bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenotyping of tumor-infiltrating lymphocytes in melanoma using tissue cytometry (46.28)
Authors: Mansfield James , Lane Kristin , van der Loos Chris , Hoyt Clifford 
Published: January 2023

Abstract
In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. An understanding of the phenotype and spatial distribution of TILs within tumor regions would be advantageous for characterizing host response. However, visual TIL assessment is prone to error and multimarker quantitation is difficult with standard methods. Here we present a multi-marker, computer-aided event-counting method for the determining the phenotypes of lymphocytes in melanoma sections using a novel multispectral imaging (MSI) approach. A section of a tissue microarray containing 120 melanoma cores was stained for CD3, S100, Foxp3 and hematoxylin. This was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. The images were analyzed using software which had been trained to recognize the tumor area based on the S100 staining pattern. Then the Foxp3 status of each CD3+ TIL was then determined. Results indicate that machine-learning software can be trained to accurately recognize tumor regions within each core. MSI enabled the accurate quantitation of three immunostains in the sample without crosstalk. Within the tumor region of each core it was possible to count the CD3+ TILs and then determine the Foxp3 status of each. This multimarker phenotyping and counting approach shows the potential for broad applicability in the assessment of TILs in many solid tumors.
An essential role for SREBP signaling in T cell blastogenesis (47.17)
Authors: Bensinger Steven , Kidani Yoko , Hock M. Benjamin , Argus Joseph , Komisopoulou Evangelia , Graeber Thomas 
Published: January 2023

Abstract
Accumulating evidence indicates that cellular metabolism is an important regulator of T cell immunity. We and others have shown that activation of T cell rapidly initiates a robust genetic and biochemical program that drives the biosynthesis of lipids. Surprisingly, inhibiting lipid synthesis markedly decreases DNA synthesis and lymphocyte proliferation, suggesting a fundamental link between lipid anabolism and cell cycle progression. To date, the molecular mechanisms underlying these intriguing observations are largely undefined. The Sterol Regulatory Element Binding Proteins (SREBP) are b-HLH transcription factors with a well-placed role in lipid homeostasis. The impact of SREBPs on T cell development and function has not been evaluated. Herein we demonstrate an essential role for SREBPs in CD8 T cell blastogenesis. Gene expression and ChIP studies coupled with metabolic flux analysis revealed an essential role for SREBP signaling in the acquisition of a lipid anabolic program by blasting T cells. In the absence of SREBP activity, mitogen-stimulated T cells do not efficiently enlarge and arrest in G0/G1 of cell cycle before undergoing apoptosis-independent cell death. Homeostatic proliferation and antigen-specific viral immunity is also compromised in the absence of SREBP activity. Taken together, these data delineate a critical role for SREBPs in T cell growth and provide a mechanistic understanding of how SREBPs regulate the acquisition of anabolic metabolism in T cells.
Visualization of bacterial dissemination and antigen presentation in gut mucosa following oral infection with murinized Listeria monocytogenes (49.15)
Authors: Xu Daqi , Sheridan Brian , Freitag Nancy , Schubert Wolf-Dieter , Lefrançois Leo 
Published: January 2023

Abstract
Listeria monocytogenes (Lm) crosses the intestinal barrier via Peyer’s patches (PP) and via intestinal epithelial cell (IEC) invasion. Most previous studies examining antigen (Ag) presentation and T cell priming with Lm infection were accomplished using systemic infection. However, Lm dissemination in the gut mucosa and its association with Ag presentation following a natural infection route is not well understood. To understand the mechanisms orchestrating intestinal T cell responses, we analyzed the kinetics and localization of Lm dissemination and Ag presentation among different mucosal tissues following oral infection with a murinized recombinant Lm expressing a modified internalin A protein that allows IEC entry. One day after infection, Lm were located in the subepithelial dome of the PP but were not visualized in the mesenteric lymph nodes (MLN). However, Ag in MLN could be as well detected as in PP by T cell proliferation assay. By 48 hours after infection, Lm had disseminated to the cortex of the MLN, apparently associated with cortical lymphatic sinuses where they co-localized with CD11b+Ly6g+ neutrophils. With time, Lm accumulated in the medulla of the MLN and were present in both PP and MLN for at least 5 days after infection. CD103+ dendritic cells (DC) surrounded the Lm and neutrophil clusters. Ongoing studies will visualize the location of Ag presentation and identify the relationship between DC subsets, bacterial dissemination and Ag presentation.
Non-depleting down-modulation of human B-cells through inhibitory coupling of FcγRIIB (CD32B) and the BCR with bi-specific DART molecules with extended pharmacokinetics. (51.8)
Authors: Chen Wei , Burke Steve , Huang Ling , Ciccarone Valentina , Zhang Tengfei , Alderson Ralph , Barat Bhaswati , Hotaling Tim , Wildjaja Lusiana , Gorlatov Sergey , Li Hua , Koenig Scott , Bonvini Ezio , Johnson Syd , Moore Paul 
Published: January 2023

