Mary Ann Mastrangelo

Increased susceptibility to liver injury after hemorrhagic shock in rats chronically fed ethanol: role of nuclear factor-kappa B, interleukin-6, and granulocyte colony-stimulating factor.

Chronic ethanol use preceding severe trauma and hemorrhagic shock (HS) is associated with an increased incidence of multiorgan failure (MOF) and death; however, the molecular basis for this increased susceptibility is unknown. We previously demonstrated that production of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), mediated by nuclear factor-κB (NF-κB), each make essential contributions to organ injury and inflammation in a rodent model of controlled HS, and we proposed in this study to examine the hypothesis that the increased susceptibility to MOF after shock/trauma in the setting of chronic ethanol use is due to an exaggerated activation of NF-κB and production of these proinflammatory cytokines. We observed increased HS-induced liver injury 4 h after resuscitation in rats fed the ethanol-containing Lieber-DeCarli liquid diet for 8 weeks compared with rats fed the control liquid diet (3-fold increase in serum alanine aminotransferase [ALT], P = 0.008, and 2-fold increase in focal liver necrosis, P = 0.005). The increased liver injury in the ethanol-fed HS rats was accompanied by a 70% increase in liver NF-κB activation (P < 0.05), a 3- to 5-fold increase in hepatocyte and Kupffer cell production of IL-6 and G-CSF (P < 0.05 for each), and a 2-fold increase in neutrophil infiltration (P < 0.005) compared with the control diet-fed HS rats. Thus, increased susceptibility to HS-induced liver injury in the setting of chronic ethanol use may be mediated, at least in part, by increased NF-κB activation resulting in increased local production of IL-6 and G-CSF and increased infiltration of neutrophils, which can damage liver cells directly and contribute to impaired sinusoidal blood flow.

Prevention of hypovolemic circulatory collapse by IL-6 activated Stat3

Half of trauma deaths are attributable to hypovolemic circulatory collapse (HCC). We established a model of HCC in rats involving minor trauma plus severe hemorrhagic shock (HS). HCC in this model was accompanied by a 50% reduction in peak acceleration of aortic blood flow and cardiomyocyte apoptosis. HCC and apoptosis increased with increasing duration of hypotension. Apoptosis required resuscitation, which provided an opportunity to intervene therapeutically. Administration of IL-6 completely reversed HCC, prevented cardiac dysfunction and cardiomyocyte apoptosis, reduced mortality 5-fold and activated intracardiac signal transducer and activator of transcription (STAT) 3. Pre-treatment of rats with a selective inhibitor of Stat3, T40214, reduced the IL-6-mediated increase in cardiac Stat3 activity, blocked successful resuscitation by IL-6 and reversed IL-6-mediated protection from cardiac apoptosis. The hearts of mice deficient in the naturally occurring dominant negative isoform of Stat3, Stat3β, were completely resistant to HS-induced apoptosis. Microarray analysis of hearts focusing on apoptosis related genes revealed that expression of 29% of apoptosis related genes was altered in HS vs. sham rats. IL-6 treatment normalized the expression of these genes, while T40214 pretreatment prevented IL-6-mediated normalization. Thus, cardiac dysfunction, cardiomyocyte apoptosis and induction of apoptosis pathway genes are important components of HCC; IL-6 administration prevented HCC by blocking cardiomyocyte apoptosis and induction of apoptosis pathway genes via Stat3 and warrants further study as a resuscitation adjuvant for prevention of HCC and death in trauma patients.

Substance P is responsible for physiological alterations such as increased chloride ion secretion and glucose malabsorption in cryptosporidiosis

ABSTRACT Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease. Cryptosporidium infection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl−) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl− secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and after Cryptosporidium parvum infection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl− secretion and glucose malabsorption, was studied using tissues derived from macaques infected with C. parvum by the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples following C. parvum infection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.

