Tumor necrosis factor related apoptosis inducing ligand (TRAIL) as a member of the TNF gene superfamily induces apoptosis primarily in tumor cells. TRAIL also plays an important role in the modulation of inflammatory responses, especially in the process of immune paralysis. The aim of the present study was to examine soluble TRAIL (sTRAIL) levels in septic patients in an attempt to explore the association between sTRAIL level and the risk of mortality.
Plasma sTRAIL levels were detected by ELISA in 50 septic patients and 20 healthy volunteers. HLA-DR expression in monocytes was detected by flow cytometry. Selective biochemical parameters were recorded, and patients were monitored in a 28-day period for mortality.
The mean plasma sTRAIL level in septic patients was significantly lower than that in healthy controls (16.9±8.3 vs. 68.3±8.6 pg/ml, P<0.01), and was significantly higher in 28-day survivors than those in non-survivors (19.4±9.8 vs. 13.9±4.7 pg/ml, P<0.05). Univariate analysis indicated that plasma sTRAIL level was positively correlated with monocyte and lymphocyte counts and HLA-DR expression level (r = 0.5, P<0.01; r = 0.3, P<0.05; r = 0.43, P<0.01, respectively). STRAIL level was negatively correlated with APACHE II score, BUN and age (r = −0.48, P<0.01; r = −0.29, P<0.05; r = −0.45, P<0.01, respectively). Multiple linear regression analysis indicated that the predictor of plasma soluble TRAIL level was HLA-DR expression (P<0.01).
Low plasma sTRAIL levels were associated with immune paralysis and a high risk of mortality in patients with septic shock. sTRAIL may prove to be a potential biomarker of immune function and predict the survival of septic patients.
Low tidal volume ventilation is beneficial in patients with severe pulmonary dysfunction and would, in theory, reduce postoperative complications if implemented during routine surgery. The study aimed to investigate whether low tidal volume ventilation and high positive end-expiratory pressure (PEEP) in a large animal model of postoperative sepsis would attenuate the systemic inflammatory response and organ dysfunction. Thirty healthy pigs were randomized to three groups: Group Prot-7h, i.e. protective ventilation for 7 h, was ventilated with a tidal volume of 6 mL x kg-1 for 7 h; group Prot-5h, i.e. protective ventilation for 5 h, was ventilated with a tidal volume of 10 mL x kg-1 for 2 h, after which the group was ventilated with a tidal volume of 6 mL x kg-1; and a control group that was ventilated with a tidal volume of 10 mL x kg-1 for 7 h. In groups Prot-7h and Prot-5h PEEP was 5 cmH2O for 2 h and 10 cmH2O for 5 h. In the control group PEEP was 5 cmH2O for the entire experiment. After surgery for 2 h, postoperative sepsis was simulated with an endotoxin infusion for 5 h. Low tidal volume ventilation combined with higher PEEP led to lower levels of interleukin 6 and 10 in plasma, higher PaO2/FiO2, better preserved functional residual capacity and lower plasma troponin I as compared with animals ventilated with a medium high tidal volume and lower PEEP. The beneficial effects of protective ventilation were seen despite greater reductions in cardiac index and oxygen delivery index. In the immediate postoperative phase low VT ventilation with higher PEEP was associated with reduced ex vivo plasma capacity to produce TNF-α upon endotoxin stimulation and higher nitrite levels in urine. These findings might represent mechanistic explanations for the attenuation of systemic inflammation and inflammatory-induced organ dysfunction.
Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in modulation of heart rate dynamics during systemic inflammation.
Sepsis caused by Gram-negative bacterial infection is characterized by extensive inflammatory cytokine production, which leads to multiple organ failure and a high lethality rate. Therefore, compounds that are able to alleviate profound inflammatory responses may have therapeutic potential in relation to sepsis. Quercetin, one of the flavonoids found widely in the human diet, has been reported to have many health benefits, but the mechanisms underlying its biological effects remain obscure. In the present study, our aim was to investigate the molecular mechanisms by which quercetin inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and to evaluate the capacity of quercetin to attenuate the mortality rate in a mice model of lethal sepsis. Our results show that quercetin significantly attenuates LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 macrophages. The LPS-stimulated phosphorylations of the inhibitors of κB kinase (IKKs), Akt, and c-Jun N-terminal kinase (JNK) are also inhibited by quercetin. Quercetin causes a significant reduction in the phosphorylation and degradation of inhibitor of κBα (IκBα) and in the nuclear level of nuclear factor-κB (NF-κB), the latter being associated with decreased NF-κB binding activity. Most importantly, acute administration of quercetin reduces the lethality rate and circulating levels of TNF-α and IL-1β in C57BL/6J mice with endotoxemia induced by LPS, whereas chronic dietary supplementation with quercetin shows no inhibitory effect on serum TNF-α and IL-1β levels. These findings provide clues that quercetin may be a promising agent for the prevention of systemic inflammatory diseases such as sepsis.
Inflammatory response during sepsis is incompletely understood due to small sample sizes and variable timing of measurements following the onset of symptoms. The vasopressin in septic shock trial (VASST) compared the addition of vasopressin to norepinephrine alone in patients with septic shock. During this study plasma was collected and 39 cytokines measured in a 363 patients at both baseline (before treatment) and 24 hours. Clinical features relating to both underlying health and the acute organ dysfunction induced by the severe infection were collected during the first 28 days of admission.
Cluster analysis of cytokines identifies subgroups of patients at differing risk of death and organ failure.
Circulating cytokines and other signaling molecules were measured using a Luminex multi-bead analyte detection system. Hierarchical clustering was performed on plasma values to create patient subgroups. Enrichment analysis identified clinical outcomes significantly different according to these chemically defined patient subgroups. Logistic regression was performed to assess the importance of cytokines for predicting patient subgroups.
Plasma levels at baseline produced three subgroups of patients, while 24 hour levels produced two subgroups. Using baseline cytokine data, one subgroup of 47 patients showed a high level of enrichment for severe septic shock, coagulopathy, renal failure, and risk of death. Using data at 24 hours, a larger subgroup of 81 patients that largely encompassed the 47 baseline subgroup patients had a similar enrichment profile. Measurement of two cytokines, IL2 and CSF2 and their product were sufficient to classify patients into these subgroups that defined clinical risks.
A distinct pattern of cytokine levels measured early in the course of sepsis predicts disease outcome. Subpopulations of patients have differing clinical outcomes that can be predicted accurately from small numbers of cytokines. Design of clinical trials and interventions may benefit from consideration of cytokine levels.
Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP−/−) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP−/− mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP−/− mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.
World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.
Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte apoptosis, indicating that it could be a potential target in clinical interventions for sepsis associated immunoparalysis.
The long pentraxin-3 (PTX3) is a key component of the humoral arm of the innate immune system. PTX3 is produced locally in response to pro-inflammatory stimuli. To investigate PTX3 levels and its use as a biomarker in patients with systemic inflammation, we developed a solid-phase enzyme-linked immunosorbent assay based on novel anti-PTX3 monoclonal antibodies detecting PTX3 with high sensitivity. The assay was applied on 261 consecutive patients admitted to an intensive care unit prospectively monitored with the systemic inflammatory response syndrome (SIRS). 100 blood donors were included as controls. PTX3 levels were elevated in patients (median = 71.3 ng/ml) compared with the controls (median = 0 ng/ml) (Mann-Whitney, p<0.0001). ROC analysis showed that PTX3 levels were significantly specific (85.0%) and sensitive (89.1%) to discriminate between healthy controls and patients (area under the curve (AUC) 0.922 (95% CI 0.892 to 0.946, p<0.0001)). Higher levels of PTX3 were associated with the development of sepsis, severe sepsis and septic shock (p = 0.0001). The serum levels of PTX3 correlated significantly with SAPS2 score (Spearman's rho 0.28, p<0.0001). Patients with high levels of PTX3 at admission did have a higher 90 day mortality rate than patients with the 25% lowest levels (Cox regression analysis, hazard ratio 3.0, p = 0.0009). In conclusion, we have established a highly sensitive and robust assay for measurement of PTX3 and found that its serum concentrations correlated with disease severity and mortality in patients with SIRS and sepsis.
