Development of a human cadaver model for training in laparoscopic donor nephrectomy.

BACKGROUND: The organ procurement network recommends a surgeon record 15 cases as surgeon or assistant for laparoscopic donor nephrectomies (LDN) prior to independent practice. The literature suggests that the learning curve for improved perioperative and patient outcomes is closer to 35 cases. In this article, we describe our development of a model utilizing fresh tissue and objective, quantifiable endpoints to document surgical progress, and efficiency in each of the major steps involved in LDN. MATERIALS AND METHODS: Phase I of model development focused on the modifications necessary to maintain visualization for laparoscopic surgery in a human cadaver. Phase II tested proposed learner-based metrics of procedural competency for multiport LDN by timing procedural steps of LDN in a novice learner. RESULTS: Phases I and II required 12 and nine cadavers, with a total of 35 kidneys utilized. The following metrics improved with trial number for multiport LDN: time taken for dissection of the gonadal vein, ureter, renal hilum, adrenal and lumbrical veins, simulated warm ischemic time (WIT), and operative time. CONCLUSION: Human cadavers can be used for training in LDN as evidenced by improvements in timed learner-based metrics. This simulation-based model fills a gap in available training options for surgeons.

Warming to 39°C but Not to 37°C Ameliorates the Effects on the Monocyte Response by Hypothermia.

OBJECTIVE: To investigate whether warming to normal body temperature or to febrile range temperature (39°C) is able to reverse the detrimental effects of hypothermia. BACKGROUND: Unintentional intraoperative hypothermia is a well-described risk factor for surgical site infections but also sepsis. We have previously shown that hypothermia prolongs the proinflammatory response whereas normothermia and especially febrile range temperature enhance the anti-inflammatory response. METHODS: Primary human monocytes were isolated from healthy volunteers. After stimulation with LPS (Lipopolysaccharide), the monocytes were exposed to 32°C for 3  hours or 6  hours and then warmed at either 37°C or 39°C for the remaining 33  hours or 36  hours, respectively. Tumor necrosis factor α, interleukin 10, and the expression of miR-155 and miR-101 were assessed at 24  hours and 36  hours. RESULTS: Warming to 37°C does not normalize monocyte cytokine secretion within 36  hours, whereas warming to 39°C partially reverses the effects of hypothermia on monocyte function. Both miR-155 and miR-101 were suppressed after the warming episode. However, 39°C had a stronger suppressive effect than 37°C. The duration of hypothermia and the warming temperature seem to be critical for a full reversibility of the effects of hypothermia. CONCLUSION: Warming to normal body temperature (37°C) does not restore normal monocyte function in vitro. These data suggest that hypothermic patients should be warmed to febrile range temperatures. Furthermore, febrile range temperatures should be investigated as a means to modulate the inflammatory response in patients with systemic infections.

MicroRNA-155 potentiates the inflammatory response in hypothermia by suppressing IL-10 production.

Therapeutic hypothermia is commonly used to improve neurological outcomes in patients after cardiac arrest. However, therapeutic hypothermia increases sepsis risk and unintentional hypothermia in surgical patients increases infectious complications. Nonetheless, the molecular mechanisms by which hypothermia dysregulates innate immunity are incompletely understood. We found that exposure of human monocytes to cold (32°C) potentiated LPS-induced production of TNF and IL-6, while blunting IL-10 production. This dysregulation was associated with increased expression of microRNA-155 (miR-155), which potentiates Toll-like receptor (TLR) signaling by negatively regulating Ship1 and Socs1. Indeed, Ship1 and Socs1 were suppressed at 32°C and miR-155 antagomirs increased Ship1 and Socs1 and reversed the alterations in cytokine production in cold-exposed monocytes. In contrast, miR-155 mimics phenocopied the effects of cold exposure, reducing Ship1 and Socs1 and altering TNF and IL-10 production. In a murine model of LPS-induced peritonitis, cold exposure potentiated hypothermia and decreased survival (10 vs. 50%; P < 0.05), effects that were associated with increased miR-155, suppression of Ship1 and Socs1, and alterations in TNF and IL-10. Importantly, miR-155-deficiency reduced hypothermia and improved survival (78 vs. 32%, P < 0.05), which was associated with increased Ship1, Socs1, and IL-10. These results establish a causal role of miR-155 in the dysregulation of the inflammatory response to hypothermia.

Video-assisted thoracoscopy as an important tool for trauma surgeons: a systematic review.

