Ori D. Rotstein

Pre–B cell colony–enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis

Pre-B cell colony-enhancing factor (PBEF) is a highly conserved 52-kDa protein, originally identified as a growth factor for early stage B cells. We show here that PBEF is also upregulated in neutrophils by IL-1beta and functions as a novel inhibitor of apoptosis in response to a variety of inflammatory stimuli. Induction of PBEF occurs 5-10 hours after LPS exposure. Prevention of PBEF translation with an antisense oligonucleotide completely abrogates the inhibitory effects of LPS, IL-1, GM-CSF, IL-8, and TNF-alpha on neutrophil apoptosis. Immunoreactive PBEF is detectable in culture supernatants from LPS-stimulated neutrophils, and a recombinant PBEF fusion protein inhibits neutrophil apoptosis. PBEF is also expressed in neutrophils from critically ill patients with sepsis in whom rates of apoptosis are profoundly delayed. Expression occurs at higher levels than those seen in experimental inflammation, and a PBEF antisense oligonucleotide significantly restores the normal kinetics of apoptosis in septic polymorphonuclear neutrophils. Inhibition of apoptosis by PBEF is associated with reduced activity of caspases-8 and -3, but not caspase-9. These data identify PBEF as a novel inflammatory cytokine that plays a requisite role in the delayed neutrophil apoptosis of clinical and experimental sepsis.

Integrity of cell-cell contacts is a critical regulator of TGF-β1-induced epithelial-to-myofibroblast transition: Role for β-catenin

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.

Results of a Randomized Trial Comparing Sequential Intravenous/ Oral Treatment with Ciprofloxacin Plus Metronidazole to Imipenem/ Cilastatin for Intra-Abdominal Infections

OBJECTIVE In a randomized, double-blind, multicenter trial, ciprofloxacin/metronidazole was compared with imipenem/cilastatin for treatment of complicated intra-abdominal infections. A secondary objective was to demonstrate the ability to switch responding patients from intravenous (IV) to oral (PO) therapy. SUMMARY BACKGROUND DATA Intra-abdominal infections result in substantial morbidity, mortality, and cost. Antimicrobial therapy often includes a 7- to 10-day intravenous course. The use of oral antimicrobials is a recent advance due to the availability of agents with good tissue pharmacokinetics and potent aerobic gram-negative activity. METHODS Patients were randomized to either ciprofloxacin plus metronidazole intravenously (CIP/MTZ IV) or imipenem intravenously (IMI IV) throughout their treatment course, or ciprofloxacin plus metronidazole intravenously and treatment with oral ciprofloxacin plus metronidazole when oral feeding was resumed (CIP/MTZ IV/PO). RESULTS Among 671 patients who constituted the intent-to-treat population, overall success rates were as follows: 82% for the group treated with CIP/MTZ IV; 84% for the CIP/MTZ IV/PO group; and 82% for the IMI IV group. For 330 valid patients, treatment success occurred in 84% of patients treated with CIP/MTZ IV, 86% of those treated with CIP/MTZ IV/PO, and 81% of the patients treated with IMI IV. Analysis of microbiology in the 30 patients undergoing intervention after treatment failure suggested that persistence of gram-negative organisms was more common in the IMI IV-treated patients who subsequently failed. Of 46 CIP/MTZ IV/PO patients (active oral arm), treatment success occurred in 96%, compared with 89% for those treated with CIP/MTZ IV and 89% for those receiving IMI IV. Patients who received intravenous/oral therapy were treated, overall, for an average of 8.6 +/- 3.6 days, with an average of 4.0 +/- 3.0 days of oral treatment. CONCLUSIONS These results demonstrate statistical equivalence between CIP/MTZ IV and IMI IV in both the intent-to-treat and valid populations. Conversion to oral therapy with CIP/MTZ appears as effective as continued intravenous therapy in patients able to tolerate oral feedings.

Ertapenem Versus Piperacillin/Tazobactam in the Treatment of Complicated Intraabdominal Infections: Results of a Double-Blind, Randomized Comparative Phase III Trial

