Flow Cytometry Applied to the Diagnosis of Primary Immunodeficiencies

Primary immunodeficiencies are the result of biological defects associated with functional immune abnormalities. It consists of a group of disorders showing a higher incidence and severity of infections, expression of immunological dysregulation such as inflammation and lymphoproliferation. The immunophenotyping and in vitro functional characterization of immunodeficient patients contribute, together with the clinical aspects, to define the underlying immune defect particularities. Flow cytometry applications in primary immunodeficiency assessment are multiple and include the study of a wide range of specific cell lymphocyte subpopulations. This chapter describes the main techniques used in the diagnosis of a wide variety of primary immunodeficiencies, in which intracellular proteins or activation markers involved in immunity are evaluated, as well as functional proliferation, cytokine production, phosphorylation of transcription factors, cytotoxic and degranulation capacity. Flow cytometry is a tool that allows rapid and accurate evaluation of multiple lymphocyte populations and immunological function, and this information is essential for the diagnosis and evaluation of patients with primary immunodeficiencies

Solid Lipid Particles for Lung Metastasis Treatment

Solid lipid particles (SLPs) can sustainably encapsulate and release therapeutic agents over long periods, modifying their biodistribution, toxicity, and side effects. To date, no studies have been reported using SLPs loaded with doxorubicin chemotherapy for the treatment of metastatic cancer. This study characterizes the effect of doxorubicin-loaded carnauba wax particles in the treatment of lung metastatic malignant melanoma in vivo. Compared with the free drug, intravenously administrated doxorubicin-loaded SLPs significantly reduce the number of pulmonary metastatic foci in mice. In vitro kinetic studies show two distinctive drug release profiles. A first chemotherapy burst-release wave occurs during the first 5 h, which accounts for approximately 30% of the entrapped drug rapidly providing therapeutic concentrations. The second wave occurs after the arrival of the particles to the final destination in the lung. This release is sustained for long periods (>40 days), providing constant levels of chemotherapy in situ that trigger the inhibition of metastatic growth. Our findings suggest that the use of chemotherapy with loaded SLPs could substantially improve the effectiveness of the drug locally, reducing side effects while improving overall survival.This research was funded by the European Regional Development Fund (ERDF) and the Spanish MINECO Refs. PI16/00496 (AES 2016), PI19/00349 (AES 2019), and DTS19/00033; IDIVAL Refs. INNVAL17/11 and INNVAL19/12. J.G. and M.B.-L. also acknowledge financial support from the Fundação para a Ciência e a Tecnologia and the ERDF through NORTE2020 (2014–2020 North Portugal Regional Operational Program) through the projects UTAP-EXPL/NTec/0038/2017 (NANOTHER) and NORTE-01-0145-FEDER-031142 (MAGTARGETON). Nano2clinics COST Action CA17140

Online Behavior of Luxury Male Grooming Products Consumer

Para analizar la credibilidad y fiabilidad que otorgan los consumidores de cosmética masculina de lujo a los sites consultados, sus motivaciones de compra y los contenidos demandados en las Webs de las marcas fabricantes, se realizó en 2014 un estudio empírico cualitativo-cuantitativo. Se constató que los consumidores consideran: a) poco creíble la información de las Webs de los fabricantes; b) más fiables las Webs de los fabricantes que las de los distribuidores para comprar; c) comprar on-line productos en promoción ya conocidos; y d) que los contenidos sobre productos son sus preferidos en las Webs de los fabricantes.To analyze, in online context, the credibility and reliability that give consumers of luxury male grooming products to sites consulted, their buying motivations, and the contents they demand in the webs of manufacturer brands, an empirical study qualitative-quantitative was conducted in 2014. It was found that consumers consider: a) little credible information of manufacturers’Web sites; (b) more reliable manufacturers’Web sites than distributors’ ones in order to buy; c) to purchase online mainly products in promotion that they have already tried; and d) that the contents about products are their favorites in the manufacturers’Webs.Depto. de MarketingFac. de Ciencias Económicas y EmpresarialesTRUEpu

Magnetic lipid nanovehicles synergize the controlled thermal release of chemotherapeutics with magnetic ablation while enabling non-invasive monitoring by MRI for melanoma theranostics

