bluuebunny/crossref_metadata_2025 huggingface dataset:
dsiRNA Inhibition of Tetraspanin-1 gene in Trichomonas vaginalis: Effect on host immune responses
Authors: Kucknoor Ashwini , Hancock Erin
Published: November 2024

Trichomonas vaginalis (TV) is a common sexually transmitted infection, that causes Trichomoniasis. Little research is done to understand the mechanisms involved in TV’s communication with host cells. TV can communicate with host cells via specialized extracellular vesicles known as exosomes. Exosomes are membrane-bound, endocytic vesicles (MVBs) that are used to traffic proteins and genetic material, to neighboring cells. The sorting of proteins in the MVBs is facilitated by a group of proteins known as tetraspanins. The tetraspanins take part in a wide range of functions. TV genome contains several tertaspanin genes. Tetraspanin-1 (TSPAN-)1 is known to be lining the MVBs when TV attaches to host cells. To understand the role of TSPAN-1 gene in eliciting host immune responses, we designed custom dsiRNA primers to inhibit the expression of TSPAN-1 in TV parasites and study its effect on TV exosome formation and changes in TLR gene expression in host epithelial cells. We successfully inhibited TSPAN-1 gene in Trichomonas. The transfected parasites when exposed to host epithelial cells, resulted in reduced number of exosomes formed. In addition, TLR 2 gene expression was downregulated, while TLR 9 was upregulated when TSPAN-1 inhibited TVs were exposed to epithelial cells. TLR 4 gene expression, however, was not altered suggesting that tetraspanin-1 in the Trichomonas play a role in host -pathogen interactions and altering the host-immune responses in TV pathogenesis.
Advancing Cryptococcal Immunity: The Indispensable Role of Batf3-cDC1 in Th1 Response Regulation
Authors: Xu Jintao , Hissong Rylan , Bareis Rachel , Creech Arianna , Goughenour Kristie , Freeman Christine , Olszewski Michal
Published: November 2024

Cryptococcus neoformans, a fungal pathogen, is responsible for severe meningoencephalitis and approximately 200,000 annual fatalities worldwide. While type I conventional dendritic cells (cDC1) are vital for generating adaptive immunity against intracellular pathogens and tumors, their role in defense against C. neoformans remains unclear. Utilizing a mouse model adept at restricting cryptococcal infection, we identified a unique transcriptional profile in the cDC1 subset, characterized by an enhanced upregulation of T-cell recruitment and activation pathways. Using Batf3-/- mice, which lack the cDC1 population, we assessed the function of these cells in antifungal immunity. The absence of Batf3-dependent cDC1 critically impairs the host's ability to mount an effective immune response against the fungal pathogen, particularly in the lung and brain. We found reduced CD4+ T cell accumulation and diminished interferon-gamma production in BATF3-/- mice, underlining cDC1's role in driving robust Th1 responses. Furthermore, Batf3-/- mice exhibited significantly weakened fungicidal activity and compromised fungal containment. This study sheds light on the vital role of a specific DC subset, Batf3-dependent cDC1, in shaping protective Th1 responses necessary for the clearance of cryptococcal infections. Our findings highlight the potential of targeting cDC1 pathways in designing interventions against C. neoformans, offering new avenues in the fight against deadly fungal pathogens.
Targeting the NS4b Antigen of West Nile Virus to the Endocytic Receptor DEC-205 to Stimulate Protective T Cell Responses to West Nile Virus Encephalitis
Authors: Herzler Joshua , Durrant Douglas , Hernandez Valeria
Published: November 2024

West Nile Virus (WNV), the primary cause of mosquito-borne neuroinvasive disease in the US, poses a significant public health threat without an approved human vaccine. DCs are pivotal in orchestrating immune responses, dictating antigen-specific effector T cell activation or tolerance based on their maturity. The endocytic receptor DEC-205, expressed by specific DC subsets, facilitates antigen uptake, processing, and cross-presentation, initiating an effective T cell response. Studies highlight DEC-205+ DCs within the CNS in reactivating antiviral effector T cell responses, restricting WNV replication, and mitigating immunopathology. We propose targeting antigens to these DCs to enhance antiviral immunity against WNV within the CNS, fusing antigens to DEC-205-specific monoclonal antibodies (mAbs). A DEC-205-targeted hybrid antibody will be developed, conjugating the WNV NS4b immunodominant peptide to a DEC-205 mAb. Subsequent purification will precede testing therapeutic efficacy in a murine WNV encephalitis model. Delivering the aDEC-205 hybrid mAb with DC maturation stimulus (poly I:C), we will assess co-stimulatory molecule expression (CD40, CD80, and CD86) on DCs and evaluate T cell activation through activation marker expression and cytokine production. This research introduces a vaccination approach against West Nile Virus encephalitis, leveraging targeted antigen delivery to DEC-205+ DCs, offering insights into potential therapeutic and preventive strategies.
ISGylation during Human Parainfluenza Virus Type 3 Infection
Authors: Miller Lindsay , Mehari Liyon , Hoffman Michael , Bose Santanu
Published: November 2024

Human parainfluenza virus type 3 (HPIV3) causes lower respiratory illnesses such as pneumonia or bronchiolitis in infants, young children, and the immunocompromised. HPIV3 associated airway inflammatory diseases manifest as a result of host mediated exaggerated inflammatory response, though limited knowledge exists regarding HPIV3-host interactions. ISGylation is a ubiquitin- like modification that plays a role in controlling various aspects of virus infections and is the process by which Interferon stimulated gene 15 (ISG15) is conjugated to target proteins by ubiquitin-like conjugating enzymes. Several respiratory viruses induce ISGylation, and this event has been shown to modulate virus-host interaction, typically leading to a restriction in viral replication. Because of its antiviral property, some viruses possess proteins which restrict ISGylation. So far, the role of ISGylation during HPIV3 infection has not been established, and as such, no HPIV3 viral protein has been identified in regulating ISGylation. We show that HPIV3 can induce ISGylation in both myeloid immune (macrophages) and non-myeloid stromal (epithelial cells) cells. Our studies also show HPIV3 encoded non-structural C protein inducing ISGylation in cells expressing the C protein. The role of ISGylation during HPIV3 infection and the mechanism by which viral C protein induces ISGylation will be discussed.
Molecular targeting of Bcl-xL during chemotherapy-induced tumor dormancy can inhibit breast cancer relapse
Authors: Koelsch Nicholas , Gupta Kinjal , Neely Victoria , Harada Hisashi , Manjili Masoud
Published: November 2024

Breast cancer (BCa) mortality is mainly due to distant recurrence of the disease associated with the establishment of tumor dormancy following initially successful treatment of early-stage breast cancer. Current clinical practice for cancer survivors is a “wait and watch” approach without any therapeutic or relapse preventing interventions, except for ER-positive BCa patients who receive hormone therapy, yet remain at risk of tumor recurrences. In attempt to identify a molecular target in dormant tumor cells, we discovered upregulation of Bcl-xL in chemotherapy-induced dormant tumor cells. To target such survival pathway in FVBN202 transgenic mouse model of neu-overexpressing breast cancer, we first established tumor dormancy by means of a low dose immunogenic chemotherapy (FAC, 5-FU + Adriamycin + Cyclophosphamide) of the neu-overexpressing mouse mammary carcinoma (MMC) while using a specific inhibitor of Bcl-xL, A-1331852. Such combination therapy in vitro resulted in the inhibition of chemotherapy-induced tumor relapse. In addition, selective knockdown of Bcl-xL in MMC by means of shRNA prevented relapse of chemotherapy-induced dormant tumor cells. Our findings suggest tumor-targeted delivery of A-1331852 during chemotherapeutic treatment of early-stage breast cancer for the prevention of distant recurrences of the disease.
B Cell Maturation Antigen Inhibits an Inflammatory Role of Testosterone in Experimental Autoimmune Encephalomyelitis in C57BL/6 mice.
Authors: Kumar Gaurav , Ko Rose , Bhatt Neal , Axtell Robert
Published: November 2024

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Sex influences both the incidence and prognosis of MS, with the disease more prevalent in women than men. Yet, men experience a more severe disease compared to women. The interplay of immune cells, hormones, and genetics may contribute to these phenomena. In experimental autoimmune encephalomyelitis (EAE) model of MS, there are no discernible effects of sex hormones in C57BL/6 mice. However, we now report an unexpected role of BCMA in controlling an inflammatory function of testosterone in C57BL/6 mice during EAE. BCMA, a cytokine receptor with established effects on B cells, was previously shown to attenuate EAE. We now show sex differences in EAE in BCMA-deficient C57BL/6 mice. Like other reports, we show no differences in the severity of EAE in male and female BCMA+/+ mice. However, the BCMA-/- males had significantly increased paralysis and CNS inflammation compared to BCMA-/- females. Remarkably, castration of BCMA-/- males significantly reduced EAE compared to sham controls, which was not observed in the BCMA+/+ males. In addition, we found that administration of testosterone pellets elevated EAE in male and female BCMA-/- mice. Previous EAE studies have shown a protective effect of testosterone in mice with genetic backgrounds other than C57BL/6. We now show a novel function for BCMA, where it inhibits a paradoxical inflammatory role for testosterone during EAE in C57BL/6 mice.
IgE Neutralization Reverses Trimellitic Anhydride-Induced Urticaria in Humanized CD34+ Mice
Authors: Yi Hao
Published: November 2024

Urticaria is a significant challenge affecting the quality of life for a substantial population worldwide, manifested by the sudden appearance of wheals, erythema, and pruritus triggered by allergic reactions, infections, or physical stimuli. Immunoglobulin E (IgE) plays a pivotal role in urticaria pathogenesis by mediating mast cell accumulation and degranulation to regulate the release of inflammatory mediators such as histamine.

We developed a humanized CD34+ mouse model of urticaria by sensitizing the depilated skin topically with Trimellitic anhydride (TMA) on four occasions, with anti-human IgE as the positive control. Four consecutive days after the final sensitization, we observed and scored animal ear conditions, and enumerated scratching frequency. Additionally, mast cell counts in the skin and ear were evaluated.

TMA-sensitized animals exhibited increased ear thickness, scoring, scratching frequency, and mast cell numbers in both skin and ear compared to normal animals. Toluidine blue staining confirmed a significant rise in mast cell numbers in TMA-sensitized mice, correlating with elevated histamine levels.

Treatment with anti-human IgE ameliorated urticaria symptoms, showcasing comparable ear thickness, mast cell numbers in the ear and skin, and scratching frequencies to the normal animals. Our findings indicate that IgE neutralization effectively reversed TMA-induced urticaria in humanized CD34+ mice.
Regulation of MHC II by OPTN, a novel regulator of MARCH1 via mTOR/AKT2/STAT3 pathway ameliorates severe outcomes during herpesvirus infection.
Authors: Kadam Rashmi , Shukla Deepak
Published: November 2024

Protective immune responses elicited by primary activation of CD4+ T cells via dendritic cells is an essential requirement to evade herpesvirus infections. Optineurin (OPTN), a selective autophagy adaptor protein, is a crucial host restriction factor for herpes simplex virus (HSV) infection. We observed a significant reduction in the basal level of MHCII expression in OPTN–/– DCs accompanied by a concomitant decrease in CD4+ T cell counts in OPTN–/– mouse model. We hypothesize that abrogation of antigen presentation via MHCII in DCs weakens adaptive immune responses, leading to severe HSV1 infection. In this study, we elucidate the mechanism of OPTN-mediated down-regulation of MHCII via AKT/mTOR/STAT3 pathway. Mechanistically, OPTN induces activation of mTORC2 sequentially upregulating AKT2 which adversely affects the activation of mTORC1 and STAT3 protein. This could be attributed to the dynamic interplay between AKT1 and AKT2. Our findings provide novel insights into how the absence of OPTN downregulates AKT2 and triggers STAT3 activation. Furthermore, aberrant activation of STAT3 influences transcriptional activity of MARCH1(an E3 ubiquitin ligase) via IL10. MARCH1 mediates ubiquitination of MHCII resulting in the reduction of MHCII expression in OPTN–/– and AKT2–/– DCs. This study represents the first comprehensive delineation of the signaling pathway involved in the regulation of MHCII, shedding light on the intricate interplay between OPTN, AKT, mTOR, STAT3, and MARCH1.
VelociT mice genetically humanized for the T cell receptor, MHCI/II and CD4/CD8 serve as a promising platform for studying HLA-restricted T cell responses in multiple inflammatory settings.
Authors: Tay Amanda , Herbin Olivier , Farrales Pamela , Moore Michael , Macdonald Lynn , Sleeman Matthew , Orengo Jamie
Published: November 2024

Mouse models of T cell-mediated immune diseases are routinely used as preclinical models for investigation of human diseases. However, it is challenging to recapitulate HLA-restricted human T cell immune responses in mice. We previously developed VelociT mice by replacing the mouse genes in the variable regions of TCRab, along with the extracellular domains of the coreceptors CD4 CD8 with corresponding human sequences, and swapped the ectodomains of mouse major histocompatibility complex class I and II proteins for the human sequences of HLA-A2 and HLA-DR2, respectively. Here, we aimed to study the development of T-cell mediated responses in VelociT mice including CD4 T cell mediated autoimmune disease, CD8 T cell mediated target killing, and allogeneic skin graft rejection. We demonstrated that VelociT mice immunized with MOG35-55 efficiently mounted an antigen-specific CD4 T-cell response and developed experimental autoimmune encephalomyelitis. We showed that immunization of VelociT mice with an HLA-A2 restricted cancer-derived peptide induced cytotoxic CD8 T cells capable of lysing peptide-pulsed target cells in an antigen-specific manner. Finally, we successfully established an allogeneic skin graft mouse model by showing that VelociT recipient mice rejected Balb/c donor skin grafts. This work established VelociT mice as a platform for investigation of T cell–mediated immunity in different disease settings including autoimmunity, antitumor responses, and allo-immunity.
Unexpected expression and function of Fc epsilon RI in breast cancer cells.
Authors: Pullen Nicholas , Ashbaugh Alexandria , Haughian James
Published: November 2024

FcεRI is typically associated with type 2 effectors such as mast cells (MC). The unique expression profile of FcεRI and evidence from pre-clinical and clinical settings have led us to study MC as potential therapeutic targets in breast cancer. Our work with the 4T1 model in vivo identified MC interacting with tumor cells, but this was complicated by a surprising finding that tumor cells intrinsically expressed FcεRI. Here we further study the expression and function of FcεRI in breast cancer cells in vitro. 4T1 cells express FcεRI similar to mouse BMMC. Two established cultures derived from human T-47D cells, estrogen dependent (E3) and estrogen withdrawn (EWD8), also express FcεRI: EWD8 cells are strong FcεRI expressers while E3 are modest, compared to LAD2 cells, a human MC line. Expression (mRNA) of all FcεRI subunits (α, β, γ) was confirmed. IgE-mediated antigen stimulation does not elicit classic Ca2+ flux in breast cancer cells as seen in respective species MC; however, it can stimulate IL-6 production from them. Preliminary analysis of primary breast cancer datasets using R2 is ongoing and discordant with our in vitro work: FcεRI mRNA abundance declines in metastatic cancers compared to non-cancerous breast tissue. We report a previously unidentified, immunologically substantive difference between breast cancer models and human primary tumors. Investigators pursuing FcεRI-relevant therapeutics in this context should be aware of this potential translational barrier.
ST8Sia6 sialylates CD44 in cancer cells to generate ligands for inhibitory Siglecs
Authors: Chen Yin Maggie , Friedman David , Saraswat Mayank , Chen Alex Shuaibo , Shapiro Michael , Wilfahrt Drew , Pandey Akhilesh , Smith Shapiro Virginia
Published: November 2024

Glycans decorate most proteins and lipids on the cell surface, and glycoproteins serve a critical role in cell-cell communication. The terminal modification of many glycans is a sialic acid. There are twenty mammalian sialyltransferases, each with a unique specificity. Recently, we demonstrated that the sialyltransferase ST8Sia6, which adds an alpha-2,8 linked sialic acid, generates ligands for the inhibitory receptor Siglec-E. ST8Sia6 overexpression accelerates tumor growth though inhibition of the immune response through Siglec-E. To understand the basis for ST8Sia6 function in tumors, proteomic analysis of murine tumor lines was performed to identify ST8Sia6 targets. Here, we demonstrate that ST8Sia6 sialylates CD44. Siglecs vary significantly across species, with the closest ortholog to murine Siglec-E is Siglec-7 in humans. We also show that ST8Sia6 sialylates CD44, generating ligands for Siglec-7. Thus, ST8Sia6 has a conserved role in both mice and humans to generate ligands for Siglecs. We also found that sialyltransferase ST6GalNac1 which adds an alpha-2,6 sialic acid to CD44, generating ligands for Siglec-7. The TCGA database showed synergistic effect of CD44 and ST8Sia6 decrease patient survival, while CD44 and ST6GalNac1 increases patient survival. Thus, CD44 is a target for ST8Sia6 and ST6GalNac1that generate ligand for inhibitory Siglecs, but the immunomodulatory effect may differ between two types of modifications, which is under current investigation.
Determining the Effects of Obesity and Type 2 Diabetes on CCR6+ Epidermal GD T Cell Number and Function in Inflammatory Disease and Wound Healing
Authors: Engel Emma , Alvizo Cristian , Castellanos Tanya , Bshara-Corson Savannah , Kasler Antolette , Lawler William , Jameson Julie
Published: November 2024

A maladaptive healing cascade causes chronic wounds. γδ T cells are affected, the dysregulation of which causes a reduction of cytokines and growth hormones. We are studying the Chemokine Receptor 6 (CCR6)/Chemokine Ligand 20 (CCL20) axis and how it functions in the wounds of obese and diabetic mice. When keratinocytes are stressed by wounding or psoriasis, CCL20 is released and interacts with CCR6 on Dermal T cells to recruit them to the epidermis. Epidermal T cells and their role in this axis are not as well understood. Upon wounding, it was found that a subset of epidermal γδ T cells express CCR6 within 24 hours, and it is downregulated by 72 hours. The role of the CCR6 epidermal γδ T cells express was also investigated. Epidermal γδ T cells, when co stimulated with CCL20 and anti-CD3, generate IL-17A, suggesting that CCR6 plays a role in TH17 responses during inflammation. Animals and humans with obesity and diabetes both exhibit elevated IL-17, so obese and diabetic mice were examined during wound healing and psoriasis.

During keratinocyte stress, epidermal gd T cells experienced significantly elevated levels of CCR6 and IL-17 in obese and diabetic mice. This shows that in chronic diseases like type 2 diabetes, resident T cell IL-17 responses are amplified. In better understanding the mechanisms used by resident T cells during their inflammatory responses to keratinocyte stress, a targeted therapy can be developed to help treat chronic wounds and psoriasis.
Unexpected modulation of T cell activity by mechanical cues
Authors: Dada Hannah , Do Kun , Cartagena-Rivera Alexander , Altan-Bonnet Grégoire
Published: November 2024

Current immunotherapies take advantage of the biochemical features of T cell activation and function, such as strengthening the antigenic responses of T cells. However, the biophysical properties and the physical environment of T cells are also relevant. Using polyacrylamide (PAA) gels to vary the mechanical landscape, we show that the mechanical environment of T cells dictates T cell capacity. Mechanical properties of PAA gels include viscoelasticity, porosity, and stiffness. Using a robotic platform that enables the automatic collection of cytokines from T cells co-cultured with tumors of varying antigenic strengths, we show that not only does antigenic quality influence T cell function, but altering the crosslinking of the PAA gel, and thus the physical environment, results in varied T cell response. Low crosslinking impairs T cell function, while increasing crosslinking gradually improves response. By using other hydrogels, we ruled out stiffness as a mediator of T cell response. Rheology and atomic force microscopy (AFM) will help uncover the physical characteristics that dictate response. Modeling these properties is essential in identifying the mechanical landscape responsible for immune response and predicting this response based on the tumor physical environment. These findings will introduce a novel approach to treating cancer by assessing the mechanical properties of the tumor and changing the physical environment to one that allows for a favorable immune response.
SLAMF7+ CD8+ T cells exhibit decreased anti-tumor responses in renal cell carcinoma
Authors: Wirth Lena , Hugaboom Miya , Street Kelly , Ruthen Neil , Jegede Opeyemi , Schindler Nicholas , McDermott David , Plimack Elizabeth , Sosman Jeffrey , Haas Naomi , Hurwitz Michael , Hammers Hans , Signoretti Sabina , Atkins Michael , Wu Catherine , Braun David
Published: November 2024

Despite the success of immune-checkpoint inhibitors (ICI) for the treatment of renal cell carcinoma (RCC), many patients fail to receive durable clinical benefits. Therefore, an understanding of resistance to ICIs in RCC is critical for the treatment of this disease. Through single-cell transcriptomic analysis of patient T cells in the HCRN GU16-260 trial, we previously identified a population of CD8+ T cells expressing the SLAMF7 immune receptor that was associated with resistance to nivolumab monotherapy. Here, we functionally interrogated the impact of SLAMF7 overexpression on CD8+ T cell effector functions and found a reduction of pro-inflammatory cytokine and Granzyme B (GzmB) production. CD3+ T cells from healthy human PBMCs were isolated, transduced with either a SLAMF7-GFP or control GFP lentiviral construct, sorted for GFP expression, and subsequently incubated with a SLAMF7 ligand or control. Cytokine and GzmB production were measured using intracellular staining by flow cytometry. SLAMF7-GFP+ CD8+ T cells incubated with a SLAMF7 ligand for 8 hours exhibited a 41.1% reduction in IFN-γ (p=0.0129), 38.3% reduction in IL-2 (p=0.0483), and a 43.8% reduction in GzmB (p=0.0044) production compared to control GFP+ CD8+ T cells. Future work will interrogate the impact of SLAMF7 expression on CD8+ T cell effector functions using patient RCC tumor-infiltrating lymphocytes and evaluate the efficacy of a therapeutic SLAMF7 blocking antibody in restoring anti-tumor functions.
Spatio-temporal Phenotyping of Murine Embryos with Ultrahigh-plex Antibody Panels for Characterization of Early Development
Authors: Tam Tsz , Cheikh Bassem , Nikulina Nadezhda , Klymyshyn Dmytro , Ma Ning , Jhaveri Niyati
Published: November 2024

The murine embryo model is one of the most common animal models for studying developmental biology. Embryonic development is a highly regulated process with spatial and temporal expression of proteins for regional specialization and tissue-specific functions. Application of spatial proteomics to developing embryos enables us to understand cellular organization and interactions within tissues along with regulatory mechanisms at the single-cell level. In this study, we applied a 36-plex panel, specifically designed for analysis of mouse embryos at different stages of development using the PhenoCycler™-Fusion (PCF) 2.0 Platform. This panel was developed by selecting biomarkers for immune cell lineages, immune activation, proliferation, structural and neuronal components. Purified antibodies were tagged to PhenoCycler barcodes and verified for specificity and reproducibility. After successful validation, the panel was deployed on the FFPE mouse embryos and ultrahigh-plex whole-slide imaging was performed using the PCF 2.0 platform. Unsupervised clustering analysis and deep phenotyping were performed to generate a high-resolution spatial atlas of mouse embryos at different stages of development. The tools developed as part of this study can be used to conduct in-depth research on various aspects of organogenesis and to study the spatio-temporal dynamics of cellular and molecular expression in developing embryos.
Alternative cell counting and viability detection using NexGreen/PI fluorescent stain on multiple low- and high-throughput image cytometry platforms
Authors: Nitta Carolina , Pierce Mackenzie , Hem Sopaul , Browe Daniel , Kuksin Dmitry , Lin Bo
Published: November 2024

In the past decade, the increase in cell and gene therapy products such as biologics, antibodies, and CAR-T has significantly increased the need for precise, robust, and consistent cell counting and viability measurements. In this work, we demonstrate a novel fluorescence-based dye, NexGreen/PI, capable of live and dead cell detection comparable to AO/PI in multiple cell buffer/media conditions. Jurkat cells of different viabilities (low, intermediate, and high) were stained in parallel with either NexGreen/PI or AO/PI for 1-2 minutes and measured on multiple image cytometer platforms including low-throughput and high-throughput instruments. Both NexGreen/PI and AO/PI dyes are used as equal volumes added to cell samples. Additionally, we tested if difficult to analyze cells in “spent” media (typically with a lower pH) or in PBS can be stained using NexGreen/PI compared to AO/PI. Results on the high-throughput platforms show that NexGreen/PI staining can discriminate between live and dead Jurkats, analogous to AO/PI. Measured viability and cell concentration of all samples using NexGreen/PI was within 5% of the paired AO/PI-stained samples. Low-throughput instrument results are also similar. Our experiments demonstrate that NexGreen/PI is scalable and can be of significant value for use in automation and high-throughput systems where multiple samples in different mediums can be precisely measured.
Generation of targeted antibody conjugates using Sortase and the AbFlex technology platform.
Authors: Jelinek Mary Anne , Tchoub Mike , Park Hyun Min , Truong Vi , Egan Brian , Weber Julie , Huang Chengyang , Bergseid Jacqueline
Published: November 2024

The miniaturization of experiments to the single cell scale and a growing interest in multiplexing analyte detection is a major driver for the development of novel antibody conjugation technologies that are robust, and which also preserve antibody function. Relative to two step detection modalities, direct primary antibody conjugates provide enhanced specificity and selectivity. We have developed the AbFlex platform, a versatile epitope tagging system for the carboxyl terminus of recombinant mouse and rabbit immunoglobulin heavy chains. It consists of a tandem array of three tags: 1) The Sortase tag recognition sequence (LPXTG); 2) The motif recognized by the BirA biotin ligase, followed by 3) the 6XHis Tag, which can be used with nickel-based purification systems.

Sortase A belongs to a family of transpeptidases found in Gram-positive bacteria that catalyze the attachment of poly-Glycine labels to the Sortase recognition sequence under physiological conditions and with reaction times of approximately one hour. We describe the use of the Staphylococcus aureus Sortase A5 pentamutant that is significantly more active than wild-type Sortase to provide a faster, more efficient labeling reaction. Performance of various recombinant mouse and rabbit antibody conjugates in a variety of applications will be presented.
Characterizing the health of transduced CAR-T cells using high-throughput image cytometry
Authors: Huang Yongyang , Guo Ning , Patel Samir , Pierce Mackenzie , Chan Leo , Lin Bo , Yang Yi
Published: November 2024

Chimeric antigen receptor (CAR)-T cell therapy is one of the novel cellular therapeutic options for cancer patients. In this work, we developed a rapid cell viability and apoptosis detection method to characterize the cell health and recovery of SubT1 cells after the plasmid-based electroporation process to create CAR expression cells using the Cellaca™ PLX Image Cytometer. Viability measurement of transduced SupT1 cells was performed by staining with acridine orange/propidium iodide (AO/PI). In addition, early and late-stage apoptosis were detected by staining transduced SupT1 cells with Annexin V/PI and Caspase3/RubyDead, respectively. Results showed that the introduction of plasmids, other than the electroporation process itself, may induce 20 – 30% additional cell death in the process, indicated by viability results measured on Day 1 post transduction. Among all the Annexin V or Caspase3 positive transduced SupT1 cells on Day 1 (22 – 34%), 70 - 80% of these cells were double positive in both Annexin V and PI, or both Caspase3 and RubyDead, and only 20 – 30% of them were in the early-stage/late-stage apoptosis. Subsequently, the SupT1 cells were quickly recovered with their viabilities increased to >90% on Day 3. With such capability of rapid detection of cell viability and apoptosis, Cellaca™ PLX Image Cytometer can potentially support the characterization of factors that affects the quality of CAR-T cells in the manufacturing process.
LAG3 regulates antibody responses following kidney transplantation
Authors: Nicosia Michael , Fan Ran , Lee Juyeun , Gorbacheva Victoria , Valenzuela Jose , Yamamoto Yosuke , Beavers Ashley , Dvorina Nina , Chuluyan Eduardo , Araki Motoo , Baldwin III William , Fairchild Robert , Min Booki , Valujskikh Anna
Published: November 2024

