Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

BACKGROUND: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. METHODS: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. RESULTS: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. CONCLUSION: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

A Whole Blood Enzyme-Linked Immunospot Assay for Functional Immune Endotyping of Septic Patients.

Sepsis initiates simultaneous pro- and anti-inflammatory processes, the pattern and intensity of which vary over time. The inability to evaluate the immune status of patients with sepsis in a rapid and quantifiable manner has undoubtedly been a major reason for the failure of many therapeutic trials. Although there has been considerable effort to immunophenotype septic patients, these methods have often not accurately assessed the functional state of host immunity, lack dynamic range, and are more reflective of molecular processes rather than host immunity. In contrast, ELISpot assay measures the number and intensity of cytokine-secreting cells and has excellent dynamic range with rapid turnaround. We investigated the ability of a (to our knowledge) novel whole blood ELISpot assay and compared it with a more traditional ELISpot assay using PBMCs in sepsis. IFN-γ and TNF-α ELISpot assays on whole blood and PBMCs were undertaken in control, critically ill nonseptic, and septic patients. Whole blood ELISpot was easy to perform, and results were generally comparable to PBMC-based ELISpot. However, the whole blood ELISpot assay revealed that nonmonocyte, myeloid populations are a significant source of ex vivo TNF-α production. Septic patients who died had early, profound, and sustained suppression of innate and adaptive immunity. A cohort of septic patients had increased cytokine production compared with controls consistent with either an appropriate or excessive immune response. IL-7 restored ex vivo IFN-γ production in septic patients. The whole blood ELISpot assay offers a significant advance in the ability to immunophenotype patients with sepsis and to guide potential new immunotherapies.

Severe immunosuppression and not a cytokine storm characterizes COVID-19 infections.

COVID-19-associated morbidity and mortality have been attributed to a pathologic host response. Two divergent hypotheses have been proposed: hyperinflammatory cytokine storm; and failure of host protective immunity that results in unrestrained viral dissemination and organ injury. A key explanation for the inability to address this controversy has been the lack of diagnostic tools to evaluate immune function in COVID-19 infections. ELISpot, a highly sensitive, functional immunoassay, was employed in 27 patients with COVID-19, 51 patients with sepsis, 18 critically ill nonseptic (CINS) patients, and 27 healthy control volunteers to evaluate adaptive and innate immune status by quantitating T cell IFN-É£ and monocyte TFN-α production. Circulating T cell subsets were profoundly reduced in COVID-19 patients. Additionally, stimulated blood mononuclear cells produced less than 40%-50% of the IFN-É£ and TNF-α observed in septic and CINS patients, consistent with markedly impaired immune effector cell function. Approximately 25% of COVID-19 patients had increased IL-6 levels that were not associated with elevations in other canonical proinflammatory cytokines. Collectively, these findings support the hypothesis that COVID-19 suppresses host functional adaptive and innate immunity. Importantly, IL-7 administered ex vivo restored T cell IFN-É£ production in COVID-19 patients. Thus, ELISpot may functionally characterize host immunity in COVID-19 and inform prospective therapies.

Development of Stable Boron Nitride Nanotube and Hexagonal Boron Nitride Dispersions for Electrophoretic Deposition.

Boron nitride nanotubes (BNNTs) represent a relatively new class of materials that provides alternative electrical and thermal properties to the carbon analogue. The high chemical and thermal stability and large band gap combined with high electrical resistance make BNNTs desirable in several thin-film applications. In this study, stable BNNT and hexagonal boron nitride (hBN) particle dispersions have been developed using environmentally friendly advanced oxidation processing (AOP) that can be further modified for electrophoretic deposition (EPD) to produce thin films. The characterization of the dispersions has revealed how the hydroxyl radicals produced in AOP react with BNNT/hBN and contaminant boron nanoparticles (BNPs). While the radicals remove the carbon contaminant present on BNNT/hBN and increase dispersion stability, they also oxidize the BNPs and the boron oxide produced, which, conversely, reduces the dispersion stability. The use of high- or low-powered ultrasonication in combination with the AOP affects the rate of the competing reactions, with low-powered sonication and AOP providing the best combination for producing stable dispersions with high concentrations. BNNT/hBN dispersions were functionalized with polyethyleneimine to facilitate EPD, where films of several micrometer thickness were readily deposited onto stainless steel and glass-fiber fabrics. BNNT/hBN films produced on glass fabrics by EPD exhibited a consistent through-thickness macroporosity that was facilitated by platelet and nanotube stacking. The film macroporosity present on the coated fabrics was suitable for use as separator layers in supercapacitors and provided improved device robustness with a minimal impact on electrochemical performance.

