Abstract INTRODUCTION Immunity in cancer patients is modified both by the cancer itself and by oncospecific treatments. Whether a patient’s adaptive immunity is impaired depends on their levels of naive lymphocytes and other cell populations. During the COVID-19 pandemic, cancer patients are at greater risk of progressing to severe forms of the disease and have higher mortality rates than individuals without cancer, particularly while they are receiving cancer-specific therapies. An individual’s protection against infection, their response to vaccines, and even the tests that determine the humoral immune response to SARS-CoV-2, depend on lymphocyte populations, meriting their study. OBJECTIVE Estimate blood concentrations of lymphocytes involved in the immune response to new pathogens in cancer patients. METHODS We carried out an analytical study of 218 cancer patients; 124 women and 94 men, 26–93 years of age, who were treated at the National Oncology and Radiobiology Institute in Havana, Cuba, March–June, 2020. Patients were divided into five groups: (1) those with controlled disease who were not undergoing cancer-specific treatment; (2) those undergoing debulking surgery; (3) patients undergoing chemotherapy; (4) patients undergoing radiation therapy and (5) patients currently battling infection. We evaluated the following peripheral blood lymphocyte subsets via flow cytometry: B lymphocytes (total, naive, transitional, memory, plasmablasts and plasma cells); T lymphocytes (total, helper, cytotoxic and their respective naive, activated, central memory and effector memory subsets); and total, secretory and cytotoxic natural killer cells and T natural killer cells. We also estimated neutrophil/lymphocyte ratios. Lymphocyte concentrations were associated with controlled disease and standard cancer therapy. For variables that did not fall within a normal distribution, ranges were set by medians and 2.5–97.5 percentiles. The two-tailed Mann–Whitney U test was used to measure the effect of sex and to compare lymphocyte populations. We calculated odds ratios to estimate lymphopenia risk. RESULTS All cancer patients had lower values of naive helper and cytotoxic T lymphocyte populations, naive B lymphocytes, and natural killer cells than normal reference medians. Naive helper T cells were the most affected subpopulation. Memory B cells, plasmablasts, plasma cells, activated T helper cells, and cytotoxic central memory T cells were increased. Patients undergoing treatment had lower levels of naive lymphocytes than untreated patients, particularly during radiation therapy. The risk of B lymphopenia was higher in patients in treatment. The odds ratio for B lymphopenia was 8.0 in patients who underwent surgery, 12.9 in those undergoing chemotherapy, and 13.9 in patients in radiotherapy. CONCLUSIONS Cancer and conventional cancer therapies significantly affect peripheral blood B lymphocyte levels, particularly transitional T helper lymphocytes, reducing the immune system’s ability to trigger primary immune responses against new antigens.

ABSTRACT Introduction: Lymphocyte immunophenotype changes in non-Hodgkin lymphoma patients are associated with prognosis and therapeutic responses. However, its association with the disease has not been systematically established. Therefor its contribution to the diagnosis process. Objective: To assess the association of lymphocyte immunophenotype in peripheral blood with the presence of non-Hodgkin lymphoma. Methods: 31 peripheral blood samples were analyzed from patients with a confirmed diagnosis of non-Hodgkin lymphoma and from 68 healthy individuals as controls, during the period 2018 to 2020. Multiparametric flow cytometry was used for immunophenotyping. The area under the curve and the Youden index were calculated to establish cut-off points in lymphocyte percentages. The association of immunophenotypic changes with non-Hodgkin's lymphoma was made using Odd ratio calculations. Results: The increase in TCD8+ and NKCD56dim lymphocytes from peripheral blood was significantly associated with the presence of non-Hodgkin lymphoma (OR= 3.4 and 2.9, respectively). Oppositely, the decrease in TCD4+, double positive T, double negative T and NKCD56bright lymphocytes was associated with the existence of non-Hodgkin lymphoma (OR= 23.0, 10.7, 6.9 and 15.8, respectively). Therefore, the decrease in the CD4/CD8 rate was also associated with the disease. Conclusion: The changes found in these lymphocytic immunophenotypes were significantly associated with the presence of non-Hodgkin lymphoma, which represents a systemic expression of the disease and suggests its diagnostic value.

RESUMEN Introducción: Los cambios en el inmunofenotipo de los linfocitos en los pacientes con linfoma no Hodgkin están asociados con el pronóstico y las respuestas terapéuticas. Sin embargo, no se ha establecido sistemáticamente la asociación con la enfermedad y por tanto su contribución al diagnóstico. Objetivo: Evaluar la asociación del inmunofenotipo linfocitario en sangre periférica con la presencia del linfoma no Hodgkin. Métodos: Se analizaron 31 muestras de sangre periférica de pacientes con diagnóstico confirmado de linfoma no Hodgkin y de 68 individuos sanos como controles, durante el período de 2018 a 2020. Se empleó la citometría de flujo multiparamétrica para el inmunofenotipado. Se calculó el área bajo la curva y el índice de Youden para establecer puntos de corte en los porcentajes linfocitarios. La asociación de los cambios inmunofenotípicos con el linfoma no Hodgkin, se realizó mediante cálculos de Odd ratio. Resultados: El aumento de linfocitos TCD8+ y NKCD56opaco se asoció significativamente con la presencia de linfoma no Hodgkin (OR= 3,4 y 2,9; respectivamente). Por el contrario, la disminución de linfocitos TCD4+, T doble positivo, T doble negativo y NKCD56brillante también se asoció con la existencia de linfoma no Hodgkin (OR= 23,0; 10,7; 6,9 y 15,8; respectivamente). Además, la disminución del índice CD4/CD8 también fue asociada con la enfermedad. Conclusiones: Los cambios encontrados en los inmunofenotipos linfocitarios se asociaron de forma significativa con la presencia del linfoma no Hodgkin, lo cual representa una expresión sistémica de la enfermedad y sugiere su valor diagnóstico.

ABSTRACT INTRODUCTION Double-negative T lymphocytes act as immunomodulators in immune response. This subpopulation is rare in blood but important in the immunopathogenesis of autoimmune diseases, viral infections, cancer and transplant rejection. These disorders have been studied in Cuba using flow cytometry, but normal values of these cells have not yet been established. OBJECTIVE Estimate preliminary reference values for double-negative T lymphocytes according to sex and age in Cuban adults. METHODS A cross-sectional study was carried out in a population of 182 healthy adult residents of Havana: 93 women and 89 men aged 18–80 years with no chronic diseases, toxic habits (smoking, excessive alcohol or caffeine intake) or medications that might alter quantity or functioning of immune-system cells. Peripheral blood was drawn to determine immunophenotype with monoclonal antibodies. The phenotype of double-negative T lymphocytes was quantified as CD45+/CD3+/CD4-/CD8-/CD56- using a Gallios flow cytometer (Beckman-Coulter, France). Medians and ranges (to the 5th and 95th percentiles) were calculated for sex and age, for both percentages and absolute values. To evaluate the effects of sex and age, both variables as well as their interaction were included in a linear model. RESULTS Respective median and range values were total percentage values 3.4 (1.6–7.4) and total absolute values (cells/μL) 57.5 (23.0–157.0). The effect of age on lymphocyte values (percentage and absolute) was significant, with lower numbers in the 51–80 years' age group (p <0.001). Percentage values according to age group were: 18–25 years, 3.8 (2.2–7.4); 26–50 years, 3.7 (1.7–8.7); and 51–80 years 2.6 (1.3–6.6). Absolute values by age group were: 18–25 years, 90 (32.6–163.7); 26–50 years, 65 (28.8–184.0); and 51–80 years 38.5 (17.9–90.1). Desegregating data by sex and age: percentage of women aged 18–25 years 5.2 (2.1–7.8), 26–50 years 4.0 (1.8–7.7), and 51–80 years 2.5 (1.3–5.8); percentage of men aged 18–25 years 3.4 (2.3–7.3), 26–50 years 3.8 (1.5–8.7), and 51–80 years 2.6 (1.2–7.3). Absolute values: women aged 18–25 years 112.0 (40.0–153.1), 26–50 years 67.0 (26.7–138.3), and 51–80 years 40 (18.6–92.0); and men aged 18–25 years 71.5 (32.1–166.7), 26–50 years 61.5 (29.9–188.7), and 51–80 years 36 (13.5–81.7). The low sex*age interaction confirms these differences occur in both men and women. Values decrease with age, with a more abrupt fall starting at 50 years. CONCLUSIONS Estimated reference values were determined for absolute values and relative proportions of double-negative T lymphocytes in healthy Cuban adults according to sex and age. Age was found to have a significant effect.

ABSTRACT Introduction: Flow cytometry allows quantification of lymphocyte subpopulations with high sensitivity, specificity and objectivity. These advantages are only achieved through the hardworking process of individualized and controlled design for each experiment. Objective: To design a flow cytometry protocol of a single polychromatic tube for peripheral lymphocyte immunophenotype. Methods: An experimental in vitro study was carried out, in March 2019, with peripheral blood samples obtained from three healthy volunteers, at the National Center for Medical Genetics. The tube was made up of six lineage markers for identifying natural B and T lymphocytes, natural killers and natural killer T cells. A protocol was developed for red blood cell lysis without washing. Fluorochrome-conjugated monoclonal antibodies were used. The optimal point of concentration corresponded to the highest staining index and preservation of the positivity percentages of each population. Progressive tube construction was performed and a logical window sequence strategy was proposed for data analysis. Results: The chosen markers allowed to carry out correct peripheral lymphocyte immunophenotype. Good discriminations between positive and negative signals were observed at the five titration points, except for anti-CD56, which presented a decreasing trend in the staining index. The total volume of conjugates required for determination of the six antigens was 3.75 μL per tube. Conclusions: A polychromatic tube was obtained that allows to carry out peripheral immunophenotype quickly and precisely by six lymphocyte antigens simultaneously, with the use of small volumes of conjugate and blood.

