bluuebunny/crossref_metadata_2025_split huggingface dataset:
A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay
Authors: Braud Veronique M , Fassy Julien , Tsalkitzi Kyriaki 
Published: January 2023

Abstract
Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard chromium 51Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentive to find alternative solutions to 51Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates using the cell-imaging multimode reader Cytation™ 5 from Biotek. We have developed classical NK cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity (ADCC). We show that in these assays, cell killing occurs within the first two hours with a half maximum killing reached after 30 minutes. This technology has numerous applications such as NK and T cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.
Using Tat-Cre to Generate Ex-Vivo Gene Knockouts in T-cells
Authors: Brown Rebecca Audrey , Byersdorfer Craig A 
Published: January 2023

Abstract
We have found unanticipated effects using CD4-driven Cre expression to generate gene knockouts, due to changes in T cells that occur between gene deletion and harvest. To bypass this, we are developing a method to delete genes in T cells just prior to experimentation using brief ex vivo exposure to a Tat-Cre fusion protein, where the TAT peptide drives translocation of Cre to excise loxP-flanked genes. We first defined an optimal concentration and length of treatment by measuring production of a knockout band in DNA extracted from treated T cells. Maximal knockout occurred with 30 minutes of Tat-Cre incubation, while longer treatment times led to no further increase in gene deletion. In contrast, increasing concentrations of Tat-Cre correlated with increasing gene deletion in a dose-dependent manner. 4.6 uM was determined to be the optimal concentration balancing deletion efficacy and resource allocation.
We next used T cells from td-Tomato (tdT)/green fluorescent protein (GFP) reporter mice, which express tdT at baseline, but delete tdT and turn on GFP when Cre is present, to determine if DNA deletions were measurable at the protein level. Immediately after Tat-Cre treatment there was no observable increase in GFP expression. However, by 2 hours post-treatment, 34% of treated cells expressed GFP, reaching 65% by 20 hours. We then sorted GFP+ T cells into a mixed leukocyte reaction, where they proliferated appropriately and where approximately 80% of treated T cells remained GFP+ while simultaneously losing tdT expression. Ultimately, this ex vivo method represents a promising approach for generating gene knockouts in T cells just prior to experimental manipulation avoiding changes that occur between deletion and harvest from donor animals.
Intravital Co-Imaging of Oxygen Tension and Immune Response in Leukemia using 2-Photon PLIM
Authors: Zal Tomasz , Rytelewski Mateusz , Harutyunyan Karine , Vinogradov Sergei , Ohtake Kazuki , Konopleva Marina 
Published: January 2023

Abstract
Tumor infiltrating lymphocytes (TILs) must compete with cancer cells for metabolic resources that become depleted in result of cancerous metabolism and compromised blood supply. In bone marrow, highly proliferative acute leukemias utilize oxidative phosphorylation and deplete local oxygen supply resulting in bone marrow hypoxia which contributes to therapeutic resistance. We aim to decipher the role that metabolic reprogramming of leukemic blasts plays in modulating the bone marrow microenvironment, particularly the impact on local oxygen tension, and how this affects the anti-leukemia immune response. To characterize the spatiotemporal dynamics of leukemia infiltrating T cells during disease progression, and determine the impact of oxygen tension, OxPhos, and OxPhos inhibition on T cell function inside the leukemic bone marrow, we have utilized a novel oxygen probe, PtP-C343, and intravital 2-photon phosphorescence lifetime microscopy (2p-PLIM). Our initial results demonstrate sufficient spatial and temporal resolution of the combination 2p-PLIM to simultaneously reveal intravital oxygen gradients and track lymphocytes in single cell resolution. The combination 2p-PLIM and in vivo cell tracking will enable to study the dynamic regulation of the migratory behavior of cancer-infiltrating T lymphocytes by the intratumoral oxygen tension gradients.
Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy
Authors: Yang Lili , Smith Drake J. , Lin Levina J. , Moon Heesung , Pham Alexander T. , Wang Xi , Liu Siyuan , Ji Sunjong , Rezek Valerie , Shimizu Saki , Ruiz Marlene , Lam Jennifer , Janzen Deanna M. , Memarzadeh Sanaz , Kohn Donald B. , Zack Jerome A. , Kitchen Scott G. , An Dong Sung 
Published: January 2023

Abstract
The humanized BLT (bone marrow-liver-thymus) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain HLA (human leukocyte antigen) haplotypes or monogene deficiencies. In order to circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ−/−) recipient mice, we were able to expand one primary BLT mouse into a colony of 4–5 proBLT (propagated BLT) mice in 6–8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to that of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2 restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable pre-clinical research tool.
IFN-λ4 induces a faster but more transient antiviral response compared to other type III interferons
Authors: Onabajo Olusegun O , Rao Nina , Obajemu Adeola A , Dilley Kari A , Sheikh Faruk , Donnelly Raymond , Shabman Reed S , Prokunina-Olsson Ludmila 
Published: January 2023

