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1.  Neonatal E. Coli Infection Causes Neuro-Behavioral Deficits Associated with Hypomyelination and Neuronal Sequestration of Iron 
The Journal of Neuroscience  2013;33(41):16334-16345.
Recent evidence indicates that inflammatory insults in neonates significantly influenced white matter development and caused behavioral deficits that manifest in young adulthood. The mechanisms underlying these developmental and behavioral complications, however, are not well understood. We hypothesize that acute brain inflammation caused by neonatal infection reduces the bioavailability of iron required for oligodendrocyte maturation and white matter development. Here, we confirm that peripheral Escherichia coli infection in neonates at postnatal day 3 (P3) caused acute brain inflammation that was resolved within 72 h. Nonetheless, transient early life infection (ELI) profoundly influenced behavior, white matter development, and iron homeostasis in the brain. For instance, mice exposed to E. coli as neonates had increased locomotor activity and impaired motor coordination as juveniles (P35) and young adults (P60). In addition, these behavioral deficits were associated with marked hypomyelination and a reduction of oligodendrocytes in subcortical white matter and motor cortex. Moreover, ELI altered transcripts related to cellular sequestration of iron in the brain including hepcidin, ferroportin, and L-ferritin. For example, ELI increased hepcidin mRNA and decreased ferroportin mRNA and protein in the brain at P4, which preceded increased L-ferritin mRNA at P12. Consistent with the mRNA results, L-ferritin protein was robustly increased at P12 specifically in neurons of E. coli infected mice. We interpret these data to indicate that neonatal infection causes significant neuronal sequestration of iron at a time point before myelination. Together, these data indicate a possible role for aberrant neuronal iron storage in neonatal infection-induced disturbances in myelination and behavior.
doi:10.1523/JNEUROSCI.0708-13.2013
PMCID: PMC3792468  PMID: 24107964
2.  HISTOLOGICAL AND BIOCHEMICAL ANALYSIS OF MECHANICAL AND THERMAL BIOEFFECTS IN BOILING HISTOTRIPSY LESIONS INDUCED BY HIGH INTENSITY FOCUSED ULTRASOUND 
Ultrasound in medicine & biology  2013;39(3):424-438.
Recent studies have shown that shock wave heating and millisecond boiling in high intensity focused ultrasound (HIFU) fields can result in mechanical fractionation or emulsification of tissue - named boiling histotripsy. Visual observations of the change in color and contents indicated that the degree of thermal damage in the emulsified lesions can be controlled by varying the parameters of the exposure. The goal of this work was to examine thermal and mechanical effects in boiling histotripsy lesions using histological and biochemical analysis. The lesions were induced in ex vivo bovine heart and liver using a 2-MHz single-element transducer operating at duty factors of 0.005–0.01, pulse durations of 5–500 ms, and in situ shock amplitude of 73 MPa. Mechanical and thermal damage to tissue was evaluated histologically using conventional staining techniques (H&E and NADH-diphorase). Thermal effects were quantified by measuring denaturation of salt soluble proteins in the treated region. According to histology, the lesions that visually appeared as a liquid, contained no cellular structures larger than a cell nucleus and had a very sharp border of 1–2 cells. Both histology and protein analysis showed that lesions obtained with short pulses (< 10 ms) did not contain any thermal damage. Increasing the pulse duration resulted in an increase in thermal damage. However, both protein analysis and NADH-diaphorase staining showed less denaturation than visually observed as whitening of tissue. The number of HIFU pulses delivered per exposure did not change the lesion shape or the degree of thermal denaturation, whereas the size of the lesion showed a saturating behaviour thus suggesting optimal exposure duration. This study confirmed that boiling histotripsy offers an effective, predictable way to non-invasively fractionate tissue into subcellular fragments with or without inducing thermal damage.
doi:10.1016/j.ultrasmedbio.2012.10.012
PMCID: PMC3570648  PMID: 23312958
high intensity focused ultrasound; HIFU; histotripsy; histology; boiling; lesion; protein analysis; thermal effects
3.  Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection 
Infection and Immunity  2013;81(9):3253-3263.
Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus, which may contribute to stressor-enhanced C. rodentium-induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium-induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium-enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium. Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection.
doi:10.1128/IAI.00278-13
PMCID: PMC3754198  PMID: 23798531
4.  B-mode Ultrasound Versus Color Doppler Twinkling Artifact in Detecting Kidney Stones 
Journal of Endourology  2013;27(2):149-153.
Abstract
Purpose
To compare color Doppler twinkling artifact and B-mode ultrasonography in detecting kidney stones.
Patients and Methods
Nine patients with recent CT scans prospectively underwent B-mode and twinkling artifact color Doppler ultrasonography on a commercial ultrasound machine. Video segments of the upper pole, interpolar area, and lower pole were created, randomized, and independently reviewed by three radiologists. Receiver operator characteristics were determined.
Results
There were 32 stones in 18 kidneys with a mean stone size of 8.9±7.5 mm. B-mode ultrasonography had 71% sensitivity, 48% specificity, 52% positive predictive value, and 68% negative predictive value, while twinkling artifact Doppler ultrasonography had 56% sensitivity, 74% specificity, 62% positive predictive value, and 68% negative predictive value.
Conclusions
When used alone, B-mode is more sensitive, but twinkling artifact is more specific in detecting kidney stones. This information may help users employ twinkling and B-mode to identify stones and developers to improve signal processing to harness the fundamental acoustic differences to ultimately improve stone detection.
doi:10.1089/end.2012.0430
PMCID: PMC3573723  PMID: 23067207
5.  Meningeal Carcinomatosis: A Metastasis from Gastroesophageal Junction Adenocarcinoma 
Case Reports in Medicine  2013;2013:245654.