ObjectiveTo examine the clinical efficacy and safety of ertapenem, a novel &bgr;-lactam agent with wide activity against common pathogens encountered in intraabdominal infection. Summary Background DataErtapenem has a pharmacokinetic profile and antimicrobial spectrum that support the potential for use as a once-a-day agent for the treatment of common mixed aerobic and anaerobic infections. MethodsThis prospective, randomized, controlled, and double-blind trial was conducted to compare the safety and efficacy of ertapenem with piperacillin/tazobactam as therapy following adequate surgical management of complicated intraabdominal infections. ResultsSix hundred thirty-three patients were included in the modified intent-to-treat population, with 396 meeting all criteria for the evaluable population. Patients with a wide range of infections were enrolled; perforated or abscessed appendicitis was most common (approximately 60% in microbiologically evaluable population). A prospective, expert panel review was conducted to assess the adequacy of surgical source control in patients who were failures as a component of evaluability. For the modified intent-to-treat groups, 245 of 311 patients treated with ertapenem (79.3%) were cured, as were 232 of 304 (76.2) treated with piperacillin/tazobactam. One hundred seventy-six of 203 microbiologically evaluable patients treated with ertapenem (86.7%) were cured, as were 157 of the 193 (81.2%) treated with piperacillin/tazobactam. ConclusionsIn this study, the efficacy of ertapenem 1 g once a day was equivalent to piperacillin/tazobactam 3.375 g every 6 hours in the treatment of a range of intraabdominal infections. Ertapenem was generally well tolerated and had a similar safety and tolerability profile to piperacillin/tazobactam. A formal process for review of adequacy of source control was found to be of benefit. The results of this trial suggest that ertapenem may be a useful option that could eliminate the need for combination and/or multidosed antibiotic regimens for the empiric treatment of intraabdominal infections.

Tertiary Peritonitis: Clinical Features of a Complex Nosocomial Infection

Abstract. The objective of this study was to define risk factors for and the clinical course of recurrent or tertiary peritonitis. Intensive supportive care of patients with life-threatening intraabdominal infections has led to the emergence of a new clinical syndrome, tertiary peritonitis, defined as the persistence or recurrence of intraabdominal infection following apparently adequate therapy of primary or secondary peritonitis. We undertook a retrospective study of 59 patients admitted with intraabdominal infection to a surgical intensive care unit (ICU). Tertiary peritonitis developed in 74% (44/59) of patients. Despite comparable premorbid health status, source of peritonitis, and admission APACHE II scores, patients with tertiary peritonitis had a significantly longer ICU stay (21.8 ± 14.9 vs. 8.5 ± 7.9 days), more advanced organ dysfunction reflected in higher organ dysfunction scores (13.3 ± 5.1 vs. 7.7 ± 3.3), and higher ICU mortality (64% vs. 33%) than patients with uncomplicated secondary peritonitis. The most common infecting organisms in patients with tertiary peritonitis wereEnterococcus,Candida, Staphylococcus epidermidis, andEnterobacter. Infectious foci were rarely amenable to percutaneous drainage and were found to be poorly localized at laparotomy. Recurrent, or tertiary, peritonitis is a common complication of intraabdominal infection in patients admitted to an ICU. It differs from uncomplicated secondary peritonitis in its microbial flora and lack of response to appropriate surgical and antibiotic therapy. Like nosocomial pneumonia in the critically ill patient, the syndrome appears to be more a reflection than a cause of adverse outcome.

The immunomodulatory effects of hypertonic saline resuscitation in patients sustaining traumatic hemorrhagic shock: a randomized, controlled, double-blinded trial.

Objective:To investigate the potential immunologic and anti-inflammatory effects of hypertonic saline plus dextran (HSD) in hemorrhagic trauma patients. Background:Unbalanced inflammation triggered by shock has been linked to multiorgan dysfunction (MOD) and death. In animal and cellular models, HSD alters the inflammatory response to shock, attenuating MOD and improving outcome. It remains untested whether HSD has similar effects in humans. Methods:A single 250-mL dose of either HSD (7.5% NaCl, 6% dextran-70) or placebo (0.9% NaCl) was administered to adult blunt trauma patients in hemorrhagic shock. The primary outcome was to measure changes in immune/inflammatory markers, including neutrophil activation, monocyte subset redistribution, cytokine production, and neuroendocrine changes. Patient demographics, fluid requirements, organ dysfunction, infection, and death were recorded. Results:A total of 27 patients were enrolled (13 HSD) with no significant differences in clinical measurements. Hyperosmolarity was modest and transient, whereas the immunologic/anti-inflammatory effects persisted for 24 hours. HSD blunted neutrophil activation by abolishing shock-induced CD11b up-regulation and causing CD62L shedding. HSD altered the shock-induced monocyte redistribution pattern by reducing the drop in “classic” CD14++ and the expansion of the “pro-inflammatory” CD14+CD16+ subsets. In parallel, HSD significantly reduced pro-inflammatory tumor necrosis factor (TNF)-α production while increasing anti-inflammatory IL-1ra and IL-10. HSD prevented shock-induced norepinephrine surge with no effect on adrenal steroids. Conclusions:This first human trial evaluating the immunologic/anti-inflammatory effects of hypertonic resuscitation in trauma patients demonstrates that HSD promotes a more balanced inflammatory response to hemorrhagic shock, raising the possibility that similar to experimental models, HSD might also attenuate post-trauma MOD.

Trial of Short-Course Antimicrobial Therapy for Intraabdominal Infection

BACKGROUND The successful treatment of intraabdominal infection requires a combination of anatomical source control and antibiotics. The appropriate duration of antimicrobial therapy remains unclear. METHODS We randomly assigned 518 patients with complicated intraabdominal infection and adequate source control to receive antibiotics until 2 days after the resolution of fever, leukocytosis, and ileus, with a maximum of 10 days of therapy (control group), or to receive a fixed course of antibiotics (experimental group) for 4±1 calendar days. The primary outcome was a composite of surgical-site infection, recurrent intraabdominal infection, or death within 30 days after the index source-control procedure, according to treatment group. Secondary outcomes included the duration of therapy and rates of subsequent infections. RESULTS Surgical-site infection, recurrent intraabdominal infection, or death occurred in 56 of 257 patients in the experimental group (21.8%), as compared with 58 of 260 patients in the control group (22.3%) (absolute difference, -0.5 percentage point; 95% confidence interval [CI], -7.0 to 8.0; P=0.92). The median duration of antibiotic therapy was 4.0 days (interquartile range, 4.0 to 5.0) in the experimental group, as compared with 8.0 days (interquartile range, 5.0 to 10.0) in the control group (absolute difference, -4.0 days; 95% CI, -4.7 to -3.3; P<0.001). No significant between-group differences were found in the individual rates of the components of the primary outcome or in other secondary outcomes. CONCLUSIONS In patients with intraabdominal infections who had undergone an adequate source-control procedure, the outcomes after fixed-duration antibiotic therapy (approximately 4 days) were similar to those after a longer course of antibiotics (approximately 8 days) that extended until after the resolution of physiological abnormalities. (Funded by the National Institutes of Health; STOP-IT ClinicalTrials.gov number, NCT00657566.).

Mechanism of decreased in vitro murine macrophage cytokine release after exposure to carbon dioxide: relevance to laparoscopic surgery.

OBJECTIVE The objective of this study was to determine the effect of carbon dioxide (CO2) on the function of peritoneal macrophages. SUMMARY BACKGROUND DATA Laparoscopic surgery is associated with minimal pain, fever, and low levels of inflammatory cytokines. To understand the mechanisms involved, the authors investigated the effect of different gases on murine peritoneal macrophage intracellular pH and correlated these alterations with alterations in LPS-stimulated inflammatory cytokine release. METHODS Peritoneal macrophages were incubated for 2 hours in air, helium, or CO2, and the effect of the test gas on immediate or next day lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) and interleukin-1 release compared. Cytosolic pH of macrophages exposed to test gases was measured using single-cell fluorescent imaging. The in vivo effects of test gases were determined in anesthetized rats during abdominal insufflation. RESULTS Macrophages incubated in CO2 produced significantly less TNF and interleukin-1 in response to LPS compared to incubation in air or helium. Cytokine production returned to normal 24 hours later. Exposure to CO2, but not air or helium, caused a marked cytosolic acidification. Pharmacologic induction of intracellular acidification to similar levels reproduced the inhibitory effect. In vitro studies showed that CO2 insufflation lowered tissue pH and peritoneal macrophage LPS-stimulated TNF production. CONCLUSIONS The authors propose that cellular acidification induced by peritoneal CO2 insufflation contributes to blunting of the local inflammatory response during laparoscopic surgery.

Definition of the role of enterococcus in intraabdominal infection: Analysis of a prospective randomized trial

BACKGROUND The role of enterococcus in intraabdominal infection is controversial. This study examines the contribution of enterococcus to adverse outcome in a large intraabdominal infection trial. METHODS A randomized prospective double-blind trial was performed to compare two different antimicrobial regimens in combination with surgical or percutaneous drainage in the treatment of complicated intraabdominal infections. A total of 330 valid patients was enrolled from 22 centers in North America. RESULTS In 330 valid patients, 71 had enterococcus isolated from the initial drainage of an intraabdominal focus of infection. This finding was associated with a significantly higher treatment failure rate than that of patients without enterococcus (28% versus 14%, p < 0.01). In addition, only Acute Physiology and Chronic Health Evaluation II score and presence of enterococcus were significant independent predictors of treatment failure when stepwise logistic regression was performed (p < 0.01 and < 0.03). Risk factors for the presence of enterococcus include age, Acute Physiology and Chronic Health Evaluation II, preinfection hospital length of stay, postoperative infections, and anatomic source of infection. There was no difference between the clinical trial treatment regimens with regard to overall failure, failure associated with enterococcus, or frequency of enterococcal isolation. CONCLUSIONS This study is the first to report enterococcus as a predictor of treatment failure in complicated intraabdominal infections. This trial also identifies several significant risk factors for the presence of enterococcus in such infections.

Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin.

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.