bluuebunny/crossref_metadata_2025 huggingface dataset:
Human α1-antitrypsin attenuates injury-induced Inflammation through interacting with high mobility group box-1 (HMGB1)
Authors: Ohayon David E , Schuster Ronen , Mizrahi Mark I , Lior Yotam , Baranovski Boris M , Shahaf Galit , Lewis Eli C
Published: January 2023

HMGB1 is involved in pathologies such as cellular injury and autoimmunity. Indeed, upon binding to immune-related receptors, HMGB1 promotes an inflammatory response, such as that might take part in pancreatic islet destruction during both autoimmune diabetes and islet transplant rejection. Specifically, elevated circulating HMGB1 levels were reported in patients with type 1 diabetes, and increased release of HMGB1 was found to correlate with injury degree and islet allograft survival. The anti-inflammatory and immune-modulatory acute-phase protein human α1-antitrypsin (hAAT) promotes islet survival in both transplantation and autoimmune diabetes models. While AAT binds to damage-released proteins, such as gp96 and HSP70, its tissue protective mechanism remains poorly understood. We now extend this observation that AAT protective activity is related to its interaction with HMGB1. Clinical-grade AAT (Glassia, Kamada Ltd., Israel), diminished recombinant HMGB1-induced inflammatory responses in mouse pancreatic islets (while restoring disrupted insulin inflammation-mediated release). Similar behavior was depicted in HMGB1-stimulated peritoneal macrophages introduced to hAAT. In addition, hAAT modulated HMGB1-inducible surface expression by altering its distribution. Lastly, we show by immunoprecipitation that HMGB1 associated proteins was positive for the presence of AAT, this is also supported by a direct ELISA. Our findings suggest that hAAT is a naturally-occurring regulator of inflammatory responses mediated by extracellular HMGB1, such that preclude outcomes of islet transplantation. hAAT may therefore be considered for therapy of cell damage-mediated pathologies in a clinically-safe manner.
c-Jun N-terminal kinase 1 defective CD4+CD25+FoxP3+ cells prolong pancreatic allograft survival in type 1 diabetic mice
Authors: Tripathi Deepak , Cheekatla Satyanarayana S. , Venkatasubramanian Sambasivan , Paidipally Padmaja , Welch Elwyn , Tvinnereim Amy R. , Vankayalapati Ramakrishna
Published: January 2023

CD4+CD25+FoxP3+ cells (Tregs) inhibit inflammatory immune responses to allografts. Here we found that co-transplantation of allogeneic pancreatic islets with Tregs that are defective in JNK1 signaling prolong the islet allograft survival in the liver parenchyma of the T1D mice. Deletion of the JNK1 in Tregs increased the expression of anti-apoptotic molecules Mcl-1, Bcl-Xl and anti-inflammatory cytokine IL-10 production. JNK1−/− Tregs persist for long period in the transplanted liver and prolong the islet allograft survival compared to WT Tregs. JNK1−/− Tregs demonstrated improved control of allo-inflammation compared to WT Tregs in the T1D liver upon co-transplantation. JNK1−/− Tregs specifically inhibit IL-17 and IL-21 medited alloimmune response. Inhibition of alloimmune response by JNK1−/− Tregs independently mediated through the LAG3 on its cell surface and the increased production of IL-10. Our study identifies a novel role of JNK1 signaling in the Tregs function and co-transplantation of JNK1 deficient Tregs enhances the islet allograft survival in the liver parenchyma of T1D mice.
Spontaneous Agglutination in Typhoid and Paratyphoid Cultures and Its Bearing Upon Absorption of Agglutinins
Authors: Teague Oscar , McWilliams Helen I.
Published: January 2023

In carrying out some experiments involving the injection of living typhoid bacilli in rabbits and the plating several hours later of the rabbits' blood in nutrient agar, we observed that some of the colonies developing upon the plates showed spontaneous agglutination when rubbed up in a drop of saline solution upon a glass slide. A number of the colonies were fished into small tubes of nutrient broth and after twenty-four hours' incubation yielded, instead of the usual turbid growth characteristic of the typhoid bacillus, flocculi throughout the broth and a flocculent sediment. We at first thought that this change in the typhoid strain had been brought about by its sojourn in the body of the rabbit; however, upon plating the original typhoid culture and fishing a number of the colonies into broth we observed a similar type of growth in some of the tubes.
The Absorption of Antigen: A Repetition of Work Reported by Doerr and Pick
Authors: Smith G. H. , Cook M. W.
Published: January 2023

The question of the parenteral absorption of antigen has been studied by Doerr and Pick (1). By comparing the reaction in normal and sensitized animals they hoped to determine whether or not in sensitized animals the anaphylactic poison was produced from the antigen. Doerr and Pick injected rabbits, sensitized and normal, with anti-cholera horse serum intravenously. The rabbits were bled at intervals and the sera tested for precipitable horse serum and for agglutinins for the cholera vibrio. Their titrations for precipitinogen and agglutinins indicate, in their opinion, that there is no essential difference in the rate of disappearance of antigen from the circulation of the normal and sensitized rabbits.

