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Individuals with systemic sclerosis (SSc) are particularly susceptible to SARS-CoV-2 infections, yet it remains to be determined if they generate humoral and cellular responses comparable to controls following SARS-CoV-2 vaccinations. Herein, we collected blood and serum after second, third, and fourth SARS-CoV-2 vaccinations in patients with SSc and controls. Following each dose, participants with SSc mounted comparable serum anti-RBD IgG, anti-RBD IgA, and spike-specific CD4 + and CD8 + T cell responses to those found in controls. At 3 months post dose 2, the frequencies of Th1, Th2, Th17, and Treg spike-specific CD4 + T cells in participants with SSc did not differ from controls. At 2-6 weeks post dose 3, participants with SSc displayed reduced frequencies, but not numbers, of Th17-polarized spike-specific CD4 + T cells. Thus, participants with SSc did not display significantly weaker humoral or cellular responses to SARS-CoV-2 vaccination than controls, enabling reassurance of vaccine immunogenicity in participants with SSc., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maggie J. Larche reports financial support was provided by Scleroderma Canada. DMEB has received honorarium for consulting on the topic of vaccines from Pfizer-Canada and AstraZeneca. MJL is on the Advisory Boards/Speakers Bureaus for AbbVie, Actelion, Amgen, Astra-Zeneca, Boehringer-Ingelheim, BMS, Fresenius-Kabi, Gilead, GSK, Lilly, Mallinckrodt, Novartis, Pfizer, Sanofi, SOBI, UCB, and Scleroderma Society of Ontario/Canada. None of the other authors have competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP affects risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2-knockout (Tet2-/-) and floxed control mice (Tet2fl/fl) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that, while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake, and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.

Aging is a complex biological process that is characterized by low-grade inflammation, called inflammaging. Aging affects multiple organs including eye and lacrimal gland. Tumor necrosis factor (TNF) is a pleiotropic cytokine that participates in inflammation, activation of proteases such as cathepsin S, and formation of ectopic lymphoid organs. Using genetic and pharmacological approaches, we investigated the role of TNF in age-related dry eye disease, emphasizing the ocular surface and lacrimal gland inflammation. Our results show the increased protein and mRNA levels of TNF in aged lacrimal glands, accompanied by increased TNF, IL1β, IL-18, CCL5, CXCL1, IL-2, IL-2 receptor alpha (CD25), IFN-γ, IL-12p40, IL-17, and IL-10 proteins in tears of aged mice. Moreover, genetic loss of the Tnf -/- in mice decreased goblet cell loss and the development of ectopic lymphoid structures in the lacrimal gland compared to wild-type mice. This was accompanied by a decrease in cytokine production. Treatment of mice at an early stage of aging (12-14-month-old) with TNF inhibitor tanfanercept eye drops for eight consecutive weeks decreased cytokine levels in tears, improved goblet cell density, and decreased the marginal zone B cell frequency in the lacrimal gland compared to vehicle-treated animals. Our studies indicate that modulation of TNF during aging could be a novel strategy for age-related dry eye disease., (Copyright © 2023 Elsevier Inc. All rights reserved.)

People exposed to adverse childhood experiences (ACEs) suffer from an increased risk of chronic disease and shorter lifespan. These individuals also tend to exhibit accelerated reproductive development and show signs of advanced cellular aging as early as childhood. These observations suggest that ACEs may accelerate biological processes of aging through direct or indirect mechanisms; however, few population-based studies have data to test this hypothesis. We analysed ACEs and biological aging data from the Canadian Longitudinal Study on Aging (CLSA; n = 23,354 adults aged 45-85) and used the BioAge R package to compute three indices of biological aging from blood-chemistry and organ-function data: Klemera-Doubal method (KDM) biological age, phenotypic age (PA), and homeostatic dysregulation (HD). Adults with ACEs tended to be biologically older than those with no ACEs, although the observed effect-sizes were small (Cohen's d<0.15), with the exception of neglect (d=0.35 for KDM and PA). Associations were similar for men and women and tended to be smaller for older as compared to midlife participants. Subtypes of ACEs perceived as being more severe (e.g., being pushed or kicked, experiencing forced sexual activity, witnessing physical violence) and more frequent and diverse exposures were associated with relatively larger effect-sizes. These findings support the hypothesis that ACEs contribute to accelerated biological aging, although replication is needed in studies with access to prospective records of ACEs and cellular-level measurements of biological aging. Furthermore, future work to better understand the degree to which associations between ACEs and biological aging are moderated by specific life-course pathways, and mediated by lifestyle and socioeconomic factors is warranted., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation., Methods: A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1β were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKβ1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot., Results: Genetic deletion of AMPKβ1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the β1 subunit., Conclusions: Together, these data suggest that the development of therapeutic agents targeting AMPKβ1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

More than 40% of HIV infections occur via female reproductive tract (FRT) through heterosexual transmission. Epithelial cells that line the female genital mucosa are the first line of defense against HIV-1 and other sexually transmitted pathogens. These sentient cells recognize and respond to external stimuli by induction of a range of carefully balanced innate immune responses. Previously, we have shown that in response to HIV-1 gp120, the genital epithelial cells (GECs) from upper reproductive tract induce an inflammatory response that may facilitate HIV-1 translocation and infection. In this study, we report that the endometrial and endocervical GECs simultaneously induce biologically active interferon-β (IFNβ) antiviral responses following exposure to HIV-1 that act to protect the epithelial tight junction barrier. The innate antiviral response was directly induced by HIV-1 envelope glycoprotein gp120 and addition of gp120 neutralizing antibody inhibited IFNβ production. Interferon-β was induced by gp120 in upper GECs through Toll-like receptor 2 signaling and required presence of heparan sulfate on epithelial cell surface. The induction of IFNβ was dependent upon activation of transcription factor IRF3 (interferon regulatory factor 3). The IFNβ was biologically active, had a protective effect on epithelial tight junction barrier and was able to inhibit HIV-1 infection in TZM-bl indicator cells and HIV-1 replication in T cells. This is the first report that recognition of HIV-1 by upper GECs leads to induction of innate antiviral pathways. This could explain the overall low infectivity of HIV-1 in the FRT and could be exploited for HIV-1 prophylaxis.