Theoretical insight on the origin of anelasticity in zinc oxide nanowires.

Anelasticity of nanowires has recently attracted attention as an interesting property for high efficiency mechanical damping materials. While the mechanism of anelasticity has so far been analysed using continuum mechanical models based on defect diffusion, the mechanisms behind anelasticity have not yet been determined on an atomic level. Such information is needed in order to be able to design and synthesise new nanomaterials having desired mechanical properties. Here we determine the potential mechanism of anelasticity in narrow zinc oxide nanowires by analyzing the bond stretching and compression within the nanowire structure based on density functional theory. Our approach shows that different local minimum structures are created when different strain patterns are applied which give rise to the anelastic behavior. These findings can be applied for the prediction of potential anelasticity of other nanowire materials.

IL-10 Has Differential Effects on the Innate and Adaptive Immune Systems of Septic Patients.

Sepsis, a disease of divergent pro- and anti-inflammatory-mediated pathways, has a high prevalence of morbidity and mortality, yet an understanding of potential unifying mediators between these pathways that may improve clinical outcomes is largely unclear. IL-10 has classically been designated an immunosuppressive cytokine, although recent data suggest that under certain conditions IL-10 can be immune stimulatory. We sought to further investigate the effect of IL-10 on innate and adaptive immunity in an in vitro human observational cohort study in patients with sepsis via modulation of IL-10 on IFN-γ production by T cells and TNF-α production and HLA-DR expression by monocytes. These results were compared with critically ill nonseptic patients and healthy volunteers. ELISpot analysis was performed using PBMC fraction from patient whole-blood samples. Finally, to provide additional potential clinical relevance, we examined the effect of IL-10 on T cell IFN-γ production in an in vivo cecal ligation and puncture model of sepsis using C57 black/J6 female mice. We found that inhibition of IL-10 significantly increased both production of T cell IFN-γ and monocyte TNF-α, whereas addition of IL-10 increased T cell IFN-γ production but decreased monocyte production of TNF-α and HLA-DR expression. There was no significant effect of IL-10 on control cohorts. IL-10-treated septic mice demonstrated increased IFN-γ production in splenocytes. Thus, IL-10 demonstrates both pro- and anti-inflammatory effects in the septic microenvironment, which is likely cell and context dependent. Further elucidation of relevant signaling pathways may direct future therapeutic targets.

Adsorption of toxic gases on silicene/Ag(111).

Silicene is a two-dimensional nanomaterial, composed of Si atoms arranged into a buckled honeycomb network. It has become of great interest in recent years due to its remarkable properties such as its natural compatibility with current silicon-based technology. Due to its extreme thinness on the nanoscale, and large lateral dimensions, it has potential applications in gas sensing, gas storage and components in modern electronic devices. In this work, density functional theory calculations and ab initio molecular dynamics simulations are used to examine the reaction of SO2, NO2 and H2S on the Si/Ag(111) surface. It was shown that each gas will adsorb on the surface in different orientations and adsorption sites. SO2 and NO2 were found to chemisorb on the surface, whereas H2S was found to physisorb. SO2 and H2S adsorb associatively, whereas NO2 readily dissociates, producing adsorbed oxygen, and gaseous NO. At elevated temperatures, the SO2 and NO2 remain strongly bound to the surface, resulting in poisoning of the silicene, while H2S readily desorbs. Ab initio molecular dynamics also show that NO2 will selectively bind before SO2 when both gases are present in the same environment. This work shows that Si/Ag(111) may provide useful properties for gas sensing and storage applications.

Tuning the work function of the silicene/4 × 4 Ag(111) surface.

Silicene, the silicon analog of graphene, is an atomically thin two-dimensional material with promising applications in gas sensing, storage and as components in modern electronic devices. Silicene epitaxially grown on the Ag(111) surface can expand the utility of the silver surface by enabling the tuning of its work function through the functionalisation of silicene. Here we examine the electronic and structural properties and the thermodynamic stability of functionalised silicene/4 × 4 Ag(111) using density functional theory calculations coupled with ab initio molecular dynamics (AIMD) simulations. We focus on 11 functional groups, namely phenyl, methyl, hydroxyl, cyano, methoxyl, amino and ethylmethylamine, in addition to 4 halogen atoms. These functional groups are shown to endow the Si/Ag(111) surface with a large variation in the work function. Our AIMD simulations confirm the thermodynamic stability of these 11 functionalised structures. This work shows the possibility of tuning the electronic structure of silicene by functionalisation, which could then be utilized in polymer solar cells and nanoelectronic circuit components.

Acceptability of financial incentives for maintenance of weight loss in mid-older adults: a mixed methods study.

BACKGROUND: Health insurers worldwide implement financial incentive schemes to encourage health-related behaviours, including to facilitate weight loss. The maintenance of weight loss is a public health challenge, and as non-communicable diseases become more prevalent with increasing age, mid-older adults could benefit from programs which motivate weight loss maintenance. However, little is understood about their perceptions of using financial incentives to maintain weight loss. METHODS: We used mixed methods to explore the attitudes and views of participants who had completed an Australian weight loss and lifestyle modification program offered to overweight and obese health insurance members with weight-related chronic diseases, about the acceptability and usefulness of different types of financial incentives to support weight loss maintenance. An online survey was completed by 130 respondents (mean age = 64 years); and a further 28 participants (mean age = 65 years) attended six focus groups. RESULTS: Both independent samples of participants supported a formalised maintenance program. Online survey respondents reported that non-cash (85.2%) and cash (77%) incentives would be potentially motivating; but only 40.5% reported that deposit contracts would motivate weight loss maintenance. Results of in-depth discussions found overall low support for any type of financial incentive, but particularly deposit contracts and lotteries. Some participants expressed that improved health was of more value than a monetary incentive and that they felt personally responsible for their own health, which was at odds with the idea of financial incentives. Others suggested ongoing program and peer support as potentially useful for weight loss maintenance. CONCLUSIONS: If financial incentives are considered for mid-older Australian adults in the health insurance setting, program planners will need to balance the discordance between participant beliefs about the individual responsibility for health and their desire for external supports to motivate and sustain weight loss maintenance.

How ionic species structure influences phase structure and transitions from protic ionic liquids to liquid crystals to crystals.

The phase behaviour of n-alkylammonium (C6 to C16) nitrates and formates has been characterised using synchrotron small angle and wide angle X-ray scattering (SAXS/WAXS), differential scanning calorimetry (DSC), cross polarised optical microscopy (CPOM) and Fourier transform infrared spectroscopy (FTIR). The protic salts may exist as crystalline, liquid crystalline or ionic liquid materials depending on the alkyl chain length and temperature. n-Alkylammonium nitrates with n ≥ 6 form thermotropic liquid crystalline (LC) lamellar phases, whereas n ≥ 8 was required for the formate series to form this LC phase. The protic ionic liquid phase showed an intermediate length scale nanostructure resulting from the segregation of the polar and nonpolar components of the ionic liquid. This segregation was enhanced for longer n-alkyl chains, with a corresponding increase in the correlation length scale. The crystalline and liquid crystalline phases were both lamellar. Phase transition temperatures, lamellar d-spacings, and liquid correlation lengths for the n-alkylammonium nitrates and formates were compared with those for n-alkylammonium chlorides and n-alkylamines. Plateau regions in the liquid crystalline to liquid phase transition temperatures as a function of n for the n-alkylammonium nitrates and formates are consistent with hydrogen-bonding and cation-anion interactions between the ionic species dominating alkyl chain-chain van der Waals interactions, with the exception of the mid chained hexyl- and heptylammonium formates. The d-spacings of the lamellar phases for both the n-alkylammonium nitrates and formates were consistent with an increase in chain-chain layer interdigitation within the bilayer-based lamellae with increasing alkyl chain length, and they were comparable to the n-alkylammonium chlorides.

