L-arginine supplementation abrogates hypoxia-induced virulence of Staphylococcus aureus in a murine diabetic pressure wound model.

Diabetic foot ulcers (DFUs) are the most common complications of diabetes resulting from hyperglycemia leading to ischemic hypoxic tissue and nerve damage. Staphylococcus aureus is the most frequently isolated bacteria from DFUs and causes severe necrotic infections leading to amputations with a poor 5-year survival rate. However, very little is known about the mechanisms by which S. aureus dominantly colonizes and causes severe disease in DFUs. Herein, we utilized a pressure wound model in diabetic TALLYHO/JngJ mice to reproduce ischemic hypoxic tissue damage seen in DFUs and demonstrated that anaerobic fermentative growth of S. aureus significantly increased the virulence and the severity of disease by activating two-component regulatory systems leading to expression of virulence factors. Our in vitro studies showed that supplementation of nitrate as a terminal electron acceptor promotes anaerobic respiration and suppresses the expression of S. aureus virulence factors through inactivation of two-component regulatory systems, suggesting potential therapeutic benefits by promoting anaerobic nitrate respiration. Our in vivo studies revealed that dietary supplementation of L-arginine (L-Arg) significantly attenuated the severity of disease caused by S. aureus in the pressure wound model by providing nitrate. Collectively, these findings highlight the importance of anaerobic fermentative growth in S. aureus pathogenesis and the potential of dietary L-Arg supplementation as a therapeutic to prevent severe S. aureus infection in DFUs.IMPORTANCES. aureus is the most common cause of infection in DFUs, often resulting in lower-extremity amputation with a distressingly poor 5-year survival rate. Treatment for S. aureus infections has largely remained unchanged for decades and involves tissue debridement with antibiotic therapy. With high levels of conservative treatment failure, recurrence of ulcers, and antibiotic resistance, a new approach is necessary to prevent lower-extremity amputations. Nutritional aspects of DFU treatment have largely been overlooked as there has been contradictory clinical trial evidence, but very few in vitro and in vivo modelings of nutritional treatment studies have been performed. Here we demonstrate that dietary supplementation of L-Arg in a diabetic mouse model significantly reduced duration and severity of disease caused by S. aureus. These findings suggest that L-Arg supplementation could be useful as a potential preventive measure against severe S. aureus infections in DFUs.

The mechanism by which cannabidiol (CBD) suppresses TNF-α secretion involves inappropriate localization of TNF-α converting enzyme (TACE).

Cannabidiol (CBD) is a phytocannabinoid derived from Cannabis sativa that exerts anti-inflammatory mechanisms. CBD is being examined for its putative effects on the neuroinflammatory disease, multiple sclerosis (MS). One of the major immune mediators that propagates MS and its mouse model experimental autoimmune encephalomyelitis (EAE) are macrophages. Macrophages can polarize into an inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2a). Therefore, elucidating the impact on macrophage polarization with CBD pre-treatment is necessary to understand its anti-inflammatory mechanisms. To study this effect, murine macrophages (RAW 264.7) were pre-treated with CBD (10 µM) or vehicle (ethanol 0.1 %) and were either left untreated (naive; cell media only), or stimulated under M1 (IFN-γ + lipopolysaccharide, LPS) or M2a (IL-4) conditions for 24 hr. Cells were analyzed for macrophage polarization markers, and supernatants were analyzed for cytokines and chemokines. Immunofluorescence staining was performed on M1-polarized cells for the metalloprotease, tumor necrosis factor-α-converting enzyme (TACE), as this enzyme is responsible for the secretion of TNF-α. Overall results showed that CBD decreased several markers associated with the M1 phenotype while exhibiting less effects on the M2a phenotype. Significantly, under M1 conditions, CBD increased the percentage of intracellular and surface TNF-α but decreased secreted TNF-α. This phenomenon might be mediated by TACE as staining showed that CBD sequestered TACE intracellularly. CBD also prevented RelA nuclear translocation. These results suggest that CBD may exert its anti-inflammatory effects by reducing M1 polarization and decreasing TNF-α secretion via inappropriate localization of TACE and RelA.

Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells.

Staphylococcal superantigens induce massive activation of T cells and inflammation, leading to toxic shock syndrome. Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 µg/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2-lacking isoform and a partial exon 3-lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.

An optimized flow cytometry panel for classifying macrophage polarization.

Macrophages are scavenger cells and a fundamental part of innate and adaptive immune responses, and they are important in wound repair and tissue remodeling. The functions of macrophages include engulfing and killing invading pathogens, processing and presenting antigens, initiation of inflammation, secreting cytokines and other inflammatory mediators, and participating in the maintenance and repair of tissues. Based on functional differences and surface and intracellular marker expression, macrophages can be generally divided into either M1 (inflammatory) or M2 (wound healing); the M2 type can be further divided into M2a, M2b, M2c, and M2d. However, due to the time, effort, and cost of establishing a panel of markers that could thoroughly assess polarization, the characterization of types and subtypes is usually done using three markers or fewer. This can lead to problems, because the expression of some of the most widely used polarization markers can be altered by commonly used inflammatory or immunological stimuli. We have developed and optimized an eleven-color polychromatic flow cytometric assay for macrophage subtype identification that prevents mischaracterization due to stimulus-induced changes in individual markers by using partially redundant markers for which at least one is not substantially affected by a commonly used inflammatory stimulus (LPS). We polarized 3 × 105 RAW 264.7 cells, a mouse macrophage cell line, with IFN-γ (± LPS), IL-4 or IL-10 to derive M1, M2a, or M2c macrophage subtypes, respectively. The TNF-α concentration in cell supernatants was tested by ELISA to verify polarization. Then polarized cells were labeled with the following antibodies and assessed by flow cytometry to identify marker expression: F4/80, Arginase 1, TLR4, CD86, VEGF, CD14, CD206, MHC Class II, and TNF-α (surface and internal). Here we have identified clear distinctions between macrophage subtypes using these markers, and we anticipate that this panel will help disclose more details of the macrophage's role in the immune response and will save investigators the time and cost usually required to identify appropriate antibodies that do not interfere with each other or lead to difficult color compensation issues.

Dosage scaling of alcohol in binge exposure models in mice: An empirical assessment of the relationship between dose, alcohol exposure, and peak blood concentrations in humans and mice.

Binge drinking is a remarkably prevalent behavior. In 2015, 27% of U.S. residents 18 years old or older reported at least one episode of binge drinking in the previous month. Rodent models for binge drinking are widely used to study the mechanisms by which alcohol causes a variety of adverse health effects in humans. Concerns have been raised that many binge-drinking studies in rodents involve alcohol doses that would be unrealistically high in humans. Allometric dosage scaling can be used to estimate the dose of a drug or chemical in mice that would be necessary to achieve similar biological effects at a realistic dose in humans. However, it has become apparent that no single allometric conversion factor is applicable for all drugs and chemicals, so it is necessary to evaluate each compound empirically. In the present study, we compared the area under the blood alcohol concentration vs. time curve (AUC) and the peak blood alcohol concentration following oral alcohol administration at various doses in mice and humans, using data from previously published studies. The results demonstrated that the oral dose of alcohol must be larger in mice (on a g of alcohol to kg of body weight basis) than in humans to achieve similar alcohol AUC values or to achieve similar peak concentrations in the blood. The dose required in mice was about 2-fold greater than the dose required in humans to achieve similar alcohol AUC and peak concentrations. The results shown here were substantially different from the average 5-12-fold difference between mice and humans calculated in previous studies using agents other than alcohol. Results shown here demonstrate that an empirical approach using data from several independent experiments provides information needed to determine the alcohol dose in mice that produces a similar level of exposure (AUC and peak concentration) as in humans. The results indicate that a single alcohol dose in the range of 5-6 g/kg, a range often used in mouse models for binge drinking, is not excessive when modeling human binge drinking. Results presented here illustrate that in mice both alcohol AUC and peak alcohol concentration correlate well with an important biological effect - activation of the hypothalamic-pituitary-adrenal axis - as indicated by increased corticosterone AUC values.

Induction of Immunosuppressive CD8+CD25+FOXP3+ Regulatory T Cells by Suboptimal Stimulation with Staphylococcal Enterotoxin C1.

Superantigens (SAgs) produced by Staphylococcus aureus at high concentrations induce proliferation of T cells bearing specific TCR Vß sequences and massive cytokinemia that cause toxic shock syndrome. However, the biological relevance of SAgs produced at very low concentrations during asymptomatic colonization or chronic infections is not understood. In this study, we demonstrate that suboptimal stimulation of human PBMCs with a low concentration (1 ng/ml) of staphylococcal enterotoxin C1, at which half-maximal T cell proliferation was observed, induced CD8+CD25+ T cells expressing markers related to regulatory T cells (Tregs), such as IFN-γ, IL-10, TGF-ß, FOXP3, CD28, CTLA4, TNFR2, CD45RO, and HLA-DR. Importantly, these CD8+CD25+ T cells suppressed responder cell proliferation mediated in contact-dependent and soluble factor-dependent manners, involving galectin-1 and granzymes, respectively. In contrast, optimal stimulation of human PBMCs with a high concentration (1 µg/ml) of staphylococcal enterotoxin C1, at which maximal T cell proliferation was observed, also induced similar expression of markers related to Tregs, including FOXP3 in CD8+CD25+ cells, but these T cells were not functionally immunosuppressive. We further demonstrated that SAg-induced TCR Vß-restricted and MHC class II-restricted expansion of immunosuppressive CD8+CD25+ T cells is independent of CD4+ T cells. Our results suggest that the concentration of SAg strongly affects the functional characteristics of activated T cells, and low concentrations of SAg produced during asymptomatic colonization or chronic S. aureus infection induce immunosuppressive CD8+ Tregs, potentially promoting colonization, propagation, and invasion of S. aureus in the host.

Binge alcohol consumption 18 h after induction of sepsis in a mouse model causes rapid overgrowth of bacteria, a cytokine storm, and decreased survival.

Alcohol abuse increases vulnerability to infections and infection-related mortality. In previous studies, we found that acute alcohol abuse in a binge-drinking model in mice decreased resistance to bacterial sepsis when alcohol was administered near the time of bacterial challenge. In the present study, we investigated the effects of alcohol administered later in the course of sepsis (18 h after injection of Escherichia coli). Our working hypothesis was that decreased production of cytokines caused by alcohol at this time would actually improve survival, because overproduction of pro-inflammatory mediators is thought to be the proximate cause of mortality in sepsis. Unexpectedly, administration of alcohol late in the course of sepsis led to a rapid increase in the number of viable bacteria in the peritoneal cavity. Significant increases in the concentrations of several cytokines and chemokines coincided with the increased number of bacteria in alcohol-treated mice and decreased survival time. These results demonstrated our working hypothesis to be incorrect, and reiterated the complexity of sepsis. Hypothermia is a consistent feature in this model of sepsis. In control mice (E. coli only), body temperature was near normal by 18 h or 21 h after administration of E. coli, but in mice treated with alcohol 18 h after E. coli, hypothermia was significant 3 h later and ultimately mortality was significantly increased. However, counteracting the hypothermic effect of alcohol by external warming of mice led to earlier mortality, demonstrating that hypothermia was not the major cause of mortality. These results, along with previous results from studies in which alcohol was given before initiation of sepsis, suggest that decreased cytokine and chemokine production may not be the key effect of alcohol that decreases resistance to sepsis. It seems more likely that suppression of mechanisms by which macrophages and neutrophils kill bacteria is critical, and this can occur even in the presence of high levels of cytokines and chemokines.

Lack of immunotoxic effects of repeated exposure to atrazine associated with the adaptation of adrenal gland activation.

