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Sepsis remains one of the leading causes of death worldwide. Oncostatin M (OSM), an interleukin (IL)-6 family cytokine, can be found at high levels in septic patients. However, little is known about its role in sepsis. This study aimed to determine if the genetic knockout of OSM receptor (OSMR) type II signaling would improve survival in a murine model of sepsis. Aged (>50 weeks) OSMR type II knockout (KO) mice and wild-type (WT) littermates received an intraperitoneal injection of fecal slurry (FS) or vehicle. The KO mice had better survival 48 h after the injection of FS than the WT mice ( p = 0.005). Eighteen hours post-FS injection, the KO mice had reduced peritoneal, serum, and tissue cytokine levels (including IL-1β, IL-6, TNFα, KG/GRO, and IL-10) compared to the WT mice ( p < 0.001 for all). Flow cytometry revealed decreased recruitment of CD11b + F4/80 + Ly6c high+ macrophages in the peritoneum of KO mice compared to WT mice (34 ± 6 vs. 4 ± 3%, P Int = 0.005). Isolated peritoneal macrophages from aged KO mice had better live E. coli killing capacity than those from WT mice ( p < 0.001). Peritoneal lavage revealed greater bacterial counts in KO mice than in WT mice (KO: 305 ± 22 vs. 116 ± 6 CFU (×10 9 )/mL; p < 0.001). In summary, deficiency in OSMR type II receptor signaling provided a survival benefit in the progression of sepsis. This coincided with reduced serum levels of pro-inflammatory (IL-1β, TNFα, and KC/GRO) and anti-inflammatory markers (IL-10), increased bacterial killing ability of macrophages, and reduced macrophage infiltration into to site of infection.

Background: Up to 40% of patients are receiving opioids at the time of total knee arthroplasty (TKA) in the United States despite evidence suggesting opioids are ineffective for pain associated with arthritis and have substantial risks. Our primary objective was to determine whether preoperative opioid users had worse knee pain and physical function outcomes 12 months after TKA than patients who were opioid-naive preoperatively; our secondary objective was to determine the prevalence of opioid use before and after TKA in Alberta, Canada., Methods: In this retrospective analysis of population-based data, we identified adult patients who underwent TKA between 2013 and 2015 in Alberta. We used multivariable linear regression to examine the association between preoperative opioid use and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and physical function scores 12 months after TKA, adjusting for potentially confounding variables., Results: Of the 1907 patients, 592 (31.0%) had at least 1 opioid dispensed before TKA, and 124 (6.5%) were classified as long-term opioid users. Long-term opioid users had worse adjusted WOMAC pain and physical function scores 12 months after TKA than patients who were opioid-naive preoperatively (pain score β = 7.7, 95% confidence interval [CI] 4.0 to 11.6; physical function score β = 7.8, 95% CI 4.0 to 11.6; p < 0.001 for both). The majority (89 ([71.8%]) of patients who were long-term opioid users preoperatively were dispensed opioids 180-360 days after TKA, compared to 158 (12.0%) patients who were opioid-naive preoperatively., Conclusion: A substantial number of patients were dispensed opioids before and after TKA, and patients who received opioids preoperatively had worse adjusted pain and functional outcome scores 12 months after TKA than patients who were opioidnaive preoperatively. These results suggest that patients prescribed opioids preoperatively should be counselled judiciously regarding expected outcomes after TKA., Competing Interests: None declared., (© 2021 Joule Inc. or its licensors.)

Background: Computed tomography (CT) scan quantifying skeletal muscle mass is the gold standard tool to identify sarcopenia. Unfortunately, high cost, limited availability, and radiation exposure limit its use. We suggest that ultrasound of the thigh muscle could be an objective, reproducible, portable, and risk-free tool, used as a surrogate to a CT scan, to help identify frail patients with sarcopenia., Materials and Methods: We included 49 patients over 64 y old, referred to the acute care surgery service. An ultrasound of thigh muscle thickness was standardized to patient thigh length (U/S whole/L ). CT skeletal muscle index (SMI) was calculated using skeletal muscle surface area of the L3 region divided by height 2 . Frailty status was assessed using the Canadian Study of Healthy Aging Clinical Frailty Scale., Results: The mean (SD) age was 76 (8) y, and 34% (n = 17) were men. CT-defined sarcopenia was identified in 65% (n = 11) of men and 75% (n = 24) of women. In general, women had longer stay in hospital than men (mean + SD 14 ± 9 versus 7 ± 3 d, P = 0.003). There was a significant positive correlation between thigh U/S whole/L and CT SMI. There was an inverse correlation between thigh U/S whole/L and frailty score; a similar relationship was observed between CT SMI and frailty. There was an association between U/S whole/L and postoperative major complications., Conclusions: This prospective observational study illustrates that the U/S whole/L index can be used as a surrogate to CT scan, whereby it can identify elderly frail patients with sarcopenia. Thigh ultrasound should be further tested as an objective tool to assess for stratifying frailty., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Acute intestinal ischemia-reperfusion injury (AIIRI) is a devastating clinical condition relevant to multiple diseases processes, including sepsis, trauma, transplantation, and burns. An AIIRI is a contributor to the development of multiple organ dysfunction syndrome (MODS). Oncostatin M (OSM)/oncostatin M receptor (OSMR) signaling is an unrecognized and novel candidate pathway for the mediation of MODS. In this study, we hypothesized that OSM mediates the injury mechanism of AIIRI leading to MODS. Methods: Wild-type (WT) and OSMR-knockout (OSMR -/- ) C57BL/6 mice underwent AIIRI using a well-established model of selective occlusion of the superior mesenteric artery (SMA). Serum cytokine concentrations were measured using a multiplex detection system. Further tissue analysis was conducted with polymerase chain reaction, enzyme-linked immunosorbent assay, Western blots, and histologic review. Results: Survival was significantly higher in WT than in OSMR -/- groups at 30 minutes of ischemia with 2 hours of reperfusion (100% versus 42.9%; P = 0.015). No significant differences in the degree of local intestinal injury was seen in the two groups. In contrast, the degree of lung injury, as evidenced by myeloperixodase activity, was lower in OSMR -/- animals in the early AIIRI groups. There was a greater degree of renal dysfunction in OSMR -/- mice. Oncostatin M mediated interleukin (IL)-10 upregulation, with WT animals having significantly lower IL-10 concentrations (52.04 ± 23.06 pg/mL versus 324.37 ± 140.35 pg/mL; P = 0.046). Conclusion: Oncostatin M signalling is essential during acute intestinal ischemia-reperfusion injury. An OSMR deficiency results in decreased early lung injury but increased renal dysfunction. There was a significantly increased mortality rate after AIIRI in mice with OSMR deficiency. Augmentation of OSM may be a novel immunomodulatory strategy for AIIRI.

Background: A significant number of patients use opioids prior to total joint arthroplasty (TJA) in North America and there is growing concern that preoperative opioid use negatively impacts postoperative patient outcomes after surgery. This systematic review and meta-analysis evaluated the current evidence investigating the influence of preoperative opioid use on postoperative patient-reported outcomes (PRO) after total joint arthroplasty., Methods: A systematic search was performed using Ovid, Embase, Cochrane Library, Scopus, Web of Science Core Collection, CINAHL on February 15th, 2018. Studies reporting baseline and postoperative PRO among those prescribed preoperative opioids and those who were not prior to total knee and hip arthroplasty were included. Standardized mean differences (SMD) in absolute difference and relative change in PRO measures between the two groups was calculated using random effect models., Results: Six studies were included (n = 7356 patients); overall 24% of patients were prescribed preoperative opioids. Patients with preoperative opioid use had worse absolute postoperative PRO scores when compared to those with no preoperative opioid use (standardized mean difference (SMD) -0.53, 95% Confidence interval (CI) -0.75, - 0.32, p < 0.0001). When relative change in PRO score was analyzed, as measured by difference between postoperative and preoperative PRO scores, there was no group differences (SMD -0.26, 95% CI -0.56, 0.05, p = 0.10)., Conclusion: Patients prescribed preoperative opioids may attain worse overall pain and function benefits after TJA when compared to opioid-naïve patients, but do still benefit from undergoing TJA. These results suggest preoperative opioid users should be judiciously counselled regarding potential postoperative pain and function improvements after TJA.

