bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of p110delta in the immunopathology of influenza virus infection (130.12)
Authors: Boesteanu Alina C , Norton Jillian A , Turner Martin , Katsikis Peter D 
Published: January 2023

Abstract
Infection of C57Bl/6 mice with influenza virus is accompanied by morbidity manifested as weight loss and lung pathology. We have found that mice that have an inactivating mutation in the leucocyte-specific phosphoinositide kinase 3 (PIK3) isoform p110delta (p110delta-/- on a C57BL/6 background manifest significantly reduced morbidity after influenza virus infection compared to C57BL/6 mice. RAG-/- mice also showed reduced morbidity compared to wild type animals, indicating a role for lymphocytes in this pathology. At day 6 postinfection activated pulmonary T cells and NK cells were greatly reduced in p110delta-/- mice. At day 10 postinfection, however, numbers of lymphocytes, B cells, CD4+ T cells, macrophages and granulocytes infiltrating the lungs of p110delta-/- mice were reduced compared to C57Bl/6 mice, without reaching statistical significance. Total number of CD8+ and virus-specific CD8+ T cells found in the lungs of infected p110delta-/- mice were significantly reduced by 2-fold compared to wild-type mice (p=0.03 for both). However, this reduction in the magnitude of the immune response did not affect viral clearance. Our data suggest that p110delta plays an important role in the lymphocyte mediated immunopathology associated with influenza virus infection and suggests that p110delta may be an important therapeutic target for influenza virus infection.
Innate immune response of bronchial epithelial cells to infection with influenza (130.7)
Authors: Wark Peter AB , Hsu Alan , Hansbro Phil 
Published: January 2023

Abstract
Bronchial epithelial cells are the natural human host for influenza infection but knowledge about the innate immune response is limited.
Study objectives; characterise the innate immune response of bronchial epithelial cells to infection with human influenza A virus and relate this to virus replication.
Methods; Influenza H3N2, A/Wellington/2004 (H3N2) was cultured and titred using plaque assays on Manine Darby Kidney Cells. Primary bronchial epithelial cells (PBECs) were obtained by bronchoscopy and cultured. Their response was compared to an epithelial cell line, Calu 3. Innate immune response was measured by the induction of mRNA by real time PCR for RIG-I, MDA-5, TLR-3, PKR, interferon (IFN)-β. Protein expression was then measured by western blot in cell lysate or supernatant.
Results; H3N2 replicated to significantly higher titres in Calu 3 cells 2.8x106 PFU/ml compared to PBECs 789 PFU/ml (p&lt;0.001). Following infection there was greater than 20 fold induction of RIG-I mRNA in both cells, strong protein expression in PBECs by 24hr, only faint expression in Calu3. A thousand fold induction in MDA-5 mRNA was seen in PBECs at 48hr less than 20 fold in Calu3. Induction of IFN-β mRNA by 24hr and protein release in the supernatant was seen in both cells by 6hr.
Conclusions; H3N2 replicates less well in PBECs and this is associated with more vigorous induction of RIG-I during early infection and later induction of MDA-5 mRNA.
In vitro modulation of influenza virus-specific CD4+ T cell responses via Dectin-1 expressed on human dendritic cell subsets (131.11)
Authors: Ni ling , Li Xiaohua , Gayet Ingrid , Kluca Peter , Li Dapeng , Dullaers Melissa , Zurawski Sandy , Zurawski Gerard , Banchereau Jacques , Oh SangKon 
Published: January 2023

Abstract
Dendritic cells (DCs) can induce and control immune responses. Types of pre-existing memory CD4+ T cells can influence the efficacy of vaccines against influenza viruses. We here show that quality of influenza-specific CD4+ T cells can be modulated by delivering antigens to subsets of human DCs via Dectin-1. A major antigen for neutralizing antibodies, HA1 of hemagglutinin (H1), was delivered to DCs by using recombinant fusion proteins, anti-Dectin-1-HA1. First, all healthy individuals tested had relatively potent HA1-specific Th1 and Th2 helper T cells. However, magnitudes of IL-10-, IL-17-, and IL-21-producing were variable in individuals and by peptide epitopes presented by DCs. Second, anti-Dectin-1-HA1 induced DCs to secrete IL-23 that upregulated IFNγ and IL-17, but downregulated IL-13. CD70 upregulated by anti-Dectin-1-HA1 contributed to the expression of IFNγ, IL-17, IL-10, and IL-13, but not IL-21. Third, lipopolysaccharide from P. gingivalis, not E. coli, permitted myeloid DCs (mDCs) to promote Th1 and Th17, but suppress Th2 type responses, in CD70-/IL-1β-dependent manners. Lastly, plasmacytoid DCs (pDCs) and mDCs expressed same variants of Dectin-1 and they presented same peptide epitopes. However, mDCs were more potent than pDCs for inducing cytokines tested, particularly IFNγ and IL-13. Thus vaccines that target Dectin-1 have a potential to enhance the immunity against influenza infections.
Characterization and the Immune Response of a Nanoencapsulated Multivalent Vaccine against Respiratory Syncytial Virus (132.2)
Authors: Subbarayan Praseetha , Pillai Shreekumar , Nyairo Elijah , Dennis Vida A , Singh Shree R 
Published: January 2023

Abstract
Development of a safe and effective vaccine is necessary to prevent RSV infection. Our project involved development of a multivalent vaccine for RSV by amplification of the antigenic regions of the RSV F, G and M2 genes which were subsequently cloned into a bacterial vector, pET-32. The recombinant clone was expressed from the transformed E. coli BL21 (DE3) cells purified and analyzed yielding a protein band of 38 kDa as predicted. The multivalent protein was encapsulated in chitosan to form protein encapsulated nanoparticle using the incorporation method. The encapsulated nanoparticles were analyzed for temperature stability, in vitro release of the protein, and also characterized using cytoviva bioimaging system as well as FTIR. The nanoencapsulated protein was stable up to a temperature of 1100C. The cytoviva bioimaging system revealed that the nanoparticles were irregular spherical shaped and the protein encapsulated nanoparticle appeared to be larger compared to the chitosan alone. The FTIR confirmed the chemical composition of the nanoparticle which consisted of polysaccharide, tripolyphosphate and protein. BALB/c female mice were immunized with the nanoencapsulated multivalent protein and the antibody response was compared to the mice vaccinated with live RSV or PBS The serum antibody response data revealed that mice immunized with the nanoencapsulated vaccine had the highest levels of antibody against RSV.
Keywords: RSV, Antibody, Vaccine, Recombinant DNA, Chitosan nanoparticle.
Sponsors: NSF-CREST, NIH-MBRS
Cross-reactive humoral epitopes of EF and LF are identified but do not constitute major anthrax toxin neutralization responses following EF and LF vaccination (132.20)
Authors: Nguyen Melissa L , Ballard Jimmy D , James Judith A , Farris Darise 
Published: January 2023

Abstract
BACKGROUND: Anthrax Lethal and Edema Toxins (LTx and ETx) are formed by binding of Lethal Factor (LF) or Edema Factor (EF) to the pore-forming moiety, Protective Antigen (PA). Immunity to LF and EF neutralize anthrax toxins.
AIM: LF and EF immune sera were used to identify cross-reactive B cell epitopes in their conserved PA-binding domains and to determine the relative contribution of such antibodies to LTx and ETx neutralization.
METHODS: A/J mice were immunized with recombinant (r) LF or EF. EF-binding antibodies were purified from LF immune sera, and LF-binding antibodies from EF immune sera by column absorption. Whole immune sera and purified cross-reactive antibodies were tested for IgG to rLF or rEF by ELISA, in vitro neutralization of LTx and ETx, and binding to solid phase, overlapping LF and EF decapeptides.
RESULTS: Cross-reactive epitopes were identified as EF aa 98-257 and 257-268 and LF aa 265-274. Whole LF and EF immune sera neutralized LTx and ETx, respectively. However, LF sera did not neutralize ETx nor did EF sera neutralize LTx. Purified cross-reactive IgG also failed to cross-neutralize.
CONCLUSIONS: Cross-reactive B cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes that likely lie outside of their PA-binding sites.
Post-exposure vaccination against influenza (132.4)
Authors: Barefoot Brice E , Sample Christopher , Ramsburg Elizabeth 
Published: January 2023