Abstract
We have previously demonstrated the ability of bi-specific Dual-Affinity ReTargeting (DART) molecules to coligate the inhibitory FcγRIIb (CD32B) receptor and the BCR component, CD79B, to inhibit B-cell activation and dampen autoimmunity (Veri et al, Arthritis. Rheum. 2010). Here we describe development and characterization of CD32BxCD79B DARTs with enhanced pharmacokinetic (PK) properties through incorporation of an albumin binding domain (ABD) or an Fc region. These second generation CD32BxCD79B DARTs, expressed in CHO cells and purified to homogeneity, demonstrated anticipated multi-specific binding properties and effectively reduced naïve and memory B-cell responses in both healthy subjects and SLE patients. Both the ABD and Fc- bearing DARTs displayed increased serum half-life resulting in prolonged ability to functionally block human B-cell responses resulting in reduced circulating IgM and IgG levels in mice engrafted with human PBMC. In contrast to rituximab, CD32BxCD79B DARTs did not induce B-cell depletion in a human fetal liver CD34+ HSC reconstituted mouse model. In summary, CD32BxCD79B DARTs with extended PK properties have been developed that inhibit human B-cell activation in the absence of depletion, forming the basis for an alternate B-cell intervention strategy for controlling autoimmune disease through convenient dosing, while avoiding the more general immune suppression associated with existing B-cell targeted strategies.
Cellular sentinels for Heat Shock Protein-peptide complexes in vivo. (52.15)
Authors: Messmer Michelle , Binder Robert 
Published: January 2023

Abstract
Heat shock proteins (HSPs), such as gp96, harvested from cancer cells elicit potent anti-tumor immunity in mice and in patients with cancer. HSPs in the extracellular environment, released by cells or introduced through vaccination, are taken up by antigen presenting cells of the immune system, which allows for cross-presentation of the HSP-chaperoned peptides to T-cells. The goal of this study is to identify the subset(s) of cells that sample HSPs in vivo prior to initiating T cell responses. By vaccinating mice with fluorescent-labeled gp96, cells in the draining lymph node (dLN) that engage extracellular HSPs have been explored in a time and dose dependent manner. Our results show that CD4+CD11b+ cells are the predominant cells that internalized the HSP in the subcapsular region of the dLN while total CD11c+ cells internalized minor amounts of HSP. Upon further subtyping, CD8+CD11c+ cells, which have been shown by other groups to be the major cross-priming dendritic cells, did not significantly internalize the HSP. The ability of the CD4+CD11b+ and CD11c+ cells to prime T cells is examined to demonstrate cross-presenting function following internalization. These studies illustrate how HSPs act to alert the immune system of cellular damage and will be useful for optimizing HSP-based vaccination regimes by identifying possible cellular targets.
Establishment of T cell stimulation by flucloxacillin in HLA-B*5701+ flucloxacillin-naïve individuals (52.21)
Authors: Wuillemin Natascha , Adam Jacqueline , Fontana Stefano , Pichler Werner , Yerly Daniel 
Published: January 2023

