Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease.

Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

Mice Fed an Obesogenic Western Diet, Administered Antibiotics, and Subjected to a Sterile Surgical Procedure Develop Lethal Septicemia with Multidrug-Resistant Pathobionts.

Despite antibiotics and sterile technique, postoperative infections remain a real and present danger to patients. Recent estimates suggest that 50% of the pathogens associated with postoperative infections have become resistant to the standard antibiotics used for prophylaxis. Risk factors identified in such cases include obesity and antibiotic exposure. To study the combined effect of obesity and antibiotic exposure on postoperative infection, mice were allowed to gain weight on an obesogenic Western-type diet (WD), administered antibiotics and then subjected to an otherwise recoverable sterile surgical injury (30% hepatectomy). The feeding of a WD alone resulted in a major imbalance of the cecal microbiota characterized by a decrease in diversity, loss of Bacteroidetes, a bloom in Proteobacteria, and the emergence of antibiotic-resistant organisms among the cecal microbiota. When WD-fed mice were administered antibiotics and subjected to 30% liver resection, lethal sepsis, characterized by multiple-organ damage, developed. Notable was the emergence and systemic dissemination of multidrug-resistant (MDR) pathobionts, including carbapenem-resistant, extended-spectrum ß-lactamase-producing Serratia marcescens, which expressed a virulent and immunosuppressive phenotype. Analysis of the distribution of exact sequence variants belonging to the genus Serratia suggested that these strains originated from the cecal mucosa. No mortality or MDR pathogens were observed in identically treated mice fed a standard chow diet. Taken together, these results suggest that consumption of a Western diet and exposure to certain antibiotics may predispose to life-threating postoperative infection associated with MDR organisms present among the gut microbiota.IMPORTANCE Obesity remains a prevalent and independent risk factor for life-threatening infection following major surgery. Here, we demonstrate that when mice are fed an obesogenic Western diet (WD), they become susceptible to lethal sepsis with multiple organ damage after exposure to antibiotics and an otherwise-recoverable surgical injury. Analysis of the gut microbiota in this model demonstrates that WD alone leads to loss of Bacteroidetes, a bloom of Proteobacteria, and evidence of antibiotic resistance development even before antibiotics are administered. After antibiotics and surgery, lethal sepsis with organ damage developed in in mice fed a WD with the appearance of multidrug-resistant pathogens in the liver, spleen, and blood. The importance of these findings lies in exposing how the selective pressures of diet, antibiotic exposure, and surgical injury can converge on the microbiome, resulting in lethal sepsis and organ damage without the introduction of an exogenous pathogen.

Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host.

Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.

A novel topoisomerase 2a inhibitor, cryptotanshinone, suppresses the growth of PC3 cells without apparent cytotoxicity.

DNA topoisomerase 2, which is ubiquitously expressed in eukaryotic cells, is an essential nuclear enzyme that promotes cell survival by regulating DNA topology and chromatid separation. This enzyme has been validated as a target for anticancer agent screening. It can be poisoned by common chemotherapeutics, such as etoposide and doxorubicin, which leads to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. However, recent studies have suggested that the topoisomerase 2a isozyme is predominantly responsible for the carcinogenic side effects associated with etoposide and doxorubicin chemotherapy. Thus, we need to find a promising topoisomerase 2-targeting anticancer agent that avoids these carcinogenic side effects. Recent studies have found that cryptotanshinone has obvious anticancer activities against diverse cancer cells. Here, we demonstrate that cryptotanshinone markedly decreases the steady-state mRNA level of topoisomerase 2a, thereby decreasing the protein and activity levels of this enzyme. Moreover, cryptotanshinone exhibited dramatic in vitro and in vivo antitumor activity with low toxicity to normal tissues. Collectively, our findings support the development of cryptotanshinone as a promising candidate for treating cancer by targeting topoisomerase 2a.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Interleukin-15 promotes intestinal dysbiosis with butyrate deficiency associated with increased susceptibility to colitis.

Dysbiosis resulting in gut-microbiome alterations with reduced butyrate production are thought to disrupt intestinal immune homeostasis and promote complex immune disorders. However, whether and how dysbiosis develops before the onset of overt pathology remains poorly defined. Interleukin-15 (IL-15) is upregulated in distressed tissue and its overexpression is thought to predispose susceptible individuals to and have a role in the pathogenesis of celiac disease and inflammatory bowel disease (IBD). Although the immunological roles of IL-15 have been largely studied, its potential impact on the microbiota remains unexplored. Analysis of 16S ribosomal RNA-based inventories of bacterial communities in mice overexpressing IL-15 in the intestinal epithelium (villin-IL-15 transgenic (v-IL-15tg) mice) shows distinct changes in the composition of the intestinal bacteria. Although some alterations are specific to individual intestinal compartments, others are found across the ileum, cecum and feces. In particular, IL-15 overexpression restructures the composition of the microbiota with a decrease in butyrate-producing bacteria that is associated with a reduction in luminal butyrate levels across all intestinal compartments. Fecal microbiota transplant experiments of wild-type and v-IL-15tg microbiota into germ-free mice further indicate that diminishing butyrate concentration observed in the intestinal lumen of v-IL-15tg mice is the result of intrinsic alterations in the microbiota induced by IL-15. This reconfiguration of the microbiota is associated with increased susceptibility to dextran sodium sulfate-induced colitis. Altogether, this study reveals that IL-15 impacts butyrate-producing bacteria and lowers butyrate levels in the absence of overt pathology, which represent events that precede and promote intestinal inflammatory diseases.