Gastroesophageal adenocarcinoma is a malignant type of cancer, which can metastasize to multiple organs. However, there have not been many case reports in the literature pertaining the relationship of gastroesophageal adenocarcinoma and carcinomatous meningitis. In this case, a 65-year-old African American male with a history of dysphagia was initially diagnosed with adenocarcinoma at gastroesophageal junction. The patient was treated with both chemotherapy and radiation, but chemotherapy was interrupted due to significant weight loss, anemia, and sudden onset of change in mental status. Patient was admitted to our facility for further evaluation of his neurological symptoms. The patient became more confused and delirious during hospital stay, and symptoms could not be explained by radiological studies and laboratory values. Therefore, a lumbar puncture was done to search for infectious and neoplastic causes that were not shown up on Computed Tomography scan (CT) and Magnetic Resonance Imaging scan (MRI) of the brain. The cerebrospinal fluid (CSF) cytology showed metastatic poorly differentiated adenocarcinoma. The patient's prognosis was poor because there is no specific treatment recommendation for primary gastroesophageal cancer at this stage. The patient passed away 4 weeks later under hospice care. The goal of our case report is to raise awareness of the rare metastatic possibility in advanced stage of gastroesophageal adenocarcinoma. In doing so, physicians can help educate and prepare family for unfavorable outcomes.
doi:10.1155/2013/245654
PMCID: PMC3880727  PMID: 24454393
6.  Ultrasonic atomization of tissue and its role in tissue fractionation by high intensity focused ultrasound 
Physics in medicine and biology  2012;57(23):8061-8078.
Atomization and fountain formation is a well-known phenomenon that occurs when a focused ultrasound wave in liquid encounters an air interface. High intensity focused ultrasound (HIFU) has been shown to fractionate tissue into submicron-size fragments in a process termed boiling histotripsy, wherein the focused ultrasound wave superheats the tissue at the focus, producing a millimetre-size boiling or vapour bubble in several milliseconds. Yet the question of how this millimetre-size boiling bubble creates submicron-size tissue fragments remains. The hypothesis of this work is that tissue can behave as a liquid such that it forms a fountain and atomization within the vapour bubble produced in boiling histotripsy. We describe an experiment, in which a 2-MHz HIFU transducer (maximum in situ intensity of 24,000 W/cm2) was aligned with an air-tissue interface meant to simulate the boiling bubble. Atomization and fountain formation were observed with high-speed photography and resulted in tissue erosion. Histological examination of the atomized tissue showed whole and fragmented cells and nuclei. Air-liquid interfaces were also filmed. Our conclusion was that HIFU can fountain and atomize tissue. Although this process does not entirely mimic what was observed in liquids, it does explain many aspects of tissue fractionation in boiling histotripsy.
doi:10.1088/0031-9155/57/23/8061
PMCID: PMC3535451  PMID: 23159812
Atomization; fountain; high intensity focused ultrasound; HIFU; histotripsy
7.  Novel High-Intensity Focused Ultrasound Clamp—Potential Adjunct for Laparoscopic Partial Nephrectomy 
Journal of Endourology  2012;26(11):1494-1499.
Abstract
Background and Purpose
Partial nephrectomy (PN) can be technically challenging, especially if performed in a minimally invasive manner. Although ultrasound technology has been shown to have therapeutic capabilities, including tissue ablation and hemostasis, it has not gained clinical use in the PN setting. The purpose of this study is to evaluate the ability of a high-intensity ultrasound clamp to create an ablation plane in the kidney providing hemostasis that could potentially aid in laparoscopic PN.
Methods
A new instrument was created using a laparoscopic Padron endoscopic exposing retractor. Ultrasound elements were engineered on both sides of the retractor to administer high-intensity ultrasound energy between the two sides of the clamp. This high-intensity focused ultrasound (HIFU) clamp was placed 2 to 2.5 cm from the upper and lower poles of 10 porcine kidneys to evaluate its effectiveness at different levels and duration of energy delivery. PN transection was performed through the distal portion of the clamped margin. Kidneys postintervention and after PN were evaluated and blood loss estimated by weighing gauze placed at the defect. Histologic analysis was performed with hematoxylin and eosin and nicotinamide adenine dinucleotide staining to evaluate for tissue viability and thermal spread.
Results
Gross parenchymal changes were seen with obvious demarcation between treated and untreated tissue. Increased ultrasound exposure time (10 vs 5 and 2 min), even at lower power settings, was more effective in causing destruction and necrosis of tissue. Transmural ablation was achieved in three of four renal units after 10 minutes of exposure with significantly less blood loss (<2 g vs 30–100 g). Nonviable tissue was confirmed histologically. There was minimal thermal spread outside the clamped margin (1.2–3.2 mm).
