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1.
J Biol Regul Homeost Agents ; 24(3): 273-85, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20846475

RESUMO

Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cbeta (PKCbeta) isoform in Jurkat cells, to explore its influence on the expression of surface antigens (CD69) and the cytokines interleukin-8 (IL-8), tumour necrosis factor (TNF)-alpha and beta, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCbeta construct (as-PKCbeta-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCbeta-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCalpha-pREP3 plasmid. IL-2 production, in contrast, was strongly inhibited in both transfectant populations stimulated by PMA plus the calcium ionophore ionomycin. Three clones (asb1/asb2/asb3), selected from as-PKCbeta-pREP3 transfectants, showed decreased PKCbeta protein levels (40 percent, 50 percent and 60 percent, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (alpha, delta, epsilon, gamma, theta, and lambda lambda/tau) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCbeta-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCbeta-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFbeta message levels were highly increased in asb2/asb3 clones, without affecting TNFalpha secretion. Analysis of the mitogen-activated protein kinase (MAP Kinase) signalling pathway showed unaltered extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation but increased activation of c-Jun N-terminal kinase (JNK1) and its substrate, the transcription factor ATF-2 (activated transcription factor-2), which are involved in IL-2 gene expression. Our results revealed new PKCbeta functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCbeta in IL-2 secretion/transcription and IL-2R regulation.


Assuntos
Linfoma de Células T/imunologia , Proteína Quinase C/fisiologia , RNA Antissenso/genética , Fator 2 Ativador da Transcrição/metabolismo , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Western Blotting , Humanos , Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/análise , Interleucina-8/biossíntese , Células Jurkat , Lectinas Tipo C/análise , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosforilação , Proteína Quinase C/análise , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Receptores de Interleucina-2/genética , Transfecção
2.
Clin Exp Allergy ; 39(7): 1088-98, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19400901

RESUMO

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Engenharia Genética , Imunoterapia/métodos , Proteínas Recombinantes de Fusão/imunologia , Adolescente , Adulto , Idoso , Alérgenos/isolamento & purificação , Alérgenos/uso terapêutico , Animais , Antígenos de Dermatophagoides/isolamento & purificação , Antígenos de Dermatophagoides/uso terapêutico , Proteínas de Artrópodes , Proliferação de Células , Clonagem Molecular , Cisteína Endopeptidases , Feminino , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Imunoglobulina E/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Testes Cutâneos , Linfócitos T/imunologia , Adulto Jovem
3.
Eur J Biochem ; 268(11): 3321-31, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11389735

RESUMO

The acid alpha-L-fucosidase is usually found as a soluble component of lysosomes where fucoglycoconjugates are degraded. In the present investigation, we have demonstrated the existence of a cell surface protein with enzymatic alpha-L-fucosidase activity that crossreacts specifically with a rabbit anti-(alpha-L-fucosidase) Ig. By different approaches, this alpha-L-fucosidase, which represents 10-20% of the total cellular fucosidase activity, was detected in all the tested human cells (hemopoietic, epithelial, mesenchymal). Two bands of approximately 43-49 kDa were observed, although theoretical data support the possibility of having the same genetic origin that the known 50 to 55-kDa Mr alpha-L-fucosidase. We speculate about an alternative traffic pathway for the plasma membrane alpha-L-fucosidase to work on the rapid turnover of glycoproteins.


Assuntos
Membrana Celular/enzimologia , alfa-L-Fucosidase/análise , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Western Blotting , Fracionamento Celular , Linhagem Celular , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/enzimologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Linfócitos/enzimologia , Monócitos/enzimologia , Neutrófilos/enzimologia , Testes de Precipitina , alfa-L-Fucosidase/imunologia , alfa-L-Fucosidase/metabolismo
5.
Immunol Lett ; 61(1): 7-13, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9580431

RESUMO

Recently, we reported that IL-12 increased expression and function of CD26/DPPIV, this may be a new cellular pathway of the Th1-like immune responses. Here, we looked for a specific subset which would respond to CD26 upregulation by IL-12. Contrary to previously described results, under our culture conditions (1 microg/ml of PHA), IL-12 enhanced preferentially the CD8 cell proliferation. By using dual fluorescence analysis, IL-12-dependent CD26 expression was found in both CD4 and CD8 (previously CD26+ or CD26-) activated T cells and, moreover, the CD45RO percentage was unaffected. However, the density of CD45RO Ag (which was reported to coexpress with CD26) was impaired. These effects can be implicated in the biological functions of IL-12 and provide some clinical possibilities.