Abstract
IFN-λ4, a recently discovered type-III interferon (IFN), is genetically regulated, and has clinical significance because of its association with impaired clearance of hepatitis C virus (HCV) infection. Type-III IFNs play a critical role in the innate immune response to viral infections. Although IFN-λ4 induces a similar antiviral profile as other type-III IFNs, its specific contribution to the innate immune response is unclear. Delineating the functional properties of IFN-λ4 that distinguish it from other type-III IFNs may give insights into its unique role during HCV, and other infections. Here we compared antiviral responses induced by all four type-III IFNs in HepG2 cells. We show that IFN-λ4 attained 50% peak antiviral activity by 4 hours while other type-III IFNs were only at 20% of peak activity at this time. The rapid antiviral response induced by IFN-λ4 was followed by a sharp decline to less than 25% of peak activity within 24 hours, while activity of other type-III IFNs were still over 60% from peak.
By comparing antiviral activity of IFN-λ4 and IFN-λ3 against Sendai virus infection in primary human hepatocytes, we show that IFN-λ4 had stronger antiviral activity than IFN-λ3 early in the course of infection, but comparable activity at later time points. We also show that despite its poor secretion, IFN-λ4 was significantly more potent than IFN-λ3 in inducible cell lines engineered to express either protein. In summary, we show that IFN-λ4 is more active during the early stages of viral infection compared to other type-III IFNs, which maybe a clinically important window for restricting viral infection in the host, and affecting disease outcome.
Interleukin-36 cytokines as countermeasures to viral immune evasion
Authors: Jensen Liselotte E 
Published: January 2023

Abstract
IL-36α, IL-36β and IL-36γ engage the same receptor, IL-36R, and are related to the pro-inflammatory IL-1α, IL-1β, IL-18 and IL-33. While there is increasing evidence that the IL-36s play important roles in host immunity, the redundancy remains an enigma. Here we explore the possibility that the IL-36s have evolved to counteract viral immune evasion mechanisms. Many skin tropic viruses inhibit IL-1β and IL-18 as immune evasion prevent protein synthesis, accelerate mRNA and protein turnover, block inflammasome activation, inhibit caspase-1, trap cytokines and/or impede signaling. IL-36γ production is induced by poly(I:C); however, IL-36γ does not appear to play a significant role in the flank skin HSV-1 infection mouse model. This outcome may be due to the release of IL-36γ being dependent upon the inflammasome pathway, which is blocked by HSV-1. Similar observations were made using IL-36α KO mice. Additional data reveal that IL-1α is released in an inflammasome independent manner from HSV-1 infected keratinocytes; an event important for early recruitment of inflammatory cells to sites of HSV-1 infection in vivo. IL-36β appears to play an additional role in the skin, as IL-36β KO mice develop larger skin lesions than wildtype mice. Furthermore, the survival rate was significantly reduced. Complementary studies involving the TLR7 ligand imiquimod were performed. Imiquimod induced skin inflammation was dependent upon IL-36α, but independent of IL-36β and IL-36γ. This could suggest an important role for IL-36α in immunity against viruses that activate TLR7 yet evade immune responses targeting IL-1/IL-18 signaling, e.g., the measles virus. Our data may suggest that the IL-36 cytokines have evolved to counteract viral immune evasion.
CXCL11 production in cerebrospinal fluid distinguishes herpes simplex meningitis from herpes simplex encephalitis
Authors: Lind Liza , Studahl Marie , Berg Linn Persson , Eriksson Kristina 
Published: January 2023

Abstract
The closely related herpes simplex viruses 1 and 2 can cause inflammations of the central nervous system (CNS), where type 1 most often manifest as encephalitis (HSE), and type 2 as meningitis (HSM). HSE is associated with severe neurological complications, while HSM is benign in adults. We proposed that the chemokine and cytokine production in cerebrospinal fluid (CSF) and serum could indicate why two closely related viruses exhibit different severity of their accompanied CNS inflammation. Secretion patterns of 30 chemokines and 10 cytokines in CSF of adult patients with acute HSE and HSM in the initial stage of disease were analyzed and compared to control subjects without viral central nervous system infections and to levels in serum. Most measured chemokines and cytokines increased in CSF of HSE and HSM patients. Overall, the CSF chemokines levels were higher in CSF of HSM patients compared to HSE patients. However, only five chemokines reached levels in the CSF that exceeded those in serum facilitating a positive CSF-serum chemokine gradient. Of these, CXCL8, CXCL9 and CXCL10 were present at high levels both in HSE and HSM whereas CXCL11 and CCL8 were present in HSM alone. Several chemokines were also elevated in serum of HSE patients but only one in HSM patients. No chemokine in- or efflux between CSF and serum was indicated as the levels of chemokines in CSF and serum did not correlate. We show that HSM is associated with a stronger and more diverse inflammatory response in the CNS compared to HSE in the initial stage of disease. The chemokine patterns were distinguished by the exclusive local CNS production of CXCL11 and CCL8 in HSM. Inflammation in HSM appear to be restricted to the CNS whereas HSE also was associated with systemic inflammation.
Investigation of Macrophages Serving as a Viral Reservoir in Pediatric SAIDS
Authors: Merino Kristen Michelle , Sugimoto Chie , Cai Yanhui , Allers Carolina , Didier Elizabeth , Kuroda Marcelo J 
Published: January 2023

