Assessment of Tumor-Associated Tissue Eosinophilia (TATE) and Tumor-Associated Macrophages (TAMs) in Canine Transitional Cell Carcinoma of the Urinary Bladder.

Transitional cell carcinoma of the urinary bladder is a significant neoplasm in dogs, characterized by a poor prognosis and a high metastatic potential. These canine spontaneous tumors share many characteristics with human transitional cell carcinoma, making them an excellent comparative model. The role of inflammatory infiltration in tumor development and progression is frequently contradictory, especially concerning tumor-associated tissue eosinophils (TATE) and tumor-associated macrophages (TAMs). This study aims to analyze TATE and TAMs in canine transitional cell carcinoma of the urinary bladder. Congo Red staining was used to identify TATE, and immunohistochemistry was performed to detect TAMs in 34 cases of canine transitional cell carcinoma of the bladder carcinomas, categorized into low and high grades. Statistically significant differences were observed between the number of eosinophils and macrophages in the two groups of tumors. The number of TATE was higher in low-grade malignant tumors, but the number of TAMs was higher in high-grade tumors. Our findings suggest the importance of TATEs and TAMs in the aggressiveness of canine transitional cell carcinoma and propose their potential use as therapeutic targets.

New-Onset Psoriatic Arthritis Following COVID-19 mRNA Vaccination in a Psoriatic Patient Under Anti-tumor Necrosis Factor Alpha Biologic Treatment: What Now?

During the COVID-19 pandemic, anti-SARS-CoV-2 vaccines were quickly developed and administered to the population worldwide. As is expected with new vaccine products, adverse reactions following immunization have been reported, namely, the development and/or exacerbation of autoimmune/autoinflammatory diseases, including rheumatic diseases. Here, we report a clinical case of a 56-year-old woman with a 44-year history of moderate-to-severe plaque psoriasis under treatment with an anti-tumor necrosis factor alpha biosimilar (adalimumab) with good control of skin disease and without rheumatic involvement to date who came to us with complaints of migratory polyarthralgia starting one week after receiving the second dose of the BNT162b2 COVID-19 mRNA vaccine. The condition progressed over the following months and a diagnosis of psoriatic arthritis was established. Biologic treatment was switched to an anti-interleukin 17A (secukinumab), with a very good clinical cutaneous and articular response, which was sustained up to the present moment. The mechanisms behind the exacerbation or new-onset of autoimmune/autoinflammatory diseases after receiving anti-COVID-19 vaccines are not yet fully understood, requiring further investigation. It is also not known whether rheumatic symptoms post-COVID-19 infection will have similar mechanisms to rheumatic symptoms post-anti-COVID-19 vaccination. With the continuing worldwide vaccination against SARS-CoV-2, clinicians need to be prepared to discuss the risks and benefits of vaccination and should be aware that it may cause or exacerbate immune disorders such as psoriatic arthritis, warranting close follow-up in terms of disease progression and treatment.

Basaloid Squamous Cell Carcinoma, an Aggressive and Rare Cancer of the Oral Cavity: Can We Prevent It at the Primary Care Setting?

