Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing

Bonanomi, Athos; Kojic, Dejan; Giger, Bettina; Rickenbach, Zaira; Jean-Richard-Dit-Bressel, Louis; Berger, Christoph; Niggli, Felix; Nadal, David

doi:10.1016/j.jim.2003.08.002

Cited by 33 publications

(20 citation statements)

References 21 publications

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“…In brief, for measuring TLR9 mRNA, we used commercially available primers and probes (Assays-on-demand; Applied Biosystems). Hydroxymethyl bilane synthase (GenBank X04217) was used as a housekeeping gene and designed as an MGB (3Ј minus groove binder) probe (9). Data generated by real-time quantitative PCR were analyzed by determining the mean normalized gene expression for every sample with the software application Q-Gene (calculation procedure 2 for mean normalized gene expression) (36).…”

Section: Methodsmentioning

confidence: 99%

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

Schlaepfer¹,

Audigé²,

Beust³

et al. 2004

J Virol

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Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4 ؉ T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4؉ T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4 ؉ T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.

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Section: Methodsmentioning

confidence: 99%

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

Schlaepfer¹,

Audigé²,

Beust³

et al. 2004

J Virol

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“…The TLDAs included HMBS, GUSB, TBP, and 18S as reference genes based on their proven role as housekeeping genes (30,31) and their uniform expression in preliminary TLDA assays in FFPE tumor samples from this series of cHL (data not shown).…”

Section: Translational Relevancementioning

confidence: 99%

A TaqMan Low-Density Array to Predict Outcome in Advanced Hodgkin's Lymphoma Using Paraffin-Embedded Samples

Sánchez-Espiridión

Sánchez-Aguilera

Montalbán

et al. 2009

Clinical Cancer Research

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Purpose: Despite major advances in the treatment of classic Hodgkin's lymphoma (cHL), f30% of patientsinadvancedstagesmayeventuallydie as resultof the disease,andcurrentmethods topredict prognosis are ratherunreliable.Thus, the applicationof robust techniques for theidentificationofbiomarkers associated with treatment response is essentialif new predictive tools are to be developed. Experimental Design: We used gene expression data from advanced cHL patients to identify transcriptional patterns from the tumoral cells and their nonneoplastic microenvironment, associated with lack of maintained treatment response. Gene-Set Enrichment Analysis was used to identify functionalpathways associated withunfavorable outcome that were significantly enrichedin either the Hodgkin's and Reed-Sternberg cells (regulation of the G 2 -M checkpoint, chaperones, histone modification, and signalingpathways) or the reactive cellmicroenvironment (mainly representedby specificT-cell populations and macrophage activation markers). Results:To explore the pathways identified previously, we used a series of 52 formalin-fixed paraffin-embedded advanced cHL samples and designed a real-time PCR-based low-density array that included the most relevant genes. A large majority of the samples (82.7%) and all selected genes were analyzed successfully with this approach. Conclusions:The results of this assay can be combined in a single risk score integrating these biologicalpathways associated with treatment response and eventually usedina larger series to develop a new molecular outcome predictor for advanced cHL.

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“…In brief, for measuring TLR7 and 8 mRNA, we used commercially available primers and probes (Assayson-demand; Applied Biosystems). Hydroxymethyl bilane synthase (GenBank X04217) was used as a housekeeping gene and designed as a 3Ј minus groove binder probe (23). Data generated by real-time qPCR were analyzed by determining the mean normalized gene expression for every sample with the software application Q-Gene (calculation procedure 2 for mean normalized gene expression) (24).…”

Section: Pseudotype Virus Preparation and Challenge Of Cells With Psementioning

confidence: 99%

TLR7/8 Triggering Exerts Opposing Effects in Acute versus Latent HIV Infection

Schlaepfer¹,

Audigé²,

Joller

et al. 2006

The Journal of Immunology

106

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TLRs trigger innate immunity by recognizing conserved motifs of microorganisms. Recently, ssRNAs from HIV and influenza virus were shown to trigger TLR7 and 8. Thus, we hypothesized that HIV ssRNA, by triggering TLR7/8, affects HIV pathogenesis. Indeed, HIV ssRNA rendered human lymphoid tissue of tonsillar origin or PBMC barely permissive to HIV replication. The synthetic compound R-848, which also triggers TLR7/8, showed similar anti-HIV activity. Loss of R-848’s activity in lymphoid tissue depleted of B cells suggested a role for B cells in innate immunity. TLR7/8 triggering appears to exert antiviral effects through soluble factors: conditioned medium reduced HIV replication in indicator cells. Although a number of cytokines and chemokines were increased upon adding R-848 to lymphoid tissue, blocking those cytokines/chemokines (i.e., IFN-α receptor, IFN-γ, MIP-1α, -1β, RANTES, and stromal cell-derived factor-1) did not result in the reversal of R-848’s anti-HIV activity. Thus, the nature of this soluble factor(s) remains unknown. Unlike lymphoid tissue acutely infected with HIV, triggering latently infected promonocytic cells induced the release of HIV virions. The anti-HIV effects of triggering TLR7/8 may inhibit rapid killing, while pro-HIV effects may guarantee a certain replication level. Compounds triggering TLR7/8 may be attractive drug candidates to purge latent HIV while preventing new infections.

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Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing

Cited by 33 publications

References 21 publications

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo

A TaqMan Low-Density Array to Predict Outcome in Advanced Hodgkin's Lymphoma Using Paraffin-Embedded Samples

TLR7/8 Triggering Exerts Opposing Effects in Acute versus Latent HIV Infection

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