bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression and Production of the Monokine Induced by IFN-γ (MIG), IFN-Inducible T Cell α Chemoattractant (I-TAC), and IFN-γ-Inducible Protein-10 (IP-10) Chemokines by Human Neutrophils
Authors: Gasperini Sara , Marchi Martina , Calzetti Federica , Laudanna Carlo , Vicentini Lucia , Olsen Henrik , Murphy Marianne , Liao Fang , Farber Joshua , Cassatella Marco A.
Published: January 2023

Monokine induced by IFN-γ (MIG), IFN-inducible T cell α chemoattractant (I-TAC), and IFN-γ-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-γ. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-γ in combination with either TNF-α or LPS. While IFN-γ, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-α, in combination with TNF-α, LPS, or IL-1β, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-γ plus either LPS or TNF-α. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.
Endothelial Targeting and Enhanced Antiinflammatory Effects of Complement Inhibitors Possessing Sialyl Lewisx Moieties
Authors: Mulligan Michael S. , Warner Roscoe L. , Rittershaus Charles W. , Thomas Lawrence J. , Ryan Una S. , Foreman Kimberly E. , Crouch Larry D. , Till Gerd O. , Ward Peter A.
Published: January 2023

The complement inhibitor soluble complement receptor type 1 (sCR1) and a truncated form of sCR1, sCR1[desLHR-A], have been generated with expression of the selectin-reactive oligosaccharide moiety, sialyl Lewisx (sLex), as N-linked oligosaccharide adducts. These modified proteins, sCR1sLex and sCR1[desLHR-A]sLex, were assessed in the L-selectin- and P-selectin-dependent rat model of lung injury following systemic activation of complement by cobra venom factor and in the L-selectin-, P-selectin-, and E-selectin-dependent model of lung injury following intrapulmonary deposition of IgG immune complexes. In the cobra venom factor model, sCR1sLex and sCR1[desLHR-A]sLex caused substantially greater reductions in neutrophil accumulation and in albumin extravasation in lung when compared with the non-sLex-decorated forms. In this model, increased lung vascular binding of sCR1sLex and sCR1[desLHR-A]sLex occurred in a P-selectin-dependent manner, in contrast to the absence of any increased binding of sCR1 or sCR1[desLHR-A]. In the IgG immune complex model, sCR1[desLHR-A]sLex possessed greater protective effects relative to sCR1[desLHR-A], based on albumin extravasation and neutrophil accumulation. Enhanced protective effects correlated with greater lung vascular binding of sCR1[desLHR-A]sLex as compared with the non-sLex-decorated form. In TNF-α-activated HUVEC, substantial in vitro binding occurred with sCR1[desLHR-A]sLex (but not with sCR1[desLHR-A]). This endothelial cell binding was blocked by anti-E-selectin but not by anti-P-selectin. These data suggest that sLex-decorated complement inhibitors have enhanced antiinflammatory effects and appear to have enhanced ability to localize to the activated vascular endothelium.
NF-κB Regulation by IκB Kinase in Primary Fibroblast-Like Synoviocytes
Authors: Aupperle Karlfried R. , Bennett Brydon L. , Boyle David L. , Tak Paul-Peter , Manning Anthony M. , Firestein Gary S.
Published: January 2023

