Extreme Dysbiosis of the Microbiome in Critical Illness.

Critical illness is hypothesized to associate with loss of "health-promoting" commensal microbes and overgrowth of pathogenic bacteria (dysbiosis). This dysbiosis is believed to increase susceptibility to nosocomial infections, sepsis, and organ failure. A trial with prospective monitoring of the intensive care unit (ICU) patient microbiome using culture-independent techniques to confirm and characterize this dysbiosis is thus urgently needed. Characterizing ICU patient microbiome changes may provide first steps toward the development of diagnostic and therapeutic interventions using microbiome signatures. To characterize the ICU patient microbiome, we collected fecal, oral, and skin samples from 115 mixed ICU patients across four centers in the United States and Canada. Samples were collected at two time points: within 48 h of ICU admission, and at ICU discharge or on ICU day 10. Sample collection and processing were performed according to Earth Microbiome Project protocols. We applied SourceTracker to assess the source composition of ICU patient samples by using Qiita, including samples from the American Gut Project (AGP), mammalian corpse decomposition samples, childhood (Global Gut study), and house surfaces. Our results demonstrate that critical illness leads to significant and rapid dysbiosis. Many taxons significantly depleted from ICU patients versus AGP healthy controls are key "health-promoting" organisms, and overgrowth of known pathogens was frequent. Source compositions of ICU patient samples are largely uncharacteristic of the expected community type. Between time points and within a patient, the source composition changed dramatically. Our initial results show great promise for microbiome signatures as diagnostic markers and guides to therapeutic interventions in the ICU to repopulate the normal, "health-promoting" microbiome and thereby improve patient outcomes. IMPORTANCE Critical illness may be associated with the loss of normal, "health promoting" bacteria, allowing overgrowth of disease-promoting pathogenic bacteria (dysbiosis), which, in turn, makes patients susceptible to hospital-acquired infections, sepsis, and organ failure. This has significant world health implications, because sepsis is becoming a leading cause of death worldwide, and hospital-acquired infections contribute to significant illness and increased costs. Thus, a trial that monitors the ICU patient microbiome to confirm and characterize this hypothesis is urgently needed. Our study analyzed the microbiomes of 115 critically ill subjects and demonstrated rapid dysbiosis from unexpected environmental sources after ICU admission. These data may provide the first steps toward defining targeted therapies that correct potentially "illness-promoting" dysbiosis with probiotics or with targeted, multimicrobe synthetic "stool pills" that restore a healthy microbiome in the ICU setting to improve patient outcomes

Alterations in Content and Localization of Defensins in Rat Ileum and Jejunum Following Ischemia-Reperfusion. Specific Peptides, in Specific Places, for Specific Jobs?

Objective: To determine alterations in quantities and distributions of natural antimicrobials following ischemia-reperfusion injury. We hypothesized that these compounds would be upregulated in areas of small intestine where changes in permeability and cellular disruption were likely and where protective mechanisms would be initiated. Methods: Rats with ischemia-reperfusion underwent superior mesenteric artery clamping and reperfusion. Shams were subjected to laparotomy but no clamping. Ileum and jejunum were harvested and sectioned, and subjected to fluorescence deconvolution microscopy for determinations of content and localization of rat beta defensins, 1, 2, 3; rat neutrophil protein-1; and cathelicidin LL-37. Modeling was performed to determine cellular location of antimicrobials. Results: Ischemia-reperfusion increased neutrophil defensin alpha (RNP-1) in jejunum; rat beta defensin 1 was increased 2-fold in ileal mucosa and slightly reduced in jejunal mucosa; rat beta defensin 2 was reduced by ischemia-reperfusion in ileum, but slightly increased in jejunum; rat beta defensin 3 was concentrated in the muscularis externa and myenteric plexus of the jejunum; ischemia-reperfusion did not alter cathelicidin LL-37 content in the small intestine, although a greater concentration was seen in jejunum compared with ileum. Conclusion: Ischemia-reperfusion injury caused changes in antimicrobial content in defined areas, and these different regulations might reflect the specific roles of jejunum versus ileum

The Splenic Injury Outcomes Trial: An American Association For the Surgery of Trauma Multi-Institutional Study

