bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering Antibody Fusion Proteins for Targeted Intracellular Therapeutic Delivery
Authors: Kaeo Kyle J , Ludwig Seth , Spangler Jamie 
Published: August 2023

Abstract
Intracellular (IC) proteins make up a significant portion of the proteome, leading to an interest in achieving immune modulation via IC targeted therapeutics. Most IC protein-targeted drugs are small molecules, which can easily pass through the cell membrane but are hindered by a lack of specificity and short half-life. Antibodies, thanks to their greater contact surface area, can have superior target specificity, and persist longer in the blood. Fusing a therapeutic to an antibody presents a promising solution, but antibodies lack a good path into the cell.
We recently discovered a multiparatopic antibody that binds multiple epitopes on the immune checkpoint protein programed death-ligand 1 (PD-L1) to induce its downregulation. This platform promotes endocytosis through clustering receptors together on the surface and enhancing internalization. We sought to exploit this mechanism for IC delivery by introducing endosomalytic peptides to induce escape of the multiparatopic antibody into the cytosol. We inserted two different endosomalytic peptides (EPs) into the downregulating antibody, one before and after the hinge region. We found that these EPs do not hinder downregulation and observed decreased lysosomal accumulation of the antibodies.
To determine the efficacy of this modified antibody as a vehicle for an IC therapeutic, we fused Cas9 to the C-terminus of the heavy chain (HC) of our anti-PD-L1 multiparatopic antibody. We further demonstrated robust PD-L1 downregulation and endosomal escape for these fusion proteins. These data generate proof-of-concept results, establishing a promising new vehicle for IC delivery, with wide-ranging applications including gene therapy, signaling modulation, and genetic engineering.
CD3ζ ITAM diversity determines chimeric antigen receptor force and function
Authors: Echelibe Hilda , Majumdar Shubhabrata , Liu Baoyu , Kolawole Elizabeth , Bettini Maria , Evavold Brian , Bettini Matthew 
Published: August 2023

Abstract
Chimeric antigen receptor (CAR) T cell immunotherapy is a powerful treatment for hematologic malignancies, however clinical responses vary depending on CAR T cell function and persistence. The optimization of CAR design has gained considerable interest with a new focus on calibrating CAR intracellular signals via the immunoreceptor tyrosine-based activation motifs (ITAMs) to increase fitness of CAR T cells. Accumulating evidence suggests that unique ITAM motifs at positions 1, 2 or 3 (ITAMs-A, B, and C respectively) of the CD3ζ signaling domain may have non-redundant roles in signal strength, T cell functionality, persistence, and memory formation. However, the individual contributions of each CD3ζ ITAM in the transmission of force and the activation of mechanosensitive signals is yet unexplored. We have found that human CARs expressing specific ITAM sequences (zeta-A only, zeta-B only or zeta-C only) generate differing amounts of Force and Bond Lifetime after interacting with CD19. Therefore, a CAR may act as a mechanosensor similar to a TCR discriminating the quality of the interaction with antigen as cellular derived forces are applied to the bond. The higher transmission of force directly correlates with the better anti-tumor efficacy in vitro and in vivo. Hence, understanding the mechanotransduction in CARs has important implications in “tuning” the activation of a CAR T cell without changing specificity.
Supported by the Seed Grant, Department of Pathology, University of Utah
CO-induced TTP improves efficacy through resetting tumor-associated macrophages toward M1 phenotype in combined CTLA-4 and PD-1 blockade immunotherapy
Authors: Gong Jeong Heon , Park Jeongmin , Joe Yeonsoo , Chung Hun Taeg 
Published: August 2023

