Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

Bouaboula, Monsif; Legoux, Pascale; Pessegue, Bernard; Delpech, B; Dumont, Xavier; Piechaczyk, Marc; Casellas, Pierre; Shire, David

doi:10.1016/s0021-9258(19)36687-6

Cited by 308 publications

(11 citation statements)

References 25 publications

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“…Control samples from nonelicited cell suspension cultures contained 2.3 × 10 -5 pmol of cad 1-C1 mRNA in 1 μg of total RNA but only 12.6 × 10 -7 pmol of c cad 1-A mRNA (Figure ). The actual number of target mRNA molecules present in each initial sample could be higher, as the efficiency of cDNA synthesis using oligo (dT) was reported to be 40−50% …”

Section: Resultsmentioning

confidence: 99%

Cloning and Heterologous Expression of a Second (+)-δ-Cadinene Synthase from Gossypium arboreum

Wang

Chen

et al. 1996

J. Nat. Prod.

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Screening of a Gossypium arboreum L. cv. Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synthase. A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DNA as a template. This second cDNA clone encodes a protein that is 80% identical to the recently described (+)-delta-cadinene synthases CAD1-C1 and C14 from G. arboreum and maintains a significant degree of homology to the other known mono-, sesqui-, and diterpene synthases. As in the case of CAD1-C1 (+)-delta-cadinene synthase from cultured cotton cells, the synthesis of the second CAD1-A mRNA was induced by treatment of cotton cell suspension cultures with a partially purified elicitor preparation from the phytopathogenic fungus Verticillium dahliae. Expression of CAD1-A mRNA was quantitated with reverse transcription PCR and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h after addition of elicitor. Heterologous expression of this second cDNA produced a 64 kD protein that catalyzed the cyclization of farnesyl diphosphate to (+)-delta-cadinene, the identical product produced by CAD1-C1. The steady-state kinetic parameters of CAD1-A were similar to CAD1-C, showing a Km of 7 mM farnesyl diphosphate and kcat of 0.039 s-1 at 30 degrees C. However, the optimal pH and Mg2+ concentration for CAD1-A activity were significantly higher than those observed for CAD1-C.

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Section: Resultsmentioning

confidence: 99%

Cloning and Heterologous Expression of a Second (+)-δ-Cadinene Synthase from Gossypium arboreum

Wang

Chen

et al. 1996

J. Nat. Prod.

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“…Oligonucleotides were synthesized by Operon Technologies. Competitive RT-PCR was carried out as previously described (13). In brief, equivalent amounts of cDNA from sorted DCs were added to each PCR reaction tube.…”

Section: Methodsmentioning

confidence: 99%

Freshly Isolated Peyer's Patch, but Not Spleen, Dendritic Cells Produce Interleukin 10 and Induce the Differentiation of T Helper Type 2 Cells

Iwasaki

Kelsall

1999

The Journal of Experimental Medicine

586

442

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Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c+ DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98–100% pure CD11c+ DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide–specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-γ (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-γ production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.

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“…The presence of cytokine transcripts of IFN-␥ , IL-2, IL-3, IL-4, IL-5, and IL-6 were tested by reverse transcriptase PCR using cytokine-specific primer sets as described (28). CD4 ϩ T cell clones were specifically stimulated with env-H19 peptide-loaded 771 cells for 2 and 16 h and total RNA and cDNA was prepared as described (29).…”

Section: Methodsmentioning

confidence: 99%

Specific T Helper Cell Requirement for Optimal Induction of Cytotoxic T Lymphocytes against Major Histocompatibility Complex Class II Negative Tumors

Ossendorp

Mengede

Camps

et al. 1998

The Journal of Experimental Medicine

537

363

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This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.

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Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

Cited by 308 publications

References 25 publications

Cloning and Heterologous Expression of a Second (+)-δ-Cadinene Synthase from Gossypium arboreum

Cloning and Heterologous Expression of a Second (+)-δ-Cadinene Synthase from Gossypium arboreum

Freshly Isolated Peyer's Patch, but Not Spleen, Dendritic Cells Produce Interleukin 10 and Induce the Differentiation of T Helper Type 2 Cells

Specific T Helper Cell Requirement for Optimal Induction of Cytotoxic T Lymphocytes against Major Histocompatibility Complex Class II Negative Tumors

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