RESUMO
Malignant transformation of human cells requires the accumulation of multiple genetic alterations, such as the activation of oncogenes and loss of function of tumor suppressor genes or those related to genomic instability. Among the genetic alterations most frequently found in human tumors are chromosomal translocations that may result in the expression of chimeric products with transforming capability or are able to change the expression of oncogenes. We show here that the adenovirus early region 1A (E1A) gene can induce a specific human fusion transcript (EWS-FLI1) that is characteristic of Ewing tumors. This fusion transcript was detected by RT-PCR in normal human fibroblasts and keratinocytes after expression of the adenovirus E1A gene, as well as in human cell lines immortalized by adenoviruses. Cloning and sequencing of the RT-PCR product showed fusion points between EWS and FLI1 cDNA identical to those detected in Ewing tumors. In addition, we detected a chimeric protein by western blot analysis and immunoprecipitation and a t(11,22) by fluorescent in situ hybridization. This association between a single viral gene and a specific human fusion transcript indicates a direct link between viral genes and chromosome translocations, one of the hallmarks of many human tumors.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Genes Virais/fisiologia , Proteínas de Fusão Oncogênica/genética , Oncogenes/genética , Sarcoma de Ewing/genética , Fatores de Transcrição/genética , Proteínas E1A de Adenovirus/genética , Adenovírus Humanos/genética , Sequência de Bases , Linhagem Celular , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 22/genética , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Genes Virais/genética , Humanos , Hibridização in Situ Fluorescente , Queratinócitos , Dados de Sequência Molecular , Peso Molecular , Mutação , Proteínas de Fusão Oncogênica/biossíntese , Oncogenes/fisiologia , Proteína Proto-Oncogênica c-fli-1 , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteína EWS de Ligação a RNA , Sarcoma de Ewing/metabolismo , Fatores de Transcrição/biossíntese , Translocação Genética/genéticaRESUMO
The synthesis of membrane-bound and secreted immunoglobulin was investigated in the human line LICR-LON-HMy2, a cell line often used for the derivation of human hybridomas. PAGE-SDS analysis of immunoprecipitates obtained from 35S-methionine labelled cell lysates shows that LICR-LON synthesize a hitherto undetected membrane form of IgG (with a heavy chain of mol. wt 62,000) in addition to the secretor form of IgG already described (55,000 heavy chain). Tunicamycin treatment, pulse-chase experiments and Western blot analysis showed that both chains are synthesized as independent proteins. Hybridomas obtained after fusion of LICR-LON and human peripheral blood lymphocytes retained the ability of the parental cell line to synthesize gamma m and gamma s. Some of these hybrids synthesize and secrete IgM which presumably originates from the parental B-lymphocytes. Precipitation and PAGE-SDS analysis of membrane proteins after iodination of intact cells revealed only one heavy chain band, corresponding in size to that of the gamma m. No indication of the synthesis of the membrane form of IgM was found in the hybrids. These data show that the parental (lymphoid) phenotype (m and s-IgG) is codominant with the more differentiated phenotype (s-IgM) of the fusion partner cell (plasma cell). These observations are compatible with a class-specific m-s regulation operating on a different chromatin structure at the expressed Ig loci of each parental chromosome.
Assuntos
Anticorpos Antineoplásicos/biossíntese , Imunoglobulina G/biossíntese , Leucemia/imunologia , Linhagem Celular , Membrana Celular/imunologia , Células Clonais/imunologia , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Glicosilação , Humanos , Hibridomas/imunologiaRESUMO
OBJECTIVES: Biclonal gammopathies are characterized by the clonal proliferation of plasma cells or their B-lymphoid progenitors and are associated with the production of abnormal immunoglobulins (M proteins or paraproteins). There are no known studies that have analyzed this disease in Spain. We studied the underlying diseases, characteristics of paraproteins and the evolution of a series of patients with biclonal gammopathy. MATERIAL AND METHODS: We reviewed clonal gammopathies at the Department of Immunology of Hospital Puerta de Hierro in Madrid, between 1970 and 2011, selecting those patients with biclonal gammopathy in one reading. We collected data on the patient's epidemiology, underlying disease, associated diseases, therapies and paraprotein and immunoglobulin levels. RESULTS: Of the 1626 cases of clonal gammapathies, 47 were biclonal gammopathy (2.89%). The median follow-up was 2 years. The main associated condition was biclonal gammopathies of undetermined significance (BGUS). The most common paraprotein combination was IgG-IgG. Upon conducting a second paraprotein reading, 81% of the patients had lost at least 1 monoclonal component. A third of the patients had not undergone treatment. CONCLUSIONS: Biclonal gammopathy are fundamentally associated with biclonal gammopathies of undetermined significance. No biclonal gammopathies of undetermined significance evolved to a malignant disease. In a high percentage of patients, at least 1 of the 2 clonal components disappeared, sometimes spontaneously.
