Expression of cytokine mRNA and protein in joints and lymphoid organs during the course of rat antigen-induced arthritis

Cytokine expression was assessed during antigen-induced arthritis (AIA) in synovial membrane (SM), inguinal lymph node (LN), and spleen using competitive RT-PCR and sandwich ELISA. In the SM, early elevations of IL-1β and IL-6 mRNA (by 6 hours; 450- and 200-fold, respectively) correlated with the joint swelling; a 6-fold increase in tumor necrosis factor α (TNFα) was not significant. Not only IL-2 and IFN-γ (which increased 10,000-fold and 200-fold, respectively), but also IL-5 and IL-10, increased acutely (6 hours – day 1; 3-fold and 35-fold, respectively) in the SM. In general, the protein levels in the SM for IL-1β, IL-6, TNFα, IFN-γ, IL-4, and IL-10 (increase from 4-fold to 15-fold) matched the course of mRNA expression. In the inguinal LN, there were early mRNA elevations of IL-6 (a 2.5-fold increase by 6 hours, which correlated positively with the joint swelling) and IL-2 (4-fold by 6 hours), as well as later rises of IL-4 and IL-5 (2.5- and 4-fold, respectively, by day 3). No significant elevations of the corresponding proteins in this tissue were observed, except for IL-1β (by day 6) and IL-10 (by day 1). In the spleen, there were significant mRNA elevations at 6 hours of IL-1β (1.5-fold), IL-6 (4-fold; positively correlated with the joint swelling), IFN-γ (3-fold), and IL-2 (7- to 10-fold). IL-5 and IL-10 (2- and 3-fold, respectively) peaked from 6 hours to day 3 in the spleen. Increases of the corresponding proteins were significant in comparison with day 0 only in the case of IL-2 (day 6). By day 6 (transition to the chronic phase), the mRNA for cytokines declined to or below prearthritis levels in all the tissues studied except for IL-1β in the SM and IL-6 in the spleen. AIA is thus characterized by four phenomena: early synovial activation of macrophages, T helper (Th)1-like, and Th2-like cells; late, well-segregated Th2-like responses in the inguinal LN; late, overlapping Th1-like/Th2-like peaks in the spleen; and chronic elevation of synovial IL-1β mRNA and spleen IL-6 mRNA

Inactivated Orf-virus shows disease modifying antiviral activity in a guinea pig model of genital herpesvirus infection

Background: Inactivated Orf virus (iORFV) has been used as a preventative as well as a therapeutic immunomodulator in veterinary medicine in different species. iORFV elicits strong effects on cytokine secretion in mice and human immune cells leading to an auto-regulated loop of initial up-regulation of inflammatory and Th1-related cytokines followed by Th2-related cytokines that attenuate immunopathology. The therapeutic potential of iORFV has been recognized in several models for difficult-to-treat disease areas such as chronic viral diseases, liver fibrosis or various forms of cancer. Methods: Guinea pigs were infected with Human Herpesvirus (HSV)-2 strain MS and treated with iORFV, Acyclovir (ACV) or placebo, respectively. Clinical score of herpes lesions and viral shedding was assessed over a period of 40 days. In addition, viral DNA in dorsal root ganglia was quantified at the end of the study. Results: Disease symptoms were minimal or absent in iORFV-treated guinea pigs but tended to be severe in animals treated with either ACV or placebo. The cumulated disease score was significantly reduced in iORFV-treated but not in ACV- or placebo-treated guinea pigs. In addition, treatment with iORFV, but not ACV or placebo, led to significant reduction of viral DNA load in dorsal root ganglia. Conclusion: iORFV effectively suppressed recurrences in guinea pigs experimentally infected with HSV. iORFV did not only reduce recurrent disease episodes but was, compared with ACV, more effective in reducing latency as measured by viral DNA detected in dorsal root ganglia of infected animals. Keywords: Immunotherapy, Immunomodulation, Orf virus, Herpesvirus, Recurrent genital herpes diseas

Inactivated ORF virus shows antifibrotic activity and inhibits human hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in preclinical models.