BACKGROUND: Delayed splenic hemorrhage after nonoperative management (NOM) of blunt splenic injury (BSI) is a feared complication, particularly in the outpatient setting. Significant resources, including angiography (ANGIO), are used in an effort to prevent delayed splenectomy (DS). No prospective, long-term data exist to determine the actual risk of splenectomy. The purposes of this trial were to ascertain the 180-day risk of splenectomy after 24 hours of NOM of BSI and to determine factors related to splenectomy. METHODS: Eleven Level I trauma centers participated in this prospective observational study. Adult patients achieving 24 hours of NOM of their BSI were eligible. Patients were followed up for 180 days. Demographic, physiologic, radiographic, injury-related information, and spleen-related interventions were recorded. Bivariate and multivariable analyses were used to determine factors associated with DS. RESULTS: A total of 383 patients were enrolled. Twelve patients (3.1%) underwent in-hospital splenectomy between 24 hours and 9 days after injury. Of 366 discharged with a spleen, 1 (0.27%) required readmission for DS on postinjury Day 12. No Grade I injuries experienced DS. The splenectomy rate after 24 hours of NOM was 1.5 per 1,000 patient-days. Only extravasation from the spleen at time of admission (ADMIT-BLUSH) was associated with splenectomy (odds ratio, 3.6; 95% confidence interval, 1.4–12.4). Of patients with ADMIT-BLUSH (n = 49), 17 (34.7%) did not have ANGIO with embolization (EMBO), and 2 of those (11.8%) underwent splenectomy; 32 (65.3%) underwent ANGIO with EMBO, and 2 of those (6.3%, p = 0.6020 compared with no ANGIO with EMBO) required splenectomy. CONCLUSION: Splenectomy after 24 hours of NOM is rare. After the initial 24 hours, no additional interventions are warranted for patients with Grade I injuries. For Grades II to V, close observation as an inpatient or outpatient is indicated for 10 days to 14 days. ADMIT-BLUSH is a strong predictor of DS and should lead to close observation or earlier surgical intervention. LEVEL OF EVIDENCE: Prognostic/epidemiological study, level III; therapeutic study, level IV

Clinically Practical Approach for Screening of Low Muscularity Using Electronic Linear Measures on Computed Tomography Images in Critically Ill Patients

This is the peer reviewed version of the following article: Avrutin, E., Moisey, L. L., Zhang, R., Khattab, J., Todd, E., Premji, T., Kozar, R., Heyland, D. K., &amp; Mourtzakis, M. (2017). Clinically practical approach for screening of low muscularity using electronic linear measures on computed tomography images in critically ill patients. Journal of Parenteral and Enteral Nutrition, 42(5), 885–891, which has been published in final form at https://doi.org/10.1002/jpen.1019. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.Background: Computed tomography (CT) scans performed during routine hospital care offer the opportunity to quantify skeletal muscle and predict mortality and morbidity in intensive care unit (ICU) patients. Existing methods of muscle cross-sectional area (CSA) quantification require specialized software, training, and time commitment that may not be feasible in a clinical setting. In this article, we explore a new screening method to identify patients with low muscle mass. Methods: We analyzed 145 scans of elderly ICU patients (≥65 years old) using a combination of measures obtained with a digital ruler, commonly found on hospital radiological software. The psoas and paraspinal muscle groups at the level of the third lumbar vertebra (L3) were evaluated by using 2 linear measures each and compared with an established method of CT image analysis of total muscle CSA in the L3 region. Results: There was a strong association between linear measures of psoas and paraspinal muscle groups and total L3 muscle CSA (R2 = 0.745, P < 0.001). Linear measures, age, and sex were included as covariates in a multiple logistic regression to predict those with low muscle mass; receiver operating characteristic (ROC) area under the curve (AUC) of the combined psoas and paraspinal linear index model was 0.920. Intraclass correlation coefficients (ICCs) were used to evaluate intrarater and interrater reliability, resulting in scores of 0.979 (95% CI: 0.940-0.992) and 0.937 (95% CI: 0.828-0.978), respectively. Conclusions: A digital ruler can reliably predict L3 muscle CSA, and these linear measures may be used to identify critically ill patients with low muscularity who are at risk for worse clinical outcomes

Modulation of Syndecan-1 Shedding after Hemorrhagic Shock and Resuscitation

The early use of fresh frozen plasma as a resuscitative agent after hemorrhagic shock has been associated with improved survival, but the mechanism of protection is unknown. Hemorrhagic shock causes endothelial cell dysfunction and we hypothesized that fresh frozen plasma would restore endothelial integrity and reduce syndecan-1 shedding after hemorrhagic shock. A prospective, observational study in severely injured patients in hemorrhagic shock demonstrated significantly elevated levels of syndecan-1 (554±93 ng/ml) after injury, which decreased with resuscitation (187±36 ng/ml) but was elevated compared to normal donors (27±1 ng/ml). Three pro-inflammatory cytokines, interferon-γ, fractalkine, and interleukin-1β, negatively correlated while one anti-inflammatory cytokine, IL-10, positively correlated with shed syndecan-1. These cytokines all play an important role in maintaining endothelial integrity. An in vitro model of endothelial injury then specifically examined endothelial permeability after treatment with fresh frozen plasma orlactated Ringers. Shock or endothelial injury disrupted junctional integrity and increased permeability, which was improved with fresh frozen plasma, but not lactated Ringers. Changes in endothelial cell permeability correlated with syndecan-1 shedding. These data suggest that plasma based resuscitation preserved endothelial syndecan-1 and maintained endothelial integrity, and may help to explain the protective effects of fresh frozen plasma after hemorrhagic shock

Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury

Protective role of p70S6K in intestinal ischemia/reperfusion injury in mice.