To approach the subject from a somewhat different point of view, Doerr and Pick measured the rate of absorption of antigen from the peritoneal cavity of normal and sensitized guineapigs. The amount of antigen absorbed into the circulation was titrated as in the rabbit experiments.
The Action of Formaldehyde on Diphtheria Toxin
Authors: Bunney William Edward
Published: January 2023

Additional evidence is offered which indicates that toxoid formation is not the result of the direct action of free formaldehyde on the diphtheria toxin. Results are obtained which suggest that the formation of toxoid by the action of formaldehyde on diphtheria toxin may depend upon a compound resulting from the reaction between formaldehyde and the amino group in an amino acid. A possible method for making acceptable toxoid from lower grade diphtheria toxins is suggested.
Advantages and Disadvantages of the Buffered Diluent for Diphtheria Toxin
Authors: Bunney William E. , White Benjamin
Published: January 2023

Previous work by Bunney and White (1) on the buffered diluent described by Glenny, Pope and Waddington (2) substantiated their description of its properties to a degree encouraging further study. Diphtheria toxin diluted in this borax-boric acid solution1 did not lose toxicity in ten weeks at room temperature. Inasmuch as such a diluent might be valuable to the research scientist interested in diphtheria toxin, as well as to those concerned with either the preparation or the use of diphtheria toxin in the Schick test the subject was investigated further. The additional data gained in this study are presented in the present paper.

I. Use of the Glenny Diluent in the Study of Diphtheria Toxin

There is need for a suitable diluent for use in studying the chemistry of diphtheria toxin, in which purified toxin could be dissolved in high or low concentrations with equal confidence, with little fear of deterioration under convenient precautions.
The Toxicity of Certain Normal Sera for the Guinea Pig
Authors: Field Madeleine E.
Published: January 2023

The effect of age upon the primary toxicity of normal serum has usually been referred to in the literature in the course of other observations. Zinsser (1), in this connection, stated that goat serum which had stood at room temperature for three days was still hemolytic and hemagglutinating when injected in doses of from 2 to 5 cc. into rabbits but had often lost its power to produce respiratory distress and death. Zipf and Wagenfeld (2) observed a fall in the systemic blood pressure of rabbits only after an intravenous injection of 2 cc. of very fresh serum. Hyde (3) noticed that ox serum became inactive for the guinea pig with age, losing its hemolytic activity and its ability to produce necrotic skin areas and cause death after standing at room temperature for ten days. DeKruif (4), also, states that normal serum which had been freed from contact with the clot will remain toxic for only about twenty-two hours.
Heredity of the Agglutinogens M and N of Landsteiner and Levine
Authors: Wiener Alexander S. , Vaisberg Maurice , Director Mas Lederer
Published: January 2023

Summary and Conclusions

Specimens of blood of 131 families with 642 children were examined for the agglutinogens A, B, M, and N. The agglutinogens M and N seem to be inherited entirely independently of the agglutinogens A and B, confirming the observation of Landsteiner and Levine.

Concerning the heredity of the agglutinogens M and N, two exceptions to the theory of a single pair of allelomorphic genes were found, but in neither case could the possibility of illegitimacy be excluded. Concerning the heredity of the agglutinogens A and B, one exception to the Bernstein theory was found, but here again the possibility of illegitimacy could not be excluded. Considering the size of the series of cases studied (642 children), a certain percentage of illegitimacies is rather to be expected. Since the agglutinogens A, B, M, and N together can detect one third of all illegitimacies, the three exceptions found would imply the presence of only nine illegitimacies. We therefore believe that our results speak well for the theories of heredity discussed.
Neurokinin A augments SynCAM expression and IL-10 release from mast cells
Authors: Sumpter Tina L. , Alwine Jeremy , Rao Mekhala , Falo Louis D. , Larregina Adriana T.
Published: January 2023

