scholarly journals Real-Time Reverse Transcription-PCR Quantification of Cytokine mRNA Expression in Golden Syrian Hamster Infected with Leishmania infantum and Treated with a New Amphotericin B Formulation

2006 ◽  
Vol 50 (4) ◽  
pp. 1195-1201 ◽  
Author(s):  
S. Rama Iñiguez ◽  
M. A. Dea-Ayuela ◽  
J. A. Sanchez-Brunete ◽  
J. J. Torrado ◽  
J. M. Alunda ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Reverse Transcription ◽  
Syrian Hamster ◽  
Leishmania Infantum ◽  
Cytokine Mrna ◽  
Golden Syrian Hamster ◽  
Cytokine Mrna Expression ◽  
Reverse Transcription Pcr ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 107 metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-γ) and, to a lesser extent, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-γ and TNF-α, with no effect on the deactivating cytokine TGF-β. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-γ and TNF-α and strong suppression of TGF-β. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-β, which in turn results in up-regulation of the Th1 cytokines IFN-γ and TNF-α.

scholarly journals Cytokine mRNA Expression in Mycobacteriam ulcerans-Infected Human Skin and Correlation with Local Inflammatory Response

2006 ◽  
Vol 74 (5) ◽  
pp. 2917-2924 ◽  
Author(s):  
R. Phillips ◽  
C. Horsfield ◽  
J. Mangan ◽  
K. Laing ◽  
S. Etuaful ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Interleukin 10 ◽  
Mycobacterium Ulcerans ◽  
Cytokine Mrna ◽  
Necrosis Factor Alpha ◽  
Tissue Samples ◽  
Local Inflammatory Response ◽  
Cytokine Mrna Expression ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT Cytokine mRNA expression in biopsies of Mycobacterium ulcerans-infected human tissue was investigated using real-time PCR, and the findings were correlated with the clinical stages of disease and histopathologies. A broad range of cytokine mRNAs were detected in 16 early nodules and 28 late-stage ulcers, including those for the Th1 cytokines tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) and the Th2 cytokine interleukin 10 (IL-10). IFN-γ was strongly expressed in both nodules and ulcers, suggesting that a Th1 response begins early in the disease. There was a significantly higher expression of IL-8 and other proinflammatory cytokines in results from 32 biopsies with neutrophilia than in those from 12 biopsies without acute inflammation. Ten tissue samples containing granulomas showed high mRNA expression for IFN-γ, IL-1β, IL-12p35, IL-12p40, IL-15, and TNF-α relative to 34 tissue samples without granulomas. These results suggest that the human immune response to M. ulcerans is similar to that seen with some other mycobacteria despite the presence of the toxin mycolactone in the tissues.

scholarly journals Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

2007 ◽  
Vol 14 (12) ◽  
pp. 1563-1571 ◽  
Author(s):  
Noel P. Harrington ◽  
Om P. Surujballi ◽  
W. Ray Waters ◽  
John F. Prescott
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequence Information ◽  
Tuberculin Skin Testing ◽  
Reverse Transcription Pcr ◽  
Ifn Γ

ABSTRACT Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-γ)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN-γ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-γ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-γ mRNA responses correlated well with IFN-γ protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-γ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-γ expression by using consensus sequences of closely related species or of other species for which IFN-γ sequence information is available.

scholarly journals Cytokine mRNA Expression in Lesions in Cats with Chronic Gingivostomatitis

1999 ◽  
Vol 6 (4) ◽  
pp. 471-478 ◽  
Author(s):  
R. Harley ◽  
C. R. Helps ◽  
D. A. Harbour ◽  
T. J. Gruffydd-Jones ◽  
M. J. Day
Keyword(s):  
Mrna Expression ◽  
Oral Mucosa ◽  
Interleukin 2 ◽  
Clinical Severity ◽  
Cytokine Mrna ◽  
Reverse Transcription Pcr ◽  
Ifn Γ ◽  
Relative Mrna Expression

ABSTRACT Semiquantitative reverse transcription-PCR assays were developed to measure feline interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, and IL-12 (p35 & p40); gamma interferon (IFN-γ); and glyceraldehyde-3-phosphate dehydrogenase mRNA concentrations in biopsies of feline oral mucosa. Biopsies were collected from 30 cats with chronic gingivostomatitis (diseased) prior to each cat receiving one of four treatments. In 23 cases replicate biopsies were collected 3 months after treatment commenced. Biopsies were also analyzed from 11 cats without clinical disease (nondiseased). Expression of IL-2, IL-10, IL-12 (p35 and p40), and IFN-γ was detected in most nondiseased biopsies, while IL-6 was detected in a minority, and IL-4 and IL-5 were both undetectable. Compared to nondiseased cats, the diseased population showed a significant increase in the relative mRNA expression of IL-2, IL-4, IL-6, IL-10, IL-12 (p35 and p40), and IFN-γ. In contrast, IL-5 mRNA expression was unchanged and was only detected in one case. No significant relationship was demonstrable between the change in relative expression of specific cytokine mRNA and the change in clinical severity of the local mucosal lesions over the treatment period. The results demonstrate that the normal feline oral mucosa is biased towards a predominantly (Th) type 1 profile of cytokine expression and that during the development of lesions seen in feline chronic gingivostomatitis there is a shift in the cytokine profile from a type 1 to a mixed type 1 and type 2 response.

scholarly journals Parasite Load Induces Progressive Spleen Architecture Breakage and Impairs Cytokine mRNA Expression in Leishmania infantum-Naturally Infected Dogs

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0123009 ◽  
Author(s):  
Amanda S. Cavalcanti ◽  
Marcelo Ribeiro-Alves ◽  
Luiza de O. R. Pereira ◽  
Gustavo Leandro Mestre ◽  
Anna Beatriz Robottom Ferreira ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Leishmania Infantum ◽  
Parasite Load ◽  
Cytokine Mrna ◽  
Cytokine Mrna Expression

scholarly journals Cytokine mRNA expression responses to resistance, aerobic, and concurrent exercise in sedentary middle-aged men

2014 ◽  
Vol 39 (2) ◽  
pp. 130-137 ◽  
Author(s):  
Cheyne E. Donges ◽  
Rob Duffield ◽  
Greg C. Smith ◽  
Michael J. Short ◽  
Johann A. Edge
Keyword(s):  
Mrna Expression ◽  
Vastus Lateralis ◽  
Receptor Expression ◽  
Middle Aged ◽  
Cytokine Mrna ◽  
Molecular Responses ◽  
Glycogen Concentration ◽  
Cytokine Mrna Expression ◽  
Tnf Α ◽  
Concurrent Exercise

Concurrent resistance and aerobic exercise (CE) is recommended to ageing populations, though is postulated to induce diminished acute molecular responses. Given that contraction-induced cytokine mRNA expression reportedly mediates remunerative postexercise molecular responses, it is necessary to determine whether cytokine mRNA expression may be diminished after CE. Eight middle-aged men (age, 53.3 ±1.8 years; body mass index, 29.4 ± 1.4 kg·m−2) randomly completed (balanced for completion order) 8 × 8 leg extensions at 70% maximal strength (RE), 40 min of cycling at 55% of peak aerobic workload (AE), or (workload-matched) 50% RE and 50% AE (CE). Muscle (vastus lateralis) was obtained pre-exercise, and at 1 h and 4 h postexercise, and analyzed for changes of glycogen concentration, tumor necrosis factor (TNF)α, TNF receptor-1 and -2 (TNF-R1 and TNF-R2, respectively), interleukin (IL)-6, IL-6R, IL-1β, and IL-1 receptor-antagonist (IL-1ra). All exercise modes upregulated cytokine mRNA expression at 1 h postexercise comparably (TNFα, TNF-R1, TNF-R2, IL-1β, IL-6) (p < 0.05). Expression remained elevated at 4 h after RE and AE (p < 0.05), though returned to pre-exercise levels after CE (p > 0.05). Moreover, AE and RE upregulated IL-1β and IL-1ra expression, whereas CE upregulated IL-1β expression only (p < 0.05). Only AE reduced muscle glycogen concentration (p < 0.05), whilst upregulating receptor expression the greatest; though, IL-6R expression remained unchanged after all modes (p > 0.05). In conclusion, in middle-aged men, all modes induced commensurate cytokine mRNA expression at 1 h postexercise; however, only CE resulted in ameliorated expression at 4 h postexercise. Whether the RE or AE components of CE are independently or cumulatively sufficient to upregulate cytokine responses, or whether they collectively inhibit cytokine mRNA expression, remains to be determined.

scholarly journals Delayed Cytokine mRNA Expression Kinetics after T-Lymphocyte Costimulation: A Quantitative Measure of the Efficacy of Cyclosporin A-based Immunosuppression

2002 ◽  
Vol 48 (12) ◽  
pp. 2225-2231 ◽  
Author(s):  
Christoph Härtel ◽  
Lutz Fricke ◽  
Nina Schumacher ◽  
Holger Kirchner ◽  
Michael Müller-Steinhardt
Keyword(s):  
T Cell ◽  
Mrna Expression ◽  
Kidney Transplant ◽  
Ex Vivo ◽  
Cytokine Mrna ◽  
Cytokine Mrna Expression ◽  
Tnf Α ◽  
Ex Vivo Study ◽  
Expression Kinetics

Abstract Background: Because cyclosporin A (CsA) and glucocorticoids inhibit the production of interleukin-2 (IL-2) and other cytokines, quantitative analysis of cytokine mRNA might constitute a pharmacodynamic measure for immunosuppressive drug effects. We investigated whether immunosuppressive drugs influence cytokine mRNA expression kinetics during T-cell costimulation. Methods: We used a human whole blood assay to determine basal (unstimulated) IL-2, IL-4, and tumor necrosis factor-α (TNF-α) mRNA concentrations and expression kinetics after anti-CD3/anti-CD28 monoclonal antibody costimulation in kidney transplant recipients undergoing CsA-based immunosuppressive triple therapy and in healthy controls (ex vivo study I). The effect of CsA on IL-2 mRNA expression kinetics was also determined ex vivo in patients undergoing CsA monotherapy (ex vivo study II) and after in vitro addition of CsA. Results: In ex vivo study I, basal TNF-α mRNA but not IL-2 and IL-4 mRNA was decreased in kidney transplant patients. We observed shifts in peak IL-2 and IL-4 (from 8 to 24 h) and TNF-α (from 4 to 8 h of costimulation) mRNA expression in kidney transplant patients after T-cell costimulation. In patients undergoing CsA monotherapy (ex vivo study II), the inhibitory effect of CsA was detectable as an individually delayed increase in IL-2 mRNA during costimulation. In vitro addition of CsA also induced a dose-independent displacement of IL-2 mRNA expression kinetics (i.e., a delay). Conclusions: A delayed increase in cytokine mRNA expression during T-cell costimulation may represent a sensitive effect of immunosuppression. The single analysis of one absolute or peak mRNA value could be misleading. For prospective studies involving measurement of cytokine mRNA, we therefore suggest the parameter “area of cytokine mRNA expression over time”, which should include absolute cytokine mRNA values at two different time points of mRNA kinetics.

Changes in urinary bladder cytokine mRNA and protein after cyclophosphamide-induced cystitis

2002 ◽  
Vol 9 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Susan E. Malley ◽  
Margaret A. Vizzard
Keyword(s):  
Urinary Bladder ◽  
Protein Expression ◽  
Mrna Expression ◽  
Fold Increase ◽  
Cytokine Expression ◽  
Cytokine Mrna ◽  
Rnase Protection ◽  
Acute Cystitis ◽  
Cytokine Mrna Expression ◽  
Tnf Α

Cyclophosphamide (CYP)-induced cystitis alters micturition function and produces reorganization of the micturition reflex. This reorganization may involve cytokine expression in the urinary bladder. These studies have determined candidate cytokines in the bladder that may contribute to the reorganization process. An RNase protection assay was used to measure changes in rat bladder cytokine mRNA [interferon-γ (IFN)-γ, interleukin-1α/β (IL-1α/β), IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, and tumor necrosis factor-α/β (TNF-α/β)] after acute (4 h), intermediate (48 h), or chronic (10 day) cystitis. The correlation between bladder cytokine mRNA and protein expression was also determined by immunoassay. Although at each time point after cystitis significant changes in bladder cytokine mRNA were observed, the magnitude differed (acute > intermediate > chronic). Acute cystitis demonstrated the most robust changes ( P ≤ 0.005; IL-1β, 330-fold increase; IL-2, 20-fold increase; IL-4, 8-fold increase; IL-6, 80-fold increase) in cytokine mRNA expression and TNF-α or TNF-β mRNA were only increased (2–10-fold) after acute cystitis. More modest increases in cytokine mRNA expression were observed after 48-h or 10-day cystitis. Cytokine protein expression generally paralleled that of mRNA. Increased cytokine expression after CYP-induced cystitis, alone or in combination with other inflammatory mediators or growth factors, may contribute to altered lower urinary tract function after cystitis.