Conclusion
In this preliminary porcine evaluation, a novel HIFU clamp induced hemostasis and created an ablation plane in the kidney. This technology could serve as a useful adjunct to laparoscopic PN in the future and potentially obviate the need for renal hilar clamping.
doi:10.1089/end.2012.0107
PMCID: PMC3495120  PMID: 22788221
8.  Overview of Therapeutic Ultrasound Applications and Safety Considerations 
Summary
Applications of ultrasound in medicine for therapeutic purposes have been an accepted and beneficial use of ultrasonic biological effects for many years. Low power ultrasound of about 1 MHz frequency has been widely applied since the 1950s for physical therapy in conditions such as tendinitis or bursitis. In the 1980s, high pressure-amplitude shockwaves came into use for mechanically resolving kidney stones, and “lithotripsy” rapidly replaced surgery as the most frequent treatment choice. The use of ultrasonic energy for therapy continues to expand, and approved applications now include uterine fibroid ablation, cataract removal (phacoemulsification), surgical tissue cutting and hemostasis, transdermal drug delivery, and bone fracture healing, among others. Undesirable bioeffects can occur including burns for thermal-based therapies and significant hemorrhage for mechanical-based therapies (e. g. lithotripsy). In all these therapeutic applications for bioeffects of ultrasound, standardization, ultrasound dosimetry, benefits assurance and side-effects risk minimization must be carefully considered in order to insure an optimal benefit to risk ratio for the patient. Therapeutic ultrasound typically has well-defined benefits and risks, and therefore presents a tractable safety problem to the clinician. However, safety information can be scattered, confusing or subject to commercial conflict of interest. Of paramount importance for managing this problem is the communication of practical safety information by authoritative groups, such as the AIUM, to the medical ultrasound community. In this overview, the Bioeffects Committee outlines the wide range of therapeutic ultrasound methods, which are in clinical use or under study, and provides general guidance for assuring therapeutic ultrasound safety.
PMCID: PMC3810427  PMID: 22441920
9.  Quantitative Assessment of Shockwave Lithotripsy Accuracy and the Effect of Respiratory Motion* 
Journal of Endourology  2012;26(8):1070-1074.
Abstract
Background and Purpose
Effective stone comminution during shockwave lithotripsy (SWL) is dependent on precise three-dimensional targeting of the shockwave. Respiratory motion, imprecise targeting or shockwave alignment, and stone movement may compromise treatment efficacy. The purpose of this study was to evaluate the accuracy of shockwave targeting during SWL treatment and the effect of motion from respiration.
Patients and Methods
Ten patients underwent SWL for the treatment of 13 renal stones. Stones were targeted fluoroscopically using a Healthtronics Lithotron (five cases) or Dornier Compact Delta II (five cases) shockwave lithotripter. Shocks were delivered at a rate of 1 to 2 Hz with ramping shockwave energy settings of 14 to 26 kV or level 1 to 5. After the low energy pretreatment and protective pause, a commercial diagnostic ultrasound (US) imaging system was used to record images of the stone during active SWL treatment. Shockwave accuracy, defined as the proportion of shockwaves that resulted in stone motion with shockwave delivery, and respiratory stone motion were determined by two independent observers who reviewed the ultrasonographic videos.
Results
Mean age was 51±15 years with 60% men, and mean stone size was 10.5±3.7 mm (range 5–18 mm). A mean of 2675±303 shocks was delivered. Shockwave-induced stone motion was observed with every stone. Accurate targeting of the stone occurred in 60%±15% of shockwaves.
Conclusions
US imaging during SWL revealed that 40% of shockwaves miss the stone and contribute solely to tissue injury, primarily from movement with respiration. These data support the need for a device to deliver shockwaves only when the stone is in target. US imaging provides real-time assessment of stone targeting and accuracy of shockwave delivery.
doi:10.1089/end.2012.0042
PMCID: PMC3412057  PMID: 22471349
10.  The SOCS Box Domain of SOCS3: Structure and Interaction with the ElonginBC-Cullin5 Ubiquitin Ligase 
Journal of molecular biology  2008;381(4):928-940.
Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3–elonginBC interaction was further characterised by determining the solution structure of the SOCS box–elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS–elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.
doi:10.1016/j.jmb.2008.06.038
PMCID: PMC3652878  PMID: 18590740
SOCS; cytokine signalling; ubiquitin ligase; elongin; cullin
11.  Prophylactic Perioperative Sodium Bicarbonate to Prevent Acute Kidney Injury Following Open Heart Surgery: A Multicenter Double-Blinded Randomized Controlled Trial 
PLoS Medicine  2013;10(4):e1001426.
In a double-blinded randomized controlled trial, Anja Haase-Fielitz and colleagues find that an infusion of sodium bicarbonate during open heart surgery did not reduce the risk for acute kidney injury, compared with saline control.
Background
Preliminary evidence suggests a nephroprotective effect of urinary alkalinization in patients at risk of acute kidney injury. In this study, we tested whether prophylactic bicarbonate-based infusion reduces the incidence of acute kidney injury and tubular damage in patients undergoing open heart surgery.
