Bobby L. Johnson

Obesity-induced hyperleptinemia improves survival and immune response in a murine model of sepsis.

Background:Obesity is a growing health problem and associated with immune dysfunction. Sepsis is defined as systemic inflammatory response syndrome that occurs during infection. Excessive inflammation combined with immune dysfunction can lead to multiorgan damage and death. Methods:The authors investigated the influence of a class 1 obesity (body mass index between 30 and 34.9) on immune function and outcome in sepsis and the role of leptin on the immune response. The authors used a long-term high-fat-diet feeding model (12 weeks) on C57Bl/6 mice (n = 100) and controls on standard diet (n = 140) followed by a polymicrobial sepsis induced by cecal ligation and puncture. Results:The authors show that class 1 obesity is connected to significant higher serum leptin levels (data are mean ± SEM) (5.7 ± 1.2 vs. 2.7 ± 0.2 ng/ml; n = 5; P = 0.033) and improved innate immune response followed by significant better survival rate in sepsis (71.4%, n = 10 vs. 10%, n = 14; P < 0.0001). Additional sepsis-induced increases in leptin levels stabilize body temperature and are associated with a controlled immune response in a time-dependent and protective manner. Furthermore, leptin treatment of normal-weight septic mice with relative hypoleptinemia (n = 35) also significantly stabilizes body temperature, improves cellular immune response, and reduces proinflammatory cytokine response resulting in improved survival (30%; n = 10). Conclusions:Relative hyperleptinemia of class 1 obesity or induced by treatment is protective in sepsis. Leptin seems to play a regulatory role in the immune system in sepsis, and treatment of relative hypoleptinemia could offer a new way of an individual sepsis therapy.

Neutrophil Derived Microvesicles: Emerging Role of a Key Mediator to the Immune Response

In response to infection and trauma, exquisite control of the innate inflammatory response is necessary to promote an anti-microbial response and minimize tissue injury. Over the course of the host response, activated leukocytes are essential for the initial response and can later become unresponsive or undergo apoptosis. Leukocytes, along the continuum of activation to apoptosis, have been shown to generate microvesicles. These vesicles can range in size from 0.1 to 1.0 μm and can retain proteins, RNA and DNA of their parent cells. Importantly, neutrophil-derived microvesicles (NDMV) are robustly increased under inflammatory conditions. The aim of this review is to summarize the research to date upon NDMVs. This will include describing under which disease states NDMVs are increased, mechanisms underlying formation, and the impact of these vesicles upon cellular targets. Altogether, increased awareness of NDMVs during the host innate response may allow for diagnostic tools as well as potential novel therapies during infection and trauma.

Characterization of microparticles after hepatic ischemia-reperfusion injury.

Background Hepatic ischemia-reperfusion (I/R) is a well-studied model of liver injury and has demonstrated a biphasic injury followed by recovery and regeneration. Microparticles (MPs) are a developing field of study and these small membrane bound vesicles have been shown to have effector function in other physiologic and pathologic states. This study was designed to quantify the levels of MPs from various cell origins–platelets, neutrophils, and endolethial cells–following hepatic ischemia-reperfusion injury. Methods A murine model was used with mice undergoing 90 minutes of partial hepatic ischemia followed by various times of reperfusion. Following reperfusion, plasma samples were taken and MPs of various cell origins were labeled and levels were measured using flow cytometry. Additionally, cell specific MPs were further assessed by Annexin V, which stains for the presence of phosphatidylserine, a cell surface marker linked to apoptosis. Statistical analysis was performed using one-way analysis of variance with subsequent Student-Newman-Keuls test with data presented as the mean and standard error of the mean. Results MPs from varying sources show an increase in circulating levels following hepatic I/R injury. However, the timing of the appearance of different MP subtypes differs for each cell type. Platelet and neutrophil-derived MP levels demonstrated an acute elevation following injury whereas endothelial-derived MP levels demonstrated a delayed elevation. Conclusion This is the first study to characterize circulating levels of cell-specific MPs after hepatic I/R injury and suggests that MPs derived from platelets and neutrophils serve as markers of inflammatory injury and may be active participants in this process. In contrast, MPs derived from endothelial cells increase after the injury response during the reparative phase and may be important in angiogenesis that occurs in the regenerating liver.

Mechanisms underlying mouse TNF-α stimulated neutrophil derived microparticle generation.

Despite advances in understanding and treatment of sepsis, it remains a disease with high mortality. Neutrophil Derived Microparticles (NDMPs) are present during sepsis and can modulate the immune system. As TNF-α is a cytokine that predominates in the initial stages of sepsis, we evaluated whether and how TNF-α can induce NDMPs in mice. We observed that TNF-α treatment results in increased NDMP numbers. We also determined that the activation of either TNF receptor 1 (TNFr1) or TNF receptor 2 (TNFr2) resulted in increased NDMP numbers and that activation of both resulted in an additive increase. Inhibition of Caspase 8 diminishes NDMPs generated through TNFr1 activation and inhibition of NF-κB abrogates NDMPs generated through activation of both TNFr1 and TNFr2. We conclude that the early production of TNF-α during sepsis can increase NDMP numbers through activation of the Caspase 8 pathway or NF-κB.

