Detection of KCNJ5 mutations in the APA tissues and cell-free DNA with a novel Taqman-based approach

Background: Primary aldosteronism (PA) is the most common endocrine form of secondary arterial hypertension with an estimated prevalence of ~ 11.2% in patients referred to specialized centers and 4% in primary care, but as high as 50% in patients with resistant hypertension. The two main forms of PA are: aldosterone-producing adenoma (APA), surgically curable with the removal of adenoma that is able to overproduce aldosterone, and bilateral adrenocortical hyperplasia (BAH), which needs drug therapy. About ~ 30% APAs have somatic mutations in KCNJ5 gene that encodes for the potassium channel (KIR3.4), which plays a crucial role in the maintenance of the cell membrane by pumping potassium (K+) out of the cells, thereby producing a negative membrane potential. The KCNJ5 channels that contain G151R, T158A, or L168R mutations cause membrane permeability to Na+, resulting in Na+ entry, cell depolarization, constitutive aldosterone production, and cell proliferation. The standard approach used to detect the mutations in KCNJ5 gene is sequencing of DNA extracted from adrenal tissue of hypertensive patients with lateralized excess in aldosterone production. However, DNA sequencing is a time consuming and rather expensive technique not feasible in all standard laboratories. Aims of our study were 1) to develop a strategy for genotyping DNA extracted from the adrenal tissue, which exploited a TAQ-MAN based PCR technology; 2) to evaluate if such Taq-Man-base technology allows detection of KCNJ5 mutations in the adrenal tissue and cf-DNA isolated from peripheral blood. Methodologic Approach: 1. Development of a novel strategy based on Taq-man probe to detect KCNJ5 mutations 2. Development of a protocol to isolate cf-DNA from blood collected in the inferior vena cava and adrenal veins. 3. Measurement of cf-DNA concentration and assessment of its fragmentation. 4. Identification of KCNJ5 mutation in cf-DNA Results: By applying the novel technology based on Taq-man probe we correctly identified 30 mutated patients in a cohort of 50 consecutive APA patients, with no misclassification. After isolating cf-DNA from the adrenal veins and inferior cava blood, and measuring its concentration, we evaluated cf-DNA fragmentation with the integrity index in 24 samples. Integrity index < 1 suggested that cf-DNA was released from the adrenal tissue through an apoptotic mechanism. HRM analysis of cf-DNA isolated from adrenal blood allowed us to identify the KCNJ5 mutation in the APA side. Conclusions and perspectives: The novel technology based on Taq-man probe allowed detection of all mutated patients in the examined cohort of APA patients, thus proving that this strategy could be used as an alternative to DNA sequencing. The results of our study also showed the feasibility to isolate cf-DNA from small amount of blood collected from the adrenal veins, and suggested that KCNJ5 mutations may be detected in cf-DNA. However, this approach needs to be verified for a larger population to determine feasibility and accuracy. If confirmed, analysis of cf-DNA isolated from the peripheral venous blood could be helpful for an early detection of KCNJ5 mutations and therefore for the selection of PA patients to be submitted to adrenal vein sampling. Furthermore, this strategy could be also useful for detecting KCNJ5 germline mutations responsible for the rare hereditary form of hyperaldosteronism FH-3

MiR-211 is essential for adult cone photoreceptor maintenance and visual function.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that play an important role in the control of fundamental biological processes in both physiological and pathological conditions. Their function in retinal cells is just beginning to be elucidated, and a few have been found to play a role in photoreceptor maintenance and function. MiR-211 is one of the most abundant miRNAs in the developing and adult eye. However, its role in controlling vertebrate visual system development, maintenance and function so far remain incompletely unexplored. Here, by targeted inactivation in a mouse model, we identify a critical role of miR-211 in cone photoreceptor function and survival. MiR-211 knockout (-/-) mice exhibited a progressive cone dystrophy accompanied by significant alterations in visual function. Transcriptome analysis of the retina from miR-211-/- mice during cone degeneration revealed significant alteration of pathways related to cell metabolism. Collectively, this study highlights for the first time the impact of miR-211 function in the retina and significantly contributes to unravelling the role of specific miRNAs in cone photoreceptor function and survival

Role for substance p-based nociceptive signaling in progenitor cell activation and angiogenesis during ischemia in mice and in human subjects

Our data highlight the role of SP in reparative neovascularization. Nociceptive signaling may represent a novel target of regenerative medicine

GSK-3 Inhibition Modulates Metalloproteases in a Model of Lung Inflammation and Fibrosis

