Outcomes of arterial vascular extremity trauma in pediatric patients.

BACKGROUND: Vascular trauma in children, although rare, carries significant risk for repair. Here we report outcomes from a single trauma center for children with extremity vascular trauma, proximal to the digits. METHODS: Retrospective chart review of patients less than age 18years with an acute, non-iatrogenic traumatic arterial vascular injury of the upper and/or lower extremity between January 2008 and December 2013. Abstracted patient demographics, injury characteristics, surgical management, and disposition were summarized and compared with nonparametric methods. RESULTS: 23 children comprised the study cohort: median age of 8years (IQR: 4.6-12), 61% (n=14) males, 100% survival. Penetrating injuries were the predominate mechanism (n=17, 74%). The median time to presentation was 154min (IQR: 65-330). Acute operations for revascularization included a primary repair (n=15, 65%) or reversed vein graft (n=7, 30%). Fasciotomies were done for 3 (13%) patients. Three amputations were done for failed revascularization. Upper extremity vascular injury (n=15, 65%) was more common. The rate of associated extremity fracture was similar between upper (21%) and lower (33%) extremities (p=0.643). Eight (35%) patients required additional surgery most commonly for debridement, washouts and dressing changes. Three patients' hospital stays were complicated by infection. Impaired function was the most common short- and long-term complication (60%, 75%). CONCLUSION: Pediatric vascular injuries are commonly associated with penetrating injuries and male gender and occurred more frequently in the upper extremities. Overall patency rates after repair were 87%. Fasciotomies were done in 13% of patients, and the overall surgical amputation rate was 13%. There was no mortality in this cohort; however, multiple operations are commonly required, including the return to OR for washouts, debridements and dressing changes. The most common short- and long-term complication was impaired function. Overall good results are achievable in pediatric vascular trauma treated with revascularization.

Outcomes of digital artery revascularization in pediatric trauma.

BACKGROUND: Digit amputation is rare in pediatric trauma but can lead to functional morbidity. The true incidence of digital arterial injury is lacking in the literature, and revascularization techniques are not well-described. METHODS: Retrospective review of a pediatric trauma registry identified patients with a digital artery injury between July 2008 and December 2013. Isolated vein injuries and arterial injuries proximal to the digits were excluded. Descriptive statistics were used. RESULTS: Twenty-five subjects met inclusion. Most were male (n=16; 64%) and the median age was 6.8 (IQR: 2.8, 11.1) years. The most common blunt (n=12) trauma was struck/crushed by object (n=4; 33%) and the most common penetrating (n=13) trauma was because of glass (n=9; 69%). All subjects were managed operatively. Initial operations for arterial repair were primary arterial repair (15; 20%), vein graft (7; 28%), thrombectomy (1; 4%), and amputation (1; 4%). Twelve patients (48%) had reported complications at initial follow-up, but only two (8%) had long-term (>24weeks) sequelae. CONCLUSION: Digital artery injury is rare among pediatric traumas. Functional outcomes after digital artery revascularization are favorable. Primary repair can successfully manage these injuries and vein grafting appears to be a suitable alternative when primary repair is not feasible.

Reactivation of multiple viruses in patients with sepsis.

A current controversy is whether patients with sepsis progress to an immunosuppressed state. We hypothesized that reactivation of latent viruses occurred with prolonged sepsis thereby providing evidence of clinically-relevant immunosuppression and potentially providing a means to serially-monitor patients' immune status. Secondly, if viral loads are markedly elevated, they may contribute to morbidity and mortality. This study determined if reactivation of herpesviruses, polyomaviruses, and the anellovirus TTV occurred in sepsis and correlated with severity. Serial whole blood and plasma samples from 560 critically-ill septic, 161 critically-ill non-septic, and 164 healthy age-matched patients were analyzed by quantitative-polymerase-chain-reaction for cytomegalovirus (CMV), Epstein-Barr (EBV), herpes-simplex (HSV), human herpes virus-6 (HHV-6), and TTV. Polyomaviruses BK and JC were quantitated in urine. Detectable virus was analyzed with respect to secondary fungal and opportunistic bacterial infections, ICU duration, severity of illness, and survival. Patients with protracted sepsis had markedly increased frequency of detectable virus. Cumulative viral DNA detection rates in blood were: CMV (24.2%), EBV (53.2%), HSV (14.1%), HHV-6 (10.4%), and TTV (77.5%). 42.7% of septic patients had presence of two or more viruses. The 50% detection rate for herpesviruses was 5-8 days after sepsis onset. A small subgroup of septic patients had markedly elevated viral loads (>104-106 DNA copies/ml blood) for CMV, EBV, and HSV. Excluding TTV, DNAemia was uncommon in critically-ill non-septic patients and in age-matched healthy controls. Compared to septic patients without DNAemia, septic patients with viremia had increased fungal and opportunistic bacterial infections. Patients with detectable CMV in plasma had higher 90-day mortality compared to CMV-negative patients; p<0.05. Reactivation of latent viruses is common with prolonged sepsis, with frequencies similar to those occurring in transplant patients on immunosuppressive therapy and consistent with development of an immunosuppressive state. Whether reactivated latent viruses contribute to morbidity and mortality in sepsis remains unknown.

L-plastin is essential for alveolar macrophage production and control of pulmonary pneumococcal infection.

We report that mice deficient for the hematopoietic-specific, actin-bundling protein L-plastin (LPL) succumb rapidly to intratracheal pneumococcal infection. The increased susceptibility of LPL(-/-) mice to pulmonary pneumococcal challenge correlated with reduced numbers of alveolar macrophages, consistent with a critical role for this cell type in the immediate response to pneumococcal infection. LPL(-/-) mice demonstrated a very early clearance defect, with an almost 10-fold-higher bacterial burden in the bronchoalveolar lavage fluid 3 h following infection. Clearance of pneumococci from the alveolar space in LPL(-/-) mice was defective compared to that in Rag1(-/-) mice, which lack all B and T lymphocytes, indicating that innate immunity is defective in LPL(-/-) mice. We did not identify defects in neutrophil or monocyte recruitment or in the production of inflammatory cytokines or chemokines that would explain the early clearance defect. However, efficient alveolar macrophage regeneration following irradiation required LPL. We thus identify LPL as being key to alveolar macrophage development and essential to an effective antipneumococcal response. Further analysis of LPL(-/-) mice will illuminate critical regulators of the generation of alveolar macrophages and, thus, effective pulmonary innate immunity.

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.

Nitric oxide delivery during cardiopulmonary bypass reduces postoperative morbidity in children--a randomized trial.

OBJECTIVE: Cardiac surgery requiring cardiopulmonary bypass and cardioplegic arrest leads to myocardial ischemic and reperfusion injury. Gaseous nitric oxide has been demonstrated to have a myocardial protective effect following ischemia-reperfusion. We hypothesized that gaseous nitric oxide administered during cardiopulmonary bypass would have similar beneficial effects. METHODS: In a prospective, randomized, blinded, placebo-controlled study, children undergoing repair of tetralogy of Fallot received either 20 ppm of gaseous nitric oxide or placebo delivered to the membrane oxygenator during cardiopulmonary bypass. RESULTS: A total of 16 children were randomized into 2 equal groups once their parents or guardians had given written informed consent. No differences were found in age, crossclamp time, cardiopulmonary bypass time, or methemoglobin between the 2 groups. The group receiving gaseous nitric oxide had a significantly shortened duration of mechanical ventilation (8.4 ± 7.6 vs 16.3 ± 6.5 hours; P < .05) and intensive care unit length of stay (53.8 ± 19.7 vs 79.4 ± 37.7 hours; P < .05) compared with the placebo group. The patients had significantly lower troponin levels at 12, 24, and 48 hours (P < .05) and lower B-type natriuretic peptide levels at 12 and 24 hours (P < .05). A trend was found toward a less positive fluid balance, with significantly less diuretic usage. The study patients had a greater mean hemoglobin at 48 hours, despite the absence of differences in chest tube output, packed red blood cell transfusion, platelet counts or transfusion requirements, fresh frozen plasma transfusion, or prothrombin time/partial thromboplastin time in the first 48 hours. CONCLUSIONS: The delivery of gaseous nitric oxide to the cardiopulmonary bypass circuit for children undergoing cardiac surgery results in myocardial protection, improved fluid balance, and an improved postoperative intensive care unit course.

