Expression of inflammatory and antiviral cytokines in pigs experimentally infected with Foot-and-Mouth disease

Transcription

1 Appendix 0 Expression of inflammatory and antiviral cytokines in pigs experimentally infected with Foot-and-Mouth disease Ciara M. Murphy, Zhidong Zhang, Melvyn Quan, Jeanette Knight and Soren Alexandersen. Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, GU ONF, U.K. Abstract The disease produced by foot-and-mouth-disease virus (FMDV) is one of major concern, both from an economical and an animal welfare point of view. Clinical disease is severe, particularly in the case of pigs, which are often affected to a greater degree by FMD than cattle or sheep. The fact that pigs from a clinical point of view become more ill than other susceptible animals lends weight to the possibility that the porcine immune response, in particular the proinflammatory cytokine response, may be significantly elevated since these cytokines, in the process of controlling the infection, can act to make acute clinical disease worse. To explore the cytokine response in pigs in response to FMDV infection, a quantitative real-time RT-PCR was developed and used to measure cytokine mrna in porcine peripheral blood mononuclear cells (PBMC) following infection of pigs with FMDV O UKG /001 virus. The cytokines studied in this paper include Interferon gamma (IFNγ), Interferon alpha (IFNα), Interferon beta (IFNβ), Interleukin-1 alpha (IL1α) and Tumor necrosis factor alpha (TNFα). The cellular housekeeping gene glyceraldehyde--phosphatedehydrogenase () is also measured as an endogenous control. PBMCs were collected from groups of pigs before and during experimental infection with FMDV in order to generate a picture of the cytokine mrna profiles of healthy animals compared with infected ones. A level of background mrna expression was noted for each of the cytokines studied. The fluctuations seen in cytokine mrna expression during the course of FMDV infection will be discussed. Introduction FMDV is an extremely rapidly replicating virus, a feature illustrated by the fact that clinical signs can become visible and quite obvious in pigs and cattle as soon as to hours after infection 1-. In sheep and goats, the clinical signs are far less obvious and as a result, disease can go unnoticed,,1. Fever, shivering, drooling and lip smacking are all characteristic signs of disease in cattle and lesions are most frequently observed on the tongue and dental pad, along the coronary band of the hoof and in the interdigital cleft. Pigs develop similar symptoms to cattle, albeit without the drooling and lip smacking and with the majority of lesions visible on the feet and sometimes around snout and lips,. Lesions may also be observed on the tip and towards the back of the tongue. Clinical disease in pigs is usually very severe, more so than disease in other species. 10

2 Both humoral and cellular responses are induced as a result of infection with FMDV. However, very little is known about the innate immune response to FMDV. The main cells responsible for innate immunity are monocytes, macrophages and neutrophils, which have the ability to phagocytose microbial pathogens. This in turn triggers the cytokine network, resulting in the development of inflammatory responses, followed by specific immune responses 1. Pigs have a larger proportion of γ/δ T cells than other species that are susceptible to FMDV, and it is possible that these cells may have a role to play in an elevated innate immune response of pigs to FMDV. Cytokines are low molecular weight soluble proteins that are involved in and can regulate the host response to infection, inflammation and immunity, cell growth and cell differentiation. They are primarily secreted by leucocytes but can also be secreted by nearly every nucleated cell type. Acting as chemical communicators between cells, cytokines bind to specific receptors on cell surfaces initiating various signal transduction pathways 1,1,1. In order to better understand the immunopathological pathways involved in the inflammatory response to FMDV in pigs, knowledge of the local cytokine profiles is critical. The use of real-time RT-PCR to achieve this provides a rapid, sensitive and quantifiable method of detecting the levels of porcine cytokine mrna. In addition, this method is capable of detecting the minute quantities of mrna (only 1% of total cellular RNA) present in cells. As a result, this method is becoming increasingly popular for the quantitation of cytokine mrna,10,1,,. Materials and Methods Animals: The animals used in these experiments were female Landrace x Large White pigs of 0 0kg body weight. Both animal experiments were carried out in biosecure isolation units. Procedure: Experiment 1: Normal control blood samples were taken for a week before inoculation. Three sets of four pigs were inoculated with different doses of FMDV inoculum intravenously starting with the most diluted inoculum first. 0. ml of diluted inoculum was inoculated IV into the Vena Cava anterior using a 1G x 1. needle and a ml syringe. The dose used was (i). logs (ii). logs and (iii). logs. After inoculation each group of pigs (low, medium and high dose) was placed in a separate room. The virus used (final titre. logs TCID/ml in BTY) was an O UKG /001 inoculum first pig passage. Body temperature and progression of disease of all pigs was recorded. Blood and serum samples were taken in heparin and plain vacutainers respectively. All samples were taken twice daily at 0:0 0:0 and 1:0-1:0 for the first three days and then daily for a week. Pigs were killed 1 days post inoculation or before if disease was very severe, i.e. pigs with loose hooves, severe secondary infection or severe inability to maintain body temperature. Experiment : Normal control blood samples were taken during the four-week period before inoculation. Three sets of four pigs were inoculated with different doses of FMDV 11

