bluuebunny/crossref_metadata_2025 huggingface dataset:
MHV68 latency modulates host immune responses to H1N1 influenza virus (49.9)
Authors: Saito Fumitake , Ito Toshihiro , Schaller Matthew , Carson William , Hogaboam Cory , Lukacs Nicholas , Standiford Theodore , Rochford Rosemary , Kunkel Steven
Published: January 2023

Human gammaherpesviruses such as Epstein-Barr virus (EBV) are clinically important pathogens, as diseases caused by gammaherpesvirus infection are a significant human health concern. Additionally, latent gammaherpesvirus infection is widespread, and can have a profound effect on subsequent immune responses. Murine gammaherpesvirus 68 (MHV68) is a natural rodent pathogen that has been used as a model to study the pathogenesis of human gammaherpesviruses. Like other herpesviruses, MHV68 causes acute infection and establishes life-long latency in the host. Recently, it has been shown that mice latently infected with MHV68 have resistance to unrelated pathogens in secondary infection models. We therefore hypothesized that latent MHV68 infection can modulate host response to H1N1 influenza virus, a serotype with high associated mortality rates in humans. To test this hypothesis, mice were infected intranasally with influenza virus following the establishment of MHV68 latency. Mice latently infected with MHV68 showed significantly higher survival of H1N1 influenza virus infection than mock-infected mice. Latent MHV68 infection led to lower influenza viral loads in the lungs, as well as decreased inflammatory pathology in the lungs. Collectively, our findings indicate that latent MHV68 infection has a protective effect against H1N1 influenza virus infection.
Regulatory T cell conversion by migratory but not lymphoid tissue resident dendritic cells (50.11)
Authors: Broggi Achille , Vitali Caterina , Mingozzi Francesca , Raimondi Giorgio , Zanoni Ivan , Granucci Francesca
Published: January 2023

There is evidence that dendritic cells (DCs) induce peripheral tolerance. Although studies on specific DC subsets have proven their ability to induce regulatory T cell conversion (iTreg cells), little is know about the DC subtypes actually able to promote peripheral CD4+ T cell tolerance to autoantigens in vivo. Here, we show that, when autoantigen presentation is not confined to specific DC subsets but is extended to all conventional DCs, steady state migratory DCs (ssmDCs) possess unique ability to induce antigen-specific iTreg cells in cutaneous lymph nodes (CLNs). Diversely, lymphoid tissue resident DCs do not show this capacity. Moreover, DC-derived CCL22 contributes to the retention of iTreg cells in CLNs and skin. Therefore migratory DCs represent the DC population devoted to the maintenance of peripheral self-tolerance.
Host defense against extracellular microbes-integrating the parts into a big picture in a clinical presentation-based medical school curriculum (51.1)
Authors: Piskurich Janet
Published: January 2023

The 2009 Scientific Foundations for Future Physicians Report of the Association of American Medical Colleges (AAMC)-Howard Hughes Medical Institute (HHMI) Committee recommends a list of specific competencies that all medical students should demonstrate before receiving the M.D. degree. Competency M4, most related to immunology, recommends students should demonstrate the ability to “Apply the principles of the cellular and molecular basis of immune and nonimmune host defense mechanisms in health and disease to determine the etiology of disease, identify preventative measures, and predict response to therapies”. An associated learning objective states that students should be able to “Apply knowledge of the mechanisms utilized to defend against intracellular or extracellular microbes to the development of immunological prevention or treatment”. Yet medical students, many learning the various parts of the immune system for the first time, may express frustration that they cannot assemble the parts into a comprehensive big picture. One integrated approach for presenting host defense mechanisms against extracellular microbes will be illustrated.
Successful integration of practical flow cytometric experience into undergraduate education. (51.4)
Authors: Rogers Clare , Gubbels Bupp Melanie , Kim Unyoung , DeKrey Gregory , Stranford Sharon , Medh Rheem , Tomich Gloria
Published: January 2023

Flow cytometry is an essential tool in almost every discipline of cell biology, and is increasingly utilized in a variety of other associated yet diverse fields, including molecular biology, bioengineering, microbiology and marine biology. For many years, access to the use of flow cytometers as teaching tools, even in large universities, was limited to graduate programs, due to the cost of instrument purchase and maintenance, and to the complexity of operation. Recently, several affordable, bench-top flow cytometers have appeared on the market. The affordability and user-friendly nature of these instruments have made them attractive to the faculty of various departments at medium- and small-sized universities and colleges for use both as teaching tools and in faculty research projects. We have compiled information here on the funding sources for both purchase and maintenance of these instruments at our various institutions, and share information on applications, protocols and lab exercises that have worked well with our students. Example protocols described include applications such as immunophenotyping, apoptosis (annexin/propidium iodide), monocytic phagocytosis, intracellular and mitochondrial reactive oxygen species detection, and detection of fluorescent protein expression in bacteria.
A forensic analysis of food poisoning using a dip-stick immunoassay (51.9)
Authors: Goudie John
Published: January 2023

Since bacteria are ubiquitous, they are likely to be present on most foods that you purchase. Proper cooking or processing of food destroys bacteria but poor personal hygiene, improper cleaning of storage and preparation areas and unclean utensils cause contamination of raw and cooked foods and allow bacteria to grow. Although cooking destroys the bacteria, the toxins produced by some are heat stable and may not be destroyed and remain in or on food when it is eaten. Students will examine food samples from a simulated (non-toxic) food poisoning outbreak for the presence of a specific antigen by utilizing a Dip-stick immunoassay. Dip-stick immunoassays are a less expensive technology for high schools and produce excellent results.
Transcriptional regulation of proteolytic enzymes in dendritic cells by effector versus regulatory T cells: a new mechanism of maintaining peripheral tolerance (52.15)
Authors: Marvel Douglas , Finn Olivera
Published: January 2023