Amplification of the proinflammatory transcription factor cascade increases with severity of uncontrolled hemorrhage in swine

INTRODUCTION Hypotension causes diffuse liver injury accompanied by increased local production of interleukin-6 (IL-6) in swine models of uncontrolled hemorrhagic shock (HS). IL-6 is transcriptionally up-regulated by nuclear factor (NF)-kappaB and results in activation of signal transducer and activator of transcription-3 (Stat3) in a murine model of controlled HS. Our objectives were: 1). to determine if increased IL-6 production and NF-kappaB and Stat3 activation occurs in a swine model of uncontrolled HS, and 2). to assess whether or not levels of IL-6 mRNA and activity of NF-kappaB and Stat3 correlate with shock severity. MATERIALS AND METHODS Swine were assigned to four groups: 1). control animals (n = 6): no intervention, 2). sham operation (n = 6): celiotomy and splenectomy, 3). uncontrolled hemorrhagic shock (UHS) (n = 6): sham plus grade V vascular liver injury and resuscitation, 4). profound uncontrolled hemorrhagic shock (PUHS) (n = 8): UHS after dilutional hypothermia. Following euthanasia at 2 h, livers were harvested, total RNA isolated, and IL-6 mRNA levels quantified by Q-RT-PCR (ABI Prism 7700, Applied Biosystems International, Foster City, CA). Protein was extracted for measurement of NF-kappaB and Stat3 activity by electrophoretic mobility shift assay (EMSA). RESULTS Compared to shams, IL-6 mRNA levels increased 4.5-fold in UHS and 90-fold in PUHS (P < 0.001). Compared with shams; NF-kappaB activity increased 2-fold in both UHS and PUHS (P < 0.05). Stat3 activity was equivalent (not significant) in UHS when compared with shams but increased 5.3-fold in PUHS. (P < 0.05). CONCLUSION These findings suggest that regional proinflammatory cytokine production results from and perpetuates a proinflammatory transcription factor cascade in a swine model of uncontrolled hemorrhagic shock and indicate that this process is proportional to the severity of shock.

Interleukin-6 infusion blunts proinflammatory cytokine production without causing systematic toxicity in a swine model of uncontrolled hemorrhagic shock.

BACKGROUND Serum elevations of interleukin-6 (IL-6) correlate with multiple organ dysfunction syndrome and mortality in critically injured trauma patients. Data from rodent models of controlled hemorrhage suggest that recombinant IL-6 (rIL-6) infusion protects tissue at risk for ischemia-reperfusion injury. Exogenous rIL-6 administered during shock appears to abrogate inflammation, providing a protective rather than a deleterious influence. In an examination of this paradox, the current study aimed to determine whether rIL-6 decreases inflammation in a clinically relevant large animal model of uncontrolled hemorrhagic shock, (UHS), and to investigate the mechanism of protection. METHODS Swine were randomized to four groups (8 animals in each): (1) sacrifice, (2) sham (splenectomy followed by hemodilution and cooling to 33 degrees C), (3) rIL-6 infusion (sham plus UHS using grade 5 liver injury with packing and resuscitation plus blinded infusion of rIL-6 [10 mcg/kg]), and (4) placebo (UHS plus blinded vehicle). After 4 hours, blood was sampled, estimated blood loss determined, animals sacrificed, and lung harvested for RNA isolation. Quantitative reverse transcriptase-polymerase chain reaction was used to assess granulocyte colony-stimulating factor (G-CSF), IL-6, and tumor necrosis factor-alpha (TNFalpha) messenger ribonucleic acid (mRNA) levels. Serum levels of IL-6 and TNFalpha were measured by enzyme-linked immunoassay (ELISA). RESULTS As compared with placebo, IL-6 infusion in UHS did not increase estimated blood loss or white blood cell counts, nor decrease hematocrit or platelet levels. As compared with the sham condition, lung G-CSF mRNA production in UHS plus placebo increased eightfold (*p < 0.05). In contrast, rIL-6 infusion plus UHS blunted G-CSF mRNA levels, which were not significantly higher than sham levels (p = 0.1). Infusion of rIL-6 did not significantly affect endogenous production of either lung IL-6 or mRNA. As determined by ELISA, rIL-6 infusion did not increase final serum levels of IL-6 or TNFalpha over those of sham and placebo conditions. CONCLUSIONS Exogenous rIL-6 blunts lung mRNA levels of the proinflammatory cytokine G-CSF. The administration of rIL-6 does not increase the local expression of IL-6 nor TNFalpha mRNA in the lung. Additionally, rIL-6 infusion does not appear to cause systemic toxicity.