3 inoculum as described previously for Experiment 1. Immediately after inoculation each pig was placed singly into a separate cubicle (no direct contact). The high dose group was kept together in a single box allowing direct contact. The inoculum used was as in Experiment 1. Blood samples for mrna anlaysis were taken at 0:0-0:0 for the duration of the experiment. Pigs were killed 1 days post inoculation or before if disease was very severe. Isolation of porcine peripheral blood mononuclear cells PBMCs (approx. 1x10 cells) were isoloated from 10ml whole heparinated blood using Histopaque - 10 (density of 10 g/dl) and suspended in 00µl of calcium/magnesium free PBS. An aliquot of 10µl of PBMCs, which was equivalent to x10 cells, was added to 10µl of lysis buffer (from Roche mrna HS kit, as per manufacturers recommendations) to a final volume of 100µl. An aliquot of 00µl from the above 100µl suspension of PBMCs in lysis buffer contained 1x10 cells the quantity of cells required for mrna extraction. The suspensions were vortexed and stored at -0 C until required. Stimulation of porcine peripheral blood mononuclear cells: In order to validate the RT-PCR assay, PBMCs were stimulated and cultured from 0- hours, RNA was extracted, reverse-transcribed into cdna, which was then amplified by real time PCR using different cytokine primer and probe sets. PBMCs were stimulated using lectin, from Phaseolus vulgaris (red kidney bean), at a concentration of µg/µl. One Falcon Multiwell tissue culture plate, ( wells, flat bottom with low evaporation lid), was prepared by aliquoting 1ml of RPMI + HEPES media containing lectin, fungizone, penicillin/streptomycin and 10% FBS into each of the wells. One well on each plate contained media only, in order to provide a negative control. Subsequently, 1x10 PBMCs were added to each well and mixed by gentle aspiration. Cells were harvested at the appropriate time and suspended in 100µl of lysis buffer (from Roche mrna HS kit). The cells in lysis buffer were vortexed and stored at -0ºC. mrna extraction and RT-PCR conditions: mrna isolated from uncultured PBMCs and lectin-activated PBMCs was extracted by the MagNA Pure LC automated instrument (Roche Molecular Biochemicals) by using the mrna HS isolation extraction kit produced by the same manufacturer. A Jenway Genova MK Life Science Analyser was used to measure RNA concentrations. mrna was then reverse transcribed into cdna using PE Biosystems TaqMan RT reagents and random primers, as described previously,0. Negative controls without RT enzyme were included with every reverse transcription to eliminate the possibility of false positive results due to DNA contamination. Primers and probes for all cytokine target sequences were designed using the computer program Primer Express (Applied Biosystems). Quantitative PCR was performed as described previously,,1. Results In order to quantify the results obtained by real-time RT-PCR, standard samples of known concentrations were prepared from vitro-transcribed RNA. These samples were 1