Abstract
Rational: Drug induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*5701 allele. To better understand this side effect, we established an in vitro model for FLUX induced T cell activation. Methods: Peripheral blood mononuclear cells from FLUX-naïve, HLA-B*5701+ healthy donors were induced by iterative in vitro restimulations with FLUX. FLUX-specific T cells (FLUX-Tc) were enriched by positive selection of CD107a expressing cells and characterized by flow cytometry and 51Cr-release assay. Results: FLUX-Tc are mainly CD8+. They react by IFNγ secretion and CD107a upregulation upon drug stimulation and are cytotoxic against autologous antigen presenting cells (APC). Cytotoxicity and reactivity was observed only if free FLUX was added to the system. FLUX-pulsed APC were not able to elicit a FLUX specific T cell response if FLUX was not additionally added. The direct addition of FLUX to FLUX-Tc resulted in their activation even in the absence of APC. This self-presentation and reactivation was still observed in the presence of the proteasome inhibitors lactacystin and bortezomib. Conclusion: The APC independent T cell reactivity and the non-involvement of the proteasome is best explained by a direct interaction of FLUX with the restricting HLA-B*5701 molecule. How and why this activation leads to a preferential damage of liver cells needs further investigation.
Specialized proresolution mediators enhance human B cell differentiation to antibody secreting cells (53.10)
Authors: Ramon Sesquile , Serhan Charles , Phipps Richard 
Published: January 2023

Abstract
The resolution of inflammation is an active and dynamic process critical in maintaining homeostasis. Newly identified lipid-derived mediators are key players during the process of inflammation resolution. These specialized proresolution mediators (SPM) constitute separate classes of compounds, which include lipoxins, resolvins, protectins and maresins, all derived from essential fatty acids. SPM regulate aspects of the immune response, including inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimulation of macrophage phagocytic activity. However, their effects on B lymphocytes are unknown. Our study shows that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA), resolvin D1 (RvD1) but not protectin D1 (PD1), strongly increase the ability of normal human B cells to produce IgM and IgG. The increased antibody levels are due to an increased number of antibody-secreting cells. Furthermore, SPM regulate the expression of the transcription factors Blimp-1, Xbp-1 and Pax-5, and promote B cell differentiation towards a CD27+CD38+ antibody secreting cell phenotype. None of the SPM affect proliferation and are non-toxic. The increase in plasma cell differentiation and antibody production coincides with the known involvement of SPM during the late stages of inflammation and pathogen clearance. These new findings highlight the potential applications of SPM as endogenous and non-toxic adjuvants and anti-inflammatory therapeutic molecules.
In vitro and in vivo characterization of nanolipoproteins (NLPs) conjugated with innate immune agonists: implications for host-based therapeutics (53.12)
Authors: Weilhammer Dina , Blanchette Craig , Fischer Nicholas , El-Etr Sahar , Loots Gabriela , Corzett Michele , Hwang Mona , Thomas Cindy , Lychak Cheri , Mohammadi Sanaz , Rasley Amy 
Published: January 2023

Abstract
Modulation of the innate immune system has the potential to provide transient, non-specific protection from a variety of infectious organisms. Activation of innate immunity is rapid and short-lived, therefore methods aimed at enhancing and prolonging the response will increase the effectiveness of innate immune targeting as a therapeutic measure. In order to enhance the stimulatory properties of innate immune agonists, we have employed a nanolipoprotein (NLP) platform. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins, analogous to reconstituted HDLs. NLPs have been successfully used in numerous biotechnology applications, including drug delivery and diagnostic imaging. Here, we show that two synthetic TLR ligands: monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. In addition, the delivery of agonist-conjugated NLPs significantly enhances immunostimulatory effects relative to agonists alone, at low doses, both in vitro and in vivo. Our data demonstrate that conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, increases cytokine production, and induces upregulation of immunoregulatory genes in peripheral organs. These data suggest that treatment with NLP:agonist conjugates may provide transient protection against lethal pathogen challenge, and experiments to determine their effectiveness at countering infection are ongoing.
Retinoids, Stilbenes and Vitis Vinifera modulate VDR-RXR cross-talk in HT22 mouse hippocampus neuron: an approach towards prevention of neuronal dysfunction in brain inflammation (54.20)
Authors: Dasgupta Subhajit , Bandyopadhyay Mausumi , Bhatt Aruna 
Published: January 2023