Background: Among older inpatients, the highest incidence of delirium is within the surgical population. Limited data are available regarding postoperative delirium risk in the acute care surgical population. The purpose of our study was to establish the incidence of and risk factors for delirium in an older acute care surgery population., Methods: Patients aged 65 years or more who had undergone acute care surgery between April 2014 and September 2015 at 2 university-affiliated hospitals in Alberta were followed prospectively and screened for delirium by means of a validated chart review method. Delirium duration was recorded. We used separate multivariable logistic regression models to identify independent predictors for overall delirium and longer episodes of delirium (duration ≥ 48 h)., Results: Of the 322 patients included, 73 (22.7%) were identified as having experienced delirium, with 49 (15.2%) experiencing longer episodes of delirium. Postoperative delirium risk factors included Foley catheter use, intestinal surgery, gallbladder surgery, appendix surgery, intensive care unit (ICU) admission and mild to moderate frailty. Risk factors for prolonged postoperative delirium included Foley catheter use and mild to moderate frailty. Surgical approach (open v. laparoscopic) and overall operative time were not found to be significant., Conclusion: In keeping with the literature, our study identified Foley catheter use, frailty and ICU admission as risk factors for delirium in older acute care surgical patients. We also identified an association between delirium risk and the specific surgical procedure performed. Understanding these risk factors can assist in prevention and directed interventions for this high-risk population., Competing Interests: None declared., (© 2019 Joule Inc. or its licensors)

We have developed a novel, intraluminal preservation solution that is tailored to the metabolic requirements of the intestine. This organ-specific solution addresses many of the problems associated with low temperature organ storage including energy, oxidative and osmotic stresses. However, conservation of energy levels remains one of the most difficult obstacles to overcome due to the inherent sensitivity of the mucosa to ischemia. Creatine-loading has become a popular and scientifically proven method of augmenting energy reserves in athletes performing anaerobic burst work activities. We hypothesized that if we could develop a method that was able to augment cellular energy levels, the structure and function of the mucosa would be more effectively preserved. The purpose of this study was to determine if creatine-loading is a feasible and effective strategy for preserving the intestine. Our data indicate that creatine loading has significant impact on energy levels during storage with corresponding improvements in mucosal structure and function. Both of our rodent models, a) continuous perfusion for 4 h and b) a single flush with our intraluminal preservation solution supplemented with 50 mM creatine, demonstrated significant improvements in creatine phosphate, ATP, Energy Charge and ATP/AMP following cold storage (P < 0.05). Notably, after 10 h creatine phosphate was 324% greater in Creatine-treated tissues compared to Controls (P < 0.05). Preferential utilization of glutathione in the Creatine group was effective at controlling oxidative injury after 10 h storage (P < 0.05). Improvements in barrier function and electrophysiology with creatine-treatment reflected superior mucosal integrity after 10 h storage; Permeability and Transepithelial resistance measurements remained at fresh tissue values. This was in stark contrast to Control tissues in which permeability rose to >300% of fresh tissue values (P < 0.005) and transepithelial resistance dropped by 95% (P < 0.005). After 10 h storage, Park's grading of histologic injury reflected extensive villus denudation (grade 4) in control tissues compared to healthy tissue (grade 0) in the Creatine group. This study demonstrates that a strategy of creatine supplementation of our intraluminal preservation solution facilitates the preservation of the intestinal mucosa during storage., (Copyright © 2018. Published by Elsevier Inc.)

Background: Frailty is a state of vulnerability to diverse stressors. We assessed the impact of frailty on outcomes after discharge in older surgical patients., Methods: We prospectively followed patients 65 years of age or older who underwent emergency abdominal surgery at either of 2 tertiary care centres and who needed assistance with fewer than 3 activities of daily living. Preadmission frailty was defined according to the Canadian Study of Health and Aging Clinical Frailty Scale as "well" (score 1 or 2), "vulnerable" (score 3 or 4) or "frail" (score 5 or 6). We assessed composite end points of 30-day and 6-month all-cause readmission or death by multivariable logistic regression., Results: Of 308 patients (median age 75 [range 65-94] yr, median Clinical Frailty Score 3 [range 1-6]), 168 (54.5%) were classified as vulnerable and 68 (22.1%) as frail. Ten (4.2%) of those classified as vulnerable or frail received a geriatric consultation. At 30 days after discharge, the proportions of patients who were readmitted or had died were greater among vulnerable patients ( n = 27 [16.1%]; adjusted odds ratio [OR] 4.60, 95% confidence interval [CI] 1.29-16.45) and frail patients ( n = 12 [17.6%]; adjusted OR 4.51, 95% CI 1.13-17.94) than among patients who were well ( n = 3 [4.2%]). By 6 months, the degree of frailty independently and dose-dependently predicted readmission or death: 56 (33.3%) of the vulnerable patients (adjusted OR 2.15, 95% CI 1.01-4.55) and 37 (54.4%) of the frail patients (adjusted OR 3.27, 95% CI 1.32-8.12) were readmitted or had died, compared with 11 (15.3%) of the patients who were well., Interpretation: Vulnerability and frailty were prevalent in older patients undergoing surgery and unlikely to trigger specialized geriatric assessment, yet remained independently associated with greater risk of readmission for as long as 6 months after discharge. Therefore, the degree of frailty has important prognostic value for readmission., Trial Registration for Primary Study: ClinicalTrials.gov, no. NCT02233153., Competing Interests: Competing interests: None declared., (© 2018 Joule Inc. or its licensors.)

Background: As populations age, more elderly patients will undergo surgery. Frailty and complications are considered to increase in-hospital cost in older adults, but little is known on costs following discharge, particularly those borne by the patient. We examined risk factors for increased cost and the type of costs accrued following discharge in elderly surgical patients., Methods: Acute abdominal surgery patients aged 65 years and older were prospectively enrolled. We assessed baseline clinical characteristics, including Clinical Frailty Scale (CFS) scores. We calculated 6-month cost (in Canadian dollars) from patient-reported use following discharge according to the validated Health Resource Utilization Inventory. Primary outcomes were 6-month overall cost and cost for health care services, medical products and lost productive hours. Outcomes were log-transformed and assessed in multivariable generalized linear and zero-inflated negative binomial regressions and can be interpreted as adjusted ratios (AR). Complications were assessed according to Clavien-Dindo classification., Results: We included 150 patients (mean age 75.5 ± 7.6 yr; 54.1% men) in our analysis; 10.8% had major and 43.2% had minor complications postoperatively. The median 6-month overall cost was $496 (interquartile range $140-$1948). Disaggregated by cost type, frailty independently predicted increasing costs of health care services (AR 1.76, 95% confidence interval [CI] 1.43-2.18, p < 0.001) and medical products (AR 1.61, 95% CI 1.15-2.25, p = 0.005), but decreasing costs in lost productive hours (AR 0.39, p = 0.002). Complications did not predict increased cost., Conclusion: Frail patients accrued higher health care services and product costs, but lower costs from lost productive hours. Interventions in elderly surgical patients should consider patient-borne cost in older adults and lost productivity in less frail patients., Trial Registration: NCT02233153 (clinicaltrials.gov).