Amitriptyline Usage Exacerbates the Immune Suppression Following Burn Injury.

ABSTRACT Currently, over 10% of the US population is taking antidepressants. Numerous antidepressants such as amitriptyline are known to inhibit acid sphingomyelinase (Asm), an enzyme that is known to mediate leukocyte function and homeostasis. Severe burn injury can lead to an immunosuppressive state that is characterized by decreased leukocyte function and numbers as well as increased susceptibility to infection. Based upon the intersection of these facts, we hypothesized that amitriptyline-treated, scald-injured mice would have an altered immune response to injury as compared with untreated scald mice. Prior to burn, mice were pretreated with amitriptyline. Drug- or saline-treated mice were subjected full thickness dorsal scald- or sham-injury. Immune cells from spleen, thymus, and bone marrow were subsequently harvested and characterized. We first observed that amitriptyline prior to burn injury increased body mass loss and spleen contraction. Both amitriptylinetreatment and burn injury resulted in a 40% decrease of leukocyte Asm activity. Following scald injury, we demonstrate increased reduction of lymphocyte precursors in the bone marrow and thymus, as well as mature leukocytes in the spleen in mice that were treated with amitriptyline. We also demonstrate that amitriptyline treatment prior to injury reduced neutrophil accumulation following peptidoglycan stimulus in scald-injured mice. These data show that Asm alterations can play a significant role in mediating alterations to the immune system after injury. The data further suggest that those taking antidepressants may be at a higher risk for complications following burn injury.

Impact of caspase-8 and PKA in regulating neutrophil-derived microparticle generation.

The morbidity and mortality from sepsis continues to remain high despite extensive research into understanding this complex immunologic process. Further, while source control and antibiotic therapy have improved patient outcomes, many immunologically based therapies have fallen short. Microparticles (MPs) are intact vesicles that serve as mediators of intercellular communication as well as markers of inflammation in various disease processes. We have previously demonstrated that MPs can be produced at the infected foci during sepsis, are predominantly of neutrophil derivation (NDMPs) and can modulate immune cells. In this study, we sought to elucidate the molecular mechanisms underlying NDMP generation. Using thioglycolate (TGA) to recruit and activate neutrophils, we first determined that intra-peritoneal TGA increase NDMP accumulation. We next utilized TGA-elicited neutrophils in vitro to investigate signaling intermediates involved in NDMP production, including the intrinsic and extrinsic caspase pathways, cAMP dependent PKA and Epac activation as well as the role myosin light chain kinase (MLCK) as a final mediator of NDMP release. We observed that NDMP generation was dependent on the extrinsic caspase apoptotic pathway (caspase 3 and caspase 8), cAMP activation of PKA but not of Epac, and on activation of MLCK. Altogether, these data contribute to an overall framework depicting the molecular mechanisms that regulate NDMP generation.

Neutrophil derived microparticles increase mortality and the counter-inflammatory response in a murine model of sepsis

Although advances in medical care have significantly improved sepsis survival, sepsis remains the leading cause of death in the ICU. This is likely due to a lack of complete understanding of the pathophysiologic mechanisms that lead to dysfunctional immunity. Neutrophil derived microparticles (NDMPs) have been shown to be the predominant microparticle present at infectious and inflamed foci in human models, however their effect on the immune response to inflammation and infection is sepsis has not been fully elucidated. As NDMPs may be a potential diagnostic and therapeutic target, we sought to determine the impact NDMPs on the immune response to a murine polymicrobial sepsis. We found that peritoneal neutrophil numbers, bacterial loads, and NDMPs were increased in our abdominal sepsis model. When NDMPs were injected into septic mice, we observed increased bacterial load, decreased neutrophil recruitment, increased expression of IL-10 and worsened mortality. Furthermore, the NDMPs express phosphatidylserine and are ingested by F4/80 macrophages via a Tim-4 and MFG-E8 dependent mechanism. Finally, upon treatment, NDMPs decrease macrophage activation, increase IL-10 release and decrease macrophage numbers. Altogether, these data suggest that NDMPs enhance immune dysfunction in sepsis by blunting the function of neutrophils and macrophages, two key cell populations involved in the early immune response to infection. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Quantifying patient improvement following sacral neuromodulation: is it time for a new scoring system for fecal incontinence?