Oral cavity cancer represents about 2%-3% of all cancers worldwide, with more than 355,000 new cases per year, one-third of which are reported in developed countries. Oral cancer is also known to be extremely aggressive when detected late, thus presenting one of the lowest cancer survival rates. It is estimated that as much as 90% of oral cancers are attributable to tobacco and/or alcohol consumption and that high-risk human papillomaviruses (HPV) infections pose an independently increased risk for their development. Therefore, it can be a preventable disease when associated with changes in lifestyle and possible modifiable risk factors, combined with early and preventive intervention. Basaloid squamous cell carcinoma (BSCC) constitutes an aggressive and rare form of oral cancer, being one of the rarest and most aggressive variants of squamous cell carcinoma (SCC, the most common), and usually presents as a high-grade disease with a poor prognosis. It is typically associated with heavy smoking and alcohol abuse, occurring most commonly in older men. Here, we report a clinical case of a 60-year-old man with excessive consumption of both tobacco and alcohol, poor oral hygiene, and partial edentulousness who came to our primary health department with complaints of odynophagia twice in a four-year time-lapse. The first time, two whitish ulcerated lesions on the left tonsil were detected and biopsied but revealed a negative histological result. After four years, he came again to our primary health care department with similar complaints of odynophagia and also sore throat with radiation to the right ear, accompanied by globus sensation and anorexia. No suspicious lesions were detected, except a globally hyperemic oropharynx. Considering the history of abusive consumption, no improvement with symptomatic treatment, and persistent clinical signs, an extended diagnostic approach was carried out. After four months, a pharyngeal mass measuring 53 mm was detected on pharyngeal-neck computed tomography (CT), and the diagnosis of a BSCC located in the right tonsillar pillar and base of the tongue was finally determined. Unlike other cancers that have been detected earlier through screening programs, oral cancer is often detected at an advanced stage, compromising survival and quality of life. The opportunity to intervene early and preventively in consumption habits, promote healthy lifestyles, and try to prevent disease is unique at the primary care level. Moreover, opportunistic screening through a thorough examination of the oral cavity is extremely important for timely diagnosis and treatment.

Management of child camps: a structured literature review and new directions / Gestão de campos de férias para crianças: uma revisão estruturada da literatura e novas direções / Gestión de campamentos infantiles: una revisión estructurada de la literatura y nuevas direcciones

The study of child camps has grown over the last years. Still, the common use of different terminology and the dispersion of the information in literature regarding camps management, makes it difficult to clarify research and hinder the improvement of managerial practices. This study aims to synthetise existent knowledge in child camps' management, identify inconsistencies and gaps in the literature, and set directions for future research and practice on child camps. A structured review of peer-reviewed articles published between 1950-2021 was conducted. Results indicate that half of the studies used the term "summer camp"; but other ten different terms were also used. Five different management areas were identified: safety, consumer behaviour, human resources, event planning and camp research. Gaps were also identified in the literature. These findings are important to set new research avenues and to improve practice. (AU)

O estudo dos campos de férias para crianças aumentou nos últimos anos. Todavia, a utilização de diferentes termos e a dispersão da informação na literatura acerca da gestão destes eventos dificulta o esclarecimento das diretrizes de pesquisa, por um lado, e dificulta a melhoria das práticas de gestão, por outro. Este estudo visa sintetizar o conhecimento existente na gestão destes eventos, identificar inconsistências e lacunas na literatura, e definir direções para futuras pesquisas e práticas. Foi realizada uma revisão estruturada de estudos publicados entre 1950-2021. Metade dos estudos utilizou o termo "campos de verão"; mas também foram utilizados outros dez termos diferentes. Foram identificadas cinco áreas de gestão: segurança, comportamento do consumidor, recursos humanos, organização de eventos e pesquisa em campos de férias. Foram ainda identificadas lacunas na literatura. Estes resultados são importantes para definir novos caminhos de pesquisa e melhorar a gestão destes eventos. (AU)

El estudio de los campamentos de verano para niños se ha incrementado en los últimos años. Pero, el uso de términos diferentes, y la dispersión de información en la literatura, sobre la gestión de estos eventos, dificulta la clarificación de las directrices de investigación, e impide que los gestores mejoren sus prácticas. Este estudio buscó sintetizar el conocimiento existente en la gestión de estos eventos, identificar inconsistencias y lagunas en la literatura y definir direcciones para futuras investigaciones y prácticas. Se realizó una revisión estructurada de estudios publicados entre 1950-2021. La mitad de los estudios utilizaron "campamento de verano"; pero también se utilizaron otros diez términos diferentes. Se identificaron cinco áreas de gestión: seguridad, comportamiento del consumidor, recursos humanos, organización de eventos y investigación en campamentos de verano. También se identificaron lagunas en la literatura. Estos resultados son importantes para definir nuevos caminos de investigación y mejorar la gestión de estos eventos. (AU)

