosis, nasal inflammatory disease, and nasal polyposis. The presence of functional epithelial sodium and chloride channels in the airway epithelium can be evaluated electrically by measuring the voltage across the nasal epithelium (Vt). Because fluticasone propionate is commonly used to treat nasal inflammatory diseases, we tested its effect on the nasal ion transport. Methods A single-blind prospective trial was performed on 12 healthy volunteers. Subjects were randomized to receive either fluticasone propionate or normal saline nasal spray twice daily for 2 weeks. We measured the nasal voltage at baseline, days 3 and 14, and 2 weeks after cessation of treatment. The basal voltage, the change in voltage after perfusion with amiloride (sodium channel blocker), and the change in voltage after perfusion with isoproterenol in a low-chloride buffer (chloride channel activator) were recorded. Saccharin clearance times were measured also. Results Two-week treatment with fluticasone propionate resulted in a significant increase in the change in Vt after perfusion with amiloride. There was no significant change in the group treated with normal saline. These findings also were observed on day 3 and were reversed completely after the 2-week washout period. The increase in amiloride-sensitive Vt did not result in a decrease in mucociliary clearance. Conclusions This study suggests that one effect of fluticasone propionate use on nasal mucosa in normal volunteers is increased epithelial sodium absorption.

O teste da diferença de potencial nasal (DPN) é um exame que mede a diferença bioelétrica através do epitélio nasal, a qual resulta do transporte iônico transepitelial dos íons sódio (Na+), pelo canal ENaC (Epitelial Na+ Channel), e cloro (Cl-), pelo canal CFTR(Cystic Fibrosis Transmembrane Conductance Regulator). DPN tem sido utilizada como teste de auxilio diagnóstico em doenças associadas à disfunção do CFTR, como a Fibrose Cística (FC). FC é uma doença genética autossômica recessiva causada por mutações que afetam o funcionamento do canal CFTR (e secundariamente do ENaC) e levam a manifestações em diversos órgãos. Normalmente a dosagem de cloro no suor acima de 60 mEq/L ou a identificação de mutações nos dois alelos confirmam diagnóstico de FC. Porém, existem casos atípicos com exames considerados inconclusivos onde as características eletrofisiológicas decorrentes da disfunção do CFTR devem ser demonstradas para estabelecimento do diagnóstico. A identificação correta destes casos é importante para instituição do tratamento adequado e definição do prognóstico. O objetivo principal deste trabalho foi padronizar a técnica da DPN para sua futura aplicação como ferramenta diagnóstica através da determinação dos seus valores de referência, de sensibilidade, de especificidade e de concordância entre os resultados das duas narinas. Secundariamente, objetivamos analisar as relações entre a presença de função residual do CFTRe a concentração de cloro no suor, fenótipo pancreático, presença de Pseudomonas aeruginosa, função pulmonar e genótipo na amostra de pacientes comFC. Foi realizado um estudo transversal com realização da DPN em um grupo de pacientes com FC (n=29, idade:15±6 anos) e dois grupos controle: não=FC (n=19, idade: 15 ± 10 anos) e sadios (n=19, idade: 17 ± 8 anos). Os resultados demonstraram que os valores da DPN são significativamente diferentes no grupo FC (FC: DPNmax: -34 ± 9mV, Δamil: -20 ± 9mV, ΔCl: 4 ± 5mV, Δamilo-iso: -19 ± 9 mV e indiceDPN: 0.85 ± 0.23; não-FC:DPNmax: -14 ± 5mV, Δamil: -6 ± 3mV, ΔCl: 17 ± 9mV, Δamilo-iso: -1 ± 4 mV e indiceDPN: 0.11 ± 0.11) e sadios: DPNmax: -15 ± 4mV, Δamil: -6 ± 3mV, ΔCl: 11 ± 7mV, Δamilo-iso: -2 ± 4 mV e indiceDPN: 0.20±0.14),com sensibilidade e especificidade de 95-96% e concordância de resultado entre as duas narinas maior para a DPNmax (r=0,934). A função residual da CFTR não mostrou relação com nenhum dos parâmetros fenotípicos avaliados. Somente mostrou relação com a gravidade do genótipo. Entretanto, foi observada relação entre os parâmetros que avaliam a hiperfunção do ENaC existente na FC e o fenótipo. Concluímos com este trabalho que foi possível reproduzir e padronizar esta técnica da DPN e demonstrar que o fenótipo da FC está mais relacionado à alteração do transporte do íon sódio através do ENaC do que à presença de função residual da CFTR.
