Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Endocrinology ; 142(1): 165-73, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11145579

RESUMO

Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.


Assuntos
Cartilagem Articular/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Idoso , Substituição de Aminoácidos , Animais , Neoplasias da Mama , Cartilagem Articular/efeitos dos fármacos , Feminino , Variação Genética , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/farmacocinética , Cinética , Taxa de Depuração Metabólica , Mutagênese Sítio-Dirigida , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Especificidade por Substrato , Sulfatos/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas
2.
Drug Metab Dispos ; 28(5): 590-7, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10772640

RESUMO

The purpose of this investigation was to characterize the stability, activity, and interactions of recombinant human nerve growth factor (rhNGF) in various biological matrices in vitro and in vivo. rhNGF (10 microg/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decrease in the recovery of rhNGF after incubation at lower concentrations (20 ng/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size exclusion HPLC analysis indicated that rhNGF forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that alpha(2)-macroglobulin (alpha(2)M) is the major plasma component that binds to rhNGF. Furthermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH(3)NH(2). Changes in the pH environment did not affect the interaction of rhNGF with alpha(2)M. We also determined that the binding of rhNGF to CH(3)NH(2)-treated pure alpha(2)M or alpha(2)M present in human plasma substantially diminished its immunoreactivity and bioactivity detection. The interaction of rhNGF with activated alpha(2)M was reversed and inhibited by coincubation with dimethyl sulfoxide. Released rhNGF under these conditions was fully bioactive. (125)I-rhNGF also binds to alpha(2)M by forming similar (125)I-rhNGF/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled material was in the form of a (125)I-rhNGF/HMW complex. These studies have provided a better understanding of the nature of the interactions of rhNGF with plasma components as well as methods to enhance, reverse, and inhibit these interactions.


Assuntos
Fatores de Crescimento Neural/metabolismo , Animais , Proteínas Sanguíneas/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Cromatografia em Gel , Dimetil Sulfóxido/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo , Camundongos , Fatores de Crescimento Neural/sangue , Fatores de Crescimento Neural/farmacologia , Células PC12 , Ligação Proteica/efeitos dos fármacos , Ratos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , alfa-Macroglobulinas/metabolismo
3.
Drug Metab Dispos ; 28(5): 598-607, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10772641

RESUMO

In this study, we have characterized the metabolism, tissue disposition, excretion routes, and plasma pharmacokinetics of recombinant human nerve growth factor after single and multiple s.c. administration in male cynomolgus monkeys. Unlabeled nerve growth factor (NGF; 2 mg/kg) was administered three times a week for 4 weeks and a full pharmacokinetic profile was obtained for doses 1 and 12. For the tissue distribution studies, 0.8 microg/kg of trace (125)I-labeled recombinant human nerve growth factor was dosed. Histological analysis of emulsion-microautoradiography indicated that specific (125)I-NGF labeling was confined to sections of nerves most frequently localized adjacent to large vessels in sections of kidney, spleen, liver, and salivary gland. A small percentage of large neurons within the sympathetic ganglia were intensely labeled, as well as large neurons within the dorsal root ganglia. We found an increased disposition of (125)I-NGF in parts of the peripheral nervous system (including sympathetic ganglia) from 8 to 24 h postdose. In contrast, radioactivity in most non-neuronal tissues declined. This suggests specific uptake in these target tissues known to express specific receptors for NGF. We also identified changes in pharmacokinetic parameters after single versus chronic s. c. administration. These studies demonstrated that s.c. administration of NGF at 0.8 microg/kg doses in monkeys is capable of accessing and localizing in the target tissues.


Assuntos
Fatores de Crescimento Neural/farmacocinética , Animais , Área Sob a Curva , Autorradiografia , Células CHO , Cricetinae , Nefropatias Diabéticas/tratamento farmacológico , Eletroforese em Gel de Poliacrilamida , Fezes/química , Meia-Vida , Humanos , Injeções Subcutâneas , Radioisótopos do Iodo , Macaca fascicularis , Masculino , Fatores de Crescimento Neural/administração & dosagem , Testes de Precipitina , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacocinética , Distribuição Tecidual
4.
Dev Biol Stand ; 97: 121-33, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10463538