Participation of the federation units in the investment of sports and leisure policies in Brazil / Participação das unidades da federação no investimento das políticas de esporte e de lazer no Brasil

The purpose of this exploratory descriptive study was to evaluate the participation of state governments (federation units) in the financing of sports and leisure public policies from 2002 to 2016. In order to better understand the regional effect of investment, the information was cut by region. Two central hypotheses were tested, the concentrator effect and the equity effect. The database was formed from the consolidated expenditure record registered with the National Treasury in the Accounting and Tax Information System of the Brazilian Public Sector - Siconfi. In order to ensure the comparability of tax information, the values were deflated by the Consumer Price Index. Although there are no constraints at the level of the federation pressing the Federation Units to be active with regard to the sports agenda, these Units have been shown to be responsive to it. The overall result produces different effects between macro-regions, regions and Federation Units, concentrating the resources, or distributing them more evenly.

O presente estudo de natureza descritivo exploratória teve por objetivo geral avaliar a participação dos governos estaduais (Unidades da Federação) no financiamento das políticas públicas de esporte e de lazer de 2002 a 2016. Para melhor compreensão do efeito regional do investimento, realizou-se o recorte das informações por região. Duas hipóteses centrais foram testadas, o efeito concentrador e o efeito equidade. O banco de dados foi formado a partir do registro do gasto consolidado registrado junto ao Tesouro Nacional no Sistema de Informações Contábeis e Fiscais do Setor Público Brasileiro ­ Siconfi. Para garantir a comparabilidade das informações fiscais, os valores foram deflacionados pelo Índice de Preços ao Consumidor (IPCA). Apesar de não haver constrangimentos no nível da federação pressionando as UF a serem ativas no que concerne à agenda esportiva, esses entes têm demonstrado serem responsivos a ela. O resultado geral produz diferentes efeitos entre as macrorregiões, regiões e Unidades da Federação. Ora concentrando os recursos, ora distribuindo de forma mais equilibrada.

Cortical deafness of following bilateral temporal lobe stroke.

Cortical deafness is an extremely rare clinical manifestation that originates mainly from bilateral cortical lesions in the primary auditory cortex. Its main clinical manifestation is the bilateral sudden loss of hearing. Diagnosis is difficulty due to its rarity and similarity with other language and communication disorders, such as Wernicke's aphasia, auditory agnosia or verbal deafness. Herein, we present a case report of a young woman with a sudden bilateral loss of auditory comprehension. Initially, a psychiatric nature of the disorder was considered, but the persistence of the symptoms, lead to the diagnosis of cortical deafness secondary to bilateral ischemic lesions in both temporal lobes. Progressive improvement occurred and three months after the initial manifestations she manifested pure verbal deafness. Cortical deafness usually has a poor functional prognosis, with limited therapeutic options. Rehabilitation and speech therapy is recommended to improve the chance of patients achieving communication skills.

Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations.

The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Here, we aimed to test the accuracy of in silico predictions by comparing them with in vitro and ex vivo functional analyses of this mutation for an accurate CF diagnosis/prognosis. To this end, we applied a new in vitro strategy using a CFTR mini-gene which includes the complete CFTR coding sequence plus intron 22 (short version) which allows the assessment of alternatively spliced mRNA levels as well as the properties of the resulting abnormal CFTR protein regarding processing, intracellular localization and function. Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl- channel function. These results recapitulate data from native tissues of a CF patient. In conclusion, the existing in silico prediction models have limited application and ex vivo functional assessment of mutation effects should be made. Alternatively the in vitro strategy adopted here can be applied to assess the disease liability of mutations for an accurate CF diagnosis/prognosis.

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.

