bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen‐specific recognition of bacterial antigens by NKT cells
Authors: Kronenberg Mitchell , Kinjo Yuki , Tupin Emmanuel , Benhnia Mohammed Rafii‐El Idrissi , Zajonc Dirk , Sellati Timothy J
Published: June 2021

The objective of these studies is to understand the biochemistry and biological significance of microbial glycolipid antigen recognition by natural killer T (NKT) cells, a natural memory lymphocyte population that expresses an invariant T cell antigen receptor (TCR) alpha chain. We have identified two types of glycolipids presented by CD1d and recognized by the NKT cell TCR. Sphingomonas, which are environmental bacteria, have antigenic glycosphingolipids (GSLs), containing a ceramide lipid moiety. The second type of lipid is a glycosylated diacyl glycerol found in Borrelia burgdorferi, a spirochete that causes Lyme disease. Interestingly, mouse and human NKT cells selectively respond to diacylglycerols with different acyl chains, as mouse CD1d prefers to present compounds with shorter and less saturated fatty acids. Therefore, the recognition of these antigens is highly sensitive to the nature of the lipid, which is buried in the CD1d groove. To establish the significance of this response, we analyzed NKT cell deficient mice and controls infected with B. burgdorferi. The NKT cell deficient mice had increased joint inflammation at five weeks after infection and reduced bacterial clearance. Our data therefore strongly suggest that the NKT cells participate in host defense against bacterial pathogens through the recognition of microbial glycolipids by their invariant TCR.
The αxβ2 integrin CD11c is a potent costimulator of CD8 T cell responses
Authors: Bose Tina , Gumpenberger Kristina , Huleatt James , Ballantyne Christie , Lefrancois Leo
Published: June 2021

Integrins are important adhesion molecules necessary for leukocyte migration, homing and cell to cell interactions. We previously showed that CD11c, the α chain of the αXβ2 integrin, is upregulated after CD8 T cell activation in vivo. However, the functional significance of CD8 T cell expression of CD11c is unknown. Here, we examined the role of CD11c during the CD8 T cell response to infection using CD11c‐deficient mice. The response was appreciably dampened in CD11c−/− mice. In order to isolate the defect to CD8 T cells, we adoptively transferred a 1:1 ratio of T cell receptor (TCR) transgenic ovalbumin‐specific wild type (WT) OT‐I and CD11c−/− OT‐I T cells into WT B6 mice and infected the recipients. Using congenic markers, we then compared the ratios of CD11c−/− versus WT OT‐I cells during the response. Initially, CD11c −/− OT‐I T cells proliferated to the same extent as WT OT‐I T cells. In stark contrast, one day later (day 5 post infection), the WT OT‐I cells greatly outnumbered the CD11c−/− OT‐I cells and this difference was maintained throughout the memory phase. Thus, these results indicated a critical role for CD11c mediated costimulation in CD8 T cell activation leading to survival. This work is supported by the NIH Grants R01‐AI41576 and P01‐AI56172.
Modulation of CD4+ T cell function by HCV containing hepatocytes
Authors: Hall Caroline H.T. , Kassel Rachel , Hahn Young S.
Published: June 2021

Hepatitis C virus (HCV) infection is not cleared by the immune system in >70% of infected individuals. Viral persistence is correlated with a defect in T cell responses. CD4+ T cells are central in directing an effective immune response by supporting the activation and proliferation of B cells and maintenance of CD8+ effector function and memory response. We want to determine the impact that the HCV‐infected liver environment may have on CD4+ T cell responses. We utilized a co‐culture system that allows for direct contact between the hepatocytes and primary CD4+ T cells. A modified HCV cell culture system composed of a hepatoma cell line, Huh 7.5, containing full‐length HCV genome or the non‐structural portion of the genome only was used. Analysis of CD4+ T cell cytokine production indicates that the complete viral genome within hepatocytes has a suppressive effect on CD4+ T cell IFN‐γ production but no significant effect on IL‐10 production as measured by ELISA. In contrast, no significant difference in cytokine production was noted in cells that contained only the non‐structural portion of the HCV genome. Additionally, the inhibition of IFN‐γ was not detectable in CD8+ T cells. This data suggests that hepatocytes containing full‐length HCV genome have a specific immunosuppressive effect on CD4+ T cell function.
Sex Differences in Clearance of HSV‐1 after Acute Ethanol Exposure
Authors: Bird Melanie D. , Milligan Gregg N. , Albright Joslyn M. , Kovacs Elizabeth J.
Published: June 2021

Sex hormones are known to regulate immunity, and a dichotomy exists in the immune response between the sexes. Ethanol is also recognized to alter the immune response and may affect clearance of infectious agents differently in men and women. To examine sex differences in ethanol's effect on virus clearance, we used an intransasal model of Herpes Simplex Virus Type I (HSV‐1) infection. Male and female C57BL/6 mice were treated with an acute dose of ethanol (2.9g/kg) designed to give blood alcohol levels of 300mg/dL at 30 minutes at which time they were inoculated with HSV‐1 (5x104pfu). The lungs were harvested and virus replication was assessed using standard plaque assays. Virus titers were significantly higher on day 2 and 6 post‐inoculation in the lungs of female mice given ethanol compared to female mice given saline (p < 0.05) and male mice given saline or ethanol (p < 0.05). Additionally, Th1 cytokines including IFN‐γ, TNF‐α, and IL‐12 were all found to be 2‐fold lower in the lungs of female mice given ethanol compared to their saline controls. No differences were observed in cytokine production between male mice given ethanol or saline. These data suggest that ethanol may inhibit IFN‐γ production in female mice leading to decreased virus clearance. Supported by NIH R01AA012034, T32AA013527, an Illinois Excellence in Academic Medicine Grant and the Dr. Ralph and Marian C. Falk Medical Research Trust.
An early and robust natural killer cell response during acute SIV infection in sooty mangabeys but not rhesus macaques is correlated with disease resistance
Authors: Pereira Lara , Ansari Aftab
Published: June 2021

