Canine distemper virus (CDV) infection of the central nervous system results in lesions of the gray and white matter. While a biphasic disease process has been discussed for leukoencephalitis with a prominent loss of viral protein expression, polioencephalitis has been associated with virus persistence. Using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), expression of pro- and anti-inflammatory cytokines such as interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-alpha (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta were studied in the cerebra of distemper dogs with white matter lesions in the cerebellum. Additionally, cytokine values were correlated with the degree of CDV infection, major histocompatibility complex class II (MHC II) expression, and infiltration of CD4-, CD8-, and CD3epsilon-positive lymphocytes. Cerebral CDV infection was not associated with detectable light microscopic lesions or infiltration of B and T lymphocytes. However, an increasing number of CDV-antigen-positive cells was associated with an upregulation of MHC II antigen. RT-PCR results revealed a significant upregulation of IL-6, IL-8, IL-12, and TNF-alpha in the cerebra of distemper dogs, whereas IL-10 and TGF-beta showed no significant increase. Elevated cytokine values were directly related to the presence of CDV antigen and MHC II upregulation. However, succeeding increases of the latter did not result in an additional proportional elevation of cytokine expression values. In summary, the present study demonstrates the expression of pro-inflammatory cytokines by resident neural cells following CDV infection. Furthermore, the lack of light microscopic changes indicates that additional factors besides cytokines are necessary for the development of a distemper-characteristic neuropathology.
Total ribonucleic acld (RNA) isolated from a continuous canine macrophage cell line (Df182) was used in reverse transcrlpuon polymerase chain reactions (RT-PCR) for the detection of transcripts of interleukin (IL)-8, -12, and tumour necrosis factor-a W F ) .Three different methods of RNA isolation (standard guanidinium-thiocyanate method with and without application of RNA matrix, and boiling) were used and compared in regard to RT-PCR results. The most suitable method was used to establish RT-PCR amplification of mRNA transcripts of IL-2, -10, and mterfcron-y (IPN) in RNA isolated from canine peripheral blood leukocytes. Integrity of RNA isolates was ensured by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or p-actin. 1L-8, -12, and TNF were amphfied from RNA isolated by vanous methods. Use of guanidinium-thiocyanate with and without RNA matrix gave the most consistent results. Boihng as a mean of RNA isolauon was quick and easy, but the RT-PCR results were extremely variable and multiple smaller bands were observed in the agarose gel in some preparations. IL-2, -10 and IFN transcripts were amplified from RNA isolated with guamidimium-thiocyanate from leukocytes stimulated wlth concanavalln A. DNase-treatment of RNA isolates was necessary to assure the destruction of genomlc DNA and to avoid amplification of genomic sequences. This was especially a problem when using primers for GAPDH, p-actin, IL-12, and TNF. Lack of DNase-treatment may lead to false posiuve results. lhis may be especially a problem when amphficatlon of so-called house-keeping genes is used as internal control for RNA integnty. These findings demonstrated that isolation of total RNA with guamdinium-thiocyanate followed by DNase-treatment gave reliable and consistent results for detection of cytokine transcrlpts by RT-PCR in a camne macrophage cell line and caninc peripheral blood leukocytes.
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