Temporal regulation of cytokine mRNA expression by tristetraprolin: dynamic control by p38 MAPK and MKP-1

2015 ◽  
Vol 308 (9) ◽  
pp. L973-L980 ◽  
Author(s):  
Pavan Prabhala ◽  
Kristin Bunge ◽  
Md. Mostafizur Rahman ◽  
Qi Ge ◽  
Andrew R. Clark ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Mrna Expression ◽  
P38 Mapk ◽  
Airway Smooth Muscle Cells ◽  
Cytokine Mrna ◽  
Temporal Regulation ◽  
Cytokine Mrna Expression ◽  
Tnf Α ◽  
Inflammatory Proteins ◽  
Anti Inflammatory

Cytokines drive many inflammatory diseases, including asthma. Understanding the molecular mechanisms responsible for cytokine secretion will allow us to develop novel strategies to repress inflammation in the future. Harnessing the power of endogenous anti-inflammatory proteins is one such strategy. In this study, we investigate the p38 MAPK-mediated regulatory interaction of two anti-inflammatory proteins, mitogen-activated protein kinase phosphatase 1 (MKP-1) and tristetraprolin (TTP), in the context of asthmatic inflammation. Using primary cultures of airway smooth muscle cells in vitro, we explored the temporal regulation of IL-6 cytokine mRNA expression upon stimulation with TNF-α. Intriguingly, the temporal profile of mRNA expression was biphasic. This was not due to COX-2-derived prostanoid upregulation, increased expression of NLRP3 inflammasome components, or upregulation of the cognate receptor for TNF-α-TNFR1. Rather, the biphasic nature of TNF-α-induced IL-6 mRNA expression was regulated temporally by the RNA-destabilizing molecule, TTP. Importantly, TTP function is controlled by p38 MAPK, and our study reveals that its expression in airway smooth muscle cells is p38 MAPK-dependent and its anti-inflammatory activity is also controlled by p38 MAPK-mediated phosphorylation. MKP-1 is a MAPK deactivator; thus, by controlling p38 MAPK phosphorylation status in a temporally distinct manner, MKP-1 ensures that TTP is expressed and made functional at precisely the correct time to repress cytokine expression. Together, p38 MAPK, MKP-1, and TTP may form a regulatory network that exerts significant control on cytokine secretion in proasthmatic inflammation through precise temporal signaling.

CYTOKINE mRNA EXPRESSION IN CHICKEN EXPERIMENTALLY INFECTED WITH DIFFERENT AVIAN REOVIRUS STRAINS

2014 ◽  
Vol 40 (01) ◽  
pp. 29-36 ◽  
Author(s):  
Pin-Chun Shen ◽  
Jia-Ling Yang ◽  
Bor-Sheu Su ◽  
Long-Huw Lee
Keyword(s):  
Mrna Expression ◽  
Post Inoculation ◽  
Avian Reovirus ◽  
Cytokine Mrna ◽  
Expression Levels ◽  
Real Time Quantitative Pcr ◽  
Cytokine Mrna Expression ◽  
Gross Lesions ◽  
Ifn Γ ◽  
Foot Pad

This study was undertaken to elucidate the cytokine response in chicken infected with strains of avian reovirus (ARV) S1133 and 2408. The expression levels of cytokine mRNA in the spleen and viral S1 RNA in various tissues at 1.5 and 2.5 days post inoculation (dpi) were examined using real-time quantitative PCR. Among the cytokines examined, the mRNA expression levels of IL-6, IFN-γ, IL-10 and iNOS at 2.5 dpi were significantly upregulated and higher in chickens infected with strain 2408 than in chickens infected with strain S1133, particularly IL-6 and IFN-γ. A significantly higher levels of viral S1 RNA were detected in the examined tissues from chickens infected with strain 2408 than with strain S1133 over the experimental course, among which the foot pad and spleen were more predominant. The highest levels of IL-6 and IFN-γ mRNA expression correlated with the viral S1 RNA levels in the spleen and the marked clinical diseases and gross lesions, suggesting that IL-6 and IFN-γ may play a role in the pathogenesis of ARV infection.

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