Assuntos
Interleucina-12/farmacologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Dipeptidil Peptidase 4/análise , Dipeptidil Peptidase 4/biossíntese , Dipeptidil Peptidase 4/genética , Humanos , Interleucina-12/fisiologia , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/metabolismo , Fenótipo , Regulação para Cima/fisiologia
6.
Biochem J ; 331 ( Pt 3): 753-61, 1998 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9560301

RESUMO

Prothymosin alpha (ProTalpha) is an acidic nuclear protein the expression of which is related to the proliferation and differentiation processes in mammalian cells. In the present study we have stably transfected HL-60 cells, a biological system that allows the study of both proliferation and differentiation, with recombinant vectors encoding sense and antisense ProTalpha mRNA. In the HL-60 cell clones overexpressing ProTalpha we observed an acceleration in the growth rate, whereas expression of the antisense orientation showed the opposite effect. Moreover, cell-cycle analysis demonstrated that the G1-phase was shortened in the cells expressing the sense construct. Before studying how ProTalpha affects differentiation, we showed that the down-regulation of ProTalpha gene during differentiation occurs in all mammalian cell lines (HL-60, K562, U937, MEL C88, N2A and PC12) analysed. The biological effect evoked by the induction of the ProTalpha sense vector was the retardation of cell differentiation, although expression of the antisense construct showed no effect on differentiation. In conclusion, our findings provide evidence that ProTalpha is directly implicated in cellular proliferation and that the maintenance of high levels of ProTalpha inside HL-60 cells is incompatible with their ability to differentiate.


Assuntos
Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Regulação da Expressão Gênica/genética , Precursores de Proteínas/genética , Timosina/análogos & derivados , Replicação do DNA/genética , Regulação para Baixo/fisiologia , Imunofluorescência , Fase G1/fisiologia , Células HL-60 , Humanos , Proteínas Nucleares/metabolismo , RNA Antissenso/genética , RNA Mensageiro/genética , Timosina/genética , Transfecção/genética
7.
Immunobiology ; 197(5): 522-33, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9413751

RESUMO

Research of a cellular pathway activated by IL-12 which may result in new therapeutical approaches for IL-12, led us to find an intriguing relationship between IL-12 and CD26/DPPIV ectopeptidase on activated T cells. Both the percentage and median fluorescence intensity (MFI) of CD26+ cells in the PHA-stimulated PBMC or lymphoblasts increased when IL-12 (optimum dose, 2 ng/ml) was present. Maximum CD26 expression was observed on day-2 cultures of lymphoblasts, the presence of IL-12 receptor probably being necessary for this upregulation. In addition, CD26 upregulation correlated with enhanced DPPIV function. Enzyme affinity and secretion of the soluble form of DPPIV were not affected by IL-12. Kinetic behaviours of Ag expression and enzymatic activity support a different CD26 regulation pathway by IL-12. These data suggest that the correlation found in vivo between the CD26 expression and Th1-like immune responses is due to this IL-12-dependent upregulation.


Assuntos
Dipeptidil Peptidase 4/biossíntese , Interleucina-12/farmacologia , Ativação Linfocitária , Linfócitos T/efeitos dos fármacos , Células Th1/enzimologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/fisiologia , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , Receptores de Interleucina/efeitos dos fármacos , Receptores de Interleucina/fisiologia , Receptores de Interleucina-12 , Células Th1/imunologia
8.
Transplantation ; 58(4): 511-7, 1994 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-8073521

RESUMO

Natural suppressor (NS) activity is mediated by cells (NS cells) of bone marrow origin with ability to suppress nonspecifically proliferative responses of lymphocytes. Here we show that pharmacologic concentrations (10(-6)-10(-8) M) of glucocorticoids (GC) greatly inhibit NS activity, as detected by coculturing bone marrow and spleen cells stimulated with B cell (LPS) or T cell (concanavalin A) mitogens. Progesterone antagonizes GC-mediated inhibition of NS activity, suggesting that GC were acting through a receptor-dependent mechanism. A prior treatment of NS cells with GC (10(-5) M) has no effect on the NS activity mediated by these cells. GC are required in culture during the first 24 hr of the suppressor assay. Addition of low amounts of IFN-gamma to GC-treated cultures fully reverses NS cell-mediated suppression. IL-2 produces a reversion as well, while addition of IL-3 or IL-4 does not prevent the GC effect. Neutralizing anti-IFN-gamma antibodies, but not anti-IL-2 or anti-TGF-beta, greatly inhibit NS activity in absence of GC. Taken together, these results indicate that GC inhibit NS activity by impairing endogenous cytokine production required to obtain successful NS cell activation, and not by acting directly on NS cells (i.e., inhibiting the secretion of putative NS factors). Among the cytokines involved in NS cell activation, IFN-gamma appears to be critical, since its addition readily overrides the GC effect and its neutralization results in strong inhibition of NS activity.


Assuntos
Células da Medula Óssea , Glucocorticoides/farmacologia , Interferon gama/farmacologia , Interleucina-2/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Medula Óssea/imunologia , Células Cultivadas , Glucocorticoides/antagonistas & inibidores , Ativação Linfocitária/imunologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes , Baço/citologia , Linfócitos T Reguladores/imunologia
9.
J Immunol ; 148(8): 2636-42, 1992 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-1560214

RESUMO

We have shown previously that an IgM mAb (A10) recognizing Ehrlich tumor (ET) cell surface carbohydrates, inhibits in vivo ET growth by a macrophage-dependent mechanism. The inhibition mechanism involving both IgM and macrophages was unclear because receptors for IgM on macrophages are controversial and another monoclonal IgM (E1), also recognizing ET cell surface carbohydrates, was completely unable to show any protective effect. Here we show that A10, but not E1, was able to promote adhesion between macrophages and ET cells by a receptor for IgM-independent mechanism. Immunofluorescence studies showed that A10, but not E1, did react with macrophages if these cells were preincubated with a source of Ag spontaneously released from ET cells. This Ag release appeared to be required for A10-mediated adhesion, because adhesion was not obtained when ET cells fixed with paraformaldehyde were used. Cytostasis studies performed with macrophages stimulated with L-929 conditioned medium and ET cells showed that A10, but not E1 nor unrelated IgM, was able to inhibit ET cell proliferation in vitro by a mechanism involving cell contact between both cell populations. Therefore, IgM inhibition of ET growth, both in vivo and in vitro, could be explained by a lectin-like mechanism, where IgM, recognizing Ag of tumor origin, bridges macrophages to tumor cells.


Assuntos
Anticorpos Monoclonais/imunologia , Comunicação Celular , Citotoxicidade Imunológica , Imunoglobulina M/imunologia , Lectinas/fisiologia , Macrófagos/fisiologia , Neoplasias Experimentais/patologia , Animais , Adesão Celular , Feminino , Camundongos , Camundongos Endogâmicos C57BL
10.
Int J Cancer ; 47(1): 86-91, 1991 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-1824690

