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1. A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood‐Brain Barrier Mouse Model

   https://doi.org/10.1002/cpim.101  

   Walter, Fruzsina R. ; Gilpin, Trey E. ; Herbath, Melinda ; Deli, Maria A. ; Sandor, Matyas ; Fabry, Zsuzsanna ( July 2020 , Current Protocols in Immunology)

   In vitro culture models of the blood‐brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to testMycobacterium tuberculosis(Mtb) dissemination across brain endothelial cells. One‐third of the global population is infected with Mtb, and in 1%‐2% of cases bacteria invade the CNS through a largely unknown process. The “Trojan horse” theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB‐granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells

   Basic Protocol 2: Isolation of primary mouse bone marrow–derived dendritic cells

   Support Protocol 1: Validation of dendritic cell purity by flow cytometry

   Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells

   Support Protocol 2: Isolation of primary mouse spleen cells

   Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells

   Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load

   Support Protocol 4: In vivo and in vitro infection with mycobacteria

   Basic Protocol 5: Assembly of the BBB co‐culture model

   Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model

    

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2. Whole blood transcriptomics identifies subclasses of pediatric septic shock

   https://doi.org/10.1186/s13054-023-04689-y  

   Yang, Jamie O. ; Zinter, Matt S. ; Pellegrini, Matteo ; Wong, Man Yee ; Gala, Kinisha ; Markovic, Daniela ; Nadel, Brian ; Peng, Kerui ; Do, Nguyen ; Mangul, Serghei ; et al ( December 2023 , Critical Care)

   Sepsis is a highly heterogeneous syndrome, which has hindered the development of effective therapies. This has prompted investigators to develop a precision medicine approach aimed at identifying biologically homogenous subgroups of patients with septic shock and critical illnesses. Transcriptomic analysis can identify subclasses derived from differences in underlying pathophysiological processes that may provide the basis for new targeted therapies. The goal of this study was to elucidate pathophysiological pathways and identify pediatric septic shock subclasses based on whole blood RNA expression profiles.

   The subjects were critically ill children with cardiopulmonary failure who were a part of a prospective randomized insulin titration trial to treat hyperglycemia. Genome-wide expression profiling was conducted using RNA sequencing from whole blood samples obtained from 46 children with septic shock and 52 mechanically ventilated noninfected controls without shock. Patients with septic shock were allocated to subclasses based on hierarchical clustering of gene expression profiles, and we then compared clinical characteristics, plasma inflammatory markers, cell compositions using GEDIT, and immune repertoires using Imrep between the two subclasses.

   Patients with septic shock depicted alterations in innate and adaptive immune pathways. Among patients with septic shock, we identified two subtypes based on gene expression patterns. Compared with Subclass 2, Subclass 1 was characterized by upregulation of innate immunity pathways and downregulation of adaptive immunity pathways. Subclass 1 had significantly worse clinical outcomes despite the two classes having similar illness severity on initial clinical presentation. Subclass 1 had elevated levels of plasma inflammatory cytokines and endothelial injury biomarkers and demonstrated decreased percentages of CD4 T cells and B cells and less diverse T cell receptor repertoires.

   Two subclasses of pediatric septic shock patients were discovered through genome-wide expression profiling based on whole blood RNA sequencing with major biological and clinical differences.

   Trial RegistrationThis is a secondary analysis of data generated as part of the observational CAF-PINT ancillary of the HALF-PINT study (NCT01565941). Registered March 29, 2012.

