bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chronic Litomosoides sigmodontis infection protects against IgE-mediated anaphylaxis in sensitized mice
Authors: Kropp Laura E. , Mitre Edward E.S. 
Published: January 2023

Abstract
While numerous animal studies have reported that helminths can prevent allergic sensitization, few have evaluated the effects of infection on pre-existing allergy. We hypothesized that chronic infection would protect against the clinical symptoms of IgE-mediated anaphylaxis in previously sensitized mice. Mice were sensitized by weekly intraperitoneal (IP) injection of either ovalbumin (OVA)/alum or PBS/alum for 3 weeks. At 5 weeks, mice were infected with Litomosoides sigmodontis (L.s.), a rodent filarial parasite, or mock infected. Mice were challenged IP with OVA during the twelfth week of infection. Following challenge, serum levels of murine mast cell protease 1 (mMCP-1) were significantly lower in sensitized mice that were L.s.-infected as compared to mock infected (212,923 vs. 447,062 pg/mL, p-value= 0.0459). Additionally, L.s.-infected mice that had been previously sensitized exhibited an average drop in core body temperature of 3.5 °C after 30 min, which was significantly less than the 5.0 °C drop observed in sensitized and mock-infected mice. Anaphylaxis was IgE and mast-cell dependent, as mice deficient in FcɛRI or mast cells mice released little to no mMCP-1 or exhibited minimal drops in core body temperature in response to challenge. Experiments to date suggest that changes in mast cell functionality were central to the protective effect. First, serum levels of OVA-IgE were not altered by L. sigmodontis infection. Second, purified mast cells from OVA-sensitized and infected mice released less histamine than OVA-sensitized and mock-infected mice in response to OVA. These results suggest that chronic helminth infection protects against anaphylaxis in part by decreasing allergen-driven mast cell degranulation.
AddaVax vs. Freund’s adjuvant use for animal immunization in hybridoma development
Authors: Hawksworth David J 
Published: January 2023

Abstract
Use of a potent adjuvant is essential for stimulating a strong, antigen specific response from immunized animals. While Freund’s continues as the gold standard for use in research animals we continuously evaluate available adjuvants as part of our improvement efforts in developing mouse and rabbit hybridomas. We recently evaluated AddaVax adjuvant for use in developing a high titer, high affinity immune response. Animals were immunized with peptides or recombinant proteins emulsified with Adjulite Freund’s adjuvant or AddaVax adjuvant. Sera samples were collected following the final immunization and the antibody evaluated for titer and relative affinity. Results show that the average serum antibody titer and relative affinity for AddaVax immunized mice were equivalent to or better than that from Freund’s mice for 3/7 peptides and 5/7 recombinant proteins. Data from two proteins also suggest AddaVax animals may show binding to antigenic epitopes not recognized by Freund’s animals. Disappointingly, rabbits immunized using AddaVax adjuvant consistently developed a much weaker response vs. Freund’s. Based on results generated, we have modified our protocols to include the use of Freund’s and AddaVax when initiating new hybridoma development projects in mice.
Structural characterization of disulfide-stabilized peptide/H2-Dd complexes
Authors: Allen Frederick M , Jiang Jiansheng , May Nathan A , Boyd Lisa F , Natarajan Kannan , Margulies David H 
Published: January 2023

Abstract
MHC class I (MHC-I) molecules fold and bind high-affinity peptides with the help of chaperones in the endoplasmic reticulum before being transported to the cell surface as stable antigen-presenting proteins. In order to understand this process, it may be necessary to artificially stabilize MHC-I molecules to behave manageably in vitro but still retain pre peptide-loaded characteristics. Others have approached this by engineering a disulfide bond in H2-Kb between residues 84 and 139, resulting in a “stitched” peptide binding grove that bypassed cellular checkpoints for misfolded proteins. We have crystallized H2-Ddwith the same Y84C/A139C mutations, as well as with W51C/G175C mutations that stitch the opposite end of the binding groove, and with all four mutations, all with the high-affinity peptide P18-I10. We determined the crystal structure of each peptide-MHC complex at 2.3, 1.7, and 1.65 Å resolution respectively. The electron density shows disulfide linkages at each expected site, and despite the mutations, the α1α2 platform domains all align to WT with an RMSD under 0.25 Å. Further efforts to measure thermal stability of each construct with high- and low-affinity peptides as well as attempts to crystallize each construct with low affinity peptides are underway. This work should contribute to a structural and mechanistic understanding of MHC-I peptide loading.
HLA-F and MHC open conformers cooperate in antigen presentation during the inflammatory response.
Authors: Geraghty Daniel , Wang Kevin L , Lee Ni , Burian Aura , Goodridge Jodie P , Ishitani Akiko , McNevin John , McElrath Margaret J 
Published: January 2023