Lymphocyte activation gene-3 (LAG3) is a coinhibitory receptor expressed by a range of immune cells. The immunomodulatory potential of LAG3 is being explored in cancer and autoimmunity fields but not in transplantation. Using our mouse kidney transplant models we sought to address the role of LAG3 in graft rejection.

Untransplanted LAG3-/- mice have elevated heterologous immunity against a panel of alloantigens. Recipient LAG3 deficiency results in rapid rejection of MHC-mismatched renal allografts that are spontaneously accepted by WT recipients, with graft histology characteristic of antibody mediated rejection (ABMR). Antagonistic LAG3 antibody treatment of WT recipients enhanced anti-donor immune responses and induced kidney damage typical of chronic rejection, indicating that LAG3 regulates de novo alloresponses. Recipient B cell depletion, but not CD8+ T cells, prevented kidney allograft rejection in LAG3-/- recipients further supporting ABMR as the mechanism of graft loss. Posttransplant, follicular T cells and plasma cells are among LAG3 expressing immune cells. To identify the mechanism we used T or B cell conditional LAG3 knockout recipients, neither reject the allograft but both had elevated levels of donor specific antibodies (DSA), indicating LAG3 expression on either T or B cells is sufficient to regulate anti-donor humoral immunity. Our results identify LAG3 as a regulator of DSA following kidney transplantation and a potential therapeutic target for ABMR.
Reversing pregnancy induced humoral sensitization to facilitate transplantation tolerance
Authors: Durgam Samarth , Wang Qiang , Yin Dengping , Alegre Maria Luisa , Chong Anita
Published: November 2024

Purpose: Pregnancy induces split tolerance with tolerization of fetus-specific effector T cells, but sensitization of B cells contributing to sex disparity in access to transplantation for multiparous women. We previously showed that, following mating with Balb/c males, post-partum sIgKO mice (mIgM-/-AID-/-) that cannot produce secreted antibodies were tolerized by aCD154 and accepted F1 heart grafts (TolRx); this was not achieved with WT mice. Here, we tested, 1) if B cells from these sIgKO mice acquired cell intrinsic hypofunction? 2) if post-partum serum inhibited the acquisition and reversed intrinsic hypofunction in post-partum B cells? Methods: Post-partum sIgKO mice were subjected to TolRx ± transfused post-partum serum. After 30 days, B cells were transferred to MD4 hosts and challenged with F1 splenocytes ± post-partum serum. F1-specific germinal center B cells were quantified on D-14. Results: 1) Post-partum B cells subject to TolRx did not differentiate into germinal center B cells in MD4 mice, in contrast to non -tolerized post-partum B cells that did. 2) Post-partum serum transfused during TolRx or into MD4 mice inhibited the induction and reversed hypofunction in post-partum B cells. Conclusion: Pregnancy-sensitized B cells develop a state of intrinsic hypofunction when tolerance was induced with aCD154 to F1 HTx but was antagonized by fetus-specific antibodies.
Toddlers have limited mucosal protection against Live Attenuated Influenza Virus in human tonsil organoids.
Authors: Sun Meng , Sola Elsa , Guo Jing , Shankar Vishnu , Kamalyan Lilit , Gupta Neha , Capasso Robson , Davis Mark
Published: November 2024

Children require separate clinical trials to vaccination and other treatments, because we know little about how their immune systems differ from adults. Notably, we developed a high-resolution in-vitro model of human immunity using tonsil organoids. These organoids have demonstrated a remarkable ability to generate specific antibody and T-cell responses to vaccines across a broad age spectrum (2-70 years old). This provides a unique opportunity to investigate the variations to vaccination between children and adults. To study the systemic cellular response to live attenuated influenza virus in human tonsil organoids, we used scRNA-seq via the BD Rhapsody platform, and validated by flow cytometry, ELISA and Luminex assays. Our data showed marked differences between toddlers (2-4 years) and adults (19-39 years). Compared to adults, toddlers exhibited lower levels of flu-specific IgA, IgG antibodies and activated cytotoxic CD8 T cells. Additionally, toddlers displayed a less comprehensive cytokines that help promote class-switched antibody production. Our data also indicated a metabolic transition within germinal centers from oxidative phosphorylation to glycolysis, as individuals progress from early childhood to adulthood. Our study can offer valuable insights to aid vaccine design and administration practices. It may also identify vulnerabilities in children's immune systems with implications for improving therapeutic treatments for infectious diseases.
Monoclonal antibody-mediated control of lyssavirus infection in the CNS requires Fc effector function
Authors: Huaman Celeste , Mastraccio Kate , Clouse Caitlyn , Rader Maddie , Swafford Isabella , Yan Lianying , Smith Ina , Markotter Wanda , Broder Christopher , Schaefer Brian
Published: November 2024