4 used to construct standard curves for each cytokine mrna and for the endogenous control,. Uniform in vitro expression of porcine : Unstimulated and stimulated PBMCs were tested for the presence of the cellular endogenous control,. As predicted, levels did not appear to change during cell stimulation, in fact the levels decreased slightly over time. An example of such an experiment is shown in Fig. 1 below. Figure 1 Expression of in lectin-stimulated cells Cytokine expression by lectin-stimulated porcine PBMCs: PBMCs were isolated from the blood of two healthy pigs and stimulated with lectin. A relatively low background level of expression of IL-1α and absence of IFNβ was noted in unstimulated PBMCs, Fig. below. For the cytokines IFNγ, IFNα and TNFα a higher level of background mrna expression was observed. PBMCs stimulated with lectin demonstrated greater levels of expression of all cytokines tested than unstimulated PBMCs, Fig. below. 1

5 IFNα Pig A Pig B Log Copies per 1.0E+.... Pig A 0 Pig A IFNβ.... IL-1α Pig B 0 Pig B 0 0 Pig A TNFα Pig B 0 0 Hours post-stimulation Figure Cytokine expression by lectin-stimulated PBMCs Background levels of inflammatory and antiviral cytokine mrnas in uninfected pigs PBMCs were isolated from a group of twelve non-infected pigs over a period of three days in Experiment 1 and a period of four weeks in Experiment. (During the four-week period prior to inoculation in Experiment, a number of pigs had a short period of 1

6 diarrhoea and coughing). These cells were then tested for the expression or nonexpression of inflammatory and antiviral cytokine mrnas, see Fig. below. Over the course of the two uninfected periods, as expected, expression levels of mrna remained relatively stable. In Experiment 1, expression levels of IFNα mrna were higher than those of the other cytokines, Fig below. The mrna expression levels of IFNγ and IFNα in Experiment were higher than those of TNFα and IL-1α, but remained relatively stable, Fig below. IFNβ mrna levels remained negative throughout both animal experiments, so IFNβ was excluded from graphs subsequent to Fig.. Average Background Expression Levels - Experiment 1 Log Copies Log per Copies 1.0E+ per cells 1.0E+ cells 1 Average Background Expression Levels - Experiment IFN beta IFN beta Day Figure Cytokine and expression by non-infected pigs Cytokine Expression in FMDV Infected Pigs: When compared with the patterns of cytokine mrna expression in uninfected pigs, see Fig. above, it is clear that cytokine mrna expression was influenced by the presence of the virus. 1

7 The preliminary results from infected pigs in experiment 1 were quite interesting, Fig.. This graph depicts the overall average cytokine mrna expression in the two experiments. There appeared to be a period of cytokine response very early in the infection at h PI, with IFNα, TNFα and IL-1α in particular rising above their background levels of expression as illustrated in Fig. above. This increased mrna expression was followed by a period of possible suppression, which included to a lesser extent the endogenous control and TNFα. Experiment also revealed a possible period of suppression of mrna expression, see Fig.. However, in this instance it occurred later, at PI and affected to a greater extent. Interestingly, mrna was expressed at a lower level than IL-1α mrna, which demonstrated a very good average response in this experiment. TNFα mrna levels were not suppressed as much as the other cytokines at h PI, but were then not detectable at h PI. It should also be noted that clinical disease occurred later in experiment than in experiment 1, perhaps due to the cubicle arrangements used in experiment. Average Cytokine Expression During FMDV Infection Experiment 1 Log Copies per 1.0E+ cells Average Cytokine Expression During FMDV Infection Experiment Hour Post Infection Figure Average Cytokine-expression by FMDV infected pigs in Experiments 1 and 1

8 Average Cytokine Expression in Low, Medium and High Dose Groups (Exp. 1) The low dose group of pigs appeared to demonstrate a similar pattern of expression for the IFNα, TNFα, IL-1α in Fig. below. There was an increase in expression at h PI, followed by a rapid decline, which extended to h PI. IFNγ demonstrated an earlier initial increase in expression h PI, which was also followed by a rapid decline up to h PI. There then followed an increase in the expression of all four cytokines, with IFNγ showing the greatest initial increase of logs at h PI. IFNα, TNFα and IL-1α then increased further at h PI. Interestingly, both of the peaks of IFNγ occurred earlier than the peaks of the other three cytokines. The medium dose group of pigs did not demonstrate the same pattern of expression as the low dose group in Fig. below. There did not appear to be an early response at h PI, as there was in the case of the low dose group. However, there was a decline in expression of IFNα, IFNγ, and TNFα mrnas at h PI. IL-1α mrna expression did not decline at this point, but was maintained. By h PI an increase in the expression of all cytokines occurred with IFNγ demonstrating the greatest degree of response once again. IFNγ again responded well in the high dose group of pigs, Fig. below, with a large increase in expression h PI that was maintained until h PI when it decreased. By h PI, IFNγ expression was elevated again. There was a poor response in the case of IFNα, but TNFα in particular and IL-1α demonstrated peaks at h and h PI. Low Dose Group Average Cytokine Expression Log Copies per 1.0E+ cells Medium Dose Group Average Cytokine Expression Hour Post Infection 1

