Selenium supplementation in patients with subclinical hypothyroidism affected by autoimmune thyroiditis: Results of the SETI study.

OBJECTIVE: The purpose of this prospective study was to assess the effects of selenium supplementation on TSH and interferon-γ inducible chemokines (CXCL9, CXCL10 and CXCL11) levels in patients with subclinical hypothyroidism due to Hashimoto's thyroiditis. PATIENTS AND METHODS: Patients with subclinical hypothyroidism due to Hashimoto thyroiditis were prospectively enrolled in the SETI study. They received 83mcg of selenomethionine/day orally in a soft gel capsule for 4 months with water after a meal. No further treatment was given. All patients were measured thyroid hormone, TPOAb, CXCL9, CXCL10, CXCL11, iodine, and selenium levels at baseline and at study end. RESULTS: 50 patients (43/7 female/male, median age 43.9±11.8 years) were enrolled, of which five withdrew from the study. At the end of the study, euthyroidism was restored in 22/45 (48.9%) participants (responders), while 23 patients remained hypothyroid (non-responders). There were no significant changes in TPOAb, CXCL9, CXCL10, CXCL11, and iodine levels from baseline to the end of the study in both responders and non-responders. TSH levels were re-tested six months after selenomethionine withdrawal: 83.3% of responding patients remained euthyroid, while only 14.2% of non-responders became euthyroid. CONCLUSIONS: The SETI study shows that short-course supplementation with selenomethionine is associated to a normalization of serum TSH levels which is maintained 6 months after selenium withdrawal in 50% of patients with subclinical hypothyroidism due to chronic autoimmune thyroiditis. This TSH-lowering effect of selenium supplementation is unlikely to be related to changes in humoral markers of autoimmunity and/or circulating CXCL9.

1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol ameliorates chemoradiation-induced oral mucositis.

OBJECTIVE: This study was designed to investigate whether necroptosis is involved in the pathogenesis of chemoradiation-induced oral mucositis in a murine model and whether 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) ameliorates this disorder. MATERIALS AND METHODS: A chemoradiation-induced oral mucositis model was established by treating mice with concurrent 5-fluorouracil (100 mg/kg, i.p.) and head and neck X-irradiation (20 Gy). Phosphate-buffered saline or PLAG (100 mg/kg or 250 mg/kg, p.o.) was administered daily. Body weights were recorded daily, and mice were sacrificed on Day 9 for tongue tissue analysis. RESULTS: On Day 9, chemoradiotherapy-treated (ChemoRT) mice had tongue ulcerations and experienced significant weight loss (Day 0:26.18 ± 1.41 g; Day 9:19.44 ± 3.26 g). They also had elevated serum macrophage inhibitory protein 2 (MIP-2) (control: 5.57 ± 3.49 pg/ml; ChemoRT: 130.14 ± 114.54 pg/ml) and interleukin (IL)-6 (control: 198.25 ± 16.91 pg/ml; ChemoRT: 467.25 ± 108.12 pg/ml) levels. ChemoRT-treated mice who received PLAG exhibited no weight loss (Day 0:25.78 ± 1.04 g; Day 9:26.46 ± 1.68 g) and had lower serum MIP-2 (4.42 ± 4.04 pg/ml) and IL-6 (205.75 ± 30.41 pg/ml) levels than ChemoRT-treated mice who did not receive PLAG. Tongue tissues of mice who received PLAG also displayed lower phosphorylation levels of necroptotic signalling proteins. CONCLUSION: 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol mitigated chemoradiation-induced oral mucositis by modulating necroptosis.

Association between soluble immune mediators and tumor responses in patients with nonsmall cell lung cancer treated with anti-PD-1 inhibitor.

