Jose M. Prince

Emerging paradigm: toll-like receptor 4-sentinel for the detection of tissue damage.

ABSTRACT The systemic inflammatory response syndrome initiated by infection shares many features in common with the trauma-induced systemic response. The toll-like receptors (TLRs) stand at the interface of innate immune activation in the settings of both infection and sterile injury by responding to a variety of microbial and endogenous ligands alike. Recently, a body of literature has evolved describing a key role for TLRs in acute injury using rodent models of hemorrhagic shock, ischemia and reperfusion, tissue trauma and wound repair, and various toxic exposures. This review will detail the observations implicating a TLR family member, TLR4, as a key component of the initial injury response.

Enterocyte TLR4 Mediates Phagocytosis and Translocation of Bacteria Across the Intestinal Barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcγRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.

IN SILICO MODELS OF ACUTE INFLAMMATION IN ANIMALS

ABSTRACT Trauma and hemorrhagic shock elicit an acute inflammatory response, predisposing patients to sepsis, organ dysfunction, and death. Few approved therapies exist for these acute inflammatory states, mainly due to the complex interplay of interacting inflammatory and physiological elements working at multiple levels. Various animal models have been used to simulate these phenomena, but these models often do not replicate the clinical setting of multiple overlapping insults. Mathematical modeling of complex systems is an approach for understanding the interplay among biological interactions. We constructed a mathematical model using ordinary differential equations that encompass the dynamics of cells and cytokines of the acute inflammatory response, as well as global tissue dysfunction. The model was calibrated in C57Bl/6 mice subjected to (1) various doses of lipopolysaccharide (LPS) alone, (2) surgical trauma, and (3) surgery + hemorrhagic shock. We tested the models predictive ability in scenarios on which it had not been trained, namely, (1) surgery ± hemorrhagic shock + LPS given at times after the beginning of surgical instrumentation, and (2) surgery + hemorrhagic shock + bilateral femoral fracture. Software was created that facilitated fitting of the mathematical model to experimental data, as well as for simulation of experiments with various inflammatory challenges and associated variations (gene knockouts, inhibition of specific cytokines, etc.). Using this software, the C57Bl/6-specific model was recalibrated for inflammatory analyte data in CD14−/− mice and was used to elucidate altered features of inflammation in these animals. In other experiments, rats were subjected to surgical trauma ± LPS or to bacterial infection via fibrin clots impregnated with various inocula of Escherichia coli. Mathematical modeling may provide insights into the complex dynamics of acute inflammation in a manner that can be tested in vivo using many fewer animals than has been possible previously.

Medium-term follow-up confirms the safety and durability of laparoscopic ventral hernia repair with PTFE

BACKGROUND Ventral abdominal wall hernias are common lesions and may be associated with life-threatening complications. The application of laparoscopic principles to the treatment of ventral hernias has reduced recurrence rates from a range of 25% to 52% to a range of 3.4% to 9%. In this study, we review our experience and assess the clinical outcome of patients who have undergone laparoscopic repair of ventral hernias. METHODS We reviewed the outcome of 79 patients with more than 1 year of follow-up who underwent laparoscopic ventral hernia repair between March 1996 and December 2001. Patient demographics, hernia characteristics, operative parameters, and clinical outcomes were evaluated. RESULTS Of the 79 patients, 37 were males. Mean age was 55.8 years (range 28-81). Sixty-eight patients had incisional hernias, including 17 with recurrent hernias. Eleven patients had primary ventral hernias. The mean defect size was 103 cm(2) (range 4-510); incarceration was present in 22 patients (27.8%), and multiple (Swiss-cheese) defects in 20 (25.3%). Laparoscopic expanded polytetrafluoroethylene mesh repair by the modified Rives-Stoppa technique was completed in 78 (98.7%). One conversion occurred because of bowel injury. The mean operating time was 110 minutes (range 45-210) and mean hospital stay was 1.7 days (range 0-20), with 46 patients (58.2%) being discharged within 24 hours of surgery. Complications included seroma formation (3), chronic pain (3), prolonged ileus (1), hematoma formation (1), and missed bowel injury (1) for a complication rate of (11.4%). There were no deaths. After a follow-up of up to 6 years (a mean of 34 months), there were 4 recurrences (5%). CONCLUSION The laparoscopic repair of ventral hernias is safe, effective, and durable with minimal morbidity. It is particularly successful in patients with recurrent lesions. The laparoscopic approach to ventral hernia repair should be considered the standard of care.

Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells

Endogenous damage‐associated molecular pattern (DAMP) molecules are released from cells during traumatic injury, allowing them to interact with pattern recognition receptors such as the toll‐like receptors (TLRs) on other cells and subsequently, to stimulate inflammatory signaling. TLR4, in particular, plays a key role in systemic and remote organ responses to hemorrhagic shock (HS) and peripheral tissue injury in the form of bilateral femur fracture. TLR4 chimeric mice were generated to investigate the cell lineage in which functional TLR4 is needed to initiate the injury response to trauma. Chimeric mice were generated by adoptive bone marrow (BM) transfer, whereby donor marrow was given to an irradiated host using reciprocal combinations of TLR4 wild‐type (WT; C3H/HeOuJ) and TLR4 mutant (Mu; C3H/HeJ) mice. After a period of engraftment, chimeric mice were then subjected to HS or bilateral femur fracture. Control groups, including TLR4‐WT mice receiving WT BM and TLR4‐Mu mice receiving Mu BM, responded to injury in a similar pattern to unaltered HeOuJ and HeJ mice, and protection was afforded to those mice lacking functional TLR4. In contrast, TLR4‐WT mice receiving Mu BM and TLR4‐Mu mice receiving WT BM demonstrated intermediate inflammatory and cellular damage profiles. These data demonstrate that functional TLR4 is required in BM‐derived cells and parenchymal cells for an optimal inflammatory response to trauma.

Early growth response 1 mediates the systemic and hepatic inflammatory response initiated by hemorrhagic shock.

Hemorrhagic shock (HS) is a major cause of morbidity and mortality in trauma patients. The early growth response 1 (Egr-1) transcription factor is induced by a variety of cellular stresses, including hypoxia, and may function as a master switch to trigger the expression of numerous key inflammatory mediators. We hypothesized that HS would induce hepatic expression of Egr-1 and that Egr-1 upregulates the inflammatory response after HS. The Egr-1−/− mice and wild-type (WT) controls (n ≥ 5 for all groups) were subjected to HS alone or HS followed by resuscitation (HS/R). Other mice were subjected to a sham procedure which included general anesthesia and vessel cannulation but no shock (sham). After the HS, HS/R, or sham procedures, mice were euthanized for determination of serum concentrations of interleukin (IL) 6, IL-10, and alanine aminotransferase. Northern blot analysis was performed to evaluate Egr-1 messenger RNA (mRNA) expression. Liver whole cell lysates were evaluated for Egr-1 protein expression by Western blot analysis. Hepatic expression of IL-6, granulocyte colony-stimulating factor, and intracellular adhesion molecule 1 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The Egr-1 DNA binding was assessed using the electrophoretic mobility shift assay. Hemorrhagic shock results in a rapid and transient hepatic expression of Egr-1 mRNA in WT mice by 1 h, whereas protein and DNA binding activity was evident by 2.5 h. The Egr-1 mRNA expression diminished after 4 h of resuscitation, whereas Egr-1 protein expression and DNA binding activity persisted through resuscitation. The Egr-1−/− mice exhibited decreased levels of hepatic inflammatory mediators compared with WT controls with a decrease in hepatic mRNA levels of IL-6 by 42%, granulocyte colony-stimulating factor by 39%, and intracellular adhesion molecule 1 by 43%. Similarly, Egr-1−/− mice demonstrated a decreased systemic inflammatory response and hepatic injury after HS/R compared with their WT counterparts. Early growth response 1 is rapidly upregulated in the liver during and after resuscitation from HS. Our results showing a blunted inflammatory response in Egr-1−/− mice provides evidence that Egr-1 functions as a proximal signal transduction mechanism responding to shock by amplifying the systemic inflammatory response.

Hypoxia activates c-Jun N-terminal kinase via Rac1-dependent reactive oxygen species production in hepatocytes.

The earliest events after the induction of hemorrhagic shock (HS) are complex and poorly understood. We have recently demonstrated that decreased tissue perfusion and hypoxia during HS lead to an increased phosphorylation of c-Jun N-terminal kinase (JNK) in vivo. The purpose of these investigations was to test the hypothesis that hypoxia activates JNK via Rac1-dependent reactive oxygen species (ROS) signaling. Mice subjected to HS and resuscitated with Ringers ethyl pyruvate solution (REPS) or N-acetylcysteine (NAC), two scavengers of ROS, demonstrated decreased levels of phosphorylated JNK. Exposure of primary mouse hepatocytes in culture to 1% oxygen led to increased production of ROS and phosphorylation of JNK. The duration of hypoxia correlated with the level of generation of ROS and JNK activation. The phosphorylation of JNK was attenuated in the presence of ROS scavengers or the nicotinamide adenosine dinucleotide phosphate [NDA(P)H] oxidase inhibitor, diphenyleneiodonium (DPI). In addition, hypoxia increased activation of Rac1. Inhibition of Rac1 activation by adenoviral gene transfer of dominant-negative Rac1 (AdRac1N17) attenuated both ROS formation and JNK activation. Together, these data suggest that ROS generation during hypoxia in the liver directly leads to JNK activation in a Rac1-dependent process.

