bluuebunny/crossref_metadata_2025_split huggingface dataset:
Validation of Immunogenicity Assessments for Non-Clinical Toxicology Evaluation
Authors: Hammonds Tiffany , Newsome Courtni , Freebern Wendy , Thompson Chris 
Published: January 2023

Abstract
To determine the immunogenic potential of biotherapeutics in nonclinical species, assays for anti-drug antibody (ADA) detection must be developed and validated before testing of samples from toxicity studies, to ensure accurate assessment of immunogenicity. ADAs can cause adverse events effecting drug exposure and efficacy complicating the interpretation of the toxicity, pharmacokinetics and pharmacodynamics data. For each compound, a validation is performed prior to study sample analysis. Parameters assessed include Inter analyst, sensitivity, accuracy, drug tolerance, precision, interference and stability of reagents and ADAs. The first step in the validation is to perform precision analysis. Precision is used to determine acceptance criteria for the assay. The inter-analyst experiment is performed by two scientists to show reproducibility. Assay acceptance criteria consists of the acceptable criteria for the pooled negative control (PNC), low quality control (LQC), high quality control (HQC) and LQC/PNC ratio to set the assay cut point. Once this criteria is determined, sensitivity, drug tolerance, interference, and stability are performed to set validation criteria for study samples, reagents, and controls. The drugs to be used on study are labeled with Biotin and Sulfotag and ADAs are assessed utilizing immuno-assay such as a standard ELISA or an electrochemiluminescent immuno-bridging assay. Once study samples are analyzed, it can be determined which subjects have an ADA response elicited by the compound and at what severity. An ADA response helps determine immunogenicity in toxicological evaluations, so it is important to assess for this response when dealing with immunotherapeutic.
Fenton-catalytic nanocomposites for reprograming macrophages towards the improved anti-biofilm activity
Authors: OJAYM ALHUSSAIN A , Yu Bing , Kim Min-Ho 
Published: January 2023

Abstract
Bacterial infections involving the formation of biofilms are associated with diminished bactericidal activity of macrophages, which has been shown to contribute to the persistence in chronic infection. We recently reported that S. aureus biofilm-induced impairment of bactericidal activities of macrophages are associated with attenuation of reactive oxygen species (ROS) generation and NF-κB activation. The objective of this study is to validate the use of biocompatible Fenton-catalytic nanocomposites composed of iron oxide nanoparticles (IONPs), a reducing agent (ascorbic acid), and hydrogen peroxide (H2O2) to take advantage of their ability to stimulate the generation of ROS and activation of NF-kB in macrophages by means of triggering a Fenton reaction. Using an in vitro culture model of NF-kB reporter-RAW 264.7 macrophages, we optimized the range of the concentrations of IONPs, ascorbic acid, and/or H2O2, along with their respective combinations, exhibiting an enhanced NF-kB activity and ROS generation. Based on the findings, we subsequently validated the effect of nanocomposite on the bactericidal activity of macrophages exposed to S. aureus biofilm. Our results revealed that RAW 264.7 cells treated with IONPs resulted in significant reduction in the number of surviving bacteria by ~50% and its efficacy further increased in combination with ascorbic acid and/or H2O2, which were in line with the extent of NF-kB activation and ROS generation. In summary, our study supports that harnessing the characteristic of Fenton-catalytic nanocomposite to tune macrophage polarization to exhibit a bactericidal activity may provide a new strategy for treating infectious diseases.
Funding support: Supported by NIH R01 NR 015674
Critical roles of TIGIT+ human regulatory B cells in the suppression of antibody-mediated allograft rejection
Authors: Hasan Md Mahmudul , Wang Yuanyuan , Blaji Uthra , Gu Jinghua , Nattamai Durga , Nyarige Verah , Thompson-Snipes LuAnn , Klintmalm Goran B. , O’Leary Jacqueline G. , Wang Junwen , Joo HyeMee , Oh SangKon 
Published: January 2023