Lyssaviruses are the causative agent for rabies, a disease that is uniformly fatal in humans. Once infection reaches the central nervous system (CNS), currently approved therapies are ineffective. Using luminescence-based longitudinal tracing of lyssavirus infection in a mouse model of lethal rabies disease, we have shown that peripheral administration of a single dose of human monoclonal antibody (mAb) F11 protects animals from lethality, even when virus is already robustly replicating in the CNS. Additionally, behavioral analyses demonstrate improved motor function of the infected mAb-treated animals compared to infected, untreated controls. Interestingly, A6, another huIgG1 mAb that targets the same lyssavirus G epitope and exhibits similar in vitro neutralization efficiency, is inferior to F11 when used for in vivo therapy. Importantly, functional inactivation of the F11 Fc domain significantly impairs protection against mortality. Thus, Fc effector function is a major determinant of mAb in vivo efficacy. Histology and flow cytometry analysis shows that mAb therapy leads to a shift in leukocyte populations that infiltrate the brain, with significant increases in MHCII+ myeloid cells and CD4+ T cells, consistent with our demonstration that CD4+ T cells are essential for mAb efficacy. Collectively, these data suggest that Fc-mediated effector functions in the periphery yield a potent antiviral immune response in the CNS.
Fractionation of mRNA vaccines improves immune responses
Authors: Sanchez Sarah , Awakoaiye Bakare , Dangi Tanushree , Irani Nahid , Penaloza-MacMaster Pablo
Published: November 2024

mRNA vaccines are used to prevent SARS-CoV-2 infection and are being explored for other infectious diseases as well as cancer. While they have shown high efficacy at preventing COVID-19, waning immunity has motivated the development of improved mRNA vaccine formulations. Vaccine fractionation has been explored previously in the context of viral vector and protein immunogens, but its effect on mRNA vaccines is still unclear. We interrogated whether fractionating an mRNA-SARS-CoV-2 spike vaccine over three days, as opposed to a single dose, would enhance immune responses in C57BL/6 mice. We compared the immune responses induced by three consecutive 1 μg doses over three days (fractionated vaccine) with a single 3 μg dose. Despite the equivalent overall dosage, the fractionated vaccine demonstrated superior CD8 T cell responses. This pattern of enhanced immune responses with the fractionated vaccine regimen was also observed with an mRNA-HIV vaccine, suggesting generalizability. Taken together, these findings suggest that prolonging the duration of antigen expression through mRNA vaccination could enhance immune responses, supporting the rationale for developing slow-release formulations for mRNA vaccines.
Sex differences in protection conferred by heterologous vaccines for pneumonic plague in an aerosol challenge model
Authors: Davies Michael , Biryukov Sergei , Rill Nathaniel , Klimko Christopher , Dankmeyer Jennifer , Hunter Melissa , Miller Jeremy , Shoe Jennifer , Talyansky Yuli , Qiu Ju , Bozue Joel , Cote Christopher
Published: November 2024

Pneumonic plague, caused by the bacterium Yersinia pestis, is a severe and rapidly progressing illness transmitted via aerosol. There are no FDA-approved vaccines, and candidate vaccines may be less effective against pneumonic than bubonic plague. Y. pestis is not known to impact males and females differently in mechanism of pathogenesis or severity. However, a recent study reported sex-biased vaccine effectiveness after intranasal challenge. In this study we observed that when male mice were given a vaccine that is protective in females challenged with aerosolized virulent Y. pestis C12, males were not protected. We then gave male and female BALB/c mice a recently developed heterologous vaccine strategy and monitored survival, bacterial burden (CFU), and immunological factors before and after aerosol challenge. This strategy consisted of two subcutaneous injections: one of Y. pestis live attenuated vaccine (LAV) strain pgm-pPst-Δcaf1 or pgm-pPst-ΔyopD/Δcaf1 and one of rF1-V protein subunit with adjuvants CpG and Alhydrogel. When primed with rF1-V followed by pgm-pPst-Δcaf1 LAV boost, 100% of females and 50% of males survived challenge. This sex difference was observed to a lesser extent with three other vaccines. Males had higher CFU titers and differing patterns of serum antibodies, lung cytokines, and splenic T cells. These data provide new knowledge about sex differences in vaccine efficacy and immunological factors that differ between males and females.
A systems vaccinology resource to develop and test computational models of immunity
Authors: Shinde Pramod , Willemsen Lisa , Orfield Shelby , Kojima Mari , Reyna Joaquin , Ha Brendan , Greenbaum Jason , Overton James , Nili Somayeh , Rasmussen Mikkel , Gygi Jeremy , Antunes Ricardo da Silva , Sette Alessandro , Olsen Lars Ronn , Grant Barry , Konstorum Anna , Guan Leying
Published: November 2024

Computational models that predict an individual's response to a vaccine offer the potential for mechanistic insights and personalized vaccination strategies. These models often stem from small cohort studies focusing on single vaccines limiting their generalizability. The ability to assess the performance of resulting models would be improved by comparing their performance on independent datasets. We established a prototype platform that evaluates Computational Models of Immunity to Pertussis Booster vaccinations (CMI-PB). It aims to generate experimental data specifically for model assessment through annual data releases and contests. In a preliminary 'dry run', over 30 existing computational models were tested to predict immune responses from pre-vaccination multi-omic profiles with only one successful model based on age. The performance of new models built using CMI-PB training data was much better but varied significantly based on the choice of pre-vaccination features used and the model-building strategy. This suggests that previously published models developed for other vaccines do not generalize well to Pertussis Booster vaccination. Overall, these results reinforced the need for comparative analysis across models and datasets, which CMI-PB aims to achieve. We seek wider community engagement for our first public prediction contest, which will open in mid 2024.
Refinement and Characterization of Feature-Based Vision Algorithm for Ship-Deck Landing under Degraded Visibility
Authors: Britcher Victoria , Datta Anubhav , Chopra Inderjit
Published: November 2024