Abstract
Vaccines currently licensed for the prevention of influenza induce antibodies against the influenza hemagglutinin (HA) and neuraminidase (NA) contained in the vaccine preparation but require at least two weeks after immunization for the development of protective immunity. We have developed a novel vaccine based on recombinant vesicular stomatitis virus which expresses the influenza hemagglutinin (rVSV HA) and protects mice from lethal influenza challenge when the vaccine is administered intramuscularly shortly after delivery of the influenza challenge virus. To our knowledge ours is the first vaccine which effectively protects animals from lethal influenza challenge when delivered by a systemic route after influenza exposure has occurred. The mechanism(s) by which rVSV HA protects remain to be determined but induction of HA-specific immune responses is essential, because animals immunized with an empty rVSV vector were not protected equally. Our data are consistent with a model in which post-exposure rVSV HA vaccination induces an immediate antiviral cytokine response, followed by expansion of HA-specific cytotoxic CD8 T cells, which circulate to the respiratory tract and clear infected cells. Vaccinated animals do not have detectable serum neutralizing Ab to influenza during the acute phase of the infection, but antibody may be required at later timepoints for complete clearance of influenza.
TRAIL regulates the intensity of T cell-mediated immune responses to influenza virus infection (133.53)
Authors: Brincks Erik L. , Legge Kevin L. , Griffith Thomas S. 
Published: January 2023

Abstract
An immune response of appropriate magnitude controls pathogen spread, while limiting immune system-induced damage to the infected host. The objective of the present study was to determine how TNF-related apoptosis-inducing ligand (TRAIL) limits influenza virus-associated immunopathology. Using TRAIL+/+ (WT) and TRAIL-/- (TKO) mice infected with a mouse-adapted influenza virus, we tested the hypothesis that TRAIL deficiency increases immunopathology associated with influenza infection by altering the T cell response. Compared to WT mice, influenza-infected TKO mice experienced increased morbidity and mortality-despite similar viral titers and rates of viral clearance. The increased mortality in the TKO mice was likely the result of increased non-specific immunopathology. Analysis of the lung-infiltrating T cells from the influenza-infected WT and TKO mice revealed similar GranzymeB, FasL, and TNF expression; however, there were increased numbers of influenza-specific T cells in the lungs of TKO mice compared to WT mice, resulting in enhanced in vivo cytotoxicity of Ag-pulsed targets in the TKO mice compared to WT mice. The enhanced T cell response in the TKO mice correlated with increased APC recruitment into the lung and lung-draining LN. Together, these data suggest that TRAIL regulates CD8 T cell responses to influenza and limits the immunopathology associated with influenza virus infections.
Natural killer like – T cells regulate induction of short- and long- lived plasma cells (134.12)
Authors: Shah Hemangi B , Lang Mark 
Published: January 2023

Abstract
Sustained serum antibody (Ab) titers are in large part maintained by bone marrow-resident long-lived plasma cells (LLPC). Although several factors governing the survival of LLPC have been identified, the mechanisms by which LLPC are induced are not fully understood. We observed that natural killer like - T (NKT) cells made substantial contributions to vaccination-induced antigen-specific Ab responses. The Ab titers were sustained and correlated with the persistence of PC in the bone marrow. In CD1d-/- mice lacking NKT cells, the PC numbers were diminished but persistent, indicating a role for NKT cells in PC induction rather than survival. We therefore used an in vivo-labeling approach with 5-bromo-2-deoxyuridine, a nucleoside analogue that labels dividing cells. In conjunction with a flow cytometric analysis of bone marrow PC we observed that CD1d-/- mice had a reduced induction of CD138+/B220lo/CD44+ precursor cells as well as BrDU+/CD138+ short-lived PC (SLPC) and BrDU-/CD138+ LLPC. Our data show that NKT cells act at a stage of induction prior to the emergence of SLPC and LLPC. Efforts are underway in our laboratory to understand how NKT cells achieve their effects on PC induction.
This work was supported by NIH Grant 5P20RR015564-07 from the National Center for Research Resources.
Over-expression of constitutively active PI3K p110γ alters NK cell development (134.4)
Authors: Orr Selinda J , Quigley Laura , McVicar Daniel W. 
Published: January 2023

