ABSTRACT
The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , HLA-A Antigens/immunology , Monitoring, Immunologic/methods , Monitoring, Immunologic/standards , CD8-Positive T-Lymphocytes/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , Europe , Flow Cytometry/methods , Flow Cytometry/standards , HLA-A Antigens/chemistry , Humans , Immunotherapy , Leukocytes, Mononuclear/immunology , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Professional Staff Committees , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Staining and LabelingABSTRACT
La expansión ex vivo de células stem y progenitoras hematopoyéticas tiene un importante potencial de aplicación clínica, incluyendo la terapia génica, purgado de células tumorales y la producción de células dendríticas para inmunoterapia. En las dos últimas décadas se han publicado numerosos trabajos demostrando expansión de células progenitoras es factible en casi todas las condiciones de cultivos ex vivo, desde cultivos estáticos en bolsas permeables a gases a cultivos de perfusión continua en bioreactores. En el presente trabajo se describen las diferentes alternativas de cultivo ex vivo de células stem y las potenciales ventajas y aplicaciones clínicas de la expansión de células de la médula ósea, sangre periférica y cordón umbilical
Subject(s)
Erythroid Precursor CellsABSTRACT
La expansión ex vivo de células stem y progenitoras hematopoyéticas tiene un importante potencial de aplicación clínica, incluyendo la terapia génica, purgado de células tumorales y la producción de células dendríticas para inmunoterapia. En las dos últimas décadas se han publicado numerosos trabajos demostrando expansión de células progenitoras es factible en casi todas las condiciones de cultivos ex vivo, desde cultivos estáticos en bolsas permeables a gases a cultivos de perfusión continua en bioreactores. En el presente trabajo se describen las diferentes alternativas de cultivo ex vivo de células stem y las potenciales ventajas y aplicaciones clínicas de la expansión de células de la médula ósea, sangre periférica y cordón umbilical (AU)
Subject(s)
Erythroid Precursor CellsABSTRACT
We report the case of a 54 year old male with an original diagnosis of chronic myeloid leukemia (CML) who developed a nodal T cell blast crisis (BC) while he was in a complete hematological remission (CR). We describe the clinical presentation and the histological, immunophenotypic and molecular characterization of the lymph node blast cells. Our case, together with other rare similar reports in the literature, argue that a T cell nodal blast crisis of CML resembles the presentation of a T-cell non-Hodgkin's lymphoma.
Subject(s)
Blast Crisis/pathology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/complications , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Lymph Nodes/pathology , Lymphoma, T-Cell/diagnosis , Blast Crisis/etiology , CD3 Complex/analysis , Cell Transformation, Neoplastic , Diagnosis, Differential , Humans , Immunophenotyping , Lymphoma, T-Cell/etiology , Lymphoma, T-Cell/genetics , Male , Middle Aged , Philadelphia ChromosomeABSTRACT
A heat-released elicitor (HRE) obtained from Sclerotinia sclerotiorum mycelium induced a transient increase in the activity of phenylalanine-ammonia lyase (PAL) in a carrot cell culture. Differential display reactions carried out on total RNA extracted from cells exposed for 7 h to the HRE revealed a complex cell response involving the induction and repression of several mRNAs. One of these elicitor-induced mRNAs encodes a 42.6-kDa protein. Northern (RNA) blot analysis and reverse-transcription polymerase chain reaction studies showed that noninduced cells have a basal expression of the 42.6-kDa protein mRNA that was stimulated five- to 10-fold by treatment with the elicitor.
Subject(s)
Ascomycota/physiology , Daucus carota/microbiology , Daucus carota/physiology , Gene Expression Regulation, Plant , Glycoproteins/biosynthesis , Phenylalanine Ammonia-Lyase/biosynthesis , Plant Proteins , Transcription, Genetic , Amino Acid Sequence , Ascomycota/genetics , Base Sequence , Cells, Cultured , Daucus carota/enzymology , Gene Expression Regulation, Enzymologic , Genes, Fungal , Glycoproteins/chemistry , Glycoproteins/genetics , Molecular Sequence Data , Open Reading Frames , RNA, Messenger/biosynthesis , Random Amplified Polymorphic DNA TechniqueABSTRACT
The study of the incidence of aflatoxins, zearalenone, and deoxynivalenol was the aim of this work. This investigation was carried out upon recently harvested corn samples collected in the Departamento of Rio Cuarto, Province of Córdoba, Argentina. 150 samples of corn were analized to investigate aflatoxins B1, G1, and zearalenone contamination. Out of these 150 samples 58 were selected and deoxynivalenol was examined.The incidence value for aflatoxin B1 was of 3.3% (5 samples), aflatoxin G1 1.3 % (2 samples), and zearalenone 6 % (9 samples).The analysis of the 58 samples showed that 24% of them were contaminated with deoxynivalenol.