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Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.

Espitia CM, Zhao W, Saldarriaga O, Osorio Y, Harrison LM, Cappello M, Travi BL, Melby PC - BMC Immunol. (2010)

Bottom Line: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression.Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression.Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter, San Antonio, Texas, USA.

ABSTRACT

Background: The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.

Results: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

Conclusions: The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

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Related in: MedlinePlus

Course of disease and parasite burden in L. panamensis infected hamsters. 6-8 week old female hamsters (n = 6) with 3 × 106 luciferase (luc)-transfected L. panamensis promastigotes in the dermis of the snout. Lesion size was measured weekly and is shown as the lesion area (mm2) in panel A. The intralesional parasite burden was determined by in vivo imaging and is shown as the total intralesional amastigotes in panel B. Images of the luc-L. panamensis burden in two representative hamsters are shown in panel C.
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Figure 2: Course of disease and parasite burden in L. panamensis infected hamsters. 6-8 week old female hamsters (n = 6) with 3 × 106 luciferase (luc)-transfected L. panamensis promastigotes in the dermis of the snout. Lesion size was measured weekly and is shown as the lesion area (mm2) in panel A. The intralesional parasite burden was determined by in vivo imaging and is shown as the total intralesional amastigotes in panel B. Images of the luc-L. panamensis burden in two representative hamsters are shown in panel C.

Mentions: We used a well-established model of localized cutaneous leishmaniasis caused by L. panamensis [1,3,4] to test the application of the standardized and validated duplex assays. In this model, at one-week post infection there is early evidence of a clinical lesion (inflammation and swelling) and parasites are easily identified in the tissue. Thereafter the lesion increases in size until 2-3 weeks post-infection when it is nodular with crusting and central necrosis (see Figure 2). After 3-4 weeks postinfection it transitions to a stable chronic lesion containing fewer parasites (data not shown). We investigated the immune response early in the course of infection because the innate and early adaptive immune responses are known to shape the clinical outcome and immunity in other models of Leishmania infection [36,37].


Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.

Espitia CM, Zhao W, Saldarriaga O, Osorio Y, Harrison LM, Cappello M, Travi BL, Melby PC - BMC Immunol. (2010)

Course of disease and parasite burden in L. panamensis infected hamsters. 6-8 week old female hamsters (n = 6) with 3 × 106 luciferase (luc)-transfected L. panamensis promastigotes in the dermis of the snout. Lesion size was measured weekly and is shown as the lesion area (mm2) in panel A. The intralesional parasite burden was determined by in vivo imaging and is shown as the total intralesional amastigotes in panel B. Images of the luc-L. panamensis burden in two representative hamsters are shown in panel C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909172&req=5

Figure 2: Course of disease and parasite burden in L. panamensis infected hamsters. 6-8 week old female hamsters (n = 6) with 3 × 106 luciferase (luc)-transfected L. panamensis promastigotes in the dermis of the snout. Lesion size was measured weekly and is shown as the lesion area (mm2) in panel A. The intralesional parasite burden was determined by in vivo imaging and is shown as the total intralesional amastigotes in panel B. Images of the luc-L. panamensis burden in two representative hamsters are shown in panel C.
Mentions: We used a well-established model of localized cutaneous leishmaniasis caused by L. panamensis [1,3,4] to test the application of the standardized and validated duplex assays. In this model, at one-week post infection there is early evidence of a clinical lesion (inflammation and swelling) and parasites are easily identified in the tissue. Thereafter the lesion increases in size until 2-3 weeks post-infection when it is nodular with crusting and central necrosis (see Figure 2). After 3-4 weeks postinfection it transitions to a stable chronic lesion containing fewer parasites (data not shown). We investigated the immune response early in the course of infection because the innate and early adaptive immune responses are known to shape the clinical outcome and immunity in other models of Leishmania infection [36,37].

Bottom Line: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression.Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression.Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter, San Antonio, Texas, USA.

ABSTRACT

Background: The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.

Results: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.

Conclusions: The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

Show MeSH
Related in: MedlinePlus