Splenocytes and thymocytes migration patterns between lymphoid organs in pregnancy.

Background: Cell migration is essential for the immune system and is frequently analyzed in adult non-pregnant animals but poorly explored in pregnant animals. However, a physiologic increased size in the spleen and periaortic lymph nodes had been reported in pregnant mice. Methods: Using a mouse model, we transferred PKH26-stained thymocytes and splenocytes from pregnant or non-pregnant animals to receptor mice in the presence or absence of pregnancy. Percentage of PKH-26 cells and Mean Fluorescence Intensity were calculated. Non-parametric ANOVA analysis was performed. Results: We detected that the percentage of PKH26+ thymocytes in the spleen, lymph nodes, and peripheral blood is higher in females than in males (p = 0.039). Our results showed a similar frequency of thymocytes and splenocytes from pregnant and non-pregnant mice located in receptor lymphoid organs (p > 0.05). Also, the location of marked cells was similar during the perinatal period (p > 0.05). Conclusions: The mobility of thymocytes and splenocytes in pregnant and non-pregnant mice is similar. Therefore, we suggest that the larger size of the spleen and periaortic lymph nodes noted previously in pregnant mice could result from the retention of leukocytes in the secondary lymphoid organs.

Evaluation of Ilex guayusa and Piper marginatum Extract Cytotoxicity on Human Dental Pulp Mesenchymal Stem Cells.

BACKGROUND: Amelogenesis imperfecta is a hereditary disorder affecting dental enamel. Among its phenotypes, hypocalcified AI is characterized by mineral deficiency, leading to tissue wear and, consequently, dental sensitivity. Excessive fluoride intake (through drinking water, fluoride supplements, toothpaste, or by ingesting products such as pesticides or insecticides) can lead to a condition known as dental fluorosis, which manifests as stains and teeth discoloration affecting their structure. Our recent studies have shown that extracts from Colombian native plants, Ilex guayusa and Piper marginatum, deposit mineral ions such as phosphate and orthophosphate into the dental enamel structure; however, it is unknown whether these extracts produce toxic effects on the dental pulp. OBJECTIVE: To assess cytotoxicity effects on human dental pulp stem cells (hDPSCs) exposed to extracts isolated from I. guayusa and P. marginatum and, hence, their safety for clinical use. METHODS: Raman spectroscopy, fluorescence microscopy, and flow cytometry techniques were employed. For Raman spectroscopy, hDPSCs were seeded onto nanobiochips designed to provide surface-enhanced Raman spectroscopy (SERS effect), which enhances their Raman signal by several orders of magnitude. After eight days in culture, I. guayusa and P. marginatum extracts at different concentrations (10, 50, and 100 ppm) were added. Raman measurements were performed at 0, 12, and 24 h following extract application. Fluorescence microscopy was conducted using an OLIMPUS fv1000 microscope, a live-dead assay was performed using a kit employing a BD FACS Canto TM II flow cytometer, and data analysis was determined using a FlowJo program. RESULTS: The Raman spectroscopy results showed spectra consistent with viable cells. These findings were corroborated using fluorescence microscopy and flow cytometry techniques, confirming high cellular viability. CONCLUSIONS: The analyzed extracts exhibited low cytotoxicity, suggesting that they could be safely applied on enamel for remineralization purposes. The use of nanobiochips for SERS effect improved the cell viability assessment.

In Vivo, In Vitro and In Silico Anticancer Activity of Ilama Leaves: An Edible and Medicinal Plant in Mexico.

Ilama leaves are an important source of secondary metabolites with promising anticancer properties. Cancer is a disease that affects a great number of people worldwide. This work aimed to investigate the in vivo, in vitro and in silico anticancer properties of three acyclic terpenoids (geranylgeraniol, phytol and farnesyl acetate) isolated from petroleum ether extract of ilama leaves. Their cytotoxic activity against U-937 cells was assessed using flow cytometry to determine the type of cell death and production of reactive oxygen species (ROS). Also, a morphological analysis of the lymph nodes and a molecular docking study using three proteins related with cancer as targets, namely, Bcl-2, Mcl-1 and VEGFR-2, were performed. The flow cytometry and histomorphological analysis revealed that geranylgeraniol, phytol and farnesyl acetate induced the death of U-937 cells by late apoptosis and necrosis. Geranylgeraniol and phytol induced a significant increase in ROS production. The molecular docking studies showed that geranylgeraniol had more affinity for Bcl-2 and VEGFR-2. In the case of farnesyl acetate, it showed the best affinity for Mcl-1. This study provides information that supports the anticancer potential of geranylgeraniol, phytol and farnesyl acetate as compounds for the treatment of cancer, particularly with the potential to treat non-Hodgkin's lymphoma.

