T-cell epitope discovery and single-cell technologies to advance food allergy research.

There is good evidence for a role of T cells in food allergy, but there is a lack of mechanistic understanding and phenotypic markers of the specific T cells contributing to pathology. Recent technologic advancements have allowed for a new experimental paradigm where we can find and pull out rare antigen-specific T cells and characterize them at the single-cell level. However, studies in infectious disease and broader allergy have shown that these techniques benefit greatly from precisely defined T-cell epitopes. Food allergens have fewer epitopes currently available, but it is growing and promises to overcome this gap. With growing use of this experimental design, it will be important to unbiasedly map T-cell phenotypes across food allergy and look for commonalities and contrasts to other allergic and infectious diseases. Once a pathologic phenotype for T cells has been established, the frequencies of these cells can be monitored with simpler techniques that could be applied to the clinic and used in diagnosis, prediction of treatment responsiveness, and discovery of targets for new treatments.

Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children.

BACKGROUND: Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges). OBJECTIVE: We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker. METHODS: Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays. RESULTS: We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA ex vivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3+ cells over FOXP3- cells. FOXP3+ cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3+ cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3+ cells were also more clonally expanded than the FOXP3- population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3+ cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127-CD25+ cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for TH2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of TH2 cytokines and pathogenic TH2/T follicular helper markers. CONCLUSIONS: Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3+ cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.

Ethanol Consumption Induces Nonspecific Inflammation and Functional Defects in Alveolar Macrophages.

Chronic alcohol drinking is associated with increased susceptibility to viral and bacterial respiratory pathogens. In this study, we use a rhesus macaque model of voluntary ethanol self-administration to study the effects of long-term alcohol drinking on the immunological landscape of the lung. We report a heightened inflammatory state in alveolar macrophages (AMs) obtained from ethanol (EtOH)-drinking animals that is accompanied by increased chromatin accessibility in intergenic regions that regulate inflammatory genes and contain binding motifs for transcription factors AP-1, IRF8, and NFKB p-65. In line with these transcriptional and epigenetic changes at the basal state, AMs from EtOH-drinking animals generate elevated inflammatory mediator responses to lipopolysaccharides and respiratory syncytial virus. However, the transcriptional analysis revealed an inefficient induction of interferon-stimulated genes with EtOH in response to the respiratory syncytial virus, suggesting disruption of antimicrobial defenses. Correspondingly, AMs from EtOH-drinking animals exhibited transcriptional shifts indicative of increased oxidative stress and oxidative phosphorylation, which was coupled with higher cytosolic reactive oxygen species and mitochondrial potential. This heightened oxidative stress state was accompanied by decreased ability to phagocytose bacteria. Bulk RNA and assay for transposase-accessible chromatin sequencing data further revealed reduced expression and chromatin accessibility of loci associated with tissue repair and maintenance with chronic EtOH drinking. Similarly, analysis of single-cell RNA sequencing data revealed shifts in cell states from tissue maintenance to inflammatory responses with EtOH. Collectively, these data provide novel insight into mechanisms by which chronic EtOH drinking increases susceptibility to infection in patients with alcohol use disorders.

Profiling of extracellular vesicle-bound miRNA to identify candidate biomarkers of chronic alcohol drinking in nonhuman primates.

BACKGROUND: Long-term alcohol drinking is associated with numerous health complications including susceptibility to infection, cancer, and organ damage. However, due to the complex nature of human drinking behavior, it has been challenging to identify reliable biomarkers of alcohol drinking behavior prior to signs of overt organ damage. Recently, extracellular vesicle-bound microRNAs (EV-miRNAs) have been found to be consistent biomarkers of conditions that include cancer and liver disease. METHODS: In this study, we profiled the plasma EV-miRNA content by miRNA-Seq from 80 nonhuman primates after 12 months of voluntary alcohol drinking. RESULTS: We identified a list of up- and downregulated EV-miRNA candidate biomarkers of heavy drinking and those positively correlated with ethanol dose. We overexpressed these candidate miRNAs in control primary peripheral immune cells to assess their potential functional mechanisms. We found that overexpression of miR-155, miR-154, miR-34c, miR-450a, and miR-204 led to increased production of the inflammatory cytokines TNFα or IL-6 in peripheral blood mononuclear cells after stimulation. CONCLUSION: This exploratory study identified several EV-miRNAs that could serve as biomarkers of long-term alcohol drinking and provide a mechanism to explain alcohol-induced peripheral inflammation.

