Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-b is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator-activated receptor (PPAR) g is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARg ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-b-stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of a-smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARg ligand, 15-deoxy-D 12,14 -prostaglandin J 2 (15 d-PGJ 2 ), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARg-independent pathway. CDDO and 15 d-PGJ 2 contain an a/b unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A 1 and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ 2 lacking the electrophilic center is much less potent. CDDO does not alter TGF-b-induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-b-responsive genes. Overall, these data indicate that certain PPARg ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-b-mediated profibrogenic activities through pathways independent of PPARg. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.

Purpose EGFR is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates anti-tumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity. Experimental Design We measured in vitro activation of cellular components of the innate and adaptive immune system. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab. Results Both mAb primarily activate NK cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate ADCC against HNSCC tumor cells, and interestingly, this effect was FcγRIIa and FcγRIIIa genotype dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa), however monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced DC maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8+ T cells in patients treated with cetuximab compared to those treated with panitumumab. Conclusions Although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering anti-tumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC.

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 days of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average more than 70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV-specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil which is associated with increasing disease severity.

Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPARgamma ligands 15d-PGJ2 and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPARgamma independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ2 contain an alpha/beta unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPARgamma ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ2 and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ2 or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ2 and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPARgamma ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.

Background Despite the success of antiretroviral therapy (ART), latent HIV-1 continues to persist in a long-lived population of resting memory CD4 + T cells within those who are infected. Finding a safe and effective means to induce latency reversal (LR) during ART to specifically expose this latent HIV-1 cellular reservoir for immune elimination has been a major barrier to a functional cure. Methods In this study, we test the use of antigen-presenting type 1-polarized, monocyte-derived dendritic cells (MDC1) generated from chronic HIV-1-infected individuals on ART as a means to induce HIV-1 latency reversal in autologous CD4 + T cells harboring replication-competent provirus. We use the same MDC1 for ex-vivo generation of autologous HIV-1 antigen-specific CD8 + cytotoxic T cells (CTL) and test their effector responses against the MDC1-exposed HIV-1- infected CD4 + T cell targets. Findings MDC1 presentation of either HIV-1 or cytomegalovirus (CMV) antigens to CD4 + T cells facilitated HIV-1 LR. This antigen-driven MDC1-mediated LR was sharply diminished with blockade of the CD40L/CD40 ‘helper’ signaling pathway. Importantly, these antigen-presenting MDC1 also activated the expansion of CTL capable of killing the exposed HIV-1-infected targets. Interpretation Inclusion of virus-associated MHC class II ‘helper’ antigens in MDC1-based HIV-1 immunotherapies could serve both as a targeted means to safely unmask antigen-specific CD4 + T cells harboring HIV-1, and to support CTL responses that can effectively target the MDC1-exposed HIV-1 cellular reservoir as a functional cure strategy. Fund This study was supported by the NIH-NAID grants R21-AI131763, U01-AI35041, UM1-AI126603, and T32-AI065380.