RESUMO
BACKGROUND: Cells respond to adverse environmental stimuli by enhancing the expression of specific genes, the products of which include a suite of proteins known as heat shock proteins (hsps), a response often attributed to cellular protection. METHODS: In this study, we characterized alterations in hsp expression in chick embryos (Hamburger-Hamilton stage 17, 72 h) exposed in ovo to arsenite (As), mercury (Hg), and cadmium (Cd), known developmental toxicants. Embryos were incubated for 2 h following exposure to 3, 10, 30, or 100 nmol metal, or for 2, 4, 12, or 24 h following treatment with 10 nmol metal. RESULTS: An enhanced de novo synthesis of 24-, 70-, and 90-kD, 70- and 90-kD, and 70-kD proteins was observed with As, Hg, and Cd treatments, respectively. These responses were transient; apparent rates of protein synthesis were maximal 2-4 h after exposure and returned to control rates by 24 h. Actinomycin D experiments demonstrated that arsenite-induced expression of these proteins is transcriptionally regulated. Immunoblotting experiments identified the 24-, 70-, and 90-kD proteins as the heat shock proteins hsp24, hsp70, and hsp90, respectively. Exposure duration-related abnormalities were noted in the neural tube with all metals and in the ganglia and somites with Cd and As. Retina, allantois, and limb defects were specific to Cd-treated embryos, and branchial arch defects were specific to As-treated embryos. CONCLUSIONS: The data support metal-induced developmental abnormalities, which are preceded by synthesis of stress proteins.
Assuntos
Arsenitos/farmacologia , Cádmio/farmacologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião não Mamífero , Proteínas de Choque Térmico/metabolismo , Mercúrio/farmacologia , Biossíntese de Proteínas , Animais , Cádmio/metabolismo , Embrião de Galinha , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Immunoblotting , Imuno-Histoquímica , Fatores de Tempo , Transcrição GênicaRESUMO
The frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+ T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+ T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+ T lymphocytes were CD95(pos) and CD27(lo) but expressed variable levels of CD45RA. A significant reduction (P < 0.05) in the frequency of CMV-specific CD4+ T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+ T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+ T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+ T lymphocytes was not predicted by the severity of CD4+ T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RA(neg) or CCR5(pos) CMV-specific CD4+ T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+ T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+ T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.