    

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3. Thermophobic Trehalose Glycopolymers as Smart C‐Type Lectin Receptor Vaccine Adjuvants

   https://doi.org/10.1002/adhm.202202918  

   Hendricksen, Aaron T. ; Ezzatpour, Shahrzad ; Pulukuri, Anunay J. ; Ryan, Austin T. ; Flanagan, Tatum J. ; Frantz, William ; Buchholz, David W. ; Ortega, Victoria ; Monreal, Isaac A. ; Sahler, Julie M. ; et al ( April 2023 , Advanced Healthcare Materials)

   Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1–2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly‐N‐isoporpylacrylamide (NIPAM) via reversible addition fragmentation chain transfer (RAFT) polymerization. The resulting thermophobic adjuvants exhibit LCSTs near 37 °C, and self‐assembled into nanoparticles with temperature‐dependent sizes (90–270 nm). Thermophobic adjuvants activate HEK‐mMINCLE and other innate immune cell lines as well as primary mouse bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs). Inflammatory cytokine production is attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvantR_gis observed by DLS, as well as glycolipid‐NIPAM shielding interactions are observed by NOESY‐NMR. In vivo, thermophobic adjuvants enhance efficacy of a whole inactivated influenza A/California/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4⁺/44⁺/62L⁺lung and lymph node central memory T cells, as well as providing better protection from morbidity after viral challenge relative to unadjuvanted control vaccine. Together, these results demonstrate the first adjuvants with potency regulated by temperature. This work envisions that with further investigation, this approach can enhance vaccine efficacy while maintaining safety.

    

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4. IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1

   https://doi.org/10.1002/JLB.1A0118-010RR  

   Elizondo, Diana M. ; Andargie, Temesgen E. ; Haddock, Naomi L. ; da Silva, Ricardo L. Louzada ; de Moura, Tatiana Rodrigues ; Lipscomb, Michael W. ( December 2018 , Journal of Leukocyte Biology)

   Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

   Adaptive immune responses are impaired in CD8+ T cells primed by DC silenced for AIF1.

    

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5. Experimental colitis promotes sustained, sex-dependent, T-cell-associated neuroinflammation and parkinsonian neuropathology

   https://doi.org/10.1186/s40478-021-01240-4  

   Houser, Madelyn C. ; Caudle, W. Michael ; Chang, Jianjun ; Kannarkat, George T. ; Yang, Yuan ; Kelly, Sean D. ; Oliver, Danielle ; Joers, Valerie ; Shannon, Kathleen M. ; Keshavarzian, Ali ; et al ( August 2021 , Acta Neuropathologica Communications)

   The etiology of sporadic Parkinson’s disease (PD) remains uncertain, but genetic, epidemiological, and physiological overlap between PD and inflammatory bowel disease suggests that gut inflammation could promote dysfunction of dopamine-producing neurons in the brain. Mechanisms behind this pathological gut-brain effect and their interactions with sex and with environmental factors are not well understood but may represent targets for therapeutic intervention.

   We sought to identify active inflammatory mechanisms which could potentially contribute to neuroinflammation and neurological disease in colon biopsies and peripheral blood immune cells from PD patients. Then, in mouse models, we assessed whether dextran sodium sulfate-mediated colitis could exert lingering effects on dopaminergic pathways in the brain and whether colitis increased vulnerability to a subsequent exposure to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We assessed the involvement of inflammatory mechanisms identified in the PD patients in colitis-related neurological dysfunction in male and female mice, utilizing mice lacking the Regulator of G-Protein Signaling 10 (RGS10)—an inhibitor of nuclear factor kappa B (NFκB)—to model enhanced NFκB activity, and mice in which CD8⁺T-cells were depleted.

   High levels of inflammatory markers includingCD8Band NFκB p65 were found in colon biopsies from PD patients, and reduced levels of RGS10 were found in immune cells in the blood. Male mice that experienced colitis exhibited sustained reductions in tyrosine hydroxylase but not in dopamine as well as sustained CD8⁺T-cell infiltration and elevatedIfngexpression in the brain. CD8⁺T-cell depletion prevented colitis-associated reductions in dopaminergic markers in males. In both sexes, colitis potentiated the effects of MPTP. RGS10 deficiency increased baseline intestinal inflammation, colitis severity, and neuropathology.

   This study identifies peripheral inflammatory mechanisms in PD patients and explores their potential to impact central dopaminergic pathways in mice. Our findings implicate a sex-specific interaction between gastrointestinal inflammation and neurologic vulnerability that could contribute to PD pathogenesis, and they establish the importance of CD8⁺T-cells in this process in male mice.

    

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