Background: The levels of circulating cytokines fluctuate with age, acute illness, and chronic disease, and are predictive of mortality; this is also true for patterns of DNA (CpG) methylation. Given that immune cells are particularly sensitive to changes in the concentration of cytokines in their microenvironment, we hypothesized that serum levels of TNF, IL-6, IL-8 and IL-10 would correlate with genome-wide alterations in the DNA methylation levels of blood leukocytes. To test this, we evaluated community-dwelling adults (n = 14; 48-78 years old) recruited to a pilot study for the Canadian Longitudinal Study on Aging (CLSA), examining DNA methylation patterns in peripheral blood mononuclear cells using the Illumina HumanMethylation 450 K BeadChip., Results: We show that, apart from age, serum IL-10 levels exhibited the most substantial association to DNA methylation patterns, followed by TNF, IL-6 and IL-8. Furthermore, while the levels of these cytokines were higher in elderly adults, no associations with epigenetic accelerated aging, derived using the epigenetic clock, were observed., Conclusions: As a preliminary study with a small sample size, the conclusions drawn from this work must be viewed with caution; however, our observations are encouraging and certainly warrant more suitably powered studies of this relationship.

Heparin-induced thrombocytopenia (HIT) is a serious drug reaction that leads to a decrease in platelet count and a high risk of thrombosis. HIT patients produce pathogenic immunoglobulin G (IgG) antibodies that bind to complexes of platelet factor-4 (PF4) and heparin. HIT immune complexes crosslink Fc-receptors resulting in platelet and monocyte activation. These events lead to the release of procoagulant chemokines and tissue factor, which together create an intensely prothrombotic state. HIT represents an atypical immune response because it has features of both T cell-dependent and T cell-independent mechanisms. The disorder is characterized by newly formed anti-PF4/heparin IgG antibodies, which are characteristic of a T cell-dependent mechanism; however, re-exposure to heparin, months after HIT, does not lead to a memory response, which is consistent with a T cell-independent mechanism. In this review, we discuss the immunobiological events that can explain these features, including the role for T cell-dependent and T cell-independent mechanisms in HIT antibody generation, the immunogenic characteristics of the PF4/heparin antigen, and the concept of a temporary loss in immune regulation contributing to the onset of HIT. We also present a novel immunobiological model to explain the atypical immune response that is characteristic of HIT., (© 2017 John Wiley & Sons Ltd.)

The Streptococcus Invasion Locus ( Sil ) was first described in Streptococcus pyogenes and Streptococcus pneumoniae , where it has been implicated in virulence. The two-component peptide signaling system consists of the SilA response regulator and SilB histidine kinase along with the SilCR signaling peptide and SilD/E export/processing proteins. The presence of an associated bacteriocin region suggests this system may play a role in competitive interactions with other microbes. Comparative analysis of 42 Streptococcus Anginosus/Milleri Group (SAG) genomes reveals this to be a hot spot for genomic variability. A cluster of bacteriocin/immunity genes is found adjacent to the sil system in most SAG isolates (typically 6-10 per strain). In addition, there were two distinct SilCR peptides identified in this group, denoted here as SilCR SAG-A and SilCR SAG-B , with corresponding alleles in silB . Our analysis of the 42 sil loci showed that SilCR SAG-A is only found in Streptococcus intermedius while all three species can carry SilCR SAG-B . In S. intermedius B196, a putative SilA operator is located upstream of bacteriocin gene clusters, implicating the sil system in regulation of microbe-microbe interactions at mucosal surfaces where the group resides. We demonstrate that S. intermedius B196 responds to its cognate SilCR SAG-A , and, less effectively, to SilCR SAG-B released by other Anginosus group members, to produce putative bacteriocins and inhibit the growth of a sensitive strain of S. constellatus .

We prepared highly oriented, multi-lamellar stacks of human red blood cell (RBC) membranes applied on silicon wafers. RBC ghosts were prepared by hemolysis and applied onto functionalized silicon chips and annealed into multi-lamellar RBC membranes. High resolution X-ray diffraction was used to determine the molecular structure of the stacked membranes. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (l o ) and liquid disordered (l d ) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the l o domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. Our results further support current models of RBC membranes as patchy structures and provide unprecedented structural details of the molecular organization in the different domains.

Herein we provide a colony forming unit (CFU)-based counting method for quantitating the bacterial binding, phagocytosis, and killing capacity of phagocytes. Although these functions can be measured by immunofluorescence and dye-based assays, quantitating CFUs is comparatively inexpensive and easy to perform. The protocol described below is easily modified for use with different phagocytes (e.g., macrophages, neutrophils, cell lines), types of bacteria or opsonic conditions.

Background:  Little is known about the immunogenicity of live-attenuated Oka/Merck varicella zoster virus (VZV)-containing vaccine (hereafter, "varicella vaccine") in frail nursing homes residents nor about immune phenotypes associated with a response., Methods:  A cohort of 190 frail nursing home residents aged 80-102 years and a cohort of 50 community-dwelling seniors aged 60-75 years (a comparison group) received varicella vaccine. Interferon γ (IFN-γ) enzyme-linked immunospot assays were performed before and 6 weeks after vaccination. Cellular markers of immunosenescence were measured in the nursing home elderly., Results:  The average number of IFN-γ spot-forming cells at baseline was significantly lower in the elderly nursing home residents than in the community-dwelling seniors. However, following vaccination, the VZV immune response increased in both cohorts, and no difference was noted in the fold difference of the response between the 2 cohorts. Upon further examination of the elderly nursing home residents, we found that higher frequencies of regulatory T cells and cytomegalovirus-specific CD4 + T cells correlated negatively with the magnitude of VZV-specific responses., Conclusions:  The Oka/Merck varicella vaccine induces VZV immunity in elderly nursing home residents that is similar to that produced in community-dwelling seniors., Clinical Trials Registration:  NCT01328548., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