Aberrant DNA methylation-induced gene inactivation is associated with the diagnosis and/or therapy of T-cell leukemias.

Aberrant hypermethylation of tumor suppressor genes is known to play an important role in the development of many tumors, and aberrant DNA hypermethylation was recently identified in hematologic malignancies, where it is thought to hold relevance in leukemogenesis. Here, we report that there are differences in the DNA methylation patterns seen in normal peripheral blood and two T-cell leukemia cell lines. We identify nine genes (CLEC4E, CR1, DBC1, EPO, HAL-DOA, IGF2, IL12B, ITGA1, and LMX1B) that are significantly hypermethylated in T-cell leukemias cell lines, and suggest that aberrant hypermethylation of these normally unmethylated genes may induce their transcriptional and expressional silencing. Furthermore, we observed that the expression levels of DNMT1 and DNMT3a were significantly decreased by 5-aza-2'-deoxycytidine (5-Aza-dC), which is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells and restore the expression levels of their target genes in Jurkat cells. This result suggests that the overexpression of DNMTs could contribute to the development of T-cell leukemias by inducing hypermethylation of the target genes. Together, our results show that aberrant hypermethylation is an important molecular mechanism in the progression of T-cell leukemias, and thus could prove useful as a prognostic and/or diagnostic marker. Moreover, 5-Aza-dC might be a promising candidate for the treatment of T-cell leukemia.

Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients.

OBJECTIVES: Celiac disease (CD)-associated duodenal dysbiosis has not yet been clearly defined, and the mechanisms by which CD-associated dysbiosis could concur to CD development or exacerbation are unknown. In this study, we analyzed the duodenal microbiome of CD patients. METHODS: The microbiome was evaluated in duodenal biopsy samples of 20 adult patients with active CD, 6 CD patients on a gluten-free diet, and 15 controls by DNA sequencing of 16S ribosomal RNA libraries. Bacterial species were cultured, isolated and identified by mass spectrometry. Isolated bacterial species were used to infect CaCo-2 cells, and to stimulate normal duodenal explants and cultured human and murine dendritic cells (DCs). Inflammatory markers and cytokines were evaluated by immunofluorescence and ELISA, respectively. RESULTS: Proteobacteria was the most abundant and Firmicutes and Actinobacteria the least abundant phyla in the microbiome profiles of active CD patients. Members of the Neisseria genus (Betaproteobacteria class) were significantly more abundant in active CD patients than in the other two groups (P=0.03). Neisseria flavescens (CD-Nf) was the most abundant Neisseria species in active CD duodenum. Whole-genome sequencing of CD-Nf and control-Nf showed genetic diversity of the iron acquisition systems and of some hemoglobin-related genes. CD-Nf was able to escape the lysosomal compartment in CaCo-2 cells and to induce an inflammatory response in DCs and in ex-vivo mucosal explants. CONCLUSIONS: Marked dysbiosis and an abundance of a peculiar CD-Nf strain characterize the duodenal microbiome in active CD patients thus suggesting that the CD-associated microbiota could contribute to the many inflammatory signals in this disorder.

Innate immunity: actuating the gears of celiac disease pathogenesis.

Celiac disease is a T cell mediated immune disorder characterized by the loss of oral tolerance to dietary gluten and the licensing of intraepithelial lymphocytes to kill intestinal epithelial cells, leading to villous atrophy. Innate immunity plays a critical role in both of these processes and cytokines such as interleukin-15 and interferon-α can modulate innate processes such as polarization of dendritic cells as well as intraepithelial lymphocyte function. These cytokines can be modulated by host microbiota, which can also influence dendritic cell function and intraepithelial lymphocyte homeostasis. We will elaborate on the role of interleukin-15, interferon-α, and the microbiota in modulating the processes that lead to loss of tolerance to gluten and tissue destruction in celiac disease.

Radioresistance in a human laryngeal squamous cell carcinoma cell line is associated with DNA methylation changes and topoisomerase II α.