Chronic ethanol consumption alters lamina propria leukocyte response to stimulation in a region-dependent manner.

Chronic heavy alcohol consumption, also referred to as chronic heavy drinking (CHD), results in intestinal injury characterized by increased permeability, dysbiosis, nutrient malabsorption, potentially higher susceptibility to infection, and increased risk of colorectal cancer. However, our understanding of the mechanisms by which CHD results in intestinal damage remains incomplete. Here, we investigated the impact of chronic drinking on transcriptional and functional responses of lamina propria leukocytes (LPLs) isolated from the 4 major gut sections. Although no significant differences were detected between LPLs isolated from the ethanol and control groups at resting state within each major gut section, our analysis uncovered key regional differences in composition and function of LPLs independent of alcohol consumption. However, in response to phorbol myristate acetate and ionomycin, duodenal LPLs from ethanol-drinking animals generated a dampened response, whereas jejunal and ileal LPLs from ethanol-drinking animals produced a heightened response. Transcriptional responses following stimulation were pronounced in ileal and duodenal LPLs from the ethanol-drinking group but less evident in jejunal and colonic LPLs compared with controls, suggesting a more significant impact of alcohol on these gut regions. The altered intestinal LPL function detected in our study reveals remarkable region specificity and novel insight into potential mechanisms of intestinal injury associated with CHD.-Barr, T., Lewis, S. A., Sureshchandra, S., Doratt, B., Grant, K. A., Messaoudi, I. Chronic ethanol consumption alters lamina propria leukocyte response to stimulation in a region-dependent manner.

Analysing the bioactive makeup of demineralised dentine matrix on bone marrow mesenchymal stem cells for enhanced bone repair.

Dentine matrix has proposed roles for directing mineralised tissue repair in dentine and bone; however, the range of bioactive components in dentine and specific biological effects on bone-derived mesenchymal stem cells (MSCs) in humans are less well understood. The aims of this study were to further elucidate the biological response of MSCs to demineralised dentine matrix (DDM) in enhancing wound repair responses and ascertain key contributing components. Dentine was obtained from human teeth and DDM proteins solubilised with ethylenediaminetetraacetic acid (EDTA). Bone marrow derived MSCs were commercially obtained. Cells with a more immature phenotype were then selected by preferential fibronectin adhesion (FN-BMMSCs) for use in subsequent in vitro assays. DDM at 10 µg/mL reduced cell expansion, attenuated apoptosis and was the minimal concentration capable of inducing osteoblastic differentiation. Enzyme-linked immunosorbent assay (ELISA) quantification of growth factors indicated physiological levels produced the above responses; transforming growth factor ß (TGF-ß1) was predominant (15.6 ng/mg DDM), with relatively lower concentrations of BMP-2, FGF, VEGF and PDGF (6.2-4.7 ng/mg DDM). Fractionation of growth factors from other DDM components by heparin affinity chromatography diminished osteogenic responses. Depletion of biglycan from DDM also attenuated osteogenic potency, which was partially rescued by the isolated biglycan. Decorin depletion from DDM had no influence on osteogenic potency. Collectively, these results demonstrate the potential of DDM for the delivery of physiological levels of growth factors for bone repair processes, and substantiate a role for biglycan as an additional adjuvant for driving osteogenic pathways.

Knockdown of the dyslexia-associated gene Kiaa0319 impairs temporal responses to speech stimuli in rat primary auditory cortex.