Idiopathic pulmonary fibrosis (IPF) is mainly characterized by aberrant extracellular matrix deposition, consequent to epithelial lung injury and myofibroblast activation, and inflammatory response. Glycogen synthase kinase 3 (GSK-3) is a serine–threonine kinase involved in several pathways, and its inhibition has been already suggested as a therapeutic strategy for IPF patients. There is evidence that GSK-3 is able to induce matrix metalloproteinase (MMP) expression and that its inhibition modulates MMP expression in the tissues. The aim of our study was to investigate the role of GSK-3 and its inhibition in the modulation of MMP-9 and -2 in an in vivo mouse model of lung fibrosis and in vitro using different cell lines exposed to pro-inflammatory or pro-fibrotic stimuli. We found that GSK-3 inhibition down-modulates gene expression and protein levels of MMP-9, MMP-2, and their inhibitors TIMP-1 and TIMP-2 in inflammatory cells harvested from bronchoalveolar lavage fluid (BALF) of mice treated with bleomycin as well as in interstitial alveolar macrophages and cuboidalized epithelial alveolar cells. To the same extent, GSK-3 inhibition blunted the increased MMP-9 and MMP-2 activity induced by pro-fibrotic stimuli in a human lung fibroblast cell line. Moreover, the αSMA protein level, a marker of fibroblast-to-myofibroblast transition involved in fibrosis, was decreased in primary fibroblasts treated with TGFβ following GSK-3 inhibition. Our results confirm the implication of GSK-3 in lung inflammation and fibrosis, suggesting that it might play its role by modulating MMP expression and activity but also pushing fibroblasts toward a myofibroblast phenotype and therefore enhancing extracellular matrix deposition. Thus, its inhibition could represent a possible therapeutic strategy

A cross-sectional study evaluating hospitalization rates for chronic limb-threatening ischemia during the COVID-19 outbreak in Campania, Italy

The expansion of coronavirus disease 2019 (COVID-19) prompted measures of disease containment by the Italian government with a national lockdown on March 9, 2020. The purpose of this study is to evaluate the rate of hospitalization and mode of in-hospital treatment of patients with chronic limb-threatening ischemia (CLTI) before and during lockdown in the Campania region of Italy. The study population includes all patients with CLTI hospitalized in Campania over a 10-week period: 5 weeks before and 5 weeks during lockdown (n = 453). Patients were treated medically and/or underwent urgent revascularization and/or major amputation of the lower extremities. Mean age was 69.2 +/- 10.6 years and 27.6% of the patients were women. During hospitalization, 21.9% of patients were treated medically, 78.1% underwent revascularization, and 17.4% required amputations. In the weeks during the lockdown, a reduced rate of hospitalization for CLTI was observed compared with the weeks before lockdown (25 vs 74/100,000 inhabitants/year; incidence rate ratio: 0.34, 95% CI 0.32-0.37). This effect persisted to the end of the study period. An increased amputation rate in the weeks during lockdown was observed (29.3% vs 13.4%; p &lt; 0.001). This study reports a reduced rate of CLTI-related hospitalization and an increased in-hospital amputation rate during lockdown in Campania. Ensuring appropriate treatment for patients with CLTI should be prioritized, even during disease containment measures due to the COVID-19 pandemic or other similar conditions

Detection of KCNJ5 mutations in the APA tissues and cell-free DNA with a novel Taqman-based approach