Abstract
The vitamin D receptor (VDR) belongs to a member of nuclear hormone receptor superfamily. The present study delineated a mechanism of VDR mode of action in prevention of neuronal dysfunction and damage in central nervous system. The in vitro treatment of HT 22 mouse hippocampus neuron with RXR agonist retinoic acid was found to modulate VDR. The stilbenes and vitis vinifera were also found to influence VDR in HT22 cells. Moreover, treatment of HT22 cells with retinoic acid rich carrot extract was found to attenuate paraquat induced damage of HT 22 cells. A study has been initiated to determine mode of action of the compounds and to evaluate mechanism of VDR-RXR interactive signals in attenuation of neuronal dysfunction in hippocampus in dementia and seizures. Acknowledgement. I (SDG) acknowledge Eileen McFadden and Christopher Riley for their support in research. The HT 22 cells were provided by Dr Mark S Kindy (MUSC). I am thankful to Dr Sebastiano Gattoni- Celli (MD) for his suggestions.We are thankful to the MUSC research endeavors.
Control of the chronic inflammatory response in a murine model of chronic granulomatous disease (54.8)
Authors: Ryan Sean , Cobb Brian 
Published: January 2023

Abstract
Chronic granulomatous disease (CGD) is a primary innate immunodeficiency associated with a defective phagocyte NADPH oxidase complex and thus increased susceptibility to recurrent bacterial and fungal infections. Although antimicrobials have proven useful in resolving primary infection, CGD patients face complications thought to stem from an unresolved hyper-inflammatory state. Using a murine model of CGD we found that a sterile inflammatory insult induces a systemic and long-lived chronic inflammatory response not seen in wild type controls. This persistent inflammatory response is characterized by an increased and sustained expansion of myeloid cells, including Gr1+Ly6G+ cells actively producing the pro-inflammatory IFNγ cytokine and a Gr1+Ly6G- myeloid-derived suppressor cell (MDSC) population. Interestingly, although the expansion of a functional MDSC population would suggest a viable mechanism to control the persistent hyper-inflammatory response, we found that the Gr1+Ly6G+ subpopulation antagonizes MDSC function which significantly affects control of adaptive T cell responses, including responses to inhaled antigen in the lung, a frequently affected organ in CGD patients. Understanding CGD immune dysregulation beyond the primary innate immunodeficiency allows for the development of targeted therapies to potentially prevent inflammatory complications in CGD patients and possibly other patients at risk of complications due to chronic inflammation.
Acinetobacter baumannii virulence requires LPS activation of TLR4 (55.12)
Authors: Lin Lin , Tan Brandon , Baquir Beverlie , Ho Tiffany , Pantapalangkoor Paul , Spellberg Brad 
Published: January 2023

Abstract
Background: New treatments are needed for lethal infections caused by extreme-drug resistant (XDR)/pan-drug resistant (PDR) Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance during non-lethal A. baumannii pulmonary infection in mice. We sought to better to understand the role of LPS-TLR4 interactions in A. baumannii pathogenesis. Methods: LPS was harvested from nine clinical isolates of A. baumannii and then assayed for TLR4 activity using the HEK-Blue cells. Wild type and TLR4 mutant mice were infected via the tail-vein with A. baumannii. Results: After A. baumannii systemic infection, 100% of wild type vs. 0% of TLR4-mutant mice died. Wild type mice developed septic shock, typified by hypothermia, acidemia, and elevated inflammatory cytokine production, wherease TLR4 mutant mice did not, despite having similar bacterial burden in organs. Density of LPS in bacteria (ng/bacillus) and TLR4 activation by LPS extracted from the bacteria did not correlate with in vivo virulence. Amount of LPS shed during growth was the key correlate with in vivo virulence. Summary: LPS activation of TLR4 is a key pathogenetic factor of A. baumannii during systemic infection. Abrogation of TLR4 function protects mice from invasive A. baumannii infection by preventing induction of septic shock. These results suggest new areas of focus for developing novel therapeutics targeting these XDR/PDR infections.
Earlier protective response and milder clinical course of Mycobacterium bovis BCG infection in mice lacking an adapter molecule CARD9 (55.6)
Authors: Kawakami Kazuyoshi , Takahashi Yurie , Yamamoto Hideki , Nakamura Yuri , Ishii Keiko , Hara Hiromitsu 
Published: January 2023