The mTOR signaling pathway plays a crucial role in the regulation of cell growth, proliferation, survival and in directing immune responses. As the intestinal epithelium displays rapid cell growth and differentiation and is an important immune regulatory organ, we hypothesized that mTOR may play an important role in the protection against intestinal ischemia reperfusion (I/R)-induced injury. To better understand the molecular mechanisms by which the mTOR pathway is altered by intestinal I/R, p70S6K, the major effector of the mTOR pathway, was investigated along with the effects of rapamycin, a specific inhibitor of mTOR and an immunosuppressant agent used clinically in transplant patients. In vitro experiments using an intestinal epithelial cell line and hypoxia/reoxygenation demonstrated that overexpression of p70S6K promoted cell growth and migration, and decreased cell apoptosis. Inhibition of p70S6K by rapamycin reversed these protective effects. In a mouse model of gut I/R, an increase of p70S6K activity was found by 5 min and remained elevated after 6 h of reperfusion. Inhibition of p70S6K by rapamycin worsened gut injury, promoted inflammation, and enhanced intestinal permeability. Importantly, rapamycin treated animals had a significantly increased mortality. These novel results demonstrate a key role of p70S6K in protection against I/R injury in the intestine and suggest a potential danger in using mTOR inhibitors in patients at risk for gut hypoperfusion

Inhibition of ERK1/2 worsens intestinal ischemia/reperfusion injury.

The role of extracellular signal-regulated protein kinase (ERK) in intestinal ischemia/reperfusion (I/R) injury has not been well investigated. The aim of the current study was to examine the effect of inhibition of the ERK pathway in an in vitro and in vivo model of intestinal I/R injury.ERK1/2 activity was inhibited using the specific inhibitor, U0126, in intestinal epithelial cells under hypoxia/reoxygenation conditions and in mice subjected to 1 hour of intestinal ischemia followed by 6 hours reperfusion. In vitro, cell proliferation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, apoptosis by DNA fragmentation, and migration using an in vitro model of intestinal wound healing. Cells were also transfected with a p70S6K plasmid and the effects of overexpression similarly analyzed. In vivo, the effects of U0126 on intestinal cell proliferation and apoptosis, intestinal permeability, lung and intestinal neutrophil infiltration and injury, and plasma cytokine levels were measured. Survival was also assessed after U0126. Activity of p70S6 kinase (p70S6K) was measured by Western blot.In vitro, inhibition of ERK1/2 by U0126 significantly decreased cell proliferation and migration but enhanced cell apoptosis. Overexpression of p70S6K promoted cell proliferation and decreased cell apoptosis. In vivo, U0126 significantly increased cell apoptosis and decreased cell proliferation in the intestine, increased intestinal permeability, intestinal and lung neutrophil infiltration, and injury, as well as systemic pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Mortality was also significantly increased by U0126. Inhibition of ERK1/2 by U0126 also abolished activity of p70S6K both in vitro and in vivo models.Pharmacologic inhibition of ERK1/2 by U0126 worsens intestinal IR injury. The detrimental effects are mediated, at least in part, by inhibition of p70S6K, the major effector of mammalian target of rapamycin pathway

Stimulation of p70S6K in vitro and in vivo and inhibition of p70S6K by rapamycin in vivo.

<p>A. Stimulation of p70S6K. Cells were cultured with FBS-free medium overnight under hypoxic conditions and then treated with 10% FBS for indicated time-points under normoxic conditions. Cells were harvested and expression of phosphorylated p70S6K (p-p70S6K) measured. The experiments were carried out triplicate. B. Stimulation of p70S6K by I/R in vivo. The superior mesenteric artery was occluded for 60 min then the intestine was harvested after indicated periods of reperfusion and Western blot analysis performed. C. Confirmation of increased p70S6K activity in intestinal epithelium by immunohistochemistry. Rapamycin or vehicle treated animals underwent intestinal I/R with 6 h of reperfusion. The intestinal tissue was harvested for immunohistochemical staining for p-p70S6K. D. Confirmation of the inhibition of p70S6K by rapamycin using Western blot. Animals were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041584#pone-0041584-g002" target="_blank">Figure 2C</a>. Intestinal tissues were harvested and Western blot analysis performed. For animal experiments, n = 5 mice per group.</p