Amy C. Fox

Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis

Systemic administration of epidermal growth factor (EGF) decreases mortality in a murine model of septic peritonitis. Although EGF can have direct healing effects on the intestinal mucosa, it is unknown whether the benefits of systemic EGF in peritonitis are mediated through the intestine. Here, we demonstrate that enterocyte-specific overexpression of EGF is sufficient to prevent intestinal barrier dysfunction and improve survival in peritonitis. Transgenic FVB/N mice that overexpress EGF exclusively in enterocytes (IFABP-EGF) and wild-type (WT) mice were subjected to either sham laparotomy or cecal ligation and puncture (CLP). Intestinal permeability, expression of the tight junction proteins claudins-1, -2, -3, -4, -5, -7, and -8, occludin, and zonula occludens-1; villus length; intestinal epithelial proliferation; and epithelial apoptosis were evaluated. A separate cohort of mice was followed for survival. Peritonitis induced a threefold increase in intestinal permeability in WT mice. This was associated with increased claudin-2 expression and a change in subcellular localization. Permeability decreased to basal levels in IFABP-EGF septic mice, and claudin-2 expression and localization were similar to those of sham animals. Claudin-4 expression was decreased following CLP but was not different between WT septic mice and IFABP-EGF septic mice. Peritonitis-induced decreases in villus length and proliferation and increases in apoptosis seen in WT septic mice did not occur in IFABP-EGF septic mice. IFABP-EGF mice had improved 7-day mortality compared with WT septic mice (6% vs. 64%). Since enterocyte-specific overexpression of EGF is sufficient to prevent peritonitis-induced intestinal barrier dysfunction and confers a survival advantage, the protective effects of systemic EGF in septic peritonitis appear to be mediated in an intestine-specific fashion.

The Role of Heat Shock Protein 70 in Mediating Age-Dependent Mortality in Sepsis

Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70−/− and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/− mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

The endogenous bacteria alter gut epithelial apoptosis and decrease mortality following Pseudomonas aeruginosa pneumonia.

ABSTRACT The endogenous bacteria have been hypothesized to play a significant role in the pathophysiology of critical illness, although their role in sepsis is poorly understood. The purpose of this study was to determine how commensal bacteria alter the host response to sepsis. Conventional and germ-free (GF) C57Bl/6 mice were subjected to Pseudomonas aeruginosa pneumonia. All GF mice died within 2 days, whereas 44% of conventional mice survived for 7 days (P = 0.001). Diluting the dose of bacteria 10-fold in GF mice led to similar survival in GF and conventional mice. When animals with similar mortality were assayed for intestinal integrity, GF mice had lower levels of intestinal epithelial apoptosis but similar levels of proliferation and intestinal permeability. Germ-free mice had significantly lower levels of tumor necrosis factor and interleukin 1&bgr; in bronchoalveolar lavage fluid compared with conventional mice without changes in systemic cytokine production. Under conventional conditions, sepsis unmasks lymphocyte control of intestinal epithelial apoptosis, because sepsis induces a greater increase in gut apoptosis in Rag-1−/− mice than in wild-type mice. However, in a separate set of experiments, gut apoptosis was similar between septic GF Rag-1−/− mice and septic GF wild-type mice. These data demonstrate that the endogenous bacteria play a protective role in mediating mortality from pneumonia-induced sepsis, potentially mediated through altered intestinal apoptosis and the local proinflammatory response. In addition, sepsis-induced lymphocyte-dependent increases in gut epithelial apoptosis appear to be mediated by the endogenous bacteria.