We previously demonstrated that altered zinc homeostasis is an important feature of pediatric sepsis, thus raising the possibility of zinc supplementation as a therapeutic strategy in sepsis. Herein we tested the hypothesis that prophylactic zinc supplementation would be beneficial in a murine model of peritoneal sepsis.
Murine model of sepsis (intraperitoneal fecal slurry injection).
Basic science research laboratory.
C57BL/6 male mice.
Intraperitoneal fecal slurry injection, with or without zinc supplementation (10 mg/kg of intraperitoneal zinc gluconate for 3 days prior to CLP).
Survival over 3 days following CLP, markers of inflammation, bacterial load studies, and immuno-phenotyping studies.
Zinc-supplemented mice demonstrated a significant survival advantage compared to control (non-supplemented) mice. Zinc-supplemented mice also demonstrated moderate reductions of inflammation and immune activation. The survival advantage primarily correlated with reduced in vivo bacterial load in zinc-supplemented mice, compared to controls. In addition, peritoneal macrophages harvested from zinc-supplemented mice demonstrated a significantly enhanced phagocytosis capacity for E. coli and S. aureus, compared to peritoneal macrophages harvested from control mice.
Prophylactic zinc supplementation reduces bacterial load and is beneficial in a murine model of peritoneal sepsis.
Volatile anesthetics are known to have immunomodulatory effects in conditions of organ injury. A recent study in an experimental sepsis model has shown remarkably improved survival when mice were exposed to volatile anesthetics. In the present study, we show that hexafluoroisopropanol – a water-soluble primary sevoflurane metabolite – has beneficial effects on the overall survival in a murine model of cecal ligation and puncture. Seven-day survival as well as tissue damage markers including transaminases and high mobility group box protein-1 were assessed as measures of end organ damage. In animals undergoing cecal ligation and puncture procedure hexafluoroisopropanol conditioning - but not late postconditioning 24 hours after sepsis induction - significantly increased survival rate (17% vs. 77%, p = 0.037) and attenuated secretion of organ damage markers. This study shows survival benefits by administration of the metabolite of a volatile anesthetic. If successfully translated, hexafluoroisopropanol might offer interesting therapeutic opportunities in the future treatment of abdominal sepsis.
Background. Secondary hospital-acquired fungal infections are common in critically-ill patients and mortality remains high despite antimicrobial therapy. Interleukin-7 (IL-7) is a potent immunotherapeutic agent that improves host immunity and has shown efficacy in bacterial and viral models of infection. This study examined the ability of IL-7, which is currently in multiple clinical trials (including hepatitis and human immunodeficiency virus), to improve survival in a clinically relevant 2-hit model of fungal sepsis.
Methods. Mice underwent cecal ligation and puncture to induce peritonitis. Four days later, surviving mice had intravenous injection with Candida albicans. Following Candida infection, mice were treated with IL-7 or saline control. The effect of IL-7 on host immunity and survival was recorded.
Results. IL-7 ameliorated the loss of immune effector cells and increased lymphocyte functions, including activation, proliferation, expression of adhesion molecules, and interferon-γ production. These beneficial effects of IL-7 were associated with an increase in global immunity as reflected by an enhanced delayed type hypersensitivity response and a 1.7-fold improvement in survival.
Conclusions. The present findings showing that IL-7 improves survival in fungal sepsis, together with its previously reported efficacy in bacterial and viral infectious models, further supports its use as a novel immunotherapeutic in sepsis.
It is well known that complement system C5a is excessively activated during the onset of sepsis. However, it is unclear whether C5a can regulate dentritic cells (DCs) to stimulate adaptive immune cells such as Th1 and Th17 in sepsis.
Sepsis was induced by cecal ligation and puncture (CLP). CLP-induced sepsis was treated with anti-C5a or IL-12. IL-12+DC, IFNγ+Th1, and IL-17+Th17 cells were analyzed by flow cytometry. IL-12 was measured by ELISA.
Our studies here showed that C5a induced IL-12+DC cell migration from the peritoneal cavity to peripheral blood and lymph nodes. Furthermore, IL-12+DC cells induced the expansion of pathogenic IFNγ+Th1 and IL-17+Th17 cells in peripheral blood and lymph nodes. Moreover, IL-12, secreted by DC cells in the peritoneal cavity, is an important factor that prevents the development of sepsis.