Abstract
TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a co-inhibitory receptor, highly expressed by Tregs, and contributes to the suppressive function of Tregs by limiting pro-inflammatory Th1 and Th17 cell responses. Herein, we report that subsets of human regulatory B cells (Bregs) express surface TIGIT and can thus play a critical role in immune regulation. Using data from human B cell surface arrays and RNA-seq, we identified two major TIGIT+ human Bregs subsets, exhibiting CD24hiCD27+CD39hiIgD+IgMhiCD1chi marginal zone (MZ)-like and CD24hiCD27+CD39+IgD−IgM+CD1c+ memory Bregs. We further demonstrated that TIGIT+ Bregs were also capable of suppressing immune responses by their ability to express not only IL-10 and PD-L1, but also granzyme B and TGFb1. In addition, TIGIT+ Bregs can also suppress immune responses by altering dendritic cell functions. In line with these observations, liver allograft recipients with donor-specific alloantibody (DSA) had significantly lower numbers of TIGIT+ Bregs than those without DSA. Moreover, the frequency of TIGIT+ Bregs correlated with that of CD4+CD25+CD127lo Tregs, while it inversely correlated with the frequency of follicular helper T cells. We thus concluded that TIGIT+ Bregs play critical roles in immune regulation in liver allograft recipients.
Antibody-drug conjugates targeting CD45 plus Janus kinase inhibitors permit allogeneic hematopoietic stem cell transplantation with minimal treatment-related toxicity
Authors: Persaud Stephen P , Cooper Matthew L. , Ritchey Julie K. , Rettig Michael P. , DiPersio John F. 
Published: January 2023

Abstract
Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative therapy for hematologic diseases. However, toxicities from chemotherapy- or irradiation-based transplant conditioning, as well as graft-versus-host disease (GvHD), limit the use of alloHSCT to the most severe diseases. Novel conditioning regimens able to achieve donor engraftment with minimal adverse effects for the patient could greatly expand the safe applicability of alloHSCT to a wider variety of diseases. Antibody-drug conjugates targeting CD45 (CD45-ADC) have been shown previously to permit engraftment in murine syngeneic HSCT models. We set out to extend this approach to the allogeneic setting, developing CD45-ADC-based regimens able to both make marrow space for donor stem cells and overcome immune barriers to their engraftment. CD45-ADC combined with CD4+ and CD8+ T cell depletion was sufficient for engraftment in MHC-matched (BALB to DBA/2) and MHC-mismatched (CB6F1 to B6) murine alloHSCT models. The selective Janus kinase inhibitor baricitinib, shown by our lab to prevent GvHD, also inhibited host-versus-graft responses and allowed allogeneic engraftment when combined with CD45-ADC. In our alloHSCT models, up to 90% donor chimerism was achieved in over 70% of mice treated with CD45-ADC plus baricitinib. Finally, unlike mice conditioned with irradiation, mice treated with CD45-ADC did not develop GvHD when infused with allogeneic splenocytes. In conclusion, CD45-ADC plus baricitinib provides a safe, effective approach to alloHSCT conditioning in mice. These studies demonstrate the promise of immunotherapeutic strategies to achieve transplant tolerance while minimizing treatment-related morbidity and mortality.
Galactin-1 favors engraftment in sickle cell disease patients after haploidentical hematopoietic stem cell transplantation and in murine MHC-mismatched allotransplantation
Authors: Shaikh Ahmad , Gangaplara Arunakumar , Olkhanud Purevdorj , Kone Abdoul , Almengo Katherine , Patel Sajni , Saxena Ankit , Lopez-Ocasio Maria , Gucek Marjan , McCoy Philip , Fitzhugh Courtney D 
Published: January 2023

Abstract
Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is associated with increased risk of allograft rejection. Understanding the mechanisms of graft rejection and tolerance induction are critical for improving HSCT outcome. Recently, we performed a proteomic analysis using plasma samples from sickle cell disease (SCD) patients who underwent haplo-HSCT and found that galectin-1 (Gal-1) levels were significantly higher in engrafted patients. Here, we studied major histocompatibility complex (MHC)-mismatched allo-HSCT murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected (cyclophosphamide/sirolimus alone) or engrafted (cyclophosphamide and sirolimus) based on our previous findings. In rejected mice we noted significantly higher frequencies of CD4+T, CD8+T, NK cells, IFN-γ TNF-α producing CD4+T cells, and also IFN-γ producing DCs and macrophages. Importantly, in engrafted mice we found significantly increased frequencies of Tregs and IL-10 producing CD4+T and Tr1 cells, and the proportion of these cells expressing Gal-1 was significantly higher. Further, we detected a significant increase in Gal-1 levels in plasma of engrafted mice, and this corroborated with our data from engrafted patients. Here, we have shown for the first time that not only Gal-1 favors engraftment in SCD patients who underwent haplo-HSCT, but Gal-1 also contributes to HSC engraftment in our MHC-mismatched murine model. Taken together, our results demonstrate that Gal-1 expressing Tregs and Tr1 cells may play an essential role in inducing immune tolerance and stable mixed chimerism after HSCT.
The Role of PPARd in Alloreactive CD8 T cell Metabolism
Authors: Brown Rebecca Audrey , Dobbs Andrea , Denby Elisabeth , Waltz Gail , Rajeevan Arathi , Byersdorfer Craig A 
Published: January 2023

Abstract
Graft-versus-host disease (GVHD) is the most common cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation. Using a pre-clinical model of GVHD, previous work has found that CD8 T cells recovered on day 7 post-transplant upregulate the transcription factor, peroxisome proliferator activated receptor delta (PPARd). While PPARd has been shown to be a key regulator of fatty acid oxidation (FAO) in other tissues, it’s role in T cells is not well studied. We hypothesize that PPARd drives FAO in alloreactive CD8 T cells and that CD8 T cells lacking PPARd will be unable to oxidize fat and thus unable to cause GVHD. To investigate this hypothesis, ex vivo FAO was measured in day 7 WT versus PPARd KO CD8 T cells by quantitating conversion of 3H-palmitate to 3H2O. Production of 3H2O decreased by 50% in KO CD8 T cells, suggesting an inability to fully oxidize fat. To understand the mechanism underlying this decrease, we performed RNA sequencing and identified thirty genes that were differentially expressed in WT versus KO CD8s including 8 genes related to FAO. Strikingly, despite a decreased ability to oxidize fat, PPARd KO CD8 T cells were recovered in equal numbers on day 7 post transplantation, suggesting that CD8 T cells lacking PPARd adopt alternative metabolic pathways to generate sufficient energy for in vivo proliferation and activation. Ultimately, our analyses indicate that PPARd is required for FAO in alloreactive CD8 T cells, but this process is dispensable for short term proliferation and/or survival of CD8 T cells during GVHD. Future directions will identify compensatory metabolic pathways utilized by PPARd KO T cells through a combination of in vitro nutrient drop out experiments and metabolomic flux analyses.
PD-1 blockade later but not at post-transplant attenuates chronic graft-versus-host disease
Authors: Rhee Stephanie , Aguilar Ethan George , Paz Katelyn , Flynn Ryan , Kuchroo Juhi , Sage Peter , Sharpe Arlene H , Freeman Gordon J , Ahmed Rafi , Blazar Bruce R 
Published: January 2023

Abstract
PD-1 and its ligand PD-L1 are widely appreciated as targets of immune checkpoint blockade. We sought to investigate the role of the PD-1 pathway in the context of chronic graft-versus-host disease (cGVHD). To induce cGVHD, mice are conditioned with Cytoxan and radiation prior to MHC disparate bone marrow (BM) + T cells (70k) and develop multi-organ symptoms with bronchiolitis obliterans lung manifestations. CGHVD mice have high survival rates past 50 days, with &lt;15% weight loss over this period. Anti-PD-1 mAb given to mice with established cGVHD resulted in disease attenuation assessed by significantly improved pulmonary function tests compared to irrelevant mAb controls. PD-1 blockade significantly reduced germinal center (GC)-promoting GC B and T follicular helper (Tfh) cell and increased GC-suppressing T-follicular regulatory (Tfr) cell frequencies. However, in studies with mice given five percent higher T cell dose, resulting in T cell rather than GC-mediated disease, anti-PD-1 mAb dramatically worsened cGVHD severity. To assess GC cell-specific effects, recipients were given PD-1-deficient cells in graft compartments. Mice receiving PD-1 KO T cells predictably exhibited marked escalation of cGVHD severity and lethality, while mice receiving PD-1 KO B cells showed diminished disease, with significant improvement in pulmonary function. Co-culture of isolated B, Tfh and Tfr cells from NP-OVA immunized mice to determine effects of individual GC cell populations on Ig class switching demonstrated significant increase in Tfr suppressive capacity with addition of anti-PD-1 mAb. Together, these data provide evidence for PD-1 support of the GC reaction and identifies the PD-1/PD-L1 axis as a potential target for cGVHD therapy.
Conventional type 2 dendritic cells and natural killer cells mediate control of early metastatic seeding
Authors: Weizman Orr-El , Krykbaeva Irina , Bosenburg Marcus , Iwasaki Akiko 
Published: January 2023