Nasal potential difference test (NPD) is a test that measures the bioelectrical difference across the nasal epithelium, which results from transepithelial ion transport of sodium (Na+), by ENaC channels (Epitelial Na+ Channel) and chloride (Cl-), by CFTR (Cystic Fibrosis Transmembrane Conductance Regulator).NPD has been used as a diagnostic tool in CFTR related disorders, such as Cystic Fibrosis (CF). CF is an autosomal recessive genetic disease caused by mutations that affect the function of the CFTR channel (andsecondarily of the EnaC)and lead to manifestations in various organs. Normally sweat chloride concentration > 60 mEq / L and identification of two CFTR mutations confirm the CF diagnosis. However there are atypical cases with inconclusive sweat chloride or genetic where the electrophysiological characteristics induced by CFTR dysfunction has to be demonstrated for diagnosis. The correct identification of these cases is important for institution of appropriate treatment and definition of prognosis. The objective of this study was to standardize the NPD for its future application as a diagnostic tool through the determination of reference values, sensibility and specificity and agreement of the results between both examined nostrils. Secondarily, we analyzed the relations between residual CFTR function and sweat chloride concentration, pancreatic phenotype, Pseudomonas aeruginosa positivity, pulmonary function and genotype in the sample of CF patients. It was a transversal study where the NPD was measured in a group of CF patients (n = 29, age: 15 ± 6 years) and two control groups: non-CF (n = 19, age: 15 ± 10 years) and healthy (n = 19, age: 17 ± 8 years). The results showed that NPD was significantly different in CF (NPDmax: -34 ± 9mV, Δamil: -20 ± 9mV, ΔCl: 4 ± 5mV, Δamilo-iso: -19 ± 9 mV e NPDindex: 0.85 ± 0.23; non-CF: NPDmax: -14 ± 5mV, Δamil: -6 ± 3mV, ΔCl: 17 ± 9mV, Δamilo-iso: -1 ± 4 mV and NPDindex: 0.11 ± 0.11) and healthy: NPDmax: -15 ± 4mV, Δamil: -6 ± 3mV, ΔCl: 11 ± 7mV, Δamilo-iso: -2 ± 4 mV and NPDindex: 0.20±0.14) with sensibility and specificity of 95-96% and agreement between both nostrils greater for NPDmax (r=0.934). The residual CFTR function did not show relation with all phenotypic parameters evaluated. It just showed relation with genotype severity. Indeed it was observed a relation between the parameters that assess the ENaC hyperfunction that occurs in CF and the phenotype. We concluded with this study that was possible to reproduce and to standardize the NPD and to demonstrate that the phenotype is more related to sodium transport alterations through ENaC than to the presence of residual CFTR function.

Orientador: Eulalia Sakano
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A fibrose cística (FC) é uma doença genética autossômica recessiva, resultante da ausência total na proteína CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), ou de alterações qualitativas ou quantitativas do gene que transcreve esta proteína, em células de diversos órgãos do corpo humano, resultando em inúmeros genótipos e fenótipos desta doença. Em muitos pacientes, o diagnóstico é difícil de ser definido, pelo método clássico de dosagem de sódio e cloro no suor, ou pelo sequenciamento genético, justificando a utilização de novas técnicas de auxílio diagnóstico, como a Diferença de Potencial Nasal (DPN). Este teste proporciona uma forma de avaliação direta e sensível, através do epitélio nasal, do transporte de sódio e cloro das membranas celulares, baseado nas propriedades bioelétricas transepiteliais. O objetivo deste trabalho foi verificar se existe diferença dos valores obtidos no exame de DPN em pacientes com FC em comparação com indivíduos controles saudáveis; e verificar se este teste permite diferenciar pacientes com FC das subclasses funcionais mais graves (I, II, III) das subclasses menos graves (IV, V, VI). Foram incluídos no estudo 15 pacientes FC, 10 com mutações mais graves (grupo A) e 5 com mutações menos graves (grupo B), e 21 controles saudáveis (grupo C). Foram considerados os seguintes parâmetros do teste da DPN: "Finger", PDMax, ?Amilorideo, ?Amilorídeo+livrecloreto e index de Wilchanski. Para a variável "Finger", foi encontrada diferença entre pacientes com FC grupo B - mutações menos graves (classe IV, V ou VI) e indivíduos saudáveis - grupo C. O valor do index de Wilchanski mostrou diferença entre pacientes com FC grupo A - mutações mais graves (classes I, II ou III) e indivíduos saudáveis - grupo C. No nosso estudo, a DPN mostrou valores estatisticamente diferentes entre FC com 2 mutações conhecidas e sujeitos saudáveis. Porém, não conseguiu diferenciar fibrocísticos com mutações mais graves (classes I, II e III) daqueles com mutações consideradas menos graves (classes IV, V e VI)