9 High Dose Group Average Cytokine Expression Log Copies per 1.0E+ cells Hour Post Infection Figure Grouped Average Cytokine Expression in FMDV Infected Pigs (Exp. 1) Average Cytokine Expression in Low, Medium and High Dose Groups (Exp. ): It is difficult to comment on the patterns of expression of the cytokines in experiment, Fig. below, because of the behaviour of, which closely follows the patterns of cytokine mrna expression, with the exception of TNFα. We think it is likely that the FMD infection induces a complex pattern of mrna induction and degradation leading to a very complex picture that not only involves inducible cytokine mrnas but perhaps other cellular constituents that may include and other RNAs. Another less likely explanation could be that no real cytokine response has been detected and that the results are merely illustrating a combination of the quality of the PBMCs isolated from each blood sample and the quality of the RNA extracted from those PBMCs. There is evidence against this, as it appeared that there was a genuine response in the case of IL-1α in all three groups of pigs. The background level of expression determined for IL- 1α was less than log copies per 1.0E+ cells, see Fig.. In contrast, the levels of expression during this experiment were as high as log copies per 1.0E+ cells at h PI, an increase in expression of logs. IL-1α and TNFα mrna levels were both elevated at h PI in the low dose group of pigs, this was followed by the possible period of suppression that occurred at h PI in experiment, hours later than the decrease in experiment 1. However, TNFα mrna levels did not decrease as much as the other cytokines at h PI, but were not detectable at h PI. The medium dose group of pigs exhibited a similar pattern of cytokine mrna expression with regard to suppression at h PI. However, there did not seem to be an equally good response from IL-1α, which peaked at h PI at. log copies per 1.0E+ cells compared with.0 log copies per 1.0E+ cells in the low dose group. The decline in mrna expression at h PI was not as severe for the medium dose group of pigs, with all mrnas remaining within the detectable range. The high dose group of pigs again demonstrated a similar pattern of cytokine expression. In this case the decline at h PI was even less pronounced and was followed by an increase in the expression of all mrnas but TNFα at h PI. 1

10 Average Low Group Cytokine Expression Average Medium Group Cytokine Expression Log Copies per 1.0E+ cells Average High Group Cytokine Expression Hour Post Infection Figure Grouped Average Cytokine Expression in FMDV Infected Pigs (Exp. ) 1