Our data suggests that C5a regulates IL-12+DC cell migration to induce pathogenic Th1 and Th17 cells in sepsis.
The co-inhibitory immune receptor carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1) and its self-ligand CEACAM1 can suppress T cell function. Suppression of T cell function in sepsis is well documented. Late-onset neonatal sepsis in VLBW-infants was associated with an increased percentage CEACAM1 positive CD4+ T-cells. Meningococcal septic shock in children was associated with increased serum soluble CEACAM1. In conclusion our data demonstrate increased surface expression of the co-inhibitory immune receptor CEACAM1 in late-onset neonatal sepsis in VLBW-infants, and increased circulating soluble CEACAM1 in children with meningococcal sepsis. Increased T-cell CEACAM1 expression and increased circulating soluble CEACAM1 may contribute to sepsis-associated immune suppression.
Periodontal diseases are inflammatory processes that occur following the influx of neutrophils into the periodontal tissues in response to the subgingival bacterial biofilm. Current literature suggests that while neutrophils are protective and prevent bacterial infections, they also appear to contribute to damage of the periodontal tissues. In the present study we compare the gene expression profile changes in neutrophils as they migrate from the circulation into the oral tissues in patients with chronic periodontits and matched healthy subjects. We hypothesized that oral neutrophils in periodontal disease patients will display a disease specific transcriptome that differs from the oral neutrophil of healthy subjects.
Venous blood and oral rinse samples were obtained from healthy subjects and chronic periodontitis patients for neutrophil isolation. mRNA was isolated from the neutrophils, and gene expression microarray analysis was completed. Results were confirmed for specific genes of interest by qRT-PCR and Western Blot analysis.
Results and Discussion
Chronic periodontitis patients presented with increased recruitment of neutrophils to the oral cavity. Gene expression analysis revealed differences in the expression levels of genes from several biological pathways. Using hierarchical clustering analysis, we found that the apoptosis network was significantly altered in patients with chronic inflammation in the oral cavity, with up-regulation of pro-survival members of the Bcl-2 family and down-regulation of pro-apoptosis members in the same compartment. Additional functional analysis confirmed that the percentages of viable neutrophils are significantly increased in the oral cavity of chronic periodontitis patients.
Oral neutrophils from patients with periodontal disease displayed an altered transcriptome following migration into the oral tissues. This resulted in a pro-survival neutrophil phenotype in chronic periodontitis patients when compared with healthy subjects, resulting in a longer-lived neutrophil. This is likely to impact the severity and length of the inflammatory response in this oral disease.
The rapid development in septic patients of features of marked immunosuppression associated with increased risk of nosocomial infections and mortality represents the rational for the initiation of immune targeted treatments in sepsis. However, as there is no clinical sign of immune dysfunctions, the current challenge is to develop biomarkers that will help clinicians identify the patients that would benefit from immunotherapy and monitor its efficacy. Using an in vitro model of endotoxin tolerance (ET), a pivotal feature of sepsis-induced immunosuppression in monocytes, we identified using gene expression profiling by microarray a panel of transcripts associated with the development of ET which expression was restored after immunostimulation with interferon-gamma (IFN-γ). These results were confirmed by qRT-PCR. Importantly, this short-list of markers was further evaluated in patients. Of these transcripts, six (TNFAIP6, FCN1, CXCL10, GBP1, CXCL5 and PID1) were differentially expressed in septic patients’ blood compared to healthy blood upon ex vivo LPS stimulation and were restored by IFN-γ. In this study, by combining a microarray approach in an in vitro model and a validation in clinical samples, we identified a panel of six new transcripts that could be used for the identification of septic patients eligible for IFNg therapy. Along with the previously identified markers TNFa, IL10 and HLA-DRA, the potential value of these markers should now be evaluated in a larger cohort of patients. Upon favorable results, they could serve as stratification tools prior to immunostimulatory treatment and to monitor drug efficacy.