Cancer causes increased mortality and is associated with altered apoptosis in murine sepsis

Objective:Whereas most septic patients have an underlying comorbidity, most animal models of sepsis use mice that were healthy before the onset of infection. Malignancy is the most common comorbidity associated with sepsis. The purpose of this study was to determine whether mice with cancer have a different response to sepsis than healthy animals. Design:Prospective, randomized controlled study. Setting:Animal laboratory in a university medical center. Subjects:C57Bl/6 mice. Interventions:Animals received a subcutaneous injection of either 250,000 cells of the transplantable pancreatic adenocarcinoma cell line Pan02 (cancer) or phosphate-buffered saline (healthy). Three weeks later, mice given Pan02 cells had reproducible, nonmetastatic tumors. Both groups of mice then underwent intratracheal injection of either Pseudomonas aeruginosa (septic) or 0.9% NaCl (sham). Animals were killed 24 hrs postoperatively or followed-up 7 days for survival. Measurements and Main Results:Mice with cancer and healthy mice appeared similar when subjected to sham operation, although cancer animals had lower levels of T- and B-lymphocyte apoptosis. Septic mice with cancer had increased mortality compared to previously healthy septic mice subjected to the identical injury (52% vs. 28%; p = .04). This was associated with increased bacteremia but no difference in local pulmonary infection. Septic mice with cancer also had increased intestinal epithelial apoptosis. Although sepsis induced an increase in T- and B-lymphocyte apoptosis in all animals, septic mice with cancer had decreased T- and B-lymphocyte apoptosis compared to previously healthy septic mice. Serum and pulmonary cytokines, lung histology, complete blood counts, and intestinal proliferation were similar between septic mice with cancer and previously healthy septic mice. Conclusions:When subjected to the same septic insult, mice with cancer have increased mortality compared to previously healthy animals. Decreased systemic bacterial clearance and alterations in intestinal epithelial and lymphocyte apoptosis may help explain this differential response.

Cecal ligation and puncture followed by methicillin-resistant Staphylococcus aureus pneumonia increases mortality in mice and blunts production of local and systemic cytokines.

ABSTRACT Mortality in the intensive care unit frequently results from the synergistic effect of two temporally distinct infections. This study examined the pathophysiology of a new model of intra-abdominal sepsis followed by methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Mice underwent cecal ligation and puncture (CLP) or sham laparotomy followed 3 days later by an intratracheal injection of MRSA or saline. Both CLP/saline and sham/MRSA mice had 100% survival, whereas animals with CLP followed by MRSA pneumonia had 67% 7-day survival. Animals subjected to CLP/MRSA had increased bronchoalveolar lavage concentrations of MRSA compared with sham/MRSA animals. Animals subjected to sham/MRSA pneumonia had increased bronchoalveolar lavage levels of interleukin 6 (IL-6), tumor necrosis factor &agr;, and granulocyte colony-stimulating factor compared with those given intratracheal saline, whereas CLP/MRSA mice had a blunted local inflammatory response with markedly decreased cytokine levels. Similarly, animals subjected to CLP/saline had increased peritoneal lavage levels of IL-6 and IL-1&bgr; compared with those subjected to sham laparotomy, whereas this response was blunted in CLP/MRSA mice. Systemic cytokines were upregulated in both CLP/saline and sham/MRSA mice, and this was blunted by the combination of CLP/MRSA. In contrast, no synergistic effect on pneumonia severity, white blood cell count, or lymphocyte apoptosis was identified in CLP/MRSA mice compared with animals with either insult in isolation. These results indicate that a clinically relevant model of CLP followed by MRSA pneumonia causes higher mortality than could have been predicted from studying either infection in isolation, and this was associated with a blunted local (pulmonary and peritoneal) and systemic inflammatory response and decreased ability to clear infection.

Prevention of Lymphocyte Apoptosis in Septic Mice with Cancer Increases Mortality