T cell-dependent IgM antibody production and natural killer cell (NKC) activity were assessed in SD rats orally administered atrazine for 28 days to males (0, 6.5, 25, or 100 mg/kg/day) or females (0, 3, 6, or 50 mg/kg/day), or 30 or 500 ppm in diet (3 or 51 mg/kg/day). Anti-asialo GM1 antibodies (NKC) and cyclophosphamide (antibody-forming cell assay [AFC]) served as positive controls. Pituitary (ACTH, prolactin), adrenal (corticosterone, progesterone, aldosterone), and gonadal (androgens, estrogens) hormones were assessed after 1, 7, and/or 28 days of treatment. Food intake and body weights were significantly reduced in the highest dosed males, and transiently affected in females. Urinary corticosterone levels were not increased in atrazine-treated groups in either sex at any time point measured (10, 22, or 24 days). Corticosterone and progesterone were elevated in males after a single atrazine dose ≥6.5 mg/kg/day, but not after 7, 14, or 28 doses. There were no effects on adrenal, pituitary, or gonadal hormones in females. Atrazine did not suppress the AFC response or decrease NKC function after 28 days in males or females. Atrazine had no effect on spleen weights or spleen cell numbers in males or females, although thymus weights were elevated in males receiving the highest dose. The lack of immunotoxic effect of atrazine was associated with diminished adrenal activation over time in males, and no effects on adrenal hormones in females.

Exposure to p,p'-DDE Alters Macrophage Reactivity and Increases Macrophage Numbers in Adipose Stromal Vascular Fraction.

Exposure to p,p'-DDE (DDE), the main bioaccumulative metabolite of the organochlorine insecticide p,p'-DDT, is associated with a higher prevalence of obesity, dyslipidemia, insulin resistance, metabolic syndrome, and immunomodulation. The present study was carried out to determine whether DDE perturbs adipose tissue homeostasis through modulation of macrophage function. Treatment with DDE or a cyclooxygenase-2 inhibitor prior to lipopolysaccharide exposure significantly decreased production of prostaglandins (PG) from J774a.1 macrophages in vitro. Similarly, J774A.1 cell lysates incubated with DDE or a specific cyclooxygenase-2 inhibitor (NS-398) produced significantly less PGE2 and PGF2α. Macrophage polarization studies revealed a pattern of DDE effects that were not fully consistent with a purely pro- or purely anti- M1 or M2 effect. However, DDE suppressed expression of two M1 markers (induced by an M1 stimulus) and enhanced expression of an M2 marker (induced by an M2 stimulus). Further studies including assessment of macrophage function are needed to fully characterize the effects of DDE on macrophage polarization. Obesity is characterized by an increase in the number of resident adipose tissue macrophages. To assess monocyte/macrophage recruitment to the adipose tissue in vivo, male C57Bl/6H mice were treated with 2 mg/kg DDE or corn oil vehicle for 5 days by gavage. Epididymal fat pads were digested and macrophage populations were analyzed by flow cytometry. In DDE-treated animals, there was a significant increase (37%) in F4/80(+)CD11b(+) macrophages/g of epididymal adipose over vehicle (P < .05). Together, these results suggest a role for DDE in the enhancement of adipose tissue macrophage recruitment and/or proliferation, as well as modulation of immune cell function that may contribute to the etiology of metabolic diseases associated with organochlorine exposure.

ERAIZDA: a model for holistic annotation of animal infectious and zoonotic diseases.

There is an urgent need for a unified resource that integrates trans-disciplinary annotations of emerging and reemerging animal infectious and zoonotic diseases. Such data integration will provide wonderful opportunity for epidemiologists, researchers and health policy makers to make data-driven decisions designed to improve animal health. Integrating emerging and reemerging animal infectious and zoonotic disease data from a large variety of sources into a unified open-access resource provides more plausible arguments to achieve better understanding of infectious and zoonotic diseases. We have developed a model for interlinking annotations of these diseases. These diseases are of particular interest because of the threats they pose to animal health, human health and global health security. We demonstrated the application of this model using brucellosis, an infectious and zoonotic disease. Preliminary annotations were deposited into VetBioBase database (http://vetbiobase.igbb.msstate.edu). This database is associated with user-friendly tools to facilitate searching, retrieving and downloading of disease-related information. Database URL: http://vetbiobase.igbb.msstate.edu.

Effects of sodium methyldithiocarbamate on selected parameters of innate immunity and clearance of bacteria in a mouse model of sepsis.

AIMS: Sodium methyldithiocarbamate (SMD), the third most widely used conventional pesticide in the United States, has been reported to inhibit several parameters associated with inflammation and to decrease resistance to infection. In a previous study, survival time was markedly decreased when mice were treated orally with SMD shortly before challenge with a high dose of Escherichia coli (E. coli) that was lethal to most of the control mice. In the present study, we evaluated selected parameters of the innate immune system using a lower challenge dose of E. coli, to determine which (if any) of these parameters reflected continued changes through 24h. MAIN METHODS: Bacterial clearance from the peritoneal cavity, production of chemokines and cytokines, and body temperature were measured. KEY FINDINGS: All these parameters were reduced by SMD up to 12h after bacterial challenge, but the concentration of the anti-inflammatory cytokine IL-10 was increased. Even so, mice in the control and SMD-treated groups cleared most bacteria by 24h. Other parameters (cytokine concentrations and body temperature) were also normal or near normal by 24h. The same dosage of SMD administered intranasally also did not significantly decrease survival. Hypothermia from 16 to 28 h correlated with lethal outcome, but SMD significantly increased hypothermia only at 2 and 4h after challenge. SIGNIFICANCE: In spite of substantial early inhibition by SMD of parameters known to be important for resistance to infection, bacterial clearance and survival were not altered, suggesting immunological reserve and/or rapid recovery after transient effects of SMD.

Contributions of nonhematopoietic cells and mediators to immune responses: implications for immunotoxicology.

Immunotoxicology assessments have historically focused on the effects that xenobiotics exhibit directly on immune cells. These studies are invaluable as they identify immune cell targets and help characterize mechanisms and/or adverse outcome pathways of xenobiotics within the immune system. However, leukocytes can receive environmental cues by cell-cell contact or via released mediators from cells of organs outside of the immune system. These organs include, but are not limited to, the mucosal areas such as the lung and the gut, the liver, and the central nervous system. Homeostatic perturbation in these organs induced directly by toxicants can initiate and alter the outcome of local and systemic immunity. This review will highlight some of the identified nonimmune influences on immune homeostasis and provide summaries of how immunotoxic mechanisms of selected xenobiotics involve nonimmune cells or mediators. Thus, this review will identify data gaps and provide possible alternative mechanisms by which xenobiotics alter immune function that could be considered during immunotoxicology safety assessment.

The effects of oil exposure on peripheral blood leukocytes and splenic melano-macrophage centers of Gulf of Mexico fishes.

In August and November 2010 we collected and examined peripheral blood and tissues from three species of Gulf of Mexico fish. Findings were compared to non-exposed control fish. The leukocyte counts of exposed alligator gar were not significantly different from controls, while exposed Gulf killifish and sea trout had significantly decreased lymphocyte counts. Liver ethoxyresorufin-O-deethylase (EROD) values from sea trout were significantly greater than control sea trout EROD values, suggesting poly aromatic hydrocarbon exposure. Splenic melano-macrophage centers (MMCs) from exposed sea trout and Gulf killifish showed a significant increase in number compared to non-exposed fish. Sea trout splenic MMCs were also significantly greater in size. These findings suggest that Gulf fish sampled were exposed to crude oil from the Macondo well and were in a lymphopenic or immuno-compromised state.

Sodium methyldithiocarbamate exerts broad inhibition of cellular signaling and expression of effector molecules of inflammation.

Sodium methyldithiocarbamate (SMD) is one of the most abundantly used conventional pesticides in the United States. At dosages relevant to occupational exposure, it causes major effects on the immune system in mice, including a decreased resistance to sepsis. This lab has identified some of the mechanisms of action of this compound and some of the immunological parameters affected, but the global effects have not previously been assessed. The purpose of the present study was to conduct transcriptomic analysis of the effects of SMD on lipopolysaccharide-induced expression of mediators important in innate immunity and inflammation. The results revealed broad effects on expression of transcription factors in both branches of Toll-like receptor 4 (TLR4) signaling (MyD88 and TRIF). However, TLR3 and interferon signaling pathways were decreased to a greater extent, and assessment of the effects of SMD on polyinosinic polycytidylic acid-induced cytokine and chemokine production revealed that these responses mediated by TLR3 were indeed sensitive to the effects of SMD, with inhibition occurring at lower dosages than required to inhibit responses to other immunological stimuli tested in our previous studies. In the downstream signaling pathways of these TLRs, functional analysis also revealed that NF-κB activation was inhibited by SMD, as indicated by gene expression analysis and a reporter construct in mice. A previously unreported effect on luteinizing hormone and follicle-stimulating hormone pathways was also observed.

Binge ethanol and liver: new molecular developments.

Binge consumption of alcohol is an alarming global health problem. Binge (acute) ethanol (EtOH) is implicated in the pathophysiology of alcoholic liver disease (ALD). New studies from experimental animals and from humans indicate that binge EtOH has profound effects on immunological, signaling, and epigenetic parameters of the liver. This is in addition to the known metabolic effects of acute EtOH. Binge EtOH alters the levels of several cellular components and dramatically amplifies liver injury in chronically EtOH exposed liver. These studies highlight the importance of molecular investigations into binge effects of EtOH for a better understanding of ALD and also to develop therapeutic strategies to control it. This review summarizes these recent developments.

Transcriptomic analysis of peritoneal cells in a mouse model of sepsis: confirmatory and novel results in early and late sepsis.

BACKGROUND: The events leading to sepsis start with an invasive infection of a primary organ of the body followed by an overwhelming systemic response. Intra-abdominal infections are the second most common cause of sepsis. Peritoneal fluid is the primary site of infection in these cases. A microarray-based approach was used to study the temporal changes in cells from the peritoneal cavity of septic mice and to identify potential biomarkers and therapeutic targets for this subset of sepsis patients. RESULTS: We conducted microarray analysis of the peritoneal cells of mice infected with a non-pathogenic strain of Escherichia coli. Differentially expressed genes were identified at two early (1 h, 2 h) and one late time point (18 h). A multiplexed bead array analysis was used to confirm protein expression for several cytokines which showed differential expression at different time points based on the microarray data. Gene Ontology based hypothesis testing identified a positive bias of differentially expressed genes associated with cellular development and cell death at 2 h and 18 h respectively. Most differentially expressed genes common to all 3 time points had an immune response related function, consistent with the observation that a few bacteria are still present at 18 h. CONCLUSIONS: Transcriptional regulators like PLAGL2, EBF1, TCF7, KLF10 and SBNO2, previously not described in sepsis, are differentially expressed at early and late time points. Expression pattern for key biomarkers in this study is similar to that reported in human sepsis, indicating the suitability of this model for future studies of sepsis, and the observed differences in gene expression suggest species differences or differences in the response of blood leukocytes and peritoneal leukocytes.

Machine learning analysis of the relationship between changes in immunological parameters and changes in resistance to Listeria monocytogenes: a new approach for risk assessment and systems immunology.

No method has been reported to predict, even approximately, the impact of mild-to-moderate changes in several immunological parameters on resistance to infection. The ability to make such predictions would be useful in risk assessment. In addition, equations that predict host resistance on the basis of changes in components of a complex biological system (the immune system) would fulfill one of the major goals of systems biology. In this study, multiple machine learning classification methods were used to predict the effects of a series of drugs and chemicals on host resistance to Listeria monocytogenes in mice on the basis of changes in several holistic immunological parameters. A data set produced under the sponsorship of the National Toxicology Program (NTP) was used in this study. The NTP data set was found to have a high percentage of missing data and to be noisy (probably due to the intrinsically stochastic nature of immune responses). Data preprocessing steps were used to mitigate these problems. In evaluating the machine learning classifiers, we first randomly partitioned the NTP data set into 10 subsets. Each time, we used nine subsets of the data to train the machine learning classifiers, and the remaining single subset to predict outcomes with regard to host resistance. This process was repeated until all 10 combinations of the 9-1 split of the subsets have been tested. The best of the classifiers predicted host resistance outcome correctly for 94.7% of cases, a result which indicates it is possible to identify mathematical expressions that will be useful for risk assessment and to establish a basis for systems immunology.

Nonclinical toxicological evaluation of occidiofungin, a unique glycolipopeptide antifungal.

Occidiofungin, a glycolipopeptide obtained from the liquid culture of Burkholderia contaminans MS14, has been identified as a novel fungicide. The present study was designed to initially assess the in vitro toxicity in a rat hepatoma (H4IIE) cell line and acute toxicological effects of occidiofungin using a mouse model. In vitro toxicity was observed in all variables at 5 µmol/L. B6C3F1 mice were given single and repeat doses of occidiofungin up to 20 mg/kg. Key effects were a reduction in body and organ weights. However, no significant decrease in body weight was noted at a dose of 1 mg/kg, which is comparable to the dose level of other cyclic glycopeptide antifungal agents currently approved for human use. Microscopic examination of treated mice did not identify any signs of organ-specific toxicity at the dose levels tested.

Investigation of the role of TNF-α converting enzyme (TACE) in the inhibition of cell surface and soluble TNF-α production by acute ethanol exposure.

Toll-like receptors (TLRs) play a fundamental role in the immune system by detecting pathogen associated molecular patterns (PAMPs) to sense host infection. Ethanol at doses relevant for humans inhibits the pathogen induced cytokine response mediated through TLRs. The current study was designed to investigate the mechanisms of this effect by determining whether ethanol inhibits TLR3 and TLR4 mediated TNF-α secretion through inhibition of transcription factor activation or post-transcriptional effects. In NF-κB reporter mice, activation of NF-κB in vivo by LPS was inhibited by ethanol (LPS alone yielded 170,000±35,300 arbitrary units of light emission; LPS plus ethanol yielded 56,120±16880, p = 0.04). Inhibition of protein synthesis by cycloheximide revealed that poly I:C- or LPS-induced secreted TNF-α is synthesized de novo, not released from cellular stores. Using real time RT-PCR, we found inhibition of LPS and poly I:C induced TNF-α gene transcription by ethanol. Using an inhibitor of tumor necrosis factor alpha converting enzyme (TACE), we found that shedding caused by TACE is a prerequisite for TNF-α release after pathogen challenge. Flow cytometry was used to investigate if ethanol decreases TNF-α secretion by inhibition of TACE. In cells treated with LPS, ethanol decreased both TNF-α cell surface expression and secretion. For example, 4.69±0.60% of untreated cells were positive for cell surface TNF-α, LPS increased this to 25.18±0.85%, which was inhibited by ethanol (86.8 mM) to 14.29±0.39% and increased by a TACE inhibitor to 57.88±0.62%. In contrast, cells treated with poly I:C had decreased secretion of TNF-α but not cell surface expression. There was some evidence for inhibition of TACE by ethanol in the case of LPS, but decreased TNF-α gene expression seems to be the major mechanism of ethanol action in this system.

Alcohol abuse and Streptococcus pneumoniae infections: consideration of virulence factors and impaired immune responses.

Alcohol is the most frequently abused substance in the world. Both acute and chronic alcohol consumption have diverse and well-documented effects on the human immune system, leading to increased susceptibility to infections like bacterial pneumonia. Streptococcus pneumoniae is the most common bacterial etiology of community-acquired pneumonia worldwide. The frequency and severity of pneumococcal infections in individuals with a history of alcohol abuse is much higher than the general population. Despite this obvious epidemiological relevance, very few experimental studies have focused on the interaction of pneumococci with the immune system of a host acutely or chronically exposed to alcohol. Understanding these host-pathogen interactions is imperative for designing effective prophylactic and therapeutic interventions for such populations. Recent advances in pneumococcal research have greatly improved our understanding of pneumococcal pathogenesis and virulence mechanisms. Additionally, a large body of data is available on the effect of alcohol on the physiology of the lungs and the innate and adaptive immune system of the host. The purpose of this review is to integrate the available knowledge in these diverse areas of for a better understanding of the how the compromised immune system derived from alcohol exposure responds to pneumococcal infections.