Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology.We report a novel method for cloning comp...Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology.We report a novel method for cloning competitor cDNAs that is rapid,efficient and inexpensive.By linking multiple competitor cDNAs in tandem,polycompetitor constructs can be created that allow the use of a single reagent for individual PCR assays.Assays can be performed on minute samples of cell culture or tissue and can be reliably quantitated after routine gel electrophoresis without the use of densitometry or labeled nucleotides.The utility of this technique lies in the ability to produce a relatively inexpensive customized reagent that is simple to use and that allows for sensitive determinations of gene expression in a rapid and convenient manner.This method should allow investigators in many areas of biology to easily quantitate a broad range of important regulatory molecules.展开更多
interleukin 12 (IL-12) is a powerful stimulus for the growth of activated T and natural killer cells,their generation of interferon γ(IFN-γ),and the differentiation of T helper type 1(Th1) effector cells from naive ...interleukin 12 (IL-12) is a powerful stimulus for the growth of activated T and natural killer cells,their generation of interferon γ(IFN-γ),and the differentiation of T helper type 1(Th1) effector cells from naive precursors in vitro. Using this characterized model of CD4 cell differentiation, we examined the immunologic effects of IL-12 administered either at the time of infection; when naive T cells are primed,or after 14 days of in fection,by which time CDt subset differentiation has occurred.Given with the inoculationof parasites,IL-12 induced IFN-γand IL-10 and markedly suppressed IL-4. Effects on IL10 and IL-4 are comparable in mice with homozygous disruption of the IFN-γgene (IFNγ%),and suppression of IL-4 was unchanged by administration of neutralizing anti-IL-10antibody.Induction of IFN-γand IL-10 mRNA by IL-12 also occurred in infected SCID mice.Given after day 14 of infection, however, IL-12 not only induced IFN-γand IL-10but also induced IL-4 in normal and IFN-γ% mice. These data demonstrate direct effects of IL-12 independent of IFN-γ IL-10,and IL-4 and demonstrate that the ineffectiveness of IL-12 administered following infection with Leishmania major correlates with resistance of differentiated Th2 cells to the IL-4 suppressing activity of IL-12.展开更多
L Leishmania major are intramacrophage parasite whose eradication requires the induction of T helper 1(Th1)effector cells.Interleukin 12(IL-12) mediates Th1 effector cells development and enhances interferon γproduct...L Leishmania major are intramacrophage parasite whose eradication requires the induction of T helper 1(Th1)effector cells.Interleukin 12(IL-12) mediates Th1 effector cells development and enhances interferon γproduction by T cells and natural killer cells.Infection of macrophages in vitro by promastigotes of L. major caused no IL-12 p40 transcripts. Using competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed.In resistant mice,the transcripts for IL-2, IL-4 and IL-10 were subsequently downregulated, whereas in susceptible mice,these transcripts were only slightly decreased, and IL4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection.Challenge of macrophages in vitro confirmed that amastigotes induced IL-12 p40 mRNA.Reexamination of the cytokine rnRNA at 4d revealed expression of IL-13 in all strains analyzed,suggesting that IL-2 and IL-13 may mediate the IL-12independent production of IFN-γ during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD+4T cells. Each of these activities may favor survival of the organism.展开更多
Parasite-specific CD8+ T cells have been shown to transfer protection against nLeishmania major in susceptible BALB/c mice.An epitope-tagged expression library was used to identify the antigen recognized by a protecti...Parasite-specific CD8+ T cells have been shown to transfer protection against nLeishmania major in susceptible BALB/c mice.An epitope-tagged expression library was used to identify the antigen recognized by a protective CD8+ T cells clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histompatibility complex class n. A conserved 36-kilodalton member of the tryptophanaspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12before injection.展开更多
Mice with homologous disruption of the interferon γ(IFN-γ) gene on the C57BL/6 background were infected witly Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice...Mice with homologous disruption of the interferon γ(IFN-γ) gene on the C57BL/6 background were infected witly Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice,deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6￣8 wk.Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-γ and lymphotoxin,tipical of T helper type 1(Th1) cells, the knockout mice developed CD+4 cells that contained transcripts for interleukin 4(IL-4),IL-5,and IL13,typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-γ or IL-4 production by T cells in similar frequencies in the respective groups of mice,and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-γ.展开更多
文摘Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology.We report a novel method for cloning competitor cDNAs that is rapid,efficient and inexpensive.By linking multiple competitor cDNAs in tandem,polycompetitor constructs can be created that allow the use of a single reagent for individual PCR assays.Assays can be performed on minute samples of cell culture or tissue and can be reliably quantitated after routine gel electrophoresis without the use of densitometry or labeled nucleotides.The utility of this technique lies in the ability to produce a relatively inexpensive customized reagent that is simple to use and that allows for sensitive determinations of gene expression in a rapid and convenient manner.This method should allow investigators in many areas of biology to easily quantitate a broad range of important regulatory molecules.
