Determining reference ranges for lymphocyte proliferation responses to phytohemagglutinin and Bacillus Calmette-Guérin in Iranian children.

PURPOSE: To establish reference ranges (RRs) for stimulation index of T cell proliferation triggered by phytohemagglutinin (PHA-SI) and Bacillus Calmette-Guérin (BCG-SI). METHODS: This study investigated data from 359 healthy children and 35 patients with cellular immunodeficiency as positive controls (2010-2021). We applied a colorimetric-based method (BrdU) to measure proliferation and determine the RRs at the 2.5th and 97.5th percentiles (95% confidence intervals). A cross-validation approach was performed. RESULTS: In healthy controls, the RRs for PHA-SI and BCG-SI ranged between 3 and 5.2 and 2.52 to 5.2, respectively. PHA-SI and BCG-SI were in Severe Combined Immunodeficiency (SCID) patients from 1.2 to 2.5 and 0 to 2, while in Mendelian susceptibility to mycobacterial diseases (MSMD) patients, 2.53 to 4.5 and 0.74 to 2.2, respectively. The thresholds' accuracy was checked for testing reference intervals with diagnostic effects. CONCLUSION: This study establishes PHA-SI and BCG-SI reference ranges to aid in diagnosing and treating congenital immunodeficiency diseases.

Phytohemagglutinin from Phaseolus vulgaris enhances the lung cancer cell chemotherapy sensitivity by changing cell membrane permeability.

Chemotherapy is still a prevalent strategy for clinical lung cancer treatment. However, the inevitable emerged drug resistance has become a great hurdle to therapeutic effect. Studies have demonstrated that the primary cause of drug resistance is a decrease in the chemotherapeutic medicine concentration. Several lectins have been confirmed to be effective as chemotherapy adjuvants, enhancing the anti-tumor effects of chemotherapy drugs. Here, we combined phytohemagglutinin (PHA), which has been reported possess anti-tumor effects, with chemotherapy drugs Cisplatin (DDP) and Adriamycin (ADM) on lung cancer cells to detect the sensitivities of PHA as a chemotherapy adjuvant. Our results demonstrated that the PHA significantly enhanced the sensitivity of lung cancer cells to DDP and ADM, and Western blot showed that PHA combined with DDP or ADM enhance cytotoxic effects by inhibiting autophagy and promoting apoptosis. More importantly, we found PHA enhanced the chemotherapeutic drugs cytotoxicity by changing the cell membrane to increase the intracellular chemotherapeutic drugs concentration. Besides, the combination of PHA and ADM increased the ADM concentration in the multidrug-resistant strain A549-R cells and achieved the drug sensitization effect. Our results suggest that PHA combined with chemotherapy can be applied in the treatment of lung cancer cells and lung cancer multidrug-resistant strains, and provide a novel strategy for clinical tumor chemotherapy and a new idea to solve the problem of drug resistance in clinical lung cancer.

Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

A modified assay for measuring thymocyte co-stimulatory activity

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays

Efecto inmunosupresor in vitro de las lipoproteínas de baja densidad / In vitro inhibition of lymphocyte proliferation by low density lipoproteins

Background: immune cells participate in the formation of atheromatous plate, however little is known about the effects of native or oxidatively modified lipoproteins on these cells. Aim: To study the effects of lipoproteins on in vitro mononuclear cell proliferation. Material and methods: peripheral blood mononuclear cells were obtained from 10 patients with type 2 diabetes mellitus (aged 52 ñ 9 years old with a disease duration of 8.2 ñ 5.7 years and a mean glycosilated hemoglobin of 9.3 ñ 2.2 percent) and 10 non diabetic healthy controls (aged 50.3 ñ 7.1 years old). These were stimulated with phytohemagglutinin (PHA) alone or in the presence of native LDLS, malondialdehyde modified LDLs or glycated LDLs. Proliferation was measured as 3H-thymidine incorporation and expressed as Stimulation Index (SI). Results: SI of patients and healthy subjects, after PHA stimulation were similar: (57.5 ñ 29.8 and 61.1 ñ 23.5) respectively LDLs did not induce proliferation in neither group. Native LDLs produced a 98 percent inhibition of PHA induced proliferation. Malondialdehyde modified and glycated LDLs caused a 50 percent inhibition. The suppressive effect was maintained when lipoproteins were incorporated to culture media 60 min prior or after PHA stimulation. Conclusions: Lipoproteins inhibit in vitro PHA induced peripheral blood mononuclear cell proliferation both in diabetic and in non diabetic subjects