The objective of this work is to refine and experimentally characterize a two-dimensional, feature-based vision algorithm for tracking a stochastically moving ship-deck under degraded visual conditions. A 2.75-kg quadrotor UAV (unmanned aerial vehicle), with only the accessories essential
for vision-based navigation, is specifically designed to establish the minimal system requirements, fabricated, and tested in-house. The algorithm was integrated into this quadrotor and was performance tested in controlled, hand-held tests as well as piloted free-flight hover. All results
were validated against Vicon ground-truth data. In the controlled tests, the algorithm was first used to estimate the motion of a Stewart platform simulating a stochastically moving ship-deck. Next, tests were conducted under visually degraded conditions, specifically glare, low illumination,
and occlusion of the landing pad. Free-flight tests were conducted with the quadrotor hovering above the landing pad at varying levels of illumination and occlusion as well as with ship-deck motion. The algorithm could accurately estimate the pose of a ship-deck undergoing Sea-state 6 motions
in visually degraded conditions in both hand-held and free-flight tests. Performance was observed to reduce slightly in free-flight compared to hand-held tests due to aircraft motion and vibration.
Triage nurse staffing: a study on the need for model improvements
Authors: Zaboli Arian , Brigo Francesco , Sibilio Serena , Brigiari Gloria , Massar Magdalena , Magnarelli Gabriele , Turcato Gianni
Published: November 2024

This study addresses the challenge of determining appropriate nurse staffing levels for triage in Emergency Departments (EDs). It evaluates whether existing nurse staffing models, typically designed for other hospital settings, can be effectively applied to triage. The research was conducted at Merano Hospital’s ED from January 1, 2021, to November 30, 2022, and involved an analysis of data from patient triage assessments. The findings indicate that standard ward staffing models are not easily adaptable to the triage environment, underscoring the need for a more systematic approach to calculating triage nurse staffing. The current Italian ministerial guidelines do not account for the unique demands and complexities of the triage setting.
Describing Zoom exhaustion and fatigue in nursing students at a university in South Africa
Authors: Emmamally Waheedha , Wentzel Dorien , Brysiewicz Petra
Published: November 2024

Background: Online platforms have gained considerable support from students and lecturers post COVID-19, however they are associated with student exhaustion and fatigue.Aim: To determine Zoom exhaustion and fatigue in nursing students at a selected university in South Africa.Methods: A quantitative cross - sectional study was conducted in KwaZulu-Natal to collect data from 146 nursing students registered at the selected university for the 4-year Bachelor of Nursing programme. The Zoom Exhaustion and Fatigue Scale collected data on responses to domains of general fatigue, visual fatigue, social fatigue, motivational fatigue, and emotional fatigue. The data were analysed using the International Business Machines, Statistical Package for Social Sciences, version 26.0. Descriptive statistics were calculated for demographics, a total score was calculated and mean scores and 95% confidence intervals for the different domains were calculated. Mann Whitney U and Kruskal Wallis Independent Tests were calculated to determine associations between demographics and Zoom usage.Results: The overall Zoom exhaustion and fatigue score was 46.71 (s.d. = 10.50). The motivational fatigue construct had the highest mean score of 3.29 (s.d. = 0.83), followed by general (3.18 s.d. = 0.81), social (3.11, s.d. = 0.88), emotional (3.06, s.d. = 0.99) and visual fatigue (2.92, s.d. = 0.94).Conclusion: The study concluded that while the overall mean score indicated moderate levels of Zoom exhaustion and fatigue among respondents, majority of the respondents scored high levels ( 48) of Zoom fatigue. Students reported higher levels of motivational fatigue compared to the other 4 constructs.
Bilateral Corneal Endotheliitis Following Corneal Cross-linking with Riboflavin and Ultraviolet A
Authors: Al-othman Ahmed Y. , Algamdi Saleh S.
Published: November 2024

This case report aims to document a case of bilateral endotheliitis occurring shortly after riboflavin-assisted cross-linking (CXL) in a young male patient with progressive keratoconus. The objective is to identify potential risk factors, treatment strategies, and outcomes while considering relevant literature on similar cases. A male in his late adolescence with bilateral progressive keratoconus underwent bilateral CXL using riboflavin and ultraviolet A light exposure. Postoperatively, the patient received moxifloxacin drops, prednisolone acetate drops, and artificial tears. Seven days later, the patient presented with decreased vision, photophobia, and corneal findings consistent with endotheliitis. Treatment with prednisolone acetate and moxifloxacin eye drops was initiated. Two months later, visual acuity deteriorated, but anterior slit-lamp findings improved, and the intraocular pressure measurement was high. Prednisolone acetate was tapered, whereas loteprednol etabonate gel, brimonidine eye drops, and cyclosporine eye drops were initiated. After 7 months, visual acuity improved, and endotheliitis resolved, although mild residual central haze persisted. This case highlights the occurrence of acute idiopathic endotheliitis following routine CXL and its successful management. Although limited cases have been reported, a standardized treatment protocol is lacking. In our patient, the utilization of loteprednol etabonate gel, cyclosporine eye drops, brimonidine eye drops, and regular follow-up examinations led to improved clinical findings and visual acuity. Further studies are warranted to establish optimal treatment approaches for similar cases of endotheliitis following CXL.