文摘interleukin 12 (IL-12) is a powerful stimulus for the growth of activated T and natural killer cells,their generation of interferon γ(IFN-γ),and the differentiation of T helper type 1(Th1) effector cells from naive precursors in vitro. Using this characterized model of CD4 cell differentiation, we examined the immunologic effects of IL-12 administered either at the time of infection; when naive T cells are primed,or after 14 days of in fection,by which time CDt subset differentiation has occurred.Given with the inoculationof parasites,IL-12 induced IFN-γand IL-10 and markedly suppressed IL-4. Effects on IL10 and IL-4 are comparable in mice with homozygous disruption of the IFN-γgene (IFNγ%),and suppression of IL-4 was unchanged by administration of neutralizing anti-IL-10antibody.Induction of IFN-γand IL-10 mRNA by IL-12 also occurred in infected SCID mice.Given after day 14 of infection, however, IL-12 not only induced IFN-γand IL-10but also induced IL-4 in normal and IFN-γ% mice. These data demonstrate direct effects of IL-12 independent of IFN-γ IL-10,and IL-4 and demonstrate that the ineffectiveness of IL-12 administered following infection with Leishmania major correlates with resistance of differentiated Th2 cells to the IL-4 suppressing activity of IL-12.
文摘L Leishmania major are intramacrophage parasite whose eradication requires the induction of T helper 1(Th1)effector cells.Interleukin 12(IL-12) mediates Th1 effector cells development and enhances interferon γproduction by T cells and natural killer cells.Infection of macrophages in vitro by promastigotes of L. major caused no IL-12 p40 transcripts. Using competitor construct to quantitate a number of transcripts, a kinetic analysis of cytokine induction during the first few days of infection by L. major was performed.In resistant mice,the transcripts for IL-2, IL-4 and IL-10 were subsequently downregulated, whereas in susceptible mice,these transcripts were only slightly decreased, and IL4 continued to be reexpressed at high levels. IL-12 transcripts were first detected in vivo by 7 d after infection.Challenge of macrophages in vitro confirmed that amastigotes induced IL-12 p40 mRNA.Reexamination of the cytokine rnRNA at 4d revealed expression of IL-13 in all strains analyzed,suggesting that IL-2 and IL-13 may mediate the IL-12independent production of IFN-γ during the first days after infection. Leishmania have evolved to avoid inducing IL-12 from host macrophages during transmission from the insect vector, and cause a striking induction of mRNAs for IL-2, IL-4, IL-10, and IL-13 in CD+4T cells. Each of these activities may favor survival of the organism.
文摘Parasite-specific CD8+ T cells have been shown to transfer protection against nLeishmania major in susceptible BALB/c mice.An epitope-tagged expression library was used to identify the antigen recognized by a protective CD8+ T cells clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histompatibility complex class n. A conserved 36-kilodalton member of the tryptophanaspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12before injection.
文摘Mice with homologous disruption of the interferon γ(IFN-γ) gene on the C57BL/6 background were infected witly Leishmania major and the immune response assessed. In contrast to wild-type or heterozygous knockout mice,deficient animals were unable to restrict growth of the parasite and suffered lethal infection over 6￣8 wk.Although wild-type and heterozygous littermates developed CD4+ cells that contained transcripts for IFN-γ and lymphotoxin,tipical of T helper type 1(Th1) cells, the knockout mice developed CD+4 cells that contained transcripts for interleukin 4(IL-4),IL-5,and IL13,typical of Th2 cells. ELISPOT assays confirmed the reciprocal patterns of IFN-γ or IL-4 production by T cells in similar frequencies in the respective groups of mice,and antibody analysis confirmed the presence of Th2-mediated isotype switching in the knockout mice. These data suggest that CD4+ T cells that normally respond to antigens by differentiation to Th1 cells default to the Th2 pathway in the absence of endogenous IFN-γ.
