Endothelial Progenitor Cells and NADPH Oxidase Enzyme Activity in the Development of an Aortic Aneurysm

Abstract Introduction: Endothelial progenitor cells (EPCs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme activity may affect the vessel wall and have a role in development of aortic aneurysms. EPCs originate from hematopoietic stem cells and can be enumerated from peripheral blood samples by flow cytometry. In this study, we aimed to evaluate the relation of EPC number and NADPH oxidase enzyme activity in the development of thoracic aortic aneurysm (TAA). Methods: Patients with TAA (n=30) and healthy individuals without TAA (control, n=10) were included in our study. Characterization and enumeration of EPC from peripheral blood samples were performed by flow cytometry with panels including markers of EPCs (CD34/CD133/CD309/CD146/CD144). Additionally, NADPH oxidase enzyme activity (capacity) was also measured by the dihydrorhodamine 123 (DHR 123) test. Results: The enumeration of EPC with CD34+/CD146+ marker showed that the number of mean EPC/106 cells was increased in the patient group (41.5/106 cells), but not in the control group (20.50/105 cells) (P<0.01). Additionally, patients with TAA presented significantly lower NADPH oxidase activity by DHR assay than healthy controls (mean stimulation index: 60.40± 7.86 and 75.10±5.21, respectively) (P<0.01). Conclusion: Our results showed that the number of EPCs is significantly higher in aortic aneurysm patients and may have a role in disease progression. The crosstalk between NADPH oxidase enzyme capacity and EPC number may be useful as a parameter to explain the clinical progression of TAA.

Enfermedad granulomatosa crónica: diagnóstico mediante el ensayo de dihidrorodamina / Chronic granulomatous disease: diagnosis by the dihydrorhodamine assay

Resumen: Introducción: La enfermedad granulomatosa crónica (EGC) se caracteriza por una alteración de la función oxidativa de neutrófilos, presentando herencia ligada al cromosoma X (EGC LX) y autosómica recesiva (EGC AR). El ensayo de dihidrorodamina (DHR) es utilizado para el diagnóstico y detección de portadoras, además proporciona información sobre patrones de herencia. Objetivo: Detectar casos de EGC en niños con infecciones recurrentes y evaluar a sus familiares femeninos mediante el ensayo de DHR, para identificar portadoras y obtener información acerca de posibles patrones de herencia. Pacientes y Método: Fueron incluidos 107 pacientes (<18 años de edad) con sospecha clínica de EGC como neumonías, linfadenopatías y abscesos, remitidos por médicos de hospitales públicos, del 2014 al 2017. Además, se incluyeron seis mujeres, familiares de los niños con EGC. A las muestras de los pacientes se aplicó el ensayo DHR, expresando los resultados como índice de estimulación de neutrófilos (IE). Resultados: La mediana de edad de los pacientes fue de 3 años y 62/107 fueron varones. El IE promedio fue 39,7 ± 13,8 y 101/107 niños exhibieron un cambio completo de fluorescencia de DHR. En 2/107 niños no se observó dicho cambio (IE = 1,0), lo cual indica posible EGC LX, y un tercer niño mostró un leve cambio (IE = 4,8), compatible con EGC AR. En 5/6 mujeres se encontró un patrón bimodal, indicando un estado de portadora. Conclusiones: Fueron detectados tres casos de EGC y cinco portadoras mediante el ensayo de DHR, realizado por primera vez en Paraguay. También se obtuvo información sobre los posibles patrones de herencia, EGC LX en dos familias y un caso probable de EGC AR.

Abstract: Introduction: Chronic granulomatous disease (CGD) is characterized by an alteration of the neutrophil oxidative function. Its inheritance patterns are linked to the X chromosome (X-linked CGD) and autosomal recessive (AR CGD). The dihydrorhodamine (DHR) assay is used for the diagnosis and detection of carriers and provides information on inheritance patterns. Objective: To detect CGD cases in chil dren with recurrent infections and to evaluate their female relatives through the DHR assay to iden tify carriers and obtain information about possible inheritance patterns. Patients and Method: 107 patients (<18 years of age) with clinical suspicion of CGD such as pneumonia, lymphadenopathies, and abscesses were included, referred by physicians from public hospitals between 2014 and 2017. Six female relatives of children with CGD were also included. The DHR assay was performed on all patient samples and the results were expressed as neutrophils stimulation index (SI). Results: The median age of patients was 3 years and 62/107 of them were male. The average SI was 39.7±13.8 and a complete shift of DHR was found in 101/107 children. In 2/107 children, no DHR shift was observed (SI=1.0) indicating possible X-linked CGD, and a third child showed a slight DHR shift (SI=4.8) compatible with AR CGD. 5/6 female relatives presented a bimodal pattern, showing a carrier status. Conclusions: Three cases of CGD and five female carriers were detected through the DHR assay, being the first time that this technique was used in Paraguay. Information on the most likely inheri tance patterns, two X-linked CGD, and one AR CGD case was also obtained.

Neutrophil oxidative burst as a diagnostic indicator of IgG-mediated anaphylaxis

BACKGROUND: IgG-mediated anaphylaxis occurs after infusion of certain monoclonal antibody-based therapeutics. New in vitro tests are urgently needed to diagnose such reactions. We investigated whether allergens trigger neutrophil oxidative burst (OB) and if neutrophil OB occurs due to allergen-specific IgG (sIgG). METHODS: Neutrophil OB was measured by dihydrorhodamine 123 flow cytometry using a leukocyte suspension spiked with a very small patch of the allergen crude extract, Dermatophagoides farinae (Der f). The mean fluorescence intensity ratio of stimulated to unstimulated samples was calculated as the neutrophil oxidative index (NOI). RESULTS: The Der f-specific NOI (Der f-sNOI) showed a time-dependent increase after Der f extract addition. At 15 min activation, higher Der f-sIgG levels were associated with lower Der f-sNOI values in 31 subjects (P < 0.05). This inverse relationship occurs due to the initial blocking effect of free Der f-sIgG. Additionally, neutrophil OB was nearly absent (Der f-sNOI of −1) in two cases: a subject with undetectable Der f-sIgG levels and washed leukocyte suspensions deprived of Der f-sIgG. CONCLUSION: Allergens can trigger neutrophil OB via preexisting allergen-sIgG. Neutrophil OB can be easily measured in a leukocyte suspension spiked with the allergen. This assay can be used to diagnose IgG-mediated anaphylaxis.