NF-κB is a key regulator of inflammatory gene transcription and is activated in the rheumatoid arthritis (RA) synovium. In resting cells, NF-κB is retained as an inactive cytoplasmic complex by its inhibitor, IκB. Phosphorylation of IκB targets it for proteolytic degradation, thereby releasing NF-κB for nuclear translocation. Recently, two related IκB kinases (IKK-1 and IKK-2) were identified in immortalized cell lines that regulate NF-κB activation by initiating IκB degradation. To determine whether IKK regulates NF-κB in primary cells isolated from a site of human disease, we characterized IKK in cultured fibroblast-like synoviocytes (FLS) isolated from synovium of patients with RA or osteoarthritis. Immunoreactive IKK protein was found to be abundant in both RA and osteoarthritis FLS by Western blot analysis. Northern blot analysis showed that IKK-1 and IKK-2 genes were constitutively expressed in all FLS lines. IKK function in FLS extracts was determined by measuring phosphorylation of recombinant IκB in vitro. IKK activity in both RA and osteoarthritis FLS was strongly induced by TNF-α and IL-1 in a concentration-dependent manner. Activity was significantly increased within 10 min of stimulation and declined to near basal levels within 80 min. Activation of IKK in FLS was accompanied by phosphorylation and degradation of endogenous IκBα as determined by Western blot analysis. Concomitant activation and nuclear translocation of NF-κB was documented by EMSA and immunohistochemistry. Transfection with a dominant negative IKK-2 mutant prevented TNF-α-mediated NF-κB nuclear translocation, whereas a dominant negative IKK-1 mutant had no effect. This is the first demonstration that IKK-2 is a pivotal regulator of NF-κB in primary human cells.
Inhibition of CCR5 Expression by IL-12 Through Induction of β-Chemokines in Human T Lymphocytes
Authors: Wang Jinhai , Guan Ennan , Roderiquez Gregory , Norcross Michael A.
Published: January 2023

IL-12 induces initiation of the differentiation of naive CD4+ T lymphocytes into Th1 cells and is important for the control of cell-mediated immunity. β-Chemokines serve to attract various types of blood leukocytes to sites of infection and inflammation. The specific receptor for the β-chemokines (macrophage-inflammatory protein (MIP)-1α, MIP-1β, and RANTES), CCR5, also functions as the primary coreceptor for macrophage-tropic isolates of HIV-1. IL-12, but not IL-4, IL-10, or IL-13, now has been shown to down-modulate the surface expression of CCR5 induced by IL-2 on both CD4+ and CD8+ T lymphocytes. Decreased CCR5 surface expression was not secondary to transcriptional inhibition, given that CCR5 mRNA was enhanced in cells cultured in IL-12/IL-2 compared with those cultured in IL-2 only. The effect of IL-12 in down-modulation of CCR5 surface expression was shown to be mediated by soluble factors secreted from the T cells. Rapid and transient intracellular Ca2+ mobilization was induced in monocytes by IL-12-induced supernatants, which desensitized the response of monocytes to MIP-1α, but not their response to stromal cell-derived factor-1α. Neutralization with specific Abs identified these factors as MIP-1α and MIP-1β from most donors. IL-4, IL-10, IFN-γ, and IL-18 primarily inhibited MIP-1β secretion and also weakly suppressed MIP-1α secretion. HIV-1 replication was inhibited in IL-2/IL-12-containing cultures that correlated with chemokine and chemokine-receptor levels. These data suggest that the effects of IL-12 on β-chemokine production and chemokine-receptor expression may contribute to the immunomodulatory activities of IL-12 and may have potential therapeutic relevance in controlling HIV-1 replication.
Emt/Itk Associates with Activated TCR Complexes: Role of the Pleckstrin Homology Domain
Authors: Ching Keith A. , Kawakami Yuko , Kawakami Toshiaki , Tsoukas Constantine D.
Published: January 2023

Expressed in mast and T-cells/inducible T cell tyrosine kinase (Emt/Itk) is a protein tyrosine kinase required for T cell Ag receptor (TCR)-induced activation and development. A physical interaction between Emt/Itk and TCR has not been described previously. Here, we have utilized laser scanning confocal microscopy to demonstrate that Ab-mediated engagement of the CD3ε chain induces the membrane colocalization of Emt/Itk with TCR/CD3. Removal of the Emt/Itk pleckstrin homology domain (ΔPH-Emt/Itk) abrogates the association of the kinase with the cell membrane, as well as its activation-induced colocalization with the TCR complex and subsequent tyrosine phosphorylation. The addition of a membrane localization sequence to ΔPH-Emt/Itk from Lck restores all of these deficiencies except the activation-induced tyrosine phosphorylation. Our data suggest that the PH domain of Emt/Itk can be replaced with another membrane localization signal without affecting the membrane targeting and activation-induced colocalization of the kinase with the TCR. However, the PH domain is indispensable for the activation-induced tyrosine phosphorylation of the kinase.
Selection of CTL Escape Mutants in Mice Infected with a Neurotropic Coronavirus: Quantitative Estimate of TCR Diversity in the Infected Central Nervous System
Authors: Pewe Lecia , Heard Stephen B. , Bergmann Conni , Dailey Morris O. , Perlman Stanley
Published: January 2023