The development of hyperglycemia and the use of early parenteral feeding are associated with poor outcomes in critically ill patients. We therefore examined the impact of exogenous glucose administration on the integrated metabolic function of endotoxemic mice using our recently developed frequently sampled intravenous glucose tolerance test (FSIVGTT). We next extended our findings using a cecal ligation and puncture (CLP) sepsis model administered early parenteral glucose support.
Male C57BL/6J mice, 8-12 weeks, were instrumented with chronic indwelling arterial and venous catheters. Endotoxemia was initiated with intra-arterial lipopolysaccharide (LPS; 1 mg/kg) in the presence of saline or glucose infusion (100 µL/hr), and an FSIVGTT was performed after five hours. In a second experiment, catheterized mice underwent CLP and the impact of early parenteral glucose administration on glucose homeostasis and mortality was assessed over 24 hrs.
And MAIN RESULTS: Administration of LPS alone did not impair metabolic function, whereas glucose administration alone induced an insulin sensitive state. In contrast, LPS and glucose combined caused marked glucose intolerance and insulin resistance and significantly impaired pancreatic insulin secretion. Similarly, CLP mice receiving parenteral glucose developed fulminant hyperglycemia within 18 hrs (all > 600 mg/dl) associated with increased systemic cytokine release and 40% mortality, whereas CLP alone (85 ± 2 mg/dL) or sham mice receiving parenteral glucose (113 ± 3 mg/dL) all survived and were not hyperglycemic. Despite profound hyperglycemia, plasma insulin in the CLP glucose-infused mice (3.7 ± 1.2 ng/ml) was not higher than sham glucose infused mice (2.1 ± 0.3 ng/ml).
The combination of parenteral glucose support and the systemic inflammatory response in the acute phase of sepsis induces profound insulin resistance and impairs compensatory pancreatic insulin secretion, leading to the development of fulminant hyperglycemia.
We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8–2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3′-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.
DNA Methylation; NFATc1; chronic lymphocytic leukemia; next-generation sequencing; reduced representation bisulfite sequencing
In sepsis, the vitamin D active metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) may play a crucial role by its action to produce cathelicidin and improve endothelial barrier function, such that a deficiency in 1,25(OH)2D is associated with poor outcome. To test our hypothesis, we performed analysis of stored plasma samples from a prospective observational study in 91 patients with sepsis, age of 59.1+/−2.0 years, 52.7% females, and 11.0% deaths at 30 days. Vitamin D status, including 25-hydroxyvitamin D (25(OH)D), 1,25(OH)2D, 24,25-dihydroxyvitamin D (24,25(OH)2D), and parathyroid hormone (PTH), were measured daily over 3 days after hospital admission. At baseline, 1,25(OH)2D was significantly different between survivors vs. non-survivors. But there was no significant difference in 25(OH)D, 24,25(OH)2D, and PTH. In a multivariable binomial logistic regression model, age, total calcium and 1,25(OH)2D were significant predictors of 30-day mortality. Kaplan Meier analysis showed that patients with mean 1,25(OH)2D measured over 3 days of < = 13.6 pg/mL had 57.1% 30-day survival compared to 91.7% in patients with 1,25 (OH)2D level >13.6 pg/mL (p<0.01). From repeated measures regression analysis, there was significant increase in 1,25(OH)2D for increases in 25(OH)D in both survivors and non-survivors. However, compared to survivors, the low 25(OH)D in non-survivors was insufficient to account for the larger decrease in 1,25(OH)2D, indicating a dysfunctional 1α-hydroxylase. Additionally, there was a significant negative correlation between PTH and 1,25(OH)2D in both survivors and non-survivors, suggesting a severe impairment in the effect of PTH to increase renal 1α-hydroxylase activity. In conclusion, low 1,25(OH)2D levels are associated with increased 30-day mortality in sepsis patients, likely due to impaired 25(OH)D hydroxylation and PTH insensitivity. Our data also suggest that the active metabolite 1,25(OH)2D may be an important therapeutic target in the design of sepsis clinical trials.
Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice.
Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival.
Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology.
When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and pancreatic metabolomics may help explain this differential response.
