Investigational new drug enabling angiotensin oral-delivery studies to attenuate pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a deadly and uncurable disease characterized by remodeling of the pulmonary vasculature and increased pulmonary artery pressure. Angiotensin Converting Enzyme 2 (ACE2) and its product, angiotensin-(1-7) [ANG-(1-7)] were expressed in lettuce chloroplasts to facilitate affordable oral drug delivery. Lyophilized lettuce cells were stable up to 28 months at ambient temperature with proper folding, assembly of CTB-ACE2/ANG-(1-7) and functionality. When the antibiotic resistance gene was removed, Ang1-7 expression was stable in subsequent generations in marker-free transplastomic lines. Oral gavage of monocrotaline-induced PAH rats resulted in dose-dependent delivery of ANG-(1-7) and ACE2 in plasma/tissues and PAH development was attenuated with decreases in right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance and pulmonary artery remodeling. Such attenuation correlated well with alterations in the transcription of Ang-(1-7) receptor MAS and angiotensin II receptor AGTRI as well as IL-1ß and TGF-ß1. Toxicology studies showed that both male and female rats tolerated ~10-fold ACE2/ANG-(1-7) higher than efficacy dose. Plant cell wall degrading enzymes enhanced plasma levels of orally delivered protein drug bioencapsulated within plant cells. Efficient attenuation of PAH with no toxicity augurs well for clinical advancement of the first oral protein therapy to prevent/treat underlying pathology for this disease.

Phosphodiesterase 4 Inhibitors Attenuate the Asthma Phenotype Produced by ß2-Adrenoceptor Agonists in Phenylethanolamine N-Methyltransferase-Knockout Mice.

Mice lacking the endogenous ß2-adrenoceptor (ß2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of ß2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various ß2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous ß2AR agonists on allergic lung inflammation can be explained by qualitative ß2AR signaling. The ß2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a ß-arrestin-dependent pathway. Previous studies suggest that ß-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the ß2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing ß2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of ß2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by ß-agonists.

Oral tolerization with cardiac myosin peptide (614-629) ameliorates experimental autoimmune myocarditis: role of STAT 6 genes in BALB/CJ mice.

INTRODUCTION: Experimental autoimmune myocarditis (EAM) is mediated by myocardial infiltration by myosin-specific T cells secreting inflammatory cytokines. MATERIALS AND METHODS: To clarify the role of cytokines in EAM, we compared STAT 6-deficient ((-/-)) with STAT 4(-/-) and wild-type (BALB/CJ) mice following immunization with cardiac myosin peptide (614-629). RESULTS: Wild-type mice developed severe disease with a small increase in severity in STAT 6(-/-) mice, while STAT 4(-/-) mice were resistant to EAM. STAT 6(-/-) mice had increased splenocyte proliferation and INF-gamma production versus wild type, while STAT 4(-/-) mice had decreased proliferation and INF-gamma. Following oral administration of myosin (614-629), tolerization was induced in wild-type mice evidenced by amelioration of myocarditis and up-regulation of IL-4. Adoptive transfer of splenocytes from orally tolerized mice resulted in inhibition of disease in STAT 6(-/-) mice. CONCLUSION: These results demonstrate that oral tolerization ameliorates EAM in BALB/CJ mice and indicate a down-regulatory role for STAT 6 genes.

Inhibition of experimental autoimmune myocarditis: peripheral deletion of TcR Vbeta 8.1, 8.2+ CD4+ T cells in TLR-4 deficient mice.

Toll-like receptors (TLR) are pattern recognition receptors that are an essential feature of host defense against pathogens. Expression of TLR-4 on dendritic cells was reported to be required for initiation of experimental autoimmune myocarditis (EAM) but the mechanism by which TLR-4 signaling affects autoimmunity is incompletely understood. To determine the role of TLR-4 in EAM, wild type and TLR-4-/- mice were immunized with myosin peptide (614-629) in CFA. TLR-4-/- mice demonstrated decreased myosin specific proliferation and decreased production of INF-gamma and IL-2. Immunization with myosin induced greater severity of myocarditis in wild type compared to TLR-4-/- mice as evidenced by lesions in the myocardium. TcR Vbeta 8.1, 8.2+ CD4+ T cells, detected in lesions were isolated from splenocytes by flow cytometry and found to undergo increased apoptosis in TLR-4-/- mice. In situ immunohistochemistry showed increased colocalization of cleaved caspase 3 and TcR Vbeta 8.1, 8.2+ CD4+ T cells in TLR-4-/- mice compared to wild type. Increased apoptosis was associated with impaired activation of NF-kB p65 and decreased cell viability in the presence of TNF-alpha. These results demonstrate that infiltrating TcR Vbeta 8.1, 8.2+ CD4+ T cells are deleted by the mechanism of apoptosis in TLR-4-/- mice with EAM.