MUC1.Tg mice express the human tumor associated antigen MUC1 in a physiological pattern that is analogous to its expression on normal human epithelial cells and tumors. Vaccine-induced responses to MUC1 peptide in these animals are weak compared to WT mice. Gene expression profiling post vaccination revealed differential regulation in spleen dendritic cells (DC) of several proteolytic enzymes that were previously thought to function exclusively in pancreatic catabolism (i.e. Trypsin-1, Trypsin-4, Carboxypeptidase B1). These enzymes were upregulated in DC when WT mice were vaccinated with the MUC1 peptide (more foreign) and highly downregulated after the same vaccination in MUC1.Tg mice (more self). Using an in vitro co-culture system, we show that this regulation is dependent on CD4 T cell interactions with DC, such that activated effector T cells (Teff), but not TLR agonists, upregulate these enzymes, whereas activated regulatory T cells (Treg) actively suppress their expression. Cell-cell contact appears to be necessary. The Treg mediated suppression is dominant and it occurs even when DC are cultured simultaneously with Treg and Teff. We propose a new mechanism of maintaining peripheral tolerance whereby self-antigen specific Treg recognize the presence of antigen on a few DC and temporarily shut down antigen processing by all DC to avoid efficient priming of effector T cells.
Prime and pull: establishing protective immunity in restrictive tissues (52.16)
Authors: Shin Haina , Iijima Norifumi , Iwasaki Akiko
Published: January 2023

Some of the world’s most pressing health concerns include sexually transmitted infections such as HIV and HSV. Thus far, vaccines against these pathogens, even those that elicit robust systemic cytotoxic responses, have been unsuccessful. The genital tract is a highly restrictive tissue that does not allow entry of activated CD8 T cells in the absence of infection or inflammation. Thus, it is possible that the failure of previous vaccination attempts reflects an inability to establish a tissue-resident population of cytotoxic T cells at the front line of infection within the genital tract. Thus, to address this issue, we have designed a novel vaccination method called ‘prime and pull’ to recruit antigen-specific effector CD8 T cells into the vaginal mucosa. After subcutaneous immunization (prime), very few effector CD8 T cells are observed in the genital tissue. However, a single topical application of the chemokines CXCL9 and CXCL10 in the vagina (pull) leads to robust recruitment of effector CD8 T cells, which is independent of CD4 T cell help. Furthermore, our ‘prime and pull’ method may generate a long-term population of memory CD8 T cells within the tissue that appears to enhance viral control after a lethal challenge. Thus, our studies suggest that ‘prime and pull’ is a promising method not only for providing protection against sexually transmitted infections, but also for directing immune responses to other restrictive microenvironments such as tumors.
The histone deacetylase inhibitor, Trichostatin A, decreases TLR-mediated cytokine production in human plasmacytoid dendritic cells by negatively affecting IRF-7 phosphorylation and nuclear translocation (52.24)
Authors: Descalzi Dante , Dai Jihong , Bocarsly-Fitzgerald Patricia
Published: January 2023

pDC produce high levels of IFN-α and TNF-α upon TLR7/9 stimulation and express high constitutive levels of the transcription factor IRF-7, which is required for IFN-α induction. Histone deacetylases (HDAC) are enzymes shown to regulate protein function by removing acetyl groups from nuclear and cytoplasmic targets. HDAC inhibitors are used clinically as cancer therapeutics and are being considered as potential anti-inflammatory agents. Our purpose was to characterize the effects of TSA, a class I/II HDAC inhibitor, on pDC cytokine production and IRF-7 protein regulation and activation. TSA treatment strongly inhibited the induction of both IFN-α and TNF-α by pDC upon stimulation with CpG-A, HSV-1, HIV-1, or Flu, as shown by intracellular flow cytometry and ELISA. TSA treatment did not induce pDC apoptosis, nor did it negatively affect cell number, arguing against general toxicity of the drug. Interestingly, although basal IRF-7 levels were not affected by TSA, upregulation of pDC IRF-7 protein expression in response to rIFN-α was inhibited. Moreover, TSA treatment inhibited virus-induced IRF-7 activation, as demonstrated by BD Phosphoflow, and nuclear translocation of IRF-7, as demonstrated by ImageStream imaging flow cytometry. These findings suggest a potential impact of acetylation on the activation and protein expression of IRF-7 in pDC and highlight a possible mechanism of cytokine inhibition by TSA in these cells.
Toll like receptor agonists stabilize MHC-II expression in macrophages thereby enhancing peptide presentation by BCG infected macrophages (52.30)
Authors: Bakhru Pearl , Sirisaengtaksin Natalie , Estrella Jaymie , Khan Arshad , Jagannath Chinnaswamy
Published: January 2023

BCG is the only human-approved vaccine used to protect against childhood tuberculosis although it fails to protect against adult tuberculosis. BCG vaccine localizes to an immature phagosomal compartment and avoids lysosomal fusion. Furthermore, it reduces the expression of MHC-II in macrophages in vitro and in phagocytic cells of mice after infection. We hypothesized that immunogenicity of BCG is reduced because of reduced MHC-II expression by APCs and the addition of Toll-like receptor (TLR) - ligands will strengthen and prolong immunity conferred by BCG vaccine and perhaps protect against adult disease. Since TLR ligands activate macrophages, we tested their effect on MHC-II expression. BCG infection down regulated the expression of MHC-II while a combination of BCG vaccine with ligands for TLR-7, and 9 markedly induced an up-regulation that correlated with the ability of macrophages to present antigens to T cells. Furthermore, we identified that TLR-7, and 9 but not TLR-1/2, TLR-3 or TLR-5 induced a down regulation of the ubiquitin ligase MARCH1 that in turn correlated with a decreased ubiquitination and enhanced clearance of MHC-II from macrophages. These data suggest that BCG down regulates immune responses while TLR-ligands can be used to up regulate MHC-II contributing to a novel mode of adjuvant action.
Immunization with a synthetic peptide vaccine directed against the loop neutralizing determinant from domain 2 of protective antigen formulated with human use adjuvants protects rabbits from inhalation anthrax (53.13)
Authors: Oscherwitz Jon , Yu Fen , Cease Kemp
Published: January 2023