RESUMEN Introducción: La citometría de flujo permite la cuantificación de las subpoblaciones de linfocitos con una elevada sensibilidad, especificidad y objetividad. Estas ventajas solo se logran con un proceso laborioso de diseño individualizado y controlado para cada experimento. Objetivo: Diseñar un protocolo de un solo tubo policromático de citometría flujo para inmunofenotipo linfocitario periférico. Métodos: Se realizó un estudio experimental in vitro con muestras de sangre periférica obtenidas de tres voluntarios sanos, en el Centro Nacional de Genética Médica, en marzo de 2019. El tubo se compuso de seis marcadores de linaje para identificar linfocitos B, T, natural killer y natural killer T. Se desarrolló un protocolo de lisis de hematíes sin lavado. Se emplearon anticuerpos monoclonales conjugados con fluorocromos. El punto óptimo de concentración correspondió al mayor índice de tinción y conservación de los porcentajes de positividad de cada población. Se realizó la construcción progresiva del tubo y se propuso una estrategia lógica de secuencia de ventanas para el análisis de datos. Resultados: Los marcadores seleccionados permitieron realizar correctamente el inmunofenotipo linfocitario periférico. En los cinco puntos de titulación se observaron buenas discriminaciones entre las señales positivas y negativas, excepto para el anti-CD56 que presentó una tendencia decreciente del índice de tinción. El volumen total de conjugados requeridos para la determinación de los 6 antígenos fue de 3,75 μL por tubo. Conclusiones: Se obtuvo un tubo policromático que permite el inmunofenotipo periférico de forma rápida y precisa por seis antígenos linfocitarios simultáneamente, con el empleo de pequeños volúmenes de conjugado y sangre.

Abstract The outbreak of coronavirus pneumonia in Wuhan, China, became a pandemic on March 11, 2020. It has caused almost 4 million confirmed cases worldwide, with more than 270,000 deaths. Coronavirus is an enveloped RNA virus of the β-coronavirus genus distributed in birds, humans, and other mammals. The World Health Organization has named the new disease COVID-19. The scientific community is looking for evidence that can lead to a better understanding of the infection and the immune response (IR), prognostic and therapeutic predictors, effective treatments and vaccines. The objective of this review was to compile updated scientific evidence of the IR to COVID-19, in order to guide professionals with solutions that have a clinical impact. The most important elements involve innate immunity with failures in the interferon system in the early stages of the infection and a sustained increase in proinflammatory interleukins. This can end in a potentially fatal cytokine storm. The infiltration of neutrophils and macrophages at the alveolar level, accompanied by neutrophilia, is very characteristic. Lymphopenia is evident at the adaptive immunity level, that, depending on the degree, can indicate the severity of the disease. Understanding the temporal sequence of the IR is crucial for choosing the appropriate and effective therapies, especially when selecting which type of anti-inflammatory drugs can be used and the frequency of the dosage. Due to the fact that it is difficult to determine when they will be clearly beneficial, not harmful to the IR and not too late, due to the irreversibility of the process.

Resumen El brote de neumonía por coronavirus en Wuhan, China, devino en pandemia el 11 de marzo del 2020. Ha provocado casi 4 millones de casos confirmados en todo el mundo, con más de 270 mil muertes. El coronavirus es un virus ARN envuelto del género β-coronavirus, distribuido en aves, humanos y otros mamíferos. La Organización Mundial de la Salud denominó a la nueva enfermedad COVID-19. La comunidad científica se ha volcado a la búsqueda de evidencias que permitan comprender mejor la infección y la respuesta inmunitaria (RI), para identificar predictores pronósticos y terapéuticos, así como tratamientos y vacunas eficaces. La presente revisión tuvo como objetivo la compilación actualizada de evidencias científicas de las características de la respuesta inmune en la COVID-19, y así orientar a los profesionales en su mejor comprensión y en algunas soluciones de impacto clínico. Los elementos más importantes involucran a la inmunidad innata, con fallos en el sistema de los interferones en etapas iniciales de la infección y el incremento sostenido de interleucinas proinflamatorias. Esto puede terminar en una tormenta de citocinas potencialmente fatal. El infiltrado de neutrófilos y macrófagos a nivel alveolar, acompañado de neutrofilia, es característico. En el área de la inmunidad adaptativa se evidencia una linfopenia que según el grado puede indicar la severidad de la enfermedad. Comprender la secuencia temporal de la RI permite elegir las terapias oportunas y eficaces, sobre todo al seleccionar los antiinflamatorios y el momento de su aplicación. Dado que resulta difícil determinar cuándo serán netamente beneficiosos, que no perjudiquen la RI y que no sea demasiado tarde, por la irreversibilidad del proceso.

ABSTRACT Introduction: Determination of lymphocytic immunophenotypes in peripheral blood is part of the evaluation of the general state of the immune system. These tests provide information about the distribution, concentration, and functionality of immune cells, which helps establish prognoses in cancer and predictions of therapeutic responses. Objective: To evaluate the distribution of circulating lymphocyte concentrations in peripheral blood of cancer patients. Methods: An analytical study was carried out with 154 cancer patients treated at the Institute of Oncology and Radiobiology in Havana, from 2017 to 2019. Multiparametric flow cytometry was used to identify lymphocyte immunophenotypes. This procedure was performed before beginning any immunotherapeutic treatment. Results: Cancer patients showed greater heterogeneity in the distribution of lymphocyte populations compared to control patients. The median for total lymphocytes and the lymphocyte subpopulations of CD3+, CD4+, CD8+ and CD19+ were significantly lower in patients. CD4+ CD8+ double-positive T lymphocytes were found to be significantly elevated. No sex differences were found. Age was negatively associated with the concentrations of T-cells populations in solid tumors, and with T- and B-cells populations in lymphomas. In prostate cancer, the lowest values of lymphocyte populations were obtained. Conclusions: Cancer patients have a lower concentration of lymphocytes in peripheral blood than healthy patients in the control group. The most affected ones were the T-cells subpopulations and B lymphocytes. Age was negatively associated with blood levels of lymphocytes, which could be related to immunosenescence.

RESUMEN Introducción: La determinación de los inmunofenotipos linfocitarios en sangre periférica forma parte de la evaluación del estado general del sistema inmunitario. Estos exámenes ofrecen informaciones sobre la distribución, concentración y funcionabilidad de las células inmunitarias, lo cual contribuye a establecer pronósticos en el cáncer y predicciones a las respuestas terapéuticas. Objetivo: Evaluar la distribución de las concentraciones linfocitarias circulantes en sangre periférica de pacientes con cáncer. Métodos: Se realizó un estudio analítico en 154 pacientes con cáncer, atendidos en el Instituto de Oncología y Radiobiología de La Habana, durante los años 2017 a 2019. Se empleó la citometría de flujo multiparamétrica para identificar los inmunofenotipos linfocitarios. Este procedimiento se realizó antes de comenzar cualquier tratamiento inmunoterapéutico. Resultados: Los pacientes con cáncer mostraron mayor heterogeneidad en la distribución de las poblaciones linfocitarias respecto a los controles. En los pacientes la mediana de los linfocitos totales y de las subpoblaciones linfocitarias CD3+, CD4+, CD8+ y CD19+ fueron significativamente menores. Los linfocitos T dobles positivos (CD4/CD8) se encontraron elevados significativamente. No se hallaron diferencias entre sexos. La edad se asoció negativamente con las concentraciones de las poblaciones T en tumores sólidos, y con T y B en los linfomas. En el cáncer de próstata se obtuvieron los valores más bajos de poblaciones linfocitarias. Conclusiones: Los pacientes con cáncer tienen menor concentración de linfocitos en sangre periférica que los controles sanos. Las células más afectadas fueron las subpoblaciones T y los linfocitos B. La edad se asoció negativamente con las concentraciones sanguíneas de linfocitos, lo cual pudiera estar en relación con la inmunosenescencia.

ABSTRACT Introduction: Epithelial ovarian cancer occupies the 6th place in incidence and mortality in women worldwide. In Cuba, it occupies the 5th place in incidence in females. This cancer is immunogenic and its malignant cells grow in interaction with multiple cells from immune system. Its clinical course depends largely on the type of inflammatory infiltrate accompanying the tumor. Cytology and histopathology are gold standard as diagnostic methods. However, flow cytometry emerges as a technology with greater sensitivity, objectivity and speed. Objective: To design a multicolored flow cytometry panel to immunophenotype the lymphocytic infiltrate of three types of samples for patients with ovarian cancer. Methods: An experimental design was carried out in vitro for the creation and evaluation of a multicolored flow cytometry panel in the Immunology laboratory of the National Institute of Oncology and Radiobiology of Cuba. The panel was designed in the blood of three healthy subjects; then it was optimized for blood in 33 healthy volunteers and blood, ascites and ovarian tumor tissue, from three patients with epithelial ovarian cancer. Several lymphocytes lineages were immunophenotypedin each sample. Results: Eleven markers were selected for the immunophenotype and the panel was made up of four multiparameter cytometry tubes. The methodology created could be applied to the samples of ascites and tumor tissue without interferences and percentages of different lymphocyte subpopulations were obtained within the expected values. Conclusions: The designed panel allowed immunophenotyping of lymphocytes in different types of ovarian cancer patient samples and reliable and reproducible results were obtained. This methodology could be employed for others diseases.

RESUMEN Introducción: El cáncer epitelial de ovario (CEO) ocupa el sexto lugar en incidencia y mortalidad a nivel mundial yen Cuba, el quinto en incidencia. Este cáncer es inmunogénicoy sus células malignas crecen en interacción conlas células inmunitarias. Su curso clínico depende del infiltrado inflamatorio acompañante del tumor. La citología e histopatología son los métodos diagnóstico de elección. Sin embargo, la citometría de flujo emerge como una tecnología de mayor sensibilidad, objetividad y rapidez. Objetivo: Diseñar un panel multicolor de citometría de flujo para inmunofenotipar el infiltrado linfocitario de tres tipos de muestras de pacientes con CEO. Métodos: Se realizó un diseño experimental, para la creación y evaluación de un panel multicolor de citometríade flujo, en el laboratorio de Inmunología del Instituto Nacional de Oncología y Radiobiología. El panel se diseñó en sangre de 3 sujetos sanos y se optimizó para sangreperiférica en 33 sujetos sanos y, en sangreperiférica, ascitis y tejido tumoral ovárico de tres pacientes con CEO. En cada muestra se inmunofenotiparonvarias poblaciones linfocitarias. Resultados: Se seleccionaron 11 marcadores antigénicospara el inmunofenotipo, el panel quedó conformado por 4 tubos de citometría. La metodología se pudo aplicar a las muestras de ascitis y tejido tumoral sin interferencias, se obtuvieron porcentajes de lassubpoblaciones linfocitarias dentro de los valores esperados. Conclusiones: El paneldiseñado permitió inmunofenotipar linfocitos en distintos tipos de muestras de pacientes con CEO, con resultados confiables y reproducibles. Esta metodología puede extenderse a la realización de inmunofenotipaje en otras enfermedades.