Detection of viruses in young children with fever without an apparent source.

OBJECTIVE: Fever without an apparent source is common in young children. Currently in the United States, serious bacterial infection is unusual. Our objective was to determine specific viruses that might be responsible. METHODS: We enrolled children aged 2 to 36 months with temperature of 38°C or greater without an apparent source or with definite or probable bacterial infection being evaluated in the St Louis Children's Hospital Emergency Department and afebrile children having ambulatory surgery. Blood and nasopharyngeal swab samples were tested with an extensive battery of virus-specific polymerase chain reaction assays. RESULTS: One or more viruses were detected in 76% of 75 children with fever without an apparent source, 40% of 15 children with fever and a definite or probable bacterial infection, and 35% of 116 afebrile children (P < .001). Four viruses (adenovirus, human herpesvirus 6, enterovirus, and parechovirus) were predominant, being detected in 57% of children with fever without a source, 13% of children with fever and definite or probable bacterial infection, and 7% of afebrile children (P < .001). Thirty-four percent of 146 viral infections were detected only by polymerase chain reaction performed on blood. Fifty-one percent of children with viral infections and no evidence of bacterial infection were treated with antibiotics. CONCLUSIONS: Viral infections are frequent in children with fever without an apparent source. Testing of blood in addition to nasopharyngeal secretions expanded the range of viruses detected. Future studies should explore the utility of testing for the implicated viruses. Better recognition of viruses that cause undifferentiated fever in young children may help limit unnecessary antibiotic use.

Evaluation of the precision of emergency department diagnoses in young children with fever.

OBJECTIVE. To characterize causes of fever in children presenting to a pediatric emergency department (ED). METHODS. One-year retrospective review of ED records. Inclusion criteria were 2 to 36 months of age with a documented temperature ≥ 39°C. Exclusion criteria were elopement, repeat visit, and underlying diagnosis with a predisposition to infection. Medical records were reviewed using a predefined, study-specific, data abstraction tool. Based on diagnosis and pathogen detection, visits were assigned to 3 groups, laboratory confirmed pathogen and focal or nonfocal diagnosis without confirmed pathogen. RESULTS. A total of 1091 visits met inclusion criteria. Fourteen percent had a pathogen detected, 56% had a focal diagnosis without a confirmed pathogen, and 30% had a nonfocal diagnosis without confirmed pathogen. CONCLUSIONS. In a cohort of febrile children 2 to 36 months of age, only 14% had a confirmed pathogen. New rapid viral diagnostic techniques may provide an opportunity to improve diagnostic certainty in young children presenting with fever.

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.

Evidence for a novel blood RNA diagnostic for pediatric appendicitis: the riboleukogram.

OBJECTIVE: To test the hypothesis that gene expression analysis of circulating white blood cells and/or plasma cytokines could be used to improve diagnostic accuracy in children being evaluated for appendicitis. METHODS: We recruited 28 children being evaluated for abdominal pain from a tertiary pediatric emergency department. Twenty patients were used as a training cohort and 8 patients as a validation cohort. After consent was obtained, blood was processed for plasma cytokine analysis and RNA gene expression. Alvarado and pediatric appendicitis scores were obtained. Principal components analysis was used to explore global differences in gene expression. The random forest method was used to classify patients into those with and without appendicitis in the prospective cohort. Comparisons were made evaluating clinical scoring systems, cytokine analysis, and gene expression analysis to accurately predict appendicitis. RESULTS: The random forest method accurately predicted appendicitis in 4 of 5 patients in the prospective cohort. Cytokine analysis was not as accurate as gene expression analysis; however, it did accurately rule out all 3 patients in the prospective cohort. Pediatric appendicitis scores and Alvarado scores were not useful for predicting appendicitis. CONCLUSIONS: Our findings provide proof of technical feasibility and support the diagnostic potential of plasma cytokines to rule out and riboleukograms to rule in the diagnosis of appendicitis.

IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis.

Sepsis is a highly lethal disorder characterized by widespread apoptosis-induced depletion of immune cells and the development of a profound immunosuppressive state. IL-7 is a potent antiapoptotic cytokine that enhances immune effector cell function and is essential for lymphocyte survival. In this study, recombinant human IL-7 (rhIL-7) efficacy and potential mechanisms of action were tested in a murine peritonitis model. Studies at two independent laboratories showed that rhIL-7 markedly improved host survival, blocked apoptosis of CD4 and CD8 T cells, restored IFN-gamma production, and improved immune effector cell recruitment to the infected site. Importantly, rhIL-7 also prevented a hallmark of sepsis (i.e., the loss of delayed-type hypersensitivity), which is an IFN-gamma- and T cell-dependent response. Mechanistically, rhIL-7 significantly increased the expression of the leukocyte adhesion markers LFA-1 and VLA-4, consistent with its ability to improve leukocyte function and trafficking to the infectious focus. rhIL-7 also increased the expression of CD8. The potent antiapoptotic effect of rhIL-7 was due to increased Bcl-2, as well as to a dramatic decrease in sepsis-induced PUMA, a heretofore unreported effect of IL-7. If additional animal studies support its efficacy in sepsis and if current clinical trials continue to confirm its safety in diverse settings, rhIL-7 should be strongly considered for clinical trials in sepsis.

Characterization and modulation of the immunosuppressive phase of sepsis.

Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had approximately 40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-gamma) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-gamma production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the host's immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.

Neutrophils are significant producers of IL-10 during sepsis.

Sepsis is a syndrome involving systemic inflammation as well as an infectious focus. Accordingly, the host immune response to sepsis involves complex leukocyte interplay that is incompletely understood. It is known that the immunoregulatory cytokine, IL-10, is rapidly expressed during the early stages of sepsis. In a murine model of sepsis, we sought to elucidate which leukocytes are early IL-10 producers. Using a novel IL-10 transcriptional reporter mouse, we observed that splenic leukocytes produced little IL-10. At the site of infection, peritoneal neutrophils produced the highest levels of IL-10 among leukocytes. Using cytokine antibody labeling, we further show that peritoneal neutrophils had high amounts of intracellular IL-10. We next depleted neutrophils and found a 40% decrease in peritoneal IL-10 levels. Altogether, this report demonstrates that among leukocytes, neutrophils are significant contributors of IL-10 at the site of infection during sepsis.

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.

An anti-apoptotic peptide improves survival in lethal total body irradiation.

Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 microM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

Sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study.