BACKGROUND: The Cleveland Clinic Florida Fecal Incontinence score is widely used to assess the severity of fecal incontinence. OBJECTIVE: We hypothesized that the Cleveland Clinic Fecal Florida Incontinence score is useful at establishing baseline disease severity, but it may underestimate the response to treatment following sacral neuromodulation because of the large number of patients who still wear a pad despite improved continence, as well as the inability to track improvements in urgency. DATA SOURCES: Data were obtained from prospectively maintained database of patients treated with sacral neuromodulation for fecal incontinence at 2 institutions beginning in 2011. DESIGN: A retrospective review of the individual components of Cleveland Clinic Fecal Florida Incontinence scores in response to treatment with sacral neuromodulation was performed. SETTINGS: The study was conducted at 1 academic medical center and 1 community medical center. PATIENTS: One hundred twenty-one consecutive patients were treated with sacral neuromodulation for fecal incontinence. INTERVENTIONS: No interventions occurred. MAIN OUTCOME MEASURES: Individual components of posttreatment Cleveland Clinic Florida Fecal Incontinence scores and subjective improvement in fecal urgency were the primary outcomes measured. RESULTS: The median preoperative Cleveland Clinic Fecal Florida Incontinence score of 14 decreased to 3 (interquartile range, 2–4) at 12 months. Of the patients, 66.1% reported still wearing a pad after the procedure. The reason for wearing a pad was residual fecal incontinence (41%), habit despite normal continence (35.3%), and urinary incontinence with complete fecal continence (23.5%). Of patients who report wearing a pad, 59% have falsely elevated Cleveland Clinic Fecal Florida Incontinence scores owing to wearing a pad despite complete fecal continence. Additionally, 96.3% of patients reported improvement in fecal urgency. LIMITATIONS: This retrospective study did not include a comparison with an alternative scoring system. CONCLUSIONS: Although the Cleveland Clinic Fecal Florida Incontinence score is a validated scale, which is simple to use for baseline disease severity, it may underestimate patient response to treatment. Additionally, it does not capture improvement in urgency. The ideal scoring system would be easy to use in clinical practice, and would account for improvement in fecal urgency.

Assessing the immune status of critically ill trauma patients by flow cytometry.

BackgroundUnintentional injury or trauma remains the leading cause of death among young adults. About one fifth of these trauma patients require care in an intensive care unit (ICU) because of severity of injuries and comorbidities. Patients hospitalized in an ICU are at increased risk for nosocomial infections, such as urinary tract infections, pneumonia, bacteremia, and wound infections. Many of these patients will develop sepsis or septic shock, and some will progress to multiple organ failure and death. The balance between the proinflammatory and counterinflammatory immune response appears to be a driving factor in this progression. At present, there is no proposed method for the timely detection of the immune status in trauma patients, making rational decisions to use immune-altering therapies difficult. ObjectiveWe demonstrate that flow cytometry, with its capabilities to characterize and/or enumerate (a) leukocyte subtypes, (b) leukocyte activation markers, (c) leukocyte-derived cytokines and microvesicles, and (d) leukocyte function is well suited to monitor the immune status of critically ill trauma patients. MethodsInformation for the review was obtained from the extant literature. DiscussionWe suggest that flow cytometry is a research method that might aid nurse scientists in investigating the immune status of critically ill patients, the recovery status of conditions such as hemorrhagic shock and tissue injury and the relationship between cancer disease progression and symptoms. Therefore, flow cytometry has the potential to broaden nursing research priority areas so that a comprehensive approach to understanding the cellular response is attained.

Postoperative predictors of early discharge following laparoscopic segmental colectomy

PurposeThere is increasing pressure to shorten length of stay (LOS) after major surgical procedures. Although laparoscopic colectomy has been shown to have shorter LOS than open colectomy, not all patients experience a short length of stay. Predictive factors for early discharge after laparoscopic colectomy have not been clearly defined. We hypothesized that patients who exhibit a brisk urine output and lack of a systemic inflammatory response on the first postoperative day would experience a shorter postoperative stay after laparoscopic colectomy.MethodsWe performed a retrospective review of patients undergoing laparoscopic segmental colectomy by one of colorectal surgeons from 2012 to 2013. Patient demographics, operative characteristics, and postoperative factors were examined. A multiple linear regression model was used to examine the impact of various factors on length of stay, while controlling for confounding variables. Systemic inflammatory response syndrome (SIRS) was defined using Society of Critical Care Medicine consensus definitions.ResultsA total of 127 patients underwent a laparoscopic segmental colectomy. When controlling for confounding variables, ileus, postoperative complication, and SIRS response were associated with 2.67, 1.16, and 0.42 additional hospital days, respectively, while each additional liter of urine output on postoperative day 1 was associated with a 0.23-day decrease in LOS (p = 0.006).ConclusionsIn the absence of postoperative ileus or overt complication, patients who do not exhibit a SIRS response, and have a brisk urine output on postoperative day (POD) 1, may be targeted for early hospital discharge after laparoscopic colectomy.