Abstract
CARD9 plays a crucial role as an adaptor molecule in signal transduction triggered through C-type lectin receptors that are known to recognize polysaccharides from various fungal microorganisms. In this study, we examined the role of CARD9 in the host defense to Mycobacterium bovis BCG that has polysaccharide cell-wall components such as lipoarabinomannan. C57BL/6 (WT) and CARD9 knockout (KO) mice were infected intratracheally with M. bovis BCG (Tokyo strain) per mouse. The number of viable bacterium, cytokine synthesis and histopathological findings in lungs were evaluated. CARD9KO mice cleared more bacteria in lungs than WT mice on day 28 after infection. In a histopathological analysis, the lung tissues from CARD9KO mice showed poorer granuloma formation and fewer bacilli in lungs than WT mice on days 14 and 28 after infection. The levels of IFN-γ, a key cytokine for the host defense to this infection, in the bronchoalveolar lavage fluids and its expression in T, NK and γδ T cells on day 7 were increased in CARD9KO mice compared to those in WT mice, although the production of this cytokine was significantly reduced in Card9KO mice on day 28. By contrast, the expression of IL-17A in lungs was not much different between WT and CARD9KO mice. These results indicate that CARD9 is dispensable for elimination of M. bovis BCG and granulomatous response, which suggests a complicated mechanism involved in the role of this molecule in the earlier host response to M. bovis BCG.
Are YFAK-specific TCR retrogenic mice whose splenocytes secrete high levels of IL-10 protected from EAE? (56.9)
Authors: Ng Sze-Ling , Strominger Jack 
Published: January 2023

Abstract
Regulatory T cells (Tregs) are important mediators of a normal immune system response, and the misregulation of Tregs can result in susceptibility to various autoimmune diseases. The random amino acid copolymer YEAK (Copaxone) is widely used in the therapy of human multiple sclerosis (MS), as well as in the amelioration of experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. Our lab has identified a more effective copolymer, YFAK. Both YEAK and YFAK ameliorate EAE by inducing regulatory T cells that secrete the immunomodulatory cytokine IL-10, which mediates bystander immunosuppression. The precise mechanisms underlying the development, function, and maintenance of regulatory T cells are unknown. Therefore, we have generated a YFAK-induced regulatory T cell line and cloned its specific T cell receptors (TCRs) in order to investigate the role of the TCR in the epigenetic change that leads to selective IL-10 secretion. Retrogenic mice expressing YFAK-specific TCRs have been generated. Splenocytes from these mice secrete high levels of IL-10, indicating that the TCR encodes information for the secretion of this cytokine. In addition, experiments in progress asking whether these TCR retrogenic mice are better protected from EAE will be reported. These studies can inform the exploitation of regulatory T cells in the treatment of autoimmune diseases such as multiple sclerosis.
Mycoplasma pneumoniae CARDS toxin exacerbates ovalbumin induced asthma in BALB/c mice (57.9)
Authors: Medina Jorge , Brooks Edward , Le Saux Claude , Chaparro Adriana , Principe Molly , Kannan Thirumalai , Baseman Joel , Dube Peter 
Published: January 2023

Abstract
Mycoplasma pneumoniae is a common human respiratory pathogen that causes acute and chronic respiratory infections that can result in a variety of pulmonary and extra-pulmonary pathologies. M. pneumoniae is associated with exacerbations of human asthma and has significant impact on pathology through unclear mechanisms. We recently demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin induces an allergic asthma-like disease in naïve mice, as evidenced by a Th2 response, mucus metaplasia and airway hyperreactivity. In this study we examined CARDS toxin’s effects on preexisting asthma using an ovalbumin mouse model of asthma. We show that CARDS toxin can exacerbate pathologies associated with ovalbumin-induced asthma in BALB/c mice both histologically and physiologically. These results were highlighted by increased collagen deposition, shown by picrosirius red staining around medium airways and an increase in eosinophils in the alveolar spaces, shown by cellular differentials. Additionally, these findings are paralleled by heightened Th2 cytokine and chemokine responses as measured by qPCR or ELISA. Finally, lung function assessment via a Flexivent system revealed increases in airway hyperreactivity demonstrated by increased resistance and decreased compliance. The data demonstrate CARDS toxin as a causal factor in the exacerbation of experimental allergic asthma and implicates CARDS toxin as a mediator of morbidity in human asthma.