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Now showing items 1 - 6 of 6

  • Synthesis and delivery of short, noncoding RNA by B lymphocytes

    Almanza, Gonzalo   Anufreichik, Veronika   Rodvold, Jeffrey J.   Chiu, Kevin T.   DeLaney, Alexandra   Akers, Johnny C.   Chen, Clark C.   Zanetti, Maurizio  

    Evolutionarily conserved short (20-30 nucleotides) noncoding RNAs (microRNAs) are powerful regulators of gene expression in a variety of physiological and pathological processes. As such, means to efficiently modulate microRNA function constitute an important therapeutic opportunity. Here we demonstrate that primary B lymphocytes can be genetically programmed with nonviral plasmid DNA for the biogenesis and delivery of antisense sequences (anti-microRNA) against microRNA-150 (miR-150). Within 18 h of transfection with an anti-miR-150 construct, primary B lymphocytes secrete similar to 3,000 copies of anti-miR-150 molecules per cell. Anti-miR-150 molecules released by B lymphocytes were internalized by CD8 T lymphocytes during cross-priming in vitro and in vivo, resulting in marked down-regulation of endogenous miR-150. However, such internalization was not observed in the absence of cross-priming. These results suggest that shuttling anti-miR-150 molecules from B lymphocytes to T cells requires the activation of receiver T cells via the antigen receptor. Finally, anti-miR-150 synthesized in B cells were secreted both as free and extracellular vesicle-associated fractions, but only extracellular vesicle-associated anti-miR-150 were apparently taken up by CD8 T cells. Collectively, these data indicate that primary B lymphocytes represent an efficient platform for the synthesis and delivery of short, noncoding RNA, paving the way for an approach to immunogenomic therapies.
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  • Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock

    Manda, Pratyusha   Feng, Yanjun   Lyons, John D.   Berger, Scott B.   Otani, Shunsuke   DeLaney, Alexandra   Tharp, Gregory K.   Maner-Smith, Kristal   Burd, Eileen M.   Schaeffer, Michelle   Hoffman, Sandra   Capriotti, Carol   Roback, Linda   Young, Cedrick B.   Liang, Zhe   Ortlund, Eric A.   DiPaolo, Nelson C.   Bosinger, Steven   Bertin, John   Gough, Peter J.   Brodsky, Igor E.   Coopersmith, Craig M.   Shayakhmetov, Dmitry M.   Mocarski, Edward S.  

    The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from cas pase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-beta-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
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  • RIPK1-dependent apoptosis bypasses pathogen blockade of innate signaling to promote immune defense

    Peterson, Lance W.   Philip, Naomi H.   DeLaney, Alexandra   Wynosky-Dolfi, Meghan A.   Asklof, Kendra   Gray, Falon   Choa, Ruth   Bjanes, Elisabet   Buza, Elisabeth L.   Hu, Baofeng   Dillon, Christopher P.   Green, Douglas R.   Berger, Scott B.   Gough, Peter J.   Bertin, John   Brodsky, Igor E.  

    Many pathogens deliver virulence factors or effectors into host cells in order to evade host defenses and establish infection. Although such effector proteins disrupt critical cellular signaling pathways, they also trigger specific antipathogen responses, a process termed "effector-triggered immunity." The Gram-negative bacterial pathogen Yersinia inactivates critical proteins of the NF-kappa B and MAPK signaling cascade, thereby blocking inflammatory cytokine production but also inducing apoptosis. Yersinia-induced apoptosis requires the kinase activity of receptor-interacting protein kinase 1 (RIPK1), a key regulator of cell death, NF-kappa B, and MAPK signaling. Through the targeted disruption of RIPK1 kinase activity, which selectively disrupts RIPK1-dependent cell death, we now reveal that Yersinia-induced apoptosis is critical for host survival, containment of bacteria in granulomas, and control of bacterial burdens in vivo. We demonstrate that this apoptotic response provides a cell-extrinsic signal that promotes optimal innate immune cytokine production and antibacterial defense, demonstrating a novel role for RIPK1 kinase-induced apoptosis in mediating effector-triggered immunity to circumvent pathogen inhibition of immune signaling.
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  • Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA

    Mowel, Walter K.   McCright, Sam J.   Kotzin, Jonathan J.   Collet, Magalie A.   Uyar, Asli   Chen, Xin   DeLaney, Alexandra   Spencer, Sean P.   Virtue, Anthony T.   Yang, EnJun   Villarino, Alejandro   Kurachi, Makoto   Dunagin, Margaret C.   Pritchard, Gretchen Harms   Stein, Judith   Hughes, Cynthia   Fonseca-Pereira, Diogo   Veiga-Fernandes, Henrique   Raj, Arjun   Kambayashi, Taku   Brodsky, Igor E.   O'Shea, John J.   Wherry, E. John   Goff, Loyal A.   Rinn, John L.   Williams, Adam   Flavell, Richard A.   Henao-Mejia, Jorge  

    Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.
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  • Increased BMP6 Levels in the Brains of Alzheimer's Disease Patients and APP Transgenic Mice Are Accompanied by Impaired Neurogenesis

    Crews, Leslie   Adame, Anthony   Patrick, Christina   DeLaney, Alexandra   Pham, Emiley   Rockenstein, Edward   Hansen, Lawrence   Masliah, Eliezer  

    During aging and in the progression of Alzheimer's disease (AD), synaptic plasticity and neuronal integrity are disturbed. In addition to the alterations in plasticity in mature neurons, the neurodegenerative process in AD has been shown to be accompanied by alterations in neurogenesis. Members of the bone morphogenetic protein (BMP) family of growth factors have been implicated as important regulators of neurogenesis and neuronal cell fate determination during development; however, their role in adult neurogenesis and in AD is less clear. We show here by qRT-PCR analysis that BMP6 mRNA levels were significantly increased in the hippocampus of human patients with AD and in APP transgenic mice compared to controls. Immunoblot and immunohistochemical analyses confirmed that BMP6 protein levels were increased in human AD brains and APP transgenic mouse brains compared to controls and accumulated around hippocampal plaques. The increased levels of BMP6 were accompanied by defects in hippocampal neurogenesis in AD patients and APP transgenic mice. In support of a role for BMP6 in defective neurogenesis in AD, we show in an in vitro model of adult neurogenesis that treatment with amyloid-beta(1-42) protein (A beta) resulted in increased expression of BMP6, and that exposure to recombinant BMP6 resulted in reduced proliferation with no toxic effects. Together, these results suggest that A beta-associated increases in BMP6 expression in AD may have deleterious effects on neurogenesis in the hippocampus, and therapeutic approaches could focus on normalization of BMP6 levels to protect against AD-related neurogenic deficits.
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  • Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice

    Gabaglia, Claudia Raja   DeLaney, Alexandra   Gee, Jennifer   Halder, Ramesh   Graham, Frank L.   Gauldie, Jack   Sercarz, Eli E.   Braciak, Todd A.  

    Background: Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. Methods: The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD. scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4(+)/FoxP3(+) T regulatory cells (Treg) in the TSLN were evaluated. Results: Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4(+)/FoxP3(+) T regulatory cell populations to be found in the TSLN. Conclusion: Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment.
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