Inactivated orf virus (iORFV), strain D1701, is a potent immune modulator in various animal species. We recently demonstrated that iORFV induces strong antiviral activity in animal models of acute and chronic viral infections. In addition, we found D1701-mediated antifibrotic effects in different rat models of liver fibrosis. In the present study, we compare iORFV derived from two different strains of ORFV, D1701 and NZ2, respectively, with respect to their antifibrotic potential as well as their potential to induce an antiviral response controlling infections with the hepatotropic pathogens hepatitis C virus (HCV) and hepatitis B virus (HBV). Both strains of ORFV showed anti-viral activity against HCV in vitro and against HBV in a transgenic mouse model without signs of necro-inflammation in vivo. Our experiments suggest that the absence of liver damage is potentially mediated by iORFV-induced downregulation of antigen cross-presentation in liver sinus endothelial cells. Furthermore, both strains showed significant anti-fibrotic activity in rat models of liver fibrosis. iORFV strain NZ2 appeared more potent compared to strain D1701 with respect to both its antiviral and antifibrotic activity on the basis of dosages estimated by titration of active virus. These results show a potential therapeutic approach against two important human liver pathogens HBV and HCV that independently addresses concomitant liver fibrosis. Further studies are required to characterize the details of the mechanisms involved in this novel therapeutic principle

iORFV NZ2 is a more potent inhibitor of HBV than strain D1701 in HBV-transgenic mice.

<p>iORFV D1701, iORFV NZ2 or placebo was administered i.p. every third day, three times in total, to HBV-transgenic mice expressing 10⁷–10⁸ HBV genome equivalents per ml plasma (n = 7 per group). HBV-specific DNA from plasma was analyzed by quantitative PCR and from livers by dot-blot hybridization as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074605#pone.0074605-Weber1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074605#pone.0074605-Chromczynski1" target="_blank">[18]</a>. The figure shows means ± standard error of means (SEM) relative to placebo-treated animals where mean HBV-DNA content was set 100%. Treatment with iORFV reduced non-chromosomal HBV-DNA in the livers as compared to placebo animals <b>(a)</b> and in the plasma with the exception of the lowest dose of iORFV D1701 <b>(b)</b>. iORFV NZ-2 was more potent in terms of its potential to inhibit HBV replication compared to strain D1701 with the two lower dosages resulting in significantly higher reduction of HBV-DNA (one-way ANOVA and <i>post hoc</i> analysis [Newman-Keuls Multiple Comparison Test] *p<0.05, **p<0.01). <b>c)</b> Immunohistological analysis of HBcAg expression in the livers of placebo-treated animals. Diffuse cytoplasmic staining in periportal areas [arrows, central veins (CV)] indicates viral capsids and ongoing HBV replication in placebo-treated mice. Cytoplasmic HBcAg as well as nuclear HBcAg-specific stain (for empty capsids) was strongly reduced in both iORFV (NZ2)-treated <b>d)</b> and iORFV (D1701)-treated mice <b>e)</b>. Figures provide typical examples of the respective group of animals treated with a dose of 1.5×10⁶ TCID₅₀ iORFV NZ 2 or D1701, respectively.</p

Both ORFV strains induce cytokines suppressing HCV replication in an <i>in vitro</i> replicon system.

<p>Figures shows relative fluorescence units (RFU) as a measure for HCV replication. <b>a)</b> 20 µl (dose 1.6×10⁵ TCID₅₀) iORFV strain D1701 or NZ2 was added to whole blood and incubated for 3 days (see methods). Supernatants of cultured blood cells were added to replicon cells and HCV replication was determined three days later. Statistical analyses were performed using the one-way ANOVA and as <i>post hoc</i> analysis the Bonferroni Test. *p<0.05 vs. placebo. RFU-value from placebo (PBS) - treated supernatants was set as 100%. ConA served as a positive control. <b>b)</b> Cell viability of replicon-bearing cells was not affected after addition of supernatants from iORFV-incubated human blood cells. <b>c)</b> iORFV inhibits HCV replication in a dose-dependent manner. D 1701 or NZ2 were used for incubation with whole blood at doses ranging from 1.6×10⁵ TCID₅₀ to 1.6×10³ TCID₅₀. Values (n = 3) show means ± standard error of means (SEM).</p

Treatment with inactivated ORFV reduces HBV core antigen (HBcAg) in HBV-transgenic mice (n = 7/group).

*<p>Scores of anti-HbcAg stain: Nuclei: grade 3: the majority of nuclei in the liver specimens revealed intense immunostaining; grade 2: the number of positive nuclei was slightly reduced predominantly at the periphery of the liver lobule; grade 1: further reduction of positive nuclei was observed. Cytoplasm: grade 3: strong staining of numerous hepatocytes was observed around the central veins; grade 2: the cytoplasmic staining was reduced; grade 1: a further reduction or even absence of cytoplasmic immunostaining was observed. The histological slides were cross-checked by an independent pathologist and the results confirmed.</p><p>The severity of HBcAG-specific stain was quantified according to a scoring from 1 to 3*.</p

iORFV (D1701) reduces antigen cross-presentation by liver sinus endothelial cells (LSEC) but enhances activation of T-cells.

<p>Activation of CD8+ T cells (B3Z) was measured by IL-2 secretion.</p