Tumor Necrosis Factor Induces Obliterative Pulmonary Vascular Disease in a Novel Model of Connective Tissue Disease-Associated Pulmonary Arterial Hypertension.

OBJECTIVE: Connective tissue disease (CTD)-associated pulmonary arterial hypertension (PAH) is the second most common etiology of PAH and carries a poor prognosis. Recently, it has been shown that female human tumor necrosis factor (TNF)-transgenic (Tg) mice die of cardiopulmonary disease by 6 months of age. This study was undertaken to characterize this pathophysiology and assess its potential as a novel model of CTD-PAH. METHODS: Histologic analysis was performed on TNF-Tg and wild-type (WT) mice to characterize pulmonary vascular and right ventricular (RV) pathology (n = 40 [4-5 mice per group per time point]). Mice underwent right-sided heart catheterization (n = 29) and micro-computed tomographic angiography (n = 8) to assess vascular disease. Bone marrow chimeric mice (n = 12), and anti-TNF-treated mice versus placebo-treated mice (n = 12), were assessed. RNA sequencing was performed on mouse lung tissue (n = 6). RESULTS: TNF-Tg mice displayed a pulmonary vasculopathy marked by collagen deposition (P < 0.001) and vascular occlusion (P < 0.001) with associated RV hypertrophy (P < 0.001) and severely increased RV systolic pressure (mean ± SD 75.1 ± 19.3 mm Hg versus 26.7 ± 1.7 mm Hg in WT animals; P < 0.0001). TNF-Tg mice had increased α-smooth muscle actin (α-SMA) staining, which corresponded to proliferation and loss of von Willebrand factor (vWF)-positive endothelial cells (P < 0.01). There was an increase in α-SMA-positive, vWF-positive cells (P < 0.01), implicating endothelial-mesenchymal transition. Bone marrow chimera experiments revealed that mesenchymal but not bone marrow-derived cells are necessary to drive this process. Treatment with anti-TNF therapy halted the progression of disease. This pathology closely mimics human CTD-PAH, in which patient lungs demonstrate increased TNF signaling and significant similarities in genomic pathway dysregulation. CONCLUSION: The TNF-Tg mouse represents a novel model of CTD-PAH, recapitulates key disease features, and can serve as a valuable tool for discovery and assessment of therapeutics.

Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice.

Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity.

Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.

Thy1 (CD90), a glycosylated, glycophosphatidylinositol-anchored membrane protein highly expressed by subsets of mesenchymal stem cells and fibroblasts, inhibits adipogenesis. The role of Thy1 on bone structure and function has been poorly studied and represents a major knowledge gap. Therefore, we analyzed the long bones of wild-type (WT) and Thy1 knockout (KO) mice with micro-computed tomography (micro-CT) and histomorphometry to compare changes in bone architecture and overall bone structure. micro-CT analysis of long bones revealed Thy1 KO and WT mice fed a high-fat diet demonstrated bone structural parameters at 4 mo that differed significantly between WT and KO mice. A significant reduction in trabecular bone volume was noted in Thy1 KO mice. The most prominent differences were observed in trabecular bone volume ratio and trabecular bone connectivity density. Consistent with micro-CT measurements, histomorphometric analysis also showed decreased bone volume in the obese Thy1 KO mice compared to obese WT mice. In vitro assays revealed that osteogenic conditions increased Thy1 expression during OB differentiation and absence of Thy1 attenuated osteoblastogenesis. Together, these findings support the concept that Thy1 serves as a major mechanistic link to regulate bone formation and negatively regulate adipogenesis.-Paine, A., Woeller, C. F., Zhang, H., Garcia-Hernandez, M. L., Huertas, N., Xing, L., Phipps, R. P., Ritchlin, C. T. Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza.

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 µl of serum) were needed, and the assay had a linear response over a four Log10 range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA's, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA's. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.