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Recurrent group A Streptococcus (GAS) tonsillitis and associated autoimmune diseases indicate that the immune response to this organism can be ineffective and pathological. TGF-[beta]1 is recognized as an essential signal for generation of regulatory T cells (Tregs) and T helper (Th) 17 cells. Here, the impact of TGF-[beta]1 induction on the T-cell response in mouse nasal-associated lymphoid tissue (NALT) following intranasal (i.n.) infections is investigated. ELISA and TGF-[beta]1-luciferase reporter assays indicated that persistent infection of mouse NALT with GAS sets the stage for TGF-[beta]1 and IL-6 production, signals required for promotion of a Th17 immune response. As predicted, IL-17, the Th17 signature cytokine, was induced in a TGF-[beta]1 signaling-dependent manner in single-cell suspensions of both human tonsils and NALT. Intracellular cytokine staining and flow cytometry demonstrated that [CD4.sup.+] IL-[17.sup.+] T cells are the dominant T cells induced in NALT by i.n. infections. Moreover, naive mice acquired the potential to clear GAS by adoptive transfer of [CD4.sup.+] T cells from immunized IL-[17A.sup.+/+] mice but not cells from IL-[17A.sup.-/-] mice. These experiments link specific induction of TGF-[beta]1 by a bacterial infection to an in vivo Th17 immune response and show that this cellular response is sufficient for protection against GAS. The association of a Th17 response with GAS infection reveals a potential mechanism for destructive autoimmune responses in humans. IL-17 | nasal-associated lymphoid tissue | Streptococcus pyogenes doi/10.1073/pnas.0904831107

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl3) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX ( cFIX) were treated with GdCl3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products. [ABSTRACT FROM AUTHOR]

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow. A peak intravascular pressure between 80 and 140 mmHg supported sufficient DNA delivery in dog liver for detection of secretable reporter proteins. Secretable reporters allowed monitoring of the time course of gene products detectable in the circulation postinfusion. Canine secreted alkaline phosphatase reporter protein levels were measured in plasma, with expression detectable for up to 6 weeks, while expression of canine erythropoietin was detectable for 7-10 days. All animals exhibited a transient increase in blood transaminases that normalized within 10 days; otherwise the treated animals were clinically normal. These results demonstrate the utility of a secreted reporter protein for real-time monitoring of gene expression in the liver in a large animal model but highlight the need for improved delivery in target tissues to support integration and long-term expression of Sleeping Beauty transposons. [ABSTRACT FROM AUTHOR]

Recurrent group A Streptococcus (GAS) tonsillitis and associated autoimmune diseases indicate that the immune response to this organism can be ineffective and pathological. TGF-β1 is recognized as an essential signal for generation of regulatory T cells (Tregs) and T helper (Th) 17 cells. Here, the impact of IGF-β1 induction on the T-cell response in mouse nasal-associated lymphoid tissue (NALT) following intranasal (i.n.) infections is investigated. ELISA and TGF-β1-luciferase reporter assays indicated that persistent infection of mouse NALIwith GAS sets the stage for TGF-β1 and IL-6 production, signals required for promotion of a Th17 immune response. As predicted, IL-17, the Th17 signature cytokine, was induced in a TGF-β1 signaling-dependent manner in single-cell suspensions of both human tonsils and NALT. Intracellular cytokine staining and flow cytometry demonstrated that CD4+ IL-17+ T cells are the dominant T cells induced in NALT by i.n. infections. Moreover, naive mice acquired the potential to clear GAS by adoptive transfer of CD4+ T cells from immunized lL-17A+/+ mice but not cells from IL-17A-/- mice. These experiments link specific induction of TGF-β1 by a bacterial infection to an in vivo Th17 immune response and show that this cellular response is sufficient for protection against GAS. The association of a Th17 response with GAS infection reveals a potential mechanism for destructive autoimmune responses in humans. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes is a major causative agent of tonsillitis or pharyngitis in children. Streptococcus pyogenes can persist in tonsils, and one-third of children treated with antibiotics continue to shed streptococci and have recurrent infections. Mouse nasal-associated lymphoid tissue (NALT) is functionally analogous to human oropharyngeal lymphoid tissues, and serves as a model for characterization of the mucosal innate immune response to S. pyogenes. Wild-type S. pyogenes induces transcription of both type I and interferon-γ (IFN-γ)-responsive genes, proinflammatory genes and acute-phase response proteins 24 h after intranasal infection. Invasion of NALT and the induction of the interferon response were not dependent on expression of antiphagocytic M protein. Intranasal infection induces a substantial influx of neutrophils into NALT at 24 h, which declines by 48 h after infection. Infection of IFN-γ−/− [IFN-γ knock-out mouse (GKO)] C57BL/6 mice with wild-type S. pyogenes resulted in local dissemination of bacteria to draining lymph nodes (LN), but did not lead to systemic infection by 48 h after infection. Infected GKO mice had an increased influx of neutrophils into NALT compared with immunocompetent mice. Thus, IFN-γ-induced responses are required to prevent local dissemination of streptococci to the draining LN. [ABSTRACT FROM AUTHOR]