Background: Primary aldosteronism (PA) is the most common endocrine form of secondary arterial hypertension with an estimated prevalence of ~ 11.2% in patients referred to specialized centers and 4% in primary care, but as high as 50% in patients with resistant hypertension. The two main forms of PA are: aldosterone-producing adenoma (APA), surgically curable with the removal of adenoma that is able to overproduce aldosterone, and bilateral adrenocortical hyperplasia (BAH), which needs drug therapy. About ~ 30% APAs have somatic mutations in KCNJ5 gene that encodes for the potassium channel (KIR3.4), which plays a crucial role in the maintenance of the cell membrane by pumping potassium (K+) out of the cells, thereby producing a negative membrane potential. The KCNJ5 channels that contain G151R, T158A, or L168R mutations cause membrane permeability to Na+, resulting in Na+ entry, cell depolarization, constitutive aldosterone production, and cell proliferation. The standard approach used to detect the mutations in KCNJ5 gene is sequencing of DNA extracted from adrenal tissue of hypertensive patients with lateralized excess in aldosterone production. However, DNA sequencing is a time consuming and rather expensive technique not feasible in all standard laboratories. Aims of our study were 1) to develop a strategy for genotyping DNA extracted from the adrenal tissue, which exploited a TAQ-MAN based PCR technology; 2) to evaluate if such Taq-Man-base technology allows detection of KCNJ5 mutations in the adrenal tissue and cf-DNA isolated from peripheral blood. Methodologic Approach: 1. Development of a novel strategy based on Taq-man probe to detect KCNJ5 mutations 2. Development of a protocol to isolate cf-DNA from blood collected in the inferior vena cava and adrenal veins. 3. Measurement of cf-DNA concentration and assessment of its fragmentation. 4. Identification of KCNJ5 mutation in cf-DNA Results: By applying the novel technology based on Taq-man probe we correctly identified 30 mutated patients in a cohort of 50 consecutive APA patients, with no misclassification. After isolating cf-DNA from the adrenal veins and inferior cava blood, and measuring its concentration, we evaluated cf-DNA fragmentation with the integrity index in 24 samples. Integrity index < 1 suggested that cf-DNA was released from the adrenal tissue through an apoptotic mechanism. HRM analysis of cf-DNA isolated from adrenal blood allowed us to identify the KCNJ5 mutation in the APA side. Conclusions and perspectives: The novel technology based on Taq-man probe allowed detection of all mutated patients in the examined cohort of APA patients, thus proving that this strategy could be used as an alternative to DNA sequencing. The results of our study also showed the feasibility to isolate cf-DNA from small amount of blood collected from the adrenal veins, and suggested that KCNJ5 mutations may be detected in cf-DNA. However, this approach needs to be verified for a larger population to determine feasibility and accuracy. If confirmed, analysis of cf-DNA isolated from the peripheral venous blood could be helpful for an early detection of KCNJ5 mutations and therefore for the selection of PA patients to be submitted to adrenal vein sampling. Furthermore, this strategy could be also useful for detecting KCNJ5 germline mutations responsible for the rare hereditary form of hyperaldosteronism FH-3.Backgouund. L’Iperaldosteronismo primario (PA) è la forma endocrina più comune d’ipertensione arteriosa secondaria con una prevalenza stimata di circa il 4% nella popolazione generale e dell’11% nei pazienti che afferiscono ai centri di riferimento per l’ipertensione. Nei pazienti con ipertensione resistente la prevalenza del PA è stimata pari al 50%, mostrando che tale patologia non è così rara come ritenuto in passato. Le due forme principali di PA sono l’adenoma producente aldosterone (APA), caratterizzato da iperproduzione lateralizzata di aldosterone, e l’iperplasia surrenalica bilaterale (BAH). La distinzione tra le due forme è di cruciale importanza poiché la prima richiede terapia chirurgica, mentre la seconda terapia medica. Considerando che la rimozione dell’APA determina la correzione del quadro biochimico-clinico di PA e la cura o il miglioramento dell’ipertensione, il riconoscimento dell’APA è fondamentale per offrire una chance di guarigione dell’ipertensione o di miglioramento del controllo dei valori pressori ai pazienti che ne sono affetti. Il 40% degli APA presenta mutazioni somatiche nel gene KCNJ5 che codifica per il canale del potassio KIR 3.4. Questo canale gioca un ruolo fondamentale nel mantenimento del potenziale di membrana pompando il K+ al di fuori della cellula, provocando in tal modo un potenziale di membrana negativo. Allorquando il gene KCNJ5 contiene le mutazioni G151R, T158A o L168R il canale KIR 3.4 acquisisce capacità di condurre Na+ all’interno della cellula. Gli effetti della mutazione a livello della cellula sono depolarizzazione cronica, produzione costitutiva di aldosterone e proliferazione cellulare. L’approccio attualmente in uso per identificare tali mutazioni è il sequenziamento, secondo Sanger, del DNA estratto dal tessuto surrenalico rimosso durante surrenectomia nei pazienti con PA e iperproduzione lateralizzata di aldosterone. Il sequenziamento del DNA, tuttavia, è costosa richiede tempo e non è disponibile di routine in tutti i laboratori. Scopo generale dello studio è stato quello di sviluppare una strategia alternativa al sequenziamento che preveda l’uso delle sonde Taq-man in Real Time PCR (Q-PCR) per il rilevamento di mutazioni KCNJ5 nel tessuto surrenalico e nel DNA circolante (cell-free DNA, cf-DNA) isolato da sangue periferico. Lo sviluppo di questa metodologia potrebbe semplificare notevolmente l’identificazione delle mutazioni KCNJ5 negli APA e, infine, permetterne la detenzione nel DNA del sangue circolante. In sintesi, l’approccio metodologico include: sviluppo di una nuova strategia basata sull’utilizzo delle sonde Taq-man per rilevare le mutazioni nel gene KCNJ5. Sviluppo di un protocollo per isolare il cf-DNA da sangue delle vene surrenaliche e dalla vena cava inferiore. Misurazione della concentrazione del cf-DNA valutandone la sua frammentazione. Identificazione di mutazioni nel gene KCNJ5 a partire dal cf-DNA Risultati. Applicando la tecnologia sviluppata nel nostro laboratorio, basata sulle sonde Taq-man, sono stati identificati correttamente 30 pazienti mutati in una coorte di 50 pazienti APA consecutivi, senza errori di classificazione. Dopo aver isolato il cf-DNA dal sangue delle vene surrenaliche e dalla vena cava inferiore, e misurato la sua concentrazione, abbiamo valutato la frammentazione del cf-DNA in 24 campioni con l'indice di integrità. I bassi valori dell’indice d’integrità riscontrati nei cf-DNA isolati da sangue venoso surrenalico suggeriscono che la ghiandola surrenalica rilasci per apoptosi frammenti di DNA. L’analisi HRM dei cf-DNA isolati dal sangue delle vene surrenaliche di un paziente con un APA sinistro contenente la mutazione L168R ha permesso d’identificare correttamente la mutazione nel cf-DNA isolato dalla vena surrenalica sinistra. Conclusioni e prospettive. La tecnologia basata sulle sonde Taq-man ha permesso d’identificare, senza errori di misclassificazione, in una coorte di 50 pazienti con APA, tutti i 30 pazienti che presentano una mutazione del gene KCNJ5. Tale strategia, pertanto, potrebbe rappresentare un’alternativa alla ben più lunga e complessa tecnica basata sul sequenziamento del DNA. I risultati del nostro studio hanno anche mostrato che è possibile isolare il cf-DNA da esigue quantità di sangue raccolto dalle vene surrenaliche permettendo l’identificazione delle mutazioni KCNJ5 usando il cf-DNA tramite approccio combinato sonde Taq-man e analisi HRM. Quest’ultimo approccio, che prevede l’uso del cf-DNA richiede, tuttavia, conferma in un ampio numero di soggetti. La stessa strategia potrebbe anche essere impiegata in futuro per la rilevazione di mutazioni germinali KCNJ5 responsabili della nota forma ereditaria d’iperaldosteronismo FH-3