Although programmed death (PD)-1 immune checkpoint therapies target the immune system, the relationship between inflammatory factors and the clinical outcome of anti-PD-1 therapy for nonsmall cell lung cancer (NSCLC) is not fully understood. Here we examined the association between soluble immune mediators and the outcome of treatment with PD-1 inhibitors in patients with advanced/recurrent NSCLC. In two independent cohorts, we assessed the levels of 88 different soluble immune mediators in peripheral blood before and after anti-PD-1 treatment, and evaluated their associations with clinical outcomes. In the training cohort, the plasma levels of chitinase 3-like-1 and GM-CSF before treatment (p = 0.006 and p = 0.005, respectively) and changes in the plasma levels of CXCL2, VEGF, IFNα2, and MMP2 after treatment (p < 0.001, p = 0.019, p = 0.019, and p = 0.012, respectively) were significantly correlated with PFS. The change in the plasma CXCL2 level was also significantly associated with treatment-related AEs (p = 0.017). In the validation cohort, however, only the changes in the plasma levels of CXCL2 and MMP2 after treatment were associated with PFS (p = 0.003 and p = 0.006, respectively), and these changes were maintained during the course of anti-PD-1 therapy in patients who showed better clinical outcomes, even in those with tumor pseudoprogression. Since CXCL2 and MMP2 can be easily measured by minimally invasive blood sampling, they could be useful for monitoring of clinical outcomes in NSCLC patients receiving PD-1 inhibitor therapy.

Inflammatory and oxidative stress biomarkers at protein and molecular levels in workers occupationally exposed to crystalline silica.

Workers chronically exposed to respirable crystalline silica (CS) are susceptible to adverse health effects like silicosis and lung cancer. This study aimed to investigate potential early peripheral biomarkers of inflammation and oxidative stress in miners. The subjects enrolled in this study were occupationally unexposed workers (OUW, n = 29) and workers exposed to crystalline silica (WECS), composed by miners, which were divided into two subgroups: workers without silicosis (WECS I, n = 39) and workers diagnosed with silicosis, retired from work (WECS II, n = 42). The following biomarkers were evaluated: gene expression of L-selectin, CXCL2, CXCL8 (IL-8), HO-1, and p53; malondialdehyde (MDA) plasma levels and non-protein thiol levels in erythrocytes. Additionally, protein expression of L-selectin was evaluated to confirm our previous findings. The results demonstrated that gene expression of L-selectin was decreased in the WECS I group when compared to the OUW group (p < 0.05). Regarding gene expression of CXCL2, CXCL8 (IL-8), HO-1, and p53, significant fold change decreases were observed in workers exposed to CS in relation to unexposed workers (p < 0.05). The results of L-selectin protein expression in lymphocyte surface corroborated with our previous findings; thus, significant downregulation in the WECS groups was observed compared to OUW group (p < 0.05). The MDA was negatively associated with the gene expression of CXCL-2, CXCL8 (IL-8), and p53 (p < 0.05). The participants with silicosis (WECS II) presented significant increased non-protein thiol levels in relation to other groups (p < 0.05). Taken together, our findings may contribute to help the knowledge about the complex mechanisms involved in the silicosis pathogenesis and in the risk of lung cancer development in workers chronically exposed to respirable CS.

Prospective Analysis of the Effects of Maternal Immune Activation on Rat Cytokines during Pregnancy and Behavior of the Male Offspring Relevant to Schizophrenia.

Influenza during pregnancy is associated with the development of psychopathology in the offspring. We sought to determine whether maternal cytokines produced following administration of viral mimetic polyinosinic-polycytidylic acid (polyI:C) to pregnant rats were predictive of behavioral abnormalities in the adult offspring. Timed-pregnant Sprague Dawley rats received a single intravenous injection of 4-mg/kg polyI:C or saline on gestational day (GD)15. Blood was collected 3 h later for serum analysis of cytokine levels with ELISA. Male offspring were tested in a battery of behavioral tests during adulthood and behavior was correlated with maternal cytokine levels. Maternal serum levels of CXCL1 and interleukin (IL)-6, but not tumor necrosis factor (TNF)-α or CXCL2, were elevated in polyI:C-treated dams. PolyI:C-treated dams experienced post-treatment weight loss and polyI:C pups were smaller than controls at postnatal day (PND)1. Various behavior alterations were seen in the polyI:C-treated offspring. Male polyI:C offspring had enhanced MK-801-induced locomotion, and reduced sociability. PolyI:C offspring failed to display crossmodal and visual memory, and oddity preference was also impaired. Set-shifting, assessed with a lever-based operant conditioning task, was facilitated while touchscreen-based reversal learning was impaired. Correlations were found between maternal serum concentrations of CXCL1, acute maternal temperature and body weight changes, neonatal pup mass, and odd object discrimination and social behavior. Overall, while the offspring of polyI:C-treated rats displayed behavior abnormalities, maternal serum cytokines were not related to the long-term behavior changes in the offspring. Maternal sickness effects and neonatal pup size may be better indicators of later effects of maternal inflammation in the offspring.