Abstract: Cystic fibrosis (CF) is an autosomal recessive genetic disease, due to the total absence of protein CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), or due to qualitative or quantitative changes in the gene that transcript this protein in cells of various organs of the human body, resulting in numerous genotypes and phenotypes of the disease. In several patients, the diagnosis is difficult to be defined by the classical method of sodium and chloride dosage in sweat, or by genetic sequencing, justifying the use of new techniques for diagnosis, as the Nasal Potential Difference (NPD). This test provides a way of direct and sensitive assessment of the transport of sodium and chloride ions in cell membranes, via the nasal epithelium, based on transepithelial bioelectric properties. The objective of this work was to verify the difference of the values obtained in the examination of NPD in patients with CF compared with healthy control subjects, and, to verify if this test allows differentiating patients with more severe CF functional subclasses (I, II , III) from patients with less severe CF subclasses (IV, V, VI). This study included 15 CF patients, 10 with more severe mutations (group A) and 5 with less severe mutations (group B), and 21 healthy controls (group C). We considered the following test parameters of NPD: "Finger", PDMax, ?Amiloride, ?Amiloríde+Chloridefree and Wilchanski index. For "Finger" values, it was found difference between patients with CF Group B - less severe mutations (class IV , V or VI) and healthy individuals - group C. The value of Wilchanski index showed difference between group A CF patients, with more severe mutations (class I, II or III) and healthy individuals - group C. In our study, NPD showed statistically different values between CF patients with two known mutations and healthy subjects. However, it was not able to distinguish between CF patients with more severe mutations (class I, II and III) of the CF patientswith less severe mutations (Class IV, V and VI)
Mestrado
Otorrinolaringologia
Mestre em Ciências Médicas

Exercise has become a vital component of the therapy regimen prescribed to cystic fibrosis (CF) patients due to its systemic benefits, such as increased sputum expectoration, attenuation of the expected 2-3% annual decline in pulmonary function, and extended life expectancy. However, exercise still is not viewed as being as beneficial as pharmacological treatments by many CF patients and can be intimidating. My aims in this study were two-fold; first, to determine the ideal exercise intensity for individuals with CF; and second, to determine if exercise at this ideal intensity could provide improvements in ion regulation in the lungs, which was measured using exhaled breath condensate (EBC) collection and nasal potential difference (NPD), that were comparable to one of their standard pharmacological therapies, albuterol. I hypothesized that with moderate intensity exercise, Na⁺ absorption would decrease from baseline due to Na⁺ channel inhibition, rather than increase or remain unchanged, as was expected with albuterol, and cause an even greater increase Cl- secretion compared to albuterol due to activation of both CF-dependent and independent Cl- efflux with exercise. CF (n=14) and healthy (n=16) subjects completed three visits, a baseline screening and two treatment visits. I collected EBC at baseline, 30- and 60-minutes post-albuterol administration on one visit, and at baseline and during three separate 15 min exercise bouts at low, moderate and high intensity on the other visit. Following the EBC collection, NPD was performed at 30- and 80-minutes post albuterol or following moderate and high intensity exercise. We also measured spirometry and diffusing capacity of the lungs for nitric oxide (DLNO) during each visit at the various time points. In CF subjects, moderate intensity exercise resulted in greater improvements in DLNO (39 ± 29vs.15 ± 22% change from baseline, exercise vs. albuterol respectively), similar levels of bronchodilation compared to 60-minutes post-albuterol administration, no change in Na⁺ absorption, and a four-fold increase in Cl- secretion. Our results suggest that moderate intensity exercise is the best dose for CF patients, and can provide comparable changes as its pharmacological counterpart albuterol, when compared over a short term duration.