Anandamide (AEA) is the prominent member of the endocannabinoid family and its biological action is mediated through the binding to both type-1 (CB1) and type-2 (CB2) cannabinoid receptors (CBR). The presence of AEA and CBR in the gastrointestinal tract highlighted their pathophysiological role in several gut diseases, including celiac disease. Here, we aimed to investigate the expression of CBR at transcriptional and translational levels in the duodenal mucosa of untreated celiac patients, celiac patients on a gluten-free diet for at least 12 months and control subjects. Also biopsies from treated celiac patients cultured ex vivo with peptic-tryptic digest of gliadin were investigated. Our data show higher levels of both CB1 and CB2 receptors during active disease and normal CBR levels in treated celiac patients. In conclusion, we demonstrate an up-regulation of CB1 and CB2 mRNA and protein expression, that points to the therapeutic potential of targeting CBR in patients with celiac disease.
The spatial conformation of a genome plays an important role in the long-range regulation of genome-wide gene expression and methylation, but has not been extensively studied due to lack of genome conformation data. The recently developed chromosome conformation capturing techniques such as the Hi-C method empowered by next generation sequencing can generate unbiased, large-scale, high-resolution chromosomal interaction (contact) data, providing an unprecedented opportunity to investigate the spatial structure of a genome and its applications in gene regulation, genomics, epigenetics, and cell biology. In this work, we conducted a comprehensive, large-scale computational analysis of this new stream of genome conformation data generated for three different human leukemia cells or cell lines by the Hi-C technique. We developed and applied a set of bioinformatics methods to reliably generate spatial chromosomal contacts from high-throughput sequencing data and to effectively use them to study the properties of the genome structures in one-dimension (1D) and two-dimension (2D). Our analysis demonstrates that Hi-C data can be effectively applied to study tissue-specific genome conformation, chromosome-chromosome interaction, chromosomal translocations, and spatial gene-gene interaction and regulation in a three-dimensional genome of primary tumor cells. Particularly, for the first time, we constructed genome-scale spatial gene-gene interaction network, transcription factor binding site (TFBS) – TFBS interaction network, and TFBS-gene interaction network from chromosomal contact information. Remarkably, all these networks possess the properties of scale-free modular networks.
Radiation combined injury (CI) is a radiation injury (RI) combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP) is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg p.o., q.d. within 2 h after CI through day 10). At day 10, CIP treatment not only significantly reduced pro-inflammatory cytokine and chemokine concentrations, including interleukin-6 (IL-6) and KC (i.e., IL-8 in human), but it also enhanced IL-3 production compared to vehicle-treated controls. Mice treated with CIP displayed a greater repopulation of bone marrow cells. CIP also limited CI-induced apoptosis and autophagy in ileal villi, systemic bacterial infection, and IgA production. CIP treatment led to LD0/10 compared to LD20/10 for vehicle-treated group after CI. Given the multiple beneficial activities of CIP shown in our experiments, CIP may prove to be a useful therapeutic drug for CI.
Dendritic cells (DCs) are major modulators in the immune system. One active field of research is the manipulation of DCs as pharmacological targets to screen novel biological modifiers for the treatment of inflammatory and autoimmune disorders. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. We assessed the capability of acetylcorynoline to regulate lipopolysaccharide (LPS)-stimulated activation of mouse bone marrow-derived DCs.
Our experimental data showed that treatment with up to 20 µM acetylcorynoline does not cause cytotoxicity in cells. Acetylcorynoline significantly inhibited the secretion of tumor necrosis factor-α, interleukin-6, and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility complex class II, CD40, and CD86 on DCs was also decreased by acetylcorynoline, and the endocytic capacity of LPS-stimulated DCs was restored by acetylcorynoline. In addition, LPS-stimulated DC-elicited allogeneic T-cell proliferation was blocked by acetylcorynoline, and the migratory ability of LPS-stimulated DCs was reduced by acetylcorynoline. Moreover, acetylcorynoline significantly inhibits LPS-induced activation of IκB kinase and mitogen-activated protein kinase. Importantly, administration of acetylcorynoline significantly attenuates 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity.
Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of DC maturation and function.
Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes.
Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre- and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP (this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice predicted to die (P-DIE) and to live (P-LIVE).
In experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1 mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice. Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2–4-fold higher than in SUR. PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE mice and those increases closely correlated with liver dysfunction.
Trauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin and was independent of endothelial injury.