Expression of low density lipoprotein receptors in lymphoblasts induced by anti-CD3 antibody in patients with hypercholesterolemia

Familial hypercholesterolemia(FH) is a disease based on defects of low-density lipoprotein receptors(LDL-R). To interrupt and control the natural course of this disease, early identification of these patients is important. The routine lipid profile tests for hypercholesterolemia can not differentiate objectively FH from secondary hypercholesterolemia. The exact diagnosis of FH heterozygotes is especially essential because it is easier to develop premature coronary heart diseases compared with secondary hyper-cholesterolemia. A simplified rapid and precise method for the mass screening of FH patients and the differentiation between FH heterozygote and secondary hyperlipidemia was needed. For the test, lymphocytes were used as target cells in LDL-R assay. After a 5 day culture with anti-CD3 Ab as a mitogen, indirect immunofluorescence stain and flow cytometric analysis were applied. The results were as follows; 74 +/- 9% of the stimulated lymphoblasts from normal controls expressed LDL-R activity. Cultured, but unstimulated, lymphocytes of normal controls showed 27 +/- 8% positivity and total cultured lymphocytes showed positivity of 46 +/- 11% positivity. Lymphoblasts, unstimulated lymphocytes, and total cultured lymphocytes from hyper-cholesterolemia without FH showed 74 +/- 10%, 25 +/- 10% and 50 +/- 17%, respectively, which showed no significant differences from normal control groups. FH Heterozygotes showed LDL-R positivity, 21 +/- 11% in lymphoblasts, 11 +/- 6% in unstimulated lymphocytes and 18 +/- 7% in total cultured lymphocytes. These data imply that adequately stimulated lymphocytes might be used for detecting defects in LDL-R and used to differentiate FH from secondary hypercholesterolemia.

Comportamiento de la respuesta a la fitohemaglutinina (PHA) de linfocitos humanos en presencia de Coralan / Behavior of response to phytohemagglutinin (PHA) of human lymphocytes in the presence of Coralan

La búsqueda de nuevos inmunomoduladores con actividad antitumoral es tarea priorizada de muchos laboratorios en el mundo. En el Instituto Nacional de Oncología y Radiobiología (INOR) se obtuvo un polisacárido de un coral blanco del Caribe, que produjo rechazo al transplante de hasta 100 por ciento del tumor ascítico de Ehrlich en ratones. Este producto evidenci ó actividad de los linfocitos T a diferentes niveles en donantes sanos, lo que podría permitir en el futuro separar los mismos por el grado de incremento de respuesta a la PHA (AU)

Produccion de Beta-endorfinas por poblaciones leucocitarias estimuladas con diferentes mitógenos / Production of beta-endorphins by hamatic leucocyte population stimulated by different mitogens