SCOPO: 1) valutare l’espressione della proteine che regola il trasporto del cloro transmembrana (CFTR) e la sua attività funzionale nei monociti; 2) creare delle linee cellulari immortalizzate a partire da linfociti B aventi genotipi differenti; 3) valutare linee cellulari immortalizzate. CONOSCENZE DI BASE: La Fibrosi Cistica (CF), la più commune e grave malattia autosomale diffusa nei Paesi Caucasici, è dovuta alla presenza di mutazioni sul gene CFTR. Sebbene la CF sia una malattia multi organo, la patologia polmonare è la principale causa di morte tra i pazienti CF. E’ caratterizzata da una infiammazione cronica, conseguenza di un’infezione batterica. La suscettibilità alle infezioni batteriche non è completamente conosciuta, anche se il coinvolgimento di CFTR nelle funzioni microbicide dei macrofagi sta emergendo in questo periodo. I macrofagi differenziano in situ a partire dai monociti infiltati nel tessuto e mostrano una marcata variabilità morfologica pur avendo comuni funzioni cellulari e molecolari. Sebbene l’espressione di CFTR nei macrofagi alveolari sia stata descritta, la sua espressione nei monociti non è ancora stata riportata anche se queste cellule risultano essere più accessibili per studi funzionali e di espressione. La valutazione dell’espressione e della funzione del CFTR nelle cellule mononucleari del sangue periferico (PBMC) è un pre-requisito per valutare il loro ruolo ed il loro potenziale utilizzo in diagnostica e nello sviluppo di nuovi farmaci con azione sul difetto molecolare del CFTR. METODI: Purificazione dei monociti e dei linfociti B da sangue intero; produzione del virus Epstein-Barr (EBV); immortalizzazione dei linfociti B mediante EBV; isolamento RNA e analisi dell’mRNA mediante PCR; Real-time PCR; Western blotting; citometria di flusso; immunofluorescenza; depolarizzazione di membrana; misurazione delle differenze dei potenziali nasali; analisi dei dati. RISULTATI: Utilizzando un anticorpo anti-CFTR policlonale e due monoclonali che riconoscono differenti epitopi, abbiamo rilevato mediante western blotting tutte le forme conosciute del CFTR. La citometria di flusso e la microscopia confocale ha confermato l’espressione di CFTR e la sua localizzazione su membrana. Abbiamo osservato che i monociti non-CF, dopo stimolazione con uno specifico agonista di CFTR, mostravano un aumento dell’intensità di fluorescenza, una variazione che non abbiamo rilevato nei monociti CF. Questi risultati hanno suggerito una correlazione dell’attività di CFTR con la depolarizzazione della membrana ed i dati sono stati confermati tramite uno specifico inibitore, CFTR (inh)-172. Questo approccio è stato comparato alla misurazione dei potenziali nasali (NPD) eseguiti sugli stessi soggetti ed la sovrapposizione dei dati ha rilevato una forte corrispondenza tra le due tecniche. I linfociti B sono stati immortalizzati mediante EBV e sono stati utilizzati come potenziali modelli cellulari per valutare l’attività del CFTR. Abbiamo rilevato la maggiore forma glicosilata di CFTR in queste linee immortalizzate utilizzando un anticorpo monoclonale anti-CFTR. Una forma a minore peso molecolare è stata anch’essa evidenziata mediante questo anticorpo ed uno policlonale. La citometria di flusso e la microscopia confocale ci hanno permesso di confermare l’espressione e la localizzazione su membrana del CFTR. Il test di depolarizzazione della membrana è stato applicato sulle cellule B immortalizzate ottenendo gli stessi risultati visti sui monociti. CONCLUSIONI: Abbiamo dimostrato l’espressione della proteina CFTR in monociti umani identificando una variante a peso molecolare corrispondente ad un basso livello di post-trasduzione della proteina. Questo è stato confermato utilizzando monociti con genotipo omozigote per la mutazione non-sense i quali perdevano l’espressione della forma di CFTR. La citometria di flusso potrebbe essere utile per valutare l’espressione di CFTR. Infatti abbiamo dimostrato che può distinguere tra non-CF ed eterozigoti da pazienti CF mediante la marcatura CD14/Rb-AF488 dei monociti. L’analisi della depolarizzazione di membrana su singola cellula ha confermato l’espressione funzionale del CFTR mostrando una elevata depolarizzazione di membrana a seguito della stimolazione delle cellule con uno specifico agonista del CFTR. Questo metodo potrebbe essere eseguito entro un paio di ore dal prelievo del sangue. Inoltre, è facilmente riproducibile con un minimo disturbo e rischio per il paziente e potrebbe permettere una valutazione in corso d’opera degli effetti di alcune particolari terapie sull’espressione e sull’attività del CFTR. Abbiamo creato uno specifico indice capace di discriminare tra CF e non-CF. La sovrapposizione dei dati NPD e di quelli sull’attività funzionale del CFTR sui monociti risultata in una perfetta corrispondenza tra le due tecniche. Poiché NPD è un test diagnostico che si applica su soggetti con test del sudore dubbio o con almeno una mutazione non conosciuta, possiamo promuovere la valutazione dell’attività del CFTR nei monociti mediante tecniche ottiche come un utile metodo per valutare l’attività di CFTR per la ricerca, includendo lo sviluppo di nuovi farmaci e la diagnosi. Dato che le cellule primarie hanno disponibilità limitata in termini quantitativi, abbiamo preso vantaggio dall’osservazione che i linfociti esprimono CFTR. Le cellule B immortalizzate potrebbero essere utilizzate come modello cellulare per studiare l’espressione e l’attività del CFTR. Abbiamo rilevato l’espressione di una forma di CFTR che probabilmente rappresenta una isoforma processata a seguito dell’attività di una specifica calpaina nei linfociti come dimostrato in letteratura. Inoltre, l’indice ottenuto mediante lo studio della depolarizzazione di membrana ci ha permesso di discriminare tra gruppi CF e non-CF come osservato nei monociti. Tutti questi risultati hanno dimostrato che il CFTR è espresso ed è attivo nei linfociti umani e nelle cellule B immortalizzate. Per tale motivo, queste cellule possono essere sfruttate per valutare la risposta di specifiche mutazioni a nuovi farmaci diretti o indiretti sul difetto di base di CF.
AIM: 1) to evaluate the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and functional activity in monocytes; 2) to create immortalized cell lines from human B-lymphocyte cells characterized by different genotypes; 3) to evaluate CFTR protein expression in immortalized B cells. BACKGROUND: Cystic Fibrosis (CF), the most common autosomal severe disorder in Caucasians, is caused by mutations in the CFTR gene. Although CF is a multi-organ disease, the lung pathology is the main cause of morbidity and mortality of CF patients. It is characterized by chronic inflammation as a consequence of persistent bacterial infections by several opportunistic pathogens. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of CFTR in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes and display a remarkable variability in cell morphology although common molecular and cellular functions. Although expression of CFTR in alveolar macrophages has been described, its expression has not been reported in monocytes that are more accessible for expression studies and functional analysis tests than macrophages. Evaluation of expression and functional activity of CFTR in peripheral blood mononuclear cells (PBMC) is a pre-requisite to evaluate their role and their potential use in diagnostic and developing new drugs acting on the molecular defect of CF. METHODS: Purification of monocytes and lymphocyte B cells from whole blood; production of Epstein-Barr Virus (EBV); immortalization of Lymphocytes B cells by EBV; RNA isolation and CFTR mRNA analysis by reverse-transcription and polymerase chain reaction (PCR); quantitative real-time PCR (RT-qPCR); western Blotting; Flow cytometry assay; immunofluorescence; cell depolarization assay; Nasal Potential Differences (NPDs) assay; analysis of cell depolarization assay data. RESULTS: In this study western blotting using a polyclonal and two monoclonal anti-CFTR antibodies that recognize different epitopes detected all known forms of CFTR. Flow cytometry and confocal microscopy analysis confirmed expression of CFTR protein expression and its membrane localization. Increased fluorescence intensity, corresponding to membrane depolarization, was observed only when non-CF monocytes were stimulated with CFTR agonist, while CF monocytes did not show fluorescence variation. These results suggested a correlation between CFTR activity and membrane depolarization and data were confirmed using a specific CFTR inhibitor, CFTR (inh)-172. This approach was compared to NPD measurements performed in a subset of the same patients subjected to this analysis. Results obtained by NPD overlapped those obtained by the analysis of monocytes from non-CF donors and CF patients. B-lymphocytes were then immortalized by EBV and were tested as potential cell models for CFTR activity assays. The major glycosylated form of CFTR was detected in immortalized non-CF EVB-transformed B cell line by a monoclonal anti-CFTR antibody, but a band with minor molecular weight was also detected with this antibody and with a polyclonal anti-CFTR antibody. Flow cytometry and confocal assay allowed us to confirm