RESUMO
Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Animais , Anticorpos Monoclonais/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Hibridomas/imunologia , Hibridomas/fisiologia , Imunoglobulina M/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Monócitos/imunologia , Monócitos/fisiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie , Baço/imunologiaRESUMO
In patients with B-cell lymphoma, only in rare cases a secreted paraprotein is found, and in very few of them an associated autoantibody activity has been demonstrated. Here we report the case of a patient with a low-grade B-cell lymphoma with a serum biclonal paraprotein (G,M)lambda and severe erythroblastopenia. Indirect immunofluorescence studies of total serum revealed cytoplasmic (Hep-2 cells) and extracellular matrix (rat tissue sections) staining, suggestive of a new specificity. After gel filtration of serum samples, only the IgM-containing fraction showed the same pattern of staining. Tumor-derived hybridomas expressed an unmutated V3-11 gene identical to that found in tumor samples and secreted an IgM immunoglobulin endowed with the same reactivity, which confirms the tumoral origin of the tissue-reactive protein. The results suggest a link between the autoimmune condition in this patient and the novel specificity displayed by the tumor-derived immunoglobulin.
Assuntos
Anticorpos Antineoplásicos/imunologia , Autoanticorpos/imunologia , Linfoma de Células B/imunologia , Linfoma Folicular/imunologia , Paraproteínas/imunologia , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/sangue , Especificidade de Anticorpos , Autoanticorpos/sangue , Sequência de Bases , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Dados de Sequência Molecular , Ratos , Células Tumorais CultivadasRESUMO
A 48-year-old patient with IgA k multiple myeloma received a BMT from his HLA-matched sibling. After transplantation, the disease relapsed. Melphalan therapy followed by reinfusion of haemopoietic blood stem cells collected from the patient led to the improvement of the clinical status, although mixed chimerism and an elevated serum IgA persisted. Successful donor immunisation against an immunogenic preparation of the recipient monoclonal protein was performed before the infusion of donor T lymphocytes (DLI) into the patient. Ten weeks after the lymphocyte infusions, no monoclonal band was evidenced and donor complete chimerism was detected. The patient did not develop GVHD. Once complete remission was achieved, the idiotype vaccine was administered to the patient. Nineteen months after DLI, the patient remains in remission. Bone Marrow Transplantation (2000).
Assuntos
Doadores de Sangue , Imunização , Imunoglobulina A/imunologia , Idiótipos de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Imunoterapia Adotiva , Transfusão de Linfócitos , Mieloma Múltiplo/terapia , Proteínas do Mieloma/imunologia , Terapia de Salvação , Linfócitos T/transplante , Antineoplásicos Alquilantes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transplante de Medula Óssea , Quimera , Terapia Combinada , Dexametasona/administração & dosagem , Doxorrubicina/administração & dosagem , Sobrevivência de Enxerto , Humanos , Masculino , Melfalan/uso terapêutico , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Indução de Remissão , Vincristina/administração & dosagemRESUMO
Intravenous infusion of gammaglobulins (IVIG) is one of the treatments of choice in patients with type II mixed cryoglobulinemia (MC). We describe the case of a patient with MC who suffered an adverse generalised reaction with severe cutaneous vasculitis accompanied by a sudden increase in cryocrit levels shortly after being treated with IVIG. When the same gammaglobulin preparation was added in vitro to a sample of the patient's serum, a strong increment in cryoglobulin precipitation and depletion of the monoclonal IgM peak resulted. We suggest that this simple method of studying the displacement of the precipitation reaction could help to predict the outcome of treatment and must be performed before starting IVIG in patients with MC.
Assuntos
Crioglobulinemia/terapia , Imunoglobulinas Intravenosas/efeitos adversos , Vasculite Leucocitoclástica Cutânea/etiologia , Adulto , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêuticoAssuntos
Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Linfoma Folicular/genética , Células da Medula Óssea/metabolismo , Vacinas Anticâncer/química , Humanos , Linfonodos/patologia , Modelos Genéticos , Mutação , Filogenia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Translocação Genética , Vacinas de DNA/químicaRESUMO
Activated mouse spleen lymphocytes have increased amounts of the glycolytic enzymes pyruvate kinase and lactate dehydrogenase. No changes were found in hexokinase and in the gluconeogenic enzyme fructose 1,6-diphosphatase. Concanavalin A-activated T cells give higher activities of those enzymes than lipopolysaccharide-activated B lymphoblasts. Insulin treatment results in a stronger increment of the enzyme activity of mitogen-activated cells. Insulin inhibits the initial proliferation induced by either Con A or LPS, but a 50% increase in antibody-forming cells was found in LPS-treated cultures. Insulin may favour the differentiation of activated cells by increasing the rate of the glycolytic pathway.
Assuntos
Insulina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/enzimologia , Mitógenos/farmacologia , Animais , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Frutose-Bifosfatase/metabolismo , Hexoquinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Baço/citologiaRESUMO
The in vitro anti-trinitrophenyl (TNP) response induced by TNP-lipopolysaccharide (TNP-LPs) in mouse spleen cells was eliminated by passage through Sephadex G10 columns. Conditioned medium obtained from peritoneal macrophages restored the response, indicating the supportive role of such cells in the response. Conversely, polyclonal B-cell activation mediated by LPS was not affected by Sephadex G10 treatment, as judged by incorporation of 3H-thymidine, generation of specific anti-TNP plaque-forming cells, and induction of polyclonal IgM-secreting cells. The failure of the LPS moiety of TNP-LPS to induce B-cell activation without macrophage help suggests a restriction in the number of available anti-LPS receptor molecules when the Ig anti-TNP receptor interacts with the haptenic (TNP) groups. This restriction can be due to the attachment of the TNP molecules to residues located in the vicinity of the lipid A mitogenic region of the carrier.
Assuntos
Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Nitrobenzenos/imunologia , Trinitrobenzenos/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Imunoglobulina M/biossíntese , Mercaptoetanol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologiaRESUMO
The correlated expression of several surface antigens (CD 1, class I HLA, CD3) was examined in human unfractionated thymocytes or selected subsets by using single or double-color flow cytometry. Prethymocyte subpopulations expressed high levels of HLA. A high proportion of cortical cells expressed low levels of either HLA or CD 3 antigens. Most of these HLA+ cortical cells corresponded to the more immature cells and did not express HLA-B loci products. CD 1 a and CD 3 antigens were expressed in a high percentage of cells and the levels of expression of each antigen in individual cells were inversely correlated. These data and the contour of double-color histograms are suggestive of the existence of a single pathway of thymocyte differentiation in which class I HLA expression is switched off around the time of the initiation of CD 3 (and Ti?) expression. We suggest that the anti-major histocompatibility complex (MHC) specificity of the unselected Ti receptor may be incompatible with the expression of MHC products on the cell membrane. At this stage, CD 1 antigens, whose expression is inversely correlated with that of HLA, may fulfill the role of MHC antigens. The latter can be re-expressed later on, once the anti-MHC specificities of the Ti receptors have been selected against. Studies on in vitro modulation of HLA molecules by interferon-alpha did not reveal any correlation to the expression of CD 1 or CD 3 antigens.
Assuntos
Antígenos de Superfície/análise , Diferenciação Celular , Antígenos HLA/imunologia , Ativação Linfocitária , Linfócitos T/fisiologia , Antígenos de Diferenciação de Linfócitos T/análise , Células Cultivadas , Pré-Escolar , Antígenos HLA/análise , Antígenos HLA-A , Antígenos HLA-B , Antígenos HLA-C , Humanos , Fenótipo , Linfócitos T/classificação , Linfócitos T/imunologia , Timo/crescimento & desenvolvimentoRESUMO
Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-alpha. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA+ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses--but not interleukin 2 responses--were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.
Assuntos
Antígenos de Superfície/biossíntese , Interferon Tipo I/farmacologia , Mitógenos/farmacologia , Timo/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T , Complexo CD3 , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Pré-Escolar , Antígenos HLA/biossíntese , Humanos , Linfócitos T/imunologia , Timo/citologia , Timo/imunologiaRESUMO
Mice were injected with DNP-derivatives of thymus dependent (DNP-OA) and thymus-independent (DNP-Ficoll and TNP-LPS) antigens, and the response to TNP-LPS in vitro was studied after several priming periods. DNP-OA priming decreases the amount of cells responding to TNP-LPS in vitro. In the case of DNP-Ficoll and TNP-LPS-primed cells, there is an initial burst of responsiveness to TNP-LPS, which progressively decreases until an abolishment of TNP-LPS responsiveness is found at day 40 after immunization. The sensitivity to TNP-LPS reappears as new precursor cells differentiate into mature cells. We suggest that B cells progressively gain the capacity to respond to thymus-independent and thymus-dependent antigens (B1-B2 differentiation) and that challenge with a particle antigen increases the ratio of maturation throughout the pathway.
