Background We recently showed that PARP-1 may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1−/− mice, IL-4 or IgE replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5. Objective To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia. Methods This study was conducted using a murine model of allergic airway inflammation and primary splenocytes. Results PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. STAT-6 protein was severely downregulated in spleens of PARP-1−/− mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1−/− mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The dowregulation of STAT-6 coincided with mRNA and protein reduction of GATA-3 and occupancy of its binding site on the IL-5 gene-promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1−/− mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes. Conclusion These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.

SummaryBackground-Identifying therapeutic drugs that block the release or effects of T-helper type 2 (Th2) cytokines after allergen exposure is an important goal for the treatment of allergic inflammatory diseases including asthma. We recently showed, using a murine model of allergic airway inflammation, that poly(ADP-ribose) polymerase (PARP) plays an important role in the pathogenesis of asthma-related lung inflammation. PARP inhibition, by single injection of a novel inhibitor, thieno [2,3-c]isoquinolin-5-one (TIQ-A), before ovalbumin (OVA) challenge, prevented airway eosinophilia in C57BL/6 mice with concomitant suppression of Th2 cytokine production and mucus secretion.

As respiratory syncytial virus (RSV) targets the mucosal surfaces of the respiratory tract, induction of both systemic and mucosal immunity will be critical for optimal protection. In this study, the ability of an intranasally delivered, formalin-inactivated bovine RSV (FI-BRSV) vaccine co-formulated with CpG oligodeoxynucleotides (ODN) and polyphosphazenes (PP) to induce systemic and mucosal immunity, as well as protection from BRSV challenge, was evaluated. Intranasal immunization of mice with FI-BRSV formulated with CpG ODN and PP resulted in both humoral and cell-mediated immunity, characterized by enhanced production of BRSV-specific serum IgG, as well as increased gamma interferon and decreased interleukin-5 production by in vitro-restimulated splenocytes. These mice also developed mucosal immune responses, as was evident from increased production of BRSV-specific IgG and IgA in lung-fragment cultures. Indeed, the increases in serum and mucosal IgG, and in particular mucosal IgA and virusneutralizing antibodies, were the most critical differences observed between FI-BRSV formulated with both CpG ODN and PP in comparison to formulations with CpG ODN, non-CpG ODN or PP individually. Finally, FI-BRSV/CpG/PP was the only formulation that resulted in a significant reduction in viral replication upon BRSV challenge. Co-formulation of CpG ODN and PP is a promising new vaccine platform technology that may have applications in mucosal immunization in humans. INTRODUCTIONHuman respiratory syncytial virus (HRSV) is a leading cause of respiratory disease in infants and young children worldwide (Heilman, 1990) and is responsible for significant economic loss; in the year 2000 alone, there were 86 000 HRSV infection-related hospitalizations in the USA, costing a total of $394 000 000 (Paramore et al., 2004). Nearly 98 % of these hospitalizations occurred in children under 5 years old. Between 1997 and 2000, HRSV bronchiolitis was the leading cause of infant hospitalization and, in 1999, an estimated 360 HRSV-associated postneonatal deaths (i.e. in children aged between 28 days and 1 year) occurred in the USA (Leader & Kohlhase, 2003).Like HRSV, bovine respiratory syncytial virus (BRSV) is an enveloped, non-segmented, single-stranded RNA pneumovirus of the family Paramyxoviridae and order Mononegavirales. BRSV is responsible for significant economic loss to the cattle industry (Stott & Taylor, 1985) and is one of the four known viral components of bovine shipping fever. HRSV and BRSV have similar clinical outcomes in their respective host species, ranging from asymptomatic infection to bronchiolitis and pneumonia, and sometimes death (Philippou et al., 2000). The distribution of both viruses is worldwide.Whilst there are several commercial BRSV vaccines currently available for immunizing cattle, better vaccines that are more efficacious in the face of maternal antibodies and that induce longer-lasting protection would be desirable. Also, there is currently no safe and effective vaccine against HRSV available for use in h...