CFTR expression and membrane location in these cell lines. Membrane depolarization test was applied in EBV-transformed B cells and the results confirmed a stimulus induced membrane depolarization in non- CF cells. CONCLUSION: We have demonstrated that CFTR proteins are expressed in human monocytes as a variant recognized by a specific antibody. Its molecular weight is consistent with a lower level of post-translational processing and its loss in patients carrying a homozygous non-sense mutation confirmed its presence in human monocytes. Flow cytometry could be also a useful method to evaluate CFTR expression. We demonstrated that it can distinguish between non-CF and HTZ subjects and CF patients analyzing stained CD14/Rb-AF488 monocytes. Single-cell membrane depolarization analysis confirmed that, upon stimulation with CFTR agonists, normal monocytes displayed a highly reproducible membrane depolarization activity consistent with the expression of functional CFTR. Single-cell depolarization assay could be performed within a few hours after blood collection. It is also easily repeatable with a minimal discomfort and risk for the patient and it could thus allow a time-course evaluation of effects of any particular therapy on CFTR expression or functional activity. A specific activity index was devised that appears capable to discriminate among CF and non-CF cells. Overlapping NPD data and functional activity data, we observed a perfect correspondence. Since NPD is a reference diagnostic test applied when a subject has borderline sweat test and at least one unidentified CFTR mutation, we might promote the evaluation of CFTR activity in monocytes by optical techniques as a useful tool to assess CFTR activity for basic and translational research, including drug development and diagnosis. As primary cells are available in limited amounts, we have taken advantage of the observation that CFTR-associated Cl- permeability has been demonstrated in lymphocytes. So, immortalized-B-cells could be useful as cellular model to study CFTR expression and activity. We observed a form of CFTR that likely represents a processed isoform possibly linked to specific calpain activity in lymphocyte cells as demonstrated in the literature. Furthermore, the index obtained by single-cell fluorescence imaging discriminated between non-CF and CF groups as shown in monocytes. All these results demonstrated that CFTR protein is expressed and is active in human lymphocytes and EBV-transformed B cells opening interesting perspectives in this field. Indeed, these cells can be exploited to evaluate the response of specific mutations to newly developed drugs acting directly or indirectly on the basic defect of CF.

Millions of children and young adults are exposed to fine particulate matter (PM2.5) and ozone, associated with Alzheimer’s disease (AD) risk. Mexico City (MC) children exhibit systemic and brain inflammation, low cerebrospinal fluid (CSF) Aβ1-42, breakdown of nasal, olfactory, alveolar-capillary, duodenal, and blood-brain barriers, volumetric and metabolic brain changes, attention and short-term memory deficits, and hallmarks of AD and Parkinson’s disease. Airborne iron-rich strongly magnetic combustion-derived nanoparticles (CDNPs) are present in young urbanites’ brains. Using transmission electron microscopy, we documented CDNPs in neurons, glia, choroid plexus, and neurovascular units of young MC residents versus matched clean air controls. CDNPs are associated with pathology in mitochondria, endoplasmic reticulum (ER), mitochondria-ER contacts (MERCs), axons,and dendrites. There is a significant difference in size and numbers between spherical CDNPs (>85%) and the angular, euhedral endogenous NPs (<15%). Spherical CDNPs (dogs 21.2 ± 7.1 nm in diameter versus humans 29.1 ± 11.2 nm, p = 0.002) are present in neurons, glia, choroid plexus, endothelium, nasal and olfactory epithelium, and in CSF at significantly higher in numbers in MC residents (p < 0.0001). Degenerated MERCs, abnormal mitochondria, and dilated ER are widespread, and CDNPs in close contact with neurofilaments, glial fibers, and chromatin are a potential source for altered microtubule dynamics, mitochondrial dysfunction, accumulation and aggregation of unfolded proteins, abnormal endosomal systems, altered insulin signaling, calcium homeostasis, apoptotic signaling, autophagy, and epigenetic changes. Highly oxidative, ubiquitous CDNPs constitute a novel path into AD pathogenesis. Exposed children and young adults need early neuroprotection and multidisciplinary prevention efforts to modify the course of AD at early stages.