Computational and Systems Biology Advances to Enable Bioagent Agnostic Signatures.

Enumerated threat agent lists have long driven biodefense priorities. The global SARS-CoV-2 pandemic demonstrated the limitations of searching for known threat agents as compared to a more agnostic approach. Recent technological advances are enabling agent-agnostic biodefense, especially through the integration of multi-modal observations of host-pathogen interactions directed by a human immunological model. Although well-developed technical assays exist for many aspects of human-pathogen interaction, the analytic methods and pipelines to combine and holistically interpret the results of such assays are immature and require further investments to exploit new technologies. In this manuscript, we discuss potential immunologically based bioagent-agnostic approaches and the computational tool gaps the community should prioritize filling.

The association of allostasis with alcohol use: A case-control study in males with and without alcohol use disorder.

BACKGROUND: Allostatic load (AL) is associated with a heightened predisposition to disease due to prolonged activation of biological stress-response systems. Alcohol use disorder (AUD) is known to activate these systems. The primary aim of the current study was to examine the relationship between AL and AUD. METHODS: Participants were males (100%) with DSM-IV Alcohol Dependence (n = 48) and healthy participants with no history of substance use disorder (n = 17). Participants with AUD were 4-6 weeks abstinent. The AL index used cortisol, interleukin-6 (IL-6), fibrinogen, tumor necrosis factor-alpha (TNFα), C-reactive protein (CRP), glucose, insulin, leptin, pulse, systolic blood pressure readings, diastolic blood pressure readings, and body mass index (BMI). Physiological dysregulation for each biological measure was determined based on values within the 25th or 75th percentiles; AL was calculated as the total number of physiologically dysregulated biological measures. RESULTS: No differences in mean AL scores between the cases and controls [t(63) = .48, p = .633] were observed. Among cases, AL was not associated with lifetime drinks per drinking day (F(2,42) = .42, p = .662), lifetime total drinks (F(2,42) = 0.48, p = .620), total drinks 6 months prior to participating in the study (F(2,43) = 0.58, p = .563), or drinks per drinking day at 3-month follow-up (F(2,35) = 1.93, p = .161). AL was negatively associated with drinks per drinking day 6 months prior to study participation (F(2,42) = 3.71, p = .033). CONCLUSIONS: The hypotheses were not supported. Given that alcohol is likely to lead to physiological dysregulation, the apparent absence of a relationship between biomarkers of cumulative stress as indicated by AL and drinking status was both unanticipated and remarkable. Based on the results, AL in the context of drinking status or drinking among males with AUD may not be applicable.

Alcohol self-administration and nicotine withdrawal alter biomarkers of stress and inflammation and prefrontal cortex changes in Gß subunits.

Background: Although alcohol and nicotine are often used together, the biological consequences of these substances are not well understood. Identifying shared targets will inform cessation pharmacotherapies and provide a deeper understanding of how co-use of alcohol and nicotine impacts health, including biomarkers of stress and inflammation.Objective: We examined the effects of nicotine exposure and withdrawal on alcohol self-administration (SA), stress and inflammatory biomarkers, and a G-protein coupled receptor subunit (Gß) in brain areas associated with drug use.Methods: Male rats were trained to SA alcohol and then received a nicotine pump (n = 7-8 per group). We assessed alcohol intake for 12 days during nicotine exposure and then following pump removal to elicit withdrawal. After the behavioral studies, we assessed plasma leptin, corticosterone, and interleukin-1ß (IL-1ß), and Gß protein expression in the amygdala, nucleus accumbens (NAc), and prefrontal cortex (PFC).Results: Nicotine exposure or withdrawal did not alter alcohol intake (p > .05). Alcohol and nicotine withdrawal elevated corticosterone levels (p = .015) and decreased Gß levels in the PFC (p = .004). In the absence of nicotine, alcohol SA suppressed IL-1ß levels (p = .039). Chronic exposure to nicotine or withdrawal during alcohol SA did not alter leptin levels or Gß expression in the amygdala or NAc (p's > .05).Conclusions: The combination of alcohol SA and nicotine withdrawal produced a persistent increase in stress biomarkers and a suppression in Gß expression in the PFC, providing an important first step toward understanding the common biological mechanisms of alcohol/nicotine misuse.

