Large-scale immunohistochemical examination for lymphoreticular prion protein in tonsil specimens collected in Britain.

There have been 173 cases of variant Creutzfeldt-Jakob disease (vCJD) in the UK, as of 5 July 2010, as a result of the bovine spongiform encephalopathy epidemic. The number of individuals subclinically infected with vCJD, and thus the eventual number of cases, remains, however, uncertain. In an attempt to address this problem, 63,007 tonsil tissue specimens were previously tested by enzyme immunoassay (EIA) for the presence of disease-related prion protein (PrP(res)) and found to be negative. To confirm the reliability of this result, all those in the birth cohort most at risk (1961-1985) and a few others, including controls, have now been tested by immunohistochemistry (IHC). Histological slides were prepared from 10,075 anonymized formalin-fixed, paraffin-embedded tissues and examined for PrP(res) with two anti-prion protein antibodies, ICMS35 and KG9. One specimen showed a single strongly positive follicle with both antibodies, on two slides from adjacent sections. As this specimen was negative when it was further investigated by EIA, IHC, and immunoblotting, it is unclear whether the patient from whom the tonsil came will go on to develop vCJD. If, however, this is the case, then a finding of 1 out of 9160 gives a prevalence of disease-related prion protein in the British population of 109 per million, with a 95% confidence interval (CI) of 3-608 per million, which is not statistically different (exact p = 0.63) from population prevalence estimates based on finding three positives out of 10 278 in a previous IHC study of appendix tissue. If this is not the case, a finding of 0 out of 9160 gives a prevalence of 0-403 per million (95% CI) for the 1961-1985 cohort, which is also not different (exact p = 0.25) from previous population prevalence estimates. Therefore, the results of this work could be summarized as finding, by IHC, no or one vCJD-positive individual.

Changes in lymphocyte subsets and cytokines during European porcine reproductive and respiratory syndrome: increased expression of IL-12 and IL-10 and proliferation of CD4(-)CD8(high).

The changes in peripheral blood mononuclear cells (PBMCs) have been studied in several reports in an attempt to determine the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, how these changes are evoked after PRRSV infection has not yet been clarified. The aim of this study was to analyze the changes seen in lymphocyte subsets and immunomodulatory cytokine expression in pigs after an acute experimental infection with a European PRRSV field isolate. Pigs were inoculated intramuscularly with PRRSV field isolate 2982. Samples from blood, medial retropharyngeal and tracheobronchial lymph nodes, and spleen were collected at different time points for flow cytometry studies and for cytokine expression by ELISA. CD21(+) cell counts increased in PBMCs and tracheobronchial lymph node cells from 17 to 24 dpi, coinciding with an increase in PRRSV-specific antibody titer in blood. CD3(+) T-cell counts increased mainly due to an enhancement of CD4(-)CD8(high) and CD4(+)CD8(+) T cells. CD4(-)CD8(low) T cells were decreased in all organs studied, whereas CD4(+)CD8(-) T cells decreased only in the spleen. The drop in viremia correlated with an enhancement of CD4(-)CD8(high) T cells, and with a higher expression of interleukin-10 (IL-10) and interleukin-12 p40 (IL-12 p40). No efficient interferon-gamma (IFN-gamma) response was detected during the acute phase of the infection, and the expression of interferon-alpha (IFN-alpha) was late and reached its maximum expression once the viremia decreased. These results point to IL-10 and IL-12 as cytokines that might play a significant role in the PRRSV immune response, as may CD4(-)CD8(high) T cells.

A method for the generation of ectromelia virus (ECTV) recombinants: in vivo analysis of ECTV vCD30 deletion mutants.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date. CONCLUSIONS/SIGNIFICANCE: We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.