Variant viruses mutated in the immunodominant cytotoxic T cell epitope surface (S) glycoprotein S-510-518 are selected in mice chronically infected with mouse hepatitis virus, strain JHM. We determined whether this selection occurred in the presence of an oligoclonal or polyclonal T cell response using soluble MHC/peptide tetramers in direct ex vivo analyses of CNS-derived lymphocytes. A total of 42% (range, 29–60%) of CD8 T cells in the CNS of mice with acute encephalitis recognized epitope S-510-518. A total of 34% (range, 18–62%) of cells from mice with hind limb paralysis (and chronic demyelination) were also epitope specific, even though only virus expressing mutated epitope is detected in these animals. Sequence analysis of the β-chain CDR3 of 487 tetramer S-510-518-positive cDNA clones from nine mice showed that a majority of clonotypes were identified in more than one mouse. From these analyses, we estimated that 300–500 different CD8 T cell clonotypes responsive to epitope S-510-518 were present in each acutely infected brain, while 100–900 were present in the CNS of each mouse with chronic disease. In conclusion, a polyclonal CD8 T cell response to an epitope does not preclude the selection of T cell escape mutants, and epitope-specific T cells are still present at high levels even after RNA-encoding wild-type sequence is no longer detectable.
Compromised Virus Control and Augmented Perforin-Mediated Immunopathology in IFN-γ-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus
Authors: Nansen Anneline , Jensen Teis , Christensen Jan Pravsgaard , Andreasen Susanne Ørding , Röpke Carsten , Marker Ole , Thomsen Allan Randrup
Published: January 2023

To define the role of IFN-γ in the control of acute infection with a noncytopathogenic virus, mice with targeted defects of the genes encoding IFN-γ, perforin, or both were infected i.v. with two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread in wild-type mice. Our results reveal that IFN-γ is pivotal to T cell-mediated control of a rapidly invasive stain, whereas it is less important in the acute elimination of a slowly invasive strain. Moreover, the majority of mice infected with the rapidly invasive strain succumb to a wasting syndrome mediated by CD8+ effector cells. The primary effector mechanism underlying this disease is perforin-dependent lysis, but other mechanisms are also involved. Wasting disease can be prevented if naive CD8+ cells from mice transgenic for an MHC class I-restricted lymphocytic choriomeningitis virus-specific TCR are adoptively transferred before virus challenge, indicating that the disease is the result of an unfortunate balance between virus replication in internal organs, e.g., liver and spleen, and the host response; resetting this balance by increasing host responsiveness will again lead to a rapidly controlled infection and limited tissue damage. Thus, the presence or absence of IFN-γ determines whether CTLs will clear infection with this noncytopathogenic virus or induce severe immunopathology.
Human RNA Helicase A Is a Lupus Autoantigen That Is Cleaved During Apoptosis
Authors: Takeda Yoshihiko , Caudell Patricia , Grady Giuia , Wang Grace , Suwa Akira , Sharp Gordon C. , Dynan William S. , Hardin John A.
Published: January 2023

Proteolytic cleavage by caspases is the central event in cells undergoing apoptosis. Cleaved proteins are often targeted by autoantibodies, suggesting that the cleavage of self Ags enhances immunogenicity and is prone to induce an autoimmune response. We found autoantibodies that immunoprecipitated a 140-kDa RNA-associated protein, provisionally designated Pa, in 11 of 350 patient sera that were positive for antinuclear Abs in an immunofluorescence test. The Pa protein gave rise to three fragments with m.w. ranging from 120–130 kDa during anti-Fas-activated apoptosis. Pure caspase-3 cleaved the Pa protein into a 130-kDa fragment corresponding to the largest of these three products. Peptide sequence analysis of a tryptic digest from immunoaffinity-purified Pa showed 100% identity to human RNA helicase A (RHA). The identity of Pa with RHA was further confirmed by immunoblotting with rabbit anti-RHA Ab using anti-Pa immunoprecipitates as substrates. All 10 anti-RHA-positive patients who were clinically analyzed were diagnosed as having systemic lupus erythematosus, and 7 of them had lupus nephritis. RHA is a multifunctional protein with roles in cellular RNA synthesis and processing. Inactivation of RHA by cleavage may be an important part of the process leading to programmed cell death. The cleaved RHA fragments that are produced during apoptosis may trigger an autoimmune response in systemic lupus erythematosus.
Determination of Glutamic Acid Decarboxylase 65 Peptides Presented by the Type I Diabetes-Associated HLA-DQ8 Class II Molecule Identifies an Immunogenic Peptide Motif
Authors: Herman Ann E. , Tisch Roland M. , Patel Salil D. , Parry Sarah L. , Olson Jennifer , Noble Janelle A. , Cope Andrew P. , Cox Brett , Congia Mauro , McDevitt2 Hugh O.
Published: January 2023

Particular HLA class II allelic sequences are associated with susceptibility to type I diabetes. To understand the mechanism, knowledge of the molecular nature of the specific TCR/peptide/class II interactions involved in the disease process is required. To this end, we have introduced the diabetes-associated human class II HLA-DQ8 allele (DQA1*0301/DQB1*0302) as a transgene into mice and analyzed T cell responses restricted by this molecule to an important Ag in human diabetes, human glutamic acid decarboxylase 65. Hybridomas were used to determine the particular peptides from this Ag presented by HLA-DQ8 to T cells and to map the core minimal epitopes required for T cell stimulation. Analysis of these core epitopes reveals a motif and relevant features for peptides that are immunogenic to T cells when presented by HLA-DQ8. The major immunogenic epitopes of glutamic acid decarboxylase 65 do not contain a negatively charged residue that binds in the P9 pocket of the HLA-DQ8 molecule. PBMC from HLA-DQ8+ diabetic and nondiabetic individuals respond to these peptides, confirming that the mouse model is a useful tool to define epitopes of autoantigens that are processed by human APC and recognized by human T cells.
Rapid formation of T cell extended processes (TCEPs) in CNS infiltrating virus specific CD8 T cells (128.21)
Authors: McDole Jeremiah R. , Danzer Steve C. , Pun Raymond Y.K. , Pirko Istvan , Johnson Aaron J.
Published: January 2023

Expansion of an effective antiviral CD8 T cell response is critical for protection from Theiler's murine encephalomyelitis virus (TMEV) induced demyelinating syndrome. However, the specific CNS cell types engaged by these protective CD8 T cells in TMEV resistant strains remain unknown. We employed confocal microscopy to visualize the morphology, migration and specific cellular interactions of GFP+ CD8 T cells specific for the Db:VP2 121-130 epitope. These epitope specific, adoptively transferred CD8 T cells polarized CD8 and granzyme B towards TMEV infected neurons. Furthermore, epitope specific CD8 T cells presented with atypical cellular morphologies, including long cellular processes with varying thickness. Using live tissue imaging we determined these T cell extended processes (TCEPs) formed rapidly and could be partly attributed to T cell migration through tissue. This study is also the first to document and measure the rapid formation of TCEPs on antiviral CD8 T cells using real time imaging in the CNS.
Reconsidering the evolutionary significance of viral immune evasion (128.33)
Authors: Hill Ann B.
Published: January 2023