Abstract
The multistep progression of transformed primary tumors to metastatic dissemination and seeding of distant site is a lethal determinant of disease. Although the role of immune cells and immune sensors in promoting or restricting metastatic growth is well described, whether immune cells are capable of detecting and controlling metastatic cells during peripheral tissue seeding is unknown. Here we examine if local immune cells can confer early anti-metastatic control in the lung using intracardiac injection to mimic metastatic spread. Using a syngeneic mouse model of YUMMER1.7 melanoma expressing GFP and luciferase (YMR1.7-G/L), we demonstrate that lung resident conventional type 2 dendritic cells (cDC2) and extravascular NK cells confer host anti-tumor immunity during the first three days following intracardiac injection. Rapid production of IFN-γ by NK cells serves an essential role to limit metastatic burden. Using mice deficient in innate sensors and signaling pathways, we demonstrate that the transcription factors IRF3 and IRF7 are required for early metastatic control in a type I IFN-independent manner. In contrast, ablation of adaptive immune cells had no effect to initial metastatic burden. Collectively our results highlight a novel cDC2 - NK cell axis that orchestrates a non-redundant innate immune response program to limit initial metastatic burden in the lung.
Response to anti-PD-1 and anti-LAG-3 immune checkpoint blockade (ICB) is associated with induction of pro-inflammatory Tregs
Authors: Sturgill Elizabeth Rowland , Mick Courtney , Kaufmann Johanna K , Redmond William L 
Published: January 2023

Abstract
T cells upregulate the immune checkpoint, LAG-3, which is associated with reduced T cell effector function following repeated antigen exposure. Further, high LAG-3 expression on Tregs has been reported in patients with varying cancer types; thus, we hypothesized that the combination of aLAG-3 with another common ICB, aPD-1, would synergize to potently reinvigorate T cells to promote tumor regression and increase survival.
Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma)and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. In-depth analysis of tumor-infiltrating lymphocytes (TIL) revealed that aPD-1/aLAG-3 therapy significantly increased the overall percentage, cytotoxicity (granzyme A), and cytokine production (TNF-a and IFN-g) of CD8+ TIL, and increased CD4+ Teff/Treg ratios compared to aPD-1 or aLAG-3 alone. These effects were enriched in responders to aPD-1/aLAG-3 therapy, which were designated as those exhibiting decreased tumor size on the day of harvest compared to maximum tumor growth post-implant. Surprisingly, Tregs isolated from tumors responding to aPD-1/aLAG-3 displayed increased proliferation, secretion of pro-inflammatory cytokines (TNF-a and IFN-g), and increased LAG-3 expression. In summary, these data suggest that aPD-1/aLAG-3 immunotherapy increased CD8+ TIL exhibiting enhanced effector function, increased CD4+ Teff/Treg ratios, and induced Treg pro-inflammatory responses. Together, these positive immunological changes led to a more immune stimulatory tumor microenvironment capable of supporting tumor regression and significantly improved tumor-free survival.
Modulation of piglet’s immune system development with fecal microbial transplantation
Authors: Blais Mylene , Verso Luca Lo , Thibodeau Alexandre , Harel Josée , Fravalo Philippe , Lessard Martin 
Published: January 2023

