Rodriguez, Moses

DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3′ modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.

Abstract Multiple sclerosis is an inflammatory, demyelinating disease of the CNS of presumed autoimmune origin. Of all the genetic factors linked with multiple sclerosis, MHC class II molecules have the strongest association. Generation of HLA class II transgenic (Tg) mice has helped to elucidate the role of HLA class II genes in chronic inflammatory and demyelinating diseases. We have shown that the human HLA-DRB1*0301 gene predisposes to proteolipid protein (PLP)–induced experimental autoimmune encephalomyelitis (EAE), whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DRB1*0301.DQ6 double-Tg mice by producing anti-inflammatory IFN-γ. HLA-DQβ1*0302 (DQ8) Tg mice were also resistant to PLP91–110–induced EAE, but production of proinflammatory IL-17 exacerbated disease in DRB1*0301.DQ8 mice. To further confirm the role of IFN-γ in protection, we generated DRB1*0301.DQ8 mice lacking IFN-γ (DRB1*0301.DQ8.IFN-γ−/−). Immunization with PLP91–110 peptide caused atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice characterized by ataxia, spasticity, and dystonia, hallmarks of brain-specific disease. Severe brain-specific inflammation and demyelination in DRB1*0301.DQ8.IFN-γ−/− mice with minimal spinal cord pathology further confirmed brain-specific pathology. Atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice was associated with increased encephalitogenicity of CD4 T cells and their ability to produce greater levels of IL-17 and GM-CSF compared with DRB1*0301.DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells, suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFN-γ in the demyelination of brain through downregulation of IL-17/GM-CSF and induction of neuroprotective factors in the brain by monocytes/microglial cells.

Multiple sclerosis and other demyelinating diseases in the pediatric population have received an increasing level of attention by clinicians and researchers. The low incidence of these diseases in children creates a need for the involvement of multiple clinical centers in research efforts. The Network of Pediatric Multiple Sclerosis Centers was created initially in 2006 to improve the diagnosis and care of children with demyelinating diseases. In 2010, the Network shifted its focus to multicenter research while continuing to advance the care of patients. The Network has obtained support from the National Multiple Sclerosis Society, the Guthy-Jackson Charitable Foundation, and the National Institutes of Health. The Network will continue to serve as a platform for conducting impactful research in pediatric demyelinating diseases of the central nervous system. This article provides a description of the history and development, organization, mission, research priorities, current studies, and future plans of the Network.

Abstract Recent studies provide a functional link between kallikrein 6 (Klk6) and the development and progression of disease in patients with multiple sclerosis (MS) and in its murine models. To evaluate the involvement of additional kallikrein family members, we compared Klk6 expression with four other kallikreins (Klk1, Klk7, Klk8, and Klk10) in the brain and spinal cord of mice infected with Theiler’s murine encephalomyelitis virus, an experimental model of progressive MS. The robust upregulation of Klk6 and Klk8 in the brain during the acute phase of viral encephalitis and in the spinal cord during disease development and progression points to their participation in inflammation, demyelination, and progressive axon degeneration. More limited changes in Klk1, Klk7, and Klk10 were also observed. In addition, Klk1, Klk6, and Klk10 were dynamically regulated in T cells in vitro as a recall response to viral antigen and in activated monocytes, pointing to their activities in the development of adaptive and innate immune function. Together, these results point to overlapping and unique roles for multiple kallikreins in the development and progression of virus-mediated central nervous system inflammatory demyelinating disease, including activities in the development of the adaptive and innate immune response, in demyelination, and in progressive axon degeneration.

Abstract TGF-β2 is a potent immunoregulatory mediator that influences B cell, T cell, and macrophage function. To test whether this cytokine alters pathology in a model of virus-induced demyelinating disease, we treated SJL/J mice with TGF-β2 either before or after infection with Theiler’s murine encephalomyelitis virus. Treatment continued three times weekly through day 35 postinfection. TGF-β2 administration resulted in significantly smaller lesions and fewer virus Ag-positive cells in the spinal cords of infected SJL/J mice. Mice treated with TGF-β2 had similar levels of virus-specific IgG as infected, control-treated mice. TGF-β2 administration significantly increased the level of non-virus-specific activated CTLs, but had no effect on virus-specific CTLs. TUNEL revealed a decrease in the number of apoptotic nuclei in the spinal cord white matter of mice treated in vivo with TGF-β2. Immunostaining with an Ab to F4/80 revealed that TGF-β2-treated mice had significantly fewer F4/80-positive cells in the white matter of the spinal cord as compared with infected control-treated mice. These data suggest that TGF-β2 may control virus-induced demyelination via an immunomodulatory mechanism that reduces macrophage infiltration.

Abstract Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2121–130 peptide inhibits the development of antiviral CTL specific for the immunodominant Db:VP2121–130 epitope expressed during acute Theiler’s murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8+ T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8+ T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.

Abstract A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.

Abstract Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.

Abstract Interaction of 4-1BB (CD137) and its ligand (4-1BBL) is thought to positively regulate cell-mediated and humoral immune responses. We have prepared transgenic mouse strains that express 4-1BBL cDNA under the control of MHC class II I-Eα promoter. The 4-1BBL-transgenic mice show progressive splenomegaly and selective depletion of B220+ B cells accompanied with low levels of circulating IgG and defective humoral responses to Ag challenge. In addition, splenocytes from the transgenic mice fail to provide stimulation for allogeneic T cells in both lymphoproliferative and CTL responses in vitro, whereas their T cells remain functionally normal. Our results reveal unexpected functions of 4-1BBL in the regulation of humoral immune responses and Ag presentation.