Abstract
In SIV-infected adult Macaca mulatta, increases in monocyte turnover indicates death of short-lived tissue macrophages and predicts SAIDS. Increased turnover also correlates with infection, not death, of long-lived tissue macrophages. Higher baseline monocyte turnover is measured in neonates, and faster disease progression. Our data shows increase in monocyte turnover and tissue macrophage infection is associated with rapid SAIDS progression in infants. We hypothesize early and high infection of macrophages results in establishment of viral reservoir in long-lived subsets, in infants not controlling viral load despite antiretroviral therapy (ART). In rhesus infants infected with SIVmac251, cell turnover is measured using BrdU uptake. Flow cytometry and confocal imaging allow phenotypic characterization of cells in blood and tissues. PCR and in situ hybridization are used to quantify SIV. Plasma cytokine analysis is performed using Milliplex bead panel. Infant macaques euthanized post-infection (no ART) are used to quantify macrophage infection. Animals euthanized after various lengths of ART provide data on reservoir seeding, viral persistence and immune activation status. ART-treated infants with discordant viral loads are evaluated to identify the source of viral replication. In treated animals, regardless of viral load, no differences are observed in activation status, absolute number, or kinetics of CD4 and CD8 T cells. Confocal imaging and viral RNA and DNA analysis of tissues and sorted cells should confirm that viral replication during ART is due to infected macrophages. Ongoing works suggest magnitude of long-lived macrophage infection may dictate effectiveness of ART in pediatric SIV infection.
The host response to murine norovirus infection induces significant engagement of IFN and TNF-a immunological programs
Authors: Levenson Eric Andrew , Martens Craig , Kanakabandi Kishore , Turner Charles , Sosnovtsev Stanislav V. , Ricklefs Stacey , Porcella Stephen , Green Kim Y. 
Published: January 2023

Abstract
Norovirus, a positive stranded RNA virus in the family Caliciviridae, is a major cause of acute gastroenteritis. Outbreaks occur primarily in locations such as schools, nursing homes, and cruise ships where individuals are in close proximity. An acute norovirus infection can become chronic in immunocompromised individuals, and an effective antiviral drug is needed. The 7.5 kb genome encodes a polyprotein in ORF1 that is co-translationally cleaved by the viral protease into six nonstructural proteins. The two structural proteins, VP1 and VP2, are encoded in ORF2 and ORF3, respectively. The viral proteins mediate replication and packaging of the genome into icosahedral capsids. The host cell provides additional proteins and building blocks for replication, but only a few essential host factors have been elucidated. To gain insight into the host response to norovirus infection, we performed next generation sequencing-based RNA sequencing on murine macrophages infected with murine norovirus. We obtained RNA from a time course of infection at 0, 8, 14, and 20 hours post infection with mock infections at 0 and 20 hours. Analysis of the host transcriptome reveals full activation of cellular immune response pathways, with NF-kβ, STAT1, and STAT3-based signaling evident. In addition, we observed transcriptional evidence of IRF3 activation with a transition to IRF3/IRF7 signaling. TNF-a-based activation is also clear with most downstream effectors up-regulated. These observations correlate well with the known cytokine response from patient serum samples, disease progression, and symptomatology of human norovirus. We are currently applying these findings toward drug discovery efforts.
B cell Intrinsic and Extrinsic Function of STING in the Antibody Response
Authors: Walker Melissa Marie , Crute Bergren W , Getahun Andrew , Cambier John C 
Published: January 2023