Francisella tularensis Infection of Mice as a Model of Sepsis.

Sepsis results from the dysregulated immune response to infection. While the stimulator and progression of the septic response is poorly understood, the systemic production of a storm of cytokines is common in all etiologies of sepsis. While the complexity of this uncontrolled cascade is difficult to replicate using single molecule agonist, for example, lipopolysaccharide (LPS), several whole organism models can stimulate this cytokine storm. Herein, we detail protocols developed to trigger and analyze the systemic septic response in mouse models using the bacterium Francisella tularensis.

Alginate Hydrogels with Embedded ZnO Nanoparticles for Wound Healing Therapy.

INTRODUCTION: In this in-vitro study, we designed a 3D printed composite of zinc oxide (ZnO) nanoparticles (NPs) with photocatalytic activities encapsulated within hydrogel (alginate) constructs, for antibacterial purposes applicable towards wound healing. We primarily sought to confirm the mechanical properties and cell compatibility of these ZnO NP infused scaffolds. METHODS: The antibacterial property of the ZnO NPs was confirmed by hydroxyl radical generation using ultraviolet (U.V.) photocatalysis. Titanium dioxide (TiO2), a well-known antibacterial compound, was used as a positive control (1% w/v) for the ZnO NP-based alginate constructs and their antibacterial efficacies compared. Among the ZnO group, 3D printed gels containing 0.5% and 1% w/v of ZnO were analyzed and compared with manually casted samples via SEM, swelling evaluation, and rheological analysis. Envisioning an in-vivo application for the 3D printed ZnO NP-based alginates, we studied their antibacterial properties by bacterial broth testing, cytocompatibility via live/dead assay, and moisture retention capabilities utilizing a humidity sensor. RESULTS: 3D printed constructs revealed significantly greater pore sizes and enhanced structural stability compared to manually casted samples. For all samples, the addition of ZnO or TiO2 resulted in significantly stiffer gels in comparison with the alginate control. Bacterial resistance testing on Staphylococcus epidermidis indicated the addition of ZnO NPs to the gels decreased bacterial growth when compared to the alginate only gels. Cell viability of STO-fibroblasts was not adversely affected by the addition of ZnO NPs to the alginate gels. Furthermore, the addition of increasing doses of ZnO NPs to the alginate demonstrated increased humidity retention in gels. DISCUSSION: The customization of 3D printed alginates containing antibacterial ZnO NPs leads to an alternative that allows accessible mobility of molecular exchange required for improving chronic wound healing. This scaffold can provide a cost-effective and durable antibacterial treatment option.

Design, synthesis, and screening of sulfonylurea-derived NLRP3 inflammasome inhibitors.

Inflammasomes are multiprotein assemblies that produce robust inflammatory responses upon stimulation with pathogen- and/or danger-associated molecular patterns. Uncontrolled inflammasome activation has been linked to the pathophysiology of a wide array of disorders including life-threatening pathogenic infections, e.g., Francisella tularensis. There has been a great deal of interest in the development of small molecule inflammasome inhibitors. Using computational modeling based on chalcone derivatives, we have developed novel tertiary sulfonylurea compounds as inhibitors of the NLRP3 inflammasome. The polar enone functional alert of chalcone was replaced with a sulfonylurea scaffold while maintaining the relative positions of the two aromatic rings. These compounds were evaluated for their ability to inhibit NLRP3 and AIM2 inflammasome activation triggered by Francisella tularensis infection.

Insulin modulates the strong reinforcing effects of nicotine and changes in insulin biomarkers in a rodent model of diabetes.