Nowadays, a number of promising strategies are being developed that aim at combining diagnostic and therapeutic capabilities into clinically effective formulations. Thus, the combination of a modified release provided by an organic encapsulation and the intrinsic physico-chemical properties from an inorganic counterpart opens new perspectives in biomedical applications. Herein, a biocompatible magnetic lipid nanocomposite vehicle was developed through an efficient, green and simple method to simultaneously incorporate magnetic nanoparticles and an anticancer drug (doxorubicin) into a natural nano-matrix. The theranostic performance of the final magnetic formulation was validated in vitro and in vivo, in melanoma tumors. The systemic administration of the proposed magnetic hybrid nanocomposite carrier enhanced anti-tumoral activity through a synergistic combination of magnetic hyperthermia effects and antimitotic therapy, together with MRI reporting capability. The application of an alternating magnetic field was found to play a dual role, (i) acting as an extra layer of control (remote, on-demand) over the chemotherapy release and (ii) inducing a local thermal ablation of tumor cells. This combination of chemotherapy with thermotherapy establishes a synergistic platform for the treatment of solid malignant tumors under lower drug dosing schemes, which may realize the dual goal of reduced systemic toxicity and enhanced anti-tumoral efficacy.Funding: This work was partially supported by NORTE 2020 (2014–2020 North Portugal Regional Operational Program), and the ERDF (European Regional Development Fund) under Grant NORTE-01-0145-FEDER-000019, by European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 686009, by “TAMs-targeted and externally controlled nanotheranostics of triple-negative-breast-cancer (Nanother)" project UTAPEXPL/NTec/0038/2017, by “Local specific treatment of triple-negativebreast-cancer through externally triggered target-less drug carriers (MagtargetON)" project NORTE-01-0145-FEDER-031142, co-funded by FCT and the ERDF through NORTE2020, and by 2014–2020 INTERREG Cooperation Programme Spain–Portugal (POCTEP) through the Project 0624_2IQBIONEURO_6_E. Co-authors also acknowledge support from Raman4clinics COST Action BM1401 and Radiomag COST action TD1402. ML-F also acknowledges the ERDF and the Spanish MINECO under project ref. PI19/00349 (AES 2019). LGH thanks the Instituto de Salud Carlos III for the Sara Borrell Grant (CD19/00035)

Expanding the clinical and genetic spectra of primary immunodeficiency-related disorders with clinical exome sequencing: expected and unexpected findings

Inmunodeficiencias primarias; Secuenciación de próxima generación; Secuenciación clínica del exomaImmunodeficiències primàries; Seqüenciació de propera generació; Seqüenciació clínica d’exomesPrimary immunodeficiencies; Next generation sequencing; Clinical exome sequencingPrimary immunodeficiencies (PIDs) refer to a clinically, immunologically, and genetically heterogeneous group of over 350 disorders affecting development or function of the immune system. The increasing use of next-generation sequencing (NGS) technology has greatly facilitated identification of genetic defects in PID patients in daily clinical practice. Several NGS approaches are available, from the unbiased whole exome sequencing (WES) to specific gene panels. Here, we report on a 3-year experience with clinical exome sequencing (CES) for genetic diagnosis of PIDs. We used the TruSight One sequencing panel, which includes 4,813 disease-associated genes, in 61 unrelated patients (pediatric and adults). The analysis was done in 2 steps: first, we focused on a virtual PID panel and then, we expanded the analysis to the remaining genes. A molecular diagnosis was achieved in 19 (31%) patients: 12 (20%) with mutations in genes included in the virtual PID panel and 7 (11%) with mutations in other genes. These latter cases provided interesting and somewhat unexpected findings that expand the clinical and genetic spectra of PID-related disorders, and are useful to consider in the differential diagnosis. We also discuss 5 patients (8%) with incomplete genotypes or variants of uncertain significance. Finally, we address the limitations of CES exemplified by 7 patients (11%) with negative results on CES who were later diagnosed by other approaches (more specific PID panels, WES, and comparative genomic hybridization array). In summary, the genetic diagnosis rate using CES was 31% (including a description of 12 novel mutations), which rose to 42% after including diagnoses achieved by later use of other techniques. The description of patients with mutations in genes not included in the PID classification illustrates the heterogeneity and complexity of PID-related disorders.This study was funded by Instituto de Salud Carlos III, grants PI14/00405 and PI17/00660, cofinanced by the European Regional Development Fund (ERDF)

First universal newborn screening program for severe combined immunodeficiency in Europe: two-years' experience in Catalonia (Spain)

Newborn screening; Severe combined immunodeficiency; T-cell receptor excision circlesCribratge de nadons; Immunodeficiència combinada greu; Cercles d'excisió de receptors de cèl·lules TCribado de recién nacidos; Inmunodeficiencia combinada grave; Círculos de escisión de receptores de células TSevere combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TRECs) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first 2 years of experience are evaluated here. All babies born between January 2017 and December 2018 were screened. TREC quantification in DBS (1.5 mm diameter) was performed with the Enlite Neonatal TREC kit from PerkinElmer (Turku, Finland). In 2018, the retest cutoff in the detection algorithm was updated based on the experience gained in the first year, and changed from 34 to 24 copies/μL. This decreased the retest rate from 3.34 to 1.4% (global retest rate, 2.4%), with a requested second sample rate of 0.23% and a positive detection rate of 0.02%. Lymphocyte phenotype (T, B, NK populations), expression of CD45RA/RO isoforms, percentage and intensity of TCR αβ and TCR γδ, presence of HLA-DR+ T lymphocytes, and in vitro lymphocyte proliferation were studied in all patients by flow cytometry. Of 130,903 newborns screened, 30 tested positive, 15 of which were male. During the study period, one patient was diagnosed with SCID: incidence, 1 in 130,903 births in Catalonia. Thirteen patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 10,069 newborns (43% of positive detections). Nine patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TRECs between 3 and 6 months of life, four infants had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months, and three patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Longer follow-up is needed to define the exact incidence of SCID in Catalonia