Frontline Science: Defects in immune function in patients with sepsis are associated with PD-1 or PD-L1 expression and can be restored by antibodies targeting PD-1 or PD-L1.

Sepsis is a heterogeneous syndrome comprising a highly diverse and dynamic mixture of hyperinflammatory and compensatory anti-inflammatory immune responses. This immune phenotypic diversity highlights the importance of proper patient selection for treatment with the immunomodulatory drugs that are entering clinical trials. To better understand the serial changes in immunity of critically ill patients and to evaluate the potential efficacy of blocking key inhibitory pathways in sepsis, we undertook a broad phenotypic and functional analysis of innate and acquired immunity in the same aliquot of blood from septic, critically ill nonseptic, and healthy donors. We also tested the ability of blocking the checkpoint inhibitors programmed death receptor-1 (PD-1) and its ligand (PD-L1) to restore the function of innate and acquired immune cells. Neutrophil and monocyte function (phagocytosis, CD163, cytokine expression) were progressively diminished as sepsis persisted. An increasing frequency in PD-L1+-suppressor phenotype neutrophils [low-density neutrophils (LDNs)] was also noted. PD-L1+ LDNs and defective neutrophil function correlated with disease severity, consistent with the potential importance of suppressive neutrophil populations in sepsis. Reduced neutrophil and monocyte function correlated both with their own PD-L1 expression and with PD-1 expression on CD8+ T cells and NK cells. Conversely, reduced CD8+ T cell and NK cell functions (IFN-γ production, granzyme B, and CD107a expression) correlated with elevated PD-L1+ LDNs. Importantly, addition of antibodies against PD-1 or PD-L1 restored function in neutrophil, monocyte, T cells, and NK cells, underlining the impact of the PD-1:PD-L1 axis in sepsis-immune suppression and the ability to treat multiple deficits with a single immunomodulatory agent.

TLR3 agonist improves survival to secondary pneumonia in a double injury model.

BACKGROUND: Toll-like receptors (TLR) can initiate various immune responses and are therefore activated under diverse infectious states. Previous studies have focused on TLR3 primarily as an antiviral pathway. However, recent research has demonstrated its efficacy in bacterial infection. Having developed a murine double injury model of cecal ligation and puncture (CLP) followed by Pseudomonas aeruginosa (Pa), we hypothesized that targeted administration of Poly I:C, a TLR3 agonist, would protect mice against secondary pneumonia. MATERIAL AND METHODS: B6 mice underwent CLP followed 4 d afterward by an intranasal dose of Pa. Animals were given Poly I:C or vehicle (phosphate-buffered saline) intranasally 24 h post CLP and every day thereafter for a total of 6 d. For acute studies, mice were sacrificed at two time points, 4 d post CLP and 1 d post pneumonia (Pa). RESULTS: Poly I:C treatment led to a significant improvement in survival (69% versus 33%). Cytokine analysis from bronchioalveolar lavage displayed significant differences both immediately before and after pneumonia. Bronchioalveolar lavage cultures taken at 24 h post double injury showed significantly higher colony counts in the lungs of control animals compared with those of Poly I:C animals. Measurements of TLR3 expression showed significant increases within both the immune and lung epithelial cells of Poly I:C-treated mice. Finally, the lungs of treated animals had significant increases in lymphocytes and innate cells. CONCLUSIONS: The prophylactic treatment applied in this clinically relevant model further illustrates the overarching hypothesis of immune dysfunction and the possibility of corrective immune modulation within the setting of sepsis.