Cutaneous mast cells (MCs) initiate innate and adaptive immune responses. In the skin, MCs reside proximal to peripheral nerve fibers, forming a functional synapse mediated, in part, by Synaptic Cell Adhesion Molecule (SynCAM). Within the synapse, MC function is likely affected by the release of soluble neuropeptides. We and others have demonstrated that the immune functions of MCs are affected by the neuropeptides from the tachykinin family. Substance P promotes MC-inflammatory functions, whereas the role of neurokinin A (NKA) in MC biology is hereto unknown. In this study, the capacity for NKA to regulate MC stasis and IgE-initiated MC activation was evaluated. In bone marrow derived (BM) MCs, NKA increased expression of SynCAM, but not expression of other cell adhesion molecules, such as ICAM or NCAM. In IgE-activated MCs, NKA did not affect SynCAM expression. However, NKA promoted IL-10 secretion resulting in the inhibition of IgE-initiated STAT5 phosphorylation and nuclear localization, leading to the inhibition of type 2 cytokine release. STAT5 phosphorylation was restored in NKA treated BMMCs by neutralizing IL-10. In vivo, intradermal administration of NKA increased the density of MCs in the skin. In spite of this increase in MCs, NKA reduced IgE-initiated passive cutaneous anaphylaxis. Our data indicates that NKA strengthens the formation of the MC-neuron synapse and down-regulates MC pro-inflammatory functions.
Resveratrol protects experimental Staphylococcus aureus enterotoxin B (SEB)-induced acute liver injury (ALI) by regulating microRNAs associated with myeloid-derived suppressor cells
Authors: Singh Narendra Prasad , Kadhim Sabah , Singh Udai P. , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Resveratrol (RES) possesses anti-inflammatory, anticancer, and antioxidant properties. Because of anti-inflammatory property of RES, we investigated whether RES can attenuate Staphylococcal enterotoxin B (SEB)-induced acute liver injury (ALI) in mice. SEB exposure causes food poisoning as well as toxic shock syndrome leading to mortality. We observed that SEB caused ALI in female mice and there was an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. RES (50 mg/kg body weight) treatment attenuated SEB-induced ALI and significantly decreased AST levels, inflammatory cytokines and chemokines, and cellular infiltration in the liver. Interestingly, RES treatment led to an increase in myeloid derived suppressor cells (MDSC) in SEB-immunized mice. Upon examination of microRNAs (miRs) profile of liver infiltrating mononuclear cells (MNCs), there was marked change in microRNA expression profile in mice exposed to SEB and treated with RES. Specifically, there was a decrease in the expression of miR-130a and miR-185 in MNCs from SEB+RES treated mice when compared to SEB+VEH treated group. Furthermore, there was an inverse relationship between miR-130a and miR-185 with CSF-1 gene expression in liver MNCs. The data presented demonstrate for the first time that RES can effectively attenuate SEB-induced ALI by inducing the proliferation of MDSC. In summary, the beneficial effects of RES may be mediated by changes in miRs regulating pathways leading to the generation of immunosuppressive cells and/or production of anti-inflammatory cytokines suppressing SEB-induced ALI.
p38 MAPK is critical for nuclear translocation of IRF-7 during CpG-induced type I IFN expression in human plasmacytoid dendritic cells
Authors: Wang Qi , Reszka-Blanco Natalia , Cheng Liang , Li Guangming , Zhang Liguo , Su Lishan
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are the major source of type I IFN (IFN-I) in response to viral and bacterial infection that are recognized through the Toll-like receptor 7 (TLR7) or TLR9 signaling pathway. TLR9 agonist, oligodeoxyribonucleotides containing unmethylated CpG motifs (CpG), stimulates pDC to produce IFN-I but the mechanism keeps incompletely defined. Here, we established a human pDC cell line with the gLuc under control of the IFNa4 promtoer/enhancer, and utilized the high throughput assay to screen for protein kinase inhibitors to identify small molecules targeting IFN-I induction pathway in pDCs. We identified p38 MAPK inhibitors that show ability to inhibit IFN-I expression in pDCs without cytotoxicity upon CpG stimulation. p38 MAPK inhibitor inhibits IFN-I expression in a dose-dependent fashion in vitro and significantly suppressed IFN-I expression in humanized mice upon CpG treatment. There was a dramatic defect in the nuclear translocation of IFN regulatory factor 7 (IRF-7) in the prescence of p38 MAPK inhibitor. Therefore, p38 MAPK enhances IRF-7 nuclear translocation and IFN-I expression in human pDCs. These findings identify p38 MAPK as a target for modulating pDC activity.
Tuning of TCR signaling pathways in chronically stimulated CD4+ T cells in vivo
Authors: Matson Courtney Alexandra , Singh Nevil J.
Published: January 2023

Prolonged antigenic stimulation is known to tune down a T cell’s ability to respond to antigen. It is not yet fully understood at what level this tuning occurs. Using an adoptive transfer model where transgenic T cells are continuously stimulated by a persistent self-antigen in vivo, we have previously found that tuned T cells show poor responses to antigen-restimulation; however, stimulation with PMA and ionomycin is mostly intact. This implies the negative regulation during tuning operates mostly on the TCR proximal signaling machinery; upstream of PKC activation and Ca2+ flux. Ex vivo, tuned T cells show decreased Zap70 kinase activity. Additionally, downstream of Zap70, chronically stimulated cells have reduced expression of both c-Fos and c-Jun leading to decreased assembly of the AP-1 transcriptional complex. Finally, chronically stimulated cells have increased expression of IκBβ, which contributes to a decrease in NFκB signaling. An important characteristic of T cells in this model is that the loss of responsiveness is mostly reversible. Tuned T cells, when adoptively transferred into an antigen-free host recover most of their functionality and can re-respond to antigen. However, acute antigen removal for at least 48 hours in vitro retains an unresponsive state suggesting the negative regulation is stable for a significant period after removal from antigen. Importantly, the stability of tuning ex vivo allows us to screen for pharmacological agents capable of improving antigen responsiveness in these T cells. Such agents may prove therapeutic for increasing T cell responses in cancer and chronic infections.
Platelet activating factor as a novel danger signal for activation of NLRP3 inflammasome
Authors: Deng Meng , Brickey W. June , Guo Haitao , Tam Jason W. , Johnson Brandon M. , New J. Stewart , Koller Beverly H. , Kearney John F. , Ting Jenny P.-Y.
Published: January 2023

Platelet activating factor (PAF) is a biologically active phospholipid that promotes pathophysiological inflammation through binding to a unique G-protein-coupled seven transmembrane receptor (PAFR). Increased PAF level has been correlated with a number of inflammatory disorders, yet the molecular mechanism underlying the pro-inflammatory function of PAF remains incompletely elucidated. Here we showed PAF activates canonical NLRP3 inflammasome, resulting in ASC oligomerization, caspase-1 processing, and IL-1β and IL-18 maturation. PAF-induced IL-1β maturation is abrogated in macrophage from Nlrp3−/−, Asc−/−, Caspase-1−/− mice, but not Nlrc4−/−, Nlrp1−/−, Aim2−/−, Caspase-11−/− mice. In addition, NEK7, a new component of the NLRP3 inflammasome, is also essential for PAF induced IL-1β maturation. PAF activation of the NLRP3 inflammasome is dependent on calcium influx, potassium efflux, but independent of lysosome cathepsin, mitochondrial reactive oxygen species (ROS), and necroptosis. Surprisingly, PAFR is not essential for PAF induced NLRP3 inflammasome activation. Physiologically, PAF induced shock is protected in NLRP3 inflammasome component deficient mice. Together, our findings reveal a new PAFR independent pathway for detecting PAF which represents an unprecedented danger signaling function of PAF, and provide new insight for understanding the pathogenesis of PAF-mediated inflammatory disorders.
The Heat Shock Protein, gp96, initiates inflammatory responses by activation of inflammasome signaling platforms in macrophages
Authors: Wang Yifei , Binder Robert J.
Published: January 2023

Several members of the heat shock protein family elicit immune responses which are, in part, a result of activation of antigen presenting cells. Activated antigen presenting cells respond to these immunogenic HSPs by up-regulating expression of co-stimulatory molecules and secretion of a panel of pro-inflammatory cytokines including IL-1b. We have tested here the requirement for activation of inflammasomes in the release of IL-1b by one immunogenic HSP, gp96. Our results show that macrophages activate NLRP3 inflammasomes in response to gp96, by K+ efflux. Using a gp96 secretion system we show that these events initiate inflammatory conditions in mice, in the absence of additional known inflammasome activators or infection. These results are important because the document a novel mechanism by which proteins of endogenous origin, the HSPs, can modulate a sterile inflammatory response following their release from cells are involved in host protection against infections by regulating IL-1b secretion via inflammsome activation.
A natural polymorphism of EsxH in Mycobacterium tuberculosis disrupts the immunodominant CD8 T cell response.
Authors: Sutiwisesak Rujapak , Carpenter Stephen , Behar Samuel M.
Published: January 2023

Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB), is an intracellular bacterium that evades antibacterial immunity. As control of Mtb requires T cell immunity, most vaccine strategies focus on eliciting protective T cells. How Mtb benefits from promoting T cell activation or evading T cell recognition is not certain. Here, we took advantage of a naturally occurring Mtb polymorphism to address this question in mouse model.

EsxH elicits an immunodominant CD8 T cell response. At the peak of immunity against Mtb in C57BL/6 mice, 30–50% of CD8 T cells are specific for EsxH4–11. Clinical Isolate 667 (CI667) is an Mtb strain containing a polymorphism in EsxH that has a single amino acid change of WT EsxH4–11 epitope from IMYNYPAM into IMYNYPTM (A10T). We hypothesized that this naturally occurring polymorphism would alter the hierarchy of CD8 T cell responses elicited by Mtb.

Following Erdman (WT) Mtb infection of C57BL/6 mice, CD8 T cells respond to three distinct epitopes, including the reported immunodominant EsxH4–11 epitope. In contrast, there was no response to any of EsxH epitopes after CI667 infection. Instead, the subdominant response to the 32C antigen became dominant. In BALB/c mice, CI667 stimulated a reduced but detectable CD8 response to EsxH20–28; the CD4 response to EsxH74–88 was similar to WT Mtb infection.

These results show that naturally occurring polymorphisms can have drastic effects on T cell immunity. We hypothesize that EsxH acts as a decoy antigen, and distracts the CD8 response from focusing on antigens that are presented by infected macrophages and protective. We are in the process of testing this hypothesis by developing isogenic Mtb strains that express EsxHErdman or EsxHCI667.
IFN-γ synergizes with polyI:C to reduce growth of murine and human cancer cell growth, with concomitant reductions in cyclinD1 and PD-L1.
Authors: Petro Thomas M. , Guinn Zachary P.
Published: January 2023

We saw that IFN-γ synergizes with poly I:C, a TLR3 agonist, in ISG54 expression. Interestingly, ISG54 induction correlated with reduced B16 melanoma growth. To study IFN-γ synergism with other PRR agonists, we treated B16 cells, as well as UM-SCC1 and UM-SCC38 human squamous cell carcinoma cells, with IFN-γ (20 ng/ml) with or without Loxoribine (Lox; TLR7), ODN1585 (TLR9), Zymosan (TLR2), cGAMP (STING), LPS (TLR4), or poly I:C (TLR3). IFN-γ or Lox alone reduced growth of each tumor cell type, poly I:C reduced growth of SCC1 cells, while Lox and poly I:C synergized with IFN-γ to reduce tumor cell growth. Moreover, SCCs required treatment with the combination of IFN- γ/poly I:C for 24 h to achieve growth cessation, while B16 cells required treatment for 48 h. IFN-γ alone induced ISG54, while no PRR agonist alone induced ISG54 in B16 cells, as determined by western blot. ISG54 was induced in SCC1 by poly I:C. All PRR agonists synergized with IFN-γ for greater levels of ISG54 in B16 cells. We also evaluated cyclin D1, required for cell cycle G1 progression, and p15, which inhibits G1 progression. IFN-γ or poly I:C alone had no effect on cyclin D1 or p15. However, IFN-γ plus poly I:C decreased cyclin D1 levels. We also evaluated PD-L1, an immune checkpoint protein, in B16 cells treated with IFN-γ ± polyI:C. IFN-γ or polyI:C alone had no effect PD-L1 levels in B16 cells, but the combination of IFN- γ plus poly I:C decreased PD-L1. Thus, IFN-γ plus polyI:C sharply reduce growth of mouse and human tumor cells, which is accompanied by reductions in Cyclin D1, which is involved in cell cycle progression, and PD-L1, which decreases antitumor immune responses. Thus IFN-γ plus polyI:C could be an effective adjuvant to cancer immunotherapy.
Molecular characterization of combination pattern recognition receptor agonist exposure on dendritic cell responses
Authors: Brown Deborah M. , Lampe Anna T. , Hain Jacey L. , Pannier Angela K.
Published: January 2023

Current Influenza A virus (IAV) vaccines induce antibody formation and therefore fail to induce heterosubtypic protection needed to overcome viral antigenic shift and drift. New vaccine strategies have used pattern recognition receptor (PRR) agonists as adjuvants to improve both B and T cell memory; however, the effect of using combined PRR agonists on vaccine induced memory is unknown. We have demonstrated that vaccination with hemagglutinin (HA) antigen with combined TLR4 and TLR9 agonists promotes protection from lethal infection more effectively than either agonist alone or HA with the oil-in-water adjuvant Addavax. To begin to dissect the molecular changes that occur in dendritic cells exposed to combination agonists, we used qPCR, multiplex assays and RNA-seq analysis to characterize the changes in gene expression in response to combined TLR4 and TLR9 stimulation. We found low dose TLR4 and TLR9 agonists in combination could enhance IL-6, IFN-β and CCL4 mRNA over either agonist alone. Analysis of in vivo early cytokine responses 6 hours post intramuscular vaccination, corroborated in vitro results, demonstrating increased levels of serum IL-6, TNF-α, CCL4 and CCL2 in response to combined agonists. To further probe the mechanistic basis for increased protection with combined agonists, transcriptome analysis was performed on dendritic cells after treatment with single or combination TLR4 and TLR9 agonists. Genes such as CXCL10, IL-12 and a number of interferon stimulated genes (ISGs) were shown to be increased following combination agonist stimulation. Further, gene ontology analysis and ingenuity pathway analysis will be performed to gain insight into the complex regulation and mechanism of dual TLR4 and TLR9 agonists.
Mucosal immunity and protective efficacy of inactivated influenza virus vaccine is improved by intranasal chitosan nanoparticle delivery in pigs
Authors: Dhakal Santosh , Renu Sankar , Ghimire Shristi , Lakshmanappa Yashavanth Shaan , Hogshead Bradley T. , Ruiz Ninoshkaly F. , Krakowka Steven , Lee Chang-Won , Gourapura Renukaradhya J.
Published: January 2023

Currently used inactivated influenza virus vaccines administered by intramuscular injection provide homologous protection, but the much required heterologous protection against constantly evolving field viruses is limited, attributed to lack of induction of mucosal IgA and cellular immune responses in the respiratory tract. We developed a novel vaccine delivery platform by encapsulating swine influenza A virus (SwIAV) killed/inactivated antigen (KAg) in mucoadhesive, biodegradable, polycationic chitosan nanoparticles (CNPs-KAg). The CNPs-KAg vaccine (<500nm in size) was administered through intranasal route as mist to nursery pigs twice and challenged with a zoonotic and virulent heterologous SwIAV. Pigs vaccinated with CNPs-KAg had enhanced mucosal IgA response throughout the respiratory tract against homologous (H1N2), heterologous (H1N1) and heterosubtypic (H3N2) SwIAV. In addition, the frequency of cytotoxic T cells and T helper/memory cells were increased in CNPs-KAg vaccinated pigs, together with increased level of systemic and mucosal recall IFNγ response. In heterologous virus challenged pigs, CNPs-KAg vaccine reduced the pneumonic lesions and virus titers in lungs and significantly reduced the nasal viral shedding. In conclusion, intranasal delivery of chitosan nanovaccine elicited better cross-protective mucosal IgA and cellular immune responses in the respiratory tract and reduced viral load and shedding, indicating that chitosan based influenza nanovaccine might be an ideal candidate vaccine against SwIAV infections under field conditions.
Metabolic activity as a surrogate marker of immune cell activation following vaccination
Authors: Waickman Adam T. , Friberg Heather , Jarman Richard , Currier Jeffrey R.
Published: January 2023

The identification and quantification of lymphocytes activated in response to vaccination is a critical first step in characterizing the efficacy of any candidate vaccine platform. Traditionally, this has been accomplished by monitoring the upregulation of defined activation markers on circulating lymphocytes by flow cytometry, or the measuring the production of effector cytokines following ex vivo stimulation. However, there is significant variability in both the kinetics and magnitude of activation marker up-regulation or cytokine production following stimulation even within the same type of lymphocyte. Therefore, traditional methodologies for identifying activated lymphocytes may offer a significantly skewed perspective on exactly which immune cells are responding to vaccination or natural infection. Herein, we examine the utility of measuring metabolic activity of circulating lymphocytes as a marker of activation following immunization with a live-attenuated dengue vaccine. Unlike any other marker, upregulation of metabolic activity is a universal indicator of lymphocyte activation which can be detected quickly after stimulation and persists throughout the functional stage of an immune response. We examine several methods of monitoring the metabolic state of both T cells and NK cells following immunization, and compare the size and diversity of the immune response generated relative to standard phenotyping protocols.
Immunomodulatory activity of 2-deoxyguanosine nanoparticles in DNA vaccines
Authors: Santos-Colón Saritza , Ramírez Maite , Miranda Eric , Reyes Nelson , Martinez Osmarie , Acosta Maxier , Rivera José , Otero Miguel
Published: January 2023