One in 15 school age children have dyslexia, which is characterized by phoneme-processing problems and difficulty learning to read. Dyslexia is associated with mutations in the gene KIAA0319. It is not known whether reduced expression of KIAA0319 can degrade the brain's ability to process phonemes. In the current study, we used RNA interference (RNAi) to reduce expression of Kiaa0319 (the rat homolog of the human gene KIAA0319) and evaluate the effect in a rat model of phoneme discrimination. Speech discrimination thresholds in normal rats are nearly identical to human thresholds. We recorded multiunit neural responses to isolated speech sounds in primary auditory cortex (A1) of rats that received in utero RNAi of Kiaa0319. Reduced expression of Kiaa0319 increased the trial-by-trial variability of speech responses and reduced the neural discrimination ability of speech sounds. Intracellular recordings from affected neurons revealed that reduced expression of Kiaa0319 increased neural excitability and input resistance. These results provide the first evidence that decreased expression of the dyslexia-associated gene Kiaa0319 can alter cortical responses and impair phoneme processing in auditory cortex.

Putting computational models of immunity to the test - an invited challenge to predict B. pertussis vaccination outcomes.

Systems vaccinology studies have been used to build computational models that predict individual vaccine responses and identify the factors contributing to differences in outcome. Comparing such models is challenging due to variability in study designs. To address this, we established a community resource to compare models predicting B. pertussis booster responses and generate experimental data for the explicit purpose of model evaluation. We here describe our second computational prediction challenge using this resource, where we benchmarked 49 algorithms from 53 scientists. We found that the most successful models stood out in their handling of nonlinearities, reducing large feature sets to representative subsets, and advanced data preprocessing. In contrast, we found that models adopted from literature that were developed to predict vaccine antibody responses in other settings performed poorly, reinforcing the need for purpose-built models. Overall, this demonstrates the value of purpose-generated datasets for rigorous and open model evaluations to identify features that improve the reliability and applicability of computational models in vaccine response prediction.

Vagus nerve stimulation during rehabilitative training improves forelimb strength following ischemic stroke.

Upper limb impairment is a common debilitating consequence of ischemic stroke. Physical rehabilitation after stroke enhances neuroplasticity and improves limb function, but does not typically restore normal movement. We have recently developed a novel method that uses vagus nerve stimulation (VNS) paired with forelimb movements to drive specific, long-lasting map plasticity in rat primary motor cortex. Here we report that VNS paired with rehabilitative training can enhance recovery of forelimb force generation following infarction of primary motor cortex in rats. Quantitative measures of forelimb function returned to pre-lesion levels when VNS was delivered during rehab training. Intensive rehab training without VNS failed to restore function back to pre-lesion levels. Animals that received VNS during rehab improved twice as much as rats that received the same rehabilitation without VNS. VNS delivered during physical rehabilitation represents a novel method that may provide long-lasting benefits towards stroke recovery.

Ex vivo antimicrobial efficacy of strong acid electrolytic water against Enterococcus faecalis biofilm.

AIM: To observe the antimicrobial effect of strong acid electrolytic water (SAEW) against an Enterococcus faecalis (E. faecalis) biofilm when used as a root canal irrigant. METHODOLOGY: The effect of SAEW, sodium hypochlorite (5.25%; NaOCl) and sodium chloride (0.9%; normal saline) on E. faecalis biofilm vitality on coverslips was observed by confocal laser scanning microscopy (CLSM). Thirty-five root canals from extracted human teeth were sterilized prior to contamination with E. faecalis for four weeks. Bacterial samples were collected with sterile paper points and plated onto BHI agar plates for 48 h. Root canal walls were observed by scanning electron microscopy before and after instrumentation, together with root canal irrigation with SAEW, NaOCl or normal saline, with or without ultrasonic vibration. Antimicrobial effectiveness was established by counting colony-forming units and analysed by two-way anova. RESULTS: Confocal laser scanning microscopy revealed that SAEW decreased E. faecalis biofilm vitality, and the proportion of dead bacteria increased in accordance with increasing treatment time. Most bacteria in the biofilms were killed after 10-min treatment. No significant difference was observed between SAEW and NaOCl groups at the same treatment time (P > 0.05) or in the susceptibility of E. faecalis to SAEW and NaOCl (P > 0.05) in extracted human teeth with or without ultrasonic activation. SAEW and NaOCl were more effective against E. faecalis biofilm than normal saline, and antimicrobial efficacy was significantly enhanced by ultrasonic vibration (P < 0.05). CONCLUSIONS: Strong acid electrolytic water effectively killed E. faecalis in a biofilm both on coverslips and in the root canals of extracted human teeth.

Oral health research funding in relation to disease burden in Australia.

BACKGROUND: This study aims to investigate and compare the major Australian government research funding schemes for oral health science with other disciplines from the burden of disease perspective. METHODS: Major government research funding scheme outcomes were identified. An innovative index of Fair Research Funding (FRF) was developed to examine the extent to which National Health and Medical Research Council funding is aligned with the disease burden. In addition to comparing different diseases, overall governmental research funding for different areas of oral health sciences was explored. RESULTS: Oral disorders with $15 million NHMRC grant funds (2017-2021) and FRF of 10.7 has the lowest and most inequitable amount of Australian government support in relation to disease burden. The share of oral health science in the Australian Research Council and Medical Research Future Fund was very minimal, with $3.43 and $1.88 million respectively. CONCLUSION: Governmental research funding for oral health sciences is inequitable according to the disease burden. More dedicated oral health sciences research funding schemes are essential. Funding for prevention-focused public oral health programmes is a vital requirement towards reducing the inequalities in population oral health.

Biological considerations of dental materials as orifice barriers for restoring root-filled teeth.

There is ample published literature regarding the technical aspects of restoring root-filled teeth, but little concerning the biological impacts, consequences, and criteria for the selection of direct restorative materials following endodontic treatment. The provision of an effective coronal seal in addition to a sound root filling is known to be important in the prevention of root canal infection. This review seeks to explore the evidence concerning the selection of dental materials in the restoration of root-filled teeth, specifically with a close examination of the properties of commonly used materials as orifice barriers. © 2023 Australian Dental Association.

Integrated single cell analysis shows chronic alcohol drinking disrupts monocyte differentiation in the bone marrow.

Chronic heavy alcohol drinking (CHD) rewires monocytes and macrophages toward heightened inflammatory states with compromised antimicrobial defenses that persist after 1-month abstinence. To determine whether these changes are mediated through alterations in the bone marrow niche, we profiled monocytes and hematopoietic stem cell progenitors (HSCPs) from CHD rhesus macaques using a combination of functional assays and single cell genomics. CHD resulted in transcriptional profiles consistent with increased activation and inflammation within bone marrow resident monocytes and macrophages. Furthermore, CHD resulted in transcriptional signatures associated with increased oxidative and cellular stress in HSCP. Differentiation of HSCP in vitro revealed skewing toward monocytes expressing "neutrophil-like" markers with greater inflammatory responses to bacterial agonists. Further analyses of HSCPs showed broad epigenetic changes that were in line with exacerbated inflammatory responses within monocytes and their progenitors. In summary, CHD alters HSCPs in the bone marrow leading to the production of monocytes poised to generate dysregulated hyper-inflammatory responses.

Integrated single cell analysis shows chronic alcohol drinking disrupts monocyte differentiation in the bone marrow niche.

Chronic alcohol drinking rewires circulating monocytes and tissue-resident macrophages towards heightened inflammatory states with compromised anti-microbial defenses. As these effects remain consistent in short-lived monocytes after a 1-month abstinence period it is unclear whether these changes are restricted to the periphery or mediated through alterations in the progenitor niche. To test this hypothesis, we profiled monocytes/macrophages and hematopoietic stem cell progenitors (HSCP) of the bone marrow compartment from rhesus macaques after 12 months of ethanol consumption using a combination of functional assays and single cell genomics. Bone marrow-resident monocytes/macrophages from ethanol-consuming animals exhibited heightened inflammation. Differentiation of HSCP in vitro revealed skewing towards monocytes expressing neutrophil-like markers with heightened inflammatory responses to bacterial agonists. Single cell transcriptional analysis of HSCPs showed reduced proliferation but increased inflammatory markers in mature myeloid progenitors. We observed transcriptional signatures associated with increased oxidative and cellular stress as well as oxidative phosphorylation in immature and mature myeloid progenitors. Single cell analysis of the chromatin landscape showed altered drivers of differentiation in monocytes and progenitors. Collectively, these data indicate that chronic ethanol drinking results in remodeling of the transcriptional and epigenetic landscapes of the bone marrow compartment leading to altered functions in the periphery.