Abstract
Combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 has improved the prognosis for many malignancies, but also occurred a high rate of immune-related adverse events (irAEs). The manifestation of these toxicities is an important limiting factor for the successful implementation of CICB, restraining its anti-tumor efficacy. Carbon monoxide (CO), a pivotal endogenous signaling molecule, is able to maintain cell and tissue homeostasis and could be used as a therapeutic regimen. In our previous reports, CO could be useful therapeutics for the regulation of various inflammatory diseases through the increase of tristetraprolin (TTP) that binds to the AU-rich elements in target mRNA. In this study, we observed that CO-induced TTP activation ameliorated colitis and hepatitis, and improved anti-tumor efficacy in CICB treatment. We found that the colitis and hepatitis in TTP deficient mice were further exacerbated by CICB treatment. Moreover, CO increases CICB efficacy through TAM to M1 polarization together with diminishes irAEs through TTP enhancement in several irAEs target organ. Thus, we suggest that CO might be efficiently decouples anti-tumor efficacy and toxicity of CICB through TTP enhancement.
Dissecting the lineage specific roles of BCL-XL in breast cancer metastasis
Authors: Carelock Madison Elizabeth , Jiao Mo , Zhang Weizhou 
Published: August 2023

Abstract
B-cell lymphoma X-large (BCL-XL) has been shown to promote breast cancer metastasis independently of its anti-apoptotic function. We recently found that proteolysis-targeting chimeras (PROTACs) against BCL-XL kill cancer cells as well as tumor-induced regulatory T cells (TI-Tregs). BCL-XL-targeting PROTACs can be potentially used to treat metastatic breast cancer – by direct killing of circulating tumor cells at their vulnerable stages and/or by eliciting anti-cancer immunity via T-reg cell depletion. Our pilot experiments showed opposing results with one model showing reduced metastasis by BCL-XL PROTAC treatment and the other model showing increased metastasis. Genetic deletion of BCL-XL (KO) in those two cell lines reproduces the effect of PROTAC on metastasis. We hypothesize that BCL-XL may play different roles in breast cancer metastasis depending on cellular cues from different cancer cells. We generated 4 other syngeneic mouse breast cancer BCL-XL-KO lines and confirmed that most of the breast cancer lines depend on BCL-XL for metastasis, whereas only 1 model exhibits an opposite phenotype. Additionally, we treated 4T1-tumor bearing mice with BCL-XL-targeting PROTAC that nearly significantly reduces metastatic spread to the lung. We also found a systematic inhibition of TI-Tregs by PROTACs indicating a lineage-specific function of BCL-XL in breast cancer metastasis. We conclude that BCL-XL is a viable therapeutic target for treating breast cancer metastasis, but further investigation into lineage-specific BCL-XL targeting is needed. Our future efforts will focus on investigating why BCL-XL inhibits metastasis in some cancer types, which can be used as an exclusion criterion for BCL-XL-targeting therapy.
Supported by grants from NIH/National Cancer Institute (R01 CA260239)
Fetal Tissues Reflect Decidual-Placental Inflammation
Authors: Vazquez Jessica , Keding Logan , Koenig Michelle R , Schotzko Michele , Antony Kathleen , Racine Jenna , Simmons Heather A , Shah Dinesh M , Wieben Oliver , Golos Thaddeus 
Published: August 2023

Abstract
Maternal inflammation can negatively impact the development of the fetus, leading to long term adverse health outcomes. We previously reported that injection of Tisseel (surgical glue) into the maternal-fetal interface led to necrosis and inflammation skewing the peripheral fetal immunome into a pro-inflammatory state. Here, we extend those observations and report that decidual inflammation leads to similar outcomes in fetal organs.
Tisseel or saline (n = 3) was injected directly into the placenta of pregnant (~GD90) rhesus macaques. C-sections were performed at ~GD150. Fetal tissues (lung, thymus, spleen, liver) were collected at necropsy, homogenates were prepared from all fetal tissues, and cytokine levels were measured by cytometric bead analysis. Mononuclear cell suspensions were prepared from thymus and spleen, labeled with antibodies against major immune cell lineages, and acquired using the Cytek Aurora. Data was analyzed using Specter (R), statistical significance was determined by Student’s t-test (p &lt; 0.05).
Tisseel group fetuses had higher levels of IL17A (lung, spleen), MCP1, and IL6 (liver). Interestingly, there was a trend towards elevated IL6 in the thymus of saline group fetuses.
Additionally RORγt+ T cells were elevated in the thymus of Tisseel group fetuses, while TRegs were elevated in saline group. The spleen from Tisseel group fetuses had a higher proportion of TH1-like cells. Interestingly, T-bet+ B cells, linked to autoimmune disorders, were also elevated in the Tisseel group.
Our preliminary results suggest that decidual/placental inflammation leads to a pro-inflammatory immunome in fetal organs. Further studies are needed to determine the functional properties of these altered immune populations.
The mucosal immune responses of the human cervix are modulated by Neisseria gonorrhoeae
Authors: Song Wenxia , Dai Yiwei , Yu Qian , Edwards Vonetta , Tettelin Herve , Stein Daniel C 
Published: August 2023