Long Non-Coding RNA LUCAT1 Promotes Proliferation and Invasion in Clear Cell Renal Cell Carcinoma Through AKT/GSK-3ß Signaling Pathway.

BACKGROUND/AIMS: Long non-coding RNAs (lncRNAs) have emerged as new regulators and biomarkers in several cancers. However, few lncRNAs have been well characterized in clear cell renal cell carcinoma (ccRCC). METHODS: We investigated the lncRNA expression profile by microarray analysis in 5 corresponding ccRCC tissues and adjacent normal tissues. Lung cancer-associated transcript 1 (LUCAT1) expression was examined in 90 paired ccRCC tissues by real-time PCR and validated by The Cancer Genome Atlas (TCGA) database. Kaplan-Meier analysis was used to examine the prognostic value of LUCAT1 and CXCL2 in ccRCC patients. Loss and gain of function were performed to explore the effect of LUCAT1 on proliferation and invasion in ccRCC cells. Western blotting was performed to evaluate the underlying mechanisms of LUCAT1 in ccRCC progression. Chemokine stimulation assay was performed to investigate possible mechanisms controlling LUCAT1 expression in ccRCC cells. Enzyme-linked immunosorbent assays were performed to determine serum CXCL2 in ccRCC patients and healthy volunteers. Receiver operating characteristic curve analysis was performed to examine the clinical diagnostic value of serum CXCL2 in ccRCC. RESULTS: We found that LUCAT1 was significantly upregulated in both clinical ccRCC tissues (n = 90) and TCGA ccRCC tissues (n = 448) compared with normal tissues. Statistical analysis revealed that the LUCAT1 expression level positively correlated with tumor T stage (P < 0.01), M stage (P < 0.01), and TNM stage (P < 0.01). Overall survival and disease-free survival time were significantly shorter in the high-LUCAT1-expression group than in the low-LUCAT1-expression group (log-rank P < 0.01). LUCAT1 knockdown inhibited ccRCC cell proliferation and colony formation, induced cell cycle arrest at G1 phase, and inhibited cell migration and invasion. Overexpression of LUCAT1 promoted proliferation, migration, and invasion of ccRCC cells. Mechanistic investigations showed that LUCAT1 induced cell cycle G1 arrest by regulating the expression of cyclin D1, cyclin-dependent kinase 4, and phosphorylated retinoblastoma transcriptional corepressor 1. Moreover, LUCAT1 promoted proliferation and invasion in ccRCC cells partly through inducing the phosphorylation of AKT and suppressing the phosphorylation of GSK-3ß. We also revealed that chemokine CXCL2, upregulated in ccRCC, induced LUCAT1 expression and might be a diagnostic and prognostic biomarker in ccRCC. CONCLUSIONS: LUCAT1 was upregulated in ccRCC tissues and renal cancer cell lines, and significantly correlated with malignant stage and poor prognosis in ccRCC. LUCAT1 promoted proliferation and invasion in ccRCC cells through the AKT/GSK-3ß signaling pathway. We also revealed that LUCAT1 overexpression was induced by chemokine CXCL2. These findings indicate that the CXCL2/LUCAT1/AKT/GSK-3ß axis is a potential therapeutic target and molecular biomarker for ccRCC.

Gastrointestinal microbiota contributes to the development of murine transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress upon blood transfusion and is the leading cause of transfusion-related fatalities. Whether the gut microbiota plays any role in the development of TRALI is currently unknown. We observed that untreated barrier-free (BF) mice suffered from severe antibody-mediated acute lung injury, whereas the more sterile housed specific pathogen-free (SPF) mice and gut flora-depleted BF mice were both protected from lung injury. The prevention of TRALI in the SPF mice and gut flora-depleted BF mice was associated with decreased plasma macrophage inflammatory protein-2 levels as well as decreased pulmonary neutrophil accumulation. DNA sequencing of amplicons of the 16S ribosomal RNA gene revealed a varying gastrointestinal bacterial composition between BF and SPF mice. BF fecal matter transferred into SPF mice significantly restored TRALI susceptibility in SPF mice. These data reveal a link between the gut flora composition and the development of antibody-mediated TRALI in mice. Assessment of gut microbial composition may help in TRALI risk assessment before transfusion.

Snail promotes ovarian cancer progression by recruiting myeloid-derived suppressor cells via CXCR2 ligand upregulation.