The current vaccines for anthrax in the U.S. and U.K. are efficacious in the two most accepted animal models of inhalation anthrax, non-human primates and rabbits, but require intensive immunization protocols.We previously demonstrated that a multiple antigenic peptide (MAP) vaccine comprising a heterologous T cell epitope from Plasmodium falciparum colinearly synthesized with a cryptic loop neutralizing determinant (LND) from domain 2 of Bacillus anthracis protective antigen can completely protect rabbits from an aerosol challenge with B. anthracis Ames strain spores when formulated with CFA. In the current study, we evaluated whether immunization of rabbits with a mixture of two MAPs, each comprising the LND peptide colinearly synthesized with one of two different heterologous T cell epitopes, could stimulate protective immunity using CFA, and in a subsequent study, using adjuvants currently evaluated for use in humans.In the initial study, all rabbits (7/7) immunized five times with the MAP vaccine formulated with CFA/IFA were completely protected from a 200 LD50-target dose of B. anthracis Ames strain. When tested with human use adjuvants, 100% (9/9), 89% (8/9), 89% (8/9) or 67% (6/9) of rabbits immunized with the MAP vaccine formulated with lipid A monophosphoryl/Alhydrogel, CpG/Alhydrogel, QS-21/Alhydrogel or Alhydrogel alone, respectively, were protected from aerosol challenge. The LND MAP vaccine may represent an adjunctive or alternative vaccine for anthrax.
Characterizing the primary B cell antibody response to HIV (53.18)
Authors: Pujanauski Lindsey , Pelanda Roberta , Torres Raul
Published: January 2023

Most individuals infected with the human immunodeficiency virus (HIV) make neutralizing antibodies (nAbs) to the virus, but this response is delayed and consequently not protective. Some nAbs that have been characterized are described as polyreactive and slightly autoreactive, two attributes of Abs produced by marginal zone (MZ) B cells. Thus, B cells capable of producing nAbs early in infection may not participate, possibly because of tolerance mechanisms. To directly examine the components of the primary Ab response to the HIV envelope protein gp120, we use a mouse model and HIV viral-like particles (VLPs). Our studies focus on the quantity and quality of the anti-HIV responses from different B cell subsets. We have found that naïve follicular (FO) and MZ B cells are both capable of producing anti-gp120 IgM in culture. Additionally, we have examined the VDJ rearrangements of gp120-reactive Abs from different populations in both the naïve and VLP-challenged repertoire. So far, it seems that there may be a preferred VDJ recombination event that is over-represented in the MZ B cell compartment. This VDJ rearrangement has not yet been found in responding cells of VLP-immunized mice, indicating that MZ B cells may not be participating in the VLP response. Abs from these subsets are being further characterized with HIV-neutralization studies. It is expected that the results from these experiments will prove informative for the development of a protective HIV vaccine.
Blockage of CD59 diminishes infectivity of HIV-1-positive plasma in vitro and in vivo (53.21)
Authors: Amet Tohti , Hu Sishun , Byrd Daniel , Yu Andy Qigui
Published: January 2023

Numerous vaccination trials have been conducted to induce neutralizing antibodies (nAbs) against HIV-1. However, high levels of Abs, including nAbs and non-nAbs, mount in vast majority of HIV-1 infected subjects, yet fail to control virus proliferation or protect patients from developing AIDS, indicating that HIV-1 virions can escape Ab immunity. Recent studies have shown that regulators of complement activation (RCA) including CD59 present on the viral envelope (Env) confer resistance to Ab-dependent complement-mediated lysis (ADCML), which may explain why HIV-1 is not neutralized or lysed even in the presence of high titer of anti-Env Abs. Here, we report that blockage of CD59 on HIV-1 Env in infected patient plasma by BRIC229, a widely used CD59-blocking Ab, or rILYd4, a novel CD59 inhibitor derived from bacterial toxin intermedilysin domain 4, significantly reduces infectivity of HIV-1-positive plasma in vitro (H9 cells) and in vivo (SCID mice reconstituted with human peripheral blood mononuclear cells or PBMCs). Similarly, BRIC229 and rILYd4 significantly enhanced ADCML of HIV-1-infected H9 cells and HIV-1-bound erythrocytes in the presence of patient plasma as a source of both complement and anti-HIV-1 Env Abs. All plasma samples from 7 HIV-1-infected patients examined contained high titer of anti-HIV-1 Env Abs. Our data suggest that blockage of CD59 on the surface of HIV-1 virions and infected cells represents a novel therapeutic approach to combat HIV-1/AIDS pandemic.
A novel combination vaccine for HIV-1 and tuberculosis is safe and immunogenic in infant rhesus macaques (53.25)
Authors: Jensen Kara , Ranganathan Uma , Van Rompay Koen , Abbott Zack , Larsen Michelle , Fennelly Glenn , Abel Kristina
Published: January 2023