Abstract
HLA-F is expressed as a protein independent of bound peptide or β2-microglobulin and surface expression is upregulated upon activation in dendritic cells, monocytes and most lymphocyte subsets. MHC class I (MHCI) is also expressed on proliferating lymphoid cells as open conformers (OCs), in addition to the ubiquitously expressed complexed form. Previous studies showed that HLA-F binds most MHCI proteins as open conformers without peptide but not as peptide bound complex. These studies were extended to show both HLA-F and MHCI OC are ligands for a subset of killer Ig-like receptors (KIRs), defining a new paradigm for MHCI function in the innate and acquired immune responses. The HLA-F/MHCI physical interaction was further implicated in the function of HLA-F and MHCI OCs in a general mode of exogenous MHCI antigen uptake and antigen presentation by activated immune cells that differs from the canonical MHCI endogenous antigen presentation. We are currently testing the hypothesis that antigen entry is governed by a synergy between specific structural characteristics of the exogenous antigen and the MHC-I allele types of target cells. New evidence is presented using HIV-1 p24 gag derived long polypeptides suggesting a requirement for HLA class I peptide epitope specificity in antigen uptake, upstream of antigen presentation, which subsequently can be presented by either or both of MHCI and MHCII. These experiments suggested that the physical proximity of HIV-1 class I and class II peptide epitopes within a p24 polypeptide can influence antigen presentation of epitopes through either MHCI or MHCII or both. A goal is to manipulate these features in designing effective immunogens for directed stimulation of antigen-specific host responses.
A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation
Authors: Chandra Shilpi , Khurana Archana , Kiosses William B. , Gray James , Hitomi Kaori , Zhao Meng , Richardson Stewart K. , Besra Gurudayal , Howell Amy R. , Sharma Sonia , Peters Bjoern , Kronenberg Mitchell 
Published: January 2023

Abstract
Although most T lymphocytes recognize peptides presented by major histocompatibility complex (MHC)-encoded class I and class II molecules, there also are significant populations of T cells that recognize nonpeptide antigens. Prominent among these T lymphocytes are the type I or invariant natural killer T cells (iNKT cells). These T lymphocytes recognize lipids presented by CD1d, a nonpolymorphic, class I-like, antigen-presenting molecule. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGal Cer, to iNKT cells. In order to stimulate iNKT cells, this antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we have identified genes that lead to altered antigen presentation in macrophages. A majority of the identified genes do not perturb surface CD1d expression, but we can demonstrate they effect the formation of surface CD1d complexes with the stimulating glycolipid. Members of the HOPS and ESCRT complexes have been identified to play an important role in Cd1d dependent antigen presentation. Interestingly, our data show that ABCC1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. CD1d and MHC-II antigen presentation pathways both depends on antigen loading in the lysosome, therefore we also analyzed the role of these genes in MHC-II antigen presentation. Although the majority of genes identified doesn’t affect MHC-II antigen presentation, there are a few molecules that affect both antigen presentation pathways.
Immuno-proteomic interrogation of antigen presentation during Dengue and Zika infections reveals novel targets for the design of broad acting T-cell vaccines
Authors: Swaminathan Kavya , Majzoub Karim , Lund Peder J , Marceau Caleb , Olsson Niclas , Davis Mark M , Carette Jan , Elias Joshua 
Published: January 2023