Snail is a major transcriptional factor that induces epithelial-mesenchymal transition (EMT). In this study, we explore the effect of Snail on tumor immunity. Snail knockdown in mouse ovarian cancer cells suppresses tumor growth in immunocompetent mice, associated with an increase of CD8+ tumor-infiltrating lymphocytes and a decrease of myeloid-derived suppressor cells (MDSCs). Snail knockdown reduces the expression of CXCR2 ligands (CXCL1 and CXCL2), chemokines that attract MDSCs to the tumor via CXCR2. Snail upregulates CXCR ligands through NF-kB pathway, and most likely, through direct binding to the promoters. A CXCR2 antagonist suppresses MDSC infiltration and delays tumor growth in Snail-expressing mouse tumors. Ovarian cancer patients show elevated serum CXCL1/2, which correlates with Snail expression, MDSC infiltration, and short overall survival. Thus, Snail induces cancer progression via upregulation of CXCR2 ligands and recruitment of MDSCs. Blocking CXCR2 represents an immunological therapeutic approach to inhibit progression of Snail-high tumors undergoing EMT.

Role of Receptor for Advanced Glycation End Products in Regulating Lung Fluid Balance in Lipopolysaccharide-induced Acute Lung Injury and Infection-Related Acute Respiratory Distress Syndrome.

Receptor for advanced glycation end products (RAGE) is implicated in inflammatory responses in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), but its role in pulmonary edema formation remains unclear, especially in infection-related ARDS mainly caused by pneumonia or sepsis. In this study, we investigated the role of RAGE in alveolar fluid regulation by using RAGE gene knockout (RAGE) mice in a murine ALI model induced by lipopolysaccharide (LPS), and by comparing soluble RAGE (sRAGE) levels in serum and bronchial alveolar lavage fluid between ARDS patients and control subjects. We found that RAGE knockout significantly improved alveolar fluid clearance and reduced pulmonary vascular albumin leakage upon LPS challenge. Furthermore, LPS-induced substantial decrease in lung expression of sodium-potassium ATPase (Na,K-ATPase), epithelial sodium channel, and zonula occluden-1 (ZO-1) were fully or partially restored by the deletion of RAGE. In addition to this, LPS-induced lung leukocyte infiltration and inflammatory cytokine and chemokine release were all attenuated in RAGE mice as compared to wide-type mice. In infection-related ARDS patients, both serum and bronchial alveolar lavage fluid levels of the sRAGE were much higher than those in control subjects, and they were positively correlated with pulmonary vascular permeability and levels of interleukin (IL)-6, IL-8, and macrophage inflammatory protein (MIP)-2. Taken together, we provided the first direct evidence for the essential role of RAGE in regulating lung fluid balance in infection-related ARDS/ALI. The underlying mechanisms may involve the downregulation of both ion-channel and tight junction proteins mediated by RAGE signaling in bacterial endotoxin-induced lung injury.

Chemokine Involvement in Lung Injury Secondary to Ischaemia/Reperfusion.

INTRODUCTION: During transplant surgeries, the lung experiences an ischaemia-reperfusion (I/R)-induced damage identified as a significant cause of morbidity and mortality. However, the mechanisms by which I/R induces leucocyte accumulation and subsequent tissue damage in lung surgeries remain unknown. Therefore, the present study aims to assess the role of monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) in leucocyte chemotaxis related to lung injury secondary to I/R. METHODS: Six pigs were subjected to an orthotopic left caudal lobe lung transplantation with a subsequent 60-min graft reperfusion (Transplant group). In addition, six animals underwent to sham surgery (Sham Group). Plasma samples and lung biopsies were collected before the beginning of pneumonectomy, before starting the reperfusion, and 30 min and 60 min after the beginning of the reperfusion. Plasma levels of intercellular adhesion molecule 1 (ICAM-1) and lung expressions of MCP-1, MIP-2, myeloperoxidase (MPO), and lung oedema were measured. RESULTS: Lung I/R caused substantial damage observed as pulmonary oedema. The oedema was evident after the ischemic insult and increased after reperfusion. After reperfusion, increased levels of MPO were observed which suggests an activation and infiltration of neutrophils into the lung tissue. After 30 min of reperfusion, MCP-1, MIP-2, and ICAM-1 levels were significantly increased compared to prepneumonectomy levels (p < 0.05) and a further increase was observed after 60 min of reperfusion (p < 0.05). CONCLUSION: The present study demonstrates that activated neutrophils, as well as MCP-1, MIP-2, and ICAM-1, are involved in inflammatory response induced by ischaemia-reperfusion-induced lung injury.