Despite improved therapies, breast milk transmission of HIV to newborns does not decline. In many resource-poor countries, these infants are also at risk for Mycobacterium tuberculosis infection. We hypothesized that a highly attenuated Mtb with improved safety and similar immunogenicity to the current BCG vaccine, but modified to harbor HIV genes, could protect infants against both HIV and Tb. We have tested 3 different rAMtb-SIV vectors in infant rhesus macaques. Vaccine safety was evaluated by immunizing 1-week old SIV-infected macaques orally (PO) and intradermally (ID) with rAMtb. Next, we tested the immunogenicity of (1) a PO prime/ID boost rAMtb-SIV, (2) a PO rAMtb-SIV prime/IM Ad5-SIV boost, and (3) an ID rAMtb-SIV prime/IM Ad5-SIV boost vaccine regimen. Our studies show that rAMtb-SIV does not cause rAMtb dissemination, even when given to immunocompromised SIV infected infants. Further, our data confirm that the oral route of immunization is effective in infant macaques. Vaccinated animals showed increased dendritic cell responsiveness to in vitro TLR stimulation, supporting our hypothesis that rAMtb is highly immunogenic in infants. While all vaccine regimens elicited cellular and humoral Mtb-specific immune responses, SIV-specific responses were greatly enhanced by the heterologous boost. Thus, rAMtb-HIV should be further developed as a pediatric combination vaccine. We will test vaccine efficacy against multiple oral SIV challenges and against Mtb exposure.
Dimeric 2G12 as a potent protection against HIV-1 (53.26)
Authors: Luo Xin , Ouyang Yong , West Anthony , Keeffe Jennifer , Gnanapragasam Priyanthi , Bjorkman Pamela , Baltimore David
Published: January 2023

We previously showed that broadly neutralizing anti-HIV-1 antibody 2G12 (human IgG1) naturally forms dimers that are more potent than monomeric 2G12 in in vitro neutralization of various strains of HIV-1. Recently, we also investigated the protective effects of monomeric versus dimeric 2G12 against HIV-1 infection in vivo using a humanized mouse model. We showed that passively transferred, purified 2G12 dimer is more potent than 2G12 monomer at preventing CD4 T cell loss and suppressing the increase of viral load following HIV-1 infection of humanized mice. In addition, we found that 2G12 dimer does not favor mutations of the HIV-1 envelope that would cause the virus to completely escape 2G12 neutralization. These results suggest dimeric 2G12 as a potent prophylactic reagent against HIV-1 in vivo, which could be used as part of an antibody cocktail to prevent HIV-1 infection. We are currently investigating whether the protective effect of 2G12 dimer against HIV-1 in humanized mice requires antibody-dependent cellular cytotoxicity (ADCC) using mutated 2G12 dimers where the ADCC function is either abolished or enhanced. Our results will suggest a mechanism of action underlying the protective effect of dimeric 2G12 against HIV-1 in vivo.
Reversal of human papillomavirus-mediated suppression of Langerhans cells function with the TLR3 agonist Poly-ICR (53.7)
Authors: Da Silva Diane , Koopman Maaike , Brand Heike , McKee Gregory , Kast W. Martin
Published: January 2023

The human papillomavirus (HPV) family of viruses establishes persistent infections because it has evolved mechanisms that allow it to evade the human immune system. We have identified HPV-mediated suppression of antigen presentation by Langerhans cells (LC) as a key mechanism through which HPV evades immune surveillance. PolyICR is a stable TLR3 agonist that is a broad inducer of innate immunity and is being developed as a vaccine adjuvant and antitumor agent. An important feature of PolyICR is its ability to functionally activate dendritic cells to induce antigen-specific T cells. In this study, we determined whether PolyICR can overcome HPV-induced immune suppression by functionally activating human LC in the presence of HPV16 and inducing HPV16-specific T cells. PolyICR was able to activate LC that had been pre-exposed to HPV16 VLP such that expression of MHC and costimulatory molecules were highly upregulated and LC secreted high amounts of Th1 and inflammatory cytokines and chemokines. Upregulation of CCR7 resulted in a significant increase in migration capacity. LC incubated with HPV16 VLP and treated with PolyICR induced an HPV16-specific CD8+ T cell response detected by interferon gamma Elispot that was absent when LC were exposed to VLP alone. These data suggest that the TLR3 agonist PolyICR is a promising therapeutic molecule that can overcome HPV-induced immune suppression of LC and result in an LC capable of stimulating an anti-HPV T-cell mediated immune response.
Antigen presentation by dendritic cells in the aortic wall triggers T helper immune responses in atherosclerosis (54.16)
Authors: Koltsova Ekaterina , Garcia Zacarias , Landau Michael , Scott Spencer , von Vietinghoff Sibylle , Galkina Elena , Acton Scott , Ley Klaus
Published: January 2023