Abstract
The recent outbreak of Zika virus (ZIKV) infection across the Americas has raised dire concerns about its potential long-term health effects including its negative impact on neuronal, psychological and motor development in affected individuals. Emerging evidence pointing to Alzheimer’s-like damage to the adult brain further highlights the urgent need for efficacious prophylactic measures. Zika-affected regions are often endemic to the related Dengue virus (DENV). Antibodies against the two viruses are crossreactive and cause antibody-mediated enhancement (ADE) of infection. This confounds the design of traditional vaccines that elicit humoral antibody-mediated responses. T cell vaccines mediated by cytotoxic CD8+ T cells that recognize conserved viral epitopes presented on the major histocompatibility complex (MHC-I) of infected cells are unencumbered by ADE and can supplement or substitute traditional vaccines. In order to identify robust T cell vaccine candidates, we first used an immunoproteomic approach and isolated over 100 novel DENV and ZIKA epitopes presented by infected B lymphocytes in vitro. These viral peptides were not predicted to be epitopes by computational programs typically used for epitope discovery, which discard &gt;95% of all predictions. Focussing on epitopes derived from polyprotein regions strictly conserved between the two viruses, we validated the presentation of these epitopes in primary dendritic cells and assayed their ability to stimulate robust T cell responses in ex vivo ELISpot assays. Together these discoveries let us assay the immunogenic potentials of an unprecedented range of DENV and ZIKV antigens which have the potential to serve as broad acting and effective Zika vaccine candidates.
Conventional influenza vaccines influence the efficacy of a candidate universal influenza vaccine in mice
Authors: Rowell Janelle , Garcia Mayra , Lo Chia-Yun , Price Graeme , Misplon Julia , Epstein Suzanne 
Published: January 2023

Abstract
Universal influenza vaccines are designed to protect against diverse strains of influenza virus. In a pandemic, universal vaccines could be life-saving. Candidate vaccines are usually tested in naïve animals, despite intended use in the human population that has a varied immune history, including responses to vaccination. To be more relevant to humans, we tested a candidate universal influenza vaccine in mice with a history of conventional vaccination. Female BALB/c mice were given two intramuscular doses of inactivated influenza vaccine (IIV) or diphtheria and tetanus toxoids vaccine (DT), one month apart. Another group had two intranasal (i.n.) doses of live attenuated influenza virus (LAIV). One month after the second dose, mice were given the universal vaccine, recombinant adenoviruses expressing influenza A NP and M2 (rAd-NP+M2, i.n.). Antibody responses to universal vaccine antigens NP and M2 were assessed by ELISA. T-cell responses to antigens were determined by IFN-γ ELISPOT. Mice were challenged with mouse-adapted A/FM/1/47 (H1N1) and subsequent weight loss determined rAd-NP+M2 efficacy. Efficacy was enhanced, inhibited or unaffected by prior history. Mice given Afluria IIV and LAIV lost less weight and had a greater antibody and T-cell response to NP than mice without prior vaccination, which are examples of enhanced rAd-NP+M2 efficacy. Inhibited vaccine efficacy was observed in mice that received Fluvirin IIV and mice had low IgG2a antibodies to NP and M2. Fluzone IIV and DT had no effect on rAd-NP+M2 efficacy. This study demonstrates that the protection by the universal influenza vaccine may be affected by a history of conventional vaccination, and suggests that performance in humans would be influenced by immune history.
Intradermal active full-length DNA Aβ42 immunization via electroporation leads to high anti-Aβ antibody levels in wild-type mice with variability in T cell responses depending on pulse parameters applied
Authors: Lambracht-Washington Doris , Fu Min , Rosenberg Roger N 
Published: January 2023