Evaluation of gastrointestinal leakage using serum (1→3)-ß-D-glucan in a Clostridium difficile murine model.

Gastrointestinal (GI) leakage in Clostridium difficile-associated diarrhea (CDAD) is well known but is not routinely assessed in clinical practice. Serum (1→3)-ß-D-glucan (BG), a fungal cell wall component used as a biomarker for invasive fungal disease, was tested in a CDAD mouse model with and without probiotics. Higher serum fluorescein isothiocyanate-dextran (FITC-dextran) and spontaneous gram-negative bacteremia, GI leakage indicators, were frequently found in CDAD mice, which died compared with those which survived. BG, serum macrophage inflammatory protein-2 and FITC-dextran but not quantitative blood bacterial count differentiated the clinical severity. Interestingly, a specific dose of Lactobacillus rhamnosus L34 attenuated CDAD and decreased serum BG and FITC-dextran, but not other parameters. BG also showed a higher area under the receiver operating characteristic curve for 7-day mortality than FITC-dextran. Fifty-five percent of CDAD mice with BG ≥ 60 pg/ml (the human negative cut-off value for invasive fungal disease) at 1 day after C. difficile gavage died within 7 days. In conclusion, S: erum BG was elevated in mice with severe CDAD, an established model of GI leakage with a strong association with mortality rate. BG monitoring in patients with CDAD is of interest as both a potential prognostic tool and a therapeutic efficacy indicator.

Budesonide ameliorates lung injury induced by large volume ventilation.

BACKGROUND: Ventilation-induced lung injury (VILI) is a health problem for patients with acute respiratory dysfunction syndrome. The aim of this study was to investigate the effectiveness of budesonide in treating VILI. METHODS: Twenty-four rats were randomized to three groups: a ventilation group, ventilation/budesonide group, and sham group were ventilated with 30 ml/kg tidal volume or only anesthesia for 4 hor saline or budesonide airway instillation immediately after ventilation. The PaO2/FiO2and wet-to-dry weight ratios, protein concentration, neutrophil count, and neutrophil elastase levels in bronchoalveolar lavage fluid (BALF) and the levels of inflammation-related factors were examined. Histological evaluation of and apoptosis measurement inthe lung were conducted. RESULTS: Compared with that in the ventilation group, the PaO2/FiO2 ratio was significantly increased by treatment with budesonide. The lung wet-to-dry weight ratio, total protein, neutrophil elastase level, and neutrophilcount in BALF were decreased in the budesonide group. The BALF and plasma tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, intercellular adhesion molecule (ICAM)-1, and macrophage inflammatory protein (MIP)-2 levels were decreased, whereas the IL-10 level was increased in the budesonide group. The phosphorylated nuclear factor (NF)-kBlevels in lung tissue were inhibited by budesonide. The histological changes in the lung and apoptosis were reduced by budesonide treatment. Bax, caspase-3, and cleaved caspase-3 were down-regulated, and Bcl-2 was up-regulated by budesonide. CONCLUSIONS: Budesonide ameliorated lung injury induced by large volume ventilation, likely by improving epithelial permeability, decreasing edema, inhibiting local and systemic inflammation, and reducing apoptosis in VILI.

Calcitriol inhibits tumor necrosis factor alpha and macrophage inflammatory protein-2 during lipopolysaccharide-induced acute lung injury in mice.

Acute lung injury is a common complication of sepsis in intensive care unit patients with an extremely high mortality. The present study investigated the effects of calcitriol, the active form of vitamin D, on tumor necrosis factor alpha (TNF-α) and macrophage inflammatory protein-2 (MIP-2) in sepsis-induced acute lung injury. Mice were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS, 1.0mg/kg) to establish the animal model of sepsis-induced acute lung injury. Some mice were i.p. injected with calcitriol (1.0µg/kg) before LPS injection. An obvious infiltration of inflammatory cells in the lungs was observed beginning at 1h after LPS injection. Correspondingly, TNF-α and MIP-2 in sera and lung homogenates were markedly elevated in LPS-treated mice. Interestingly, calcitriol obviously alleviated LPS-induced infiltration of inflammatory cells in the lungs. Moreover, calcitriol markedly attenuated LPS-induced elevation of TNF-α and MIP-2 in sera and lung homogenates. Further analysis showed that calcitriol repressed LPS-induced p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) phosphorylation. In addition, calcitriol blocked LPS-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65 and p50 subunit in the lungs. Taken together, these results suggest that calcitriol inhibits inflammatory cytokines production in LPS-induced acute lung injury.