Accumulating evidence suggests that changes in methylation patterns may help mediate the sensitivity or resistance of cancer cells to ionizing radiation. The present study provides evidence for the involvement of radioresistance-induced DNA methylation changes in tumor radioresistance. We established radioresistant laryngeal cancer cells via long-term fractionated irradiation, and examined differences in DNA methylation between control and radioresistant laryngeal cancer cells. Interestingly, we found that the promoter-CpG islands of 5 previously identified radioresistance-related genes (TOPO2A, PLXDC2, ETNK2, GFI1, and IL12B) were significantly altered in the radioresistant laryngeal cancer cells. Furthermore, the demethylation of these gene promoters with a DNA methyltransferase inhibitor (5-aza-2'-deoxycytidine) increased their transcription levels. Treatment with 5-aza-2'-deoxycytidine also sensitized the radioresistant laryngeal cancer cells to irradiation, indicating that changes in DNA methylation contributed to their radioresistance. Of the tested genes, the expression and activity levels of TOPO2A were tightly associated with the radioresistant phenotype in our system, suggesting that the hypermethylation of TOPO2A might be involved in this radioresistance. Collectively, our data suggest that radiation-induced epigenetic changes can modulate the radioresistance of laryngeal cancer cells, and thus may prove useful as prognostic indicators for radiotherapy.

Phosphate-containing polyethylene glycol polymers prevent lethal sepsis by multidrug-resistant pathogens.

Antibiotic resistance among highly pathogenic strains of bacteria and fungi is a growing concern in the face of the ability to sustain life during critical illness with advancing medical interventions. The longer patients remain critically ill, the more likely they are to become colonized by multidrug-resistant (MDR) pathogens. The human gastrointestinal tract is the primary site of colonization of many MDR pathogens and is a major source of life-threatening infections due to these microorganisms. Eradication measures to sterilize the gut are difficult if not impossible and carry the risk of further antibiotic resistance. Here, we present a strategy to contain rather than eliminate MDR pathogens by using an agent that interferes with the ability of colonizing pathogens to express virulence in response to host-derived and local environmental factors. The antivirulence agent is a phosphorylated triblock high-molecular-weight polymer (here termed Pi-PEG 15-20) that exploits the known properties of phosphate (Pi) and polyethylene glycol 15-20 (PEG 15-20) to suppress microbial virulence and protect the integrity of the intestinal epithelium. The compound is nonmicrobiocidal and appears to be highly effective when tested both in vitro and in vivo. Structure functional analyses suggest that the hydrophobic bis-aromatic moiety at the polymer center is of particular importance to the biological function of Pi-PEG 15-20, beyond its phosphate content. Animal studies demonstrate that Pi-PEG prevents mortality in mice inoculated with multiple highly virulent pathogenic organisms from hospitalized patients in association with preservation of the core microbiome.

Mitogen-activated protein kinase phosphatase-1 inhibition and sustained extracellular signal-regulated kinase 1/2 activation in camptothecin-induced human colon cancer cell death.

Camptothecins are commonly used chemotherapeutics; in some models, they enhance signaling via the mitogen-activated protein kinase (MAPK) pathway through effects on upstream kinases. To evaluate the impact of camptothecin (CPT) on MAPKs in human colon cancer, we studied HCT116 and CaCo2 colon cancer cells. We found that HCT116 cells highly express mitogen-activated protein kinase phosphatase-1 (MKP1), which selectively inactivates extracellular signal-regulated kinase (ERK), whereas MKP1 levels were undetectable in CaCo2 cells. CPT did not affect ERK activity in CaCo2 cells, but did induce a striking increase in ERK activity in HCT116 cells in association with a corresponding decrease in MKP1. The reduction in MKP1 expression occurred at a posttranscriptional level and was blocked by the proteasome inhibitor MG132, whereas that CPT-induced downregulation of MKP1 was not due to proteasome-mediated degradation. Treatment of HCT116 cells with CPT induced a sustained activation of nuclear ERK, which was required for CPT-induced apoptosis. P38 and JNK activity were unaffected by CPT, suggesting that the effects of CPT are mediated specifically by ERK. These results suggest that targeting dual-specificity MAPK phosphatases in colon cancer cells may be a viable strategy for optimizing camptothecin-based therapeutic protocols.

InkTag: Secure Applications on an Untrusted Operating System.

InkTag is a virtualization-based architecture that gives strong safety guarantees to high-assurance processes even in the presence of a malicious operating system. InkTag advances the state of the art in untrusted operating systems in both the design of its hypervisor and in the ability to run useful applications without trusting the operating system. We introduce paraverification, a technique that simplifies the InkTag hypervisor by forcing the untrusted operating system to participate in its own verification. Attribute-based access control allows trusted applications to create decentralized access control policies. InkTag is also the first system of its kind to ensure consistency between secure data and metadata, ensuring recoverability in the face of system crashes.