Se acepta generalmente que los péptidos opioides endógenos juegan un papel fundamental en la modulación de la respuesta inmune en procesos dolorosos. Durante tres años, nuestro Departamento ha investigado una técnica capaz de realizar estudios semi-cuantitativos de las Beta-endorfinas presentes en fa superficie de células leucocitarias hemáticas. Los trabajos previos, indican que la cantidad de Beta-endorfina presente en la superficie de estas células leucocitarias no depende exclusivamente de fa cantidad de Beta-endorfina existente en el plasma de los pacientes. Existiría una tracción de la misma que sería sintetizada por las propias células inmunocompetentes. El objetivo es conocer la capacidad de producción de Beta-endorfinas por células leucocitarias hemáticas cultivadas in vitro y estimuladas con diferentes mitógenos ( Fitohematoglutinina y Concanavalina A). La población de estudio estuvo formada por 16 sujetos sanos. A todos, se les extrajo una muestra de sangre, en la que se separaron fas células de fa serie blanca mediante una técnica de gradiente de densidad. Con las células leucocitarias obtenidas de cada sujeto se hicieron tres alicuotas ajustándose fa suspensión a 2.5 millones de células/250 microlitros en medio de PBS. Una de las muestras quedó como muestra control, la segunda se estimuló con PHA y la tercera muestra con Con A. Todas las muestras se incubaron durante 24 horas y se determinó la cantidad de Beta-endorfina existente en el sobrenadante celular mediante la técnica comercial de Nichols Institute. Se observaron concentraciones de Beta-endorfinas significativamente superiores en las muestras estimuladas con PHA en relación a las muestras controles y a las muestras estimuladas con ConA (AU)

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Human purified alpha-fetoprotein: I. In vitro effects on lymphocyte proliferation to mitogens and on mixed lymphocyte reactivity

Estudou-se o efeito de alfafetoproteína (AFP) sobre a proliferaçäo de linfócitos periféricos humanos estimulados por fitohemaglutinina (PHA), concanavalina A (Con A), pokeweed mitogênio (PWM) e por células alogênicas em culturas mistas de linfócitos, ao medir-se e comparar-se a incorporaçäo de timidina triciada (parâmetros de crescimento celular) em culturas contendo AFP humanas purificada e em culturas sem a proteína (controles). Nenhuma alteraçäo quantitativa na reatividade linfocitária foi verificada nas culturas com AFP

Correlation between the lack of mononuclear cell cytotoxicity against schistosomula in vitro with schistosomiasis

A comparison was made of the reactivity of mononuclear cells from subjects and from S. mansoni-infected patients. The following parameters were evaluated: 1) ability of mononuclear cells to kill schistosoma in the presence of complement; 2) [3H]-inositol incorporation into phosphatidylinositol (PI) and the rate of inositolphosphates (IPx) released. Cells from normal subjects, but not from S. mansoni infected patients, were able to kill schistosomula in vitro. A decrease in inositolpolyphosphates (IPx) was observed for phytohemagglutinin (PHA)-stimulated mononuclear cells from infected patients when compared with mononuclear cells from normal subjects after 24 h of incubation. The results suggest that the reactivity of mononuclear cells from infected patients is altered under conditions of nonspecific stimulation with PHA when compared with normal cells

Cell-mediated immunity in alcoholic liver cirrhosis

Se efectuaron estudios de inmunidad mediada por células en 25 pacientes con cirrosis hepática alcohólica (CA) activa e inactiva. Fueron observadas diversas alteraciones inmunológicas: aumento de los linfocitos B y disminución de los linfocitos T. Sólo en los enfermos con CA activa la relación H/S era más baja que los valores normales. Los linfocitos NK (Leu 7 +) y la función citotóxica natural killer estaban aumentados; sin embargo el índice NK/Leu 7 estaba disminuido. En los enfermos con CA activa la proliferación blástica a la PHA estaba descendida. Se observó un déficit de la función supresora inducida por Con A en los pacientes con CA. En los cocultivos alogeneicos de linfocitos supresores de los pacientes y células efectoras normales, se observó un descenso de la función supresora T-T con respecto a los cultivos autólogos normales. No se encontraron diferencias cuando los linfocitos de normales fueron inducidos con Con A y se cocultivaron con linfocitos efectores de los enfermos. Estos resultados indican que los linfocitos de los pacientes con CA presentan anomalías en la inducción de supresión por Con A (AU)

The influence of aldosterone on the phytohemagglutinin - induced human lymphocyte blastogenesis

Este trabalho investigou a capacidade da aldosterona, cuja concentraçäo no sangue de mulheres grávidas está aumentada em até 10 vezes quando comparada com os níveis séricos em mulheres näo grávidas, em inibir a proliferaçäo in vitro de linfócitos periféricos humanos estimulados por fitohemaglutinina (PHA), medida através da incorporaçäo de timidina triciada pelo DNA celular. As doses de aldosterona utilizadas nas culturas de linfócitos foram aquelas existentes em soros de mulheres näo grávidas, em soros de mulheres grávidas no final da gestaçäo e dose duas vezes maiores que essas últimas. Três concentraçöes diferentes de PHA, subótimas, ótimas e supraótimas, foram usadas nos testes. O hormônio e o mitogênio foram adicionados no início das incubaçöes. Nossos resultados mostraram que aldosterona nas doses acima referidas näo tem qualquer efeito sobre a proliferaçäo de linfócitos estimulados por PHA. Além disso, foi também demonstrado que incubaçäo prévia dos linfócitos com aldosterona por 24 horas, que faz as células reexpressarem receptores para outros hormônios, seguida da adiçäo do mitogênio, näo provoca qualquer efeito da aldosterona sobre este teste in vitro de imunidade mediada por células

Efeito da fitohemaglutinina na proliferaçäo celular do epitélio do intestino delgado de ratos / Effect of phytohemagglutinins in cell proliferation of the epithelium of the small intestine of rats

Vinte ratos albinos, foram divididos em dois grupos de dez: grupo e, que recebeu dieta sólida contendo 4% de fitohemaglutinina (FHG) ativa e água "ad libitum" e grupo P controle pareado iso-calórico, que recebeu a mesma dieta sólida porém com fitohemaglutinina inativada pelo calor e água "ad livitum". Diariamente os animais foram pesados, as quantidades de raçäo e água foram anotados. No 14§ de experimento, os animais foram sacrificados e colhidos fragmentos de jejuno e íleo para estudo morfocinético. Os resultados mostraram que: a ingestäo hídrica foi semelhante em ambos os grupos estudados e o grupo E ganhou menos peso corporal do que o grupo P. As populaçöes de enterócitos das vilosidades jejunais do grupo E, foram menores em termos estatísticos quando comparadas ao grupo P, e o contrário se deu no íleo, onde o grupo E foi maior que o P. A altura da vilosidade do grupo E foi semelhante ao do grupo P, mas o íleo do grupo E foi maior do que o do grupo P. As profundidades, as populaçöes e as taxas de produçäo celular nas criptas jejunais do grupo E foram significativamente maiores que no grupo P. Concluindo, os achados no presente estudo fornecem evidências de que a ingestäo de fitohemaglutinina lesa a mucosa jejunal proxima, diminuindo a populaçäo celular da vilosidade e estimulando a hiperplasia da cripta, desenvolvendo adaptaçäo localizada. Este modelo adaptativo é semelhante ao que acontece na doença celíaca. Esta lesäo proximal estimula a hiperplasia da unidade cripta-vilosidade do epitéileal, desenvolvendo adaptaçäo à distância. Estas adaptaçöes aconteceram em animais que ingeriram fitohemaglutinina, mesmo na vigência de desnutriçäo multicarencial

Comportamiento de la respuesta a la fitohemaglutinina (PHA) de linfocitos humanos en presencia de Coralan / Behavior of response to phytohemagglutinin (PHA) of human lymphocytes in the presence of Coralan

La búsqueda de nuevos inmunomoduladores con actividad antitumoral es tarea priorizada de muchos laboratorios en el mundo. En el Instituto Nacional de Oncología y Radiobiología (INOR) se obtuvo un polisacárido de un coral blanco del Caribe, que produjo rechazo al transplante de hasta 100 por ciento del tumor ascítico de Ehrlich en ratones. Este producto evidenci ó actividad de los linfocitos T a diferentes niveles en donantes sanos, lo que podría permitir en el futuro separar los mismos por el grado de incremento de respuesta a la PHA