Methods and Findings
In a multicenter, double-blinded (patients, clinical and research personnel), randomized controlled trial we enrolled 350 adult patients undergoing open heart surgery with the use of cardiopulmonary bypass. At induction of anesthesia, patients received either 24 hours of intravenous infusion of sodium bicarbonate (5.1 mmol/kg) or sodium chloride (5.1 mmol/kg). The primary endpoint was the proportion of patients developing acute kidney injury. Secondary endpoints included the magnitude of acute tubular damage as measured by urinary neutrophil gelatinase-associated lipocalin (NGAL), initiation of acute renal replacement therapy, and mortality. The study was stopped early under recommendation of the Data Safety and Monitoring Committee because interim analysis suggested likely lack of efficacy and possible harm. Groups were non-significantly different at baseline except that a greater proportion of patients in the sodium bicarbonate group (66/174 [38%]) presented with preoperative chronic kidney disease compared to control (44/176 [25%]; p = 0.009). Sodium bicarbonate increased urinary pH (from 6.0 to 7.5, p<0.001). More patients receiving bicarbonate (83/174 [47.7%]) developed acute kidney injury compared with control patients (64/176 [36.4%], odds ratio [OR] 1.60 [95% CI 1.04–2.45]; unadjusted p = 0.032). After multivariable adjustment, a non-significant unfavorable group difference affecting patients receiving sodium bicarbonate was found for the primary endpoint (OR 1.45 [0.90–2.33], p = 0.120]). A greater postoperative increase in urinary NGAL in patients receiving bicarbonate infusion was observed compared to control patients (p = 0.011). The incidence of postoperative renal replacement therapy was similar but hospital mortality was increased in patients receiving sodium bicarbonate compared with control (11/174 [6.3%] versus 3/176 [1.7%], OR 3.89 [1.07–14.2], p = 0.031).
Conclusions
Urinary alkalinization using sodium bicarbonate infusion was not found to reduce the incidence of acute kidney injury or attenuate tubular damage following open heart surgery; however, it was associated with a possible increase in mortality. On the basis of these findings we do not recommend the prophylactic use of sodium bicarbonate infusion to reduce the risk of acute kidney injury. Discontinuation of growing implementation of this therapy in this setting seems to be justified.
Trial registration
ClinicalTrials.gov NCT00672334
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Open heart surgery is a type of cardiac surgery that is used to treat patients with severe heart disease, where the patient's chest is cut open and surgery is performed on the internal structures of the heart. During open heart surgery, surgeons may use a technique called cardiopulmonary bypass to temporarily take over the function of the heart and lungs. This type of surgery may be used to prevent heart attack or heart failure in patients with conditions such as angina, atherosclerosis, congenital heart disease, or valvular heart disease. There are a number of complications associated with open heart surgery and one of these is the rapid loss of kidney function, known as acute kidney injury (AKI), and formerly known as acute renal failure. Symptoms of AKI can be variable, with diagnosis of AKI based on laboratory findings (such as elevated blood urea nitrogen and creatinine), or clinical signs such as inability of the kidneys to produce sufficient amounts of urine. Globally, more than 10 million people are affected by AKI each year. AKI occurs in about one quarter of patients undergoing cardiac surgery and is associated with longer stays in the hospital and an increased risk of death. Treatment of AKI includes administration of intravenous fluids, diuretics, and, in severe cases, patients may require kidney dialysis.
Why Was This Study Done?
The mechanism for why AKI occurs during cardiac surgery is complex and thought to involve multiple factors relating to blood circulation, the immune system, and toxins released by the kidneys. In addition to treating AKI after it occurs, it is important to identify patients who are at risk for developing AKI prior to cardiac surgery and then apply techniques to prevent AKI during cardiac surgery. A number of interventions have been tested for preventing AKI during cardiac surgery, but there is currently no strong evidence for a standard way to prevent AKI. One intervention that has potential for preventing AKI is the administration of sodium bicarbonate during cardiac surgery. Sodium bicarbonate causes alkalinization of the urine, and it is thought that this could reduce the effect of toxins in the kidneys. A previous pilot study showed promising effects for sodium bicarbonate to reduce the likelihood of AKI. In a follow-up to this pilot study, here the researchers have performed an international randomized controlled trial to test whether administration of sodium bicarbonate compared to sodium chloride (saline) during cardiac surgery can prevent AKI.
What Did the Researchers Do and Find?
350 patients undergoing open heart surgery with at least one risk factor for developing AKI were recruited across four sites in different countries (Germany, Canada, Ireland, and Australia). These patients were randomly assigned to receive either sodium bicarbonate (treatment) or saline control solution, given as a continuous infusion into the blood stream for 24 hours during surgery. Neither the researchers nor the patients were aware of which patients were assigned to the treatment group. The researchers measured the occurrence of AKI within the first 5 days after surgery and they found that a greater proportion of those patients receiving sodium bicarbonate developed AKI, as compared to those patients receiving saline control. On the basis of these findings the study was terminated before planned recruitment was completed. A key issue with this study is that a greater proportion of the patients in the sodium bicarbonate group had chronic kidney disease prior to open heart surgery. After adjusting for this difference in the statistical analysis, the researchers observed that the difference between the groups was not significant—that is, it could have happened by chance. The authors also observed that a significantly greater proportion of patients receiving sodium bicarbonate died in the hospital after surgery compared to patients receiving saline control.
What Do These Findings Mean?
These findings suggest that giving an infusion of sodium bicarbonate to induce alkalinization of the urine during open heart surgery is not a useful treatment for preventing AKI. Furthermore, this treatment may even increase the likelihood of death. The researchers do not recommend the use of sodium bicarbonate infusion to reduce the risk of AKI after open heart surgery and stress the need for discontinuation of this therapy. Key limitations of this research study are the early termination of the study and the greater proportion of patients with chronic kidney disease prior to surgery.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001426.
The Renal Association, a professional association for kidney doctors and researchers, provides information about acute kidney injury
The International Society for Nephrology and the International Federation of Kidney Foundations provide information about preventing acute kidney injury around the world and jointly initiated World Kidney Day
MedlinePlus has information on open heart surgery
doi:10.1371/journal.pmed.1001426
PMCID: PMC3627643  PMID: 23610561
12.  Stressor-Induced Increase in Microbicidal Activity of Splenic Macrophages Is Dependent upon Peroxynitrite Production 
Infection and Immunity  2012;80(10):3429-3437.