RESUMO

We have developed a novel strategy for a rapid bioassay that is accurate, precise, sensitive, and high capacity. It is capable of quantifying ligand bioactivity by measuring ligand-induced receptor tyrosine kinase activation in terms of receptor-phosphorylation. The assay, termed << Kinase Receptor Activation>> or KIRA, uses two separate microtiter plates, one for ligand stimulation of intact cells, and the other for receptor capture and phosphotyrosine ELISA. The assay makes use of either endogenously expressed receptors or stably transfected receptors with a polypeptide flag. KIRA assays for the ligands IGF-I and NGF were compared to their corresponding endpoint bioassays (3T3 cell proliferation for IGF-I and PC12 cell survival for NGF). The KIRA assays showed excellent correlation with the more classical endpoint bioassays. Further, they were highly reproducible, minimizing the requirement for repeat assays. The KIRA assay format has great potential as a rapid, accurate and precise bioassay, both for potency determination as well as stability-indicating analyses.


Assuntos
Bioensaio/métodos , Receptores Proteína Tirosina Quinases/metabolismo , Células 3T3 , Animais , Bioensaio/estatística & dados numéricos , Células CHO , Divisão Celular , Linhagem Celular , Cricetinae , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Humanos , Ligantes , Camundongos , Células PC12 , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor IGF Tipo 1/metabolismo , Receptor trkA , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transdução de Sinais , Transfecção
5.
J Pharm Biomed Anal ; 19(6): 883-91, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10698554

RESUMO

We have developed a novel strategy for a rapid bioassay that is accurate, precise, sensitive, and high capacity. It is capable of quantifying ligand bioactivity by measuring ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a 'kinase receptor activation' or KIRA, utilizes two separate microtiter plates, one for ligand stimulation of intact cells, and the other for receptor capture and phosphotyrosine ELISA. The assay makes use of either endogenously expressed receptors or stably transfected receptors with a polypeptide flag. KIRA assays for the ligands IGF-I and NGF were compared to their corresponding endpoint bioassays (3T3 cell proliferation for IGF-I and PC12 cell survival for NGF). The KIRA assays showed excellent correlation with the more classical endpoint bioassays. Further, they were highly reproducible, minimizing the requirement for repeat assays. The KIRA assay format has great potential as a rapid, accurate and precise bioassay, both for potency determination as well as stability-indicating analyses.


Assuntos
Bioensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Receptores Proteína Tirosina Quinases/análise , Ativação Enzimática , Humanos , Fator de Crescimento Insulin-Like I/análise , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkA/análise , Células Tumorais Cultivadas , Proteínas do Envelope Viral/análise
6.
Biochemistry ; 37(25): 8870-8, 1998 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-9636028

RESUMO

IGF-1 (insulin-like growth factor 1) is a 70-residue protein hormone which has both metabolic and mitogenic activities mediated through IGF-1 binding to cell surface receptors. However, an unrelated class of proteins, the IGF-binding proteins (IGFBPs) also bind IGF-1 in the serum and tissues and block or modulate its activity in vivo. Therefore, inhibitors of the IGFBPs can alter the distribution between free and bound IGF-1 [Loddick, S. A., Liu, X.-J., Lu, Z.-X., Liu, C., Behan, D. P., Chalmers, D. C., Foster, A. C., Vale, W. W., Ling, N., and De Souza, E. B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898] and potentially affect the distribution of IGF-1 among body tissues. We report here that phage-displayed peptide libraries have yielded a peptide that binds IGFBP-1 and produces IGF-like activity at sub-micromolar concentrations. The 14-residue peptide has an extremely well-defined solution conformation that can aid in the design of smaller, orally active compounds. Interestingly, the peptide structure contains a helix, as does one region of IGF-1 previously implicated in IGFBP binding, yet displays side chains different from those of the IGF-1 helix I. Furthermore, an IGF-1 variant lacking receptor-signaling activity in vitro is shown here to produce IGF-like mitogenic and metabolic activity in vivo. These results suggest that small antagonist mimetics of protein ligands, identified by binding selection to otherwise inhibitory factors, may be useful as indirect agonists for a variety of therapeutic applications.