Interaction Between Macrophages and Cryptococcus neoformans: Distinguishing Phagocytosed Versus External Fungi.

The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.

Repurposing discarded leukodepletion filters as a source of mononuclear cells for advanced in vitro research.

In light of advancements in the field of immuno-oncology, the demand for obtaining mononuclear cells for in vitro assays has surged. However, obtaining these cells from healthy donors remains a challenging task due to difficulties in donor recruitment and the requirement for substantial blood volumes. Here, we present a protocol for isolating peripheral blood mononuclear cells (PBMCs) from leukodepletion filters used in whole blood and erythrocytes by apheresis donations at the Hemonucleus of the Barretos Cancer Hospital, Brazil. The method involves rinsing the leukodepletion filters and subsequent centrifugation using a Ficoll-Paque concentration gradient. The isolated PBMCs were analyzed by flow cytometry, which allowed the identification of various subpopulations, including CD4+ T lymphocytes (CD45+CD4+), CD8+ T lymphocytes (CD45+CD8+), B lymphocytes (CD45+CD20+CD19+), non-classical monocytes (CD45+CD64+CD14-), classical monocytes (CD45+CD64+CD14+), and granulocytes (CD45+CD15+CD14-). In our comparative analysis of filters, we observed a higher yield of PBMCs from whole blood filters than those obtained from erythrocytes through apheresis. Additionally, fresh samples exhibited superior viability when compared to cryopreserved ones. Given this, leukodepletion filters provide a practical and cost-effective means to isolate large quantities of pure PBMCs, making it a feasible source for obtaining mononuclear cells for in vitro experiments. SUMMARY: Here, we provide a detailed protocol for the isolation of mononuclear cells from leukodepletion filters, which are routinely discarded at the Barretos Cancer Hospital's Hemonucleus.

Comparative study of two laboratory techniques for the detection of HLA-B27 in patients with axial spondyloarthritis: a cross-sectional analysis.

BACKGROUND: The diagnostic and prognostic relevance of Human Leukocyte Antigen B-27 (HLA-B27) in Axial Spondyloarthritis (AxSpA) is undeniable, with 70% of Ankylosing Spondylitis (AS) patients carrying the B27 gene, contrasted with a mere 4.35% in the general population. Flow cytometry (FC) and Polymerase Chain Reaction (PCR) have emerged as the predominant techniques for routine HLA-B27 typing. While various studies have compared these methods, none have catered to the unique characteristics of the Brazilian demographic. Therefore, this research aims to compare FC and PCR in a Brazilian cohort diagnosed with AxSpA. METHODS: An analytical cross-sectional study was undertaken involving 62 AxSpA outpatients from a Brazilian University Hospital. Both FC and PCR-SSP assays were utilized to ascertain HLA-B27 typing. The outcomes (either confirming or refuting the allele's presence) underwent rigorous scrutiny. Agreement between the methodologies was assessed using the kappa statistic. A p-value of < 0.05 was deemed statistically significant. RESULTS: Of the participants, 90.3% (n = 56) were HLA-B27 positive according to FC, while 79% (n = 49) were identified as positive using the PCR method. FC exhibited a sensitivity rate of 98% paired with a specificity of 38.5%. The Positive Predictive Value for FC stood at 85.7%, and the Negative Predictive Value was 83.5%. Consequently, the overall accuracy of the FC method was gauged at 85.5%. A kappa coefficient of κ = 0.454 was derived. CONCLUSIONS: FC demonstrated noteworthy sensitivity and satisfactory accuracy in HLA-B27 detection, albeit with a reduced specificity when contrasted with PCR-SSP. Nevertheless, given its cost-effectiveness and streamlined operation relative to PCR, FC remains a pragmatic option for preliminary screening in clinical practice, especially in low-income regions. To optimize resource allocation, we advocate for a refined algorithm that initiates by assessing the relevance of HLA-B27 typing based on Choosing Wisely recommendations. It then leans on FC, and, if results are negative yet clinical suspicion persists, advances to PCR. This approach aims to balance diagnostic accuracy and financial prudence, particularly in regions contending with escalating medical costs.

Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulata.

Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 µL of physiological crustacean (PS), while the rest received 10 µL of LPS [0.1 mg.kg-1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.

Phenotypic characteristics and differentiation potential of gingival mesenchymal stem cells in hyperglycemia ­ an ex vivo exploratory study / Características fenotípicas y potencial de diferenciación de las células madre mesenquimales gingivales en la hiperglucemia: un estudio exploratorio ex vivo

Background: The therapeutic use of gingival mesenchymal stem cells (GMSCs) as autologous cells may pose the challenge of alterations inflicted by the hyperglycemic environment. Objective: This study aims to assess the effects of hyperglycemia on the characteristics of GMSCs in diabetics. Materials and Methods: 10 patients who consented and fulfilled the criteria for inclusion and exclusion were recruited and categorized as test (HbA1c > 6.5) and control (HbA1c < 6.0). Gingival explants were obtained from gingival collar of teeth, washed, digested and cultured. The cells were subjected to microscopic observation to assess phenotype characteristics, and flow cytometry and qRT-PCR to assess differentiation potential. Stem cell markers CD90, CD73, CD105, CD34, CD45, HLA DR & HLA ABC, osteogenic differentiation markers RUNX2 & OCN, adipogenic differentiation markers PPARG2 & FABP4 and chondrogenic differentiation markers SOX9 & AGCN were evaluated. Results: Microscopic appearance of spindle shaped cells was found to be comparable in both groups. Flow cytometry results demonstrated comparable expressions with both groups, samples being positive for CD90, CD73, CD105, HLA ABC and negative for CD34, CD45 & HLA DR. There were variations in the expression of markers when assessed for differentiation potentials. Conclusions: The hyperglycemic environment did not manifest any changes in the phenotypic characteristics of GMSCs among diabetics. However, the expression of certain differentiation markers was significantly altered in the diabetic test population included. Further research is being conducted to understand the GMSCs in a hyperglycemic environment with an aim to develop strategies to optimize its clinical implications. Keywords: Gingiva; Mesenchymal stem cells; Diabetes mellitus; Cell Differentiation; Hyperglycemia; Flow cytometry.

Antededentes: El uso terapéutico de células madre mesenquimales gingivales(GMSC) como células autólogas puede plantear el desafío de las alteraciones infligidas por el entorno hiperglucémico. Objetivo: Este estudio tiene como objetivo evaluar los efectos de la hiperglucemia sobre las características de las GMSC en diabéticos. Materiales y Métodos: Se reclutaron y categorizaron 10 pacientes que dieron su consentimiento y cumplieron los criterios de inclusión y exclusión como prueba (HbA1c > 6,5) y control (HbA1c < 6,0). Los explantes gingivales se obtuvieron del cuello gingival de los dientes, se lavaron, digirieron y cultivaron. Las células se sometieron a observación microscópica para evaluar las características fenotípicas y a citometría de flujo y qRT-PCR para evaluar el potencial de diferenciación. Se evaluaron los marcadores de células madre CD90, CD73, CD105, CD34, CD45, HLA DR y HLA ABC, marcadores de diferenciación osteogénica RUNX2 y OCN, marcadores de diferenciación adipogénica PPARG2 y FABP4 y marcadores de diferenciación condrogénica SOX9 y AGCN. Resultados: Se encontró que la apariencia microscópica de las células fusiformes era comparable en ambos grupos. Los resultados de la citometría de flujo demostraron expresiones comparables en ambos grupos, siendo las muestras positivas para CD90, CD73, CD105, HLA ABC y negativas para CD34, CD45 y HLA DR. Hubo variaciones en la expresión de los marcadores cuando se evaluaron los potenciales de diferenciación. Conclusiones: El entorno hiperglucémico no manifestó ningún cambio en las características fenotípicas de las GMSC entre los diabéticos. Sin embargo, la expresión de ciertos marcadores de diferenciación se alteró significativamente en la población de prueba de diabetes incluida. Se están realizando más investigaciones para comprender las GMSC en un entorno hiperglucémico con el objetivo de desarrollar estrategias para optimizar sus implicaciones clínicas.