DNA vaccines induce protective immune responses against several pathogens and tumor cells, but strategies to improve their effectiveness are still under study. Only two adjuvants have been licensed by the FDA, which increases the needs for the discovery of novel adjuvants for HIV vaccines and other infectious diseases. In this regard, we study the immunomodulatory properties of 2-Deoxyguanosine-based nanoparticles (SHS) in DNA vaccines coding for HIV Gag (NL4-3 clone) and Vaccinia Virus Western Reserve (VVWR) A27 proteins. The sequence of A27L was codon optimized and linked to the Outer Domain-1 (OD1) of gp120 HIV-1 protein sequence serving as an additional molecular adjuvant. In this study, female Balb/c mice were immunized three times intramuscularly with plasmids (pGag and pOD1-A27L). Then, seven days after the last dose, cellular and humoral immunity were evaluated through ELISpot and ELISA assays. We found significantly higher frequencies of IFN-γ-producing cells in mouse immunized with pGag and pOD1-A27L co-formulated with SHS versus controls. We also found higher significant levels of IFN-γ over IL-4 in supernatant of splenocytes isolated from mice receiving pGag and pOD1-A27L combined with SHS. Moreover, administration of pOD1-A27L+SHS stimulated a significant higher production of total IgG in mice when compared to Naïve group. Here, we demonstrated that pGag and pOD1-A27L vaccines co-formulated with SHS nanoparticles successfully activate adaptive immunity through production of Th-1 cytokines and/or IgG antibodies. As this type of immunity is essential to clear many viral infections, these data support the evaluation of SHS nanoparticles as a novel adjuvant for DNA vaccines against multiple viral infectious agents.
Safety, tolerance, and effects on alveolar macrophages of a nebulized TLR agonist combination in foals
Authors: Bordin Angela Ilha , Bray Joselyne , Rocha Joana , Markesich Diane , Cohen Noah
Published: January 2023

The intracellular pathogen R. equi causes pneumonia in foals and people that closely resembles that caused by Mycobacterium tuberculosis (Mtb). Development of resistance to antimicrobials by R. equi and Mtb represents a major health problem for veterinary and human medicine. There is a need for control methods and treatment alternative to the use of antibiotics. Our alternative approach is to use a TLR2/6 and TLR9 agonist combination known as PUL-042 to stimulate innate immunity in the lungs. Our objectives were to evaluate safety and ex vivo efficacy of PUL-042 (Pulmotect Inc, Houston, TX) in newborn foals. Ten newborn foals were divided in 2 groups (PUL-042 and saline control) and submitted to a bronchoalveolar lavage (BAL) on day 2 of age, 6 nebulizations in a 2-week interval and a second BAL 24 h after the last nebulization. BAL cells were infected with a virulent strain of R. equi, and then lysed at either 0 h (T0) and after 48 h of culture (T48). Supernatants were saved for determination of INF-γ, IL-1β, and IL-10 concentration by ELISA. PUL-042 did not induce either adverse effects or airway inflammation in foals, as assessed by clinical examination and BAL cytology. At the tested doses, nebulization of PUL-042 did not significantly (P > 0.05) affect ex vivo killing capacity of R. equi by AMs of foals at 2 and 21 days of age relative to controls. Results of cytokines are pending. Repeat nebulization of newborn foals with PUL-042 did not induce any adverse effects nor an inflammatory response in the lungs. At the dose tested, PUL-042 did not affect the killing capacity of R. equi by AMs, but its effects on other immune cells, and at other doses, need further evaluation.
ICOS-mediated phosphoinositide 3-kinase signaling controls induction and maintenance of collagen-induced arthritis
Authors: Panneton Vincent , Yazdchi Sahar Bagherzadeh , Witalis Mariko , Chang Jinsam , Suh Woong-Kyung
Published: January 2023

The inducible T-cell costimulator (ICOS) is a key costimulatory receptor facilitating differentiation and function of follicular helper T cells and inflammatory T cells. Rheumatoid arthritis patients were shown to have elevated levels of ICOS+ T cells in the synovial fluid suggesting a potential role of ICOS-mediated T cell costimulation in autoimmune joint inflammation. In this study, using ICOS knockout and knockin mouse models, we found that ICOS-mediated phosphoinositide 3-kinase (PI3K) signaling is required for the induction and maintenance of collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. For the initiation of CIA, ICOS-mediated PI3K signaling was critical for antibody production and expansion of inflammatory T cells. Further, we found that ICOS-PI3K signaling is important for maintenance of CIA in an antibody independent manner. Importantly, we found that a small molecule inhibitor of glycolysis, 3-bromopyruvate, ameliorates established CIA suggesting a potential link between ICOS-PI3K signaling and glucose metabolism. Thus, we identified ICOS-PI3K axis as a key signaling pathway that controls induction and maintenance of CIA and provide evidence that T cell glycolytic pathways can be potential therapeutic targets for rheumatoid arthritis.
The immune adaptor ADAP plays a pivotal role invariant natural killer T cell functions
Authors: Das Rupali , Gohl Trevor , Lee Hyun Hee , Mack Ryan , Abdelaziz Sami , Griffin Ryan
Published: January 2023

Invariant natural killer T cells (iNKTs) comprise a sub-lineage of T lymphocytes that express an “invariant” T cell receptor (TCR) with specificity for glycolipid antigens (Ag) such as a-galactosyl ceramide (aGC), when presented in complex with the MHC class I molecule CD1d. Following TCR engagement, iNKTs rapidly secrete cytokines and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells as well as directly kill tumor cells. Based on their dual functions as potent cytokine producers and tumor killers, iNKTs serve as a powerful tool for use in cell-based immunotherapies for cancer. Although TCR-CD1d interactions are generally required for iNKT cell functions, the signaling mechanisms that co-operate with the TCR to promote maximal iNKT cell responses remain poorly understood. Our previous studies demonstrate that the adaptor protein SAP and its signaling partner, Fyn (a Src-tyrosine kinase) is critical for iNKT cell functions. We recently investigated the role of ADAP (adhesion and degranulation promoting adaptor protein), a Fyn-binding protein in iNKT functions. We observe that aGC-induced iNKT cell activation, cytokine production, and bystander immune cell activation as well as anti-tumor response are remarkably reduced in ADAP-deficient (Adap−/−) mice. These data suggest that ADAP is a critical regulator of Ag-induced iNKT cell functions. Studies are currently underway to elucidate the mechanism(s) by which ADAP regulates iNKT cell functions. These studies will have an impact by establishing new paradigms for iNKT cell signaling and offering insights into how iNKTs can be best activated to enhance host immunity and treat cancer.
Suppressed plasmablast responses in febrile infants, including children with Kawasaki disease.
Authors: Hoffman Jonathon , Martin Meghan , Wrotniak Brian H. , Hicar Mark D.
Published: January 2023

Kawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Future studies on KD immune responses are hampered by a limited understanding of normal immune responses in these ages. Plasmablasts are a transitional form of B cells that lead to long-term plasma cells, and their levels rise in the peripheral blood after exposure to a foreign antigen. In adults, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD.