Assuntos
Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/metabolismo , Mimetismo Molecular , Sequência de Aminoácidos , Animais , Bacteriófago M13/metabolismo , Ligação Competitiva/efeitos dos fármacos , Insulina/farmacologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/farmacologia , Ligantes , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Receptor IGF Tipo 1/deficiência
7.
Exp Cell Res ; 234(2): 354-61, 1997 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9260905

RESUMO

A rapid, sensitive, and high-capacity assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a "kinase receptor activation" or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC50 of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fatores de Crescimento Neural/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Células CHO , Cricetinae , Ativação Enzimática , Ligantes , Fatores de Crescimento Neural/sangue , Fatores de Crescimento Neural/farmacologia , Células PC12 , Fosforilação , Ratos , Ratos Wistar , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Proteínas Recombinantes de Fusão , Sensibilidade e Especificidade , Simplexvirus , Proteínas do Envelope Viral/genética
8.
Anal Biochem ; 235(2): 207-14, 1996 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-8833330

RESUMO

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.


Assuntos
Proteínas de Transporte/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/farmacologia , Neuregulina-1 , Receptor ErbB-2/metabolismo , Animais , Western Blotting , Humanos , Fosforilação , Coelhos , Receptores Proteína Tirosina Quinases/metabolismo , Sensibilidade e Especificidade , Células Tumorais Cultivadas
10.
J Infect Dis ; 167(2): 411-7, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8421174

RESUMO

Patients from across the spectrum of clinical manifestations of Leishmania chagasi infection were evaluated for in vitro correlates of immunity. Peripheral blood mononuclear cells were assayed for parasite-specific lymphoproliferation, cytokine generation, and the capacity to activate autologous macrophages to kill intracellular amastigotes. Patients with acute kala-azar were generally unreactive in each of these assays. Children with subclinical infection demonstrated relatively low levels of proliferation and interferon-gamma production, but none went on to develop overt kala-azar during the study. Patients evaluated after therapy for kala-azar demonstrated yet higher levels of lymphoproliferation and cytokine generation and produced low but significant levels of cytokines in vitro in response to parasite antigens, but not during the activation of infected macrophages. Finally, peripheral blood mononuclear cells from adults with positive delayed-type hypersensitivity responses and no history of kala-azar showed the broadest reactivity in vitro. These patients' cells generated the largest amounts of activating cytokines in vitro during the activation of autologous macrophages to a leishmanicidal state.


Assuntos
Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leucócitos Mononucleares/imunologia , Doença Aguda , Adolescente , Adulto , Animais , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Citocinas/biossíntese , Humanos , Hipersensibilidade Tardia , Interferon gama/biossíntese , Interleucina-5/biossíntese , Ativação Linfocitária , Ativação de Macrófagos , Linfócitos T/imunologia
11.
Infect Immun ; 59(12): 4710-4, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1937832

RESUMO

Neutralization of interleukin 4 (IL-4) at the time of infection with Leishmania major allowed susceptible BALB/c mice to heal. Recombinant IL-4, however, had little effect on the course of L. major infection in resistant C57BL/6 mice, nor did coinfection with Nippostrongylus brasiliensis, despite marked elevation of endogenous IL-4 levels.


Assuntos
Interleucina-4/fisiologia , Leishmania tropica , Leishmaniose Cutânea/imunologia , Animais , Interferon gama/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Nematoides/imunologia , Nippostrongylus
12.
J Immunol ; 147(5): 1653-8, 1991 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-1831830

RESUMO

Leishmania major disseminates in genetically susceptible BALB/c mice to cause fatal disease. Progressive infection has been linked to the failure of parasite-specific Th1, IFN-gamma-producing, CD4+ T lymphocytes to expand and direct macrophage activation and control of intracellular parasitism. In contrast, Th2 CD4+ cell expansion accompanies disease progression. Immunomodulation using CD4 cell depletion at the time of infection results in control of infection and Th1 CD4+ cell expansion. A Th1-like cell line, H1A, was established from the draining lymph nodes of an anti-CD4-pretreated BALB/c mouse infected with L. major, H1A was CD4, TCR(+)-alpha/beta, and released IL-2 and IFN-gamma in response to parasite Ag. A Th2-like cell line, U1A, was established from the lymph node cells of an infected BALB/c mouse that was also CD4, TCR(+)-alpha/beta but released IL-4 and IL-5 after stimulation. Mice with severe combined immunodeficiency were reconstituted with H1A and U1A before infection with L. major. Non-reconstituted mice were unable to restrict parasite growth. Mice reconstituted with H1A healed infection, whereas mice reconstituted with U1A suffered exacerbation of disease. Analysis of spleen cells by flow cytometry confirmed the reconstitution of CD4+ cells in both instances, and stimulation with mitogen established that the lymphokine profile of the donor cells had been maintained during 6 to 8 wk of infection. Histologic analysis of the lesions confirmed migration of donated cells to sites of infection. Neutralization of IFN-gamma in H1A-reconstituted mice and IL-4 in U1A-reconstituted mice reversed the disease phenotype mediated by the two cell lines. These data demonstrate the capacity of CD4+ T cells alone to modulate both positively and negatively the course of leishmaniasis in a lymphokine-dependent manner.