Protocol to evaluate the antiviral effect of FDA-approved drugs against dengue virus in Huh7 cells and AG129 mice.

Finding an effective therapy against diseases caused by flaviviruses remains a challenge. Here, we present a protocol to test Food and Drug Administration-approved drugs that inhibit host nuclear protein import, promoting a reduction of dengue infection. We describe steps for analyzing the drug effect on nuclear import inhibition of cellular and viral proteins by confocal microscopy or western blotting. We then describe procedures for measuring the antiviral drug effects on virus-infected cells by flow cytometry and testing drug efficacy in dengue-infected AG129 mice by survival assays. For complete details on the use and execution of this protocol, please refer to Palacios-Rápalo et al.1.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

A flow cytometry-based assay to measure neutralizing antibodies against SARS-CoV-2 virus.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has highlighted the need for serological assays that can accurately evaluate the neutralizing efficiency of antibodies produced during infection or induced by vaccines. However, conventional assays often require the manipulation of live viruses on a level-three biosafety (BSL3) facility, which presents practical and safety challenges. Here, we present a novel, alternative assay that measures neutralizing antibodies (NAbs) against SARS-CoV-2 in plasma using flow cytometry. This assay is based on antibody binding to the S protein and has demonstrated precision in both intra- and inter-assay measurements at a dilution of 1:50. The cut-off was determined using Receiver Operating Characteristic (ROC) analysis and the value of 36.01% has shown high sensitivity and specificity in distinguishing between pre-pandemic sera, COVID-19 patients, and vaccinated individuals. The efficiency significantly correlates with the gold standard test, PRNT. Our new assay offers a safe and efficient alternative to conventional assays for evaluating NAbs against SARS-CoV-2.

[Immunophenotyping by spectral cytometry reveals a profile of lymphopenia associated with deregulation with an increase in effector memory lymphocytes in a patient with a mutation in the ITPR3 gene]. / Inmunofenotipo por citometría espectral, revela un perfil de linfopenia asociado a desregulación con aumento de linfocitos de memoria efectora en paciente con mutación en el gen ITPR3.

BACKGROUND: Variants in intracellular calcium transport genes have been associated with syndromic immunodeficiencies with a SCID phenotype. CASE REPORT: Seven-year-old girl of non-consanguineous parents, in Cartagena-Colombia. At two months of age, he presented hematochezia and was diagnosed with alimentary proctolitis without improvement with restriction to milk, wheat and eggs, and malnutrition developed. At eight months, a colon biopsy shows chronic lymphoid hyperplasia, presenting with anemia, eosinophilia, but total and specific IgE to normal foods. After four years, the Immunology Service found her asymptomatic, nutritionally recovered and without allergic sensitization, but eosinophilia and elevated calprotectin persisted, suggesting an early-onset inflammatory bowel disease. Immunoglobulins were normal, lymphocyte populations with CD3, CD4 and CD8 lymphopenia. At six years old, she presented atopic dermatitis, still had elevated calprotectin and was lymphopenic. Immunophenotyping by spectral cytometry using Cytek®cFluor®Immunoprofiling-Kit14 showed lymphopenia and CD4/CD8 inversion. Naïve CD4+ and CD8+ T lymphocytes were decreased, while T-CD8+CD45RA-CCR7- and T-CD8+CD45RA+CCR7- effector memory populations were expanded. Effector and central memory CD4+ T-lymphocytes were also increased1 (Image 1). The exome revealed a heterozygous variant in the ITPR3 gene (carrier father), c.7571G>A, p.(Arg2524His); predictors classify it as having a potential eliminating effect. CONCLUSIONS: The clinical features and immunophenotype of this candidate variant differ from others related to intracellular calcium transport. They are functional studies necessary to validate their causality. A patient with a potentially deleted variant presents an immunophenotype with CD3 lymphopenia and persistent lymphocyte activation.