The intestinal microbiota is essential for nutrient acquisition, immune development, and exclusion of invading pathogens. The upper respiratory tract (URT) microbiota is less well studied and does not appear to abide by many of the paradigms of the gastrointestinal tract. Decades of carriage studies in children have demonstrated that microbe-microbe competition and collusion occurs in the URT. Whether colonization with common pathogens (e.g., Staphylococcus aureus and Streptococcus pneumoniae) alters immune development or susceptibility to respiratory conditions is just beginning to be understood. Herein, we discuss the biogeography of the URT microbiota, the succession and evolution of the microbiota through the life course, and discuss the evidence for microbe-microbe interactions in colonization and infection., (© 2016 Federation of European Biochemical Societies.)

Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection., Importance: The present study provides evidence that broadly neutralizing HA stalk-specific antibodies induce downstream Fc-mediated neutrophil effector functions. In addition to their ability to neutralize, this class of antibodies has been shown to rely on Fc-Fc receptor interactions for optimal protection in vivo Curiously, neutralizing antibodies that bind the HA head domain do not require such interactions. Our findings build on these previous observations and provide a more complete picture of the relationship between stalk-specific antibodies and cells of the innate immune compartment. Furthermore, our data suggest that the ability of HA stalk-specific antibodies to mediate Fc-Fc receptor engagement is epitope dependent. Overall, this work will inform the rational design of improved influenza virus vaccines and therapeutics., (Copyright © 2016 Mullarkey et al.)

Motivation: Evolutionarily conserved amino acids within proteins characterize functional or structural regions. Conversely, less conserved amino acids within these regions are generally areas of evolutionary divergence. A priori knowledge of biological function and species can help interpret the amino acid differences between sequences. However, this information is often erroneous or unavailable, hampering discovery with supervised algorithms. Also, most of the current unsupervised methods depend on full sequence similarity, which become inaccurate when proteins diverge (e.g. inversions, deletions, insertions). Due to these and other shortcomings, we developed a novel unsupervised algorithm which discovers highly conserved regions and uses two types of information measures: (i) data measures computed from input sequences; and (ii) class measures computed using a priori class groupings in order to reveal subgroups (i.e. classes) or functional characteristics., Results: Using known and putative sequences of two proteins belonging to a relatively uncharacterized protein family we were able to group evolutionarily related sequences and identify conserved regions, which are strong homologous association patterns called Aligned Pattern Clusters, within individual proteins and across the members of this family. An initial synthetic demonstration and in silico results reveal that (i) the data measures are unbiased and (ii) our class measures can accurately rank the quality of the evolutionarily relevant groupings. Furthermore, combining our data and class measures allowed us to interpret the results by inferring regions of biological importance within the binding domain of these proteins. Compared to popular supervised methods, our algorithm has a superior runtime and comparable accuracy., Availability and Implementation: The dataset and results are available at www.pami.uwaterloo.ca/∼ealee/files/classification2015 CONTACT: akcwong@uwaterloo.ca, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Probiotics are live microorganisms that may confer health benefits when ingested. Randomized trials suggest that probiotics significantly decrease the incidence of ventilator-associated pneumonia (VAP) and the overall incidence of infection in critically ill patients. However, these studies are small, largely single-center, and at risk of bias. The aim of the PROSPECT pilot trial was to determine the feasibility of conducting a larger trial of probiotics to prevent VAP in mechanically ventilated patients in the intensive care unit (ICU)., Methods: In a randomized blinded trial, patients expected to be mechanically ventilated for ≥72 hours were allocated to receive either 1 × 10(10) colony-forming units of Lactobacillus rhamnosus GG or placebo, twice daily. Patients were excluded if they were at increased risk of L. rhamnosus GG infection or had contraindications to enteral medication. Feasibility objectives were: (1) timely recruitment; (2) maximal protocol adherence; (3) minimal contamination; and (4) estimated VAP rate ≥10 %. We also measured other infections, diarrhea, ICU and hospital length of stay, and mortality., Results: Overall, in 14 centers in Canada and the USA, all feasibility goals were met: (1) 150 patients were randomized in 1 year; (2) protocol adherence was 97 %; (3) no patients received open-label probiotics; and (4) the VAP rate was 19 %. Other infections included: bloodstream infection (19.3 %), urinary tract infections (12.7 %), and skin and soft tissue infections (4.0 %). Diarrhea, defined as Bristol type 6 or 7 stools, occurred in 133 (88.7 %) of patients, the median length of stay in ICU was 12 days (quartile 1 to quartile 3, 7-18 days), and in hospital was 26 days (quartile 1 to quartile 3, 14-44 days); 23 patients (15.3 %) died in the ICU., Conclusions: The PROSPECT pilot trial supports the feasibility of a larger trial to investigate the effect of L. rhamnosus GG on VAP and other nosocomial infections in critically ill patients., Trial Registration: Clinicaltrials.gov NCT01782755 . Registered on 29 January 2013.

Macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor (cA-SR) that recognizes and phagocytoses a wide variety of pathogens. Most cA-SRs that contain a C-terminal scavenger receptor cysteine-rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signaling is less clear. The discovery of a naturally occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signaling and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant-negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-like receptor 2 (TLR2)-mediated pro-inflammatory signaling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, whereas MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signaling and MARCO-mediated cellular adhesion.

Aging is accompanied by changes in hematopoiesis and consequently in leukocyte phenotype and function. Although age-related changes in bone marrow hematopoiesis are fairly well documented, changes in extramedullary hematopoiesis are less well described. We observed that 18-22-mo-old mice had larger spleens than young controls and found that the enlargement was caused by increased monopoiesis. Because extramedullary hematopoiesis is often driven by inflammation, we hypothesized that the chronic, low-level inflammation that occurs with age is a causal agent in splenomegaly. To test this theory, we compared the number of monocytes in 18-mo-old tumor necrosis factor-knockout mice, which are protected from age-associated inflammation, and found that they did not have increased extramedullary monopoiesis. To determine whether increased splenic monopoiesis is caused by intrinsic changes in the myeloid precursors that occur with age or by the aging microenvironment, we created heterochronic bone marrow chimeras. Increased splenic monopoiesis occurred in old recipient mice, regardless of the age of the donor mouse, but not in young recipient mice, demonstrating that these cells respond to signals from the microenvironment. These data suggest that decreasing the inflammatory microenvironment with age would be an effective strategy for reducing inflammatory diseases propagated by cells of myeloid lineage, which increase in number with age., (© Society for Leukocyte Biology.)

Effector T cells (TEFF) are a barrier to booster vaccination because they can rapidly kill Ag-bearing APCs before memory T cells are engaged. We report in this study that i.v. delivery of rhabdoviral vectors leads to direct infection of follicular B cells in the spleen, where the earliest evidence of secondary T cell responses was observed. This allows booster immunizations to rapidly expand CD8(+) central memory T cells (TCM) during the acute phase of the primary response that is dominated by TEFF Interestingly, although the ablation of B cells before boosting with rhabdoviral vectors diminishes the expansion of memory T cells, B cells do not present Ags directly. Instead, depletion of CD11c(+) dendritic cells abrogates secondary T cell expansion, suggesting that virus-infected follicular B cells may function as an Ag source for local DCs to subsequently capture and present the Ag. Because TCM are located within B cell follicles in the spleen whereas TEFF cannot traffic through follicular regions, Ag production and presentation by follicular APCs represent a unique mechanism to secure engagement of TCM during an ongoing effector response. Our data offer insights into novel strategies for rapid expansion of CD8(+) T cells using prime-boost vaccines by targeting privileged sites for Ag presentation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Nasopharyngeal colonization by the Gram-positive bacterium Streptococcus pneumonia is a prerequisite for pneumonia and invasive pneumococcal diseases. Colonization is asymptomatic, involving dynamic and complex interplay between commensals, the host immune system, and environmental factors. The elderly are at an increased risk of developing pneumonia, which might be due to changes in the respiratory microbiota that would impact bacterial colonization and persistence within this niche. We hypothesized that the composition of the upper respiratory tract (URT) microbiota changes with age and subsequently can contribute to sustained colonization and inefficient clearance of S. pneumoniae To test this, we used a mouse model of pneumococcal colonization to compare the composition of the URT microbiota in young, middle-aged, and old mice in the naive state and during the course of colonization using nasal pharyngeal washes. Sequencing of variable region 3 (V3) of the 16S rRNA gene was used to identify changes occurring with age and throughout the course of S. pneumonia colonization. We discovered that age affects the composition of the URT microbiota and that colonization with S. pneumoniae is more disruptive of preexisting communities in older mice. We have further shown that host-pathogen interactions followingS. pneumonia colonization can impact the populations of resident microbes, including Staphylococcus and Haemophilus. Together, our findings indicate alterations to the URT microbiota could be detrimental to the elderly, resulting in increased colonization of S. pneumonia and decreased efficiency in its clearance., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6-8 wk) and old (18-22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age.

The global population is rapidly aging. Currently, 566 million people are ≥65 years old worldwide, with estimates of nearly 1.5 billion by 2050, particularly in developing countries. Infections constitute a third of mortality in people ≥65 years old. Moreover, lengthening life spans correlate with increased time in hospitals or long-term care facilities and exposure to drug-resistant pathogens. Indeed, the risk of nosocomial infections increases with age, independent of duration spent in healthcare facilities. In this review, we summarize our understanding of how the aging immune system relates to bacterial infections. We highlight the most prevalent infections affecting aging populations including pneumonia, urinary tract infections, and wound infections and make recommendations for future research into infection in aging populations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: We report a case of Mycoplasma pneumoniae infection as one possible trigger for Weston Hurst syndrome (acute hemorrhagic leukoencephalitis), a rare disorder of microvascular injury often described as a postinfectious complication of an upper respiratory illness., Methods: This is a case of a 27-year-old man presenting with a Glasgow Coma Scale score of 3 and an acute head CT revealing extensive vasogenic edema in the right hemisphere associated with mass effect in the context of a recent upper respiratory illness. Right frontal biopsy was performed on day 2, which showed acute cerebritis, and the patient was aggressively treated with antibiotics. However, over the next 5 days from presentation, the vasogenic edema increased, leading ultimately to brain herniation and death., Results: A full autopsy was performed at 5 days from presentation, which showed areas of vessel wall fibrinoid necrosis throughout the right hemisphere as well as, but less so, in the left frontal lobe and pons. Chest x-ray on presentation revealed atypical pneumonia, blood tests were positive for cold agglutinins, and at full autopsy, there was myocarditis, all in keeping with recent M pneumoniae infection. DNA obtained from lung and diseased brain (postmortem) was positive for Mycoplasma providing more direct evidence for brain invasion by this organism as the ultimate trigger for Weston Hurst syndrome., Conclusions: This is a rare case report of Weston Hurst syndrome having both initial brain biopsy on day 2 and full autopsy results on day 5 of presentation revealing important clinical clues about the pathogenesis of this often fatal disorder.