Atherosclerosis is an immune-mediated disease associated with lipid accumulation and formation of atherosclerotic plaques in the major arteries of the body. Vascular dendritic cells (DC) are present throughout the adventitia of the normal mouse aorta and expand under atherosclerotic conditions, but their function remains incompletely understood. To test whether DC can mediate productive antigen presentation directly in the aortic wall, we used two-photon microscopy. CD11cYFP tagged DC were motile in mouse aortas and interacted with T cells in an antigen dependent manner. In atherosclerotic Apoe-/- CD11cYFP mice, DC interacted with transferred T cells from Apoe-/-, but not C57BL/6 mice. Interaction with DC decreased T cell migration velocity and induced secretion of IFN-γ, TNF-α and IL-17, which enhanced oxLDL uptake by primary macrophages. Using flow cytometry of digested whole aorta from C57BL/6 and Apoe-/- mice fed either Western diet or regular chow diet, we found CD11b+CD11c+ and CD11b-CD11c+ DC in the aortic wall of C57BL/6 mice, and this population expanded in Apoe-/- mice on western diet. Under these conditions, CD8a+ and CD103 DC appear among the CD11c+ DC population. Taken together, our data show the emergence of distinct subsets of DC in the mouse aorta during the progression of atherosclerosis and demonstrate that the aortic wall can be a site of active antigen presentation leading to the induction of a pro-atherogenic immune response.
Multicolor flow cytometry analysis of peripheral blood lymphocytes in pediatric patients with inflammatory bowel disease (54.7)
Authors: Abramson Tzvia , Tarlton Nicole , Green Caroline , Jandu Jagmohan , Abramson Oren , Wong Anthony , Lazarus Nicole , Rott Lusijah , Ulloa E. , Butcher Eugene
Published: January 2023

While the etiology and mechanism of Inflammatory Bowel Disease (IBD) is not fully understood, immunologic abnormalities play a role in these diseases. Monitoring of disease progression and response to therapeutics is typically invasive and lacking. Increased recruitment of lymphocytes to the gut has been demonstrated in IBD, leading to inflammation and ultimately to gut tissue damage. Lymphocyte circulation is tightly controlled by trafficking receptors (TRs) that act in combination to govern migration to the target organ. Plasmablasts (PBs) are immature circulating B cells that may reflect tissue localized plasma cells. We hypothesize that the PB blood levels and trafficking patterns can inform on the location of tissue damage and the clinical condition of the patients. Using multicolor flow cytometry we examined PB (IgA+/CD38high) and memory B cell (IgA+/CD19+) blood levels, and TRs expressed on these cells. We show that an increase in IgA+ PB blood levels reflect an aggravated clinical state, and a significantly greater amount of these cells in ulcerative colitis patients express colon associated TRs (CCR10+) as opposed to small intestine associated TRs (CCR9+). Additionally, these trafficking patterns are more evident on IgA+ PBs rather than memory B cells. This work in progress suggests that the analyses of the TR phenotype of circulating immature and mature B lymphocytes may provide valuable information for monitoring disease progression via a less invasive process.
Hydrogen sulfide: improved hemodynamics and mitigation of ischemia-reperfusion injury in a large animal model (54.8)
Authors: Satterly Steven , Salgar S. , Stallings J. , Martin M.
Published: January 2023

Hemorrhagic shock can lead to ischemia-reperfusion (IR) injury followed by multi-organ failure and death. We tested intravenous Hydrogen sulfide (H2S) in a porcine hemorrhagic shock-resuscitation model evaluating IR injury. Experimental design: Sham (n=3); H2S untreated group (n=5); and H2S treated group (n=14). Animals were splenectomized, induced 35% hemorrhage and ischemia by cross-clamping the supraceliac aorta for 50 minutes. Intravenous fluid and H2S were administered at cross-clamp. Serial hemodynamic measurements and blood plasma analysis were followed for 6 h post-cross clamp release. The mean lactate levels (a measure of injury) were markedly lower in H2S group (7.0±1.6 mmol/L) compared to untreated group (9.3±1.2 mmol/L). Epinephrine requirement was significantly (P<0.05) lower in H2S group (727±645 µg) compared to untreated group (3052±1700 µg). Cardiac output was significantly (P<0.05) lower in H2S treated pigs. The intravenous fluid, hematocrit, and body temperature did not differ significantly (P<0.05). Blood plasma pro-inflammatory cytokines (IL1-β, IL-6, IFN-γ, TNF-α) at 6 h post-cross clamp release were significantly lower (P<0.05) compared to baseline in both H2S treated (n=4) and untreated groups (n=3). Organ (liver, kidney, heart, lung, small bowel, aorta) histopathology and gene expression (iNOS, eNOS, HO-1, HIF-1α, TNFα, IL-6, IFN-γ, IL1-β, etc.,) analyses are in progress. H2S improved hemodynamics and appears to be protective against IR injury.
Chronic estradiol exposure in oophorectomized mice induces a systemic inflammatory disorder that is mediated via TNFα. (54.9)
Authors: Halder Ramesh , Shinyashiki Brenda , Chaudhuri Gautam , Singh Ram
Published: January 2023

Estradiol (E2) administration to oophorectomized (Ovx) mice has been shown to induce the expression of IL-1β, IL-6 and TNFα and inducible NO synthase mRNA in macrophages. Here, we investigated the effect of chronic E2 supplementation on immune responses and cytokines. We found that long-term E2 exposure in Ovx mice led to a marked increase in granulocytes in the liver and spleen. Although, E2 exposed Ovx mice had reduced total cellularity of thymus and spleen and reduced CD4+ and CD8+ T cells in the spleen, more splenic T cells from these mice produced IFN-γ and IL-17 as compared to sham-operated and placebo-treated Ovx mice. IFN-γ and IL-17 were also abundantly made by T, NK and NKT cells in the liver of E2-exposed Ovx mice compared to animals in the other two groups. Intriguingly, E2 exposure in Ovx mice led to significant weight loss as compared to sham-operated mice and placebo-treated Ovx mice. Strikingly, the pro-inflammatory cytokine (IFN-γ and IL-17) response, granulocyte accumulation in liver, and weight loss elicited by chronic E2 exposure in Ovx mice were largely abolished by TNFα deficiency. Finally, 54% of chronically E2-exposed Ovx wild-type B6 mice, as compared to none of sham and placebo-Ovx wild-type mice and none of E2-exposed Ovx TNFα-/- mice, died during the study period. Thus, chronic E2 exposure in Ovx mice elicits an inflammatory condition that is mediated, at least in part, by TNFα.
Anti-malarial drug Artesunate ameliorates oxidative lung damage in experimental allergic asthma (55.12)
Authors: Ho Wanxing , Cheng Chang , Peh Hong Yong , Xu Fengguo , Tannenbaum Steven , Ong Choon Nam , Wong Wai-Shiu
Published: January 2023