ABSTRACT Introduction: When non-Hodgkin lymphomas infiltrate the central nervous system increases mortality. The diagnosis of this infiltration can be made by the study of cerebrospinal fluid using flow cytometry, with a higher sensitivity than conventional cytology. Objective: To estimate the relationship between the cellular immunophenotype of the cerebrospinal fluid and the overall survival of patients with non-Hodgkin lymphoma and neurological symptoms. Methods: An analytical and prospective study was conducted in 15 patients with confirmed diagnosis of non-Hodgkin lymphoma and neurological symptoms, with negative cytology of the cerebrospinal fluid. Patients cared at Oncology Department of the National Institute of Oncology and Radiobiology, during the years 2017-2018. The immunophenotype was characterized by multiparametric flow cytometry. Results: 60.0% of the patients was female and 53.4% older than 60 years. There was an overall mortality of 26.7%. 17 immunophenotypes were found, 58.9% of them was normal, 23.4% reactive and 17.7% suspected of malignancy. Overall survival advantage was obtained in patients with cerebrospinal fluid with normal immunophenotype (HR 0.04). Conclusions: Flow cytometry could discriminate cells suspected of malignancy, in patients whose cytologies were negative. The presence in the cerebrospinal fluid of atypical cells, pleocytosis and a high lymphocyte-monocyte index were associated with a lower overall survival.

RESUMEN Introducción: Los linfomas no-Hodgkin pueden infiltrar el sistema nervioso central y producir síntomas neurológicos, lo cual incrementa la mortalidad. El diagnóstico de esta infiltración se puede realizar mediante el estudio del líquido cefalorraquídeo por la técnica de citometría de flujo, con una mayor sensibilidad que la citología convencional. Objetivo: Estimar la supervivencia global de pacientes con Linfoma no-Hodgkin y síntomas neurológicos según el inmunofenotipo celular del líquido cefalorraquídeo. Métodos: Se realizó un estudio analítico y prospectivo en 15 pacientes con diagnóstico confirmado de linfoma no-Hodgkin y síntomas neurológicos, con citología negativa del líquido cefalorraquídeo, tratados en el servicio de oncología del Instituto Nacional de Oncología y Radiobiología, durante los años 2017 y 2018. El inmunofenotipo fue caracterizado mediante citometría de flujo multiparamétrica. Resultados: El 60,0 % de los pacientes fue del sexo femenino y el 53,4 % mayor de 60 años. Hubo una mortalidad del 26,7 %. Se realizaron 17 inmunofenotipos, el 58,9 % fue normal, el 23,4 % reactivo y el 17,7 % sospechoso de malignidad. La supervivencia global fue mayor en pacientes con líquido cefalorraquídeo con inmunofenotipo normal (HR. 0.04). Conclusiones: La citometría de flujo pudo discriminar células sospechosas de malignidad, en pacientes cuyas citologías fueron negativas. La presencia en el líquido cefalorraquídeo de células atípicas, de pleocitosis y de un índice de linfocito-monocito alto se asoció con una supervivencia global menor.

ABSTRACT INTRODUCTION Quantification of lymphocyte subpopulations is useful for evaluating immune response in states of health and disease, including immunodeficiencies, autoimmunity, infections and cancer. Studies have found that concentrations and proportions of different cell subpopulations vary with geographic location, age, sex and ethnicity. Knowing the normal values of these cells and their variation in healthy populations will contribute to improved clinical practice and scientific research. OBJECTIVE Estimate normal absolute concentrations and percentages of the most abundant lymphocyte subpopulations in peripheral blood and their relation to sex and age. METHODS A cross-sectional analysis was conducted in 129 healthy adults, 61 men and 68 women aged 18–80 years; 89 aged <50 years and 40 ≥50 years. We included individuals who agreed to participate by written informed consent. Exclusion criteria were chronic disease, or use of tobacco, alcohol or medications that can alter immune system cell numbers and functions. Through dual platform flow cytometry, we determined absolute and percentage values for T lymphocyte subsets CD3+, CD3+/CD4+T, CD3+/CD8+T, CD19+ B cells and CD3-/CD56+ natural killer cells in peripheral blood, using an 8-color flow cytometer. We estimated medians and the 2.5 and 97.5 percentiles and calculated the Pearson correlation coefficient to evaluate associations. Significance tests were also used to compare groups. The significance threshold was p = 0.05 in all cases. RESULTS Ranges of absolute values and percentages (%) were: total lymphocytes: 1200–3475 cells/μL (20.2–49.3); CD3+ T cells: 880–2623 cells/μL (56.5–84.7); CD3+/CD4+ T cells: 479–1792 cells/μL (30.3–55.7); CD3+/CD8+ T cells: 248–1101 cells/μL (13.2–42.9); CD19+ B cells: 114– 1491 cells/μL (5.4–49.5); CD3-/CD56+ natural killer cells: 70–652 cells/μL (3.7–28.0); and the CD4+:CD8+ index: 0.80–3.92. Absolute numbers–– but not percentages––of lymphocytes and CD3+ T cells were higher in those <50 years (p = 0.025 and 0.020, respectively). Absolute values and relative percentages of CD3+/CD8+ and relative values of CD3+/CD4+ T cells were significantly higher in the younger subgroup (p = 0.004 and p = 0.047). Age was not associated significantly with B lymphocytes or natural killer cells. Absolute and relative values of CD3+/CD4+ T lymphocytes were significantly higher in women (p = 0.009 and 0.036, respectively). CONCLUSIONS Absolute numbers of total lymphocytes and T and CD3+/CD8+ T lymphocytes are higher in younger individuals. In percentage values, CD3+/CD4+ T lymphocytes are lower in older persons. Absolute and percentage values of CD3+/CD4+ T phenotype are higher in women. These differences justify adjusting clinical analyses to different values by age and sex.

ABSTRACT INTRODUCTION Flow cytometry allows immunophenotypic characterization of important lymphocyte subpopulations for diagnosis of diseases such as cancer, autoimmune diseases, immunodeficiencies and some infections. Normal values of rare lymphoid cells in blood, quantified by cytometry, vary among different populations; so it is indispensable to obtain normal national values that can be used in clinical practice. OBJECTIVE Characterize distribution of rare T-lymphocyte populations in peripheral blood, specifically double-positive T, natural killer T and activated T lymphocytes, as well as their relationship to sex and age. METHODS A cross-sectional study was carried out in 129 adults (68 women, 61 men) aged >18 years, without chronic diseases or unhealthy habits, who signed informed consent. Peripheral blood was collected for immunophenotyping of lymphocyte subpopulations with monoclonal antibodies specific for CD4+CD8+ double-positive T cells, CD3+CD56+ natural killer T cells, and CD3+CD25+HLA-DR+ activated T cells. An eight-color flow cytometer (Beckman Coulter Gallios) was used. The analytic strategy was modified, associating variables of interest in a single graphic, using conventional monoclonal labeling antibodies. Medians and minimum and maximum percentiles (2.5 and 97.5, respectively) were used as descriptive statistics, stratified by sex, for cell counts and percentages. A linear regression model was applied to assess age effects and a two-tailed Mann-Whitney U test for independent samples was used to assess sex differences. The significance threshold was set as p ≤0.05. RESULTS Median percentages of total lymphocytes: natural killer T cells 6.3% (1.4%–23%) in men and 4.7% (0.8%–11.3%) in women (p = 0.003); activated T cells 1.0% (0.2%–2.2%) in men and 1.2% (0.4%–3.1%) in women, without statistical significance; and double positives 0.8% (0.1%–4.2%) in men and 0.9% (0.3–5.1) in women, also without statistical significance. Median cell counts (cells/μL) were: natural killer T cells, 126 (27–580) in men and 105 (20–279) in women (p = 0.023); activated T cells: 20 (4–46) in men and 25 (7–75) in women, (p = 0.013) and double-positive T cells: 17 (2–85) in men and 21 (7–154) in women, without statistical significance. Sex influenced natural killer T cells, but age did not. CONCLUSIONS Age does not affect counts and percentages of rare T lymphocyte subpopulations in the blood of healthy Cuban adults. Sex differences found for some phenotypes suggest the need for different reference values for women and men.

ABSTRACT Paleolimnological studies require a broad understanding of the geometry and depth of the sedimentary fill of lake basins prior to coring campaigns. Seismic methods are routinely employed for the indirect characterization of lake-bottom sediments. However, the use of seismic methods might occasionally be limited due to surface conditions or poor seismic contrasts between the stratigraphic units, which lead to data quality that is too poor to provide sufficient information for the selection of drill sites. Sources of cultural noise are one of the main reasons why seismic methods fail in study areas near or within large cities, such as Mexico City. Under certain conditions, electrical and electromagnetic methods might be a suitable substitute or complement to seismic exploration, as they respond to different physical properties of the subsurface. To evaluate the applicability of such methods, here we present two recent case studies from the desiccated lakes Chalco and Xochimilco both located in urban areas within the Basin of Mexico where electrical resistivity tomography (ERT), transient electromagnetic (TEM) and magnetotelluric (MT) soundings were conducted for the characterization of lake basement and lacustrine sediments. In both examples, the results of the geophysical exploration contributed valuable stratigraphic information for the following drilling and core recovery, which highlights the potential of electrical and electromagnetic methods in paleolimnological studies.