Abstract
The aim of the study was to investigate fecal microbial transplantation (FMT) as a novel strategy to modulate the postnatal development of porcine immune system and the establishment of the intestinal microbiota in newborn piglets. Ten litters were used for the experiment. At birth, four piglets were identified in each litter and randomly assigned to the control (CTRL) and the FMT groups. At 3,4,8,9, and 10 days of age, piglets in the FMT group were orally administered with FMT material suspended in 10% bovine colostrum solution. The FMT was prepared with an equal mixture of fecal material harvested from healthy suckling and fully weaned piglets. Control group was inoculated with the vehicle solution. Animals were then euthanized at d 22 to identify the leukocyte subsets in mesenteric lymph nodes (MLN) and blood by flow cytometry and to characterize the microbiota from ileum, caecum and colon by 16S rRNA amplicon sequencing. Results showed that FMT reduced the overall number of T lymphocytes (CD3+) both in blood and MLN (P&lt;0.05). Blood percentages of Th lymphocytes (CD3+CD4+CD8α−) were also reduced in piglets receiving FMT (P&lt;0.05), as well as γδ T lymphocyte (CD3+γδ+) and monocyte/macrophage (SWC3+CD14+) concentrations in the MLN (P&lt;0.05). On the other hand, FMT increased the blood percentage of NK cells (CD3−CD4−CD8α+CD16+) (P&lt;0.01). Results obtained from the microflora analysis confirmed that FMT administration affected piglet’s colon microbiota. In conclusion, although it had a mild impact on hindgut microflora, we observed that FMT has the potential to modulate the immune system development in newborn piglets. Further studies are necessary to fully understand the long-lasting effects of FMT on animal health.
Identifying autoantibody biomarkers for Addison’s disease in dogs
Authors: Treeful Amy , Friedenberg Steven 
Published: January 2023

Abstract
Addison’s disease is a life-threatening endocrinopathy caused by the immune-mediated destruction of the adrenal cortex. Only dogs and humans develop the disease naturally. In humans, autoantibodies against the adrenocortical steroidogenic enzyme 21-hydroxylase can predict patients at high risk of developing the disease. No comparable biomarker exists for canine Addison’s disease. We hypothesized that, similar to humans, newly diagnosed dogs with Addison’s disease will have consistently detectable autoantibodies against adrenocortical steroidogenic enzymes. Serum was collected from dogs in four study cohorts (diagnosed within 30 days and healthy age-matched controls; long-term cases and healthy age-matched controls) in three dog breeds with a high prevalence of Addison’s disease. These samples were evaluated for anti-adrenocortical autoantibodies using both immunofluorescence assays and Western blotting. Initial results from immunofluorescence assays on bovine adrenal sections probed with serum IgGs from the recently diagnosed cohort showed positive staining in the adrenal cortex and negative staining for the unaffected cohorts. Additionally, preliminary data from Western blots on canine adrenal homogenates probed with serum IgGs identified a band at ~42 kD unique to the recently diagnosed group from each breed. These findings have been replicated using five canine adrenal homogenates, as well as bovine and porcine homogenates. Our results suggest that dogs with a new diagnosis of Addison’s disease may have autoantibodies targeting a ~42 kD adrenal protein. Ongoing work includes testing reproducibility and identifying the specific antigenic target using 2D gels with spot cutting followed by LC-MS/MS.
Structural and functional divergence of duplicated mosquito Toll and Spz compared to Drosophila
Authors: Saucereau Yoann , Marcaida Maria Jose , Gangloff Monique 
Published: January 2023

Abstract
Mosquitoes are vectors of many infectious diseases that constitute a risk to human health around the globe. Arthropod-borne viruses (arboviruses) such as Dengue and Zika viruses, are mainly transmitted by Aedes mosquito species, while plasmodium is spread by members of the Anopheles genus. The vectorial capacity of mosquito is based on a complex balance between the mosquito’s immune control and evasion mechanisms by the pathogen. The Toll signalling pathway is a key component of insect innate immunity that underwent duplication in mosquitoes compared to Drosophila melanogaster (Dm). Here we produced duplicated Spz isoforms and ectodomains of Toll1A and Toll5A in order to study binding specificity and signalling in Aedes aegypti (Aa). Surprisingly, Toll1A, the closest structural homolog to DmToll, did not bind to DmSpz, AaSpz1B or AaSpz1C, while Toll5A specifically bound to Spz1C. In Aag2 cells, however, DmSpz triggered gambicin production upon proteolytic processing, in contrast to mosquito Spz isoforms, which lead to a distinct set of antimicrobial peptides. Computational pairing of Toll and Spz failed to predict isoform and species-specificity, probably due to conformational changes upon ligand binding as observed by SAXS analysis of Toll5A-Spz1C complex. This study sheds light on the molecular basis of Toll and Spz neofunctionalization in the mosquito.