Oxidative stress is an important clinical feature of asthma, resulting in oxidative damage to lung proteins, DNA and lipids. Our previous study has shown that artesunate, an anti-malarial drug, has potent anti-inflammatory effects in a murine asthma model. As oxidative stress plays a critical role in the pathogenesis of asthma, we further investigated the effects of artesunate on oxidative lung damage in this asthma model. BALB/c mice sensitized and challenged with ovalbumin, developed airway inflammation and oxidative lung damage with increased levels of nitrotyrosine (protein), 8-isoprostane (lipid) and 8-hydroxy-2-deoxy-guanosine (DNA). Superoxide dismutase (SOD) expression and activity were increased, while catalase expression and activity were suppressed in asthmatic mice. Treatment with artesunate showed comparable anti-inflammatory effects to dexamethasone, a potent corticosteriod, significantly suppressing airway inflammation and oxidative lung damage. Mice treated with artesunate had reduced levels of nitrotyrosine, 8-isoprostane and 8-hydroxy-2-deoxy-guanosine. SOD expression and activity were returned to basal levels, while catalase gene expression and activity were promoted with artesunate treatment. Gluthathione peroxidase expression and activity were unaltered in all treatment groups. Our findings indicate that artesunate has potent anti-inflammatory effects, comparable to dexamethasone, by ameliorating oxidative lung damage in experimental allergic airways.
Multiple drug hypersensitivity: role of T regulatory cells (55.15)
Authors: Daubner Barbara , Groux-Keller Monika , Kawabata Thomas , Naisbitt Dean J. , Park Kevin B. , Thomas Wendland , Marianne Lerch , Pichler Werner J.
Published: January 2023

Introduction: Up to 10% of patients with severe immune-mediated drug hypersensitivity tend to develop multiple drug hypersensitivities (MDH), and react clinically and immunologically to various drugs. Objective: Evaluating the role of T regulatory (T reg) cells in drug reactions and developing an improved clinical and immunological definition of MDH. Methods: T cell reactivity to various drugs was assessed by expression analysis of activation markers and cross-reactivity of drug specific T cell clones to various drugs was analyzed. The role of T regs (Foxp3+/CD25high) was analyzed functionally in T reg depletion assays. The phenotype of reacting T cells was characterized by analysis of activation and cell exhaustion markers. Results: T regs from MDH patients are functionally highly active and suppress T cell proliferation similar to T regs from monoallergic patients and healthy controls. Removal of T regs enhances the reactivity to involved drugs, but does not facilitate an immune response to not exposed drugs in vitro. Drug reactive T cells from monoallergic patients reside in the resting CD4+CD25neg T cell fraction while in MDH patients they reside in an in vivo activated cell fraction (CD4+/CD25dim/CD38+/HLA-DR+/PD-1+). Conclusion: T regs are able to regulate drug specific immune responses. In MDH patients, the drug reactive T cells are contained in a pre-activated cell fraction. This may enhance their responsiveness to drugs and thus explain MDH.
The eicosanoid hepoxilin A3 plays critical role in transmigration of PMNs and concomitant invasion of bacteria in Streptococcus pneumoniae lung infection (56.11)
Authors: Bhowmick Rudra , Gronert Karsten , McCormick Beth , Leong John
Published: January 2023

The hallmark of pneumonia caused by Streptococcus pneumoniae (pneumococcus) is massive recruitment of polymorphonuclear cells (PMNs) from the circulation into lung alveolar spaces, causing considerable tissue damage. Moreover, pneumococcal translocation across lung epithelium into blood can lead to potentially lethal septicemia. The PMN influx caused by the bacterial insult, might lead to the disruption of the barrier function of the respiratory epithelium, thus contributing to the transepithelial migration of S. pneumoniae into the bloodstream. Our results show that S. pneumoniae triggered PMN migration from basolateral to apical side of polarized monolayers of lung epithelial cell line H292, and this PMN migration was associated with elevated levels of bacterial migration in the reverse direction. HPLC/MS studies of infected H292 culture supernatants showed increased levels of hepoxilin A3 (HXA3), a 12-LOX-derived eicosanoid that can act as a PMN chemoattractant. Administration of a 12-LOX inhibitor significantly reduced both pneumococcus-induced PMN migration and translocation of bacteria itself. Interestingly, 12-LOX knockout mice, or wild type mice treated with a 12-LOX inhibitor, showed diminished lung inflammation and bacteremia, and showed 100% survival in an otherwise lethal lung challenge. Our results indicate that during S. pneumoniae lung infection, HXA3 plays important roles in both pulmonary inflammation and bacterial invasion of the bloodstream.
CD73-generated adenosine is critical for immune regulation during Toxoplasma gondii infection (56.13)
Authors: Mahamed Deeqa , Mills Jeffrey , Bynoe Margaret
Published: January 2023