Common Variable Immunodeficiency and Neurodevelopmental Delay Due to a 13Mb Deletion on Chromosome 4 Including the NFKB1 Gene: A Case Report

Chromosomal rearrangements; Primary immunodeficiencies; Syndromic immunodeficienciesReordenacions cromosòmiques; Immunodeficiències primàries; Immunodeficiències sindròmiquesReordenamientos cromosómicos; Inmunodeficiencias primarias; Inmunodeficiencias sindrómicasSyndromic immunodeficiencies are a heterogeneous group of inborn errors of immunity that can affect the development of non-immune organs and systems. The genetic basis of these immunodeficiencies is highly diverse, ranging from monogenic defects to large chromosomal aberrations. Antibody deficiency is the most prevalent immunological abnormality in patients with syndromic immunodeficiencies caused by chromosomal rearrangements, and usually manifests as a common variable immunodeficiency (CVID)-like phenotype. Here we describe a patient with a complex phenotype, including neurodevelopmental delay, dysmorphic features, malformations, and CVID (hypogammaglobulinemia, reduced pre-switch and switch memory B cells, and impaired vaccine response). Microarray-based comparative genomic hybridization (aCGH) revealed a 13-Mb deletion on chromosome 4q22.2-q24 involving 53 genes, some of which were related to the developmental manifestations in our patient. Although initially none of the affected genes could be linked to his CVID phenotype, subsequent reanalysis identified NFKB1 haploinsufficiency as the cause. This study underscores the value of periodic reanalysis of unsolved genetic studies performed with high-throughput technologies (eg, next-generation sequencing and aCGH). This is important because of the ongoing incorporation of new data establishing the relationship between genes and diseases. In the present case, NFKB1 had not been associated with human disease at the time aCGH was performed. Eight years later, reanalysis of the genes included in the chromosome 4 deletion enabled us to identify NFKB1 haploinsufficiency as the genetic cause of our patient’s CVID. In the future, other genes included in the deletion may be linked to human disease, allowing us to better define the molecular basis of our patient’s complex clinical phenotype.This study was funded by Instituto de Salud Carlos III, grants PI17/00660 and PI20/00761, cofinanced by the European Regional Development Fund (ERDF). This study was also funded by the Jeffrey Modell Foundation. This work is supported by the European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases Network (ERN-RITA)

Early Diagnosis and Treatment of Purine Nucleoside Phosphorylase (PNP) Deficiency through TREC-Based Newborn Screening

Newborn screening; Severe combined immunodeficiencyCribatge nounat; Immunodeficiència combinada severaCribado neonato; Inmunodeficiencia combinada gravePurine nucleoside phosphorylase (PNP) deficiency is a rare inherited disorder, resulting in severe combined immunodeficiency. To date, PNP deficiency has been detected in newborn screening only through the use of liquid chromatography tandem mass spectrometry. We report the first case in which PNP deficiency was detected by TREC analysis.This research was funded by Jeffrey Modell Foundation

Programa educativo sobre higiene de manos en escolares primarios

Proper hand hygiene can reduce the incidence of many infectious respiratory and intestinal diseases, among others. A quasi-experimental study was carried out to implement an educational program on hand hygiene in 45 students of 4th level of the Guerrillero Heróico Elementary School, Cienfuegos, Cuba, during the months of October 2018 - December 2019. The design of a range of actions responded to the identified learning needs, producing a significant change with a value of p <0.05. In this study, the 80% had a low level of theoretical knowledge about hand hygiene before the application of the educational program and only 6.6% were rated as good. Among the reasons stated for not washing their hands, infants indicated the lack of resources for it and the insufficient perception of the risk that this behavior entails. The majority stated that they had received information on the matter at school and on television.La adecuada higiene de manos puede reducir la incidencia de muchas enfermedades infecciosas respiratorias e intestinales, entre otras. Se realizó un estudio cuasiexperimental, con el objetivo de implementar un programa educativo acerca de la higiene de manos en 45 niños de 4to grado de la Escuela Primaria Guerrillero Heróico, Cienfuegos, Cuba, durante los meses de octubre 2018 - diciembre 2019. El diseño de un conjunto de acciones respondió a las necesidades de aprendizaje identificadas, produciéndose un cambio significativo con un valor p<0,05. El 80% de los involucrados tuvo un nivel bajo de conocimientos teóricos sobre higiene de manos antes de la aplicación del programa educativo y solo un 6,6 % resultó calificado como bueno. Entre las razones declaradas por las que no se lavan las manos, los infantes señalaron la falta de recursos para eso y la insuficiente percepción del riesgo que conlleva esa conducta. La mayoría declaró que recibían información al respecto en el ambiente escolar y a través de la televisión