Abstract
Aβ immunotherapy has high potential as possible prevention treatment for Alzheimer’s disease, and anti-Aβ antibodies will help in the removal of excess amyloid from brain. We have previously shown that active DNA Aβ1-42 immunization via gene gun delivery led to a non-inflammatory immune response with efficient antibody levels which resulted in decreased Aβ levels in brains of the immunized AD mouse model mice. To optimize the DNA delivery and make DNA vaccination more applicable for clinical use, we have investigated here the use of intradermal electroporation using a variety of different voltages and electropulse parameters in wild-type mice. In the direct comparison to DNA delivery via gene gun, higher levels of anti-Aβ42 antibodies were detected already after three DNA immunizations. The antibody isotype profile following DNA electroporation was mixed with IgG1 and IgG2a antibodies. With a fine tuning of the electropulse protocol the T cell responses could be adjusted to similar findings we published earlier for DNA Aβ1-42 immunization via gene gun delivery with low levels of inflammatory cytokines (IFNγ, IL-17) in the in-vitro Aβ42 peptide re-stimulation assays. We found that short pulse durations (5 ms) were better than long pulse durations (10 ms) in regard to obtaining a Th2 type of immune response, and that a higher rate of pulses (16–20 pulses) increased the antibody response in our sets of experiments. Full-length DNA Aβ1-42 immunization delivered via electroporation with a fine tuning of the electropulse conditions has potential to be used in the clinical setting.
mBM-Mesenchymal Stem Cells as a vehicle for immunization of mice against Bordetella pertussis
Authors: MAROLI SREE LAKSHMI VELANDI , Molla Touhidul , Llontop Peter , Abramson Tzvia 
Published: January 2023

Abstract
Mesenchymal Stem Cells (MSC) derived from bone marrow is well documented to lack immunogenic features and demonstrate anti-inflammatory characteristics. Additionally, in vitro treatment of MSCs with IFNγ was previously shown to increase expression of antigen-presenting molecules such as MHC class II. Therefore in this study, we hypothesize that MSC can serve as an immunization vehicle to deliver Bordetella pertussis (Bp) antigens.
MSC were isolated from bone marrow. To confirm MSC characteristics, cells were differentiated into chondrocytes, adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to demonstrate that the propagated MSCs express high level of MHC II when pretreated with IFNγ. IFNγ treated MSC were then pulsed overnight with heat-killed B. pertussis and intranasally administered (one million/mouse) twice with one-week interval. Mice were then challenged with Bp infection. Mice immunized with MSC/Bp were 100% protected from the infection challenge as measured by the bacterial load in lungs at 5dpi.
Th17 are required for effective resolution of B. pertussis infection, but those immune responses are mounted only late p.i. In contrast, mice immunized with INFγ+/MSC/Bp, display a high frequency of Th17 cells in the lungs that was detected as early as 5dpi. These preliminary data suggest that BM-MSCs can serve as a vector to protect mice from Bp infection. We will further investigate for the precise immune responses induced by MSC/Bp vaccine.
Chitosan nanoparticle delivery of Influenza A Virus DNA vaccine enhances antibody class switching and abrogates weight loss post IAV challenge
Authors: Brown Deborah M , Lampe Anna T. , Farris Eric , Williams James A. , Pannier Angela K. 
Published: January 2023

Abstract
DNA vaccination presents a promising new approach for Influenza A Virus (IAV) vaccines, as they can be generated quickly in response to the viral antigenic shift and drift characteristic of IAV pandemic outbreaks. A DNA vaccination approach for IAV was examined using a plasmid encoding PR8 H1N1 hemagglutinin (HA) protein as a potential IAV vaccine. In addition to the HA antigen sequence the plasmid also encoded a sequence that when transcribed, activates the pattern recognition receptor RIG-I, improving innate immune activation. To increase the potential of this IAV DNA vaccine, our study focused on delivery of the plasmid to the respiratory tract using chitosan (CS) nanoparticles. CS, a mucoadhesive/mucopenetrating derivative of chitin, forms complexes with DNA to improve uptake and transfection of the plasmid by immune cells in the lung. In vitro, plasmid/CS complexes containing unbound CS induced bone marrow derived dendritic cell (BMDC) death. BMDC death was accompanied by a robust increase in inflammatory cytokine mRNA. Although BMDC death was abrogated by free CS removal, cytokine expression was also reduced. Alternative complexation methods are being explored to achieve an appropriate balance between cytokine production and cell death. In vivo intranasal prime-boost vaccination with low dose PR8 HA plasmid/CS nanoparticles resulted in PR8 specific IgM and total IgG antibody formation. Although weight loss was abrogated in plasmid/CS vaccinated mice, protection from lethal PR8 infection did not correlate with antibody level. By optimizing this vaccine approach, including dose, regime, and CS nanoparticle modification, critical insights into the development of rapidly deployable IAV vaccines could be gained.
Type III interferons restrict neutrophil activity in response to Staphylococcus aureus
Authors: Pires Silvia D , Parker Dane 
Published: January 2023