As do other herpesviruses, cytomegaloviruses (CMVs) all encode genes that interfere with the MHC class I pathway of antigen presentation to CD8 T cells. An extraordinary variety of molecular mechanisms have evolved to interfere with this pathway, and abundant experimental evidence indicates that these genes effectively impair the ability of CD8 T cells to recognize and kill virus-infected cells. The ubiquity of these genes argues that the virus is under strong selective pressure to keep them, and hence that they must provide a real evolutionary advantage. Paradoxically, however, viruses lacking MHC I immune evasion genes appear almost completely unimpaired in their ability to establish acute infection, and to persist for the life of the host. I propose that the apparent paradox results from considering immune evasion in terms of an evolutionary struggle between viruses and their hosts. Most individuals are infected with multiple strains of CMV. I suggest that immune evasion has evolved because it confers selective advantage to CMV strains in their intense evolutionary competition with other strains for access to an ultimately limiting resource. CD8 T cells provide some impediment to accessing this resource, but in most circumstances are not limiting. Hence, avoiding CD8 T cells is primarily of selective advantage when there is competition for access to resources. This concept leads us to focus on identifying limiting resources, and quantifying the degree of hindrance imposed by immune elements, when modeling viral pathogenesis.
The Role of Complement Receptor CD21/35 in a Murine Model of Chronic Wasting Disease (129.21)
Authors: Michel Brady Allen , Zabel Mark
Published: January 2023

Prion accumulation and replication occur in lymphoid follicles or inflammatory foci containing follicular dendritic cells (FDCs). FDCs accumulate immune complexes through their complement receptors CD21/35 and Fc receptors. These receptors, specifically CD21/35, have also been shown to play a critical role in peripheral prion pathogenesis in mouse scrapie models. In this study we examined feces from mice at 0, 1, 3, 6, and 8 days after oral inoculation for PrPCWD to determine whether prion retention and shedding depend on complement receptors CD21/35. We also plan to evaluate if CD21/35 can co-localize with CWD prions in the spleen at time points 0, 1, 2, 4, and 16 hours post i.p. inoculation using conventional Western Blotting and confocal microscopy.
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

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

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<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

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

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

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

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

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

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

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

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

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

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

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

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)
C5b-9 activated, Kv1.3 channel-sensitive Akt pathway mediate oligodendrocyte proliferation (135.7)
Authors: Tegla Cosmin A , Cudrici Cornelia , Singh Anil , Rozycka Monica , Rus Violeta , Judge Susan I.V. , Rus Horea
Published: January 2023

Voltage-gated Potassium (Kv) channels are involved in regulation of growth factors induced cell proliferation. Previously we showed that C5b-9 induced cell cycle activation in oligodendrocytes (OLG) but had yet to investigate the role of Kv channels. Here, we examined the effect of C5b-9 on Kv channel expression and function. OLG Kv channels were identified by recording whole-cell currents and their expression confirmed by western blotting. OLG exposure to C5b-9 significantly increased Kv1.3 and Kv1.4 expression while C5b6 had no effect. However, C5b-9 induced expression of OLG Kv1.3 and Kv1.4 was blocked by 4-aminopyridine, while pretreatment with the recombinant scorpion toxin rOsK-1, a specific Kv1.3 inhibitor blocked the expression of Kv1.3 alone. Since both ERK and Akt pathways are known to be induced by C5b-9 we assessed the possible role of Kv1.3 in activation of these kinases. rOsK1 inhibited Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. These data strongly suggest a role for Kv1.3 in controlling Akt activation induced by C5b-9. rOsK-1 significantly inhibited DNA synthesis induced by C5b-9 in OLG, indicating that Kv1.3 plays an important role in C5b-9 induced cell cycle. In conclusion, our data suggest that Kv1.3 channel plays an important role in controlling cell cycle activation by affecting the activation of Akt induced by C5b-9.
The role of Eri-1 as a small RNA regulator in the immune system (136.43)
Authors: Abdul-Wajid Sarah , Thomas Molly , Ansel K. Mark
Published: January 2023

