Immature dendritic cells generated from cryopreserved human monocytes show impaired ability to respond to LPS and to induce allogeneic lymphocyte proliferation.

Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.

Dendritic cells derived from metastatic cancer patients vaccinated with allogeneic dendritic cell-autologous tumor cell hybrids express more CD86 and induce higher levels of interferon-gamma in mixed lymphocyte reactions.

Dendritic cells (DCs) are highly effective antigen-presenting cells that, when derived from cancer patients, seem to be functionally deficient. Herein, we show that vaccination with allogeneic DC-autologous tumor cell hybrids affects the phenotype and improves the function of monocyte-derived DCs (Mo-DCs) from cancer patients. Mononuclear cells were isolated from patients' peripheral blood by density gradient centrifugation, and adherent cells were cultured in medium containing GM-CSF plus IL-4 and, after 5 days, TNF-alpha. After 2 more days, Mo-DCs were harvested and their CD80, CD86, and CD83 expression was assessed by flow cytometry. They were also used as stimulators in mixed lymphocyte reactions (MLR), where IFN-gamma production was measured by ELISA. Mo-DCs from unvaccinated patients expressed significantly lower levels of CD86, and tended to express lower levels of CD83 than Mo-DCs from healthy donors. However, Mo-DCs generated after hybrid cell vaccination presented increased expression of the same markers and induced significantly higher levels of IFN-gamma in MLR. These results indicate that the use of allogeneic DC-based cancer vaccines induces recovery of DC function in metastatic cancer patients and, therefore, could precede the use of autologous DCs for vaccine preparation. Such an approach could be relevant and should be investigated in clinical trials.

Correlation of mixed lymphocyte culture with chronic graft-versus-host disease following allogeneic stem cell transplantation.

The purpose of the present study was to evaluate the mixed lymphocyte culture as a predictive assay of acute and chronic graft-versus-host disease (GVHD). We studied 153 patients who received a first bone marrow transplantation from human leukocyte antigen-identical siblings. Acute GVHD was observed in 26 of 128 (20.3%) patients evaluated and chronic GVHD occurred in 60 of 114 (52.6%). One-way mixed lymphocyte culture (MLC) assays were performed by the standard method. MLC results are reported as the relative response (RR) from donor against patient cells. The responses ranged from -47.0 to 40.7%, with a median of 0.5%. The Kaplan-Meier probability of developing GVHD was determined for patients with positive and negative MLC. There was no significant difference in incidence of acute GVHD between the groups studied. However, the incidence of chronic GVHD was higher in recipients with RR >4.5% than in those with RR < or =4.5%. The Cox Proportional Hazards model was used to examine the effect of MLC levels on incidence of chronic GVHD, while adjusting for the potential confounding effect of others suspected or observed risk factors. The relative risk of chronic GVHD was 2.5 for patients with positive MLC (RR >4.5%), 2.9 for those who received peripheral blood progenitor cells as a graft, and 2.2 for patients who developed previous acute GVHD. MLC was not useful for predicting acute GVHD, but MLC with RR >4.5% associated with other risk factors could predict the development of chronic GVHD, being of help for the prevention and/or treatment of this late complication.

Addition of exogenous cytokines in mixed lymphocyte culture for selecting related donors for bone marrow transplantation.

CONTEXT: Mixed lymphocyte culturing has led to conflicting opinions regarding the selection of donors for bone marrow transplantation. The association between a positive mixed lymphocyte culture and the development of graft-versus-host disease (GVHD) is unclear. The use of exogenous cytokines in mixed lymphocyte cultures could be an alternative for increasing the sensitivity of culture tests. OBJECTIVE: To increase the sensitivity of mixed lymphocyte cultures between donor and recipient human leukocyte antigen (HLA) identical siblings, using exogenous cytokines, in order to predict post-transplantation GVHD and/or rejection. TYPE OF STUDY: Prospective study. SETTING: Bone Marrow Transplantation Unit, Universidade Estadual de Campinas. PARTICIPANTS: Seventeen patients with hematological malignancies and their respective donors selected for bone marrow transplantation procedures. PROCEDURES: Standard and modified mixed lymphocyte culturing by cytokine supplementation was carried out using donor and recipient cells typed for HLA. MAIN MEASUREMENTS: Autologous and allogenic responses in mixed lymphocyte cultures after the addition of IL-4 or IL-2. RESULTS: In comparison with the standard method, average responses in the modified mixed lymphocyte cultures increased by a factor of 2.0 using IL-4 (p < 0.001) and 6.4 using IL-2 (p < 0.001), for autologous donor culture responses. For donor-versus-recipient culture responses, the increase was by a factor of 1.9 using IL-4 (p < 0.001) and 4.1 using IL-2 (p < 0.001). For donor-versus-unrelated culture responses, no significant increase was observed using IL-4, and a mean response inhibition of 20% was observed using IL-2 (p < 0.001). Neither of the cytokines produced a significant difference in the unrelated control versus recipient cell responses. CONCLUSION: IL-4 supplementation was the best for increasing the mixed lymphocyte culture sensitivity. However, IL-4 also increased autologous responses, albeit less intensively than IL-2. Thus, with this loss of specificity we believe that it is not worth modifying the traditional mixed lymphocyte culture method, even with IL-4 addition.

Efeito imunossupressor dos gangliosídeos: estudo in vivo / Immunosupressive effect of gangliosides: in vivo study

Imunomodulação mais específica e eficaz é uma meta importante a ser atingida na área de órgão. Neste sentido, foi estudado previamente o papel imunomodulador dos gangliosídeos "in vitro". No presente trabalho objetivou-se avaliar este efeito agora "in vivo", mimetizando a situação do transplante alogênico. Foram utilizados 26 ratos Wistar 1 EPM, machos, com 3 meses de idade, pesando cerca de 250g, procedentes do Centro de Desenvolvimento de Pesquisa Experimental em Medicina e Biologia. Os animais fora mantidos por 5 dias, para adaptação, no biotério setorial da Disciplina de Técnica Operatória e Cirurgia Experimental da UNIFESP-EPM, recebendo água e ração própria para a espécie. Os animais foram distribuídos em grupos conforme segue: grupos experimento (que receberam 1, 3 e 6 mg/kg/dia de gangliosídeos) e um grupo controle que recebeu veículo, todos por via intramuscular durante 7 dias consecutivos. No 8º dia, com os animais anestesiados com éter etílico foi feita a remoção cirúrgica do baço de todos os animais, os quais foram sacrificados por exsanguinação, ainda sob efeito anestésico. Os baços removidos foram processados para a obtenção de linfócitos os quais foram cultivados em placa de cultura com 96 poços, distribuídos da seguinte forma: 1,5x10(5) linfócitos viáveis de cada animal dos grupos experimento e controle foram cultivados com 1,5x10(5) linfócitos viáveis de um rato não tratado, sendo assim realizada a reação mista de linfócitos. Os linfócitos provenientes dos animais dos grupos controle e 1 mg apresentaram aumento da proliferação sem nenhuma alteração. Por outro lado, foi observada uma taxa de inibição ao redor de 70 por cento sobre a proliferação linfocitária dos animais dos grupos 3 e 6 mg comparados aos animais dos grupos controle e 1 mg. O resultado desta investigação estimula a utilização dos gangliosídeos no tratamento da rejeição alogênica.

Histocompatibilidad / Histocompatibility

El Complejo Mayor de Histocompatibilidad humano consiste en una serie de genes fuertemente enlazados presentes en el brazo corto del cromosoma 6, normalmente heredados en bloque y constituyen el haplotipo HLA. La mayoría presenta alto polimorfismo, lo que permite la presencia de numerosas variantes alélicas. La herencia de este segmento puede seguirse dentro de una familia por tipificación de ADN celular o de los antígenos HLA expresados en las membranas celulares, permitiendo estudios de filiación o poblacionales. La función más importante es el reconocimiento de la identidad: las células se reconocen entre sí como propias de un individuo. Juegan un papel esencial en la selección clonal de linfocitos, en la presentación antigénica y en la regulación del sistema inmune. El estudio de su asociación con enfermedades representó un importante avance en la Inmunología clínica. En la actualidad numerosos trabajos reafirman su papel en los procesos autoinmunes, en la respuesta inmune según la interacción del sitio de unión del HLA y el epitope antigénico presentado y probablemente en la susceptibilidad a determinados tumores. Objetivos: Introducir al conocimiento del Sistema Mayor de Histocompatibilidad y su aplicación clínica, comprender los fundamentos de los estudios más frecuentes fomentando la autoevaluación y educación continua (AU)

Estudos citogenéticos e de resposta à fito-hemaglutimina em cultura de linfócitos de sangue periférico de paciente portadora de linfoma maligno näo-Hodgkin e de sua irmä gêmea fraterna / Cytogenetic and phytohemagglutinins response studies in lymphocyte culture of peripheral blood in a patient with malignant no-Hodgkin's lymphoma and her fraternal twin

Culturas de linfócitos do sangue periférico de paciente portadora de linfoma näo-Hodgkin e de sua gêmea fraterna clinicamente normal foram realizadas, para estudos citogenéticos e de resposta à PHA. Na cultura da paciente foram encontradas anomalias cromossômicas, numéricas e estruturais, sugerindo que importantes alteraçöes estäo em franco processamento. Embora a cultura tenha ainda mostrado comportamento bastante peculiar e sugestivo de alto metabolismo das células, a índice mitótico obtido foi baixo em relaçäo à cultura controle, enquanto que o índice de blastos transformados pela PHA foi mais elevado. Fato a se notar, tanto na cultura da paciente quanto na de seu controle - gêmea fraterna -, foi a forte associaçäo entre cromossomos satelitados