This study examined whether the strong reinforcing effects of nicotine and changes in insulin biomarkers observed in diabetic rats are modulated via insulin. A model of diabetes was employed involving administration of streptozotocin (STZ), which produces hypoinsulinemia in rats. The present study included vehicle- or STZ-treated rats that received sham surgery or insulin pellets. Two weeks later, the rats were given extended access to intravenous self-administration (IVSA) of saline or nicotine. Concomitant changes in food intake, water responses, and body weight were assessed during 12 days of IVSA. After the last session, plasma levels of insulin, leptin, amylin, and glucagon-like peptide-1 (GLP-1) were assessed using Luminex® technology. In a separate cohort, phosphorylated insulin receptor substrate-2 (pIRS-2) and insulin growth factor-1 receptor ß (IGF-1Rß) were assessed in the nucleus accumbens (NAc) and ventral tegmental area (VTA) of vehicle- or STZ-treated rats that received sham surgery or an insulin pellet. STZ-treated rats displayed an increase in glucose levels, a decrease in body weight, and an increase in nicotine, food, and water intake relative to controls. STZ-treated rats also displayed a decrease in plasma insulin and leptin levels and an increase in amylin and GLP-1 levels relative to controls. Importantly, all of the STZ-induced changes in behavior and insulin biomarkers were prevented by insulin supplementation. STZ-treated rats also displayed a decrease in pIRS-2 and IGF-1Rß in the NAc (but not VTA), an effect that was also prevented by insulin. These data suggest that insulin systems in the NAc modulate the strong reinforcing effects of nicotine in male diabetic rats.

Obesity Exacerbates the Cytokine Storm Elicited by Francisella tularensis Infection of Females and Is Associated with Increased Mortality.

Infection with Francisella tularensis, the causative agent of the human disease tularemia, results in the overproduction of inflammatory cytokines, termed the cytokine storm. Excess metabolic byproducts of obesity accumulate in obese individuals and activate the same inflammatory signaling pathways as F. tularensis infection. In addition, elevated levels of leptin in obese individuals also increase inflammation. Since leptin is produced by adipocytes, we hypothesized that increased fat of obese females may make them more susceptible to F. tularensis infection compared with lean individuals. Lean and obese female mice were infected with F. tularensis and the immunopathology and susceptibility monitored. Plasma and tissue cytokines were analyzed by multiplex ELISA and real-time RT-PCR, respectively. Obese mice were more sensitive to infection, developing a more intense cytokine storm, which was associated with increased death of obese mice compared with lean mice. This enhanced inflammatory response correlated with in vitro bacteria-infected macrophage cultures where addition of leptin led to increased production of inflammatory cytokines. We conclude that increased basal leptin expression in obese individuals causes a persistent low-level inflammatory response making them more susceptible to F. tularensis infection and heightening the generation of the immunopathological cytokine storm.

Personalized Proteomics: The Future of Precision Medicine.

Medical diagnostics and treatment has advanced from a one size fits all science to treatment of the patient as a unique individual. Currently, this is limited solely to genetic analysis. However, epigenetic, transcriptional, proteomic, posttranslational modifications, metabolic, and environmental factors influence a patient's response to disease and treatment. As more analytical and diagnostic techniques are incorporated into medical practice, the personalized medicine initiative transitions to precision medicine giving a holistic view of the patient's condition. The high accuracy and sensitivity of mass spectrometric analysis of proteomes is well suited for the incorporation of proteomics into precision medicine. This review begins with an overview of the advance to precision medicine and the current state of the art in technology and instrumentation for mass spectrometry analysis. Thereafter, it focuses on the benefits and potential uses for personalized proteomic analysis in the diagnostic and treatment of individual patients. In conclusion, it calls for a synthesis between basic science and clinical researchers with practicing clinicians to design proteomic studies to generate meaningful and applicable translational medicine. As clinical proteomics is just beginning to come out of its infancy, this overview is provided for the new initiate.

A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components.

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB.

Mycobacterium-Specific γ9δ2 T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity.

Numerous pathogens, including Mycobacterium tuberculosis, can activate human γ9δ2 T cells to proliferate and express effector mechanisms. γ9δ2 T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We prepared M. tuberculosis-specific γ9δ2 T cell lines to study their direct protective effects and APC functions for M. tuberculosis-specific αß T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αß T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ9δ2 T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ9δ2 T cells enhance the expansion of M. tuberculosis-specific αß T cells and increase the ability of αß T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ9δ2 T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ9δ2 T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4(+) αß T cells and γ9δ2 T cells provide similar immune enhancing/APC functions for M. tuberculosis-specific T cells. These effector and helper effects of γ9δ2 T cells further indicate that these T cells should be considered important new targets for new TB vaccines.

Viral infection causes a shift in the self peptide repertoire presented by human MHC class I molecules.

PURPOSE: MHC class I presentation of peptides allows T cells to survey the cytoplasmic protein milieu of host cells. During infection, presentation of self peptides is, in part, replaced by presentation of microbial peptides. However, little is known about the self peptides presented during infection, despite the fact that microbial infections alter host cell gene expression patterns and protein metabolism. EXPERIMENTAL DESIGN: The self peptide repertoire presented by HLA-A*01;01, HLA-A*02;01, HLA-B*07;02, HLA-B*35;01, and HLA-B*45;01 (where HLA is human leukocyte antigen) was determined by tandem MS before and after vaccinia virus infection. RESULTS: We observed a profound alteration in the self peptide repertoire with hundreds of self peptides uniquely presented after infection for which we have coined the term "self peptidome shift." The fraction of novel self peptides presented following infection varied for different HLA class I molecules. A large part (approximately 40%) of the self peptidome shift arose from peptides derived from type I interferon-inducible genes, consistent with cellular responses to viral infection. Interestingly, approximately 12% of self peptides presented after infection showed allelic variation when searched against approximately 300 human genomes. CONCLUSION AND CLINICAL RELEVANCE: Self peptidome shift in a clinical transplant setting could result in alloreactivity by presenting new self peptides in the context of infection-induced inflammation.

Discovering naturally processed antigenic determinants that confer protective T cell immunity.

CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection - information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I-transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.

Sculpting MHC class II-restricted self and non-self peptidome by the class I Ag-processing machinery and its impact on Th-cell responses.

It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery - which supplies peptides for presentation by class I molecules - plays no role in class II-restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)-cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4(+) T-cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II-restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4(+) T-cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4(+) T-cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II-restricted peptidome, impacting the CD4(+) T-cell repertoire, and ultimately altering Th-cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II-restricted epitopes that would otherwise be capable of eliciting functional Th-cell responses.

Granzyme A produced by γ(9)δ(2) T cells induces human macrophages to inhibit growth of an intracellular pathogen.

Human γ(9)δ(2) T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ(9)δ(2) T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ(9)δ(2) T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ(9)δ(2) T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ(9)δ(2) T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ(9)δ(2) T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ(9)δ(2) T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.

IL-15 regulates homeostasis and terminal maturation of NKT cells.

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

Live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse T-cell responses in young children.

BACKGROUND: Two doses of either trivalent live attenuated or inactivated influenza vaccines (LAIV and TIV, respectively) are approved for young children (≥ 24 months old for LAIV and ≥ 6 months old for TIV) and induce protective antibody responses. However, whether combinations of LAIV and TIV are safe and equally immunogenic is unknown. Furthermore, LAIV is more protective than TIV in children for unclear reasons. METHODS: Children 6-35 months old were administered, 1 month apart, 2 doses of either TIV or LAIV, or combinations of LAIV and TIV in both prime/boost sequences. Influenza-specific antibodies were measured by hemagglutination inhibition (HAI), and T cells were studied in flow cytometric and functional assays. Highly conserved M1, M2, and NP peptides predicted to be presented by common HLA class I and II were used to stimulate interferon-γ enzyme-linked immunospot responses. RESULTS: All LAIV and/or TIV combinations were well tolerated and induced similar HAI responses. In contrast, only regimens containing LAIV induced influenza-specific CD4(+), CD8(+), and γδ T cells, including T cells specific for highly conserved influenza peptides. CONCLUSIONS: Prime/boost combinations of LAIV and TIV in young children were safe and induced similar protective antibodies. Only LAIV induced CD4(+), CD8(+), and γδ T cells relevant for broadly protective heterosubtypic immunity. CLINICAL TRIALS REGISTRATION: NCT00231907.