Abstract
The cervix is the gate to the upper female reproductive tract (FRT), and the mucosal surface is the first line of defense against pathogens. The sexually transmitted pathogen Neisseria gonorrhoeae (GC) infects from the lower tract and does not induce inflammation until GC ascends through the cervix to the upper FRT, suggesting immune evasion. We examined local immune responses in the human cervix to GC infection during the first 24 hours using a human tissue explant model. GC inoculation increased the secretion of the pro-inflammatory cytokines IL-1β, TNF-α, and TNF-β, the anti-inflammatory cytokines IL-10 and LIF, and the chemokines IL-8, CXCL1, CXCL2, and CCL3. GC expressing opacity-associated protein Opa52 (binds to CEACAM) enhanced anti-inflammatory cytokines production. The elevated cytokine production was concurrent with increased nuclear staining of NF-κB p65. RNAseq analysis found increases in mRNA levels of cytokines and NF-κB pathway genes in GC-inoculated cervical tissues, compared to no GC controls. Inhibiting NF-κB activation by Bay reduced GC-induced secretion of pro-inflammatory but not anti-inflammatory cytokines. While macrophages and T-cells were abundant in the human cervix, colonization of GC expressing Opa52 did not recruit the immune cells, despite the induction of chemokines and cytokines. However, colonization of Opa-deleted GC recruited macrophages to the subepithelium of the cervical transformational zone and increased the number of CD3+ T-cells contacting Langerhans cells in the ectocervical epithelium. Together these results suggest that GC infection modulates the balance of the cervical cytokine environment towards anti-inflammatory to suppress local inflammatory responses.
Supported by grants from NIH RO1 AI141894 and RO1 AI123340
SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process
Authors: Eshetie Kirubel , Najafova Nurana , Graefen Bahar , Aliyeva Leyla , Tauseef Mohammad , Fazal Nadeem 
Published: August 2023

Abstract
Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as diabetes and atherosclerosis. Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions. Vascular injury is associated with activation of the coagulation cascade and release of thrombin, which increases endothelial permeability by activating endothelial cell surface thrombin receptor. This increase in endothelial permeability is typically followed by a recovery period of ≈2 hours, during which barrier integrity is restored. It has been surmised that thrombin signaling stimulates intrinsic repair mechanisms that restore barrier function. To validate our finding we determined endothelial barrier function using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Our data show that 50mM thrombin increase endothelial permeability followed by complete recovery and reannealing of adherens junction in control endothelial cells. However, SPHK1 depleted cells showed significantly decrease in barrier disruption, and barrier failed recover completely compared to control monolayer. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease.
CTRE Seed Grant to Professor Nadeem Fazal, MD, PhD
Sex-specific and immune-cell-specific contributions of CD18 in myocardial ischemia-reperfusion injury
Authors: Fan Zhichao , Liu Wei , Cronin Chunxia G , Wen Lai , Ley Klaus , Liang Bruce T 
Published: August 2023