Ultrasound therapy and transcutaneous electrical neuromuscular stimulation for management ofpost-mastectomy upper limb lymphedema / Terapia de ultrassom e estimulação elétrica transcutânea neuromuscular para a tratamento do linfedema de membro superior pós-mastectomia

Objetivo: Avaliar o efeito da estimulação elétrica transcutânea ou terapia de ultra-som no tratamento de pós-mastectomia linfedema do membro superior. Método: revisão sistemática da literatura foi realizada 1980-2012 do MedLine, Cochrane Library, LILACS e SciELO. Os termos utilizados na pesquisa foram (neoplasia de mama ou câncer de mama ou de linfedema) e (hipertermia, induzido ou diatermia ou terapia de ultra-som ou ultra-som ou a estimulação elétrica nervosa transcutânea ou dezenas). As seleções dos estudos eram de pacientes mulheres com linfedema pós-mastectomia membro superior que foram submetidos a diatermia por terapia de ultra-som e estimulação elétrica nervosa transcutânea. Só randomizado (RCT) e projetos quase randomizados do estudo foram incluídos (ambos estreita e Broad Therapy). Somente estudos publicados no formato de artigo completo foram incluídos. Depois de analisar os 2.158 resumos resultantes da pesquisa, foram selecionados apenas dois artigos. Dois pesquisadores analisaram os dois artigos, usando o Van Tulder e JADAD escalas para avaliação da qualidade. Resultados: Ambos os trabalhos avaliaram o uso da terapia de ultra-som e estimulação elétrica para o tratamento do linfedema pós-mastectomia. Um total de 132 indivíduos foram incluídos em ambos os estudos, e pouca melhora foi observada em redução ou a qualidade de vida da dor. Somente o estudo usando a terapia de ultra-som identificada uma pequena redução nos sintomas de linfedema. No entanto evidências que suportam a aplicação deste método está faltando. Conclusão: Mais estudos são necessários para avaliar o uso da terapia de ultra-som ou eletroterapia para o tratamento de linfedema pós-mastectomia e para avaliar o efeito potencial dessas terapias no desenvolvimento posterior da doença metastática.

Objective: This article aims to assess the effect of transcutaneous electrical stimulation or ultrasound therapy in the treatment of post-mastectomy upper limb lymphedema. Method: A systematic literature review was performed from 1980 to 2012 from the MedLine, Cochrane Library, LILACS and SciELO databases. The terms used in the search were (breast neoplasm OR breast cancer OR lymphedema) and (hyperthermia, induced OR diathermy OR ultrasonic therapy OR ultrasound OR transcutaneous electrical nerve stimulation OR TENS). The selections of the studies concerned female patients with post-mastectomy upper limb lymphedema who underwent diathermy by ultrasound therapy and transcutaneous electric nerve stimulation. Only randomized (RCT) and quasi-randomized study designs were included (both Narrow and Broad Therapy). Only studies published in the full paper format were included. After reviewing the 2,158 abstracts resulting from the search, only two papers were selected. Two researchers analyzed the two articles, using the Van Tulder and JADAD scales for quality assessment. Results: Both papers evaluated the use of ultrasound therapy and electric stimulation for treatment of post-mastectomy lymphedema. A total of 132 subjects were included in these two studies, and little improvement was observed in pain reduction or quality of life. Only the study using ultrasound therapy identified a small reduction in lymphedema symptoms; however, evidence supporting the application of this method is lacking. Conclusion: Further studies are needed to evaluate the use of ultrasound therapy or electrotherapy for treatment of post-mastectomy lymphedema and to evaluate the potential effect of these therapies on later development of metastatic disease.

Kinematical Analysis along Maximal Lactate Steady State Swimming Intensity.