Substance P Receptor Antagonism for Treatment of Cryptosporidiosis in Immunosuppressed Mice

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, causes self-limited diarrhea in normal hosts but can cause life-threatening diarrhea for immunosuppressed patients. There is an urgent need for new drugs to treat this chronic disease. Cryptosporidium parvum infection is associated with intestinal structural and pathophysiologic changes, including villi blunting and glucose malabsorption. Substance P (SP), a neuropeptide and pain transmitter, is associated with the gastrointestinal tract and is elevated in humans and macaques after experimental C. parvum challenge. To examine the relevance of SP in the pathogenesis of cryptosporidiosis, and to determine if SP receptor antagonism can be employed for treatment of cryptosporidiosis in immunosuppressed hosts, we used an immunosuppressed murine model (dexamethasone-immunosuppressed mice) that is frequently utilized for examining chemotherapeutic potential of drugs. Quantitative ELISA was used to measure intestinal SP levels in immunosuppressed mice with, and without, C. parvum infection. Intestinal physiological alterations, as studied by the Ussing chamber technique, plus weight change, fecal oocyst shedding, and villi measurements, were compared in infected mice with, and without, SP receptor antagonist (aprepitant) treatment. Immunosuppressed mice infected with C. parvum demonstrated increased SP levels as well as physiological alterations (glucose malabsorption), weight loss, fecal oocyst shedding, and structural alterations (increased intestinal villi blunting) compared to uninfected mice. Each of these defects was significantly inhibited by aprepitant treatment. These studies demonstrate the potential of SP receptor antagonism for treatment of pathogenesis of cryptosporidiosis in immunosuppressed hosts.

Interleukin-6 treatment reverses apoptosis and blunts susceptibility to intraperitoneal bacterial challenge following hemorrhagic shock*

Background:Resuscitation from hemorrhagic shock (HS) predisposes to subsequent infections. Susceptibility to infection following sepsis has been attributed to apoptosis. Interleukin (IL)-6 has been shown to have antiapoptotic properties and to decrease postresuscitation inflammation in rodent and porcine models of HS. Objective:The objective was to determine if HS increases host susceptibility to infection, if IL-6 administration at resuscitation reduces this susceptibility, and if changes in susceptibility to infection are accompanied by parallel changes in apoptosis. Subjects and Interventions:Mice were randomized into three groups—HS, sham, and no-surgery control—and each group was further randomized to receive either IL-6 (3 &mgr;g/kg; HS/IL-6) or placebo (HS/P) at the start of resuscitation. In the HS-infection protocol, each mouse was challenged intraperitoneally the next day with a sublethal dose of Staphylococcus aureus (4 × 107 colony-forming units); 24 hrs later, the peritoneal cavity was lavaged and the major organs were harvested for culture. In the HS-apoptosis protocol, the livers were harvested the next day and analyzed by means of the terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) assay. Results:HS/P mice had a six- to eight-fold increase in total bacterial counts in comparison with sham and control mice that was attributable to a seven- to nine-fold increase in liver burden. IL-6 treatment reduced total and liver bacterial counts in HS/IL-6 mice by 62% and 69%, respectively, to levels statistically indistinguishable from IL-6-treated sham and control mice. The number of TUNEL-positive liver cells in the HS/P group was increased eight-fold vs. that in the sham group (p = .002); IL-6 resuscitation completely reversed the HS-induced increase in TUNEL-positive cells in the HS/IL-6 group (p = .002). Conclusions:IL-6 treatment at resuscitation eliminated the HS-mediated increase in total and liver bacterial burden and protected the liver from HS-induced apoptosis. Reduced liver apoptosis may explain the ability of IL-6 to blunt the HS-induced increase in susceptibility to bacterial challenge.