Sepsis and septic shock are the leading causes of death in critically ill patients. Acute intestinal ischemia/reperfusion (AII/R) is an adaptive response to shock. The high mortality rate from AII/R is due to the severity of the disease and, more importantly, the failure of timely diagnosis. The objective of this investigation is to use nuclear magnetic resonance (NMR) analysis to characterize urine metabolomic profile of AII/R injury in a mouse model. Animals were exposed to sham, early (30 min) or late (60 min) acute intestinal ischemia by complete occlusion of the superior mesenteric artery, followed by 2 hrs of reperfusion. Urine was collected and analyzed by NMR spectroscopy. Urinary metabolite concentrations demonstrated that different profiles could be delineated based on the duration of the intestinal ischemia. Metabolites such as allantoin, creatinine, proline, and methylamine could be predictive of AII/R injury. Lactate, currently used for clinical diagnosis, was found not to significantly contribute to the classification model for either early or late ischemia. This study demonstrates that patterns of changes in urinary metabolites are effective at distinguishing AII/R progression in an animal model. This is a proof-of-concept study to further support examination of metabolites in the clinical diagnosis of intestinal ischemia reperfusion injury in patients. The discovery of a fingerprint metabolite profile of AII/R will be a major advancement in the diagnosis, treatment, and prevention of systemic injury in critically ill patients.

Background: Acute mesenteric ischemia (AMI) has a high morbidity and mortality and often presents as a diagnostic challenge. Currently, there is no blood, urine, or radiologic tests that provide a definitive diagnosis of AMI. The aim of this study was to evaluate the clinical accuracy of urine intestinal fatty acid-binding protein (I-FABP) to diagnosis AMI., Materials and Methods: Twenty patients referred to the Acute Care Surgery service at University of Alberta Hospital with suspected AMI taken to the operating room for definitive diagnosis were recruited. Pathologic findings from surgical specimens confirmed a gold standard diagnosis for intestinal ischemia. The patients found to be nonischemic became the internal controls. Conventional clinical markers were examined in blood including white blood cell count, lactate, and creatinine. Blood was also examined by enzyme-linked immunosorbent assays (ELISAs) for I-FABP and interleukin-6. Urine was examined preoperatively and 6 and 24 h postoperatively for I-FABP., Results: Thirteen patients were pathologically diagnosed with AMI while five patients were nonischemic; two were excluded due to missing biologic specimens. There was no difference in age or gender between ischemic and nonischemic patients (56 ± 5 versus 66 ± 11 years old, respectively; six females with ischemic and three females in the nonischemic group). There was no difference in serum lactate and creatinine between the two groups. Serum interleukin-6 levels in patients with AMI were significantly higher than nonischemic controls (0.4 ± 0.2 ng/mL versus 0.2 ± 0.07 ng/mL, respectively, P = 0.03). There was a nonstatistically significant increase in serum I-FABP in AMI patients compared to internal controls (9 ± 3 ng/mL versus 2.4 ± 0.9 ng/mL, respectively, P = 0.2). Urine I-FABP was significantly higher in patients diagnosed with AMI than in controls (7 ± 1 ng/mL versus 2 ± 1 ng/mL, respectively, P = 0.007). The receiver operating characteristic curve illustrated that urine I-FABP discriminates significantly between patients with AMI and controls (area under receiver operating characteristic = 0.88, P = 0.03)., Conclusions: The traditional clinical markers lactate and white blood cell count were not able to differentiate AMI from nonischemic bowel. However, we found that urine I-FABP was a noninvasive biomarker with high specificity and sensitivity for accurately diagnosing AMI in patients. A noninvasive accurate tool for AMI would facilitate for a rapid treatment, while preventing unnecessary surgical interventions in high-risk patient populations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Acute mesenteric ischemia (AMI) is a life-threatening condition that can result in multiple organ injury and death. A timely diagnosis and treatment would have a significant impact on the morbidity and mortality in high-risk patient population. The purpose of this study was to investigate if intestinal fatty acid binding protein (I-FABP) and α-defensins can be used as biomarkers for early AMI and resultant lung injury. C57BL/6 mice were subjected to intestinal ischemia by occlusion of the superior mesenteric artery. A time course of intestinal ischemia from 0.5 to 3 h was performed and followed by reperfusion for 2 h. Additional mice were treated with N-acetyl-cysteine (NAC) at 300 mg/kg given intraperitoneally prior to reperfusion. AMI resulted in severe intestinal injury characterized by neutrophil infiltrate, myeloperoxidase (MPO) levels, cytokine/chemokine levels, and tissue histopathology. Pathologic signs of ischemia were evident at 1 h, and by 3 h of ischemia, the full thickness of the intestine mucosa had areas of coagulative necrosis. It was noted that the levels of α-defensins in intestinal tissue peaked at 1 h and I-FABP in plasma peaked at 3 h after AMI. Intestinal ischemia also resulted in lung injury in a time-dependent manner. Pretreatment with NAC decreased the levels of intestinal α-defensins and plasma I-FABP, as well as lung MPO and cytokines. In summary, the concentrations of intestinal α-defensins and plasma I-FABP predicted intestinal ischemia prior to pathological evidence of ischemia and I-FABP directly correlated with resultant lung injury. The antioxidant NAC reduced intestinal and lung injury induced by AMI, suggesting a role for oxidants in the mechanism for distant organ injury. I-FABP and α-defensins are promising biomarkers, and may guide the treatment with antioxidant in early intestinal and distal organ injury.

Among the intraabdominal organs, the intestine is the most susceptible to storage injury and as a consequence its safe cold ischemic time in the clinic is restricted to below 10 hours. The current practice for the intestinal preservation (IP) consists of an in-situ vascular flush with iced University of Wisconsin or Histidine-Tryptophan-Ketoglutarate solution followed by cold storage at 4°C. Mucosal injury is initiated within 1 hour and rapidly progresses to mucosal breakdown; tissue injury worsens upon reperfusion and further impairs the mucosal barrier, favoring bacterial translocation and sepsis. In addition of releasing danger signals, an advanced ischemia-reperfusion injury (IRI) may increase graft immunogenicity and promote rejection. Several alternative approaches have been tested as alternatives to the static storage. The aim of this review is to summarize and discuss the various intraluminal interventions as additional strategies aiming to reduce the IP/reperfusion injury and highlight the underlying pathophysiological mechanisms.

Acute intestinal ischaemia/reperfusion injury (AII/R) is an adaptive physiologic response during critical illness, involving mesenteric vasoconstriction and hypoperfusion. Prevention of AII/R in high risk patient populations would have a significant impact on morbidity and mortality. The purpose of this study was to investigate the protective effects of VSL#3 probiotic treatment in a murine model of AII/R. Adult 129/SvEv mice were subjected to an experimental AII/R model using superior mesenteric artery occlusion. Animals were pre-treated with either three days or two weeks of VSL#3 probiotics. Local tissue injury markers were assessed by levels of myeloperoxidase and activation of nuclear factor kappa B (NFкB). Systemic and local cytokines, including interleukin (IL)-1β, IL- 10, TNFα, and interferon gamma were measured by ELISA and multiplex fluorescent detection. VSL#3 probiotics reduced local tissue inflammation and injury due to AII/R. A two-week course of VSL#3 was more effective than a shorter three-day course. The reduction in local inflammation from the two-week course of VSL#3 is correlated to a significant reduction in levels of active IL-1β, and tissue levels of myeloperoxidase. Levels of active NFкB were significantly elevated in the vehicle-fed AII/R mice, corroborating with tissue inflammation, which were attenuated by VSL#3 administrations. VSL#3 did not cause any systemic inflammation or lung injury. VSL#3 probiotics are effective in reducing local tissue injury from AII/R by down-regulating pro-inflammatory mediators and immune cell recruitment. This study highlights a potential role for VSL#3 in management of patients at high risk for AII/R.