Inducible nitric oxide synthase (iNOS) inhibition was recently shown to exert no effect on allergen challenge in human asthma, raising serious concerns about the role of the protein in the disease. The present study investigated the role of iNOS in ovalbumin-induced eosinophilia from the perspective of its relationship with poly(ADP-ribose) polymerase-1 (PARP-1) and oxidative DNA damage.A mouse model of ovalbumin-induced eosinophilia was used to conduct the studies. iNOS-associated protein nitration and tissue damage were partially responsible for allergeninduced eosinophilia. iNOS expression was required for oxidative DNA damage and PARP-1 activation upon allergen challenge. PARP-1 was required for iNOS expression and protein nitration, and this requirement was connected to nuclear factor-kB. PARP-1 was an important substrate for iNOS-associated by-products after ovalbumin-challenge. PARP-1 nitration blocked its poly(ADPribosyl)ation activity. Interleukin-5 re-establishment in ovalbumin-exposed PARP-1 -/-mice reversed eosinophilia and partial mucus production without a reversal of iNOS expression, concomitant protein nitration or associated DNA damage.The present results demonstrate a reciprocal relationship between inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 and suggest that expression of inducible nitric oxide synthase may be dispensable for eosinophilia after interleukin-5 production. Inducible nitric oxide synthase may be required for oxidative DNA damage and full manifestation of mucus production. Such dispensability may explain, in part, the reported ineffectiveness of inducible nitric oxide synthase inhibition in preventing allergen-induced inflammation in humans.

We recently used a murine model of allergic airway inflammation to show that poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in the pathogenesis of asthma-related lung inflammation. In this study, we show that PARP-1 inhibition, by a novel inhibitor (TIQ-A) or by gene deletion, prevented eosinophilic infiltration into the airways of OVA-challenged mice. Such impairment of eosinophil recruitment appeared to take place after IgE production. OVA challenge of wild-type mice resulted in a significant increase in IL-4, IL-5, IL-10, IL-13, and GM-CSF secretions. Although IL-4 production was moderately affected in OVA-challenged PARP-1−/− mice, the production of IL-5, IL-10, IL-13, and GM-CSF was completely inhibited in ex vivo OVA-challenged lung cells derived from these animals. A single TIQ-A injection before OVA challenge in wild-type mice mimicked the latter effects. The marked effect PARP-1 inhibition exerted on mucus production corroborated the effects observed on the Th2 response. Although PARP-1 inhibition by gene knockout increased the production of the Th1 cytokines IL-2 and IL-12, the inhibition by TIQ-A exerted no effect on these two cytokines. The failure of lung cells derived from OVA-challenged PARP-1−/− mice to synthesize GM-CSF, a key cytokine in eosinophil recruitment, was reestablished by replenishment of IL-5. Furthermore, intranasal administration of IL-5 restored the impairment of eosinophil recruitment and mucus production in OVA-challenged PARP-1−/− mice. The replenishment of either IL-4 or IgE, however, did not result in such phenotype reversals. Altogether, these results suggest that PARP-1 plays a critical role in eosinophil recruitment by specifically regulating the cascade leading to IL-5 production.

No abstract

Vaccination of calves with formalin-inactivated bovine respiratory syncytial virus (FI-BRSV) induces low levels of cellular immunity that may not be protective. Since inactivated and subunit vaccines formulated with CpG oligodeoxynucleotides (ODNs) have been shown to induce cellular immune responses, we studied the ability of a FI-BRSV vaccine formulated with CpG ODN to elicit cellular immunity against BRSV. Neonatal calves were immunized with FI-BRSV, FI-BRSV formulated with CpG ODN or medium and challenged with BRSV after two immunizations. Calves vaccinated with FI-BRSV formulated with CpG ODN developed increased numbers of IFN-gamma secreting cells in the peripheral blood and broncho-tracheal lymph nodes and enhanced BRSV-specific serum IgG2 in comparison to FI-BRSV immunized animals. Calves that received the FI-BRSV vaccine formulated with CpG ODN also experienced a reduction in the amount of BRSV in the lung tissue. Based on these observations, CpG ODN appears to be a suitable candidate adjuvant for inactivated BRSV vaccines.

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV