Diseño y optimización de un tubo policromático de citometría de flujo para inmunofenotipo linfocitario periférico / Design and optimization of a polychromatic tube of flow cytometry for peripheral lymphocyte immunophenotype

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(AU)

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(AU)

Effects of polysaccharide from Ophiopogon japonicus on immune response to Newcastle disease vaccine in chicken

In order to investigate the immune enhancement effects of Ophiopogon japonicus polysaccharide Ophiopogon japonicus (OJPS) on Newcastle disease (ND) live vaccine, chickens vaccinated against ND live vaccine was orally administered with the OJPS at high, medium and low concentrations respectively. In negative control group, chickens were given orally equal volume of physiological saline. On day 14, 21 and 28, the serum antibody titer, erythrocyte-C3b receptor rosette rate (E-C3bRR), erythrocyte-C3b immune complex rosette rate (E-ICRR) and peripheral lymphocyte proliferation were measured. The results showed that at most time points, the antibody titer, peripheral lymphocyte proliferation, E-C3bRR and elimination rate of immune complex of three OJPS administrating groups were significantly higher (P<0.05) than those in negative control group. It indicated that OJPS could significantly improve the immune efficacy of Newcastle disease live vaccine, Ophiopogon japonicus polysaccharide possessed synergistical immunoenhancement.(AU)

Overexpression of Cell Cycle Proteins of Peripheral Lymphocytes in Patients with Alzheimer's Disease

OBJECTIVE: Biological markers for Alzheimer's disease (AD) will help clinicians make objective diagnoses early during the course of dementia. Previous studies have suggested that cell cycle dysregulation begins earlier than the onset of clinical manifestations in AD. METHODS: We examined the lymphocyte expression of cell cycle proteins in AD patients, dementia controls (DC), and normal controls (NC). One-hundred seventeen subjects (36 AD, 31 DC, and 50 NC) were recruited. The cell cycle proteins CDK2, CDK4, CDK6, cyclin B, and cyclin D were measured in peripheral lymphocytes. Cell cycle protein expression in the three groups was compared after adjusting for age and sex. RESULTS: The levels of cell cycle proteins CDK2, CDK4, CDK6, cyclin B, and cyclin D were significantly higher in AD patients than in the NC subjects. The DC group manifested intermediate levels of cell cycle proteins compared with the AD patients and the NC subjects. The present study indicates that cell cycle proteins are upregulated in the peripheral lymphocytes of AD patients. CONCLUSION: Cell cycle dysregulation in peripheral lymphocytes may present a promising starting point for identifying peripheral biomarkers of AD.

Effect of Cysteamine on Human Peripheral Blood Mononuclear Cells-Chemically Injured Keratocytes Reaction

PURPOSE: To investigate the effect of cysteamine on mixed peripheral blood mononuclear cells (PBMCs)-chemically injured keratocytes reaction (mixed lymphocyte-keratocyte reaction; MLKR). METHODS: PBMC stimulation assay was performed after keratocytes were chemically injured with 0.05 N NaOH for 60 seconds. MLKR was treated with various concentrations of cysteamine (0-10 mM). Intracellular reactive oxygen species (ROS) formation was measured using the oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescein diacetate (DCF-DA). Proliferation rate of PBMCs stimulated by NaOH-treated keratocytes and secretion profiles of matrix metalloprotease-9 (MMP-9), transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), and macrophage migration inhibitory factor (MIF) were determined using the bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively. RESULTS: Proliferation rate of PMBCs was suppressed by cysteamine in a dose-dependent manner (p = 0.019). Fluorescence of DCF-DA decreased depending on cysteamine concentration (p < 0.001). MMP-9, IL-6 and TGF-beta1 levels were suppressed by cysteamine in a dose-dependent manner (p < 0.05), whereas MIF levels increased with cysteamine concentration of 0.5-10 mM (p = 0.008). CONCLUSIONS: These study results indicate that cysteamine induced the ROS-mediated inhibition of inflammatory cytokine release and proliferation of PBMCs stimulated by chemically injured keratocytes. Thus, cysteamine can be used in the treatment of chemical corneal burns.

Comparison of Radiation Adaptive Responses in Peripheral Lymphocytes of Patients Undergoing Tc-99m MDP and Tc-99m DTPA Scintigraphies / 대한핵의학회잡지

PURPOSE: The purpose of this study was to compare the radiation adaptive response (RAR) in peripheral lymphocytes (PL) of patients induced by Tc.-99m MDP and Tc-99m DTPA scintigraphies. MATERIALS AND METHODS: Lymphocytes from 45 patients (25 males, 20 females, mean age 44+/-18 years) were collected before and after scintigraphies using 740 MBq Tc-99m MDP (n=22) or Tc-99m L)TPA (n=23). Lympho-cytes from 20 controls (12 males, 8 females, mean age 43+/-7 years) were also callected. They were exposed to challenge dose of 2 Gy gamma-rays using a Cs-137 cell irradiator, Number of ring-form (R) and dicentric (D) chromosomes was counted under the light microscope. From them a representative score, Ydr, was calculated as Ydr=(D+R)/cells. Adaptation index (AI) was defined as difference of Ydr between unconditioned and conditioned lymphocytes. Ydr was also measured after an administration of cyclohexi-mide (CHM), a protein synthesis inhibitor, before challenge dose. RESULTS: RAR was induced in both groups of patients. CHM abolished the adaptive response in both groups. AI of Tc-99m MDP group was significantly higher than that of Tc-99m DTPA group. CONCLUSION: Tc-99m MDP induced RAR was more prominent than those induced by Tc-99m DTPA.

Change of Dopamine Receptor mRNA Expression in Lymphocyte from Schizophrenic Patients / 신경정신의학

OBJECTIVES: Schizophrenia, commonly developed in adolescence and young adulthood, is one of the most common mental diseases in psychiatry. The etiology or pathogenesis of this disease is uncertain yet, but the dysfunction of dopaminergic system in the brain has been proposed. However, there is no direct evidence of dysfunction of brain dopaminergic systems in schizophrenic patients because the direct assessment of brain dopaminergic systems is almost impossible at present. To overcome this problem, recently founded dopamine receptors and their mRNAs in peripheral lymphocytes was used. The purpose of this study was to define whether the dopamine receptors of peripheral lymphocytes can be the peripheral markers of schizophrenia or not. METHODS: The total numbers of subjects are 87 schizophrenic patients. Among them 44 patients were schizophrenics who had been taking antipsychotic drugs for more than 3 years(medicated patients) 43 schizophrenics who recently are not taken antipsychotic drugs for more than 3 months(drugfree patients) For controls age and sex matched 31 healthy persons were enrolled. Sequential reverse transcription and quantitative polymerase chain reaction of the mRNA were used to investigate the expression of dopamine receptors in peripheral lymphocytes. The gene expression of dopamine receptors were compared in each groups and after starting antipsychotic medicines in drug-free patients, the clinical scales and the dopamine receptors of peripheral lymphocytes were sequentially studied before, 2 weeks after and 8 weeks after medication. RESULTS: 1)In drug-free schizophrenics, D3 dopamine receptor mRNA expression of peripheral lymphocytes was significantly increased comparing with that of controls and drug-medicated schizophrenics and D5 dopamine receptor mRNA expression was increased comparing with only that of drug-medicated schizophrenics. There was no difference between controls and drug-medicated schizophrenics. 2)After antipsychotic medication, dopamine receptors of lymphocytes were increased tendency. 3)Drug-free patients were divided into two groups according to dopamine receptor expression in controls to evaluate the significance of increased dopamine receptor expression. The group of patients with increased dopamine receptor expression had following clinical characteristics. (1)More severe psychiatric symptoms (2)Poor pharmacological response of pBPRS on medicines (3)Severe extrapyramidal side effects after pharmacological treatment CONCLUSION: These results revealed that the molecular biologically determined dopamine receptors of peripheral lymphocytes were reactive, and increased expression of dopamine receptor in peripheral lymphocyte had clinical significance for subgrouping and prognostication. These findings suggested that dopamine receptors of peripheral lymphocyte may represent dopamine receptors of the brain, even the location cannot be determined.

Effect of Radiation Therapy for Urogenital Malignant Tumors on Peripheral Lymphocyte Count / 대한비뇨기과학회지

The effect of prophylactic postoperative radiotherapy on peripheral lymphocyte count was determined for 20 cases of urogenital malignant tumors (bladder cancer-13 cases, prostatic cancer-4 cases, renal cancer-3 cases). The results were obtained as follows, l) Radiotherapy decreased the white blood cell count by a mean percentage of 16.7% (14.0% for bladder cancer, 16.4% for prostatic cancer. 36.8% for renal cancer, respectively) during the period of first month, but did not alter the granulocyte count. 2) Radiotherapy brought about high degree of decrease of peripheral lymphocyte count by a mean percentage of 47.9% (47. 3% for bladder cancer. 50.0% for prostatic cancer. 32.3% for renal cancer, respectively). 3) The recovery of the radiation-induced lymphocyte deficiency to pre-irradiation level was observed in 1-2 years after completion of radiation therapy (2 years for bladder cancer. 1 1/2 years for prostatic cancer. 1 year for renal cancer. respectively).