Background & Aims: Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC., Methods: We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb + sirolimus., Results: Anti-DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosis of the liver, but 100 μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups., Conclusions: Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The aim of this study was to assess recovery, cell death, and cell composition of post-thaw cultured human islets. Cryopreserved islets were provided by the Clinical Islet Transplant Program, Edmonton, Canada. Islets were processed using media prepared in accordance with Pre-Edmonton and Edmonton protocols. Cryopreserved islets were rapidly thawed and cultured for 24 h, 3 d, 5 d, and 7 d, following which they were processed for histology. Islet quantification, integrity, morphology and tissue turnover were studied via hematoxylin and eosin stained sections. Ultrastructure was studied by electron microscopy and endocrine cell composition by immunohistochemistry. Using the Pre-Edmonton protocol, islet recovery was 50.1% and islet survival was 50% at 24 h while for the Edmonton protocol, the islet recovery was 69.4% (p<0.001) and islet survival, 50% at ≈2.5 d. With an increasing culture duration although the physical integrity was retained there was an increasing loss of cohesivity both at light microscopic and at ultrastructure level regardless of the protocols used. Percentage islet survival and tissue turnover correlated negatively with culture duration in both protocols. The Edmonton protocol appears to preserve the islets better. However, culture duration adversely affects islet survival and quality, indicating the need for more optimal cryopreservation and culture techniques.

Metabolomic profiling can be used to study disease-induced changes in inflammatory bowel diseases (IBD). The aim of this study was to investigate the difference in the metabolomic profile of males and females as they developed IBD. Using the IL-10 gene-deficient mouse model of IBD and wild-type mice, urine at age 4, 6, 8, 12, 16, and 20 weeks was collected and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Multivariate data analysis was employed to assess differences in metabolomic profiles that occurred as a consequence of IBD development and severity (at week 20). These changes were contrasted to those that occurred as a consequence of gender. Our results demonstrate that both IL-10 gene-deficient and wild-type mice exhibit gender-related changes in urinary metabolomic profile over time. Some male-female separating metabolites are common to both IL-10 gene-deficient and control wild-type mice and, therefore, appear to be related predominantly to gender maturation. In addition, we were able to identify gender-separating metabolites that are unique for IL-10 gene-deficient and wild-type mice and, therefore, may be indicative of a gender-specific involvement in the development and severity of the intestinal inflammation. The comparison of the gender-separating metabolomic profile from IL-10 gene-deficient mice and wild-type mice during the development of IBD allowed us to identify changes in profile patterns that appear to be imperative in the development of intestinal inflammation, but yet central to gender-related differences in IBD development. The knowledge of metabolomic profile differences by gender and by disease severity has potential clinical implications in the design of both biomarkers of disease as well as the development of optimal therapies.

Aim: Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV., Methods: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 × 10(6)  cells/mouse, i.v.) 1 day prior to HCMV inoculation., Results: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, non-transplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up., Conclusion: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of hepatocytes., (© 2012 The Japan Society of Hepatology.)

Background: Crohn's disease recurrence after an ileocecal resection is common; yet, its pathophysiology is poorly understood and available treatment is suboptimal. The purpose of this study was to examine the bacterial, local, and systemic immune changes that follow ileocolonic anastomosis in a rodent model of Crohn's disease, the interleukin-10 gene-deficient (IL-10 null) mice., Materials and Methods: We divided wild-type and IL-10 null mice into three treatment groups: ileocolonic anastomosis, sham operation (ileo-ileal anastomosis), and control group without an operation. We sacrificed mice at 6 and 15 wks after the operation. At 6 wks, we assessed bacterial changes using the denaturing gel electrophoresis and similarity coefficient calculation. At both time points, we examined the small bowel for inflammation and fibrosis with histology. We measured the interferon gamma secretion by splenocytes stimulated with gastrointestinal bacterial antigens and splenocyte composition as a marker of systemic response., Results: At 6 wks, ileocolonic anastomosis resulted in increased similarity in bacterial species between the ileum and colon. The ileocolonic anastomosis did not lead to significant inflammation in the small intestine, but it resulted in an increased collagen deposition in all animals undergoing surgery, the most pronounced fibrosis of which was present in IL-10 null mice 15 wks after ileocolonic anastomosis. Furthermore, this was associated with significantly increased interferon gamma secretion by bacterial antigen-stimulated splenocytes and a decreased number of CD11+ cells in the same experimental group., Conclusions: Ileocolonic anastomosis leads to bacterial changes in the terminal ileum. In the genetically susceptible host, it is associated with small bowel fibrosis and systemic immune alterations. The composition of immune cells in the spleen is altered and splenocytes hypersecrete proinflammatory cytokine (interferon gamma) when challenged with gastrointestinal bacterial antigens., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The etiology of inflammatory bowel diseases (IBD) is largely unknown, but appears to be perpetuated by uncontrolled responses to antigenic components of the endogenous flora. Tolerance to antigenic stimulation can be achieved by exposure to a given antigen in high amounts (high dose tolerance). Colitis induced by feeding of Dextran Sodium Sulfate (DSS) is an often-used animal model mimicking clinical and histological features of human IBD., Aims: We investigated whether treatment with high doses of endogenous bacterial components can affect the response to these antigenic components and thus impact the course of the inflammatory response induced by DSS., Methods: 129/SvEv mice were injected intravenously in the tail vein with lysates prepared from fecal material of conventionally-raised mice. Control mice received a solution of bacterial antigen-free lysates prepared from fecal material of germ-free mice. Seven days later, colitis was induced in these mice by introducing DSS (3.5%) in the drinking water for 5 days. Onset and course of the inflammatory response was monitored by assessment of weight loss. Mice were sacrificed at day 7 post colitis induction and tested for histopathologic injury, intestinal cytokine release, and systemic response to bacterial antigens., Results: Intravenous injection with fecal lysates reduced intestinal and antigen-stimulated systemic pro-inflammatory cytokine release and prevented DSS-induced weight loss and intestinal injury., Conclusion: Pretreatment with high amount of endogenous bacterial components has a profound tolerogenic effect on the systemic and mucosal immune responses resulting in reduced intestinal inflammation and abrogates colitis-induced weight loss.