Lymphocyte apoptosis is thought to have a major role in the pathophysiology of sepsis. However, there is a disconnect between animal models of sepsis and patients with the disease, because the former use subjects that were healthy prior to the onset of infection while most patients have underlying comorbidities. The purpose of this study was to determine whether lymphocyte apoptosis prevention is effective in preventing mortality in septic mice with preexisting cancer. Mice with lymphocyte Bcl-2 overexpression (Bcl-2-Ig) and wild type (WT) mice were injected with a transplantable pancreatic adenocarcinoma cell line. Three weeks later, after development of palpable tumors, all animals received an intratracheal injection of Pseudomonas aeruginosa. Despite having decreased sepsis-induced T and B lymphocyte apoptosis, Bcl-2-Ig mice had markedly increased mortality compared with WT mice following P. aeruginosa pneumonia (85 versus 44% 7-d mortality; p = 0.004). The worsened survival in Bcl-2-Ig mice was associated with increases in Th1 cytokines TNF-α and IFN-γ in bronchoalveolar lavage fluid and decreased production of the Th2 cytokine IL-10 in stimulated splenocytes. There were no differences in tumor size or pulmonary pathology between Bcl-2-Ig and WT mice. To verify that the mortality difference was not specific to Bcl-2 overexpression, similar experiments were performed in Bim−/− mice. Septic Bim−/− mice with cancer also had increased mortality compared with septic WT mice with cancer. These data demonstrate that, despite overwhelming evidence that prevention of lymphocyte apoptosis is beneficial in septic hosts without comorbidities, the same strategy worsens survival in mice with cancer that are given pneumonia.

Inhibition of Intestinal Epithelial Apoptosis Improves Survival in a Murine Model of Radiation Combined Injury

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

Erythropoietin in sepsis: a new use for a familiar drug?