Platelet secretion of CXCL4 is Rac1-dependent and regulates neutrophil infiltration and tissue damage in septic lung damage.

BACKGROUND AND PURPOSE: Platelets are potent regulators of neutrophil accumulation in septic lung damage. We hypothesized that platelet-derived CXCL4 might support pulmonary neutrophilia in a murine model of abdominal sepsis. EXPERIMENTAL APPROACH: Polymicrobial sepsis was triggered by coecal ligation and puncture (CLP) in C57BL/6 mice. Platelet secretion of CXCL4 was studied by using confocal microscopy. Plasma and lung levels of CXCL4, CXCL1 and CXCL2 were determined by elisa. Flow cytometry was used to examine surface expression of Mac-1 on neutrophils. KEY RESULTS: CLP increased CXCL4 levels in plasma, and platelet depletion reduced plasma levels of CXCL4 in septic animals. Rac1 inhibitor NSC23766 decreased the CLP-enhanced CXCL4 in plasma by 77%. NSC23766 also abolished PAR4 agonist-induced secretion of CXCL4 from isolated platelets. Inhibition of CXCL4 reduced CLP-evoked neutrophil recruitment, oedema formation and tissue damage in the lung. However, immunoneutralization of CXCL4 had no effect on CLP-induced expression of Mac-1 on neutrophils. Targeting CXCL4 attenuated plasma and lung levels of CXCL1 and CXCL2 in septic mice. CXCL4 had no effect on neutrophil chemotaxis in vitro, indicating it has an indirect effect on pulmonary neutrophilia. Intratracheal CXCL4 enhanced infiltration of neutrophils and formation of CXCL2 in the lung. CXCR2 antagonist SB225002 markedly reduced CXCL4-provoked neutrophil accumulation in the lung. CXCL4 caused secretion of CXCL2 from isolated alveolar macrophages. CONCLUSIONS AND IMPLICATIONS: Rac1 controls platelet secretion of CXCL4 and CXCL4 is a potent stimulator of neutrophil accumulation in septic lungs via generation of CXCL2 in alveolar macrophages. Platelet-derived CXCL4 plays an important role in lung inflammation and tissue damage in polymicrobial sepsis.

Age Impacts Pulmonary Inflammation and Systemic Bone Response to Inhaled Organic Dust Exposure.

Agricultural workers have high rates of airway and skeletal health disease. Studies recently demonstrated that inhaled agricultural organic dust extract (ODE)-induced airway injury is associated with bone deterioration in an animal model. However, the effect of age in governing these responses to organic dusts is unclear, but might be important in future approaches. Young (7-9 wk) and older (12-14,o) male C57BL/6 mice received intranasal (i.n.) inhalation exposure to ODE from swine confinement facilities once or daily for 3 wk. Acute ODE-induced neutrophil influx and cytokine and chemokine (tumor necrosis factor [TNF]-α, interleukin [IL]-6, keratinocyte chemoattractant [CXCL1], macrophage inflammatory protein-2 [CXCL2]) airway production were reduced in older compared to young mice. Repetitive ODE treatment, however, increased lymphocyte recruitment and alveolar compartment histopathologic inflammatory changes in older mice. Whole lung cell infiltrate analysis revealed that young, but not older, mice repetitively treated with ODE demonstrated an elevated CD4:CD8 lymphocyte response. Acute inhalant ODE exposure resulted in a 4-fold and 1.5-fold rise in blood neutrophils in young and older mice, respectively. Serum IL-6 and CXCL1 levels were elevated in young and older mice i.n. exposed once to ODE, with increased CXCL1 levels in younger compared to older mice. Although older mice displayed reduced bone measurements compared to younger mice, younger rodents demonstrated ODE-induced decrease in bone mineral density, bone volume, and bone microarchitecture quality as determined by computed tomography (CT) analysis. Collectively, age impacts the airway injury and systemic inflammatory and bone loss response to inhalant ODE, suggesting an altered and enhanced immunologic response in younger as compared to older counterparts.

Lung necrosis and neutrophils reflect common pathways of susceptibility to Mycobacterium tuberculosis in genetically diverse, immune-competent mice.