The purpose of this study was to conduct a kinematical analysis during swimming at the intensity corresponding to maximal lactate steady state (MLSS). Thirteen long distance swimmers performed, in different days, an intermittent incremental protocol of n x 200 m until exhaustion and two to four 30-min submaximal constant speed bouts to determine the MLSS. The video analysis, using APAS System (Ariel Dynamics Inc., USA), allowed determining the following relevant swimming determinants (in five moments of the 30-min test: 0, 25, 50, 75, and 100%): stroke rate, stroke length, trunk incline, intracyclic velocity variation, propelling efficiency, index of coordination and the time allotted to propulsion per distance unit. An ANOVA for repeated measures was used to compare the parameters mean values along each moment of analysis. Stoke rate tended to increase and stroke length to decrease along the test; a tendency to decrease was also found for intracyclic velocity variation and propelling efficiency whereas the index of coordination and the propulsive impulse remained stable during the MLSS test. It can be concluded that the MLSS is not only an intensity to maintain without a significant increase of blood lactate concentration, but a concomitant stability for some biomechanical parameters exists (after an initial adaptation). However, efficiency indicators seem to be more sensitive to changes occurring during swimming at this threshold intensity. Key PointsIn MLSS swimming intensity, stability of the stroke length and stroke frequency occurs after an initial adaptation.Efficiency indicators seem to be more sensitive to possible changes occurring through time at MLSS intensity.MLSS is a useful and practical swimming intensity to be maintained for a long period of time, but some constraints in technique can occur.

Influence of prior exercise on VO2 kinetics subsequent exhaustive rowing performance.

Prior exercise has the potential to enhance subsequent performance by accelerating the oxygen uptake (VO2) kinetics. The present study investigated the effects of two different intensities of prior exercise on pulmonary VO2 kinetics and exercise time during subsequent exhaustive rowing exercise. It was hypothesized that in prior heavy, but not prior moderate exercise condition, overall VO2 kinetics would be faster and the VO2 primary amplitude would be higher, leading to longer exercise time at VO2max. Six subjects (mean ± SD; age: 22.9±4.5 yr; height: 181.2±7.1 cm and body mass: 75.5±3.4 kg) completed square-wave transitions to 100% of VO2max from three different conditions: without prior exercise, with prior moderate and heavy exercise. VO2 was measured using a telemetric portable gas analyser (K4b(2), Cosmed, Rome, Italy) and the data were modelled using either mono or double exponential fittings. The use of prior moderate exercise resulted in a faster VO2 pulmonary kinetics response (τ1 = 13.41±3.96 s), an improved performance in the time to exhaustion (238.8±50.2 s) and similar blood lactate concentrations ([La(-)]) values (11.8±1.7 mmol.L(-1)) compared to the condition without prior exercise (16.0±5.56 s, 215.3±60.1 s and 10.7±1.2 mmol.L(-1), for τ1, time sustained at VO2max and [La(-)], respectively). Performance of prior heavy exercise, although useful in accelerating the VO2 pulmonary kinetics response during a subsequent time to exhaustion exercise (τ1 = 9.18±1.60 s), resulted in a shorter time sustained at VO2max (155.5±46.0 s), while [La(-)] was similar (13.5±1.7 mmol.L(-1)) compared to the other two conditions. Although both prior moderate and heavy exercise resulted in a faster pulmonary VO2 kinetics response, only prior moderate exercise lead to improved rowing performance.

High-content siRNA screen reveals global ENaC regulators and potential cystic fibrosis therapy targets.

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.

Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

Rectal forceps biopsy procedure in cystic fibrosis: technical aspects and patients perspective for clinical trials feasibility.

BACKGROUND: Measurements of CFTR function in rectal biopsies ex vivo have been used for diagnosis and prognosis of Cystic Fibrosis (CF) disease. Here, we aimed to evaluate this procedure regarding: i) viability of the rectal specimens obtained by biopsy forceps for ex vivo bioelectrical and biochemical laboratory analyses; and ii) overall assessment (comfort, invasiveness, pain, sedation requirement, etc.) of the rectal forceps biopsy procedure from the patients perspective to assess its feasibility as an outcome measure in clinical trials. METHODS: We compared three bowel preparation solutions (NaCl 0.9%, glycerol 12%, mannitol), and two biopsy forceps (standard and jumbo) in 580 rectal specimens from 132 individuals (CF and non-CF). Assessment of the overall rectal biopsy procedure (obtained by biopsy forceps) by patients was carried out by telephone surveys to 75 individuals who underwent the sigmoidoscopy procedure. RESULTS: Integrity and friability of the tissue specimens correlate with their transepithelial resistance (r = -0.438 and -0.305, respectively) and are influenced by the bowel preparation solution and biopsy forceps used, being NaCl and jumbo forceps the most compatible methods with the electrophysiological analysis. The great majority of the individuals (76%) did not report major discomfort due to the short procedure time (max 15 min) and considered it relatively painless (79%). Importantly, most (88%) accept repeating it at least for one more time and 53% for more than 4 times. CONCLUSIONS: Obtaining rectal biopsies with a flexible endoscope and jumbo forceps after bowel preparation with NaCl solution is a safe procedure that can be adopted for both adults and children of any age, yielding viable specimens for CFTR bioelectrical/biochemical analyses. The procedure is well tolerated by patients, demonstrating its feasibility as an outcome measure in clinical trials.