Cellular and humoral immunogenicity of the COVID-19 vaccine and COVID-19 disease severity in individuals with immunodeficiency.

Background: A well-coordinated adaptive immune response is crucial for limiting COVID-19 disease. Some individuals with immunodeficiency are at a high risk of developing severe COVID-19. Therefore, the development of standardized methods for measuring different arms of the vaccine response in the setting of immunodeficiency is of particular interest. In this study, we compared the vaccine response of individuals living with immunodeficiency with healthy controls in terms of interferon gamma (IFN-γ) production and spike protein-specific antibody level post primary COVID-19 vaccination and booster vaccines. Additionally, the disease severity of those individuals who contracted COVID-19 was assessed. Methods: Whole blood was stimulated overnight from 71 participants and 99 healthy controls. Commercially available PepTivator® peptide pool and trimeric spike protein stimulation were used. ELISA was used to analyze IFN-γ levels. The total SARS-CoV-2 spike protein antibody titre was measured using a Roche Elecsys® S total antibody assay. Patient characteristics, COVID-19 infection status and IDDA 2.1 'Kaleidoscope' scores were recorded. Vaccine responses were scored from zero to three. Results: 99% of healthy controls, 89% of individuals with IEI and 76% with secondary immunodeficiency (SID) had an IFN-γ level above the validated reference range after peptide mix stimulation following primary vaccination. There was an increase in IFN-γ levels in patients with inborn errors of immunity (IEI) following the booster vaccine (p = 0.0156). 100% of healthy controls, 70% of individuals living with IEI and 64% of individuals living with SID had detectable spike protein-specific antibody levels following the primary vaccination. 55% of immunodeficiency patients who had mild COVID-19 and 10% with moderate/severe COVID-19 had detectable antibody and IFN-γ levels post vaccine. The mean pre-infection IDDA 2.1 scores were higher in individuals who developed moderate/severe COVID-19 (25.2 compared to 9.41). Conclusions: Covid whole-blood IGRA is a highly accurate, straightforward and robust assay and can be easily adapted to measure cellular response to COVID-19. A complete evaluation of the vaccine response may be particularly important for individuals living with immunodeficiency. A clinical immunodeficiency score and a validated vaccine response score may be valuable tools in estimating COVID-19 disease risk and identifying individuals living with immunodeficiency who may benefit from enhanced vaccination schedules.

HMGB2 regulates the differentiation and stemness of exhausted CD8+ T cells during chronic viral infection and cancer.

Chronic infections and cancers evade the host immune system through mechanisms that induce T cell exhaustion. The heterogeneity within the exhausted CD8+ T cell pool has revealed the importance of stem-like progenitor (Tpex) and terminal (Tex) exhausted T cells, although the mechanisms underlying their development are not fully known. Here we report High Mobility Group Box 2 (HMGB2) protein expression is upregulated and sustained in exhausted CD8+ T cells, and HMGB2 expression is critical for their differentiation. Through epigenetic and transcriptional programming, we identify HMGB2 as a cell-intrinsic regulator of the differentiation and maintenance of Tpex cells during chronic viral infection and in tumors. Despite Hmgb2-/- CD8+ T cells expressing TCF-1 and TOX, these master regulators were unable to sustain Tpex differentiation and long-term survival during persistent antigen. Furthermore, HMGB2 also had a cell-intrinsic function in the differentiation and function of memory CD8+ T cells after acute viral infection. Our findings show that HMGB2 is a key regulator of CD8+ T cells and may be an important molecular target for future T cell-based immunotherapies.

Chronic alcohol consumption dysregulates innate immune response to SARS-CoV-2 in the lung.