Monocyte phenotype and output changes with age, but why this occurs and how it impacts anti-bacterial immunity are not clear. We found that, in both humans and mice, circulating monocyte phenotype and function was altered with age due to increasing levels of TNF in the circulation that occur as part of the aging process. Ly6C+ monocytes from old (18-22 mo) mice and CD14+CD16+ intermediate/inflammatory monocytes from older adults also contributed to this "age-associated inflammation" as they produced more of the inflammatory cytokines IL6 and TNF in the steady state and when stimulated with bacterial products. Using an aged mouse model of pneumococcal colonization we found that chronic exposure to TNF with age altered the maturity of circulating monocytes, as measured by F4/80 expression, and this decrease in monocyte maturation was directly linked to susceptibility to infection. Ly6C+ monocytes from old mice had higher levels of CCR2 expression, which promoted premature egress from the bone marrow when challenged with Streptococcus pneumoniae. Although Ly6C+ monocyte recruitment and TNF levels in the blood and nasopharnyx were higher in old mice during S. pneumoniae colonization, bacterial clearance was impaired. Counterintuitively, elevated TNF and excessive monocyte recruitment in old mice contributed to impaired anti-pneumococcal immunity since bacterial clearance was improved upon pharmacological reduction of TNF or Ly6C+ monocytes, which were the major producers of TNF. Thus, with age TNF impairs inflammatory monocyte development, function and promotes premature egress, which contribute to systemic inflammation and is ultimately detrimental to anti-pneumococcal immunity.

dsRNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and after release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As) mediate dsRNA entry, it is unknown whether they contribute to signaling beyond ligand internalization. In this study, we investigated whether SR-As contribute to innate immune signaling independent of the classic TLR and retinoic acid-inducible gene-I-like receptor (RLR) pathways. We generated a stable A549 human epithelial cell line with inducible expression of the hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IFN-β promoter stimulator 1, adaptors for TLR3 and the RLRs, respectively. Cells expressing NS3/4A and TLR3/MyD88/IFN-β promoter stimulator 1(-/-) mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I IFN. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, whereas downstream signaling leading to IFN production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Current flow cytometry (FCM) reagents and instrumentation allow for the measurement of an unprecedented number of parameters for any given cell within a homogenous or heterogeneous population. While this provides a great deal of power for hypothesis testing, it also generates a vast amount of data, which is typically analyzed manually through a processing called "gating." For large experiments, such as high-content screens, in which many parameters are measured, the time required for manual analysis as well as the technical variability inherent to manual gating can increase dramatically, even becoming prohibitive depending on the clinical or research goal. In the following article, we aim to provide the reader an overview of automated FCM analysis as well as an example of the implementation of FLOw Clustering without K, a tool that we consider accessible to researchers of all levels of computational expertise. In most cases, computational assistance methods are more reproducible and much faster than manual gating, and for some, also allow for the discovery of cellular populations that might not be expected or evident to the researcher. We urge any researcher who is planning or has previously performed large FCM experiments to consider implementing computational assistance into their analysis pipeline.

The class A scavenger receptor (cA-SR) family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria, such as Streptococcus pneumoniae and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a scavenger receptor cysteine-rich (SRCR) domain while the remaining two receptors lack the domain. Previous work has suggested that the macrophage-associated receptor with collagenous structure (MARCO) shares a recent common ancestor with the non-SRCR-containing receptors; however, the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO's SRCR domain, which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together, these findings indicate a common origin of the SRCR domain within the cA-SRs; however, different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.

Although the microneutralization (MN) assay has been shown to be more sensitive than the hemagglutination inhibition (HAI) assay for the measurement of humoral immunity against influenza viruses, further evidence relating MN titres to protective efficacy against infection is needed. Serum antibodies against seasonal H1N1 and H3N2 influenza were measured in children and adolescents (n = 656) by MN and hemagglutination inhibition (HAI) assays. Compared to HAI, the MN assay is more sensitive in detecting serum antibodies and estimates of protective effectiveness against PCR-confirmed infection were higher for both subtypes. Given our findings, the MN assay warrants further consideration as a formal tool for the routine evaluation of vaccine-induced antibody responses.

Background: Probiotics are defined as live microorganisms that may confer health benefits when ingested. Meta-analysis of probiotic trials suggests a 25 % lower ventilator-associated pneumonia (VAP) and 18 % lower infection rates overall when administered to patients in the intensive care unit (ICU). However, prior trials are small, largely single center, and at high risk of bias. Before a large rigorous trial is launched, testing whether probiotics confer benefit, harm, or have no impact, a pilot trial is needed. The aim of the PROSPECT Pilot Trial is to determine the feasibility of performing a larger trial in mechanically ventilated critically ill patients investigating Lactobacillus rhamnosus GG. A priori, we determined that the feasibility of the larger trial would be based on timely recruitment, high protocol adherence, minimal contamination, and an acceptable VAP rate., Methods/design: Patients ≥18 years old in the ICU who are anticipated to receive mechanical ventilation for ≥72 hours will be included. Patients are excluded if they are at increased risk of probiotic-associated infection, have strict enteral medication contraindications, are pregnant, previously enrolled in a related trial, or are receiving palliative care. Following informed consent, patients are randomized in variable unspecified block sizes in a fixed 1:1 ratio, stratified by ICU, and medical, surgical, or trauma admitting diagnosis. Patients receive 1 × 10 10 colony forming units of L. rhamnosus GG (Culturelle, Locin Industries Ltd) or an identical placebo suspended in tap water administered twice daily via nasogastric tube in the ICU. Clinical and research staff, patients, and families are blinded., Discussion: The primary outcomes for this pilot trial are the following: (1) recruitment success, (2) ≥90 % protocol adherence, (3) ≤5 % contamination, and (4) ~10 % VAP rate. Additional clinical outcomes are VAP, other infections, diarrhea (total, antibiotic associated, and Clostridium difficile), ICU and hospital length of stay, and mortality. The morbidity, mortality, and cost of VAP underscore the need for cost-effective prophylactic interventions. The PROSPECT Pilot Trial is the initial step toward rigorously evaluating whether probiotics decrease nosocomial infections, have no effect, or actually cause infections in critically ill patients., Trial Registration: ClinicalTrials.gov. NCT01782755.

Tumor necrosis factor (TNF), a potent inflammatory cytokine, and mitochondrial DNA (mtDNA), a product of inflammation-induced tissue damage, increase with age ("inflammaging") and many chronic diseases. Peripheral blood neutrophils, a critical component of innate immunity, have also been shown to be altered with age, and are exceptionally sensitive to external stimuli. Herein, we describe that the phenotype of neutrophils from the advanced-age, frail elderly (ELD) is determined by levels of circulating TNF and mtDNA. Neutrophils from ELD donors are morphologically immature, and have higher levels of intracellular reactive oxygen species (ROS) and expression of the activation markers CD11b and HLA-DR. The frequency of CD11b(++) neutrophils correlated with plasma TNF, and recombinant TNF elevated neutrophil CD11b ex vivo and in vivo. Furthermore, neutrophils from aged TNF-deficient mice expressed CD11b similar to young counterparts. The frequency of HLA-DR(+) neutrophils, on the other hand, positively correlated with circulating mtDNA, which increased neutrophil HLA-DR expression in a dose-dependent manner ex vivo. Cell-surface TLR-9 expression, however, was unaltered on neutrophils from ELD donors. In summary, we provide novel evidence that products of age-related inflammation modulate neutrophil phenotype in vivo. Given this, anti-inflammatory therapies may prove beneficial in improving neutrophil functionality in the elderly., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cell adhesion to biomaterials can be mediated in part by mechanisms aside from the traditionally recognized opsinization and integrin binding mechanisms. In this study, we investigated the role of scavenger receptor A (SR-A) in leukocyte binding to a series of well-controlled polyanionic and uncharged hydrogels based on a poly(N-isopropylacrylamide) backbone. The hydrogels were injected in the peritoneal cavity of SR-A knockout (KO) and wild-type mice using a minimally invasive procedure and allowed to set in situ. After 24 h, the hydrogels were recovered and analyzed, the peritoneal cavity was lavaged, and cytokine concentrations were assessed by ELISA. The polyanionic hydrogels retrieved from the KO animals were found to be completely devoid of adherent leukocytes, which were present in other materials regardless of the mouse strain in which they were injected. Results from a subsequent in vitro cellular adhesion study with a RAW264.7 cell line failed to yield a similarly definitive role for SR-A in the cellular binding of a polyanionic hydrogel. Taken together, the results of this study show that SR-A mediates leukocyte adhesion to a polyanionic hydrogel in the peritoneal cavity, but other adhesion mechanisms contribute to cellular binding in vitro., (© 2014 Wiley Periodicals, Inc.)

The upper respiratory tract (URT) is a crucial site for host defense, as it is home to bacterial communities that both modulate host immune defense and serve as a reservoir of potential pathogens. Young children are at high risk of respiratory illness, yet the composition of their URT microbiota is not well understood. Microbial profiling of the respiratory tract has traditionally focused on culturing common respiratory pathogens, whereas recent culture-independent microbiome profiling can only report the relative abundance of bacterial populations. In the current study, we used both molecular profiling of the bacterial 16S rRNA gene and laboratory culture to examine the bacterial diversity from the oropharynx and nasopharynx of 51 healthy children with a median age of 1.1 years (range 1-4.5 years) along with 19 accompanying parents. The resulting profiles suggest that in young children the nasopharyngeal microbiota, much like the gastrointestinal tract microbiome, changes from an immature state, where it is colonized by a few dominant taxa, to a more diverse state as it matures to resemble the adult microbiota. Importantly, this difference in bacterial diversity between adults and children accompanies a change in bacterial load of three orders of magnitude. This indicates that the bacterial communities in the nasopharynx of young children have a fundamentally different structure from those in adults and suggests that maturation of this community occurs sometime during the first few years of life, a period that includes ages at which children are at the highest risk for respiratory disease.

MVs, which can be subgrouped into exosomes, SVs, and OMVs, are secreted by eukaryotic and prokaryotic cells. Many previously inexplicable phenomena can be explained by the existence of these vesicles, as they appear to be important in a wide range of biologic processes, such as intercellular communication and transfer of functional genetic information. In this review, we discuss the immunologic roles of MVs during sterile insult and infectious disease. MVs contribute to clotting initiation, cell recruitment, and neovascularization during wound healing. In the context of pathogen infection, both the host and the pathogen use MVs for communication and defense. MVs are exploited by various viruses to evade the host immune response and contribute to viral spread. Bacteria produce MVs that contain virulence factors that contribute to disease pathology and antibiotic resistance. This review summarizes the role of MVs in the pathology and resolution of disease., (© Society for Leukocyte Biology.)

Problem: Semen is the primary medium for sexual transmission of HIV-1 and contains high concentrations of TGF-β1, but its role in regulating HIV-mediated immune activation is unclear., Method of Study: TGF-β1 and sCD14 were compared in blood plasma (BP) and seminal plasma (SP) from HIV-uninfected and infected, antiretroviral therapy (ART)-naive and ART-treated men and in THP-1 cells following exposure to HIV-1. The relationship between TGF-β1 and sCD14 was determined by Spearman correlation., Results: Active and latent forms of TGF-β1 were compartmentalized between BP and SP. Highest active TGF-β1 levels were present in SP of ART-naïve chronic-infected men and decreased following ART treatment. Latent TGF-β1 was upregulated in BP following HIV infection, and highest levels were observed in BP of acute-infected men. Similar expression trends were observed between latent TGF-β1 and sCD14 in BP. A significant negative correlation was observed between active TGF-β1, sCD14, and semen viral load in ART-naive men., Conclusion: TGF-β1 is compartmentalized between blood and semen, possibly co-expressed with sCD14 by activated monocytes/macrophages in BP as a result of HIV infection. Conversion of latent TGF-β1 into its active form could contribute to regulation of viral load and immune activation in the male genital tract, but depends on the stage of infection., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Elevated levels of serum cytokines, a marker of immune activation and chronic inflammation, are commonly associated with age and are a significant risk factor for all-cause mortality in the elderly. This phenomenon is very similar to that exhibited by individuals with diseases of inflammatory etiology and chronic viral infections such as human immunodeficiency virus (HIV). Although the origin of chronically elevated cytokines with age is unknown, for chronic diseases and viral infections, a role for circulating bacterial products and other pattern recognition receptor (PRR) ligands has been suggested. Given this, we sought to examine whether the levels of circulating cytokines (tumour necrosis factor (TNF), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12) in the advanced-age, frail elderly (n = 135) correlated with plasma levels of lipopolysaccharide (LPS), muramyl dipeptide (MDP), 16S ribosomal DNA, total cell-free DNA and host-derived mitochondrial DNA. After adjusting for multiple testing, no associations between circulating products and donor age, sex or comorbidities were observed. However, a significant negative correlation between MDP and IL-10 was identified. Given the anti-inflammatory nature of IL-10, a negative relationship with a potent inflammatory agonist such as MDP is not surprising and suggests a potential role for circulating MDP in the propagation of age-related immune activation.

Rising rates of antibiotic resistance in bacterial pathogens is a medical crisis of global concern that necessitates the development of new treatment strategies. We have isolated a natural product with macrophage-stimulating activity from a screen of microbially produced bioactive molecules. Streptazolin increased bacterial killing and elaboration of immunostimulatory cytokines by macrophages in vitro. Furthermore, we show that streptazolin stimulates the macrophage nuclear factor κB (NF-κB) pathway via phosphatidylinositide 3-kinase (PI3K) signaling, and that the conjugated diene moiety is essential for stimulatory activity. Immunostimulatory molecules like streptazolin represent entries into new treatment paradigms to address the challenge of antibiotic resistance.

The advanced-age, frail elderly are especially vulnerable to developing pneumococcal infection and disease. Macrophages are critical mediators in the defence against Streptococcus pneumoniae at the upper respiratory tract, however, little is known of their anti-pneumococcal capacity in the elderly. Herein we demonstrate that monocyte-derived macrophages (MDMs) from the advanced-age, frail elderly produce less TNF, IL-6, IL-1β and IL-8 in response to heat-killed S. pneumoniae, which does not appear to be related to mRNA stability or decay. Furthermore, despite maintaining the ability to bind and phagocytose bacteria, MDMs from these individuals have a reduced capacity to kill S. pneumoniae. These defects parallel reduced PI3K-AKT signaling, which can significantly abrogate bacterial killing, but does not affect cytokine responses. Since macrophages are critical in the defence against S. pneumoniae, this study adds valuable insight into the susceptibility of the elderly to pneumococcal disease and highlights the PI3K-AKT signaling pathway as a potential therapeutic target., (Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Pneumonia caused by Streptococcus pneumoniae is a major cause of death and an economic burden worldwide. S. pneumoniae is an intermittent colonizer of the human upper respiratory tract, and the ability to control asymptomatic colonization determines the likelihood of developing invasive disease. Recognition of S. pneumoniae by resident macrophages via Toll-like receptor 2 (TLR-2) and the macrophage receptor with collagenous structure (MARCO) and the presence of interleukin-17 (IL-17)-secreting CD4(+) T cells are required for macrophage recruitment and bacterial clearance. Despite the fact that the primary cellular effectors needed for bacterial clearance have been identified, much of the underlying regulatory mechanisms are unknown. Herein, we demonstrate that the small, noncoding RNA microRNA-155 (mir-155) is critical for the effective clearance of S. pneumoniae. Our studies show that mir-155-deficient mice maintain the ability to prevent acute invasive pneumococcal infection but have significantly higher bacterial burdens following colonization, independently of macrophage recognition by TLR-2, MARCO expression, or bactericidal capacity. The observed defects in bacterial clearance parallel reduced IL-17A and gamma interferon CD4(+) T-cell responses in vivo, lower IL-17A mRNA levels in the nasopharynx, and a reduced capacity to induce Th17 cell polarization. Given that knockout mice are also limited in the capacity to generate high-titer S. pneumoniae-specific antibodies, we conclude that mir-155 is a critical mediator of the cellular effectors needed to clear primary and secondary S. pneumoniae colonizations., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: The Streptococcus Milleri/Anginosus Group (SMG) colonize mucosal surfaces, especially the airways, and are considered to be normal mucosal microbiota; however, they are a major cause of abscesses, pneumonia and pleural empyema. The production of exoenzymes and virulence factors do not correlate with SMG pathogenicity. Since SMG infections are associated with robust inflammatory responses, we hypothesized that host immune responses might distinguish strains associated with asymptomatic carriage and those associated with fulminant disease., Methods: We measured IL1β, TNF, IL10, IL12, IL23, IL17, and IL4 production from human peripheral blood mononuclear cells (PBMCs) stimulated with a panel of clinical isolates from the airways and infections and measured the ability of these isolates to stimulate TLR2., Results: Isolates were categorized based on the levels of cytokines they induced from PBMCs (high, intermediate, low). Airway isolates predominantly induced low levels of cytokines and isolates from invasive disease induced higher levels, although about 10% of the strains produced divergent cytokine responses between donors. Interestingly, the donors were most divergent in their production of IL17, IL12 and IL23., Conclusions: We propose that the ability to inhibit or avoid an inflammatory response is associated with carriage in the airways and variability in responses between isolates and donors might contribute to susceptibility to disease.

Background: Circulating myeloid cells are important mediators of the inflammatory response, acting as a major source of resident tissue antigen presenting cells and serum cytokines. They represent a number of distinct subpopulations whose functional capacity and relative concentrations are known to change with age. Little is known of these changes in the very old and physically frail, a rapidly increasing proportion of the North American population., Design: In the following study the frequency and receptor expression of blood monocytes and dendritic cells (DCs) were characterized in a sample of advanced-age, frail elderly (81-100 yrs), and compared against that of adults (19-59 yrs), and community-dwelling seniors (61-76 yrs). Cytokine responses following TLR stimulation were also investigated, as well as associations between immunophenotyping parameters and chronic diseases., Results: The advanced-age, frail elderly had significantly fewer CD14(++) and CD14(+)CD16(+), but not CD14(++)CD16(+) monocytes, fewer plasmacytoid and myeloid DCs, and a lower frequency of monocytes expressing the chemokine receptors CCR2 and CX3CR1. At baseline and following stimulation with TLR-2 and -4 agonists, monocytes from the advanced-age, frail elderly produced more TNF than adults, although the overall induction was significantly lower. Finally, monocyte subset frequency and CX3CR1 expression was positively associated with dementia, while negatively associated with anemia and diabetes in the advanced-age, frail elderly., Conclusions: These data demonstrate that blood monocyte frequency and phenotype are altered in the advanced-age, frail elderly and that these changes correlate with certain chronic diseases. Whether these changes contribute to or are caused by these conditions warrants further investigation.

Eosinophils natively inhabit the small intestine, but a functional role for them there has remained elusive. Here, we show that eosinophil-deficient mice were protected from induction of Th2-mediated peanut food allergy and anaphylaxis, and Th2 priming was restored by reconstitution with il4(+/+) or il4(-/-) eosinophils. Eosinophils controlled CD103(+) dendritic cell (DC) activation and migration from the intestine to draining lymph nodes, events necessary for Th2 priming. Eosinophil activation in vitro and in vivo led to degranulation of eosinophil peroxidase, a granule protein whose enzymatic activity promoted DC activation in mice and humans in vitro, and intestinal and extraintestinal mouse DC activation and mobilization to lymph nodes in vivo. Further, eosinophil peroxidase enhanced responses to ovalbumin seen after immunization. Thus, eosinophils can be critical contributors to the intestinal immune system, and granule-mediated shaping of DC responses can promote both intestinal and extraintestinal adaptive immunity., (© 2014 Chu et al.)

The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H2O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2% of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6% of the metabolome by a combination of BD polar (59.4%) and BD nonpolar (53.9%) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages.

Rationale: The microbial communities inhabiting the upper respiratory tract protect from respiratory infection. The maturity of the immune system is a major influence on the composition of the microbiome and, in youth, the microbiota and immune system are believed to mature in tandem. With age, immune function declines and susceptibility to respiratory infection increases. Whether these changes contribute to the microbial composition of the respiratory tract is unknown., Objectives: Our goal was to determine whether the microbes of the upper respiratory tract differ between mid-aged adults (18-40 yr) and the elderly (>65 yr)., Methods: Microbiomes of the anterior nares and oropharynx of elderly individuals were evaluated by 16S rRNA gene sequencing. These communities were compared with data on mid-aged adults obtained from the Human Microbiome Project., Measurements and Main Results: The microbiota of the elderly showed no associations with sex, comorbidities, residence, or vaccinations. Comparisons of mid-aged adults and the elderly demonstrated significant differences in the composition of the anterior nares and oropharynx, including a population in the anterior nares of the elderly that more closely resembled the oropharynx than the anterior nares of adults. The elderly oropharyngeal microbiota were characterized by increased abundance of streptococci, specifically, Streptococcus salivarius group species, but not Streptococcus pneumoniae, carriage of which was low (<3% of participants), as demonstrated by PCR (n = 4/123)., Conclusions: Microbial populations of the upper respiratory tract in mid-aged adults and the elderly differ; it is possible that these differences contribute to the increased risk of respiratory infections experienced by the elderly.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream(1). This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates(2), or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models. Our mouse model of intranasal colonization is adapted from human models(3) and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx(4-7). In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice(8), however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Bioinformatics includes a suite of methods, which are cheap, approachable, and many of which are easily accessible without any sort of specialized bioinformatic training. Yet, despite this, bioinformatic tools are under-utilized by immunologists. Herein, we review a representative set of publicly available, easy-to-use bioinformatic tools using our own research on an under-annotated human gene, SCARA3, as an example. SCARA3 shares an evolutionary relationship with the class A scavenger receptors, but preliminary research showed that it was divergent enough that its function remained unclear. In our quest for more information about this gene - did it share gene sequence similarities to other scavenger receptors? Did it contain conserved protein domains? Where was it expressed in the human body? - we discovered the power and informative potential of publicly available bioinformatic tools designed for the novice in mind, which allowed us to hypothesize on the regulation, structure, and function of this protein. We argue that these tools are largely applicable to many facets of immunology research.

Although women constitute half of all HIV-1-infected people worldwide (UNAIDS World AIDS Day Report, 2011), the earliest events in the female reproductive tract (FRT) during heterosexual HIV-1 transmission are poorly understood. Recently, we demonstrated that HIV-1 could directly impair the mucosal epithelial barrier in the FRT. This suggested that the HIV-1 envelope glycoprotein gp120 was being recognized by a membrane receptor on genital epithelial cells, leading to innate immune activation. In this study, we report that pattern-recognition receptors TLR2 and -4 bind to HIV-1 gp120 and trigger proinflammatory cytokine production via activation of NF-κB. The gp120-TLR interaction also required the presence of heparan sulfate (HS). Bead-binding assays showed that gp120 can bind to HS, TLR2, and TLR4, and studies in transfected HEK293 cells demonstrated that HS and TLR2 and -4 were necessary to mediate downstream signaling. Exposure to seminal plasma from HIV-1-infected and uninfected men with gp120 added to it induced a significant proinflammatory cytokine response from genital epithelial cells and disruption of tight junctions, indicating a role for gp120 in mucosal barrier disruption during HIV-1 heterosexual transmission. These studies provide, for the first time to our knowledge, a possible mechanism by which HIV-1 gp120 could directly initiate innate immune activation in the FRT during heterosexual transmission.