Assuntos
Linfócitos B/imunologia , Lipopolissacarídeos/imunologia , Nitrobenzenos/imunologia , Trinitrobenzenos/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Antígenos , Cruzamentos Genéticos , Dinitrobenzenos/imunologia , Feminino , Ficoll/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mitomicinas/farmacologia , Ovalbumina/imunologia , Baço/imunologiaRESUMO
Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.
Assuntos
Complexo CD3/análise , Ativação Linfocitária , Linfócitos T/fisiologia , Antígenos de Superfície/análise , Separação Celular , Pré-Escolar , DNA/análise , Citometria de Fluxo , Humanos , Lactente , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Linfócitos T/química , Linfócitos T/imunologiaRESUMO
The CD45RA and CD45RO isoforms have been reported to define complementary subsets among CD4+ T cells: CD45RA CD4+ T cells are considered "virgin T cells" and CD45RO "primed T cells." We investigated the secretion of lymphokines by human CD4+ CD45RO and CD4+ CD45RA T helper cells after mitogen stimulation. CD45RA and CD45RO CD4+ T cells were isolated by negative immunoselection using magnetic beads. CD45RO cells, but not CD45RA cells, proliferate well in response to pokeweed mitogen (PWM) or insoluble anti-CD3. Both subpopulations produced interleukin (IL)-2, IL-6, and interferon (IFN)-gamma when stimulated with PWM for 1-4 days. Only Day 1 supernatants from CD45RO cells contained moderate amounts of IL-4. After 14 days of continuous culture and stimulation with PWM, the CD45RA subset had lost the expression of CD45RA and gained that of CD45RO. When long-term cultured CD45RA or CD45RO cells were treated with insoluble anti-CD3, they incorporated [3H]thymidine at similar levels, but only CD45RO cells secreted IL-4 and significantly increased their secretion of IFN-gamma. These data indicate that despite phenotype conversion, the two subpopulations maintain functional differences in the secretion of lymphokines, thus suggesting that circulating CD45RA and CD45RO cells may represent different lines of differentiation.
Assuntos
Antígenos CD/imunologia , Antígenos CD4/imunologia , Antígenos de Histocompatibilidade/imunologia , Interleucinas/metabolismo , Subpopulações de Linfócitos T/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Antígenos Comuns de Leucócito , Ativação Linfocitária/imunologia , Fenótipo , Subpopulações de Linfócitos T/metabolismoRESUMO
Treatment of unfractionated human thymocytes in culture with the synthetic glucocorticoid dexamethasone induced cell death, as measured by trypan blue exclusion, after several hours of incubation. In purified subsets of human cortical and medullary thymocytes dexamethasone caused cell lysis with similar kinetics in both populations; 50% of thymocytes were killed after 20-24 h of incubation with the steroid. The mechanism of dexamethasone-induced cell death seems to correspond to apoptosis since degradation of DNA into oligonucleosome-sized fragments could be observed in the cultures treated with the steroid. A certain degree of DNA fragmentation and cell death could also be observed in control cultures of thymocytes. In contrast, peripheral T lymphocytes were resistant to the cytolytic effect of glucocorticoid hormone. The killing of human thymocytes by dexamethasone was inhibited by cycloheximide, suggesting that this cell death program requires a fully operating protein synthesis machinery and perhaps the induction of new proteins.