Immunosuppression in patients who die of sepsis and multiple organ failure.

CONTEXT: Severe sepsis is typically characterized by initial cytokine-mediated hyperinflammation. Whether this hyperinflammatory phase is followed by immunosuppression is controversial. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting. OBJECTIVES: To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression. DESIGN, SETTING, AND PARTICIPANTS: Rapid postmortem spleen and lung tissue harvest was performed at the bedsides of 40 patients who died in intensive care units (ICUs) of academic medical centers with active severe sepsis to characterize their immune status at the time of death (2009-2011). Control spleens (n = 29) were obtained from patients who were declared brain-dead or had emergent splenectomy due to trauma; control lungs (n = 20) were obtained from transplant donors or from lung cancer resections. MAIN OUTCOME MEASURES: Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. RESULTS: The mean ages of patients with sepsis and controls were 71.7 (SD, 15.9) and 52.7 (SD, 15.0) years, respectively. The median number of ICU days for patients with sepsis was 8 (range, 1-195 days), while control patients were in ICUs for 4 or fewer days. The median duration of sepsis was 4 days (range, 1-40 days). Compared with controls, anti-CD3/anti-CD28-stimulated splenocytes from sepsis patients had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5361 (95% CI, 3327-7485) pg/mL vs 418 (95% CI, 98-738) pg/mL; interferon γ, 1374 (95% CI, 550-2197) pg/mL vs 37.5 (95% CI, -5 to 80) pg/mL; interleukin 6, 3691 (95% CI, 2313-5070) vs 365 (95% CI, 87-642) pg/mL; and interleukin 10, 633 (95% CI, -269 to 1534) vs 58 (95% CI, -39 to 156) pg/mL; (P < .001 for all). There were similar reductions in 5-hour lipopolysaccharide-stimulated cytokine secretion. Cytokine secretion in sepsis patients was generally less than 10% that in controls, independent of age, duration of sepsis, corticosteroid use, and nutritional status. Although differences existed between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of sepsis patients vs cancer patients and vs transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells. CONCLUSIONS: Patients who die in the ICU following sepsis compared with patients who die of nonsepsis etiologies have biochemical, flow cytometric, and immunohistochemical findings consistent with immunosuppression. Targeted immune-enhancing therapy may be a valid approach in selected patients with sepsis.

Increased susceptibility to Candida infection following cecal ligation and puncture.

Secondary infection following septic insult represents a significant cause of morbidity and mortality in hospitalized patients. Sepsis induced immunosuppression is a major factor in the host's susceptibility to nosocomial infections and Candida albicans accounts for a growing number of these. Given the importance of improving our understanding of the immune response to sepsis and the increasing rates of C. albicans infections, we sought to develop a murine model of double injury consisting of primary peritonitis, i.e., cecal ligation and puncture (CLP), followed by a secondary challenge of C. albicans. As observed in previous work, after primary injury the immune profile of the host changes over time. Therefore, while keeping the mortality rates from the respective individual injuries low, we altered the timing of the secondary injury between two post-CLP time points, day two and day four. Mice subjected to C. albicans infection following CLP have significantly different survival rates dependent upon timing of secondary injury. Animals challenged with C. albicans at two days post CLP had 91% mortality whereas animals challenged at four days had 47% mortality. This improvement in survival at four days was associated with restoration of innate cell populations and as evidenced by stimulated splenocytes, increases in certain inflammatory cytokines. In addition, we show that susceptibility to C. albicans infection following CLP is dependent upon the depth of immunosuppression. Although at four days post-CLP there is a partial reconstitution of the immune system, these animals remain more susceptible to infection compared to their single injury (C. albicans alone) counterparts. Collectively, these studies demonstrate that immunosuppression following initial septic insult changes over time. This novel, two hit model of CLP followed by Candida provides additional insight into the immune compromised state created by primary peritonitis, and thereby opens up another avenue of investigation into the causes and possible cures of an emerging clinical problem.