Background: Prosthetic mesh is used frequently in abdominal wall hernia reconstruction but is prone to postoperative adhesion formation. Complications resulting from intra-abdominal adhesions represent a considerable clinical and cost burden. We, herein, investigate the antiproliferative and antiadhesiogenic properties of sirolimus and hydrogel-impregnated, drug-eluting mesh to decrease such complications in a mouse model of abdominal wall hernia repair., Methods: A 1 × 1cm(2) polypropylene mesh from 1 of 3 groups (group 1, plain control; group 2, hydrogel [2% agarose]; and group 3, hydrogel + 10 mcg sirolimus) was implanted operatively into the peritoneal cavity of BALB/c mice and followed for up to 4 weeks. Adhesions were scored by percent surface area of mesh (range, 0-100%), severity (range, 0-3), and tenacity (range, 0-4). Representative samples were assessed by scanning electron microscopy., Results: Mesh impregnated with the combination of hydrogel and sirolimus led to a significant decrease in adhesion formation. The percent surface area of adhesional attachment to mesh was decreased from 100.0 ± 0% in the plain mesh control group versus 18 ± 8% (P < .001) in the combined impregnated mesh group. Similarly, adhesion severity scores were decreased from a score of 2.9 ± 0.1 (plain mesh) versus 1.4 ± 0.1 (sirolimus/hydrogel-impregnated mesh) (P < .001). Scores for tenacity were also decreased markedly from 3.5 ± 0.2 (plain mesh) versus 1.5 ± 0.1 (sirolimus/hydrogel-impregnated mesh (P < .001)., Conclusion: Creation of a sirolimus drug-eluting and hydrogel-impregnated polypropylene mesh resulted in marked decrease of adhesion formation in this mouse model, was well tolerated without side effects, and has potential for clinical application., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Aim: To investigate the effects of dextrans of various molecular weights (Mw) during a 12 h cold storage time-course on energetics, histology and mucosal infiltration of fluorescein isothiocyanate (FITC)-dextran., Methods: Rodent intestines were isolated and received a standard University of Wisconsin vascular flush followed by intraluminal administration of a nutrient-rich preservation solution containing dextrans of varying Mw: Group D1, 73 kdal; Group D2, 276 kdal; Group D3, 534 kdal; Group D4, 1185 kdal; Group D5, 2400 kdal., Results: Using FITC-labeled dextrans, fluorescent micrographs demonstrated varying degrees of mucosal infiltration; lower Mw (groups D1-D3: 73-534 kdal) dextrans penetrated the mucosa as early as 2 h, whereas the largest dextran (D5: 2400 kdal) remained captive within the lumen and exhibited no permeability even after 12 h. After 12 h, median injury grades ranged from 6.5 to 7.5 in groups D1-D4 (73-1185 kdal) representing injury of the regenerative cryptal regions and submucosa; this was in contrast to group D5 (2400 kdal) which exhibited villus denudation (with intact crypts) corresponding to a median injury grade of 4 (P < 0.05). Analysis of tissue energetics reflected a strong positive correlation between Mw and adenosine triphosphate (r(2) = 0.809), total adenylates (r(2) = 0.865) and energy charge (r(2) = 0.667)., Conclusion: Our data indicate that dextrans of Mw > 2400 kdal act as true impermeant agents during 12 h ischemic storage when incorporated into an intraluminal preservation solution.

Introduction: Our lab has developed an effective nutrient-rich solution that facilitates energy production and control of oxidative stress during static cold storage of the intestine; however, the requirement for oncotic agents, such as hydroxyethylstarch (HES), has not been evaluated. This study investigated the effectiveness and requirement for HES in an intraluminal preservation solution during a clinically relevant period of cold storage., Methods: Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a 'back table' intraluminal flush with a nutrient-rich preservation solution containing varying amounts of HES (n=6 per group): Group 1, 0%; Group 2, 2.5%; Group 3, 5%; Group 4, 10%. Energetics, oxidative stress, and morphology were assessed over a 24h time-course of cold storage., Results: Overall, the 5% HES solution, Group 3, demonstrated superior energetic status (ATP and total adenylates) compared to all groups, P<0.05. Malondialdehyde levels indicated a reduction in oxidative stress in Groups 3 and 4 (P<0.05). After 12h, median modified Parks' grades for Groups 2 and 3 were significantly lower than Groups 1 and 4, P<0.05., Conclusion: Our data suggests that when employing an intraluminal preservation solution for static organ storage, oncotic support is a fundamental requirement; 5% HES is optimal., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Introduction: Our lab has developed a novel strategy for intestinal preservation involving the intraluminal delivery of a nutrient-rich preservation solution. The aim of this study was to compare the effectiveness of two impermeant agents for use in our solution: Dextran 70 (D70; Mw=70 kDa) and Hydroxyethyl starch (HES; Mw=2200 kDa)., Methods: Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a 'backtable' intraluminal flush with: UW solution (group 1, UW), or an amino acid-based nutrient-rich preservation solution (AA solution) containing either 5% D70 (group 2, AA-D70) or HES (group 3, AA-HES). Tissue samples (n=6) were taken at 2, 4, 8, and 12 h cold storage; histology, energetic, end-product, and oxidative parameters were assessed. In separate groups (n=4), D70 and HES were fluorescently labeled with fluorescein isothiocyanate (FITC) in order to directly observe mucosal penetration of the starch and dextran., Results: Over the 12 h storage time-course, direct visualization of the fluorescently labeled D70 showed penetration of the mucosal layer as early as 2 h and progressively continued to do so throughout the 12 h period. In contrast, HES did not cross the mucosal barrier and remained captive within the lumen. As time of storage progressed, grade of injury increased in all groups, however, at 4 and 12 h the AA-HES treated tissues exhibited significantly less injury compared to UW and AA-D70, P < 0.05. AA-HES group showed on moderate villus clefting (median grade 2; P < 0.05) while the AA-D70 group exhibited complete villus denudation (grade 4) and the UW group had extensive injury into the regenerative cryptal regions (grade 6). Metabolic parameters revealed a preferential maintenance of ATP and Energy Charge; increases in lactate, alanine and ammonium supported the involvement of aerobic and anaerobic pathways for energy production., Conclusion: The results of this study challenge the idea that oncotic support is not a fundamental requirement of static organ storage. Furthermore, our data suggests that HES is an effective oncotic agent for use in our intraluminal nutrient-rich preservation solution, while Dextran 70 is not., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The severe combined immunodeficiency/albumin linked-urokinase type plasminogen activator (SCID/Alb-uPA) human liver chimeric mouse model has added a new dimension to studies of liver based human diseases and has important potential for study of human hepatic drug metabolism. However, it remains unclear if natural killer (NK) cell in SCID/Alb-uPA mice has an important negative impact on engraftment and expansion of human hepatocytes after transplantation. Here, we explore the role of mouse NK cells in the rejection of transplanted human hepatocytes in SCID/Alb-uPA mice. We assessed NK cell activity in vivo, using (125)I-iodo-2'-deoxyuridine incorporation assay. Low serum human alpha-1 antitrypsin (hAAT, <10 microg/ml) recipients, representing graft failure, showed resistance to engraftment of MHC class I knockout marrow (indicating high NK cell activity), while NK cell-depleted low hAAT recipients and high hAAT (>100 microg/ml) recipients accepted MHC class I knockout marrow, indicating a correlation between low NK cell activity, in vivo, and high level human hepatocyte engraftment. We also showed that higher level engraftment of human hepatocytes was achieved in both NK cell-depleted SCID/Alb-uPA mice and Rag2(-/-)gammac(-/-)/Alb-uPA (T,B and NK cell deficient) mice compared with untreated SCID/Alb-uPA mice. These results support a critical role for mouse NK cells in the rejection of human hepatocytes xenotransplanted to immunodeficient mice.

Human hepatocyte transplantation is an alternative treatment for acute liver failure and liver diseases involving enzyme deficiencies. Although it has been successfully applied in selected recipients, both isolation and transplantation outcomes have the potential to be improved by better donor selection. This study assessed the impact of various donor variables on isolation outcomes (yield and viability) and posttransplant engraftment, using the SCID/Alb-uPA (severe combined immunodeficient/urokinase type plasminogen activator under the control of an albumin promoter) human liver chimeric mouse model. Human hepatocytes were obtained from 90 human liver donor specimens and were transplanted into 3942 mice. Multivariate analysis revealed improved viability with younger donors (P = 0.038) as well as with shorter warm ischemic time (P = 0.012). Hepatocyte engraftment, assessed by the posttransplant level of serum human alpha1-antitrypsin, was improved with shorter warm ischemia time. Hepatocytes isolated from older donors (>or=60 years) had lower viability and posttransplant engraftment (P

Curcumin is a tumeric-derived, water-insoluble polyphenol with potential beneficial health effects for humans. It has been shown to have preventive as well as therapeutic effects in chemically induced murine models of colitis. To investigate whether curcumin exerts a similar effect on the spontaneous colitis in interleukin (IL)-10 gene-deficient mice, we gavaged these mice daily for 2 weeks with 200 mg/kg per day curcumin emulsified in carboxymethyl cellulose, a food additive generally used as a viscosity modifier. Mice fed the curcumin/carboxymethyl cellulose mixture and those receiving carboxymethyl cellulose alone demonstrated similar reductions in histological injury score and colon weight/length ratio compared to water-fed controls. However, significant reductions in pro-inflammatory cytokine release in intestinal explant cultures were only seen in mice treated with the curcumin mixture. Our data demonstrate that in IL-10 gene-deficient mice, both oral curcumin and carboxymethyl cellulose, appear to have modifying effects on colitis. However, curcumin has additional anti-inflammatory effects mediated through a reduced production of potent pro-inflammatory mucosal cytokines.