Cell-mediated immunity in alcoholic liver cirrhosis

Se efectuaron estudios de inmunidad mediada por células en 25 pacientes con cirrosis hepática alcohólica (CA) activa e inactiva. Fueron observadas diversas alteraciones inmunológicas: aumento de los linfocitos B y disminución de los linfocitos T. Sólo en los enfermos con CA activa la relación H/S era más baja que los valores normales. Los linfocitos NK (Leu 7 +) y la función citotóxica natural killer estaban aumentados; sin embargo el índice NK/Leu 7 estaba disminuido. En los enfermos con CA activa la proliferación blástica a la PHA estaba descendida. Se observó un déficit de la función supresora inducida por Con A en los pacientes con CA. En los cocultivos alogeneicos de linfocitos supresores de los pacientes y células efectoras normales, se observó un descenso de la función supresora T-T con respecto a los cultivos autólogos normales. No se encontraron diferencias cuando los linfocitos de normales fueron inducidos con Con A y se cocultivaron con linfocitos efectores de los enfermos. Estos resultados indican que los linfocitos de los pacientes con CA presentan anomalías en la inducción de supresión por Con A

Evaluacion de la producion de interleuquina 1 por celulas adherentes en pacientes con diabetes tipo II / Production of interleukin 1 by adherent cells in patients with Diabetes Mellitus type II

Se evaluó la actividad comitogénica de la interleuquina 1 producida en cultivos de células adherentes mononucleares de pacientes con diabetes mellitus (DM) tipo II o insulino no dependente. Dicha actividad fue medida por la incorporación de timidina en cultivos de timocitos de ratones (C3H/Hej, en presencia de fitohemaglutinina (PHA). Se observó que los sobrenadantes de los cultivos de células adherentes de los pacientes con DM tipo II no produjeron efecto sinergista con la PHA, obteniéndose niveles de linfoproliferación semejantes a los obtenidos en ausencia de dicha lectina. Esta falta de efecto comitogénico (p < 0,001) con respecto a la respuesta obtenida con la interleuquina 1 de sujetos normales más PHA, podría deberse a la liberación de algún/os factor/es solubles que podieron interferir con los receptores glicosilados para mitógenos o con los procesos de activación celular (AU)

Alteraciones inmunológicas en pacientes con púrpura trombocitopenica idiopática crónica / Immunological changes in patients with chronic idiopathic thrombocytopenic purpura

En este trabajo se valoró la inmunidad mediada por células y los niveles de inmunocomplejos circulantes en 30 pacientes con púrpura trombocitopénica idiopática crónica (PTI). Se hallaron diversas alteraciones inmunológicas: los pacientes con PTI tenían valores relativos de linfocitos B aumentados y disminuidos los de linfocitos T. La transformación blástica inducida por PHA estaba disminuida al igual que la función supresora T-T inespecífica inducida por Con A. Se rencontró una correlación directa entre los recuentos plaquetarios y la función supresora (r=0,61, p<0,01). El nivel de IgG asociada a la membrana plaquetaria era más alto en aquellos pacientes con marcada depresión de la actividad supresora. Estos resultados no estaban relacionados a la edad y sexo de los pacientes ni al tratamiento o evaluación de la enfermedad (AU)

Evaluacion de la producion de interleuquina 1 por celulas adherentes en pacientes con diabetes tipo II / Production of interleukin 1 by adherent cells in patients with Diabetes Mellitus type II

Se evaluó la actividad comitogénica de la interleuquina 1 producida en cultivos de células adherentes mononucleares de pacientes con diabetes mellitus (DM) tipo II o insulino no dependente. Dicha actividad fue medida por la incorporación de timidina en cultivos de timocitos de ratones (C3H/Hej, en presencia de fitohemaglutinina (PHA). Se observó que los sobrenadantes de los cultivos de células adherentes de los pacientes con DM tipo II no produjeron efecto sinergista con la PHA, obteniéndose niveles de linfoproliferación semejantes a los obtenidos en ausencia de dicha lectina. Esta falta de efecto comitogénico (p < 0,001) con respecto a la respuesta obtenida con la interleuquina 1 de sujetos normales más PHA, podría deberse a la liberación de algún/os factor/es solubles que podieron interferir con los receptores glicosilados para mitógenos o con los procesos de activación celular