As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii evades immune system-mediated clearance by undergoing stage differentiation to persist indefinitely in susceptible hosts. Previously, we found that mice deficient in the ecto-enzyme CD73, which generates adenosine in the extracellular matrix, were significantly resistant to acute and chronic toxoplasmosis after oral infection with the T. gondii type II ME49 strain. Resistance in CD73-knockout mice was due to a reduction in immune-mediated pathology in the intestine, and a delay in parasite differentiation in the CNS. To further clarify the role of CD73 and extracellular adenosine in T. gondii pathogenesis, we infected wildtype and CD73KO mice with ME49 cysts by the intraperitoneal (IP) route. In contrast to oral infection, IP-infected CD73KO mice were highly susceptible to immune-mediated pathology, with significantly increased infiltration of neutrophils and T cells into the peritoneal cavity. Peritoneal exudate cells from infected CD73KO mice generated higher levels of the inflammatory mediators nitric oxide, TNFα, and IL1β, without enhanced parasite killing or clearance. In addition, mice deficient in the adenosine receptors A1 or A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii, compared to wildtype mice. Thus extracellular adenosine is a key molecule that regulates the immune response to an intracellular pathogen and promotes host survival.
Increased neutrophil migration rescues impaired function and susceptibility to Pseudomonas pneumonia after burn injury (56.25)
Authors: Neely Crystal , Wolfgang Matthew , Vadlamudi Sivaram , Maile Robert , Cairns Bruce
Published: January 2023

Bacterial lung infection is a major cause of mortality late after burn injury. Neutrophil dysfunction is implicated in the susceptibility of wound infection with Pseudomonas aeruginosa. We hypothesized that burn-induced impaired neutrophil migration and function within the lungs would increase susceptibility to intratracheal P. aeruginosa infection. Female C57BL/6 mice were anesthetized and subjected to a 20% full thickness contact burn or sham injury. Fourteen days later, mice were inoculated with 1x106 CFU of P. aeruginosa. After 24 hours, pulmonary bacterial load was evaluated. Flow cytometric analysis was used to quantify neutrophils and evaluate their function by dihydrorhodamine (DHR) oxidation. Contrary to our hypothesis, burn mice had a significant increase in bacterial clearance compared to sham. This correlated with an increased percentage and absolute number of lung neutrophils in burn mice (mean±SEM, burn: 2.5±1.4x108, sham: 2.7±0.8x107cells, p=0.007). However, these neutrophils had reduced reactive-oxygen production compared to sham (burn: 1044±21, sham: 51431±182 MFI of oxidized DHR, p=0.0131). As predicted, lung neutrophil function was significantly impaired after burn injury. However, burn mice exhibited a larger neutrophil influx than sham. The net result was that burn mice cleared the infection quicker than sham mice. This implies a fine balance exists between neutrophil migration and function after burn injury.
Development of functionally active recombinant form of mouse IL-35 (57.11)
Authors: Lee Hyun-Ku , Pascual David , Sehy David , Chitta Sriram , Singh Gita , Quintel Payton , Andrew Kody , Wardle Lauren , Rosenberg Jonathan , Singh Sujay
Published: January 2023

As a novel heterodimeric cytokine and one of the IL-12 family members, IL-35 is composed of IL-12p35 subunit and Epstein-Barr virus-induced gene 3 (EBI3) protein. IL-35 is known to play an essential role in immune regulation of the CD4+CD25+ Treg cells, alleviating inflammatory responses. We have developed the recombinant fusion construct of mouse IL-35 (mIL-35)-hIgG1 Fc (hFc), in which EBI3 and IL-12p35 are joined by a flexible linker of (Gly4Ser)3. The mIg k-chain leader sequence at the amino terminus also allows secretion of the fusion protein. The mIL-35 construct was stably expressed in HEK 293 cells, and culture supernatants of single clones were screened by ELISA using a combination of anti-hFc and anti-mEBI3 antibodies, among which the most positive clone 35-5 was selected and adapted in a serum-free culture system. Functional activity of the secreted mIL-35 from this clone was tested using the [3H]thymidine-based cell proliferation assay. When CD4+ T cells purified from TCR-transgenic DO11.10 mice were stimulated with a combination of syngeneic irradiated APCs and OVA peptide in the presence of the mIL-35 culture supernatants, the mIL-35 significantly suppressed CD4+ T cell proliferation. Furthermore, the mIL-35-mediated inhibition of CD4+ T cell proliferation was reversed by anti-mIL-35 antibody. Taken together, these results demonstrate that the recombinant mIL-35 is functionally active and can be a useful research tool to study T cell-based immune regulations.
Progesterone, IL-10 and TGF-β act in synergy to impair plasmacytoid dendritic cell functions in cord blood (57.19)
Authors: Harnois Michael , Charrier Emily , Cordeiro Paulo , Herblot Sabine , Duval Michel
Published: January 2023

Plasmacytoid dendritic cells (pDC) are specialized dendritic cells, also known as IFN-α producing cells. pDC are key inducers of immune responses upon viral infections; the environment although modulate their phenotype and functions. We have shown that cord blood (CB) pDC are unable to produce IFN-α after stimulation while they retain the capacity to maturate in antigen presenting cells. To get insights into the mechanisms of pDC regulation in CB, we first investigated the levels of immune regulators secreted by the placenta in CB. We show that progesterone (PG), IL-10 and TGF-β are present at high levels in CB as compared to adult blood. Using in vitro differentiated pDCs we analysed the effect of these molecules, separately or in combination, on pDC differentiation and activation. We reveal that physiological concentrations of these three factors individually have low impact on pDCs while in combination they inhibit pDC differentiation and IFN-α production. Higher concentrations of PG, IL-10 or TGF-β are also able to inhibit IFN-α production, but these conditions are not relevant to physiological levels in CB. Our results therefore shed new light on the synergy of immune regulators secreted by the placenta that have an impact on foetal and neonatal immune responses.
EFNB1 and EFNB2 physically bind to IL-7R-alpha and retard its internalization from the cell surface (57.2)
Authors: Luo Hongyu , Qi Shijie , Wu Zenghui , Han Bing , Wang Xuehai , Wu Jiangping
Published: January 2023

IL-7 plays vital roles in thymocyte development, T cell homeostasis and the survival of these cells. IL-7 receptor alpha (IL-7R-alpha) on the thymocytes and T cells is rapidly internalized upon IL-7 ligation. Ephrins (EFN) are cell surface molecules and are ligands of the largest family of receptor kinases, Eph kinases. We discovered that T cell-specific double deletion of EFNB1 and EFNB2 (double KO) in mice led to reduced IL-7R-alpha expression in thymocytes and T cells, and that IL-7R-alpha downregulation in double KO CD4 cells upon IL-7 treatment was accelerated. On the other hand, EFNB1 and EFNB2 overexpression in T cell line EL4 cells retarded IL-7R-alpha downregulation. The EFNB1/EFNB2 double KO T cells manifested compromised homeostatic proliferation, which is an IL-7-dependent process. Using fluorescent resonance energy transfer (FRET), we demonstrated that EFNB1 and EFNB2 physically interacted with IL-7R-alpha. Such interaction likely retarded IL-7R-alpha internalization, as EFNB1 and EFNB2 were not subjected to internalization. Therefore, we revealed a novel function of EFNB1 and EFNB2 in stabilizing the IL-7R-alpha expression at the post-translational level, and a previously unknown modus operandi of EFNBs in the regulation of the expression of other vital cell surface receptors.
Type I interferon inhibits expression of the alternative GATA3 transcript in human Th2 cells (57.22)
Authors: Huber Jonathan , Farrar J.
Published: January 2023

The GATA3 transcription factor contributes to multiple immune processes, including the differentiation of T helper type 2 (Th2) cells. Th2 cells regulate many aspects of allergic disorders and express GATA3 in response to IL-4 signaling. Subsequently, GATA3 promotes transcription of the Th2 cytokines while also creating a feed-forward loop that stabilizes Th2 commitment. A previous study in mice has shown that Th2 commitment is accompanied by increased utilization of an alternate first exon (exon 1A) in the GATA3 transcript via an upstream promoter. The resulting transcript contains the same protein coding sequence but permits cell-specific control of expression levels. We previously demonstrated that the antiviral cytokine type I interferon (IFN-α/β) suppresses GATA3 mRNA and protein levels both in developing Th2 cells and in fully committed Th2 cells, leading to a decrease in Th2 cytokine expression. Here, we show that IL-4 significantly enhances utilization of exon 1A in the GATA3 transcripts in human Th2 cells. IFN-α/β preferentially inhibits expression of the exon 1A transcript, suggesting that IFN-α/β can regulate Th2 cells without affecting the basal levels of GATA3 required for other immune processes. Thus, IFN-α/β suppresses GATA3 in human Th2 cells and may be useful as a novel therapeutic for asthma and allergy.
SUMOylation of chromosomal protein HMGN2 negatively influences its binding to nucleosomes (57.24)
Authors: Wu Jie , Kwak Man , Shin Jeon-Soo
Published: January 2023

The high mobility group-17 (HMG-17, HMGN2) is a small and unique non-histone protein that partakes in many key cellular processes, such as architectural element, transcription regulation and DNA repair. Recently, more functions of HMGN2 have been suggested, including antimicrobial activity, homing to special cells, cytokine release. The biochemical properties of HMGN2 protein are regulated by post-transcription modification like acetylation and phosphorylation, but it is unknown whether HMGN2 properties can also be affected by SUMOylation. In the present study, we showed for the first time that HMGN2 is modified by the covalent attachment of small ubiquitin-related modifier (SUMO)-1 and we identified the major SUMOylated lysine residues, located at HMGN2 nucleosome binding domain. Using a basal SUMOylation system in Escherichia coli, we were able to successfully separated sumo-conjugated HMGN2. Finally, we demonstrated that SUMOylated HMGN2 decreases binding ability to nucleosome particle core in comparison to its unSUMOylated form. These observations potentially provide a new perspective for a better understanding of the functions of HMGN2.
STING regulates IFN-β expression during endoplasmic reticulum stress (57.4)
Authors: Smith Judith , Tian Austin , Zeng Ling , Gutman Delia , Barber Glen , Liu Yi-Ping
Published: January 2023

STING (TMEM173, MPYS,MITA) located in the endoplasmic reticulum (ER), plays a critical role in type I IFN responses to intracellular DNA. STING activates TBK1, resulting in phosphorylated IRF3 that regulates IFN-β transcription. Our previous work showing markedly enhanced LPS-induced IFN-β during ER stress implicated increased IRF3 recruitment to the ifnb1 promoter. In this study, utilizing STING-/- mouse embryonic fibroblasts (MEFs) and RNAi (in MEFs and macrophages) we determined that STING was essential for synergistic induction of IFN-β mRNA in response to LPS and ER stress (thapsigargin/TPG treatment). TPG treatment alone induced re-localization and clustering of STING and TBK1 in MEFs. Although ER stress resulted in nuclear translocation of IRF3 in a STING-independent manner, TPG and TPG+LPS-induced IRF3 phosphorylation (as detected by immunofluorescence and western blot respectively) depended upon STING. In comparison, STING was not required for LPS or LPS+TPG induced NF-κB phosphorylation. These results indicate that ER stress alone results in IRF3 nuclear translocation and phosphorylation. In mice, treatment with the ER stress inducer tunicamycin was sufficient for induction of serum-detectable IFN-β, comparable with low dose LPS. Our results also suggest that the synergistic IFN-β responses during LPS and ER stress involve STING-dependent IRF3 phosphorylation.