This study is focused on characterizing the normal plasmablast responses in the predominant age range of KD presentation. Children presenting to an urban pediatric emergency room who had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls.

Plasmablast numbers and timing of response are similar between infectious disease control and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children. Using deep sequencing, immunoglobluin variable gene usage and sequences will be compared between infants and older children.

In this single-time point survey, we demonstrate that infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed poor serum immune responses and maintenance of such responses in this age group.
Mitochondrial dynamics and metabolic programming in Th17 cell fate
Authors: Decker Carl William , Choi Clarice , Cave Si , Hernandez Jeniffer B.
Published: January 2023

Th17 cells have quickly become a paradigm of intense research interest, owing in part to their central role in mediating tissue-immune system communication, their demonstrable anti-tumor capacity, and their involvement in the pathogenesis of several autoimmune disorders. As with other effector Th1 and Th2 cells, they are members of a lineage derived from CD4 naïve cells, differentiating from their progenitors in response to immunological need. Recent research has revealed that differentiation of memory T-cells is dependent on the morphology of fluid mitochondrial networks, and in particular on remodeling dynamics that yield fused mitochondrial superstructures that favor oxidative phosphorylation-driven metabolism. In a similar fashion, our data suggest that Th17 T-cell differentiation is contingent upon highly fissed mitochondria network morphology, wherein a more independent arrangement of mitochondria and increased aerobic glycolsysis is employed. An important component of this research is the modulation of DRP-1, a GTPase that when phosphorylated in the cytosol initiates fission mitochondrial dynamics. Specifically, our data suggests that attenuation of DRP-1 action may play an important role in Th17 differentiation, and thus a better understanding of Th17 metabolism and how it affects immune cell fate, which at this time is poorly characterized.
Unilateral corneal nerve cutting induces bilateral dry eye disease in a murine model.
Authors: Kim Kyoung Woo , Lee Hyo Kyung , Ryu Jin Suk , Jeong Hyun Jeong , Yoon Chang Ho , Kim Mee Kum
Published: January 2023

Purpose

To investigate the effect of the severing of corneal basal nerve plexus in one eye on bilateral dry eye disease in a murine model.

Methods

6 week-old female BALB/c mice were divided into control (n=30) and nerve-cutting (NC) groups (n=30). The left cornea was partially incised with a 2.0mm circular trephine, through the upper two-thirds of the cornea stromal layer. Lissamine green corneal staining and tear volume measurements were performed every week for 4 weeks. At 2 and 4 weeks, dendritic cells, CD4+/CD8+ T cells, regulatory T cells in cervical lymph nodes, conjunctivocornea and spleens were analyzed by flow cytometry. With real-time RT-PCR, neuropeptides were also analyzed in intra-orbital lacrimal glands at 2 weeks.

Results

In bilateral eyes of mice in NC group, corneal staining grades were increased and tear volumes were decreased. CD11c+CD86hi, CD11c+MHCIIhi cells in conjunctivocornea were increased in trephined eyes at 2 weeks and in contralateral eyes at 4 weeks. CD69 were highly expressed in CD4+/CD8+ T cells in conjunctivocornea of trephined eyes at 4 weeks. CD8+IFNγ+ T cells were increased in contralateral cervical lymph nodes in NC group. Meanwhile, regulatory T cells in spleens were significantly suppressed in NC group. In addition, neuropeptide Y and neutrin were increased in intra-orbital lacrimal glands in NC group

Conclusions

It suggests that unilateral corneal nerve cutting may induce bilateral dry eye disease by entering activated dendritic cells into the bilateral eyes with a disturbance of immune privilege.
Intestinal epithelium intrinsic RARα signaling regulates gut barrier integrity via IL-18
Authors: Iyer Namrata R. , Vaishnava Shipra
Published: January 2023