11 Discussion The aim of this study was to gain a deeper understanding into the pathobiology of FMDV infection in pigs and into the pathogenesis of the disease through the quantitation of the mrna levels of a variety of antiviral and proinflammatory cytokines. The very severe nature of FMD infection in pigs leads us to believe that the porcine immune system is particularly activated in order to eliminate the virus, and that this activation may cause some of the general clinical signs observed in pigs. By studying cytokine expression, an insight into the innate immune response to FMDV can be gained. In the present preliminary study, cytokines were quantitated by real-time RT-PCR, an increasingly popular method for the detection of cytokine mrna,,11,1,1,. The assay utilised in this study was validated by determining the magnitude of induction of cytokine mrnas by activation of porcine PBMCs. This work demonstrated that the assay could effectively be applied to the detection of cytokine mrna fluctuations in porcine PBMCs. The housekeeping gene, was used as an endogenous control to demonstrate that cellular mrna was being efficiently extracted, reverse transcribed into cdna and amplified by real-time PCR. expression remained relatively constant in the in vitro PBMC experimental system, however there was evidence of a degree of fluctuation of in the in vivo system. In our in vivo system average levels were determined to be approximately 10 copies of per 10 cells (the number of cells from which mrna was extracted). A fluctuation of mrna expression of almost logs was seen in these uninfected pigs, Fig.. However, the observed fluctuations of cytokine and mrna levels were much greater in FMDV infected pigs, Fig.. The effect of FMDV infection on was mainly observed as a severe decrease in expression, Fig.. There is evidence that transcription can be inducible in some systems,10. Nevertheless, we believe that it is more likely that the suppression effect seen on and the cytokines studied was as a direct result of FMDV infection or possibly as a result of the effects of fever and other host processes induced by the virus. Neither of the two animal experiments carried out revealed a positive result for the expression of IFNβ. However, IFNβ was expressed (albeit at a relatively low level, see Fig. ) by the lectin-stimulated PBMCs. The likely reason for this is the cells being assayed in this study; IFNβ is predominantly a product of fibroblasts, not PBMCs as were sampled here. The expression of IFNβ by lectin-stimulated PBMCs may be the result of their over-stimulation to the degree that more signalling pathways were activated than occurs during FMDV infection. The overall average cytokine expression for the two animal experiments revealed a similar pattern in both cases, see Fig. above. In experiment 1 an initial peak of cytokine mrna expression was seen from as early as h to h PI. This was followed, by a decline in mrna expression at h PI. Expression appeared to recover by h PI, it is possible that the translation of early expressed cytokine mrna into protein communicators led to a second round of cytokine mrna expression. Experiment (with animals in groups 1 and housed individually) revealed a slightly different pattern of cytokine mrna expression The first peak of cytokine mrna expression was not seen before h PI, but was followed by a rapid decline at h PI, similar to the decline observed in experiment 1 at h PI. Levels of mrna expression in experiment did not begin to recover until h PI. As previously mentioned, this may be an effect of the cubicle arrangements in the second experiment, where pigs were 10

12 housed individually and developed disease later than pigs in experiment 1. Cytokine mrna expression appeared to recover more rapidly in the case of experiment 1, this may be due to higher levels of virus in the environment, or possibly because the pigs in experiment had experienced unrelated health problems in the period immediately before FMD infection. The high dose group of pigs in experiment were housed as a group like those in experiment 1, but still exhibited a later peak of cytokine mrna expression, see Fig.. However, in comparison with the other two groups in experiment, the recovery of cytokine mrna expression in the high dose group occurred earlier and was elevated by h PI, see Fig.. FMD infection appears to induce a very complex picture of mrna stimulation and degradation. If mrna is indeed degraded at certain points in the disease process, this may be due to a direct effect of the virus. However, we think it is more likely that this is due to the effects of cytokines released as a result of infection. For instance through the induction of intra-cellular RNase-L by interferons, which produces extensive cleavage of both host and viral mrna leading to the inhibition of protein synthesis 1. More work is required both at the mrna and the protein level so that virus and host effects during infection can be elucidated. Reference List 1. Alexandersen, S., I. Brotherhood, and A. I. Donaldson. 00. Natural aerosol transmission of foot-and-mouth disease virus to pigs: minimal infectious dose for strain O1 Lausanne. Epidemiol. Infect. 1: Alexandersen, S. and A. I. Donaldson. 00. Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs. Epidemiol. Infect. 1:1-.. Alexandersen, S., M. B. Oleksiewicz, and A. I. Donaldson The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative timecourse study using TaqMan RT-PCR. J. Gen. Virol. :-.. Alexandersen, S., Z. Zhang, and A. Donaldson. 00. Aspects of the persistence of foot-and-mouth disease virus in animals-the carrier problem. Microbes. Infect. :10.. Alexandersen, S., Z. Zhang, S. M. Reid, G. H. Hutchings, and A. I. Donaldson. 00. Quantities of infectious virus and viral RNA recovered from sheep and cattle experimentally infected with foot-and-mouth disease virus O UK 001. J Gen. Virol. : Bachrach, H. L. 1. Foot-and-mouth disease. Annu Rev Microbiol :01-.. Barroso, I., B. Benito, C. Garci-Jimenez, A. Hernandez, M. J. Obregon, and P. Santisteban. 1. Norepinephrine, tri-iodothyronine and insulin upregulate glyceraldehyde--phosphate dehydrogenase mrna during Brown adipocyte differentiation. Eur. J. Endocrinol. 11:1-1.. Blaschke, V., K. Reich, S. Blaschke, S. Zipprich, and C. Neumann Rapid quantitation of proinflammatory and chemoattractant cytokine expression in small tissue samples and monocyte-derived dendritic cells: validation of a new real-time RT- PCR technology. J. Immunol. Methods :-0. 11

13 . Brieland, J. K., C. Jackson, S. Hurst, D. Loebenberg, T. Muchamuel, R. Debets, R. Kastelein, T. Churakova, J. Abrams, R. Hare, and A. O'Garra Immunomodulatory role of endogenous interleukin-1 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection. Infect. Immun. : Bustin, S. A Absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology : Casteels, K. M., C. Mathieu, M. Waer, D. Valckx, L. Overbergh, J. M. Laureys, and R. Bouillon. 1. Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1,-dihydroxyvitamin D in combination with a short induction course of cyclosporin A. Endocrinology 1: Goodbourn, S., L. Didcock, and R. E. Randall Interferons: cell signalling, immune modulation, antiviral responses and virus countermeasures. Journal of General Virology 1: Janeway, C. A., Jr. and R. Medzhitov. 00. Innate immune recognition. Annu Rev Immunol. 0: Le Page, C., P. Genin, M. G. Baines, and J. Hiscott Interferon activation and innate immunity. Rev Immunogenet. :-. 1. Liu, Z., K. Geboes, S. Colpaert, L. Overbergh, C. Mathieu, H. Heremans, M. de Boer, L. Boon, G. D'Haens, P. Rutgeerts, and J. L. Ceuppens Prevention of experimental colitis in SCID mice reconstituted with CDRBhigh CD+ T cells by blocking the CD0-CD1 interactions. J. Immunol. 1: Nandi, S., B.S.Negi, and A.K.Sen. 1. Epidemiology of foot and mouth disease in sheep and goats. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases 0: O'Neill, L. A The interleukin-1 receptor/toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci. STKE. 000:RE1. 1. Oleksiewicz, M. B., A. I. Donaldson, and S. Alexandersen Development of a novel real-time RT-PCR assay for quantitation of foot- and-mouth disease virus in diverse porcine tissues. J. Virol. Methods :-. 1. Overbergh, L., D. Valckx, M. Waer, and C. Mathieu. 1. Quantification of murine cytokine mrnas using real time quantitative reverse transcriptase PCR. Cytokine 11: Reid, S., N. Ferris, G. Hutchings, Z. Zhang, G. Belsham, and S. Alexandersen. 00. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay J Virol. Methods 10:. 1. Schutze, S., K. Wiegmann, T. Machleidt, and M. Kronke. 1. TNF-induced activation of NF-kappa B. Immunobiology 1:1-0. 1

14 . Sen, G. C. and P. Lengyel. 1. The interferon system. A bird's eye view of its biochemistry. J. Biol. Chem. : Sirlin J.L. 1. Molecular Species of RNA, p. 1-. Biology of RNA. Academic Press, London.. Sobrino, F., M. Saiz, M. A. Jimenez-Clavero, J. I. Nunez, M. F. Rosas, E. Baranowski, and V. Ley Foot-and-mouth disease virus: a long known virus, but a current threat. Vet. Res. :1-0.. Stordeur, P., L. F. Poulin, L. Craciun, L. Zhou, L. Schandene, A. de Lavareille, S. Goriely, and M. Goldman. 00. Cytokine mrna quantification by real-time PCR. J. Immunol. Methods :-.. Takamatsu, H. H., M. S. Denyer, and T. E. Wileman. 00. A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells. Vet. Immunol. Immunopathol. :-.. Yang, H. and R. M. Parkhouse Characterization of the porcine gammadelta T- cell receptor structure and cellular distribution by monoclonal antibody PPT Immunology :0-0.. Yin, J. L., N. A. Shackel, A. Zekry, P. H. McGuinness, C. Richards, K. V. Putten, G. W. McCaughan, J. M. Eris, and G. A. Bishop Real-time reverse transcriptasepolymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mrna expression with fluorogenic probes or SYBR Green I. Immunol. Cell Biol. :1-1. 1