Induction of oral tolerization in CD86 deficient mice: a role for CD86 and B cells in the up-regulation of TGF-beta.

Feeding myelin oligodendrocyte glycoprotein (MOG) followed by immunization results in induction of oral tolerance evidenced by the amelioration of experimental autoimmune encephalomyelitis (EAE). Oral tolerization is characterized by the suppression of Th1 responses and up-regulation of Th2 responses and TGF-beta. To identify the costimulatory molecules and cell types involved in cytokine-mediated suppression we examined wild type mice and mice deficient for either CD86 (CD86-/-) or B cells (muMT). Oral tolerance was found in CD86-/- mice evidenced by amelioration of disease severity, decreased proliferative responses and IFN-gamma production and increased IL-4. TGF-beta was not up-regulated in CD86-/- or muMT mice but was increased in wild type mice. Analysis of the gut associated lymphoid tissue (GALT) of different mouse strains (C57BL/6 and PLJxSJL F1) fed distinct myelin antigens (MOG and myelin basic protein, MBP) showed that TGF-beta was increased in wild type mice of both strains by 3 days post-immunization and further increased with time. In contrast, no up-regulation of TGF-beta was found in the GALT of CD86-/- or muMT mice. These results demonstrate that CD86 is not required for oral tolerization and that both CD86 and B cells are important for the up-regulation of TGF-beta following oral antigen.

Induction of low dose oral tolerance in IL-10 deficient mice with experimental autoimmune encephalomyelitis.

IL-10 has been shown to be an important anti-inflammatory mediator that has both down-regulatory and immunomodulatory effects. Utilizing IL-10(-/-) mice we demonstrate the induction of low dose oral tolerance characterized by the up-regulation of TGF-beta and IL-4 and the suppression of Ag specific proliferation with little suppression of INF-gamma. More severe EAE was found in IL-10(-/-) mice than in wild type controls, however, feeding resulted in amelioration of disease severity in both groups. Orally tolerized IL-10(-/-) mice had greater disease severity compared to orally tolerized wild type mice. IL-4 was present in the GALT of IL-10(-/-) mice and up-regulation of TGF-beta was detected in the lamina propria of fed mice. These results demonstrate that IL-10 is not required for the induction of low dose oral tolerance but is required for the regulation of INF-gamma which affects severity of disease in tolerized mice.

Induction of low dose oral tolerance in monocyte chemoattractant protein-1- and CCR2-deficient mice.

The chemokine monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR2 have been shown to play an important role in the migration and trafficking of macrophages and Th1 effector cells in experimental autoimmune encephalomyelitis. Also, MCP-1 has been reported to regulate oral tolerance induction by inhibition of Th1 cell-related cytokines and by the ability of Abs to MCP-1 to inhibit oral tolerance. This study demonstrates that neither MCP-1 nor its receptor CCR2 is required for the induction of oral tolerance. Mice deletional for either MCP-1 or CCR2 had suppressed cell-proliferative and Th1 responses following oral administration and immunization with myelin oligodendrocyte glycoprotein (MOG(35-55)). TGF-beta was up-regulated in fed and immunized deletional mice, while IL-4 was absent from deletional mice, but up-regulated in controls. Decreased experimental autoimmune encephalomyelitis severity was found in MOG(35-55)-fed MCP-1 deletional mice, indicating induction of oral tolerance. These results demonstrate that MCP-1 is not required for induction of oral tolerance and that MCP-1 and CCR2 are essential for up-regulation of IL-4 in tolerized mice.