Assuntos
Síndromes de Imunodeficiência/imunologia , Leishmaniose/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos CD4/análise , Antígenos CD8 , Linhagem Celular , Feminino , Interferon gama/fisiologia , Interleucina-4/fisiologia , Camundongos , Camundongos Endogâmicos BALB C
13.
Proc Natl Acad Sci U S A ; 88(16): 7011-5, 1991 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-1908085

RESUMO

The expression of interleukin (IL) 2, IL-4, IL-10, and interferon gamma (IFN-gamma) by lymphocyte subsets was examined during infection of resistant C57BL/6 and susceptible BALB/c mice with the protozoan parasite Leishmania major. CD4+ and CD8+ T lymphocytes and B lymphocytes were isolated from the lymph nodes draining infectious lesions, and their RNA was examined for lymphokine transcripts. Distinct patterns of CD4+ cell cytokine expression were apparent: C57BL/6 CD4+ cells contained IFN-gamma and IL-2 mRNA, whereas BALB/c CD4+ cells expressed IL-4 and IL-10 message. CD8+ cells contributed little lymphokine expression during disease, but B cells were a major source of IL-2 mRNA in both strains of mice. BALB/c mice made resistant by treatment with anti-CD4 antibody at the time of infection repopulated lymph nodes with CD4+ cells that expressed IL-2 and IFN-gamma. Protective treatment with anti-IL-4 antibody in vivo also resulted in the appearance of CD4+ cells with increased IFN-gamma and diminished IL-4 and IL-10 expression. These data establish CD4+ cells as the primary source of IFN-gamma in healing mice and of IL-4 and IL-10 during progressive infection and confirm that the spectral extremes of this disease are characterized by the presence of CD4+ cells expressing Th1 or Th2 phenotypes in vivo.


Assuntos
Subpopulações de Linfócitos B/imunologia , Antígenos CD4/análise , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Interleucinas/biossíntese , Leishmaniose/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Feminino , Interferon gama/genética , Interleucina-10 , Interleucina-2/genética , Interleucina-4/genética , Interleucinas/genética , Leishmaniose/fisiopatologia , Linfonodos/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , RNA Mensageiro/genética , Baço/imunologia
15.
J Exp Med ; 171(1): 115-27, 1990 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-2104918

RESUMO

BALB/c mice infected with Leishmania major develop fatal, progressive disease, despite an immune response characterized by expansion of CD4+ T cells in the draining lymph nodes. The immune response has been further characterized by a lack of IFN-gamma mRNA, but increased IL-4 mRNA in lymphoid tissues, and striking elevation of serum IgE. Treatment of infected BALB/c mice with rIFN-gamma at doses shown to be beneficial in other protozoan infections was insufficient to ameliorate L. major infection. In contrast, neutralization of IL-4 by six weekly injections of mAb 11B11 led to attenuation of disease in 100% of animals, and complete cure in 85%. Resolution of disease required the presence of T cells, and recovered mice remained resistant to reinfection at 12 wk. This immunity was adoptively transferable and was dependent on both CD4+ and CD8+ cells. Although administration of anti-IL-4 was associated with fourfold increase in IFN-gamma mRNA in lymph node cells draining the lesion, the coadministration of neutralizing R4 6A2 anti-IFN-gamma mAb had no effect on resistance to disease. This was in marked contrast to resolution of disease in both resistant C57BL/6- and GK1.5-pretreated BALB/c mice that was abrogated by in vivo treatment with anti-IFN-gamma. These data suggest a novel mechanism of cellular immunity established by interference with the development of Th2 cells during infection.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Interferon gama/uso terapêutico , Interleucina-4/imunologia , Leishmaniose/terapia , Animais , Anticorpos Monoclonais/imunologia , Northern Blotting , Feminino , Hibridomas/imunologia , Imunização Passiva , Leishmania tropica/imunologia , Leishmaniose/imunologia , Leishmaniose/patologia , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Ratos , Proteínas Recombinantes , Baço/imunologia , Baço/patologia
16.
J Exp Med ; 169(1): 59-72, 1989 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-2521244

RESUMO

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.


Assuntos
Interferon gama/fisiologia , Interleucinas/fisiologia , Leishmaniose/fisiopatologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos de Diferenciação de Linfócitos T/imunologia , Regulação da Expressão Gênica , Imunoglobulina E/sangue , Interleucina-4 , Leishmaniose/parasitologia , Linfonodos/fisiopatologia , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/genética , Baço/fisiopatologia
17.
J Clin Invest ; 82(6): 2097-105, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3264291

RESUMO

Infection of monocyte-macrophages with human immunodeficiency virus may be central to the pathogenesis of the acquired immunodeficiency syndrome. The ability of infected macrophages to prime T cells through IL-1 production was investigated in vitro. Purified human monocytes maintained in suspension culture were infected with strain HIV-DV. Intracellular expression of virus p24 antigen increased from undetectable levels immediately after infection to 13-59% of cells by 10-14 d; infected macrophages remained viable for up to 60 d. Supernatants collected between 14 and 20 d after infection were examined in the murine thymocyte co-mitogenesis assay and demonstrated to contain a potent IL-1 inhibitor, designated contra-IL-1. Contra-IL-1 activity was present in all supernatants examined after 4 d of infection, and peaked coincident with peak p24 antigen expression. Inhibitory activity was not present in uninfected cells. Contra-IL-1 activity eluted after gel filtration with an approximate molecular weight of 9 kD. Inhibitory activity was removed by exposure to heat or acid pH, or by incubation with chymotrypsin or staphylococcal V8 protease. Contra-IL-1 did not inhibit IL-2- or IL-4-dependent proliferation of murine T cell lines. Despite its ability to inhibit IL-1 activity, contra-IL-1 did not interfere with the binding of recombinant IL-1 beta to a fibroblast cell line. Contra-IL-1 inhibited the proliferation of normal peripheral blood mononuclear cells to both concanavalin A and tetanus toxoid; inhibition could be attenuated by the addition of exogenous IL-1. Messenger RNA extracted from infected macrophages was examined by Northern analysis for the presence of message to IL-1 beta. No message was apparent, suggesting that the presence of contra-IL-1 was not obscuring the concomitant release of IL-1. Infected macrophages stimulated with endotoxin generated readily detectable message for IL-1 beta. Spleen macrophages purified from two patients with AIDS complicated by immune thrombocytopenia spontaneously expressed p24 antigen in vitro and released contra-IL-1 activity into the media. Contra-IL-1 may contribute to the immune dysfunction of AIDS.


Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Interleucina-1/antagonistas & inibidores , Linfocinas/metabolismo , Macrófagos/imunologia , Células Cultivadas , Quimotripsina , Humanos , Interleucina-2/metabolismo , Interleucina-4 , Interleucinas/metabolismo , Macrófagos/microbiologia , Monócitos/imunologia , RNA Mensageiro/metabolismo , Serina Endopeptidases , Linfócitos T/imunologia
18.
J Immunol ; 141(6): 2132-7, 1988 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-3262649

RESUMO

BALB/cJ mice, which are homozygous for Lshs on chromosone 1, are genetically susceptible to Leishmania donovani (S3), but spontaneously reduce their parasite burdens late in the course of infection. Spleens from chronically infected mice (5 to 8 mo) were found to have T cells that responded to leishmanial Ag by proliferating and secreting immune IFN. An IFN-secreting T cell line derived from the spleen of a chronically infected mouse, after boosting with leishmanial Ag and treatment with Pentostam to kill residual parasites, was able to activate macrophages to kill amastigotes in vitro. Furthermore, after adoptive transfer of this cell line, naive BALB/cJ mice challenged with amastigotes demonstrated a 42-fold reduction in parasite burden compared to controls. Four of five clones derived from the protective T cell line secreted IFN and proliferated between days 7 and 14 after Ag stimulation. All clones were Ly1+2-, L3T4+. Another T cell line, which had Ly1+2-, L3T4+ phenotype, was derived from the lymph nodes of s.c. immunized, uninfected mice. It multipled but never produced IFN in response to leishmanial Ag and failed to protect macrophages against L. donovani infection in vitro or in vivo. This nonprotective T cell line and the fifth clone from the protective line, which also did not secrete IFN, proliferated between days 2 and 7 after Ag stimulation. In summary, leishmania-responsive helper/inducer T cells, which produced IFN but were slow to proliferate in response to Ag in vitro and which were able to activate macrophages to reduce amastigotes in vitro and in vivo, are present in chronically infected BALB/cJ mice and may mediate the decrease in parasite burden during chronic infection.