Abstract
Leukocyte recruitment and their mediated inflammatory responses are critical for cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury. Blocking leukocyte recruitment in mouse knockouts (KO) of beta2 integrin (CD18) or blocking CD18 with antibodies in multiple animals significantly reduced infarct size after myocardial I/R injury. However, the cell-specific contribution of leukocyte CD18 to I/R injury is unknown. In this study, we used the newly established CD18flox/flox (hITGB2 KI) mice to address this knowledge gap. We crossed them to CSF1R-cre (CD115) and MRP8-cre (S100A8) and tested the KO of CD18 in different leukocytes. Interestingly, CSF1R-cre CD18flox/flox unexpectedly deleted CD18 in all peripheral blood leukocytes, including neutrophils, monocytes, CD4 T cells, CD8 T cells, B cells, and NK cells. It also elevated the cell number of these leukocytes in peripheral blood. In MRP8-cre CD18flox/flox mice, CD18 was only knocked out in neutrophils but not other peripheral blood leukocytes. And only neutrophil number was elevated in peripheral blood. After 35 minutes of myocardial ischemia and 24 hours of reperfusion, we found both CSF1R-cre CD18flox/flox and MRP8-cre CD18flox/flox mice have significantly reduced infarct size compared to cre-controls. However, if we distinguish the sex in analysis, we only found a significant alleviation in female but not male CSF1R-cre CD18flox/flox mice. In contrast, we observed a significant alleviation only in male but not female MRP8-cre CD18flox/flox mice. These results suggested sex-specific and immune-cell-specific contributions of CD18 in myocardial ischemia-reperfusion injury and provided new insights into CD18 targeting therapies.
This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454, R41HL156322, and R44HL152710) and a startup fund from UConn Health.
Effects of cannabidiol on the differentiation of human monocytes into macrophages
Authors: Vigano MariaLuisa , Samarani Suzanne , Ahmad Ali , Costiniuk Cecilia Theresa 
Published: August 2023

Abstract

Background
Innate immune myeloid cells, such as macrophages, contribute to chronic inflammation when consistently activated. Recently, cannabidiol (CBD), an active non-psychoactive constituent of the Cannabis sativa plant (i.e., Marijuana), has sparked interest as a safe and effective anti-inflammatory and immunomodulatory agent. This study aimed to investigate the CBD’s effects on the differentiation of human macrophages and their cell surface receptors.

Methods
Peripheral blood was collected from healthy human donors. Monocytes, isolated from peripheral blood mononuclear cells, were differentiated with macrophage colony stimulating factor into monocyte-derived macrophages (MDM) with and without CBD (5 μM). Differentiated MDMs were then stained for different cell surface markers and analyzed by multicolor flow-cytometry. Proportions of anti-inflammatory (M2: CD206, CD71, CD163) and pro-inflammatory (M1: CD86, CD163) MDMs, and expression of myeloid lineage markers (CD14, CD16), chemokine receptor 5 (CCR5) and endocannabinoid type 2 receptor (CB2) were determined.

Results
CBD promoted a shift towards a greater proportion of M2-type vs. M1-type MDMs (p&lt;0.05, Fisher’s exact test). Cells displayed a significantly reduced expression of CD14, CD163, CD86, CCR5 and CB2 (p&lt;0.05, Student t-test).

Conclusions
CBD appears to promote differentiation of MDMs into anti-inflammatory phenotypes. Further studies are ongoing to determine the mechanistic pathways implicated in these processes, as these findings may have implications for the use of CBD in various disease conditions.
cd13 controls myeloid cell-cell fusion
Authors: Ghosh Mallika , Mcgurk Fraser , Shapiro Linda 
Published: August 2023

Abstract
Macrophages have the ability to undergo phenotypic and functional changes in response to microenvironmental cues. One such change is the formation of multinucleated giant cells (MGCs) via intercellular fusion triggered by certain bacterial or parasitic infections or implantation of medical devices that induces deleterious MGC formation in response to the foreign body. Despite advances in biomaterials, implants ultimately corrode to produce particles that initiate a foreign body response characterized by macrophage fusion into pro-inflammatory, Foreign Body Giant Cells that directly define the pathology of loosening of medical implants. We have shown that the transmembrane protein CD13 is a multifunctional molecule that regulates diverse processes such as cell-cell adhesion, receptor endocytosis and recycling and organization of the actin cytoskeleton, all of which are critical in cell-cell fusion. We demonstrate that subcutaneous foreign implants in CD13KO mice produce significantly more MGCs with increased levels of plasma pro-inflammatory cytokines. In vitro induction of fusion in CD13-deficient myeloid progenitors generated from bone marrow resulted in hyperfusion to generate MGCs that were considerably larger in size and contained many more nuclei than those from wild type progenitors, suggesting that CD13 regulates MGC formation at the stage of fusion. While expression of the key fusion proteins are typically downregulated in mature cells post-fusion, expression of these proteins is sustained at high levels in multinucleated cells lacking CD13. Thus, CD13 acts as a brake to prevent aberrant myeloid cell-cell fusion and may be a novel therapeutic target in pathological conditions mediated by abnormal cell fusion.
Supported by grants from NIH (1R21 AI15)
RELT is overexpressed in human inflammatory bowel disease and breast cancer
Authors: Cusick John K , Moua Pachai , Wong Stephanie , Chang Esther , Chou Emily , Shyu Yennie , Abram Alyssa , Alcaide Jessa , Talbott George , Yanumula Anusri , Qureshi Vaniya , Budhiraja Anuj , Cusick Gianne , Shi Yihui , Atapattu Dhammika , Fitzpatrick Leo 
Published: August 2023