A need for defining optimal reagents and assays to identify the NK cell lineage in non‐human primates (NHP) is required because recent data clearly implicate an important role for NK cells in contributing to disease progression in HIV‐1 infected individuals. This cell lineage is particularly involved in the acute phase of infection which can optimally be studied in SIV infected NHP. A study of the role of NK cells in SIV infected disease resistant sooty mangabeys (SM) as compared with SIV infected disease susceptible rhesus macaques (RM) may provide important clues as to the contributing role of NK cells in the polarized clinical outcome in these two species. The use of previously identified reagents along with newly defined reagents and multi‐color flow cytometry has revealed that a gated population of CD3− CD8+ NKG2A+ cells identify 4 NK cell subsets with a predominant CD16+ CD56− subset primarily contributing to functional NK cytolytic activity. Cross sectional studies revealed a significant decline in this cytolytic subset in SIV‐infected RM but not in SIV‐infected SM. Importantly, longitudinal studies of individual animals pre and post SIV infection showed that while infection induces transient increases in NK cells in both species, there was a marked difference in the kinetics by which such increases occurred with SIV‐infected SM exhibiting a much earlier and more robust NK response than SIV‐infected RM.
Impairment of lung dendritic cell antigen presenting capacity by human paramyxovirus infections
Authors: Guerrero‐Plata Maria Antonieta , Kolli Deepthi , Ivanciuc Teodora , Casola Antonella , Garofalo Roberto P.
Published: June 2021

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are major etiologic agents of severe respiratory tract infections. We have recently shown that both CD4+ and CD8+ T cells are virtually absent in lung of fatal cases of RSV‐positive lower respiratory tract infections, suggesting that this pathogen can severely inhibit T cell responses. Pulmonary dendritic cells (DC) are critical in regulating T cell‐mediated immune response in the respiratory tract. Therefore, we examined the effect of RSV and hMPV infections on the capacity of lung dendritic cells to stimulate T cells. RSV and hMPV induced a peak of CD11C+/MHC‐II+ DC recruitment (>3×106) to the lung by day 8 and 10, respectively. DC remained elevated by day 18 (1×106), when no virus is detected, compared to DC recruited in control mice (1×105). The capacity of lung DC to present antigen to T cells was impaired by RSV or hMPV infection as DC from infected mice showed a reduced capacity (> 65%) to present OVA peptide to CD4+ T cells, compared to DC from control mice. The impairment was observed despite the fact that MHC‐II and CD80 were upregulated. Importantly, we observed that expression of the negative T cell regulator PDL‐1 was also upregulated on DC from infected mice. In conclusion, this study demonstrates that RSV and hMPV may evade immune response by suppressing DC‐mediated T cell proliferation.

NIH P01 062885, Flight Attendant Medical Research Institute
Characterizations of the virus‐induced differentiation of bone marrow plasmacytoid dendritic cells into CD11b+ dendritic cells
Authors: Liou Li‐Ying , Zuniga Elina I. , Oldstone Michael B.A.
Published: June 2021

Plasmacytoid dendritic cells (pDCs), so called natural interferon‐producing cells (IPCs), can produce large amount of type‐I interferon (IFN‐I) upon virus infection. Intensive studies have shown that pDCs are also involved in activation and modulation of adaptive immunity. However, less is known about the regulation of their developmental plasticity during virus infection.

Recently we demonstrated that bone marrow (BM) pDCs isolated from lymphocytic choriomeningitis virus (LCMV) infected mice differentiated into CD11b+ DCs in vitro. Here we provide in vivo evidence to prove the biological significance of our in vitro finding. We accomplished this by adoptive transferring pDCs isolated from LCMV infected mice and analyzing the immunoglobulin D‐J rearrangements (a genetic marker of pDCs) in CD11b+ DCs isolated from LCMV infected mice. Additionally, we demonstrated that the differentiation of pDCs into CD11b+ DCs does not rely on the infection of BM pDCs by LCMV, instead, is mediated by IFN‐I signaling. In preliminary studies we used pDC specific monoclonal antibodies against Ly49Q and Siglec‐H to characterize the pDC subtypes that differentiate into CD11b+ DCs. Collectively, our work indicates that BM pDCs constitute an important source for CD11b+ DCs during viral infection and further delineates the biological and phenotypic features of this novel DC developmental pathway.

This work was supported by US. Public Health Service grant (AI 045927) and a training grant (NS041219) to L.L. from the National Institutes of Health.
B cell‐Dendritic Cell interaction during influenza infection
Authors: Chen Ching‐I , Lerche Jacquelyn , Baumgarth Nicole
Published: June 2021

Strong antibody responses against influenza virus are mounted rapidly in the respiratory tract following an infection. These B cell responses contribute to immune protection, but how they are regulated is still incompletely understood. Antigen‐presenting cells (APC) can directly support B cell activation and class switch recombination in the absence of T cells. We aimed to determine whether influenza infection‐induced respiratory tract APC support virus‐specific B cell responses. We conducted co‐culture experiments of enriched influenza virus‐specific B cells with FACS‐purified dendritic cells (DC) and macrophages from three‐day infected lung and draining lymph nodes. B cells from MCMV‐infected mice served as controls. B cell proliferation was assessed by CFSE‐staining, total and virus‐specific antibody‐secretion was measured by ELISA and the Ig isotype profile was determined by bead array. Overall, respiratory tract APC did not support clonal B cell expansion but had significant effects on virus‐specific IgG‐secretion. Surprisingly, lung APC, particularly DC, were superior in their ability to provide help compared to lymph node APC. The effects were virus‐specific and required no additional antigen. Our data provide evidence that respiratory tract APC can drive specific B cell differentiation and they suggest an important role for tissue‐resident APC in B cell response induction. NIH A1055881.
Changes in Lung Antigen Presenting Cell Populations in a Mouse Model of Influenza Infection
Authors: Ho Adrian WS , Kemeny David Michael
Published: June 2021

Antigen preseting cells (APCs) in the lung serve as sentinels against airborne pathogens. Infection of the respiratory tract by Influenza A virus induces a recruitment of APCs to the lung. Of the various populations of APCs, lung dendritic cells play an essential role in the induction of adaptive immune responses to pulmonary infection. Using a mice model of influenza, we report the changes in the lung APC populations that occur over time. We focused our analysis on three groups of APCs, the CD11c+CD11b+MHCII+ lung dendritic cell, the CD11c+CD11b−MHCIIlow/int alveolar macrophage and the CD11c+CD11b−MHCIIhigh cell, which remains relatively uncharacterised in literature. We note a significant increase in the number of CD11c+CD11b+MHCII+ cells over time, but not in the other two populations. This is despite the detection of high CCL2 levels in brochoalveolar lavage. Finally, we show that lung dendritic cells upregulate expression of co‐stimulatory molecules even before migration to lymph nodes draining the lung, where co‐stimulatory molecule expression is further enhanced.
Mechanisms of social stress enhancement of virus‐specific immune memory
Authors: Mays Jacqueline Wiesehan , Powell Nicole Damico , Bailey Michael T , Padgett David A , Sheridan John Francis
Published: June 2021

Murine studies have demonstrated that the experience of social defeat (social disruption stress, SDR) before a primary influenza virus challenge augmented memory CD8+T cell responses to viral re‐challenge. Patterning of immune memory is established by the immune response to a primary infection, and the present study was designed to investigate potential mechanisms for memory enhancement during a primary challenge. C57BL/6 mice underwent 2 hrs of SDR per night for 6 nights (SDR‐INF group), which entailed introducing an aggressive mouse that physically defeated all resident mice. After night 6 of SDR, mice were intranasally infected with 1 HAU of influenza A/PR/8. Clonal expansion was significantly enhanced in the SDR‐INF group (p<0.05) for the immunodominant DbNP366+T cell subset, but not the DbPA224+ subset. SDR‐INF mice terminated viral replication significantly earlier than INF mice, and expressed more IFNγ mRNA prior to and during A/PR/8 infection. This study suggests that social defeat‐induced enhancement of virus‐specific immune memory occurs at the activation/clonal expansion phase of a primary infection, and is mediated by both cellular and environmental factors.

Supported by NIH grants F30DE17068‐02, RO1MH46801‐13, T32DE014320‐04.
Social disruption stress enhances the primary response to influenza infection through the activation of dendritic cells
Authors: Powell Nicole Damico , Mays Jaqueline W , Hanke Mark L , Stiner LaTonia M , Bailey Michael T , Padgett David A , Sheridan John F
Published: June 2021

Psychological stress has a significant impact on the anti‐viral immune response. In previous studies, social disruption stress (SDR) enhanced the adaptive response to influenza A/PR/8 infection, particularly at the level of the immunodominant DbNP366+T cell subset. This study was designed to determine the effect of SDR‐primed dendritic cells (DCs) on the development of enhanced influenza‐specific immune responses. Mice were infected with A/PR/8 24 hours after the transfer of purified DCs from the spleens of HCC or SDR mice. Mice receiving SDR‐primed DCs had significantly increased DbNP366+T cells in the lung 9 days post‐infection when compared to mice receiving HCC DCs. In addition, recipients of SDR‐primed DCs had decreased levels of M1 mRNA expression and increased levels of IFN‐γ mRNA expression in the lung 9 days post‐infection when compared to HCC DC recipients. Companion in vitro cultures showed increased IFN‐γ in the supernatants of SDR‐primed DCs co‐cultured with influenza peptide NP366–74 and CD8+T cells from influenza‐infected mice, compared to cultures containing HCC DCs. Taken together, these data indicate that SDR‐primed DCs enhance the immune response to A/PR/8 infection through the increase in DbNP366+T cell frequency and overall IFN‐γ secretion, which leads to enhanced viral clearance in the SDR‐primed DC recipient mice. Supported by NIH grants NIMH/RO1MH046801–15 and NIDCR/T32DE014320–6.
A Detrimental Role for IL‐10 During Host Immune Responses to Influenza Virus Infection
Authors: Sun Keer , Torres Luisa , Metzger Dennis W.
Published: June 2021

IL‐10 is an important anti‐inflammatory molecule that can cause immunosuppression, leading to long‐term viral persistence during chronic infection of mice with viruses such as lymphocytic choriomeningitis virus. However, its specific role during acute viral infections has not been documented. We now report that IL‐10 appears to also play a detrimental role during host responses to acute influenza virus infection since absence of IL‐10 led to improved viral clearance, weight recovery and survival rate in influenza‐infected mice. There were similar levels of CD4+ and CD8+ T cell recruitment into the lungs of infected WT and IL‐10−/− mice, and depletion of CD8+ T cells did not significantly influence viral clearance in IL‐10−/− mice. Although viral infected IL‐10−/− mice exhibited different kinetics of neutrophil recruitment compared to WT mice, neutrophil depletion did not hamper viral clearance. On the other hand, H1N1‐specific antibody levels in BALF were slightly increased in viral infected IL‐10−/− mice compared to WT mice. The mechanism of IL‐10 mediated suppression of host recovery from influenza virus infection is under further investigation. (Supported by NIH grant RO1 AI 41715)
Increased Circulating Proinflammatory Cytokines in Older Women with Persistent HPV Infection
Authors: Pinto Ligia A , Kemp Troy J , Hildesheim Allan , Garcia‐Pineres Alfonso , Williams Marcus C , Rodriguez Ana Cecilia , Schiffman Mark , Burk Robert , Freer Enrique , Bonilla Jose , Herrero Rolando
Published: June 2021

We have recently observed a generalized decrease in lymphoproliferative responses to PHA, Flu, and HPV‐16 L1 VLP from PBMCs isolated from women >45 years old who were persistently infected with type‐specific HPV. To better understand the mechanism of decreased lymphoproliferation in HPV persistently infected women, we examined plasma and PBMC culture supernatants from 100 women selected from a natural history cohort in Costa Rica for 16–24 different cytokines/chemokines using multiplex bead assays. Plasma cytokines associated with an inflammatory response (IL‐6, IL‐8, TNF‐a, and MIP‐1a) were significantly increased in persistently infected individuals as compared to controls. Supernatants from unstimulated PBMCs had an increase in IL‐6, TNF‐a, and MIP‐1a within the persistence group, consistent with our plasma findings. In contrast, PHA‐stimulated cultures from the HPV persistent group had a significant decrease in cytokines/chemokines, suggesting possible anergy and/or apoptosis of cells from women with persistent HPV infection upon stimulation. These findings suggest that cervical HPV persistence in older women is associated with an increase in systemic inflammatory cytokines. Analyses of mechanism(s) of systemic inflammation and poor lymphoproliferation in persistent HPV infection are in progress. (Funded by NCI Contract N01‐CO‐12400)
T cell‐intrinsic TGF‐beta signaling is essential for CD8 T cell exhaustion and viral persistence in vivo
Authors: Zuniga Elina Isabel , Sauer Karsten , Poling Matthew C.
Published: June 2021

Chronic viral infections kill millions of people annually worldwide. Loss of T cell function has been noticed for long time during chronic infections but the underlying factors are not completely understood. Here, we evaluate the role of TGF‐β in the establishment of a persistent viral infection. For that, we infected mice expressing a dominant negative form of TGF‐β receptor II from a modified CD4 promoter (dnTGFBRII) with a persistent variant of lymphocytic choriomeningitis virus, Clone 13 (Cl 13). We found that attenuated TGF‐β signaling in T cells allows the expansion of virus specific CD8 T cells in Cl 13 infected mice resulting in potent cytokine production, enhanced cytotoxic capacity and down‐regulated levels of προγραμμεδ χελλ δεατη−1. Μορεοϖε, ιν χοντραστ το τηειρ ωιλδ τψπε χουντερπαρτσ, Χλ 13−ινφεχτεδ−δνΤΓΦΒΡΙΙ μιχε ραπιδλψ ελιμινατε τηε ϖιρυσ φρο μ βλοοδ ανδ σεϖ εραλ τισσυεσ. Ουρ δατα ιδεντιφψ α Τ χελλ−ιντρινσιχ ινηιβιτο ρψ πατηωαψ ρεσπονσιβλε φορ ΧΔ8 Τ χελλ εξηαυστιον ανδ ϖιραλ περσιστενχε ινδιχατινγ τηατ σελεχτιϖε μο δυλατιον οφ ΤΓΦ−β signaling on T cells holds a strong promise for alternative treatments of chronic viral infections.
Marked Expansion of Foxp3+, Vbeta5+ Regulatory T cells during Chronic Lymphocytic Choriomeningitis Virus (LCMV) Infection
Authors: Punkosdy George , Ahmed Rafi , Shevach Ethan
Published: June 2021

In order to analyze the potential role that Treg play in viral infection, we have compared the phenotypic changes in Treg during the course of acute (Armstrong) and chronic (Clone 13) LCMV infection in mice. Foxp3+ Treg increased in frequency among CD4+ cells and expressed increased levels of CTLA‐4, GITR, ICOS, and PD‐1 specifically during Clone 13 infection suggesting an increase in Treg activation during chronic infection. Analysis of TCR Vbeta usage revealed a marked increase in a subpopulation of Treg expressing the TCR Vbeta5 during Clone 13 infection (from 7% to ∼25% of total Foxp3+ cells in uninfected or Armstrong infected and Clone 13 infected mice, respectively). Surprisingly, no change in the frequency of CD4+ Vbeta5+Foxp3− was noted during either Armstrong or Clone 13 infections. The Vbeta5+Foxp3+ Treg expressed additional markers that have been associated with Treg activation including high levels of CD101, CD103 and granzyme B. The increase in frequency of Vbeta5+ Treg was due to the preferential expansion of this population early in infection, as determined by BrdU uptake and Ki‐67 staining, and not differentiation of Vbeta5+Foxp3− T cells. We are now in the process of determining the antigen specificity of the Vbeta5+Foxp3+ T cells and determining the specific role these cells may play in the regulating the immune response during chronic viral infection.
Which comes first Tregs or TB?
Authors: Green Angela M. , Bongers Kale , Flynn JoAnne L.
Published: June 2021

Worldwide, Mycobacterium tuberculosis (Mtb) is responsible for ~ 2 million deaths per year. While a majority of individuals control the infection, 5–10% develop active disease. Recent studies in human TB patients indicate patients with active disease have increased frequency of regulatory T cells (Tregs) in peripheral blood when compared to latent controls. However, whether a higher frequency of Tregs is responsible for the development of active disease or whether the frequency of these cells increases is in response to active disease is not known. To determine which may be the case, we used a non‐human primate model of disease, the only animal model to mimic human latent infection. Cynomolgus macaques (30) were infected with low dose M.tb and followed until disease state (active or latent) was declared (6–8 months). Regardless of infection outcome, all animals exhibited a significant drop in the frequency of Tregs (CD4+foxP3+) during the first 8 weeks of infection (p<0.05). Concurrent with the decrease seen in the peripheral blood the frequency of CD4 T cells and Tregs increased in the airways of these animals. Interestingly, monkeys with active disease exhibited a trend toward higher Tregs by 20 weeks post infection. Taken together, these data indicate that Tregs may increase in the periphery in response to inflammation from active disease. NIH grant: RO1 AI50732‐01
Prophylaxis with cationic liposome‐DNA complexes protects hamsters from phleboviral disease: importance of CpG motifs?
Authors: Gowen Brian B. , Fairman Jeff , Wong Min‐Hui , Jung Kie‐Hoon , Bailey Kevin W. , Morrey John D.
Published: June 2021

Cationic liposome‐DNA complexes formed with plasmid DNA (CLDC), CpG‐free plasmid DNA, or CpG‐containing oligodeoxynucleotides (ODN) were evaluated for their ability to elicit protective immunity against Punta Toro virus (PTV) challenge in the hamster infection model. Liposome‐DNA formulations were administered by the intraperitoneal route 4 h prior to inoculation of a highly lethal PTV inoculum. Survival outcomes, as well as reduction of viral burden and liver disease, were assessed. A dose of CLDC containing 30 μg of plasmid significantly improved survival outcome, decreased systemic and liver viral loads, and reduced liver pathology compared to animals receiving placebo. Known mouse‐ and human‐reactive ODNs delivered in liposome complexes failed to protect challenged hamsters. Treatment with liposomes containing non‐CpG motif‐containing plasmid reduced liver virus titers compared with untreated or CpG treated, but did not protect hamsters from death. This suggests that while elements other than CpG motifs may stimulate a response sufficient to control viral replication in the target organ, the empty vector plasmid DNA backbone with CpG islands appears to be critical to stimulating protection against mortality in the PTV hamster model. Supported by contract grant NO1‐AI‐15435 from the Virology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Viridans streptococcal carbohydrates and their salivary receptor proteins
Authors: Khagi Simon , Quinn Tim , Herndon Betty
Published: June 2021

Streptococcal colonization of damaged heart surfaces in infective endocarditis depends on the recognition of host lectin‐receptors by bacterial glycoprotein. Our group has studied the viridans streptococcal glycoproteins of 30 bacteremic patient isolates both by carbo‐HPLC and by endocarditis production in the rat model. Two strains, endocarditis virulent Streptococcus#15 (Strep 15)and endocarditis negative #4 (Strep 4) were used to measure salivary receptor protein titer for the bacterial sugars. Tests were run on 16 samples (96 tests). Mixed saliva was stimulated by chewing sterile paraffin, collected on ice, sterile filtered and stored at −70C until serially diluted for in vitro agglutination with washed, killed streps 4 and 15, suspended at 0.6 absorbance. Triplicate twofold dilutions of each saliva sample were incubated 24 hr with each fixed streptococcus, and aggregation was scored both visually and microscopically. Individual patients showed an agglutination titer representing the subject's salivary mucins and platelet surface sialic acids, and ranged from 1:8 for strep 4 to 1:2048 for strep15 with young, healthy subjects and 1:2 and 1:4 in other subjects. Although salivary titer differed significantly between subjects, the ratio of virulent to non‐virulent titer was constant between individuals. These experiments suggest that host recognition of bacterial sugars relates more to the bacterial sugar (and its virulence?) than to individual differences in salivary target lectins. This concept, if shown in a larger population, may provide important insights into the pathogenesis of infective endocarditis induced by oral streptococci.
Beta‐glucan activates microglia through Dectin‐1 without inducing cytokine production
Authors: Shah Vaibhav , Keshvara Lakhu
Published: June 2021

Microglia, the resident mononuclear phagocytic cells, are critical for immune responses within the CNS. They recognize and are activated by various Pathogen Associated Molecular Patterns (PAMPs). β‐glucans are PAMPs present within fungal cell walls that are known to trigger protective responses in a number of immune cells. To better understand microglial responses to β‐glucans and the underlying signaling pathways, we sought to determine if Dectin‐1, a major β‐glucan receptor recently identified in leukocytes, mediates β‐glucan induced activation of microglia. Here, we report that Dectin‐1 is expressed on the surface of murine primary microglia, and engagement of the receptor with particulate β‐glucan resulted in an increase in tyrosine phosphorylation of Syk, a hallmark feature of the Dectin‐1 signaling pathway. Moreover, phagocytosis of β‐glucan particles and subsequent intracellular production of ROS were also mediated by Dectin‐1. Further, interaction of β‐glucan with Dectin‐1 resulted in activation of Vav and PI3K/Akt pathways, suggesting that β‐glucan induce multiple signaling pathways in microglia. However, unlike in leukocytes, β‐glucan mediated microglial activation did not result in production of cytokines. Thus, the interaction of microglial Dectin‐1 with glucan elicits a unique response, suggesting that the Dectin‐1 pathway may play an important role in antifungal immunity in the CNS.
Tolerance to rat liver allograft after total lymphoid irradiation is mediated by CD4+CD25+ regulatory T cells
Authors: Fujiki Masato , Esquivel Carlos O , Martinez Olivia M , Strober Samuel , Krams Sheri M
Published: June 2021

We have previously determined that post transplant total lymphoid Irradiation (TLI) induces tolerance in an experimental model of liver transplantation. The purpose of this study is to characterize the mechanism of TLI induced tolerance.

TLI significantly prolonged survival in Lewis recipients of fully‐histoincompatible DA liver allografts with 88% of the recipients surviving long‐term (>100 days). CD4+CD25+Foxp3+ T cells were markedly increased in the liver grafts, PBMC, and spleen by day 35 post‐transplant and remained high in the spleen >100 days post‐transplant. Splenocytes from long‐term surviving recipients have a reduced alloresponse in MLR. Moreover, isolated splenic CD4+CD25+ T cells inhibited the MLR of naive Lewis splenocytes to DA splenocytes. Importantly, adoptive transfer of whole splenocytes into naive Lewis recipients of DA heart grafts significantly prolonged cardiac graft survival (MST whole splenocytes 33 days vs. control 18.5 days:p<0.01), whereas depletion of CD4+CD25+ T cells from splenocytes completely abrogated the prolongation of cardiac allograft survival (MST 16 days; p<0.01, vs. whole splenocytes).

TLI induces tolerance to liver allografts via a mechanism involving CD4+CD25+Foxp3+ T cells that accumulate in the liver early after transplant and remain in the spleen >100 days. Studies using TLI to induce tolerance in clinical liver transplantation are currently underway.
Pancreatic Islets Engineered to Display on Their Surface an Exogenous FasL Protein Survive Indefinitely in Allogeneic Recipients
Authors: Zhao Hong , Yolcu Esma S. , Bandura‐Morgan Laura , Lacelle Chantale , Askenasy Nadir , Shirwan Haval
Published: June 2021

Objective: To engineer pancreatic islets in a rapid and efficient manner with a novel form of FasL protein and test the efficacy of engineered islets for long‐term survival in allogeneic hosts.

Methods: BALB/c islets were engineered by cell surface modification with biotin followed by the display of a chimeric form of FasL protein with core streptavidin (SA‐FasL). SA‐FasL‐engineered islets were transplanted into streptozotocin diabetic C57BL/6 mice under transient cover of rapamycin.

Results: All the islets showed effective engineering with SA‐FasL, which persisted on the surface of islets for weeks in vitro as assessed by confocal microscopy. All the islets (n=23) with SA‐FasL survived over 100–360 days without detectable signs of rejection. In marked contrast, all the unmodified (n=9) and SA‐engineered (n=14) islets underwent acute rejection within 40 days. The observed tolerance was localized to the engineered islets as unmodified second set of islets transplanted under contralateral kidney of long‐term (>90 days) graft recipients were rejected in a normal tempo (MST=17 ± 9 days) without any effect on the survival of primary islets.

Conclusion: Engineering pancreatic islets with exogenous immunomodulatory molecules, such as SA‐FasL, in a rapid and efficient manner represents a novel means of immunomodulation with considerable therapeutic potential. NIH (R21 DK61333, R01 AI47864, R21 AI057903, R21 HL080108), JDRF (1‐2001‐328), and ADA (1‐05‐JF‐56).
Embryonic stem cells cultured in serum free medium acquire bovine apolipoprotein B‐100 from feeder cell layers and serum replacement medium
Authors: Sakamoto Norihisa , Sakamoto Michiko , Rosenberg Amy S.
Published: June 2021

Previous studies have demonstrated that cell populations that are cultured with heterologous animal products can acquire xenoantigens, potentially limiting their clinical utility because of immune responses. Embryonic stem cells (ESCs) are an attractive source of multiple potential cellular therapies and are typically derived and routinely cultured on murine embryonic fibroblast (MEFs) feeder cell layers in commercially available serum replacement (SR) medium or fetal calf serum (FCS) containing medium. Recently we found that a strong antibody response was generated in human subjects after the second infusion of therapeutic cells cultured in FCS containing medium. This response was specific for bovine apolipoprotein B‐100 (apoB‐100) which is the major protein component of LDL and targets its binding to abundant low density lipoprotein receptors on the cell surface from which it is internalized. Here we have shown that ESCs cultured on MEFs in SR medium acquired bovine apoB‐100 from MEFs and from the SR medium as well. Our findings also suggest that bovine low density lipoproteins (LDL) are used as critical nutrients for ESC propagation.
Vascularized bone marrow transplant (VBMT) serves as a novel surgical approach to stem cell transplantation and stimulates normal repopulation of lymphocytes in the spleens of irradiated (XRT) rat hosts
Authors: Licata Lauren , Rojavin Yuri , Patel Mitul , Tai Chau , Strande Louise , Matthews Martha , Hewitt Charles
Published: June 2021

Our lab demonstrated that VBMT, the microsurgical transplantation of a femur with its vascular pedicle, can induce immune tolerance across a semi‐allogeneic barrier without graft versus host‐disease. We hypothesized that VBMT alone is potentially superior to cellular stem cell transplantation and sufficient to induce normal repopulation of the spleens of XRT rat recipients.

Lewis Brown Norway rats received 9 Gy radiation. Rats then received either a conventional cellular (cBMT, n=5) or VBMT (n=4) bone marrow transplant from Lewis donors. Control groups were: non‐transplanted, non‐irradiated (CT, n= 5) or XRT only (n= 4). Stained histologic sections of spleens were analyzed with digital image analysis (DIA).

XRT rats died 9±1 days post irradiation. Other groups were sacrificed: cBMT at 110 days; VBMT at 102 ±1 days. Compared to CT, XRT spleens showed white pulp atrophy; cBMT had less white pulp but appeared hypercellular and VBMT spleens appeared normal. DIA results can be seen in the table below:

VBMT is capable of reconstituting spleens of XRT semi‐allogeneic recipients similar in appearance to normal rats. This model has clinical relevance for both stem cell transplantation and solid organ transplantation, in that VBMT induces chimerism and tolerance.
ST2L upregulation promotes maturation resistance in rapamycin‐conditioned dendritic cells
Authors: Turnquist Heth Roderick , Sumpter Tina L. , Nau Gerard J. , Thomson Angus W.
Published: June 2021

Objective: Maturation resistance and tolerogenicity can be conferred on dendritic cells (DC), ‐crucial regulators of T cells, by exposure to rapamycin (RAPA), a tolerance‐sparing immunosuppressant. The mechanisms underlying this acquired unresponsiveness, typified by diminished responses to Toll‐like receptor (TLR) ligation, have not been identified. Thus, our objective was to elucidate a molecular basis for RAPA‐induced DC maturation resistance.

Methods: RAPA administration was used to condition splenic DC in vivo and bone‐marrow derived DC in vitro. DC maturation was monitored by assessment of co‐stimulatory molecule expression and allostimulatory capacity. To identify negative regulators of maturation, microarray analysis and quantitative RT‐PCR was completed, and findings confirmed via Western and flow cytometric analyses.

Results: DC conditioning with RAPA promotes overexpression of the IL‐1 receptor(R) family member, ST2L, and enhances its surface expression. ST2L is the receptor for IL‐33 and has also been implicated as a negative regulator of TLR signaling. Consistent with this regulatory function, induced ST2L expression suppressed the responsiveness of RAPA‐DC to TLR ligation.

Conclusion: This work identifies a novel mechanism by which a clinically‐important immunosuppressant impedes the capacity of DC to mature and consequently stimulate effector/adaptive T cell responses.
The Evolution and Diversity of IGG
Authors: Butler John , Wertz Nancy , Kacskovics Imre
Published: June 2021

Immunoglobulin G (IgG) is the flagship mammalian antibody with evolutionary roots in amphibian IgF. Cartilaginous and bony fish have only two antibody isotypes; IgM and IgD. IgD is structurally diverse and seems to have disappeared in birds, in egg‐laying and marsupial mammals and even some eutherian mammals. Meanwhile, IgG emerged as the “mammalian antibody”. Like is often true, evolutionary emergence is associated with evolutionary divergence making mammalian IgG an interesting topic for studies on the evolutionary diversification of a gene family. Gene duplication produced an array of IgG subclasses ranging from only one (rabbits) to seven (horses) with all other possibilities in between. Swine have six putative subclasses of IgG with at least two alleles known for five of these. IgG3 is monomorphic and closest to the “stem IgG lineage” among mammals. Alternative splicing is common in the CH2 domain, potentially allowing swine to express >20 different IgG antibodies.

Understanding the evolution of IgG among mammals has practical implications. Since speciation preceded subclass diversification investigators should not extrapolate the function of e.g. IgG1 in one species to an IgG of the same name in another. All of this begs the question as to why this antibody class evolved and diversified exclusively among mammals. Perhaps it is their ability to utilize Fc receptor‐mediated events like opsonization, ADCC, transepithelial transport or cellular re‐cycling which protects IgG from catabolism extending its half‐life. All of these provide survival advantage to especially offspring that have not yet developed their own adaptive immune response.
A unique process of variable disulfide polymerization facilitates affinity maturation in salmonids
Authors: Ye Jianmin , Bromage Erin S. , Kaattari Stephen L.
Published: June 2021

Affinity maturation within the salmonid IgM antibody response has been amply demonstrated; however, we have, now, additionally found that this process is linked, to the non‐uniform disulfide cross‐linking of the antibodies¡− monomeric subunits. This relationship was demonstrated by three different routes; 1) isolation and characterization of high and low affinity antibodies from the same source, 2) isolation of antibodies during the process of affinity maturation, and 3) via specific induction of high and low affinity antibodies in vitro. Each method demonstrated a direct relationship between affinity and the degree of disulfide polymerization, with the most cross‐linked forms possessing the highest affinity. Half‐life studies performed by transferring labeled immunopurified antibody to naive hosts, revealed the most rapid removal of the more reduced forms, which possessed the lowest affinity, from the circulation. It appears that the process of intermolecular disulfide polymerization of the antibody molecule, or some directly related or contingent physical feature must be acting to facilitate the process of affinity maturation in salmonid fish.
Identification CD79a and CD79b homologs in channel catfish, Ictaulurus punctatus
Authors: Sahoo Manoranjan , Edholm Eva‐stina , Stafford James , Bengten Eva , Miller Norman W. , Wilson Melanie
Published: June 2021

The catfish (Ip) B cell receptor accessory molecules CD79a and CD79b homologs have been identified. Each are encoded by single copy genes that encode proteins containing a signal peptide, an extracellular immunoglobulin domain, a transmembrane region and a cytoplasmic tail containing an immunoreceptor tyrosine kinase activation motif (ITAM). At the message level, both IpCD79a and IpCD79b correlate well with IgM message expression since they are highly expressed in membrane IgM+ peripheral blood leukocytes (PBL) and in catfish clonal B cell lines, but not in IgM− PBL or in clonal T cells or macrophages. Such findings indicate that IpCD79a and IpCD79b expression is B cell restricted. Furthermore, studies using catfish clonal B cells (3B11) transfected with constructs encoding epitope‐tagged IpCD79 proteins revealed that IpCD79a is expressed as a 45 kDa protein and IpCD79b is expressed as a 32 kDa protein. These correlate with the molecular weights of putative tyrosine phosphorylated accessory proteins that are co‐immunoprecipitated with surface IgM following anti‐Ig stimulation.Supported by NIH grant R01 AI19530.
Infection of dendritic cells with the American, but not the European, PRRSv genotype induces Treg (Foxp3+CD25+) cells.
Authors: Hernandez Jesus , Silva Erika , Reséndiz Mónica , Flores Lilian , Fraile Lorenzo , Montoya Maria
Published: June 2021

The potential of PRRSv‐infected dendritic cells (DCs) to induce Foxp3+CD25+ T‐regulatory cells was evaluated in vitro using two American and five European strains. Monocyte‐derived DCs were infected, cultured for 24 h and in some cultures were treated with IFN‐α. Infected DCs were co‐cultured with autologous non‐adherent cells for 5 days and some cells stained with CFSE. Cytokines were quantified by qPCR and by ELISA, whereas the expression of Foxp3+ and CD25+ was analyzed by FACS. Our results showed that American, but not European, type viruses reduced T cell proliferation. The addition of IFN‐α did not affect T cell proliferation but it inhibited the ability of PRRSv to replicate. DCs infected with American type increased the number of T cells expressing Foxp3+CD25+ phenotype and IFN‐α reduced the generation of this cells. Surprisingly, European type did not induce T cells with Foxp3+CD25+ phenotype. The expression of IL‐10 mRNA was 3 fold higher in DCs infected with American type as compared to DCs infected with the European type. Finally, American type increased the expression of TGF‐β; mRNA five fold. In conclusion, these results suggest that American, but not European type PRRSv, induce the expression of T‐regs cells (Foxp3+CD25+), and this induction could be dependent on TGF‐β.

Funded by SEP‐CONACyT (43602) Mexico, and SMES (AGL2005‐07073‐C02‐02) Spain.
Comparative innate immune gene expression in chicken intestine after in vivo challenge with commensal (Campylobacter) and pathogenic (Salmonella) bacteria
Authors: Shaughnessy Ronan Gerard , Meade Kieran G. , Cahalane Sarah , Allan Brenda , Reiman Carla , Farrelly Cliona O'
Published: June 2021

Campylobacter jejuni is a commensal bacterium in chickens and a major cause of food poisoning in humans. It is unknown whether the intestinal immune response has a role in establishing the commensal state. The innate immune responses of 4 week‐old broiler chickens to C. jejuni challenge (108 bacteria) were studied and compared with an avian pathogen, Salmonella enterica. Challenged and control birds were sacrificed 6 hours post‐challenge, RNA was extracted and cDNA synthesised. Selected genes involved in pathogen detection and innate immune response to bacterial infection were profiled in the caecum by quantitative real‐time PCR.

A significant increase in the expression of TLR21 was observed after challenge with both types of bacteria (P < 0.05) compared to control birds. TLR4 expression was increased 4‐fold after C. jejuni challenge only (P = 0.02). IL6 was increased in response to both commensal and pathogenic challenges (P < 0.02). In contrast, IL8 mRNA expression was decreased 4‐fold in response to C. jejuni but increased 5‐fold to S. enterica challenge (P = 0). Antimicrobial peptides were differentially expressed in both models. Specifically, AvBD6 was increased by 24‐fold in response to S. enterica infection (P = 0).

Results indicate that C. jejuni induces early innate immune gene expression changes, some similar to and others distinct from pathogenic challenge

Funding: FIRM, Ireland
Model to estimate in vivo enrichment of beef muscle and liver with 14C‐vitamin B12 (14C‐B12)
Authors: Garrod Marjorie G , Johnson Heidi A , Calvert Christopher C , Allen Lindsay H , Green Ralph , Miller Joshua W
Published: June 2021

Beef enriched in vivo with 14C‐B12 may be used for assessing B12 bioavailability and absorption in humans. However, B12 transfer rates between plasma and tissues in bovines are not well characterized. We created a model of B12 kinetics based on data collected from two 500 lb calves: calf 1 received two I.V. pulse doses of 14C‐B12 (7.5 μg, 250 nCi/dose) given 24h apart; calf 2 received a continuous infusion of 14C‐B12 (22.5 μg, 750 nCi) for 21d using an Alzet pump. 14C content of plasma, liver and muscle was determined by accelerator mass spectrometry. The model is composed of 4 state variables representing B12 pools in blood, liver, muscle, and other tissues. Exchanges between pools were predicted based on mass action kinetics. Using pulse dose data, uptake rates from plasma into muscle and liver were concentration dependent and estimated to be 5.5%/d and 3.9%/d, respectively. Using continuous infusion data, uptake rates from plasma into muscle and liver were concentration independent and estimated to be 0.072 nmol/d and 0.141 nmol/d, respectively. Model estimates of 14C‐B12 accumulation in liver and muscle were within 30% of measured values. SDs of the estimated exchange rates were low indicating rate predictions were unique. This model can provide estimates of B12 enrichment in meat and liver using different dosing protocols.

Funding: National Cattlemen's Beef Association
On the obesity paradox
Authors: Childers Douglas Karl , Allison David B.
Published: June 2021

Regarding body mass index (BMI; kg/m2) and mortality, two well established observations are (1) there is a U‐shaped (i.e., concave) association ‐ people with intermediate BMIs tend to outlive people with unusually high or low BMIs; and (2) The nadirs of these curves tend to increase monotonically with age. Whether these associations represent the true shape of the causal effects of BMI remain subject to debate and inquiry. Multiple hypotheses have been advanced to explain either of these two observations. Here we introduce a new hypothesis that may explain both phenomena, by drawing on the so‐called obesity paradox. We evaluate the plausibility that the concave relation and the increase in the nadir with age are mathematical consequences of the relationships among age, BMI, and disease; and mortality as a function of the prior two variables.

We incorporate a hypothetical relationship between BMI and disease risk into the Weibull function, F(t) = exp(−λtk), to model the probability of an individual to survive until age t, with a given BMI; by replacing the constant λ with a function of age and BMI, that represents the effects of these two variables on disease and mortality. The resulting function can be viewed as a survival curve that now depends on both BMI and age. We consider the behavior of this survival curve as the individual's BMI varies, while holding t constant, to find the optimal BMI at age t. We then consider the behavior of the optimal BMI for an individual as age increases. In particular, we establish that under the assumptions of our model, the optimal BMI at age t increases as a function of t.