Exposing mice to a social stressor called social disruption (SDR) that involves repeated social defeat during intermale aggression results in increased circulating cytokines, such as interleukin-1α (IL-1α) and IL-1β, and increased reactivity of splenic CD11b+ macrophages to inflammatory stimuli. For example, upon lipopolysaccharide stimulation, macrophages from stressor-exposed mice produce higher levels of cytokines than do cells from nonstressed controls. Moreover, the SDR stressor enhances the ability of these macrophages to kill Escherichia coli both in vitro and in vivo, through a Toll-like receptor 4-dependent mechanism. The present study tested the hypothesis that stressor-enhanced bacterial killing is due to increases in the production of peroxynitrite. Male mice were exposed to the SDR stressor or were left undisturbed. Upon stimulation with E. coli, splenic macrophages from SDR-exposed mice expressed significantly increased levels of inducible nitric oxide synthase mRNA and produced higher levels of peroxynitrite. Blocking the production of peroxynitrite abrogated the SDR-induced increase in microbicidal activity. Studies in IL-1 receptor type 1 knockout mice indicated that the increased microbicidal activity and peroxynitrite production was dependent upon IL-1 signaling. These data confirm and extend the importance of IL-1 signaling for stressor-induced immunopotentiation; the finding that inhibiting superoxide or nitric oxide production inhibits both peroxynitrite production and killing of E. coli demonstrates that peroxynitrite mediates the stressor-induced increase in bacterial killing.
doi:10.1128/IAI.00714-12
PMCID: PMC3457565  PMID: 22825446
13.  The Intestinal Microbiota Are Necessary for Stressor-Induced Enhancement of Splenic Macrophage Microbicidal Activity 
Brain, Behavior, and Immunity  2011;26(3):371-382.
The indigenous microbiota impact mucosal, as well as systemic, immune responses, but whether the microbiota are involved in stressor-induced immunomodulation has not been thoroughly tested. A well characterized murine stressor, called social disruption (SDR), was used to study whether the microbiota are involved in stressor-induced enhancement of macrophage reactivity. Exposure to the SDR stressor enhanced the ability of splenic macrophages to produce microbicidal mediators (e.g., inducible nitric oxide synthase (iNOS), superoxide anion, and peroxynitrite) and to kill target Escherichia coli. Exposure to the SDR stressor also increased cytokine production by LPS-stimulated splenic macrophages. These effects, however, were impacted by the microbiota. Microbicidal activity and cytokine mRNA in splenic macrophages from Swiss Webster germfree mice that lack any commensal microbiota were not enhanced by exposure to the SDR stressor. However, when germfree mice were conventionalized by colonizing them with microbiota from CD-1 conventional donor mice, exposure to the SDR stressor again increased microbicidal activity and cytokine mRNA. In follow up experiments, immunocompetent conventional CD-1 mice were treated with a cocktail of antibiotics to disrupt the intestinal microbiota. While exposure to the SDR stressor enhanced splenic macrophage microbicidal activity and cytokine production in vehicle-treated mice, treatment with antibiotics attenuated the SDR stressor-induced increases in splenic macrophage reactivity. Treatment with antibiotics also prevented the stressor-induced increase in circulating levels of bacterial peptidoglycan, suggesting that translocation of microbiota-derived peptidoglycan into the body primes the innate immune system for enhanced activity. This study demonstrates that the microbiota play a crucial role in stressor-induced immunoenhancement.
doi:10.1016/j.bbi.2011.11.002
PMCID: PMC3288745  PMID: 22100833
Microbiota; Stress; Social Disruption; Macrophage; Microbicidal Activity; Peroxynitrite; IL-1β; TNF-α; iNOS; Superoxide; Peptidoglycan
14.  Stress and the anti-influenza immune response: repeated social defeat augments clonal expansion of CD8+T cells during primary influenza A viral infection 
Journal of Neuroimmunology  2012;243(1-2):34-42.
Social disruption stress (SDR) prior to primary influenza A virus (IAV) infection augments memory to IAV re-challenge in a T cell-specific manner. However, the effect of SDR on the primary anti-viral immune response has not been elucidated. In this study, SDR-infected (INF) mice terminated viral gene expression earlier and mounted an enhanced pulmonary IAV-specific CD8+T cell response versus controls. Additionally, SDR-INF mice had a more pro-inflammatory lung profile prior to and during infection and an attenuated corticosterone response. These data demonstrate neuroendocrine modification of the lung microenvironment and increased antigen-specific T cell activation, clonal expansion and viral control in stress-exposed mice.
doi:10.1016/j.jneuroim.2011.12.011
PMCID: PMC3287073  PMID: 22244573
psychosocial stress; glucocorticoid; mice; virus; influenza; T cell; immune response; HPA axis; sympathetic nervous system; tetramer; inflammation; neuroendocrine
15.  Viral Pneumonitis Is Increased in Obese Patients during the First Wave of Pandemic A(H1N1) 2009 Virus 
PLoS ONE  2013;8(2):e55631.
Introduction
There is conflicting data as to whether obesity is an independent risk factor for mortality in severe pandemic (H1N1) 2009 influenza (A(H1N1)pdm09). It is postulated that excess inflammation and cytokine production in obese patients following severe influenza infection leads to viral pneumonitis and/or acute respiratory distress syndrome.
Methods
Demographic, laboratory and clinical data prospectively collected from obese and non-obese patients admitted to nine adult Australian intensive care units (ICU) during the first A(H1N1)pdm09 wave, supplemented with retrospectively collected data, were compared.