Mouse Eri-1 is a 3' to 5' exoribonuclease whose C.elegans counterpart has been shown to inhibit RNAi. We have explored a similar role for murine Eri-1 in T-cells; siRNA efficacy and miRNA levels are increased in Eri-1 deficient T-cells. Reconstitution with wild-type Eri-1 via a retroviral construct returned miRNA levels to their wild-type quantities. In addition to validating a small RNA regulatory role for mouse Eri-1, we are exploring various immunological effects of Eri-1 deficiency. Eri-1 deficient T-cells exhibit an increased propensity to differentiate into Th2 cells in vitro and germinal center formation is altered in the absence of Eri-1.
CD8+ T cells induce severe thyroid epithelial cell hyperplasia (TEC H/P) and fibrosis (137.10)
Authors: Yu Shiguang , Sharp Gordon C. , Braley-Mullen Helen
Published: January 2023

IFN-γ -/-NOD.H-2h4 mice develop TEC H/P and fibrosis characterized by abnormal proliferation of thyrocytes and infiltration of thyroids by CD4+ and CD8+ T cells. Mice with severe TEC H/P produce anti-mouse thyroglobulin (MTg) autoantibodies, and their splenocytes transfer severe TEC H/P to SCID recipients, indicating that TEC H/P has an autoimmune basis. The goal of this study was to determine what cells transfer severe TEC H/P and fibrosis. Splenocytes from donors with severe TEC H/P cultured 72 hr with MTg consistently transferred more severe TEC H/P than the same number of freshly isolated cells, indicating that cells able to transfer severe TEC H/P can be activated in vitro. To determine what cells are important for transfer of TEC H/P, spleens of IFN-γ -/- mice with severe TEC H/P were activated in vitro, and CD4+ and CD8+ T cells were isolated and transferred to SCID recipients. Recipients of CD8+ cells had more severe TEC H/P and more fibrosis than recipients of CD4+ T cells or unseparated splenocytes. They had few infiltrating CD4+ cells, suggesting that CD8+ T cells are primary effector cells for severe TEC H/P and fibrosis. Recipients of CD4+ T cells developed mild hyperplasia and minimal fibrosis. Studies are in progress to determine what molecules secreted by CD8+ T cells mediate TEC H/P and fibrosis, and if CD4+ T cells are required for activation of CD8+ T cells. (Supported by VA Merit Review and NIH R56 AI074857).
The Adaptor Protein Shc Plays a Key Role in Early B Cell Development (138.22)
Authors: Deusterman Amber Jin , Bender Timothy P , Ravichandran Kodi S
Published: January 2023

B cell development is a highly ordered process that requires immature cells to pass through multiple checkpoints to advance through development. Successful expression and productive signaling via antigen and cytokine receptors are required at specific stages of development. The adaptor protein Shc is phosphorylated on three critical tyrosine residues and facilitates signaling downstream of multiple receptors, including the pre-T cell receptor. However, its role in B cell development is currently unknown.

Here, using a Cre-loxP mediated approach, we inducibly expressed as transgenes either wild type Shc or a mutant form Shc (ShcFFF, where three critical tyrosines have been mutated). Igα-Cre-mediated induction of ShcFFF expression (but not wt Shc) at the common lymphoid progenitor stage resulted in a severe block at the pre-pro-B to pro-B transition, with diminished numbers maintained throughout B development. Expression of ShcFFF at later stages of B cell development (via CD19-Cre) showed no defect in B cell numbers, emphasizing the importance of Shc during early B development. Mechanistically, Shc appears to affect B cell development at the level of cytokine dependent proliferation, as well as pre-BCR signaling. These results suggest a non-redundant role for Shc in early B development.