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis in susceptible humans. Here, we infected Diversity Outbred (DO) mice with ∼100 bacilli by aerosol to model responses in a highly heterogeneous population. Following infection, 'supersusceptible', 'susceptible' and 'resistant' phenotypes emerged. TB disease (reduced survival, weight loss, high bacterial load) correlated strongly with neutrophils, neutrophil chemokines, tumor necrosis factor (TNF) and cell death. By contrast, immune cytokines were weak correlates of disease. We next applied statistical and machine learning approaches to our dataset of cytokines and chemokines from lungs and blood. Six molecules from the lung: TNF, CXCL1, CXCL2, CXCL5, interferon-γ (IFN-γ), interleukin 12 (IL-12); and two molecules from blood - IL-2 and TNF - were identified as being important by applying both statistical and machine learning methods. Using molecular features to generate tree classifiers, CXCL1, CXCL2 and CXCL5 distinguished four classes (supersusceptible, susceptible, resistant and non-infected) from each other with approximately 77% accuracy using completely independent experimental data. By contrast, models based on other molecules were less accurate. Low to no IFN-γ, IL-12, IL-2 and IL-10 successfully discriminated non-infected mice from infected mice but failed to discriminate disease status amongst supersusceptible, susceptible and resistant M.-tuberculosis-infected DO mice. Additional analyses identified CXCL1 as a promising peripheral biomarker of disease and of CXCL1 production in the lungs. From these results, we conclude that: (1) DO mice respond variably to M. tuberculosis infection and will be useful to identify pathways involving necrosis and neutrophils; (2) data from DO mice is suited for machine learning methods to build, validate and test models with independent data based solely on molecular biomarkers; (3) low levels of immunological cytokines best indicate a lack of exposure to M. tuberculosis but cannot distinguish infection from disease.

Enterococcus faecalis Glycolipids Modulate Lipoprotein-Content of the Bacterial Cell Membrane and Host Immune Response.

In this study, we investigated the impact of the cell membrane composition of E. faecalis on its recognition by the host immune system. To this end, we employed an E. faecalis deletion mutant (ΔbgsA) that does not synthesize the major cell membrane glycolipid diglycosyl-diacylglycerol (DGlcDAG). Proteomic analysis revealed that 13 of a total of 21 upregulated surface-associated proteins of E. faecalis ΔbgsA were lipoproteins. This led to a total lipoprotein content in the cell membrane of 35.8% in ΔbgsA compared to only 9.4% in wild-type bacteria. Increased lipoprotein content strongly affected the recognition of ΔbgsA by mouse macrophages in vitro with an increased stimulation of TNF-α production by heat-fixed bacteria and secreted antigens. Inactivation of the prolipoprotein diacylglycerol transferase (lgt) in ΔbgsA abrogated TNF-α induction by a ΔbgsA_lgt double mutant indicating that lipoproteins mediate increased activation of mouse macrophages by ΔbgsA. Heat-fixed ΔbgsA bacteria, culture supernatant, or cell membrane lipid extract activated transfected HEK cells in a TLR2-dependent fashion; the same was not true of wild-type bacteria. In mice infected intraperitoneally with a sublethal dose of E. faecalis we observed a 70% greater mortality in mice infected with ΔbgsA compared with wild-type-infected mice. Increased mortality due to ΔbgsA infection was associated with elevated plasma levels of the inflammatory cytokines TNF-α, IL-6 and MIP-2. In summary, our results provide evidence that an E. faecalis mutant lacking its major bilayer forming glycolipid DGlcDAG upregulates lipoprotein expression leading to increased activation of the host innate immune system and virulence in vivo.

Effects of storage conditions on the stability of serum CD163, NGAL, HMGB1 and MIP2.