Abstract
PI3K is critical in NK cell responses such as cytokine production and cytotoxicity. The roles for the individual p110γ/δ isoforms in NK cell development and signaling are controversial. Over-expression of the p110γ/δ PI3K isoforms would help to dissect their roles in NK cells, however it is difficult to stably express proteins in NK cells. Therefore, we developed a novel system to over-express p110 in primary NK cells. IL2Rγc-/- mice lack NK cells and reconstitution of IL2Rγc-/- bone marrow (BM) with IL2Rγc restores NK cell development. Here, we describe a novel approach to produce NK cells in vivo, all of which express a "cargo" gene. This involves retroviral infection of IL2Rγc-/-/Rag2-/- BM with a construct containing a cargo gene followed by an IRES and IL2Rγc to permit selective NK cell development. Using EGFP as a control cargo protein, we showed that we can deliver EGFP to 100% of reconstituted NK cells. Constitutively active p110γ expressing NK cells display an immature phenotype and these cells do not produce IFNγ in response to NK1.1 stimulation or induce cytotoxicity in response to Yac1 cells. In contrast, constitutively active p110δ expressing NK cells display normal development, NK1.1-induced IFNγ production and cytotoxity. These data highlight the necessity for tight regulation of p110γ during NK cell development and function whereas regulation of p110δ is not as important for these processes.
Effect of Mycobacterium tuberculosis trapped in NETs on macrophage proinflammatory response (134.83)
Authors: Ramos-Kichik Victoria , Silva-Garcia Raul , Serafin-Lopez Jeanet , Rojas-Espinosa Oscar , Rivera-Gutierrez Sandra , Estrada-Parra Sergio , Estrada-Garcia Iris 
Published: January 2023

Abstract
We have previously shown that M. tuberculosis induce neutrophil extracellular traps (NETs) formation. Since NETs could capture M. tuberculosis bacilli but were unable to kill them, we hypothesized that these structures might be involved in maintaining the infectious focus localized and at the same time allowing macrophages to arrive at the infection site where they probably would phagocyte mycobacteria trapped in NETs. In this study we tested if phagocytosis of M. tuberculosis trapped in NETs versus M. tuberculosis alone had differential effect on macrophage proinflammatory cytokine gene expression. Methods: human monocyte-derived macrophages were left unstimulated or infected either with NETs, M. tuberculosis H37Rv trapped in NETs or M. tuberculosis H37Rv alone for 24h and proinflammatory cytokine gene expression was analyzed using SABioscience nylon membrane macroarrays. Results: M. tuberculosis trapped in NETs induced a lower gene expression profile of proinflamatory cytokines in comparison with that induced by M. tuberculosis alone in 2 of 3 subjects analyzed. In the other subject, NETs acted as an adjuvant, increasing the proinflammatory response against M. tuberculosis.
Triggering human TLR2/1-induces distinct IL-1β- and VDR-dependent antimicrobial peptides required for host defense β (135.42)
Authors: Liu Philip T , Schneck Mirjam , Walker Valencia P , Kanchanapoomi Melissa , Cheng Genhong , Modlin Robert 
Published: January 2023

Abstract
Activation of Toll-like receptor 2/1 (TLR2/1) on human monocytes induces a vitamin D receptor (VDR) dependent antimicrobial pathway against intracellular mycobacteria, involving the induction of the antimicrobial peptide cathelicidin. Here, we demonstrate a novel TLR-induced antimicrobial pathway leading to induction of DEFB4, requiring synergy between IL-1β- and VDR- dependent transcriptional activation. Strikingly, TLR2/1 induction of DEFB4, but not cathelicidin required IL-1β activity despite both requiring VDR activation. TLR2/1L activation triggered the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions, the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in activating DEFB4 but not cathelicidin. Knockdown of either DEFB4 or cathelicidin resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacterial infection in primary monocytes. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for TLR-induced antimicrobial pathway against intracellular pathogens.
THE Class IA PI3-Kinase signaling Pathway: A Potential Target for Enhancing the Proinflammatory Cytokines in the Aged Macrophages (135.53)
Authors: Fallah Mosoka P , Chelvarajan Ralph L , Bondada Subarrao 
Published: January 2023