A new cytological approach to improve the final diagnosis of Eosinophilic Granulomatosis with Polyangiitis (EGPA)

Introduction: Eosinophilic granulomatosis with polyangiitis (EGPA) is an uncommon systemic necrotizing vasculitis that affects small to medium sized vessels and is associated with severe asthma, allergic rhinitis, nasal polyposis and blood and tissue eosinophilic infiltration. Antineutrophil cytoplasmic autoantibodies (ANCA) are present in about 40% of cases. The presence of four or more of the above described findings yields a sensitivity of 85% and a specificity of 99.7% for the final diagnosis of EGPA. The aim of the study was to develop a new diagnostic tool to support the diagnostic and prognostic work-up of EGPA patients with the possibility to evaluate extracellular mediators in nasal secretion. Methods: Nasal secretions were gained from 40 patients of which 20 EGPA, 10 suspected EGPA and 10 controls after positioning the cotton pieces in the nasal middle meatus where they were left for 10 min. Subsequently the cotton pieces were put in 5 ml of saline solution and left in ice until processing. The liquid obtained from the squeezing of the cottons was centrifuged two times at 1600 RPM for 10 min. Supernatant was frozen after first centrifugation. Trypan Blue was added to 10 \u3bcl of sample to calculate the number of cells and their vitality. Finally, 100 \u3bcl of sample with containing 1.5\u20132.00 7 105 cells were centrifuged with Cytospin at 300 RPM for 15 min. The slides were stained with May Grunwald/Giemsa and cell population analysed at microscope with objective oil immersion 40 7 of magnification. The remaining cells were frozen or cultured. Results: The mean number of cells obtained after centrifugation was 2.00\u20134.08 7 106 cells/ml for EGPA patients and 6.42\u20138.62 7 105 cells/ ml for control group and suspected EGPA. Cytologic analysis allowed us to count an increase in the percentage of eosinophils in EGPA patients with active ENT disease and in some of the patients with a suspect of EGPA, where this percentage correlated with histologic evidence of disease. Conclusions: This method seems preliminarily a reliable cytologic examination allowing fresh cells isolation and analysis of extracellular mediators. Compared to the direct slither of the mucus on the surface of the glass, cytospin significantly reduced any dye accumulation. Further studies are ongoing to support the described method, in order to confirm the cytologic diagnostic reliability to identify prognostic biomarkers of EGPA