Intestinal epithelium, overlying mucus layer and anti-microbial peptides together form a physico-chemical barrier crucial for homeostasis with the gut microbiota. Intestinal epithelial cells (IECs) are at the frontline of host-microbe interactions in the gut, yet how they modulate barrier function in response to dietary cues is poorly understood. Vitamin A and its key metabolite retinoic acid (RA), are crucial regulators of gut immune homeostasis. In this study, we employ a mouse model that expresses a dominant negative retinoic acid receptor exclusively in IECs (dnRARVillin-Cre) to establish a novel role for RA signaling in regulating the mucus barrier. The absence of RA signaling in colonic epithelium resulted in significant increase in mucus layer thickness indicating that RA regulates goblet cell function. Concomitant with this, we observed that in dnRARVillin-Cre mice there are more goblet cells and an enhanced spatial separation between host and the gut microbiota. Further, we showed that dnRARVillin-Cre mice have a diminished sensitivity to chemically induced colitis in comparison to wild-type mice. Quantitative RNA and protein analysis uncovered that gene transcription and protein activation and secretion of key pro-inflammatory cytokine IL-18 was severely impaired in epithelium of dnRARVillin-Cre mice. IL-18 has previously been shown to act as a negative regulator of goblet cell function in the intestinal epithelium. We now show that intestinal epithelium intrinsic RA signaling can modulate IL-18 levels to regulate the mucus barrier. Our results have thus disclosed a novel link between a diet derive metabolite and intestinal epithelial cell function that can be harnessed to modulate mucosal immunity.
The metabolic basis of resistance to Adoptive T Cell Therapy (ACT) in patients with solid tumors
Authors: Peng Weiyi , Cascone Tina , McKenzie Jodi , Mbofung Rina , Punt Simone , Wang Zhe , Xu Chunyu , Williams Leila , Wang Zhiqiang , Bristow Christopher , Carugo Alessandro , Peoples Michael , Li Lerong , Karpinets Tatiana , Huang Lu , Malu Shruti , Creasy Caitlin , Leahey Sara , Chen Jiong , Bernatchez Chantale , Gopal Vashisht , Heffernan Timothy , Hu Jianhua , Wang Jing , Amaria Rodabe , Wistuba Ignacio , Woodman Scott , Roszik Jason , Davis Eric , Davies Michael , Heymach John , Hwu Patrick
Published: January 2023

Adoptive T cell therapy (ACT) has produced impressive responses in a subset of patients with advanced malignancies. However, majority of patients still failed to respond. Thus, there is an urgent need to understand the resistant mechanisms in non-responders and develop more effective ACT strategies. Here, we employed two independent and unbiased approaches to identify novel molecular determinants of immune resistance. We generated gene expression profiles on an immune resistant melanoma cell line to identify alternative immunosuppressive mechanisms. In addition, we utilized a new high-throughput shRNA screening platform developed by our group to functionally interrogate immune resistance in patient-derived melanoma cells. Results from both analyses implicated tumor-associated glycolysis as a critical pathway that enables tumor cells to evade T cell-mediated antitumor activity. By using samples from melanoma and non-small cell lung cancer patients, we showed that increased expression of glycolysis-related genes is associated with poor T cell infiltration of tumors. Moreover, we found that increasing tumor glycolysis impaired T cell killing of melanoma cells, while inhibiting glycolysis restored T cell-mediated apoptosis of tumor cells. More importantly, from two non-overlapping ACT-treated patient cohorts, we discovered that tumor glycolytic activity in patients who experienced disease progression following ACT was significantly higher compared to those patients who were responsive to therapy. Taken together, our results demonstrate that tumor glycolytic metabolism is associated with the efficacy of ACT and identify glycolysis as a candidate target for combinatorial therapeutic intervention.
Effect of 3,3′-Diindolylmethane on Gut Microbiome in Colorectal Cancer
Authors: Alrafas Haider Rasheed Daham , Busbee Brandon , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world, and currently there is no cure for CRC. In the current study we investigated the effects of 3,3′-diindolylmethane (DIM) on the murine azoxymethane-dextran sodium sulfate (AOM-DSS) CRC model. Our data shows that administration of DIM alleviates symptoms associated with CRC. Endoscopy and histopathology showed DIM decreased colonic tissue damage and tumor growth. In addition, DIM was able to decrease myeloid-derived suppressor cells (MDSCs) in the blood and spleen in the AOM-DSS model. DIM treatment also significantly altered T cells populations which included decreases in Tregs and IL-10 producing T cells while increasing in Th1 and Th17 cells. We performed 16S rRNA sequencing of cecal flushes and validation with PCR revealed that AOM-DSS administration led to significant decreases in bacterial species Ruminococcus gnavus, Akkermansia muciniphila, Bifidobacterium pseudolongum, Haemophilus parainfluenza, and Acineobacter lwoffi, while increasing species such as Bacteroides acidifaciens, Bacteriodes ovalus, and Parabacteroides distasonis. However, mice that were treated with DIM showed a reversal in these gut microbial alterations caused by AOM-DSS CRC induction. We quantified short chain fatty acids (SCFA). AOM-DSS administration led to a significant decrease in i-butyric and n-butyric acids, however, mice that were treated with DIM increased these levels significantly. Finally we performed fecal transfer experiments to confirm that the alterations in gut microbiome by DIM mediated the anti-cancer effects. Collectively, these data show that DIM can ameliorate CRC by preventing cancer-associated gut microbial dysbiosis
Brain tumor-induced neuronal stress orchestrates adaptive immune surveillance through fractalkine
Authors: Nwajei Felix , Shanmugasundaram Meenakshi , Paine Dana , Zal Anna , Beceren-Braun Figen , Gabrisiewicz Konrad , Zhou Shouhao , Lee Sungho , Rodriguez Benjamin , Heimberger Amy , Zal Tomasz
Published: January 2023

Tissue damage contributes to initiation and modulation of an immune response. Tumor progression generally causes distress to the surrounding tissue. However, how tumor-induced parenchymal damage regulates anti-tumor immune response remains to be understood. We found that tumors that invaded brain parenchyma compressed the surrounding neurons causing increased expression of the neuronal chemokine CX3CL1/fractalkine in the peritumoral margin. Intravital two-photon microscopy revealed perivascular recruitment of monocyte-derived CD11c+ dendritic cells and T cells that interacted and killed individual cancer cells in tumor margins. Immune surveillance of brain tumors became inefficient in mice lacking the receptor for fractalkine, CX3CR1, resulting in more aggressive tumor progression. Our results identify tissue stress and associated chemokine signaling as a potential target to orchestrate anti-tumor immune surveillance in the brain.