Control of TMEM16A by INO-4995 and other inositolphosphates.

BACKGROUND AND PURPOSE: Ca(2+)-dependent Cl(-) secretion (CaCC) in airways and other tissues is due to activation of the Cl(-) channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca(2+) -activated Cl(-) channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates. EXPERIMENTAL APPROACH: The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques. KEY RESULTS: We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF(-1) in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P(4) or Ins(1,3,4,5)P(4) and enzymes controlling levels of InsP(4) or PIP(2) and PIP(3) had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl(-) currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl(-) currents activated by ionomycin or ATP, while intracellular Ca(2+) signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways. CONCLUSIONS AND IMPLICATIONS: These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease.

HGF stimulation of Rac1 signaling enhances pharmacological correction of the most prevalent cystic fibrosis mutant F508del-CFTR.

Cystic fibrosis (CF), a major life-limiting genetic disease leading to severe respiratory symptoms, is caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride (Cl(-)) channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection, and ultimately lung failure. The most frequent mutation, F508del, causes the channel to misfold, triggering its premature degradation and preventing it from reaching the cell surface. Recently, novel small-molecule correctors rescuing plasma membrane localization of F508del-CFTR underwent clinical trials but with limited success. Plausibly, this may be due to the mutant intrinsic plasma membrane (PM) instability. Herein, we show that restoration of F508del-CFTR PM localization by correctors can be dramatically improved through a novel pathway involving stimulation of signaling by the endogenous small GTPase Rac1 via hepatocyte growth factor (HGF). We first show that CFTR anchors to apical actin cytoskeleton (via Ezrin) upon activation of Rac1 signaling through PIP5K and Arp2/3. We then found that such anchoring retains pharmacologically rescued F508del-CFTR at the cell surface, boosting functional restoration by correctors up to 30% of wild-type channel levels in human airway epithelial cells. Our findings reveal that surface anchoring and retention is a major target pathway for CF pharmacotherapy, namely, to achieve maximal restoration of F508del-CFTR in patients in combination with correctors. Moreover, this approach may also translate to other disorders caused by trafficking-deficient surface proteins.

Measurements of CFTR-mediated Cl- secretion in human rectal biopsies constitute a robust biomarker for Cystic Fibrosis diagnosis and prognosis.

BACKGROUND: Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl(-)) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. METHODOLOGY/PRINCIPAL FINDINGS: To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl(-) secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n=51), individuals with clinical CF suspicion (n=49) and age-matched non-CF controls (n=18). Conclusive measurements were obtained for 96% of cases. Patients with "Classic CF", presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl(-) secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl(-) secretion (10-57%) and non-CF controls show CFTR-mediated Cl(-) secretion ≥ 30-35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in "CF suspicion" individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl(-) secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. CONCLUSIONS/SIGNIFICANCE: Determination of CFTR-mediated Cl(-) secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.

Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

Folding and rescue of a cystic fibrosis transmembrane conductance regulator trafficking mutant identified using human-murine chimeric proteins.

Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys(584) interacts with residue Leu(581) in human CFTR Glu(584) interacts with Phe(581) in mouse CFTR. Introduction of the murine residue (Phe(581)) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.