Abstract
Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR member that activates NF-kB and is implicated in several cancers. We therefore sought to characterize the association of RELT with inflammatory bowel disease (IBD) and breast cancer (BC). Additionally, we further characterized the association of RELT with Filamin A (FLNA), an actin-binding protein that can promote tumorigenesis, or counteract tumors, depending on its cellular location. Female CB-17 SCID mice were injected with anti-CD40 monoclonal antibody to induce colitis and euthanized seven days later. Immunohistochemistry (IHC) was utilized to compare RELT expression in the mouse colitis model with uninjected control mice. IHC was utilized to compare the expression of RELT in biopsies of human IBD and non-inflamed tissue, as well as human BC versus non-malignant tissue. Luciferase assays and an X-gal morphology assay were utilized to examine cell death in the BC cell line MDA-MB-231 (231). RELT immunostaining is significantly increased in both mouse colitis and human IBD biopsies in comparison to that of species-matched non-inflamed tissue. An apparent association between colonic histological damage and RELT immunostaining was observed in both mice and human specimens. RELT staining was also increased in human BC biopsies versus patient-matched non-malignant tissue. RELT overexpression induced death in 231 cells, an effect that was not abrogated by a RELT mutation in the binding site for the OXSR1 kinase. However, co-expression of a S2152A mutant of FLNA did abrogate the ability of RELT to induce death in 231 cells. Collectively, these results implicate RELT as being important for human disease and further our understanding of the interaction between RELT and FLNA.
Dynamics of immune cells in the fetal brain upon viral inflammation
Authors: Arreguin Montserrat Carolina , Wu Vincent , Betts Michael , Jurado Kellie Ann 
Published: August 2023

Abstract
The fetal immune system undergoes many developmental milestones, with several immune cell subsets emerging during pregnancy. Recent evidence has shown that maternal infections during pregnancy can influence offspring immunity in the long term. Moreover, maternal infections are associated with an increased risk for neurodevelopmental disorders in the offspring. Yet, our knowledge of the immune cell dynamics in the fetal brain throughout gestation and their response to inflammation remains limited. We sought to characterize the phenotype of immune cells in murine implantation sites at mid-gestation, a time point equivalent to the beginning of the second trimester in humans. Our preliminary studies revealed specific and robust populations of maternal and fetal immune cells in murine fetal brains. Quite unexpectedly, one of these populations expressed NK1.1, a marker for natural killer cells and innate lymphoid cells. Given that the presence of maternal and fetal NK1.1+ cells in the fetal brains at mid-gestation has not been previously reported, our goal is to determine the phenotype and kinetics of the maternal and fetal NK1.1+ immune cells in the fetal brain during a healthy pregnancy and upon congenital infection. To address this, I will use a high-dimensional sequencing technology, CITE-seq, that allows me to simultaneously study surface markers and gene expression of cells. Maternal immune responses critically impact fetal development during gestation and have long-term health outcomes in offspring. Understanding maternal and fetal immune responses to inflammation will inform the origin of fetal inflammation-driven neurodevelopmental disorders and ultimately improve offspring’s health outcomes.
Supported by Packard Foundation Fellowship for Science and Engineering