Abstract
Generation of protective immune responses requires coordinate stimulation of innate and adaptive immune systems. An important mediator of innate immunity is STimulator of INterferon Genes (STING), a ubiquitously expressed adaptor molecule that functions in the relay of signals initiated by sensing of cytosolic DNA and bacterial ci-di-nucleotides. Allelic variants of STING, HAQ-STING and R232H-STING, found in 15–20% of humans appear, based on in vitro studies, to be defective in signal relay, suggesting that carriers may be predisposed to certain infections. Our laboratory is engaged in studying the functions of STING, HAQ-STING and R232H-STING.
Here we show that STING promotion of in vivo IgG1 and IgA antibody responses requires both systemic and B cell expression of STING. Analyses of the possible direct effects ci-di-GMP on B cells revealed that CDNs stimulate upregulation of activation markers (CD69 and CD86) as well as apoptotic death, and these responses are dependent on STING expression. Coordinate BCR signals synergize in induction of B cell activation but partially spare cells from apoptosis.
Consistent with defective function of HAQ-STING, B cells from a newly-produced human HAQ and R231H knockin mouse were defective in mounting responses to ci-di-GMP.
Taken together, these data indicate that STING and BCR function coordinately in B cells to promote cell activation and antibody responses. Human carriers of HAQ and R231H STING may be defective in responses to certain immunogens.
Spontaneous Translocation of a Gut Pathobiont Drives Autoimmunity that is Preventable by Intramuscular Vaccination
Authors: Vieira Silvio Manfredo , Hiltensperger Michael , Kumar Varun , Dehner Carina , Zegarra-Ruiz Daniel , Ruff William , Khan Nafeesa , Goodman Andrew , Kriegel Martin A 
Published: January 2023

Abstract
The gut microbiota contributes to the pathogenesis of autoimmunity. The mechanisms and sites of host-microbiota interactions are, however, largely unknown. Dietary antigens reach the mesenteric lymph node (MLN) via the lymphatics and the liver via the portal vein. We hypothesized that commensals reach lymphocytes beyond the gut barrier in autoimmune-prone hosts. We first altered the gut microbiota with single antibiotics in the (NZWxBXSB)F1 model of systemic autoimmunity that is characterized by a leaky barrier based on oral gavage with FITC-dextran. To test commensal translocation, we cultured anaerobically MLN and livers. Compared to controls, vancomycin alone lowered autoantibodies, Th17 and Tfh cells, and protected mice from autoimmune deaths. Cultures of MLN and liver of (NZWxBXSB)F1 mice revealed significant anaerobic growth, which was suppressed by vancomycin. Sequencing of colonies identified E. gallinarum, a human gut commensal, that grew uniquely in tissues of autoimmune but not control mice. Species-specific PCR of liver tissues from autoimmune hepatitis patients also revealed E. gallinarum. 16S rDNA sequencing confirmed the presence of Enterococcus genus in the gut of (NZWxBXSB)F1 mice. Germ-free C57BL/6 mice monocolonized with E. gallinarum also led to translocation and induction of autoantibodies as well as Th1/Th17 responses. Remarkably, an intramuscular vaccine against E. gallinarum prevented autoimmune deaths in (NZWxBXSB)F1 mice. These data support that an innocuous gut commensal translocates spontaneously to initiate autoimmunity that is preventable by a vaccine targeted at the translocating pathobiont. This approach represents a novel therapeutic avenue for immune-mediated diseases.
Engineered immunomodulatory activity in therapeutic nucleic acids
Authors: Johnson Morgan B , Khisamutdinov Emil F , Afonin Kirill A. , Marriott Ian 
Published: January 2023

Abstract
Therapeutic nucleic acids (TNAs) can be engineered to deliver functional moieties such as siRNAs to target cells. An additional benefit of TNAs is that they can be complexed with amphiphilic molecules for delivery into sites such as the central nervous system (CNS) that are difficult to access due to the blood-brain barrier. However, previous TNA constructs have failed clinical trials due to the induction of severe systemic inflammatory responses. We have developed a new generation of TNAs in which immunomodulatory activity, in addition to chemical and thermodynamic stability, can be engineered. In this study, we have assessed the immunomodulatory activity of various TNAs on human CNS cell types. We have determined the ability of 16 TNAs of differing nucleic acid composition and polygon type to elicit cytokine and chemokine production by transfected human microglia and astrocyte-like cells. Our results indicate that, in general, our TNA formulations do not elicit immune responses in human astrocyte-like cells, but do induce significant release of the type I interferon, IFN-β, by cultured microglia with minimal release of the inflammatory cytokine IL-6 and the chemokine IL-8. Interestingly, we observed that the TNA nucleic acid composition and polygon type affect the immune mediator amount and profile released by human microglia-like cells. Specifically, IFN-β responses to TNAs composed of RNA are robust while those composed of DNA are minimal. Furthermore, triangle and pentagon TNA structures stimulate more IFN-β release than square and hexagon compositions. Together these results suggest these novel TNAs may be specifically engineered to minimize detrimental inflammatory responses while promoting beneficial host immunity.