IL-15 prevents apoptosis, reverses innate and adaptive immune dysfunction, and improves survival in sepsis.

IL-15 is a pluripotent antiapoptotic cytokine that signals to cells of both the innate and adaptive immune system and is regarded as a highly promising immunomodulatory agent in cancer therapy. Sepsis is a lethal condition in which apoptosis-induced depletion of immune cells and subsequent immunosuppression are thought to contribute to morbidity and mortality. This study tested the ability of IL-15 to block apoptosis, prevent immunosuppression, and improve survival in sepsis. Mice were made septic using cecal ligation and puncture or Pseudomonas aeruginosa pneumonia. The experiments comprised a 2 x 2 full factorial design with surgical sepsis versus sham and IL-15 versus vehicle. In addition to survival studies, splenic cellularity, canonical markers of activation and proliferation, intracellular pro- and antiapoptotic Bcl-2 family protein expression, and markers of immune cell apoptosis were evaluated by flow cytometry. Cytokine production was examined both in plasma of treated mice and splenocytes that were stimulated ex vivo. IL-15 blocked sepsis-induced apoptosis of NK cells, dendritic cells, and CD8 T cells. IL-15 also decreased sepsis-induced gut epithelial apoptosis. IL-15 therapy increased the abundance of antiapoptotic Bcl-2 while decreasing proapoptotic Bim and PUMA. IL-15 increased both circulating IFN-gamma, as well as the percentage of NK cells that produced IFN-gamma. Finally, IL-15 increased survival in both cecal ligation and puncture and P. aeruginosa pneumonia. In conclusion, IL-15 prevents two immunopathologic hallmarks of sepsis, namely, apoptosis and immunosuppression, and improves survival in two different models of sepsis. IL-15 represents a potentially novel therapy of this highly lethal disorder.

Bim siRNA decreases lymphocyte apoptosis and improves survival in sepsis.

To assess the degree of lymphocyte apoptosis and survival in mice treated with small interfering RNA (siRNA) targeted to Bim, a proapoptotic molecule from the Bcl-2 family, within a clinically relevant model of sepsis. C57BL/6 mice were treated with a single dose of Bim siRNA complexed in cationic liposomes via tail vein injection. Approximately 24 h later, mice were subjected to either cecal ligation and puncture (CLP) or sham surgery. Animals were killed at 20 h postsurgery, and spleens were harvested for fluorescence-activated cell sorting analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling as a marker for apoptosis. A second cohort of mice was followed for survival for 7 days. The degree of lymphocyte apoptosis in Bim siRNA-treated mice was markedly decreased compared with controls. Fluorescent activated cell sorter analysis demonstrated 13.1% +/- 1.2% B-cell apoptosis and 11.5% +/- 1.5% T-cell apoptosis in control mice compared with 2.7% +/- 0.4% B-cell apoptosis and 3.9% +/- 0.3% T-cell apoptosis in Bim siRNA-treated mice after CLP (P < 0.001 and P < 0.01, respectively). This striking difference in lymphocyte apoptosis correlated with a significant survival advantage in Bim siRNA-treated mice. At 7 days, there was 90% overall survival in Bim siRNA-treated septic mice compared with 50% overall survival in control septic mice (P < 0.05). Treatment with Bim siRNA in vivo has the potential to be an effective therapy in the treatment of sepsis.

Splenic CD4+ T cells have a distinct transcriptional response six hours after the onset of sepsis.