RESUMO

C57BL/6J mice immunized with devitalized Ehrlich tumor (ET) cells produce high serum levels of IgM antibodies to ET cell-surface carbohydrates that are critical in the observed resistance against this tumor. However, this response is not found in ET-bearing mice at any stage of tumor development. Since previous studies had shown splenic natural suppressor (NS) cells in ET-bearers, their role in such IgM impairment was assessed. Here we show that tumor-bearers' spleen cells (TBSC) are unable to produce IgM in vitro in response to LPS, due to the presence of NS cells. Nevertheless, TBSC do produce IgM antibodies to ET cell-surface carbohydrates in increasing amounts as the tumor progresses. Yet these antibodies are not detected in sera of ET-bearers and are greatly decreased in immunized mice with a growing tumor. Moreover, increasing amounts of circulating carbohydrates, able to absorb most specific IgM, are found in ET-bearing sera associated with a large molecular size structure(s). These carbohydrates are also found in ET cell-culture supernatants and cell-free ascites fluid derived from this tumor, indicating their tumor origin. Taken together, our results indicate that lack of specific IgM antibodies in ET-bearing mice is not due to faulty production, but to in vivo absorption by carbohydrates shed from ET cells in increasing amounts as the tumor progresses. Thus, NS cells are unable to suppress this IgM production in vivo, despite the strong suppressor activity they show for many responses in vitro.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Carcinoma de Ehrlich/imunologia , Imunização , Linfócitos T Reguladores/imunologia , Animais , Formação de Anticorpos , Antígenos de Superfície/biossíntese , Carcinoma de Ehrlich/terapia , Cromatografia em Gel , Imunoglobulina M/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Baço/metabolismo
11.
Cancer Res ; 50(22): 7301-6, 1990 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-2224860

RESUMO

We have shown previously that IgM from Ehrlich tumor (ET)-immunized mice, recognizing ET cell surface carbohydrates, protects control mice to a subsequent tumor challenge. The factors involved in such IgM-mediated protection were unknown, since it was independent of complement activation. Here, we have extended these in vivo studies by means of monoclonal IgM antibodies. Two of them (A10 and E1), strongly recognizing ET cells and with specificity to ET cell surface carbohydrates, were selected. The results show that A10 (but not E1 or unrelated IgM antibodies) is able to protect nonimmunized mice against ET growth. Protection by A10 was also seen by reducing 800-fold the initial dose; however, E1 was unsuccessful whatever the dose used. A10-mediated protection was observed in C3-defective mice (cobra venom factor treated) or in C5-deficient DBA/2, but not in silica-treated animals. Endotoxin removal did not affect the protection afforded by A10 while specific IgM depletion prevented any protective effect. In addition, the relationship between natural antibodies of IgM isotype recognizing ET cell surface carbohydrates and mouse strain resistance to this tumor is established. Similarly, this natural resistance seems to be complement independent but macrophage mediated. Therefore, these results indicate that some IgM molecules recognizing cell surface carbohydrates may participate in in vivo tumor suppression by a macrophage-dependent mechanism.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Carcinoma de Ehrlich/imunologia , Macrófagos/imunologia , Animais , Divisão Celular , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Feminino , Imunidade Celular , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos
12.
Int J Cancer ; 44(2): 307-14, 1989 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-2527208

RESUMO

Spleen cells from C57BL/6J mice bearing Ehrlich carcinoma growing as a solid tumor show progressive unresponsiveness to concanavalin A (Con A) and lipopolysaccharide (LPS) mitogens. This is accompanied by striking spleen enlargement with marked hematopoietic activity. Lymphoproliferative assays of normal spleen cells in co-culture with tumor-bearing spleen cells (TBSC) show that: (a) TBSC contain non-specific suppressor cells able to abrogate both Con A and LPS responses, or mixed lymphocyte reaction, of normal spleen cells and (b) suppression by TBSC is MHC-unrestricted, non-prostaglandin-mediated and greatly enhanced by Con A supernatants. Suppressor cells associated with TBSC are large, low-density cells without markers of mature B or T lymphocytes or of the mononuclear phagocyte system. Most appear to be asialo-GM1-negative, as suppression was only partially inhibited by treatment with anti-asialo-GM1 and complement. Since NK activity is lacking in TBSC, our data strongly suggest that these "null" suppressor cells are related to the natural suppressor (NS) cells found described in normal bone-marrow and neonatal spleens, or induced in adult spleens by total lymphoid irradiation, graft-vs.-host disease, or cyclophosphamide treatment.


Assuntos
Carcinoma de Ehrlich/imunologia , Gangliosídeo G(M1) , Baço/imunologia , Linfócitos T Reguladores/imunologia , Animais , Concanavalina A/farmacologia , Glicoesfingolipídeos/análise , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos , Prostaglandinas/biossíntese
13.
Rev Clin Esp ; 183(2): 55-6, 1988 Jun.
Artigo em Espanhol | MEDLINE | ID: mdl-3175164
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