Abstract
Staphylococcus aureus is an important human pathogen, in particular the methicillin resistant S. aureus (MRSA) strain USA300 that is epidemic in the United States. Type III interferons (IFN-λ) signal through the IL-28 receptor (IFNLR) located on epithelial cells and neutrophils. Neutrophil-mediated killing is a crucial defence system against S. aureus. Compared to WT mice, Ifnlr−/− mice had significantly improved clearance of S. aureus from the airway and lung tissue (&gt;90%; P&lt;0.001) following a 24-hour infection. In a mortality model of infection, all Ifnlr−/− mice survived, whereas 75% of WT mice succumbed to infection (P&lt;0.05). Despite a lack of difference in neutrophil recruitment to the airway in WT and Ifnlr−/− mice in response to S. aureus, Ifnlr−/− infected mice showed a significant increase in the neutrophil related cytokine G-CSF (38%; P&lt;0.05). Moreover, expression of the neutrophil surface markers, Ly6C and Ly6G, were higher (39% and 20%, respectively; P&lt;0.01) on infected Ifnlr−/− neutrophils compared to WT infected mice. Neutrophils isolated from Ifnlr−/− mice compared to WT neutrophils also displayed a 2-fold increase in MPO activity (peroxidation) (p&lt;0.05) and a 2.1-fold increase in Ca2+ flux when compared to WT neutrophils (P&lt;0.001), while WT neutrophils stimulated with purified IFN-λ displayed a 0.5-fold decrease in Ca2+ flux (p&lt;0.005). In addition, stimulation of WT neutrophils with purified IFN-λ led to a 2-fold decrease of their S. aureus killing capacity (P&lt;0.05). Controlling neutrophil recruitment and activation is important to provide a balanced response to infection. Our data suggests that type III IFN signalling can impair neutrophil activation and their killing capacity in response to S. aureus.
RLR-mediated immunity is unique and critical in the response to West Nile Virus
Authors: Stone Amy E.L. , Wilkins Courtney , Green Richard , Gale Michael J 
Published: January 2023

Abstract
RIG-I-Like Receptors (RLRs) include RIG-I, MDA5, and LGP2. RLRs are vital pathogen recognition receptors in the defense against RNA viruses. West Nile Virus (WNV) is an emerging, neurotropic flavivirus that infects the central nervous system to cause disease and death. RIG-I and MDA5 work in tandem to initiate innate immune responses against WNV infection, though the role of LGP2 in this process is not well defined. To gain a deeper understanding of the contributions of each RLR in the innate immune response to WNV, we conducted a systems biology approach to assess the role of each RLR in immune programming. We conducted genome-wide RNAseq and bioinformatics analysis of WNV infection in bone marrow derived macrophages from the RLR-deficient mice. Here we show that the RLRs drive distinct immune gene activation and polarization of the immune response. In our data, the RLR-dependent, WNV-induced immune response polarization overshadows the classical drivers of viral innate immune responses, interferon I (IFN) and IFN-stimulated genes, thus underscoring the importance of innate immune activation for channeling the adaptive immune system into specific effector pathways. Bioinformatics analysis and computational modeling was used to define a unique gene signature of each RLR following WNV infection. Further, we dissected the complex virus-induced cytokine response utilizing multiplex analyses to link gene induction and cytokine protein output. These analyses reveal that distinct RLR-driven transcriptional programs lead to differential functional output in immune response initiation against WNV infection. Our study demonstrates a clear, defined role for RIG-I, MDA5, and LGP2 in immune programming against virus infection.