BACKGROUND: In animal and human autopsy studies of sepsis, CD4+ splenocytes either undergo apoptosis or are polarized to the Th2 effector subtype. In mice, these changes occur within 24 hours of the onset of sepsis. Preventing the loss of CD4+ T cells and the Th2-polarization of CD4+ T cells provides a significant survival advantage in mouse models of sepsis. The molecular mechanism(s) for the phenotypic changes of splenic CD4+ T cells in sepsis are not well understood. STUDY DESIGN: CD4+ splenocytes were enriched by negative selection from disaggregated spleens of septic and sham-operated mice at 6 and 24 hours after surgery. Phenotypic analysis using cell surface markers (CD25, CD44, CD62L, CD69), cytokine secretion in response to CD3/CD28 coligation, and whole genome microarray gene expression profiles were obtained for these cells. RESULTS: Consistent with previous reports, sepsis induced a progressive decrease in the number of CD4+ splenocytes and a time-dependent alteration in CD4+ T-cell phenotype. At 6 hours, when no differences in cell number or surface marker expression were observed, significant alterations in RNA abundance were measured for 498 probe sets. Ontologic classification of these genes indicated changes in cellular physiology. Pathway analysis indicated that T-cell receptor signaling and mitogen-activated protein kinase signaling were significantly altered by sepsis. CONCLUSIONS: These data demonstrated a sepsis-specific transcriptional program that precedes sepsis-induced phenotypic changes in CD4+ splenocytes.

Antibiotics improve survival and alter the inflammatory profile in a murine model of sepsis from Pseudomonas aeruginosa pneumonia.

Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.

Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive transfer of necrotic splenocytes improves survival in sepsis.

In sepsis, both necrotic and apoptotic cell death can occur. Apoptotic cells induce anergy that could impair the host response, whereas necrotic cells cause immune activation that might result in enhanced antimicrobial defenses. We determined whether adoptive transfer of apoptotic or necrotic cells impacted survival in a clinically relevant sepsis model. We also evaluated the effects of adoptive transfer of apoptotic or necrotic cells on the prototypical TH1 and TH2 cytokines IFN-gamma and IL-4, respectively. C57BL6/J mice had adoptive transfer of apoptotic (irradiated) or necrotic (freeze thaw) splenocytes. Controls received saline. Apoptotic cells greatly increased mortality, whereas necrotic splenocytes markedly improved survival, P < or = 0.05. The contrasting effects that apoptotic or necrotic cells exerted on survival were mirrored by opposite effects on splenocyte IFN-gamma production with greatly decreased and increased production, respectively. Importantly, either administration of anti-IFN-gamma antibodies or use of IFN-gamma knockout mice prevented the survival benefit occurring with necrotic cells. This study demonstrates that the type of cell death impacts survival in a clinically relevant model and identifies a mechanism for the immune suppression that is a hallmark of sepsis. Necrotic cells (and likely apoptotic cells) exert their effects via modulation of IFN-gamma

Effects of age on mortality and antibiotic efficacy in cecal ligation and puncture.

The incidence and mortality of sepsis increase with age, consequently, 80% of the clinical mortality from sepsis occurs in patients over age 65. Despite this aged clinical population, most research models of sepsis use 6- to 16-week-old mice as patient surrogates. This age range of mice corresponds to human ages 10 to 17 years. To assess the influence of age on rodent CLP and on antibiotic therapy, we studied young (4 month), mature (12 month), and aged (24 month) mice. Male C57BL/6 mice (n = 27-30 in each age group) were subjected to cecal ligation and puncture (CLP), two punctures with a 25-gauge needle. Mice were observed untreated for 10 days. Young mice had 20% mortality, mature mice had 70% mortality (P = 0.0013 vs. young), and aged mice had 75% mortality (P = 0.0001 vs. young). To assess the effects of age on antibiotic therapy, mice were subjected to CLP as above (n = 38-40 in each age group). Mice were then randomized to treatment with intraperitoneal injections of ceftriaxone and metronidazole or normal saline. Therapy was initiated 12 h after CLP, and injections were repeated every 12 h for 7 days. Young mice saw a 56% decrease in mortality from CLP with antibiotic therapy (P = 0.001), and mature mice had a 30% decrease in mortality (P = 0.06). Aged mice saw no benefit from antibiotic therapy. We also compared plasma cytokine levels between young and aged mice after CLP. When compared with young mice, aged mice had higher levels of IL-6 and TNF-alpha 24 h after CLP. However, high IL-6 was predictive of mortality at any age. Mice appear to have age-dependent responses to intra-abdominal sepsis and to appropriate therapy.