Assuntos
Separação Celular , Leishmaniose Visceral/imunologia , Linfócitos T/imunologia , Animais , Antígenos de Protozoários/administração & dosagem , Separação Celular/métodos , Células Cultivadas , Doença Crônica , Células Clonais/classificação , Cricetinae , Imunidade Inata , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Baço/patologia , Linfócitos T/classificação , Linfócitos T/parasitologia
19.
Exp Parasitol ; 65(2): 258-68, 1988 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-3127233

RESUMO

Genetically susceptible BALB/c and resistant C57BL/6 mice were infected with Leishmania major and the phenotypes of the responding cells in the draining lymph nodes and cutaneous lesions were analyzed. As early as 1 week, significantly increased numbers of L3T4+ cells as compared to Lyt-2+ cells were present in BALB/c mice lymph nodes (P less than 0.005). Increases in L3T4+ and Lyt-2+ cells were comparable in C57BL/6 mice, resulting in threefold lower L3T4/Lyt-2 ratio than in BALB/c mice. T cell subsets were activated in both strains to express interleukin-2 receptor (IL2R) above resting values, although greater numbers of activated L3T4+ cells were present in the draining lymph nodes from BALB/c at 1 and 3 weeks of infection than in C57BL/6 (P = 0.02). Despite the presence of activated L3T4+ cells in both strains, macrophages differed in the expression of immunologically important surface molecules during infection. Tissue macrophages from BALB/c mice were IgG1/G2b Fc receptor (FcR)+ and Ia- late in disease, whereas macrophages in C57BL/6 became FcR and Ia during healing. BALB/c mice, treated with monoclonal antibody GK1.5 to transiently deplete L3T4+ cells, became resistant to subsequent infection and developed a macrophage phenotype that was FcR- and Ia+. These differences in macrophage phenotype were closely linked to susceptibility during infection with L. major and may play a role in the pathophysiology of murine leishmaniasis.


Assuntos
Leishmania tropica/imunologia , Leishmaniose/imunologia , Linfonodos/imunologia , Linfócitos/imunologia , Macrófagos/imunologia , Animais , Linfócitos B/imunologia , Contagem de Células , Suscetibilidade a Doenças , Antígenos de Histocompatibilidade Classe II/análise , Imunidade Inata , Leishmania tropica/genética , Leishmaniose/genética , Leishmaniose/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Receptores Fc/análise , Receptores Imunológicos/análise , Receptores de Interleucina-2 , Linfócitos T/imunologia
20.
Infect Immun ; 56(2): 336-42, 1988 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2828237

RESUMO

Leishmania species are obligate intracellular pathogens of mononuclear phagocytes. Successful infection depends on sequestration of the promastigote (insect form) within host cells, allowing transformation into the relatively hardy amastigote stage. Promastigotes are killed readily by circulating phagocytes and nonimmune serum, suggesting that cutaneous infection is initiated within a permissive cell in the epidermis or dermis. From large sections of primate skin dermal macrophages and epidermal Langerhans cells were isolated, and their interaction with promastigotes of Leishmania major was investigated in vitro. Dermal macrophages were readily infected with promastigotes, and successful transformation to and replication of amastigotes was observed. Ingestion of promastigotes by dermal macrophages was not associated with a significant respiratory burst, in contrast to that by other macrophage populations, and was associated with significantly greater survival of parasites. Stimulation of these cells with phorbol myristate acetate or opsonized zymosan revealed that those cells were generally oxidatively deficient. Langerhans cells could not be successfully infected by promastigotes under similar conditions. Examination of these cells for expression of CR3, which has been identified as a potential Leishmania receptor, revealed that Langerhans cells did not express the alpha M subunit of CR3, whereas dermal macrophages were CR3 positive. These data support the concept that dermal macrophages are the site of initiation of Leishmania infection.


Assuntos
Células de Langerhans/imunologia , Leishmania tropica/imunologia , Leishmaniose/imunologia , Macrófagos/imunologia , Pele/imunologia , Animais , Anticorpos Monoclonais , Células Epidérmicas , Epiderme/imunologia , Imunidade Celular , Receptores de Complemento/metabolismo , Receptores de Complemento 3b , Pele/citologia , Superóxidos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...