Results
Of 173 patients, 100 (57.8%), 73 (42.2%) and 23 (13.3%) had body mass index (BMI) <30 kg/m2, ≥30 kg/m2 (obese) and ≥40 kg/m2 (morbidly obese) respectively. Compared to non-obese patients, obese patients were younger (mean age 43.4 vs. 48.4 years, p = 0.035) and more likely to develop pneumonitis (61% vs. 44%, p = 0.029). Extracorporeal membrane oxygenation use was greater in morbidly obese compared to non-obese patients (17.4% vs. 4.7%, p = 0.04). Higher mortality rates were observed in non-obese compared to obese patients, but not after adjusting for severity of disease. C-reactive protein (CRP) levels and hospital length of stay (LOS) were similar. Amongst ICU survivors, obese patients had longer ICU LOS (median 11.9 vs. 6.8 days, p = 0.017). Similar trends were observed when only patients infected with A(H1N1)pdm09 were examined.
Conclusions
Among patients admitted to ICU during the first wave of A(H1N1)pdm09, obese and morbidly obese patients with severe infection were more likely to develop pneumonitis compared to non-obese patients, but mortality rates were not increased. CRP is not an accurate marker of pneumonitis.
doi:10.1371/journal.pone.0055631
PMCID: PMC3572103  PMID: 23418448
16.  Sedation depth and long-term mortality in mechanically ventilated critically ill adults: a prospective longitudinal multicentre cohort study 
Intensive Care Medicine  2013;39(5):910-918.
Purpose
To ascertain the relationship among early (first 48 h) deep sedation, time to extubation, delirium and long-term mortality.
Methods
We conducted a multicentre prospective longitudinal cohort study in 11 Malaysian hospitals including medical/surgical patients (n = 259) who were sedated and ventilated ≥24 h. Patients were followed from ICU admission up to 28 days in ICU with 4-hourly sedation and daily delirium assessments and 180-day mortality. Deep sedation was defined as Richmond Agitation Sedation Score (RASS) ≤−3.
Results
The cohort had a mean (SD) age of 53.1 (15.9) years and APACHE II score of 21.3 (8.2) with hospital and 180-day mortality of 82 (31.7 %) and 110/237 (46.4 %). Patients were followed for 2,657 ICU days and underwent 13,836 RASS assessments. Midazolam prescription was predominant compared to propofol, given to 241 (93 %) versus 72 (28 %) patients (P < 0.0001) for 966 (39.6 %) versus 183 (7.5 %) study days respectively. Deep sedation occurred in (182/257) 71 % patients at first assessment and in 159 (61 %) patients and 1,658 (59 %) of all RASS assessments at 48 h. Multivariable Cox proportional hazard regression analysis adjusting for a priori assigned covariates including sedative agents, diagnosis, age, APACHE II score, operative, elective, vasopressors and dialysis showed that early deep sedation was independently associated with longer time to extubation [hazard ratio (HR) 0.93, 95 % confidence interval (CI) 0.89–0.97, P = 0.003], hospital death (HR 1.11, 95 % CI 1.05–1.18, P < 0.001) and 180-day mortality (HR 1.09, 95 % CI 1.04–1.15, P = 0.002), but not time to delirium (HR 0.98, P = 0.23). Delirium occurred in 114 (44 %) of patients.
Conclusion
Irrespective of sedative choice, early deep sedation was independently associated with delayed extubation and higher mortality, and thus was a potentially modifiable risk in interventional trials.
doi:10.1007/s00134-013-2830-2
PMCID: PMC3625407  PMID: 23344834
Sedation depth; Mechanical ventilation; Delirium; Critically ill; Mortality
17.  OBSERVATIONS OF TRANSLATION AND JETTING OF ULTRASOUND-ACTIVATED MICROBUBBLES IN MESENTERIC MICROVESSELS 
Ultrasound in medicine & biology  2011;37(12):2139-2148.
High-speed photomicrography was used to study the translational dynamics of single microbubbles in microvessels of ex vivo rat mesenteries. The microbubbles were insonated by a single 2 μs ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 0.8–4 MPa. The microvessel diameters ranged from 10 – 80 μm. The high-speed image sequences show evidence of ultrasound-activated microbubble translation away from the nearest vessel wall; no microbubble showed a net translation toward the nearest vessel wall. Microbubble maximum translational displacements exceeded 20 μm. Microjets with the direction of the jets identifiable were also observed; all microjets appear to have been directed away from the nearest vessel wall. These observations appear to be characteristic of a strong coupling between ultrasound-driven microbubbles and compliant microvessels. Although limited to mesenteric tissues, these observations provide an important step in understanding the physical interactions between microbubbles and microvessels.
doi:10.1016/j.ultrasmedbio.2011.09.013
PMCID: PMC3223323  PMID: 22036639
Microbubbles; Microvessels; Ultrasound; Microbubble translation; Microjets; Inertial cavitation; Acoustic cavitation; Mechanical bioeffects; Ultrasound contrast agent; Mesentery blood vessels; High speed photomicrography; Microbubble dynamics
18.  Long-term quality of life in patients with acute respiratory distress syndrome requiring extracorporeal membrane oxygenation for refractory hypoxaemia 
Critical Care  2012;16(5):R202.
Introduction
The purpose of the study was to assess the long term outcome and quality of life of patients with acute respiratory distress syndrome (ARDS) receiving extracorporeal membrane oxygenation (ECMO) for refractory hypoxemia.