Abstract
The elderly suffer from increased infections with Streptococcus pneumonia due to a reduced ability to produce antibodies to the capsular polysaccharide (CPS) antigens. The antibody response to CPS is T-independent but needs the macrophage-derived cytokines such as IL-12, IL-6 and IL-1beta to induce anti-CPS antibody response. We found that production of these cytokines via TLR4 or TLR2 stimulation is dysregulated in both splenic (SM) and bone marrow derived macrophages (BMDM) from the aged (20-24 months) in comparison to young adult (8-12 weeks) BALB/c mice: a decrease in IL-12, IL-6 and IL-1beta but an increase in IL-10 in the aged. We hypothesized that the aged macrophages have an intrinsic signaling defect leading to cytokine dysregulation. A microarray analysis showed that aged SM expresses more P110delta and p85beta subunits of Class IA PI3-Kinase than the young adult. This was confirmed, at the mRNA levels, via qt-RTPCR in both SM and BMDM and at the protein levels for P-AKT (a downstream target of PI3-kinase) in BMDM. Inhibition of the PI3-kinase with LY294002, shifted the TLR2/4 induced cytokine phenotype of the aged to that of the young adult. Therefore, targeting PI3-Kinase could rescue the intrinsic signaling defect in the aged macrophages and enhance anti-CPS antibody responses.
Agonistic signaling via the neurokinin 1 receptor and CD40 have a synergistic effect to promote dendritic cell survival and potent CTL responses (135.56)
Authors: Janelsins Brian M , Mathers Alicia R , Tkacheva Olga A , Erdos Geza , Shufesky William J , Morelli Adrian E , Larregina Adriana T 
Published: January 2023

Abstract
Immune competent organs are richly innervated and pro-inflammatory neuropeptides released by nerve endings favor the initiation of innate and adaptive immune responses. By binding the neurokinin 1 receptor (NK1R), the pro-inflammatory neuropeptide substance P promotes immune cell survival and potent cellular immunity. Dendritic cells (DCs) express surface NK1R and the priming of T cell responses requires Ag presentation by DCs able to withstand apoptotic signaling. We hypothesized that signaling DCs via the NK1R prevents apoptosis of DCs favoring sustained DC-T cell contact and robust CTL responses. Using murine bone marrow-derived DCs (BMDCs) cultured with the NK1R agonist [Sar9Met(02)11]-SP (SarSP-DCs), we demonstrate that signaling via the NK1R utilizes the PI3K-Akt pathway to prolong the expression of anti-apoptotic molecules. Additionally, SarSP-DCs showed increased expression of surface CD40. Adoptive transfer of Ag-loaded SarSP-DCs showed enhanced longevity in local draining lymph nodes vs. control DCs, which was a result of a combination of signaling via the NK1R and CD40 molecules. Importantly, SarSP-DCs elicited potent CTL responses compared to control DCs. We conclude that agonistic signaling via the NK1R and CD40 have a synergistic effect that enhances DC survival and favors the generation of potent CTL responses.
Supported by NIH grant: R01 CA100893 (ATL)
Toll-like Receptor 9: The road to endolysosomes is through the cell surface (135.59)
Authors: Chockalingam Annapoorani , Brooks James C , Leifer Cynthia A 
Published: January 2023

Abstract
TLR9 recognizes its ligand, CpG DNA, in the endocytic compartment. Consistent with this, TLR9 is not expressed on the surface of most cells. Forced surface expression of TLR9 results in lack of response to CpG DNA. However, in intestinal epithelial cells TLR9 is at the surface and responds to CpG DNA. Whether surface localization is a normal trafficking intermediate for TLR9 or certain cells are able to differentially localize TLR9 to the cell surface is unknown. We hypothesize that, in most cells, TLR9 traffics to the cell surface and is then rapidly internalized for signaling. To test this we treated cells with the endocytosis inhibitor dynasore. Treatment with dynasore caused the accumulation of TLR9 at the cell surface and inhibition of CpG DNA induced phosphorylation of ERK1/2. This suggests that proper regulation of TLR9 internalization is critical for signaling, consistent with previous studies. Mutation of a tyrosine motif (YXXΦ, X = any and, Φ = hydrophobic amino acid) resulted in failure to respond to CpG DNA and accumulation of TLR9 at the cell surface suggesting that this tyrosine motif is involved in surface internalization and in initiation of signaling. These data support the hypothesis that TLR9 traffics to endolysosomes via the cell surface and that the tyrosine motif plays a key role in regulating endocytosis of TLR9 from the cell surface. K22CA113705 and AI076588 (CAL)