ANTECEDENTES: Las variantes en genes del transporte de calcio intracelular han sido asociadas a inmunodeficiencias sindrómicas con un fenotipo IDCG. REPORTE DE CASO: Niña de siete años, de padres no consanguíneos, en Cartagena-Colombia. A los dos meses de vida, presenta hematoquecia y se diagnostica con proctolitis alimentaria sin mejoría con restricción a leche, trigo y huevo, desarrollando desnutrición. A los ocho meses, una biopsia de colon muestra hiperplasia linfoide crónica, cursa con anemia, eosinofilia, pero IgE total y específica a alimentos normales. A los cuatro años, el Servicio de Inmunología la encuentra asintomática, recuperada nutricionalmente y sin sensibilización alérgica, pero persiste eosinofilia y calprotectina elevada, sugiriendo una enfermedad inflamatoria intestinal de inicio temprano. Las inmunoglobulinas fueron normales, poblaciones linfocitarias con linfopenia CD3, CD4 y CD8. A los seis años, presenta dermatitis atópica, sigue con calprotectina elevada y linfopénica. El inmunofenotipo por citometría espectral mediante Cytek®cFluor®Immunoprofiling-Kit14, mostró linfopenia e inversión CD4/CD8. Los linfocitos T-vírgenes CD4+ y CD8+ estaban disminuidos, en cambio las poblaciones de memoria efectora T-CD8+CD45RA-CCR7- y T-CD8+CD45RA+CCR7­ estaban expandidas. Los linfocitos T-CD4+ de memoria efectora y central, también estaban aumentados1 (Imagen 1). El exoma reveló una variante heterocigótica en el gen ITPR3 (padre portador), c.7571G>A, p.(Arg2524His); los predictores la clasifican como de potencial efecto deletéreo. CONCLUSIONES: La clínica y el inmunofenotipo de esta variante candidata difiere de otras relacionadas con el transporte del calcio intracelular. Son necesarios estudios funcionales para validar su causalidad. Una paciente con una variante potencialmente deletérea, presenta un inmunofenotipo con linfopenia CD3 y activación persistente de los linfocitos.

[Cytokine measurement using a multiplex technique with spectral flow cytometry: application in the study of the immunobiology of ascariasis]. / Medición de citoquinas mediante una técnica multiplex por citometría espectral: aplicación en el estudio de la inmunobiología de la ascariasis.

OBJECTIVE: To quantify the production of Th1/Th2/Th17 cytokines induced by Ascaris lumbricoides antigens in peripheral blood mononuclear cells (PBMCs) using a multiplex technique. METHODS: PBMCs were cultured from individuals with mild A. lumbricoides infection (n = 20) and uninfected individuals (n = 21) and stimulated with A. lumbricoides extract (ExtAscaris), a mix of anti-CD2/CD3/CD28 (CDmix) as a positive control, and only medium (negative control). Cytokines in the supernatants were measured using the BD™ Cytometric Bead Array Human Th1/Th2/Th17 kit, to identify IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2, and IL-17A. Readings were taken on a spectral cytometer (Northern Lights, Cytek, USA), and analysis was performed using R software with packages "tidyverse," "beadplexr," "flowCore," and "arsenal." Cytokine concentrations were calculated using a 5-parameter logistic curve. The t-test was used to compare cases and controls, and statistical significance was set at p < 0.05. The study was approved by the Ethics Committee of the University of Cartagena and the participants provided informed consent. This study was financially supported by the Colombian Sistema General de Regalías under the BPIN2020000100405 - BPIN2020000100364. RESULTS: Efficient fluorescence intensity extraction for each cytokine was achieved using detection channel R8 and the "mclust" clustering model (Figure 1). No significant differences were found in the levels of the seven cytokines between cases and controls (Figure 2). Although the IFN-γ response to ExtAscaris was higher in cases than in controls (252.5 ng/mL vs. 173.1 ng/mL), the difference was not significant. IL-17A (detection limit: 18.9 pg/mL) was more detectable in cases than controls (5 cases, 23% vs. 2 controls, 9.5%). IL-4 was only detected in the supernatants from CDmix-stimulated cultures but not with the Ascaris extract (Figure 2). CONCLUSIONS: The multiplex technique using spectral flow cytometry combined with open-source software analysis proved applicable for quantifying cytokines induced by A. lumbricoides antigens in PBMCs. However, a more sensitive method is needed to evaluate IL-4 response in the context of ascariasis. The results did not reveal significant differences in cytokine production between cases and controls for the evaluated stimuli.

OBJETIVOS: Cuantificar la producción de citoquinas Th1/Th2/Th17, inducida por antígenos de Ascaris lumbricoides en PBMCs, utilizando una técnica de multiplex. MÉTODOS: Se realizaron cultivos de PBMCs de personas con infección leve por A. lumbricoides (n = 20), y no infectadas (n = 21), y se estimularon con extracto de A. lumbricoides (ExtAscaris), un mix de anti-CD2/CD3/CD28 (CDmix), como control positivo, y solo medio (control negativo). Las citoquinas en los sobrenadantes, se midieron usando el estuche BD™ Cytometric Bead Array Human Th1/Th2/Th17, para identificar IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2 e IL-17A. La lectura se realizó en un citómetro espectral (Northern Lights, Cytek, USA), y el análisis en software R, usando los paquetes tidyverse, beadplexr, flowCore y arsenal. Se calculó la concentración de citoquinas mediante ajuste de curva logística de cinco parámetros. Se empleó la prueba t para comparar casos y controles y una p < 0,05, se consideró como significativa. Se contó con autorización del Comité de Ética de la Universidad de Cartagena para hacer la investigación y con el consentimiento informado por parte de los participantes. Este trabajo fue financiado por el Sistema General de Regalías de Colombia, bajo el BPIN2020000100405 - BPIN2020000100364. RESULTADOS: Al utilizar el canal de detección R8 para identificar las citoquinas y el modelo de agrupamiento mclust, se extrajo eficientemente la intensidad de fluorescencia para cada citoquina (Figura 1). No se encontraron diferencias significativas en los niveles de las siete citoquinas entre casos y controles (Figura 2). Aunque la respuesta de IFN-, γ hacia ExtAscaris fue más alta en los casos de controles (252,5 ng/mL vs 173,1 ng/mL), la diferencia no fue significativa. La IL-17A (límite de detección: 18,9 pg/mL) fue más detectable en casos que en controles (cinco casos, 23% vs dos controles, 9,5%). La IL-4 solo se detectó en los sobrenadantes de cultivos estimulados con CDmix, pero no con el extracto de Ascaris (Figura 2). CONCLUSIONES: La técnica multiplex por citometría espectral, combinada con el análisis en software de licencia libre, se mostró aplicable para cuantificar citoquinas inducidas por antígenos de A. lumbricoides en PBMCs. Sin embargo, se requiere de un método más sensible para evaluar la respuesta de IL-4 en el contexto de la ascariasis. Los resultados no revelaron diferencias significativas en la producción de citoquinas entre casos y controles para los estímulos evaluados.

[Characterization of peripheral blood cellular populations in relation to Ascaris Lumbricoides infection in rural areas of Bolívar]. / Caracterización de poblaciones celulares en sangre periférica, con relación a la infección por Ascaris lumbricoides en zonas rurales de Bolívar.

OBJECTIVE: To compare the relative frequencies of immune cell populations in the peripheral blood according to A. lumbricoides infection status. METHODS: Peripheral blood samples were collected from participants infected (n = 35) and uninfected with A. lumbricoides (n=27) residing in different rural municipalities of Bolívar. Infection was diagnosed using two coprological examinations and the Kato-Katz technique. Immunophenotyping was performed using two panels of markers and staining in fresh blood. The flow cytometry reading was performed on a spectral cytometer (Northern Lights, Cytek, USA). The populations identified in the first panel (Figure 1) were T lymphocytes (CD45+ CD3+), CD4+ or CD8+, B lymphocytes (CD45+ SSClow CD3- CD19+), neutrophils (CD45+ SSChi CD3- CD16+), and eosinophils (CD45+ SSChi CD3- CD16low). Monocytes were identified in another panel (Figure 2): classical (CD14++ CD16 -), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++). Dendritic cells, including CD123 + + CD303 + (plasmacytoid), HLA-DR + + CD1c + (myeloid CD1c +), and CD14-CD141 + + (myeloid CD141 +), were also identified. The study received approval from the Ethics Committee of the University of Cartagena, and participants provided informed consent. Funding was provided by the Colombian Sistema General de Regalías under BPIN2020000100405 - BPIN2020000100364. RESULTS: No significant differences were observed in age [mean cases: 35.69 (SD: 17.7) vs. controls: 37.04 (SD: 15.6) years] or sex (cases: 62.9% vs. controls: 74.1%) (Table 1). All infections were mild, with a median of 96 eggs (IQR, 48-216). A marginally significant difference was observed only in the percentage of neutrophils (45.37% in cases vs. 54.79% in controls, p=0.041) (Figure 3). Although the frequency of eosinophils was higher in the cases (8.1% vs. 6%), this difference was not significant (p=0.138) (Figure 3). No significant differences were observed in the populations of monocytes or dendritic cells between cases and controls (Figure 4). CONCLUSION: Mild A. lumbricoides infection appears to affect the number of neutrophils in peripheral blood. The low infection intensity in the studied samples may explain the lack of a significant impact on other cellular populations.

OBJETIVO: Comparar las frecuencias relativas de poblaciones de células inmunes en sangre periférica de acuerdo con el estado de infección por A. lumbricoides. MÉTODOS: Se recolectaron muestras de sangre periférica de participantes infectados (n=35) y no infectados con A. lumbricoides (n=27), residentes en distintos municipios rurales de Bolívar. La infección se diagnosticó por dos métodos coprológicos y la técnica de Kato-Katz. El inmunofenotipo se determinó con dos baterías de marcadores y tinciones en sangre fresca. La lectura fue realizada en un citómetro espectral (Northern Lights, Cytek, USA). Las poblaciones identificadas en la primera batería (Figura 1) fueron linfocitos T (CD45+ CD3+) CD4+ o CD8+, linfocitos B (CD45+ SSClow CD3- CD19+), neutrófilos (CD45+ SSChi CD3- CD16+), y eosinófilos (CD45+ SSChi CD3- CD16low). Los monocitos se identificaron en otra batería (Figura 2): clásicos (CD14++ CD16­), intermedios (CD14++ CD16+), y no clásicos (CD14+ CD16++). También se identificaron células dendríticas, tales como: CD123++ CD303+ (plasmocitoides), HLA-DR++ CD1c+ (mieloides CD1c+), y CD14- CD141++ (mieloides CD141+). El estudio recibió la aprobación del Comité de Ética de la Universidad de Cartagena, y los participantes otorgaron su consentimiento informado. La financiación fue proporcionada por el Sistema General de Regalías de Colombia, bajo el BPIN2020000100405 - BPIN2020000100364. RESULTADOS: No se observaron diferencias significativas en edad [media = casos: 35,69 (DE: 17,7) vs controles: 37,04 (DE: 15,6 años] o sexo (casos: 62,9% vs. controles: 74,1%). Todas las infecciones fueron leves con una mediana de huevos de 96 (RIC: 48 - 216). Solo se encontró diferencia significativa marginal en el porcentaje de neutrófilos (45,37% en los casos vs 54,79% en controles, p=0,041). Si bien la frecuencia de eosinófilos fue más alta en los casos (8,1% vs. 6%), esta diferencia no alcanzó la significancia (p=0,138). No se observaron diferencias significativas en las poblaciones de monocitos o células dendríticas entre casos y controles (Figura 4). CONCLUSIÓN: La infección leve por A. lumbricoides parece afectar el número de neutrófilos en sangre periférica. Es posible que por la baja intensidad de la infección en la muestra estudiada, no se detecte un impacto importante de la misma sobre el resto de las poblaciones celulares. Palabras claves: Helmintos; Ascaris lumbricoides; Citometría de flujo; Inmunofenotipado; Neutrófilos.

Investigating the Role of SNAI1 and ZEB1 Expression in Prostate Cancer Progression and Immune Modulation of the Tumor Microenvironment.

Prostate cancer (PCa) is an immunologically cold tumor and the molecular processes that underlie this behavior are poorly understood. In this study, we investigated a primary cohort of intermediate-risk PCa (n = 51) using two NanoString profiling panels designed to study cancer progression and immune response. We identified differentially expressed genes (DEGs) and pathways associated with biochemical recurrence (BCR) and clinical risk. Confirmatory analysis was performed using the TCGA-PRAD cohort. Noteworthy DEGs included collagens such as COL1A1, COL1A2, and COL3A1. Changes in the distribution of collagens may influence the immune activity in the tumor microenvironment (TME). In addition, immune-related DEGs such as THY1, IRF5, and HLA-DRA were also identified. Enrichment analysis highlighted pathways such as those associated with angiogenesis, TGF-beta, UV response, and EMT. Among the 39 significant DEGs, 11 (28%) were identified as EMT target genes for ZEB1 using the Harmonizome database. Elevated ZEB1 expression correlated with reduced BCR risk. Immune landscape analysis revealed that ZEB1 was associated with increased immunosuppressive cell types in the TME, such as naïve B cells and M2 macrophages. Increased expression of both ZEB1 and SNAI1 was associated with elevated immune checkpoint expression. In the future, modulation of EMT could be beneficial for overcoming immunotherapy resistance in a cold tumor, such as PCa.