Surgical Management of Ovarian Disease in Infants, Children, and Adolescents: A 15-Year Review

BACKGROUND Despite the reported efficacy and the presumed benefits of minimally invasive surgery (MIS) for ovarian lesions in adults, questions remain as to the surgical indications, results, and outcomes for these procedures across pediatric age groups. The aim of this study was to review our experience with the management of ovarian disease in children to determine if there has been a shift in the management of these lesions from open surgery (OS) to an MIS approach in the pediatric population. METHODS An institutional review board (IRB)-approved retrospective chart review included all patients who underwent surgical management of ovarian disease from January 1, 1992 to July 10, 2007. Patients with ectopic pregnancy, known pelvic inflammatory disease, or concomitant illness requiring operative management at the time of ovarian surgery were excluded. Demographics, clinical signs and symptoms, diagnosis, surgical outcomes, and history of prior abdominal procedures were obtained. Statistical analysis included comparison of means, paired t-test, chi-squared test, and multivariate analysis, where indicated. RESULTS A total of 231 patients were evaluated in this study, with a mean age of 12.8 years (range, 3 weeks to 20 years). There were 221 (95.7%) benign lesions and 10 (4.3%) were malignant. There were 156 simple or hemorrhagic cysts (70.5%) and 46 mature teratomas (20.8%). Three complications (1.3%) occurred, which were associated with surgery and no mortalities. Abdominal pain (82.3%), nausea or vomiting (24.2%), and abdominal tenderness (10.0%) were the most common presenting symptoms or signs. Operative outcomes for benign disease (n = 221) were compared between MIS and open cases over the entire time period as well as within three consecutive 5-year time intervals. CONCLUSIONS There was a notable shift toward the management of benign ovarian disease in using MIS techniques over the course of three different 5-year intervals. This approach was also associated with shorter hospital stay, less operative blood loss, and shorter operative times, when compared to an open approach. When indicated, a laparoscopic approach should be performed for presumed benign ovarian disease in children.

A review of laparoscopic Nissen fundoplication in children weighing less than 5 kg

PURPOSE Minimally invasive procedures in small infants and neonates are being performed in increasing numbers. In this study, we describe our institutions experience with laparoscopic Nissen fundoplications (LNFs) in children weighing less than 5 kg. METHODS All cases of LNF attempted in children weighing less than 5 kg since January 2003 at a tertiary-care pediatric hospital were reviewed after Institutional Review Board approval. RESULTS One hundred twenty-two children weighing less than 5 kg underwent LNF during the study period. They ranged from 2 weeks to 3 years of age (mean, 94 +/- 61.3 days) and weighed 1.94 to 4.99 kg (mean, 3.68 +/- 0.77 kg). Twenty-nine percent (n = 35) were neurologically impaired. Eighty-eight percent (n = 107) had concurrent gastrostomy tube placement. Eight (7%) were converted to laparotomy. The average operative time was 112 +/- 46 minutes. Seventy-one percent (n = 87) required intensive care unit use for an average of 14.3 +/- 17.4 days. The average time to start enteral feeds was 2.6 +/- 2.6 days. Thirty-one percent (n = 38) required postoperative mechanical ventilation for an average of 12.0 +/- 20.6 days. The average hospital length of stay was 36.6 +/- 36.0 days (range, 3-175 days). Six patients (5%) had a complication or recurrent gastroesophageal reflux. Three patients had recurrent reflux, one of which underwent another LNF. One patient had a gastric perforation. Another required a redo LNF after a disrupted wrap was noted at a recurrent hiatal hernia repair. Lastly, one patient had bleeding from an accessory hepatic artery with liver retractor placement. CONCLUSIONS Laparoscopic Nissen fundoplication can safely and effectively be performed in small children (<5 kg) with similar outcomes and rates of complication as previously published reports in larger children. These children, however, do have prolonged intensive care unit and mechanical ventilation use associated with their prematurity and significant comorbidities.

Transcriptomic response of murine liver to severe injury and hemorrhagic shock: a dual-platform microarray analysis

Trauma-hemorrhagic shock (HS/T) is a complex process that elicits numerous molecular pathways. We hypothesized that a dual-platform microarray analysis of the liver, an organ that integrates immunology and metabolism, would reveal key pathways engaged following HS/T. C57BL/6 mice were divided into five groups (n = 4/group), anesthetized, and surgically treated to simulate a time course and trauma severity model: 1) nonmanipulated animals, 2) minor trauma, 3) 1.5 h of hemorrhagic shock and severe trauma (HS/T), 4) 1.5 h HS/T followed by 1 h resuscitation (HS/T+1.0R), 5) 1.5 h HS/T followed by 4.5 h resuscitation (HS/T+4.5R). Liver RNA was hybridized to CodeLink and Affymetrix mouse whole genome microarray chips. Common genes with a cross-platform correlation >0.6 (2,353 genes in total) were clustered using k-means clustering, and clusters were analyzed using Ingenuity Pathways Analysis. Genes involved in the stress response and immunoregulation were upregulated early and remained upregulated throughout the course of the experiment. Genes involved in cell death and inflammatory pathways were upregulated in a linear fashion with elapsed time and in severe injury compared with minor trauma. Three of the six clusters contained genes involved in metabolic function; these were downregulated with elapsed time. Transcripts involved in amino acid metabolism as well as signaling pathways associated with glucocorticoid receptors, IL-6, IL-10, and the acute phase response were elevated in a severity-dependent manner. This is the first study to examine the postinjury response using dual-platform microarray analysis, revealing responses that may enable novel therapies or diagnostics.