Methods
A retrospective observational study with prospective health related quality of life (HRQoL) assessment was conducted in ARDS patients who had ECMO as a rescue therapy for reversible refractory hypoxemia from January 2009 until April 2011 in a tertiary Australian centre. Survival and long-term quality of life assessment, using the Short-Form 36 (SF-36) and the EuroQol health related quality of life questionnaire (EQ5D) were assessed and compared to international data from other research groups.
Results
Twenty-one patients (mean age 36.3 years) with ARDS receiving ECMO for refractory hypoxemia were studied. Eighteen (86%) patients were retrieved from external intensive care units (ICUs) by a dedicated ECMO retrieval team. Eleven (55%) had H1N1 influenza A-associated pneumonitis. Eighteen (86%) patients survived to hospital discharge. Of the 18 survivors, ten (56%) were discharged to other hospitals and 8 (44%) were discharged directly home. Sequelae and health related quality of life were evaluated for 15 of the 18 (71%) long-term survivors (assessment at median 8 months). Mean SF-36 scores were significantly lower across all domains compared to age and sex matched Australian norms. Mean SF-36 scores were lower (minimum important difference at least 5 points) than previously described ARDS survivors in the domains of general health, mental health, vitality and social function. One patient had long-term disability as a result of ICU acquired weakness. Only 26% of survivors had returned to previous work levels at the time of follow-up.
Conclusions
This ARDS cohort had a high survival rate (86%) after use of ECMO support for reversible refractory hypoxemia. Long term survivors had similar physical health but decreased mental health, general health, vitality and social function compared to other ARDS survivors and an unexpectedly poor return to work.
doi:10.1186/cc11811
PMCID: PMC3682304  PMID: 23082772
19.  Antibody-mediated Neutralization of Ebola Virus Can Occur by Two Distinct Mechanisms 
Virology  2010;401(2):228-235.
Human Ebola virus (EBOV) causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the non-essential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.
doi:10.1016/j.virol.2010.02.029
PMCID: PMC3351102  PMID: 20304456
Virus; Ebola; Immunity; Neutralization; Antibody; Human; Nonhuman Primate; Rodent
20.  Stressor-Induced Alterations of Adaptive Immunity to Vaccination and Viral Pathogens 
doi:10.1016/j.iac.2010.09.002
PMCID: PMC3339561  PMID: 21094924
Psychoneuroimmunology; Stress Influenza vaccine; Antibody response
21.  Shock Wave Technology and Application: An Update☆ 
European Urology  2011;59(5):784-796.
Context
The introduction of new lithotripters has increased problems associated with shock wave application. Recent studies concerning mechanisms of stone disintegration, shock wave focusing, coupling, and application have appeared that may address some of these problems.
Objective
To present a consensus with respect to the physics and techniques used by urologists, physicists, and representatives of European lithotripter companies.
Evidence acquisition
We reviewed recent literature (PubMed, Embase, Medline) that focused on the physics of shock waves, theories of stone disintegration, and studies on optimising shock wave application. In addition, we used relevant information from a consensus meeting of the German Society of Shock Wave Lithotripsy.
Evidence synthesis
Besides established mechanisms describing initial fragmentation (tear and shear forces, spallation, cavitation, quasi-static squeezing), the model of dynamic squeezing offers new insight in stone comminution. Manufacturers have modified sources to either enlarge the focal zone or offer different focal sizes. The efficacy of extracorporeal shock wave lithotripsy (ESWL) can be increased by lowering the pulse rate to 60–80 shock waves/min and by ramping the shock wave energy. With the water cushion, the quality of coupling has become a critical factor that depends on the amount, viscosity, and temperature of the gel. Fluoroscopy time can be reduced by automated localisation or the use of optical and acoustic tracking systems. There is a trend towards larger focal zones and lower shock wave pressures.
Conclusions
New theories for stone disintegration favour the use of shock wave sources with larger focal zones. Use of slower pulse rates, ramping strategies, and adequate coupling of the shock wave head can significantly increase the efficacy and safety of ESWL.
doi:10.1016/j.eururo.2011.02.033
PMCID: PMC3319085  PMID: 21354696
Extracorporeal shock wave; lithotripsy; Lithotripter; Shock wave generation; Urolithiasis
22.  Exposure to a Social Stressor Alters the Structure of the Intestinal Microbiota: Implications for Stressor-Induced Immunomodulation 
Brain, behavior, and immunity  2010;25(3):397-407.
The bodies of most animals are populated by highly complex and genetically diverse communities of microorganisms. The majority of these microbes reside within the intestines in largely stable but dynamically interactive climax communities that positively interact with their host. Studies from this laboratory have shown that stressor exposure impacts the stability of the microbiota and leads to bacterial translocation. The biological importance of these alterations, however, is not well understood. To determine whether the microbiome contributes to stressor-induced immunoenhancement, mice were exposed to a social stressor called social disruption (SDR), that increases circulating cytokines and primes the innate immune system for enhanced reactivity. Bacterial populations in the cecum were characterized using bacterial tag-encoded FLX amplicon pyrosequencing. Stressor exposure significantly changed the community structure of the microbiota, particularly when the microbiota were assessed immediately after stressor exposure. Most notably, stressor exposure decreased the relative abundance of bacteria in the genus Bacteroides, while increasing the relative abundance of bacteria in the genus Clostridium. The stressor also increased circulating levels of IL-6 and MCP-1, which were significantly correlated with stressor-induced changes to three bacterial genera (i.e., Coprococcus, Pseudobutyrivibrio, and Dorea). In follow up experiments, mice were treated with an antibiotic cocktail to determine whether reducing the microbiota would abrogate the stressor-induced increases in circulating cytokines. Exposure to SDR failed to increase IL-6 and MCP-1 in the antibiotic treated mice. These data show that exposure to SDR significantly affects bacterial populations in the intestines, and remarkably also suggest that the microbiota are necessary for stressor-induced increases in circulating cytokines.
doi:10.1016/j.bbi.2010.10.023
PMCID: PMC3039072  PMID: 21040780
Social Stress; Gastrointestinal Microbiota; Pyrosequencing; Inflammation; Immunomodulation
23.  The Effects of Terlipressin on Regional Hemodynamics and Kidney Function in Experimental Hyperdynamic Sepsis 
PLoS ONE  2012;7(2):e29693.
Background and Aims
Although terlipressin (TP) may improve renal function in cirrhotic patients, its use in sepsis remains controversial due to concerns about regional ischemia. We investigated the effects of TP on regional hemodynamics and kidney function in experimental hyperdynamic sepsis.
Methods
We studied thirteen merino ewes in a university physiology laboratory using a randomized controlled cross over design. We implanted flow probes around the pulmonary, circumflex coronary, superior mesenteric, renal and iliac arteries. We injected live Escherichia coli and induced hyperdynamic sepsis. We treated animals with either bolus vehicle or a single dose of TP (sTP = 1 mg). In a second group, after 1 mg of TP, two additional bolus injections (mTP) of 0.5 mg were given at 2 hourly intervals.
Main Results
sTP (1 mg) significantly increased mean arterial pressure (MAP) (74 to 89 mmHg; P<0.0001) creatinine clearance (31 to 85 mL/min; P<0.0001) and urine output (24 to 307 mL/hr) (P<0.0001). However, it decreased CO (5.7 to 3.9 L/min; p<0.0001), coronary blood flow (CBF) (43 to 32 mL/min; p<0.0001) and mesenteric blood flow (MBF) (944 to 625 mL/min; p = 0.004) and increased blood lactate (2.1 to 4.0 mmol/L; p<0.0001). Extra doses of TP caused little additional effect.
Conclusions
In hyperdynamic sepsis, bolus TP transiently improves MAP and renal function, but reduces CO, CBF and MBF, and increases blood lactate. Caution should be applied when prescribing bolus TP in septic patients at risk of coronary or mesenteric ischemia.
doi:10.1371/journal.pone.0029693
PMCID: PMC3280248  PMID: 22355305
24.  Immunogenic Dendritic Cells Primed by Social Defeat Enhance Adaptive Immunity to Influenza A Virus 
Brain, behavior, and immunity  2010;25(1):46-52.
Dendritic cells (DCs) sample their surrounding microenvironment and consequently send immunogenic or regulatory signals to T cells during DC/T cell interactions, shaping the primary adaptive immune response to infection. The microenvironment resulting from repeated social defeat increases DC co-stimulatory molecule expression and primes DCs for enhanced cytokine responses in vitro. In this study we show that social disruption stress (SDR) results in the generation of immunogenic DCs, capable of conferring enhanced adaptive immunity to influenza A/PR/8/34 infection. Mice infected with influenza A/PR/8/34 virus 24h after the adoptive transfer of DCs from SDR mice had significantly increased numbers of DbNP366-74CD8+ T cells, increased IFN-γ and IFN-α mRNA, and decreased influenza M1 mRNA expression in the lung during the peak primary response (9 days post-infection), compared to mice that received DCs from naïve mice. These data demonstrate that repeated social defeat is a significant environmental influence on immunogenic DC activation and function.
doi:10.1016/j.bbi.2010.07.243
PMCID: PMC2991426  PMID: 20656014
25.  Recombinant Adenovirus Serotype 26 (Ad26) and Ad35 Vaccine Vectors Bypass Immunity to Ad5 and Protect Nonhuman Primates against Ebolavirus Challenge▿ 
Journal of Virology  2011;85(9):4222-4233.
The use of adenoviruses (Ad) as vaccine vectors against a variety of pathogens has demonstrated their capacity to elicit strong antibody and cell-mediated immune responses. Adenovirus serotype C vectors, such as Ad serotype 5 (Ad5), expressing Ebolavirus (EBOV) glycoprotein (GP), protect completely after a single inoculation at a dose of 1010 viral particles. However, the clinical application of a vaccine based on Ad5 vectors may be hampered, since impairment of Ad5 vaccine efficacy has been demonstrated for humans and nonhuman primates with high levels of preexisting immunity to the vector. Ad26 and Ad35 segregate genetically from Ad5 and exhibit lower seroprevalence in humans, making them attractive vaccine vector alternatives. In the series of studies presented, we show that Ad26 and Ad35 vectors generate robust antigen-specific cell-mediated and humoral immune responses against EBOV GP and that Ad5 immune status does not affect the generation of GP-specific immune responses by these vaccines. We demonstrate partial protection against EBOV by a single-shot Ad26 vaccine and complete protection when this vaccine is boosted by Ad35 1 month later. Increases in efficacy are paralleled by substantial increases in T- and B-cell responses to EBOV GP. These results suggest that Ad26 and Ad35 vectors warrant further development as candidate vaccines for EBOV.
doi:10.1128/JVI.02407-10
PMCID: PMC3126236  PMID: 21325402

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