RESUMEN Los estudios paleolimnológicos requieren de una base amplia de información sobre la geometría y la profundidad de los paquetes sedimentarios de las cuencas lacustres antes de la extracción de núcleos. Comúnmente, se emplean métodos sísmicos para la caracterización indirecta de los sedimentos acumulados en los fondos de los lagos. Sin embargo, en ocasiones el uso de métodos sísmicos puede ser limitado debido a las condiciones superficiales, contrastes sísmicos reducidos entre las unidades estratigráficas de interés o a una calidad de datos insuficiente para la selección de los sitios de perforación. Las fuentes de ruido cultural son la principal razón por la que métodos sísmicos fallan en sitios de estudio ubicados cerca de o dentro de ciudades grandes, como la Ciudad de México. Bajo ciertas condiciones, métodos eléctricos y electromagnéticos pueden sustituir o complementar la exploración sísmica, ya que responden a otras propiedades físicas del subsuelo. Para evaluar la aplicabilidad de estos métodos, se presentan dos estudios de caso realizados en los antiguos lagos de Chalco y Xochimilco, ambos ubicados en áreas urbanas dentro de la Cuenca de México, en los que se realizaron mediciones de tomografía de resistividad eléctrica (TRE), sondeos electromagnéticos por transitorios (TEM) y sondeos magnetotelúricos (MT) para caracterizar el basamento y los sedimentos lacustres. En ambos ejemplos, los resultados de la exploración geofísica brindaron información estratigráfica valiosa para la posterior perforación y recuperación de núcleos, lo cual evidencia el potencial de los métodos eléctricos y electromagnéticos para estudios paleolimnológicos.

Metformin is a widely used biguanide in the treatment of type 2 diabetes mellitus. Recent evidence indicates new uses for this old drug. Cancer is a worldwide health problem that develops more frequently in type 2 diabetic people with worse prognosis. However, it has been demonstrated that when they are treated with metformin, the incidence, complications and mortality rates are lower. For these reasons, a number of epidemiological, basic, preclinical research studies as well as clinical assays have been conducted for almost a decade in order to substantiate evidence from the protective and antitumor effect of this drug. It was found that it has some antineoplastic effects due to insulin sensitizing mechanisms and other pro-apoptotic ones that impair the cancer stem cell development and interfere in carcinogenesis. This has shown a wide range of applications of metformin in the treatment of cancer including the preventive and coadjuvant effects of the conventional treatments.

La metformina es una biguanida ampliamente usada en el tratamiento de la diabetes mellitus tipo 2. Evidencias recientes proponen nuevos usos para esta vieja droga. El cáncer, que constituye un problema de salud mundial, se desarrolla con más frecuencia y peor pronóstico en los pacientes con diabetes tipo 2. Sin embargo, se ha comprobado que cuando estos son tratados con metformina presentan menor incidencia, complicaciones y mortalidad por cáncer. Por tal motivo, durante casi una década, se llevaron a cabo numerosas investigaciones epidemiológicas, básicas, preclínicas y ensayos clínicos, para fundamentar las evidencias del efecto protector y antitumoral de este medicamento. En tal sentido, se encontró que posee efectos antineoplásicos, por mecanismos sensibilizadores de la insulina, y otros, como que son pro-apoptóticos, que alteran el desarrollo de la célula madre cancerosa e interfieren con la carcinogénesis. Esto ha abierto una amplia gama de aplicaciones de la metformina en la terapéutica del cáncer, que abarcan desde los efectos preventivos, hasta las coadyuvantes de los tratamientos estándares.

Abstract Nowadays there are two accepted theories explaining the origin and development of the cancer: Tumor Immunoediting and the inflammation associated to the cancer. In both, the participation of regulatory T cells (Treg) in the carcinogenesis, progression and metastatic processes is evident but most of the studies locate these cells in advanced stages of the immunopathogenesis of the neoplastic process. However, these cells participate in the peripheral tolerance and homeostasis of the immune system. Therefore their role is not limited to contribute in the tumor cell evasion, but rather they may participate before the first cancer cell emerges. Their most important function is probably the one involved in the equilibrium stage of the immunoediting, because in this phase the adaptive immune system is decisive for the progression into the escape stage, being able in some cases to induce a spontaneous regression of the tumor. A study in depth of the dynamics of these cells in the initial stages of the cancer can help with the identification of biomarkers for diagnosis and prognosis, as well as therapeutic targets that could change the course of cancer. In this revision an approach to the participation of the Treg in the development of cancer through inflammation and immunoediting is described.

Resumen En la actualidad existen 2 teorías inmunológicas, mayormente aceptadas, que explican el origen y desarrollo del cáncer: la inmunoedición tumoral y la inflamación asociada al cáncer. En ambas participan los linfocitos T reguladores (Treg) en el proceso de carcinogénesis, progresión y metástasis. Pero la mayoría de los estudios las ubican en etapas avanzadas dentro de la inmunopatogenia. La teoría de la inmunoedición del tumor propone 3 etapas inmunopatogénicas: eliminación, equilibrio y escape. Sin embargo, estas células participan en la tolerancia periférica y homeostasis del sistema inmunitario (SI). Por lo tanto, su papel no se limita a contribuir al escape de los tumores, sino que pueden participar aún antes de la aparición de la primera célula neoplásica. Quizá la función más importante se encuentre en la etapa de equilibrio de la inmunoedición, pues en ella el papel del SI adaptativo es decisivo para el control del paso a la etapa de escape, pudiendo en algunos casos provocar la regresión espontánea. Profundizando en los conocimientos de la dinámica que muestran estas células en las etapas iniciales del cáncer, se podrán describir biomarcadores diagnósticos y pronósticos, así como blancos terapéuticos que cambien el curso de la enfermedad. En esta revisión se hace una aproximación a la participación de las Treg en el desarrollo del cáncer a través de los procesos de la inflamación e inmunoedición.

Summary: Introduction: The magnitude of the HIV infection transcends the health problem that holds, since it is the fertile ground to generate other, emerging and reemerging diseases, of equal or greater impact. In this regard, the cancer increases its incidence, severity and mortality in HIV positive patients. This association is given in the areas of epidemiology, pathogenesis, pathophysiology, clinical and therapeutic. Methods: To update these relations are conducted an extensive literature review; apply theoretical methods such as analysis and synthesis and the historical logic. Develop: The nexus most evident in the genesis and development of cancer was the dysfunction of the immune system, using the immunodeficiency and chronic inflammation that induces HIV. The complexity of both diseases presupposes a challenge, to the hour of handle clinic and therapeutically, requiring a paradigm shift on the part of the services of health. However, not all the elements which have emerged from the review are negative, several findings in the conduct shown benefits for HIV-positive patient with cancer. Likewise, the prevention of cancer in them is possible. Conclusions: The relationship between HIV and cancer are supported epidemiologically, moreover, there is evidence of causality of HIV as conducive to the emergence of malignant neoplasms to cellular and molecular level in their pathogenesis. However not everything is negative there is evidence of mutual benefits to address these health problems.

Resumen: Introducción: La magnitud de la infección por el virus de inmunodeficiencia humana (VIH) trasciende el problema de salud que encierra, ya que constituye el terreno propicio para generar otros, emergentes y reemergentes, de igual o mayor repercusión. En tal sentido, el cáncer incrementa su incidencia, gravedad y mortalidad en los pacientes VIH positivos. Esta asociación se da en las áreas de la epidemiología, patogenia, fisiopatología, clínica y terapéutica. Métodos: Para actualizar estas relaciones se realizó una amplia revisión documental, aplicándose métodos teóricos como análisis y síntesis y el histórico lógico. Desarrollo: El nexo más evidenciado en la génesis y desarrollo del cáncer, fue la disfunción del sistema inmune, mediante la inmunodeficiencia e inflamación crónica que induce el VIH. La complejidad de ambas enfermedades presupone un desafío a la hora de manejarlas clínica y terapéuticamente, por lo que se requiere un cambio de paradigma por parte de los servicios de salud. Sin embargo, no todos los elementos surgidos de la revisión son desfavorecedores, varios hallazgos en la conducta muestran beneficios para el paciente VIH positivo con cáncer. Asimismo, la prevención del cáncer en ellos es posible. Conclusiones: Las relaciones entre el VIH y el cáncer están sustentadas epidemiológicamente, además, existen evidencias de causalidad del VIH como favorecedor de la aparición de neoplasias malignas a nivel celular y molecular en sus patogenias. Sin embargo, no todo es negativo, hay evidencias de beneficios mutuos al abordar estos problemas de salud.

Regulatory T cells (Treg) lymphocytes are a subpopulation of lymphoid cells whose function is the regulation of the immune system. These cells have suppressor functions involved in the development and monitoring of malignancies. In ovarian cancer, an increase of Treg in tumor microenvironment and systemic circulation has been shown. The objective of this paper is to update the knowledge related to immune system involvement in ovarian cancer. Document review was used, through information search engines available on Hinari. The increase of Treg lymphocytes in the tumor microenvironment and systemic level is one of the mechanisms allowing the tumor to evade the immune response. This mechanism does not have the same impact in all tumors, however, ovarian cancer itself determines the existence of the disease progress. The Treg cells inhibit effector cells by mechanisms dependent on cell-cell contact and the release of cytokines such as interleukin-10 and beta transforming growth factor. The result of this increase is helping to trigger the tolerance mechanisms to the immune system action, thereby associating with poor prognostic parameters. There is scientific evidence on the involvement of Treg lymphocytes in ovarian cancer, which have allowed the understanding the impact they have on this disease clinical features and prognosis, offering attractive new therapeutic targets that will improve this disease course.

Los linfocitos T Reguladores (Treg) son una subpoblación de células linfoides cuya función es la regulación del sistema inmune. Estas células tienen funciones supresoras que intervienen en la evolución y control de los tumores malignos. En el cáncer de ovario se ha evidenciado un incremento de las Treg en microambiente del tumor y en circulación sistémica. El objetivo de este trabajo es actualizar los conocimientos relacionados con la participación del sistema inmune en el cáncer de ovario. Se empleó la revisión documental, a través de buscadores de información disponibles en Hinari. El incremento de los linfocitos Treg, en el microambiente del tumor y a nivel sistémico, constituye uno de los mecanismos que le permiten al tumor evadir la respuesta inmune. Este mecanismo no tiene la misma repercusión en todos los tumores, sin embargo, en el cáncer de ovario su existencia sí determina el avance de la enfermedad. Las Treg inhiben a las células efectoras por mecanismos dependientes del contacto célula-célula y por la liberación de citoquinas como la interleucina 10 y factor transformador del crecimiento beta. El resultado de este incremento es que contribuye a desencadenar los mecanismos de tolerancia a la acción del sistema inmune, asociándose por ende a parámetros de mal pronóstico. Existen evidencias científicas sobre la participación de los linfocitos Treg en el cáncer de ovario, que han permitido comprender la repercusión que ejercen en las manifestaciones clínicas y pronóstico de esta enfermedad, proponiendo nuevos y atractivos blancos terapéuticos que mejorarán el curso de esta.