BACKGROUND: Several cytokines have been involved in the diagnosis and prognosis for the pathogenesis and severity of chronic hepatitis B (CHB) such as cluster of differentiation 163 (CD163), neutrophil gelatinase-associated lipocalin (NGAL), high-mobility group box 1 (HMGB1) and macrophage inflammatory protein-2 (MIP-2). Nevertheless, the stability and reliability of these cytokines can be greatly influenced by handling and storage processes. METHODS: In this study, potential utility of serum samples of a CHB cohort was evaluated to investigate several processes that might impact cytokine profiles such as temperature, storage time and number of freeze-thaw cycles. Blood samples collected from 100 patients with CHB were separated immediately and divided into two groups. In one group, samples (n=50) stored at -80 °C were subject to 1-3 freeze-thaw cycles. In the other group, samples (n=50) were stored at 4 °C and 25 °C for 3 h, 9 h, 24 h, 48 h, 72 h, and 7 d time points, respectively. To assess the influence of different storage conditions on cytokines, the levels of CD163, NGAL, HMGB1 and MIP-2 were measured using enzyme-linked immunosorbent assays (ELISA) kits. RESULTS: No significant differences of these four cytokines after 1-3 repeated freeze-thaw cycles. Significant differences of NAGL levels were seen between 9 h and 7 d (P<0.05), and also in HMGB1 at 25 °C, while the other cytokines were relatively stable at the two storage temperatures over the various time points. CONCLUSION: This study indicated that these four cytokines remained stable within three freeze-thaw cycles and 7 d at 4 °C. No perceptible effects on CD163 and MIP-2 levels were presented under the storage condition of 7 d at room temperature, whereas the degradation of NGAL and HMGB1 were notable.

Exaggerated Acute Lung Injury and Impaired Antibacterial Defenses During Staphylococcus aureus Infection in Rats with the Metabolic Syndrome.

Rats with Metabolic Syndrome (MetaS) have a dysregulated immune response to the aseptic trauma of surgery. We hypothesized that rats with MetaS would have dysregulated inflammation, increased lung injury, and less effective antibacterial defenses during Staphylococcus (S.) aureus sepsis as compared to rats without MetaS. Low capacity runner (LCR; a model of MetaS) and high capacity runner (HCR) rats were challenged intravenously with S. aureus bacteria. After 48 h, inflammatory mediators and bacteria were quantified in the blood, bronchoalveolar lavage fluid (BALF), and lung homogenates. Lungs were analyzed histologically. BALF protein and lung wet-dry ratios were quantified to assess for vascular leak. Endpoints were compared in infected LCR vs HCR rats. LCR rats had higher blood and lung S. aureus counts, as well as higher levels of IL-6 in plasma, lungs and BALF, MIP-2 in plasma and lung, and IL-17A in lungs. Conversely, LCR rats had lower levels of IL-10 in plasma and lungs. Although lactate levels, and liver and renal function tests were similar between groups, LCR rats had higher BALF protein and lung wet-dry ratios, and more pronounced acute lung injury histologically. During S. aureus bacteremia, as compared with HCR rats, LCR (MetaS) rats have heightened pro-inflammatory responses, accompanied by increased acute lung injury and vascular leak. Notably, despite an augmented pro-inflammatory phenotype, LCR rats have higher bacterial levels in their blood and lungs. The MetaS state may exacerbate lung injury and vascular leak by attenuating the inflammation-resolving response, and by weakening antimicrobial defenses.

Hypercapnic acidosis prolongs survival of skin allografts.

BACKGROUND: Evidence reveals that hypercapnic acidosis (HCA) modulates immune responses. However, the effect of HCA on allogenic skin graft rejection is unknown. We examined whether HCA might improve skin graft survival in a mouse model of skin transplantation. METHODS: A major histocompatibility-complex-incompatible BALB/c to C57BL/6 mouse skin transplantation model was used. Animals were divided into sham control, air, and HCA groups. Mice in the HCA group were exposed daily to 5% CO2 in air for 1 h. Skin grafts were harvested for histologic analyses. Nuclear factor (NF)-κB activation was determined in harvested draining lymph nodes. Spleen weights and serum levels of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 2 were serially assessed after skin transplantation. RESULTS: Skin allografts survived significantly longer in the HCA group of mice than those in the air group. Allografted mice in the air group underwent a 2.1-fold increase in spleen weight compared with a 1.1-fold increase in the mice with HCA on day 3. There were increased inflammatory cell infiltration, folliculitis, focal dermal-epidermal separation, and areas of epidermal necrosis in the air group that were reduced with HCA treatment. In the HCA group, CD8(+) T cell infiltration at day 7 decreased significantly but not CD4(+) T cell infiltration. In addition, HCA significantly suppressed serum tumor necrosis factor-α on days 1 and 3 and chemokine (C-X-C motif) ligand 2 on days 1 and 10. Furthermore, the HCA group had remarkably suppressed NF-κB activity in draining lymph nodes. CONCLUSIONS: HCA significantly prolonged the survival of incompatible skin allografts in mice by reducing proinflammatory cytokine production, immune cell infiltration, and NF-κB activation.