Autophagy is the regulated process cells use to recycle nonessential, redundant, or inefficient components and is an adaptive response during times of stress. In addition to its function in enabling the cell to gain vital nutrients in times of stress, autophagy can also be involved in elimination of intracellular microorganisms, tumor suppression, and antigen presentation. Because of difficulty in diagnosing autophagy, few clinical studies have been performed. This study examined whether autophagy occurs in hepatocytes during sepsis. Electron microscopy (EM) was performed on liver samples obtained from both an observational clinical cohort of six septic patients and four control patients as well as liver specimens from mice with surgical sepsis (by cecal ligation and puncture) or sham operation. EM demonstrated increased autophagic vacuoles in septic vs nonseptic patients. Randomly selected fields (3000 microm(2)) from control and septic patients contained 1.2+/-1.5 vs 5.3+/-3.3 (mean+/-s.d.) complex lysosomal/autophagolysosomal structures per image respectively (P<0.001). In rare instances, hepatocytes with autophagic vacuoles appeared to be unequivocally committed to death. Membrane alterations (membrane vacuoles, invagination into adjacent organelles, and myelin figure-like changes) occur in a subpopulation of mitochondria in sepsis, but other hepatocyte organelles showed no consistent ultrastructural injury. Findings in murine sepsis paralleled those of patients, with 7.2+/-1.9 vs 38.7+/-3.9 lysosomal/autophagolysosomal structures in sham and septic mice, respectively (P=0.002). Quantitative RT-PCR demonstrated that sepsis induced the upregulation of select apoptosis and cytokine gene expression with minimal changes in the core autophagy genes in liver. In conclusion, hepatocyte autophagic vacuolization increases during sepsis and is associated with mitochondrial injury. However, it is not possible to determine whether the increase in autophagic vacuolization is an adaptive response or a harbinger of cell death.

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.

Lymphocyte phenotyping to distinguish septic from nonseptic critical illness.

BACKGROUND: Clinical signs and symptoms of sepsis are nonspecific and often indistinguishable from those of nonseptic critical illness. This ambiguity frequently delays the diagnosis of sepsis until culture results can confirm the presence or absence of an infectious organism. Lymphocyte phenotyping can be conducted rapidly and may provide information on the presence of infection before culture results are available. In this study, we hypothesized that lymphocyte phenotype can distinguish between septic and nonseptic critical illness. STUDY DESIGN: C57Bl/6 mice were subjected to either P aeruginosa pneumonia or lipopolysaccharide-induced acute lung injury (ALI). Animals were sacrificed 24 hours postinjury and splenic lymphocytes were harvested. Additionally, 13 patients in a surgical ICU were enrolled in the study. Whole blood was obtained and lymphocytes were isolated by density gradient centrifugation. Lymphocyte phenotype was identified through flow cytometry after labeling lymphocytes for CD3, CD4, CD8, CD20, CD40, CD69, and CD86 with fluorochrome-conjugated antibodies. RESULTS: CD69 expression on B cells and CD8+ splenocytes from septic mice was significantly increased compared with acute lung injury mice (p < 0.001 and p < 0.05, respectively). Similarly, CD4+ and CD8+ lymphocytes from septic patients had a two- to threefold increase in the expression of CD69 compared with nonseptic critically ill patients (p < 0.05). CONCLUSIONS: These data indicated that CD69 expression on lymphocytes may be useful in distinguishing between septic and nonseptic critical illness. Continued investigation into the expression of CD69 during sepsis is warranted.

Peptide-mediated activation of Akt and extracellular regulated kinase signaling prevents lymphocyte apoptosis.

Lymphocyte apoptosis is a hallmark of sepsis and contributes to disease mortality. In other acute injuries, such as myocardial and cerebral ischemia/reperfusion, apoptosis plays a significant role in disease-associated morbidity and mortality. We previously showed that constitutive activation of the potent antiapoptotic Akt/protein kinase B signaling pathway in lymphocytes both reduces sepsis-induced lymphocyte apoptosis and confers a significant survival advantage compared to wild-type littermates. Here, we demonstrate a therapeutic approach to acutely augment Akt activity in a wild-type animal. A cell-permeable peptide conjugated to the Akt-binding domain of the endogenous Akt coactivator, Tcl-1, prolongs Akt activity, activates extracellular regulated kinase (ERK) signaling and protects lymphocytes from numerous apoptotic stimuli both in vitro and in vivo. Molecular approaches to activate the antiapoptotic Akt and ERK signaling pathways may provide a novel tool to study these signaling pathways, as well as a new antiapoptotic strategy for the treatment of sepsis and other acute injuries.

Anti-apoptotic peptides protect against radiation-induced cell death.

The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15Gy radiation. In mice exposed to 5Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues.