Human parvovirus B19 (B19V) causes a variety of human diseases. Disease outcomes of bone marrow failure in patients with high turnover of red blood cells and immunocompromised conditions, and fetal hydrops in pregnant women are resulted from the targeting and destruction of specifically erythroid progenitors of the human bone marrow by B19V. Although the ex vivo expanded erythroid progenitor cells recently used for studies of B19V infection are highly permissive, they produce progeny viruses inefficiently. In the current study, we aimed to identify the mechanism that underlies productive B19V infection of erythroid progenitor cells cultured in a physiologically relevant environment. Here, we demonstrate an effective reverse genetic system of B19V, and that B19V infection of ex vivo expanded erythroid progenitor cells at 1% O(2) (hypoxia) produces progeny viruses continuously and efficiently at a level of approximately 10 times higher than that seen in the context of normoxia. With regard to mechanism, we show that hypoxia promotes replication of the B19V genome within the nucleus, and that this is independent of the canonical PHD/HIFα pathway, but dependent on STAT5A and MEK/ERK signaling. We further show that simultaneous upregulation of STAT5A signaling and down-regulation of MEK/ERK signaling boosts the level of B19V infection in erythroid progenitor cells under normoxia to that in cells under hypoxia. We conclude that B19V infection of ex vivo expanded erythroid progenitor cells at hypoxia closely mimics native infection of erythroid progenitors in human bone marrow, maintains erythroid progenitors at a stage conducive to efficient production of progeny viruses, and is regulated by the STAT5A and MEK/ERK pathways.

Peripheral T cells can be polarized towards type 1 or type 2 cytokine immune responses during TCR engagement. Because T cell selection by peptide plus self-MHC in the thymus requires TCR engagement, we hypothesized that type 1 cytokines may polarize developing T cells. We cultured thymi from BBDR rats in adult thymus organ cultures (ATOC) under type 1 cytokine conditions in the absence of exogenous antigen. Type 1 cytokine-conditioned ATOC generated cells that spontaneously secreted high levels of IFNgamma, but not IL-4. A second exposure to type 1 cytokines further increased IFNgamma secretion by these cells, most of which were blasts that expressed the activation markers CD25, CD71, CD86, and CD134. Studies using blocking antibodies and pharmacological inhibitors suggested that both IL-18 and cognate TCR-MHC/ligand interactions were important for activation. Blocking anti-MHC class I plus anti-MHC class II antibodies, neutralizing anti-IL-18 antibody, and the p38 MAP-kinase inhibitor SB203580 each reduced IFNgamma production by approximately 75-80%. Cyclosporin A, which prevents TCR signaling, inhibited IFNgamma production by approximately 50%. These data demonstrate that exposure to type 1 cytokines during intrathymic development can polarize differentiating T cells, and suggest a mechanism by which intrathymic exposure to type 1 cytokines may modulate T cell development.

Tissue repair is driven by migratory macrophages and fibroblasts that infiltrate injury sites and secrete growth factors. We now report the enhancement of skeletal muscle repair by targeting transgene delivery to these repair cells using matrix-immobilized gene vectors. Plasmid and adenovirus vectors immobilized in collagen-gelatin admixtures were delivered to excisional muscle wounds, and when encoding either fibroblast growth factor-2 (FGF2) or FGF6 transgenes, produced early angiogenic responses that subsequently remodeled into arteriogenesis. FGF2 gene delivery enhanced the number of CD31(+) endothelial cells present at treatment sites > 6-fold by day 14, and muscular arteriole density up to 11-fold by day 21 (P<0.0001). Muscle repair was also enhanced, as FGF gene-treated wounds filled with regenerating myotubes expressing the marker CD56 (an average 20-fold increase in CD56 expression versus controls, P<0.0001). These responses required transfection of a threshold level of repair cells, achievable only in injured muscles, and were transgene-driven, as neither platelet-derived growth factor-B (PDGFB) gene nor FGF2 protein delivery produced equivalent responses. In conclusion, using biomatrices to direct gene delivery to repair cells allows for relatively complex regenerative processes such as arteriogenesis and myogenesis, and therefore represents a promising approach to treating injured and ischemic muscle.

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide-major histocompatibility complex (MHC) class I or class II ligands recognized by alphabeta T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide-MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46-61)-Ak or HEL-(116-129)-Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide-MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46-61)-Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.