BACKGROUND: Alcohol consumption is widespread with over half of the individuals over 18 years of age in the U.S. reporting alcohol use in the last 30 days. Moreover, 9 million Americans engaged in binge or chronic heavy drinking (CHD) in 2019. CHD negatively impacts pathogen clearance and tissue repair, including in the respiratory tract, thereby increasing susceptibility to infection. Although, it has been hypothesized that chronic alcohol consumption negatively impacts COVID-19 outcomes; the interplay between chronic alcohol use and SARS-CoV-2 infection outcomes has yet to be elucidated. METHODS: In this study we employed luminex, scRNA sequencing, and flow cytometry to investigate the impact of chronic alcohol consumption on SARS-CoV-2 anti-viral responses in bronchoalveolar lavage cell samples from humans with alcohol use disorder and rhesus macaques that engaged in chronic drinking. FINDINGS: Our data show that in both humans (n = 6) and macaques (n = 11), the induction of key antiviral cytokines and growth factors was decreased with chronic ethanol consumption. Moreover, in macaques fewer differentially expressed genes mapped to Gene Ontology terms associated with antiviral immunity following 6 month of ethanol consumption while TLR signaling pathways were upregulated. INTERPRETATION: These data are indicative of aberrant inflammation and reduced antiviral responses in the lung with chronic alcohol drinking. FUNDING: This study was supported by NIH 1R01AA028735-04 (Messaoudi), U01AA013510-20 (Grant), R24AA019431-14 (Grant), R24AA019661 (Burnham), P-51OD011092 (ONPRC core grant support). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Chronic alcohol consumption dysregulates innate immune response to SARS-CoV-2 in the lung.

Alcohol consumption is widespread with over half of the individuals over 18 years of age in the U.S. reporting alcohol use in the last 30 days. Moreover, 9 million Americans engaged in binge or chronic heavy drinking (CHD) in 2019. CHD negatively impacts pathogen clearance and tissue repair, including in the respiratory tract, thereby increasing susceptibility to infection. Although, it has been hypothesized that chronic alcohol consumption negatively impacts COVID-19 outcomes; the interplay between chronic alcohol use and SARS-CoV-2 infection outcomes has yet to be elucidated. Therefore, in this study we investigated the impact of chronic alcohol consumption on SARS-CoV-2 anti-viral responses in bronchoalveolar lavage cell samples from humans with alcohol use disorder and rhesus macaques that engaged in chronic drinking. Our data show that in both humans and macaques, the induction of key antiviral cytokines and growth factors was decreased with chronic ethanol consumption. Moreover, in macaques fewer differentially expressed genes mapped to Gene Ontology terms associated with antiviral immunity following 6 month of ethanol consumption while TLR signaling pathways were upregulated. These data are indicative of aberrant inflammation and reduced antiviral responses in the lung with chronic alcohol drinking.

A critical review of recent knowledge of alcohol's effects on the immunological response in different tissues.

Alcohol misuse contributes to the dysregulation of immune responses and multiorgan dysfunction across various tissues, which are associated with higher risk of morbidity and mortality in people with alcohol use disorders. Organ-specific immune cells, including microglia in the brain, alveolar macrophages in the lungs, and Kupffer cells in the liver, play vital functions in host immune defense through tissue repair and maintenance of homeostasis. However, binge drinking and chronic alcohol misuse impair these immune cells' abilities to regulate inflammatory signaling and metabolism, thus contributing to multiorgan dysfunction. Further complicating these delicate systems, immune cell dysfunction associated with alcohol misuse is exacerbated by aging and gut barrier leakage. This critical review describes recent advances in elucidating the potential mechanisms by which alcohol misuse leads to derangements in host immunity and highlights current gaps in knowledge that may be the focus of future investigations.

Complex mixture discrimination and the role of contaminants.

Rats were trained in a 2-alternative odor choice task to discriminate between a 10-component odor mixture and the same mixture with one component removed and replaced with 1 of 3 concentrations of a different monomolecular odor (contaminant). All stimuli were presented within a training session, thus the rat essentially had to learn to discriminate the 10-component mixture from "not" the 10-component mixture. Rats performed most poorly discriminating the complete mixture from the mixture with one component removed and no contaminant added. As the concentration of the contaminant increased from 10 ppm to a concentration equal to the other components (100 ppm), discrimination improved linearly. In analyses of individual differences, rats that spent more time in the sampling port (sampling and making a decision) were more accurate than rats that spent less time. Together, these results emphasize the balance between perceptual stability and perceptual discrimination expressed by the olfactory system dealing with dynamic mixtures and the robust effects of contamination on those processes. In addition, they provide further support that modification of sampling/decision time is a strategy used by rats to deal with difficult discriminations of complex odors.