Despite significant advances in understanding the pathophysiology of sepsis (1), mortality from the disease remains unacceptably high (2). Management of sepsis is largely supportive, and outside of antibiotics, no therapy aimed at treating sepsis is universally accepted (3). There is a huge gap between rapid scientific advances at the bench and translating these advances into clinical practice. The literature is replete with therapies showing marked mortality benefits in animal trials. Unfortunately, when these same agents are tried in patients, the vast majority have turned out to be no better than placebo. While the reasons behind this are complex (4), it has proven much easier to cure sepsis in inbred rodents under controlled laboratory settings than genetically diverse humans with significant co-morbidities. Despite this frustrating reality, the search for novel therapies that can improve outcomes in septic shock remains of paramount importance. Usually, a novel therapeutic comes in the form of a new agent that is previously unfamiliar to the medical community. However, sometimes it can proverbially be right under everyone’s nose, in the form of a new indication for a widely-familiar drug already readily available. In this issue of Critical Care Medicine, Dr. Aoshiba and colleagues report that administration of erythropoietin improves survival in a murine model of sepsis (5). Erythropoietin acts as the primary regulator of erythropoiesis and is used clinically to treat anemia resulting from chronic kidney disease (6) or chemotherapy (7). Erythropoietin has previously been studied in two large-scale trials in critically ill patients. One showed a 19% decrease in blood transfusion with erythropoietin (8) while the other showed no impact on transfusion practices (9). Neither trial showed an overall benefit in mortality, although a similar trend toward improved survival was noted in both studies in trauma patients. While erythropoietin is best known for its clinical efficacy in anemia, it has a number of effects outside of the hematopoietic system. Erythropoietin receptors have been identified in a variety of cells including neurons, endothelial cells, cardiocytes, muscle, and hair follicles, and erythropoietin has been demonstrated to play a role in protecting, and potentially reversing pathologic changes in response to stressors such as ischemia/reperfusion, hypoxia, or metabolic injury (10;11). In theory, these tissue protective properties could make erythropoietin an attractive agent for modulating the host response in sepsis. To address this, Dr. Aoshiba and colleagues gave erythropoietin to inbred mice at varying timepoints after a lethal dose of lipopolysaccharide (LPS). Animals given erythropoietin 30 minutes, 1 or 2 hours after LPS had improved survival; however, the survival benefit was lost when erythropoietin was initiated 3 or more hours after LPS. Interestingly, pretreatment with erythropoietin also failed to confer a survival advantage. The survival benefit with erythropoietin administration 1 hour after LPS was associated with attenuated apoptosis in multiple organs, reduced nitric oxide production and tissue hypoxia. In contrast, it was not associated with inflammatory changes. This study is exciting because it identifies a potential new therapy for treating sepsis. However, it has a number of limitations. First, erythropoietin was efficacious only in a very narrow time window. Sepsis usually presents over the course of a number of hours to a few days, and it is frequently difficult to pinpoint exactly when a patient gets septic. It would therefore be difficult to know when the drug could be started and expected to be useful, which minimizes its theoretical utility. Additionally erythropoietin’s lack of efficacy in pretreatment argues against its usage as prophylaxis in patients at risk for developing sepsis (i.e. ventilated burn patients who may develop a secondary pneumonia). The results are also limited by the model used. LPS does not accurately replicate the features seen in human sepsis. Endotoxemia causes a rapid, intense pro-inflammatory response with rapid death or recovery. In contrast, sepsis has a more indolent timecourse with a complicated, lower intensity, mixed inflammatory response with prolonged immunoparalysis (12). The marked difference in the immunopathologic response between LPS and more authentic models of sepsis such as cecal ligation and puncture (CLP) (13) has been proposed as one reason why positive animal studies using endotoxemia as a surrogate for sepsis have not translated into clinical benefit in human trials. Recognizing this shortcoming, the authors also gave erythropoietin beginning 1 hour after a lethal model of CLP. While erythropoietin extended survival by 12 hours, all animals died within three days. While it is possible that this modest prolongation in time until death could translate into a meaningful survival advantage if a less lethal sepsis model were used, this would have to be convincingly demonstrated prior to proceeding with clinical trials as there would be no utility to bringing an agent to the bedside if survival is extended by less than 1 day. Another concern of this study was the high dose of erythropoietin used. Clinical trials of erythropoietin in critically ill patients with anemia used 40,000 units/week (600 IU/kg). In contrast, the authors used a dose of 1000 IU/kg/day. Since four doses of erythropoietin were given, mice received a total of 4000 IU/kg over the course of the study, a greater than 6-fold increase over doses used in ICU patients. A dose of 40,000 units/week has been associated with an increased incidence of thrombosis in critically ill patients, and it is unclear whether the higher dose used in this study would be associated with a further increase in the rate of clinically significant thrombosis. This risk as well as the fact that erythropoietin is associated with shorter time to death and more rapid tumor growth in patients with cancer suggests the risk/benefit ratio would have to be carefully considered in patients prone to thrombosis or those with cancer. Despite these limitations, this study represents a triumph of imagination. It takes a common agent and looks at it in a new way, resulting in improved survival in experimental sepsis, accompanied by exciting mechanistic insights. However, multiple hurdles need to be overcome before erythropoietin could be considered for clinical trials. Erythropoietin thus represents a new member of the ever growing list of agents that improve survival in inbred rodents but whose utility is currently limited by the chasm between preclinical studies and the difficulties encountered when trying to translate these studies into effective therapeutics.

Synanthropic Mammals as Potential Hosts of Tick-Borne Pathogens in Panama.

Synanthropic wild mammals can be important hosts for many vector-borne zoonotic pathogens. The aim of this study was determine the exposure of synanthropic mammals to two types of tick-borne pathogens in Panama, spotted fever group Rickettsia (SFGR) and Borrelia relapsing fever (RF) spirochetes. One hundred and thirty-one wild mammals were evaluated, including two gray foxes, two crab-eating foxes (from zoos), four coyotes, 62 opossum and 63 spiny rats captured close to rural towns. To evaluate exposure to SFGR, serum samples from the animals were tested by indirect immunofluorescence assay (IFA) using Rickettsia rickettsii and Candidatus Rickettsia amblyommii antigen. Immunoblotting was performed using Borrelia turicatae protein lysates and rGlpQ, to assess infection caused by RF spirochetes. One coyote (25%) and 27 (43%) opossums showed seroreactivity to SFGR. Of these opossums, 11 were seroreactive to C. R. amblyommii. Serological reactivity was not detected to B. turicatae in mammal samples. These findings may reflect a potential role of both mammals in the ecology of tick-borne pathogens in Panama.