PURPOSE: Trauma patients frequently have serious chest injuries. Retained hemothoraces and persistent pneumothoraces are among the most frequent complications of chest injuries which may lead to major, long-term morbidity and mortality if these complications are not recognized and treated appropriately. Video-assisted thoracoscopy (VATS) is a well-established technique in surgical practice. The usefulness of VATS for treatment of complications after chest trauma has been demonstrated by several authors. However, there is an ongoing debate about the optimal timing of VATS. METHODS: A computerized search was conducted which yielded 450 studies reporting on the use of VATS for thoracic trauma. Eighteen of these studies were deemed relevant for this review. The quality of these studies was assessed using a check-list and the PRISMA guidelines. Outcome parameters were successful evacuation of the retained hemothorax or treatment of other complications as well as reduction of empyema rate, length of hospital stay, and hospital costs. RESULTS: There was only one randomized trial and two prospective studies. Most studies report case series of institutional experiences. VATS was found to be very successful in evacuation of retained hemothoraces and seems to reduce the empyema rate subsequently. Furthermore, the length of hospital stay and costs can be drastically reduced with the early use of VATS. CONCLUSION: Early VATS is an effective treatment for retained hemothoraces or other complications of chest trauma. We propose a clinical pathway, in which VATS is used as an early intervention in order to prevent serious complications such as empyemas or trapped lung.

Macrophage genetic reprogramming during chronic peritonitis is augmented by LPS pretreatment.

BACKGROUND: Persistent and tertiary chronic peritonitis is a clinically challenging problem especially in those who are critically ill. This could be attributed to a state of immune-paralysis, known as microbial tolerance. Microbial tolerance is the diminished pro-inflammatory protein response following repeated stimulation by numerous pathogen-associated molecular patterns (PAMPs) of varying origins, which we have shown in this novel model of chronic peritonitis. We aimed in this study to investigate the molecular mechanisms behind microbial tolerance and the early innate immune response resolution in this model. METHODS: C57BL/6 mice were pretreated intra-peritoneally (IP) with saline or endotoxin LPS 10 mg/kg (LPS 10). Following pretreatment, peritonitis was induced 24 h later injecting 10(3)Klebsiella pneumonia CFU IP. Gentamicin was administered 4 h prior to infection and BID thereafter. Peritoneal exudate cells (PEC) were obtained through peritoneal lavage and RNA was isolated (n = 3) at 4, 24, and 48 h following infection. SA Biosciences© RT2-Profiler PCR array mouse Toll-like receptor signaling pathway (PAMM_018A) data were further analyzed by Ingenuity Pathway Inc. analysis (IPA). RESULTS: Of the 89 genes studied, 26 were significantly up-regulated (fold change > 1.2 and P value < 0.05) in the saline pretreated group at 4, 24, and 48 h after infection. There were no down-regulated genes. In the LPS-pretreated group, 35 genes were significantly up-regulated; of these genes, 13 were not increased in the saline pretreated infected mice. This left 22 up-regulated genes in both infected groups. At 4 h, 6 of these 22 genes (CHUK, HMGB1, HSPD1, IRAK2, LY96, and TLR4) were further 2-fold increased in the LPS pretreatment group compared with the saline pretreatment group. Only IRAK2 was 2-fold increased at 24 h. By 48 h, no LPS effect was seen. When applying IPA analysis, six main canonical pathways were constantly dysregulated in the same significance order in both the saline and LPS group at 4, 24, and 48 h. These were: Toll-like receptor and NF-κB signaling, hepatic cholestasis, interleukin-6, and LPS-mediated MAPK signaling pathways, and pattern recognition receptors of bacterial pathway. CONCLUSION: Peritonitis increased PEC gene expression associated with sepsis and a pro-inflammatory response, which was further augmented by LPS pretreatment over 24 h only. Prior exposure to LPS did not induce PEC gene tolerance to subsequent infection with Klebsiella at the mRNA level. Post-transcriptional modification as microRNA down-regulation of inflammatory cytokines could possibly explain such phenomena.

Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study.

We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery-extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.

Unintentional perioperative hypothermia is associated with severe complications and high mortality in elective operations.

INTRODUCTION: Hypothermia occurs in as many as 7% of elective colorectal operations and is an underestimated risk factor for complications and death. Rewarming of hypothermic patients alone is not sufficient to prevent such adverse events. We investigated the outcomes of patients who became hypothermic (<35°C) after elective operations and compared them with closely matched, nonhypothermic operative patients to better define the impact of hypothermia on surgical outcomes, as well as to identify independent risk factors for hypothermia. METHODS: We queried the University HealthSystem Consortium (UHC) database for elective operative patients who became unintentionally hypothermic from October 2008 to March 2012, and identified 707 patients. Exclusion criteria were deliberate hypothermia, age <18 years, or death on day of admission. Separately, to validate the accuracy of hypothermia coding, we reviewed the hospital charts of all University of Louisville Hospital patients with hypothermia whose data were submitted to UHC. RESULTS: All patients from UHC with a code for hypothermia were indeed unintentionally hypothermic. Hypothermic patients undergoing elective operations experienced a 4-fold increase in mortality (17.0% vs 4.0%; P < .001) and a doubled complication rate (26.3% vs 13.9%; P < .001), in which sepsis and stroke increased the most. Several independent risk factors for hypothermia were amenable to preoperative improvement: anemia, chronic renal impairment, and unintended weight loss. Severity of illness on admission, age >65 years, male sex, and neurologic disorders also were risk factors. CONCLUSION: Hypothermia is associated with an increased rate of mortality and complications. Preventive treatment of these risk factors before operation and aggressive warming measures in the "at risk" population may decrease hypothermia-related morbidity and mortality in elective operations. Randomized-controlled trials should be conducted to evaluate the impact of aggressive warming measures in the at-risk population.

Differential microRNA (miRNA) expression could explain microbial tolerance in a novel chronic peritonitis model.

We observed persistent peritoneal bacteria despite a transient early innate immune response to intraperitoneal (IP) Klebsiella pneumoniae. Pretreatment with LPS prior to peritonitis induced a tolerant pattern of pro-inflammatory cytokine protein production over 72 h, but not at the mRNA level. MicroRNAs (miRNAs) regulate inflammatory cytokines and may explain this paradox. After pretreatment with IP LPS or saline, C57BL/6 mice were given 10(3) CFU of K. pneumoniae IP. Total RNA was isolated from peritoneal exudate cells (4 h, 24 h and 48 h following infection). mRNA and miRNA expression levels were detected and bioinformatics pathway analysis was performed, followed by measuring TNF-α, IL-1ß, IL-6 and High-mobility Group Box 1 (HMBG1) protein levels. Of 88 miRNAs studied, 30 were significantly dysregulated at all time points in the LPS-pretreated group, including MiR-155, -146a, -142-3p, -299, and -200c -132 and -21. TNF-α, regulated by miR-155 and miR-146a, was decreased in the LPS-pretreated group at all time points (P < 0.05), as were HMGB1, a key alarmin regulated by miR-146, -142-3p, -299 and -200c (P < 0.05), and IL-1ß and IL-6, both regulated by miR-132and miR-21 respectively (P < 0.05). Specific dysregulation of miR-155, -146a, -142-3p, -299, and -200c -132 and -21 with their corresponding effects on the TLR and NF-κB signaling pathways during inflammation, suggests a plausible mechanism for tolerance in this novel chronic model with persistent peritoneal infection.

MicroRNA as a new factor in lung and esophageal cancer.

Lung cancer is the most lethal cancer due to late detection in advanced stages; early diagnosis of lung cancer allows surgical treatment and improves the outcome. The prevalence of gastroesophageal reflux-related adenocarcinomas of the esophagus is increasing; repetitive surveillance endoscopies are necessary to detect development of cancer. A blood-based biomarker would simplify the diagnosis and treatment of both diseases. MicroRNAs (miRNAs) are short RNA strands that interfere with protein production. miRNAs play pivotal roles in cell homeostasis, and dysregulation of miRNAs can lead to the development of cancer. miRNAs can be found in all body fluids and have been proposed to serve as messengers between closely localized cells but also distant organs. Cancer cells actively secrete miRNAs, and these miRNA profiles can be found in blood. We outline, here, how these miRNAs may aid in diagnosis and treatment of lung and esophageal cancers, as well as their apparent limitations.

Does clinically relevant temperature change miRNA and cytokine expression in whole blood?

Unintentional hypothermia is a well-described risk factor for death and complications after elective and emergency surgery. The molecular mechanisms by which hypothermia exerts its detrimental effects are not well understood. Differences in cytokine production and the overall cell function have been reported under hypothermic conditions. We investigated the effect of a range of clinically relevant temperatures on cytokine production and microRNA (miRNA) expression in a whole-blood model. We found that there was a wide variation in tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 production among different subjects, ranging from low to high TNF-α producers. The intersubject variation can also be found on the transcriptional level: high producers had higher upregulation of TNF-α messenger RNA than intermediate and low producers. This variation in TNF-α was reproducible in each individual. Temperature seems to modulate TNF-α production among these different groups. miRNA expression was modulated by temperature. miRNA-181a might control, or be a part of the mechanism which controls, TNF-α production. However, an analysis of whole-leukocyte RNA does not allow the investigation of mechanisms in a specific leukocyte subpopulation such as monocytes, because these changes may be concealed by miRNA expression changes in the other leukocyte subsets. In conclusion, TNF-α, IL-6, and IL-10 production is highly variable among different persons, but temperature affects the expression of miRNAs, which may consequently alter the production of TNF-α.