Different response of body weight change according to ketonuria after fasting in the healthy obese.

The relationship between obesity and ketonuria is not well-established. We conducted a retrospective observational study to evaluate whether their body weight reduction response differed by the presence of ketonuria after fasting in the healthy obese. We used the data of 42 subjects, who had medical records of initial urinalysis at routine health check-up and follow-up urinalysis in the out-patient clinic, one week later. All subjects in the initial urinalysis showed no ketonuria. However, according to the follow-up urinalysis after three subsequent meals fasts, the patients were divided into a non-ketonuria group and ketonuria group. We compared the data of conventional low-calorie diet programs for 3 months for both groups. Significantly greater reduction of body weight (-8.6 ± 3.6 kg vs -1.1 ± 2.2 kg, P < 0.001), body mass index (-3.16 ± 1.25 kg/m(2) vs -0.43 ± 0.86 kg/m(2), P < 0.001) and waist circumference (-6.92 ± 1.22 vs -2.32 ± 1.01, P < 0.001) was observed in the ketonuria group compared to the non-ketonuria group. Fat mass and lean body mass were also more reduced in the ketonuria group. In addition, serum free fatty acid concentration after intervention in the ketonuria group showed significant more increment than in the non-ketonuria group. The presence of ketonuria after fasting may be a predicting factor of further body weight reduction.

Eternal Sunshine of the Spotless Machine: Protecting Privacy with Ephemeral Channels.

Modern systems keep long memories. As we show in this paper, an adversary who gains access to a Linux system, even one that implements secure deallocation, can recover the contents of applications' windows, audio buffers, and data remaining in device drivers-long after the applications have terminated. We design and implement Lacuna, a system that allows users to run programs in "private sessions." After the session is over, all memories of its execution are erased. The key abstraction in Lacuna is an ephemeral channel, which allows the protected program to talk to peripheral devices while making it possible to delete the memories of this communication from the host. Lacuna can run unmodified applications that use graphics, sound, USB input devices, and the network, with only 20 percentage points of additional CPU utilization.

Changes of body weight and inflammatory markers after 12-week intervention trial: results of a double-blind, placebo-control pilot study.

PURPOSE: Low grade inflammation is a well-known characteristic in obese subjects. We investigated body weight changes and inflammatory markers after 12-week intervention trial. MATERIALS AND METHODS: Twenty-six obese subjects were enrolled and 19 (13 men and 6 women) completed the study. Sibutramine is an FDA-approved drug for body weight control; therefore, we chose this drug as the standard treatment medication in this study. Patients were randomly allocated to receive an anti-inflammatory agent (Diacerein treatment group; n = 12) or placebo (n = 7) for 12 weeks. Anthropometry, body proportion by dual-energy X-ray absorptiometry, and metabolic parameters at the beginning and end of study were measured and compared. RESULTS: The treatment group had a tendency towards more reduction in anthropometry as compared to the placebo group, in body weight reduction (-7.0 kg vs. -4.6 kg), body mass index (-2.51 kg/m² vs. -1.59 kg/m²), and waist circumference (-7.3 cm vs. -4.4 cm). These reductions were not statistically significant. Changes in levels of high-sensitivity C-reactive protein and adiponectin in the treatment group were more favorable than in the placebo group. CONCLUSION: This small pilot study showed no statistical difference for changes in anthropometry, and inflammatory markers between the two groups. Therefore, we could not find any additional effects of Diacerein on weight loss and inflammatory variables in this study.

Ketonuria after fasting may be related to the metabolic superiority.

Obese individuals are less able to oxidize fat than non-obese individuals. Caloric reduction or fasting can detect ketonuria. We investigated the differences of metabolic parameters in the presence of ketonuria after a minimum 8 hr fast in a cross-sectional analysis of 16,523 Koreans (6,512 women and 10,011 men). The relationship between the presence of ketonuria of all subjects and prevalence of obesity, central obesity, metabolic syndrome, and obesity-related metabolic parameters were assessed. The ketonuria group had lower prevalence of obesity, central obesity, and metabolic syndrome than the non-ketonuria group. In addition, all metabolic parameters (including body weight, waist circumference, blood glucose, high-density lipoprotein, triglyceride, blood pressure, and insulin) were favorable in the ketonuria group than in the non-ketonuria group, even after adjustment for age, tobacco use, and alcohol consumption. The odds ratios of having obesity (odds ratio [OR]=1.427 in women, OR=1.582 in men, P<0.05), central obesity (OR=1.675 in women, OR=1.889 in men, P<0.05), and metabolic syndrome (OR=3.505 in women, OR=1.356 in men, P<0.05) were increased in the non-ketonuria group compared to the ketonuria group. The presence of ketonuria after at least an 8 hr fast may be indicative of metabolic superiority.