Flow cytometry protocol for GLUT4-myc detection on cell surfaces.

Insulin and muscle contraction trigger GLUT4 translocation to the plasma membrane, which increases glucose uptake by muscle cells. Insulin resistance and Type 2 diabetes are the result of impaired GLUT4 translocation. Quantifying GLUT4 translocation is essential for comprehending the intricacies of both physiological and pathophysiological processes involved in glucose metabolism. The most commonly used methods for measuring GLUT4 translocation are the ELISA-type assay and the immunofluorescence assay. While some reports suggest that flow cytometry could be useful in quantifying GLUT4 translocation, this technique is not frequently used. Much of our current understanding of the regulation of GLUT4 has been based on experiments using the rat myoblast cell line (L6 cell) which expresses GLUT4 with a myc epitope on the exofacial loop. In the present study, we use the L6-GLUT4myc cell line to develop a flow cytometry-based approach to detect GLUT4 translocation. Flow cytometry offers the advantages of both immunofluorescence and ELISA-based assays. It allows easy identification of separate cell populations in the sample, similar to immunofluorescence, while providing results based on a population-level analysis of multiple individual cells, like an ELISA-based assay. Our results demonstrate a 0.6-fold increase with insulin stimulation compared with basal conditions. Finally, flow cytometry consistently yielded results across different experiments and exhibited sensitivity under the tested conditions.

Platelet-Derived Microvesicles Contribute to the Pathophysiogenesis of Human Cutaneous Leishmaniasis: A Nano-Flow Cytometric Approach in Plasma Samples from Patients before and under Antimonial Treatment.

Cutaneous leishmaniasis is a neglected tropical disease caused, in Brazil, mainly by Leishmania braziliensis, which is a protozoan transmitted during the blood feeding of infected female sandflies. To control leishmaniasis, the participation of CD4+ Th1 cells together with macrophages, neutrophils, and other peripheral blood cells, including platelets, is necessary. These anuclear fragments, when activated, produce microvesicles (MVs) that can reach locations outside the blood, carrying molecules responsible for activating pro-inflammatory responses and antigen presentation. Using flow cytometry, this current study evaluated the frequency and concentration of platelet-derived MVs (pMVs) in plasma samples obtained from patients in the acute phase and undergoing treatment, as well as from healthy volunteers. Our results revealed a higher frequency and concentration of pMVs in the plasma of patients with acute CL when compared to all other groups studied. These results highlight the impact of pMVs in modulating the immune response of CL patients, correlating their higher concentrations and frequencies in CL-patient plasmas, with the acute inflammatory status of the disease and their reduction with beneficial results of systemic treatment with antimony. This knowledge is essential to define potential treatment protocols, as well as highlight pMVs as biomarkers for the different clinical stages of CL.

Simple protocol for measuring CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells from a minimum volume of mouse peripheral blood.

Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of myeloid origin and immature state, whose hallmark is the capacity to suppress T cells and other immune populations. In mice, the first approach to identify MDSCs relies in the measurement of their phenotypical markers: CD11b and GR-1. In addition, two main subtypes of MDSCs have been defined based on the expression of the following markers: CD11b+ Ly6G- Ly6C+ (monocytic-MDSCs, M-MDSCs) and CD11b+ Ly6G+ Ly6C+/low (polymorphonuclear-MDSCs, PMN-MDSCs). Since CD11b+ GR-1+ (Ly6C+/Ly6G+) MDSCs can increase significantly in peripheral blood during numerous acute or chronic processes, measuring alterations in the phenotypic markers CD11b and GR-1 could be important as a first step before assessing the suppressive function of the cells. In many cases it could be necessary to measure CD11b+ Gr-1+ cells from a minimum volume of peripheral blood cells without greatly affecting animal viability, since this approach would allow for further studies to be conducted on subsequent days, such as measuring parameters of the immune response or even survival in the context of the pathology under study. The following protocol describes a simple and optimized protocol for measuring the presence of CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells using 2+ channel flow cytometry, from a minimum volume of mouse peripheral blood obtained by facial vein puncture.

Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.