Assuntos
Dexametasona/toxicidade , Linfócitos T/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Pré-Escolar , Cicloeximida/farmacologia , Dano ao DNA , Dexametasona/antagonistas & inibidores , Humanos , Inibidores da Síntese de Proteínas/farmacologia , Timo/citologiaRESUMO
In recent years, several studies have confirmed the clonal elimination of thymocytes with receptors that recognize Ag and MHC molecules present on the membrane of thymic stromal cells, a process that may be relevant to the establishment of self-tolerance. In our work, we show that anti-CD3 treatment of single positive CD4+ or CD8+ human medullary thymocytes (obtained by anti-CD1a plus C) induces their apoptotic death. Some events commonly associated with the early steps of normal activation (IL-2R expression, increase in cytoplasmic Ca2+) are also induced after anti-CD3 treatment. Nevertheless, IL-2 is not secreted by these activated cells. The addition of exogenous IL-2 inhibits the apoptosis induced by anti-CD3. We suggest that the lack of secretion of IL-2 by medullary thymocytes may be a physiologic mechanism implicated in the process of negative selection that leads to tolerance.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Tolerância Imunológica , Interleucina-2/farmacologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Timo/citologia , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3 , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8 , Cálcio/fisiologia , Sobrevivência Celular , Citoplasma/metabolismo , DNA/análise , Humanos , Técnicas In Vitro , Ativação Linfocitária , Receptores de Interleucina-2/metabolismo , SolubilidadeRESUMO
We have studied the effects of phorbol-dibutyrate (PBu2), a protein kinase C (PKC) activator, on the proliferation of peripheral human T cells and thymocyte subpopulations selected by treatment with monoclonal antibodies and complement: pre-thymocytes (CD1a-CD3-CD4-CD8-), cortical thymocytes (CD3-, class I- antigens) and medullary thymocytes (enriched as CD1a- cells). PBu2 induces a dose-dependent proliferative response in human peripheral blood T cells at concentrations greater than 6 ng/ml, this proliferation being mediated by the autocrine interleukin 2 (IL2)/IL2 receptor (IL2R) pathway. Pre-thymocytes respond to PBu2 in a way similar to T cells, being able to secrete IL2 in significant amounts and express the p55 chain of IL2R. On the other hand, cortical thymocytes are not induced to proliferate after PKC activation and neither expression of the p55 chain of IL2R nor IL2 secretion is observed. Human medullary thymocytes, phenotypically identical to peripheral blood T cells, show no proliferation in response to PBu2 at any concentration tested unless IL2 is supplied to the cultures. The activation of PKC induces the expression of IL2R in these cells, but not IL2 secretion. The implications of PKC activation in thymic maturation, the role of IL2 and the relevance of the differences between medullary thymocytes and peripheral blood T cells are discussed.
Assuntos
Ativação Linfocitária , Proteína Quinase C/metabolismo , Linfócitos T/fisiologia , Diferenciação Celular , Separação Celular , Ativação Enzimática , Humanos , Interleucina-2/fisiologia , Dibutirato de 12,13-Forbol/farmacologia , Receptores de Interleucina-2/fisiologia , Linfócitos T/enzimologia , Timo/citologiaRESUMO
Alkaline phosphatase activity was assayed by a microtiter assay (with p-nitrophenylphosphate used as substrate) in the plasma membrane of mouse spleen cells activated in vitro by the B cell mitogen LPS and the T cell-dependent B cell mitogen, PWM. No activity was detected in spleen cells cultured in the presence of the T cell mitogens PHA and Con A. This alkaline phosphatase activity was detected in the blast-enriched 30 to 40% fraction of discontinuous Percoll gradients in LPS-treated cultures, and the enzymatic activity assayed was susceptible to inhibition by the alkaline phosphatase inhibitors EDTA and L-phenylalanine. These data support the idea that the membrane alkaline phosphatase activity could be used as a marker for activated B cells.
Assuntos
Fosfatase Alcalina/metabolismo , Linfócitos B/enzimologia , Ativação Linfocitária , Animais , Linfócitos B/imunologia , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/imunologia , Ácido Edético/farmacologia , Concentração de Íons de Hidrogênio , Linfoma/enzimologia , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos AKR , Fenilalanina/farmacologia , Plasmocitoma/enzimologia , Plasmocitoma/imunologiaRESUMO
Specific immunological responses to the idiotypic epitopes present in the surface immunoglobulin (Ig) of the clonal tumour population can be induced for active immunotherapy in patients with B-cell non-Hodgkin lymphoma (NHL). The clonality of the tumour cells should have important implications for the success of the implemented therapy. Here we report on the case of a patient enrolled in a protocol of active idiotypic immunotherapy in which previous cytofluorometric analysis showed a major IgM+, kappap+ population in the tumoral cell suspensions. However, sequence analysis of both tumour sample and tumour-derived hybrids revealed the presence of two unrelated clones that used different VH and VK gene segments. It was possible to obtain hybridomas secreting these two different IgM, kappap idiotypic proteins. The patient was immunised with a mixture of these two idiotypic Igs conjugated to keyhole limpet haemocyanin. Anti-idiotypic antibodies directed against both tumour-associated proteins were detected. This is the first case of anti-idiotypic therapy in a patient with a biclonal NHL. Our work calls attention to the question of clonality in the context of idiotypic vaccination in NHL patients.