Introduction: This study investigated the role of a novel nutrient-rich preservation solution in alleviating intestinal ischemia-reperfusion (IR) injury in a large animal model., Materials and Methods: Porcine intestines were treated in vivo with the following intraluminal flush solutions: group 1, none; group 2, University of Wisconsin solution; group 3, an amino acid-based solution, previously shown to be effective in reducing IR injury in rodent models. Intestinal ischemia was induced in vivo for 60 min, followed by 180 min reperfusion. Key metabolic aspects were assessed in relation to two fundamental kinase mechanisms that govern cell fate, AMP kinase, and Jun kinase., Results: After 180 min reperfusion, groups 1 and 2 exhibited clefting, denudation, and mucosal hemorrhage, whereas injury was markedly reduced in group 3 (median grades 4.5 and 5 vs. 0; P<0.05). In contrast to groups 1 and 2, group 3 tissues exhibited a full recovery of adenylates (ATP, total adenylates) and an effective control of oxidative stress throughout reperfusion. Neutrophil-mediated inflammation was abrogated in group 3. An up-regulation of two key enzymes (glutaminase and alanine aminotransferase) provided a mechanism for the superior recovery of energetics and the preservation of mucosal integrity in group 3. A strong activation of AMP-activated protein kinase resulting in the up-regulation of a primary proapoptotic kinase mechanism, Jun kinase, was evident in groups 1 and 2., Discussion: A strategy of intraluminal administration of a nutrient-rich solution represents a potential therapy for alleviating intestinal IR injury; these findings suggest a more effective method for the ischemic storage of intestine.

The objective of the present investigation was to evaluate the effects of acidic pH of the perfusate and presence of lipopolysaccharide (LPS) on permeability of rumen and colon mucosal tissues to mannitol and LPS using the Ussing chamber system. Rumen and colon tissues (n = 8), obtained from slaughtered feedlot steers, were tested for changes in permeability to (3)H-mannitol under pH of 4.5, 5.5, and 6.5 for rumen and at 5.5, 6.5, and 7.4 for colon, with or without LPS from Escherichia coli B:055 at 500 microg/mL. The (3)H-Mannitol was added at 10 microL (525.4 GBq/mmol) on the mucosal side of the Ussing chamber to detect changes in permeability, and 4 samples were taken at 20, 25, 30, and 35 min from the serosal side. Permeability of rumen and colon mucosa to (3)H-mannitol increased 6- and 5-fold, respectively, at acidic pH values of 4.5 and 5.5 and in the presence of 500 micro/mL of LPS. In contrast, LPS did not affect rumen and colon permeability at pH that ranged from 5.5 and 7.4. Translocation of LPS across the rumen and colon mucosa of cattle was not pH dependent. The LPS translocated through these tissues if present at the mucosal side. In conclusion, the permeability of rumen and colon tissues to (3)H-mannitol increased in presence of LPS and under acidic pH, whereas LPS permeated through mucosal tissues independently of the pH of the perfusate. Further research is warranted to understand the mechanism(s) by which acidic pH of the rumen digesta and presence of LPS make rumen and colon tissues "leaky".

Freezing survival was assessed in three species of terrestrial slugs, a holarctic but native North American species, Deroceras laeve, and two species introduced from Europe, D. reticulatum and Arion circumscriptus. The introduced species showed very poor freezing survival. Supercooling points of the introduced species were quite high ( approximately -3 degrees C) and their freezing survival was very poor, limited to short-term freezing at -1.2 to -1.5 degrees C and low ice contents (23-44%). D. laeve showed a significant elevation of supercooling point between slugs collected in the autumn (-4.8 degrees C +/- 0.5) and those collected early in the spring (-3.1 degrees C +/- 0.4). This species also showed substantial freezing survival which was greater for spring-collected slugs (100% survival of 1 h freezing at -2 degrees C with an ice content of 65%) than for autumn animals (100% survival for 1 h at -1 degrees C with approximately 40% ice). Carbohydrate and amino acid responses to freezing and anoxia exposures were compared in the two Deroceras species. D. laeve showed a strong hyperglycemic response to freezing, a 100-fold increase in glucose levels that suggested that glucose may have a cryoprotective function in this species. D. reticulatum did not accumulate glucose and neither species produced glycerol or lactate. Both species showed typical responses to anoxia (aspartate and glutamate catabolism, alanine and succinate accumulation) and D. laeve also showed this pattern during freezing, suggesting a natural switch to anaerobiosis to support freezing survival.

Background: In the IL-10 gene-deficient mouse model, development of intestinal inflammation is associated with a defect in epithelial barrier integrity that is thought to allow sufficient passage of bacteria or bacterial antigens to initiate a mucosal immune response. Microbial monoassociation experiments into axenic animals have shown that some, but not all, endogenous bacteria will initiate an intestinal inflammatory response. For instance, Bacteroides vulgatus does not initiate intestinal inflammation in axenic IL-10 gene-deficient mice. We investigated whether B. vulgatus requires concomitant disruption of the intestinal epithelial barrier integrity in order to initiate an inflammatory response., Methods: We first identified a dose of the indomethacin that would cause a primary disruption of the epithelial barrier without causing intestinal inflammation. IL-10 axenic mice were then administered this dose of indomethacin in their drinking water for 7 days and concomitantly monoassociated, by oral gavage, with B. vulgatus., Results: Indomethacin treatment (2 microg/g/d) for 7 days resulted in disruption of epithelial barrier integrity, but it caused neither a systemic inflammatory response nor a mucosal inflammatory response in the colon or cecum. Monoassociation with B. vulgatus alone did not lead to a mucosal inflammatory response, despite a measurable systemic response. In contrast, administration of indomethacin plus B. vulgatus-monoassociation resulted in a marked intestinal inflammatory response in colon and cecum., Conclusions: Our data show that, in a genetically predisposed animal model, the nondisease-causing endogenous bacteria, B. vulgatus, is able to cause an intestinal inflammatory response provided that disruption of the intestinal epithelial barrier has occurred.

Background: A recent study from our laboratory documented significant improvements in post-transplant viability in an experimental model of intestinal transplantation when a novel, nutrient-rich preservation solution was used during cold storage. The current study investigated the relationship between energetic/oxidative stress responses and fundamental kinase signaling events during the period of organ storage. This relationship may be a key factor contributing to improved graft viability after storage in a nutrient-rich preservation solution., Methods: Rat small intestine was harvested and flushed intraluminally with University of Wisconsin (UW) solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush (clinical control); Group 2, luminal UW solution; Group 3, luminal AA solution. Energetics (ATP, total adenylates), oxidative stress (malondialdehyde), histology, and MAPK (P38, JNK, ERK)/AMPK/Caspase-3 were assessed throughout 12-hour cold storage., Results: P38 and JNK were upregulated strongly in Group 2 after 1- and 12-hour storage. Group 3 exhibited a delayed activation and subsequent downregulation of these pre-apoptotic signals. Between 6 to 12 hours, a strong upregulation of ERK was observed in Group 3. AMPK downregulation correlated with a reduction in AMP/ATP ratio, ERK upregulation, and P38/JNK downregulation in Group 3. After 12-hour storage, histology indicated superior preservation of mucosal architecture in Group 3 tissues., Conclusions: A nutrient-rich preservation solution abrogates pre-apoptotic signaling (JNK and P38) and upregulates cytoprotective signals (ERK). Our data support the concept of a concerted effort facilitating cellular protection in response to ischemic stress.

Colonic epithelial cells are constantly exposed to high levels of bacterial DNA in the intestinal lumen and must recognize and respond appropriately to pathogens, while they maintain a tolerance to nonpathogenic commensal bacterial strains. Bacterial DNA is recognized by Toll-like receptor 9 (TLR9). The aim of this study was to investigate TLR9 expression and localization in colonic epithelial cells under basal conditions and in response to bacterial DNA. HT-29 cells were exposed to DNA from various strains of commensal and pathogenic microbes. TLR9 mRNA expression was determined by real-time reverse transcription-PCR, and interleukin-8 (IL-8) secretion was measured by an enzyme-linked immunosorbent assay. Localization of TLR9 was determined by flow cytometry in HT-29 cells and by immunofluorescence in HT-29 cells and mouse colonic tissue. Immunofluorescence and flow cytometric analyses demonstrated that there was intracellular and surface expression of TLR9 in HT-29 cells under basal conditions. Exposure of cells to DNA from pathogenic strains of Salmonella and Escherichia coli resulted in a significant increase in TLR9 mRNA expression. Salmonella enterica serovar Dublin DNA increased surface TLR9 protein and IL-8 secretion. There was no change in mRNA levels or localization of TLR9 in response to Bifidobacterium breve. Chloroquine did not block IL-8 secretion in response to S. enterica serovar Dublin DNA. TLR9 was expressed on the colonic apical surface in wild-type mice but not in germfree mice. These results demonstrate that intestinal epithelial cells recognize pathogenic bacterial DNA and respond by increasing surface localization and expression of TLR9, suggesting that the epithelial inflammatory response to pathogenic DNA is mediated at least in part by increased TLR9 expression.

Object: Hydrocephalus results from abnormal cerebrospinal fluid (CSF) volumes or flow patterns. The absorption of CSF is determined largely by pressures within veins and venous sinuses in the head and adjacent to the spine. Most surgical solutions for hydrocephalus involve diversion of excess CSF into alternative absorption sites, and most of these solutions are still suboptimal. The focus of this work has been to recreate more normal CSF absorption into the dural venous sinuses without having to directly access the superior sagittal sinus (SSS)., Methods: Intraosseous skull infusion for the purpose of accessing the SSS and the systemic venous system was tested by experimental skull infusions of tracer fluids into living large animals (14 adult pigs). Compared with control injections into an ear vein, infusions into the skull through specially designed infusion devices had similar systemic absorption characteristics. This suggested that intraosseous skull infusion in a living large animal was successful in gaining access to the SSS and systemic venous system., Conclusions: This study constitutes the first demonstration of the success of intraosseous skull infusion in gaining rapid access to the systemic venous system and it thus opens the possibility of using this strategy for diversion of CSF back into the intracranial venous system for the treatment of hydrocephalus.

Rodent models have been essential to understanding the immune-mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at 4 degrees C for 48 h/buffer containing 0.5% Triton X-100 followed by a 72 h washout in phosphate-buffered saline. Osmotic decellularization was performed in similar manner to the detergent-based technique except without the addition of Triton X-100. Enzymatic decellularization consisted of trypsin/EDTA at 37 degrees C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular elements, and hydroxyproline assays. Detergent-based methodology effected near-complete decellularization of both the leaflets and aortic wall in addition to preservation of the extracellular matrix (ECM). Osmotic lysis was associated with preservation of ECM and moderate decellularization. Enzymatic decellularization resulted in complete decellularization but extensive degeneration and fragmentation of the ECM. When implanted into the infrarenal aorta of allogeneic rats for 1 week, valves decellularized with detergent-based and osmotic methodology failed to stimulate an allogeneic immune response as evidenced by an absence of T cell infiltrates. Osmotic lysis protocols with low dose detergent appear to be most effective at both removing antigenic cellular elements and preserving ECM.

Background: Glutaraldehyde fixation (G-F) decreases but likely does not eliminate the antigenicity of bioprosthetic heart valves. Rejection (with secondary dystrophic calcification) may be why G-F xenograft valves fail, especially in young patients, who are more immunocompetent than the elderly. Therefore, we sought to determine whether rejection of G-F xenograft occurs and to correlate this with graft calcification., Methods and Results: Ascending aortas/valves (from rats [syngeneic] or guinea pigs [xenogeneic]) were transplanted (fresh or after 48 hour of G-F) into the infrarenal aortas of young rat recipients for 20 days. A xenogeneic group was also treated with steroids until graft harvest. The valves and media/adventitia were scored blindly for inflammation (0 to 4). Percent graft infiltration by T cells/macrophages was determined (immunohistochemistry), and rat IgG ELISAs were performed. There was >3 times more valve inflammation, >10 times more valve T-cell/macrophage infiltrate, and >3 times antibody rise in the G-F xenogeneic groups compared with the fresh syngeneic or the G-F syngeneic groups (P<0.05). There was >2 times more adventitial inflammation and T-cell/macrophage infiltrate in the xenogeneic groups (P<0.05). Steroid treatment decreased inflammation and antibody rise in the xenogeneic groups (P<0.05). Correlation analysis revealed media/adventitia inflammation (P=0.02) and percent macrophage (P=0.01) infiltration to be predictors of calcification., Conclusions: G-F xenografts have cellular/humoral rejection and calcify secondarily.

Background: Oxidative stress during cold small bowel (SB) storage has not been investigated because oxygen is depleted rapidly after procurement. We hypothesized that oxidative catabolism facilitated by a proven amino acid-based (AA) storage solution promotes oxidative stress; furthermore, there is an important role for antioxidant supplementation during cold storage., Methods: SB from Sprague-Dawley rats (n = 6 in each group) were procured according to standardized procedures involving vascular flush with modified University of Wisconsin solution and luminal treatment with an AA-based solution proven previously to aid preservation. SB were assigned randomly to the following antioxidant treatment groups: group 1, none; group 2, superoxide dismutase/catalase; group 3, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble analogue of vitamin E). Energetics, oxidative stress, electrophysiology, and histology were assessed over 24 hours at 4 degrees C., Results: The addition of Trolox in group 3 resulted in a significant reduction in malondialdehyde levels compared with all other groups throughout 24 hours of cold storage. Tissue energetics correlated well with reduced oxidative injury; over the first 12 hours, adenosine triphosphate and total adenylates were superior in tissues treated with Trolox (group 3) versus AA solution alone (group 1). Functional assessment showed relatively normal permeability in all groups, however, Trolox-treated tissues showed significantly higher short-circuit current compared with control group (17.7 vs 5.5 microA/cm(2)). Histologic integrity was improved in group 3 after 24 hours of cold storage., Conclusions: Oxidative stress appears to be a determinant in the pathogenesis of mucosal injury during cold storage. Trolox effectively abrogates storage-related oxidative stress in SB.

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4 degrees C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30-40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.

Background: This study tested the effectiveness of a nutrient-rich preservation solution in a small animal model of orthotopic whole small bowel transplantation., Methods: Lewis rats received syngeneic total orthotopic small bowel graft after cold storage for 6 h. Donor small bowel was flushed vascularly with University of Wisconsin (UW) solution and flushed luminally with UW solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush; Group 2, UW solution; Group 3, AA solution. Biopsies were taken over 14 days posttransplant; energetics, oxidative stress, neutrophil recruitment and histologic injury were assessed., Results: All animals in Groups 1 and 2 failed to survive 12 h posttransplant due to hemorrhagic shock and fluid loss. In contrast, all animals in Group 3 survived the operation; survival after 14 days was 80% (4/5). In Group 3, full recovery of tissue adenylates (ATP and energy charge) to freshly isolated tissue values occurred within 3 days. Oxidative stress as assessed by malondialdehyde (MDA) levels was low in Group 3 throughout 14 d; Groups 1 and 2 exhibited high oxidative stress over the initial 35 min reperfusion (P<0.05). Neutrophil recruitment (myeloperoxidase activity) was significantly reduced in Group 3 tissues, as was histologic injury (P<0.05 compared to Groups 1 and 2). By day 14, Group 3 exhibited complete mucosal restoration., Conclusions: The data presented in this communication supports the use of an intraluminal preservation solution that is tailored to the metabolic requirements of the small bowel.

HLA-B27 transgenic rats develop spontaneous colitis under specific pathogen-free conditions (SPF) but germ-free rats remain disease-free, emphasizing a role for intestinal bacteria in the pathogenesis of chronic intestinal inflammation. Prebiotics are dietary substances that affect the host by stimulating growth and/or activity of potentially health promoting bacteria. The aims of this study were to investigate whether prebiotics can prevent colitis in SPF HLA-B27 rats, and secondly, to explore mechanisms of protection. SPF HLA-B27 transgenic rats received orally the prebiotic combination long-chain inulin and oligofructose (Synergy 1), or not, prior to the development of clinically detectable colitis. After seven weeks, cecal and colonic tissues were collected for gross cecal scores (GCS), histologic inflammatory scores (scale 0-4), and mucosal cytokine measurement. Cecal and colonic contents were collected for analysis of the gut microbiota by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH), and analysis of short-chain fatty acid composition. Prebiotic treatment significantly decreased GCS and inflammatory histologic scores in the cecum and colon. Prebiotic treatment also decreased cecal IL-1beta, but increased cecal TGF-beta concentrations. Inulin/oligofructose altered the cecal and colonic PCR-DGGE profiles, and FISH analysis showed significant increases in cecal Lactobacillus and Bifidobacterium populations after prebiotic treatment compared with water-treated rats. In conclusion, the prebiotic combination Synergy 1 reduced colitis in HLA-B27 transgenic rats, which effect was associated with alterations to the gut microbiota, decreased tissue proinflammatory cytokines and increased immunomodulatory molecules. These results show promise for prebiotics as primary or adjuvant maintenance therapy for chronic inflammatory bowel diseases.

Objectives: Cryopreserved valve allografts used in congenital cardiac surgery are associated with a significant cellular and humoral immune response. This might be reduced by removal of antigenic cellular elements (decellularization). The aim of this study was to determine the immunologic effect of decellularization in a rat allograft valve model., Methods: Brown Norway and Lewis rat aortic valves were decellularized with a series of hypotonic and hypertonic buffers, protease inhibitors, gentle detergents (Triton X-100), and phosphate-buffered saline. Valves were implanted into Lewis rats in syngeneic and allogeneic combinations. Cellular (CD3 and CD8) infiltrates were assessed with morphometric analysis, and the humoral response was assessed with flow cytometry., Results: Morphometric analysis identified a significant reduction in CD3 + cell infiltrates (cells per square millimeter of leaflet tissue) in decellularized allografts compared with that seen in nondecellularized allografts at 1 (79 +/- 29 vs 3310 +/- 223, P < .001), 2 (26 +/- 11 vs 109 +/- 20, P = .004), and 4 weeks (283 +/- 122 vs 984 +/- 145, P < .001). Anti-CD8 staining confirmed the majority of infiltrates were cytotoxic T cells. Flow cytometric mean channel fluorescence intensity identified a negative shift (abrogated antibody formation) for decellularized allografts compared with nondecellularized allografts at 2 (19 +/- 1 vs 27 +/- 3, P = .033), 4 (35 +/- 2 vs 133 +/- 29, P = .001), and 16 weeks (28 +/- 2 vs 166 +/- 54, P = .017)., Conclusions: Decellularization significantly reduces the cellular and humoral immune response to allograft tissue. This could prolong the durability of valve allografts and might prevent immunologic sensitization of allograft recipients.

Introduction: Donor nutritional status may be a determinant of small bowel (SB) quality following storage. In this study, we investigated the effect of donor nutritional status and a proven nutrient-rich preservation solution on graft quality following cold storage., Methods: Rats were fasted (12-14 h) or non-fasted. SB (n=6) was flushed vascularly with modified University of Wisconsin (UW) solution and flushed luminally with UW or an amino acid-rich (AA) solution as follows: Fasted. UWV, none; UWL, UW solution; AAL, AA solution. Non-fasted. UWV, none; UWL, UW solution; AAL, AA solution. Energetics, peroxidation (malondialdehyde; MDA), glutathione and histology were assessed over 24 h at 4 degrees C., Results: Energetics (ATP, ATP/ADP, and energy charge) were significantly higher in AAL (fasted and non-fasted) groups than other groups. However, there were no differences in energetics parameters between fasted and non-fasted animals in all groups. MDA was higher in fasted groups than non-fasted tissues; interestingly, AAL values were up to 10-fold lower than other groups. Higher glutathione levels were detected in non-fasted AAL tissues. Mucosal integrity was markedly superior in luminally treated tissues (UWL and AAL) in fasted and non-fasted states. Most noteably, AAL tissues from fasted animals exhibited grade 2 injury (villus clefting), whereas normal mucosa was observed in non-fasted tissues (grade 0)., Conclusion: Luminal flushing and a nutrient-rich preservation solution improve energetics, oxidative stress, and mucosal integrity during storage. Poorer donor nutritional status does not affect energetics throughout storage, but causes mucosal injury as a result of increased oxidative stress, even after a brief period of donor fasting.

The aim of this study was to improve small bowel (SB) quality during cold storage by combining two proven preservation strategies involving perfluorocarbon (PFC) and a novel luminal amino acid-rich solution. Rodent SB was flushed vascularly with UW solution and flushed luminally as follows: Group 1 (control)--no luminal flush, stored in UW; Group 2--luminal UW solution, stored in PFC; Group 3--luminal amino-acid (AA) solution, stored in PFC; and Group 4--luminal AA solution, stored in AA solution. Energetics, histology and mucosal function/electrophysiology were assessed over 24 h at 4 degrees C. ATP was consistently greater in Groups 2-4 than in the Control Group. Groups 3 and 4 exhibited significantly greater ATP, ATP/ADP ratios and energy charge levels after 12-h storage than in the other Groups. Histologic injury was generally lower in the AA-treated tissues (Groups 3 and 4); after 24 h, only minor epithelial clefting (Park's median grade 2) was present in Group 4; and consistent transmural infarction (grade 8) was evident in Groups 1 and 2. Combined treatment with luminal amino acid solution and oxygenated storage solution (PFC or AA solution) significantly improves energetics and mucosal function. This strategy may have implications for successful SB preservation in the clinic., (Copyright 2004 Blackwell Munksgaard)

Successful preservation of small bowel (SB) is closely correlated with the maintenance of cellular energetics. This study was designed to assess the ability of a modified UW solution supplemented with buffer and glucose to facilitate ATP production during cold storage. In part A, rats SB (n = 4) were flushed vascularly as follows: Group 1, UW solution (control); Group 2, HUW solution (UW+90 mM histidine). Inclusion of histidine resulted in a >3-fold increase in buffering capacity over the pH range 7.4-6.8. Positive effects of histidine on ATP and energy charge were apparent after 4-10h storage. Examination of the key regulatory enzyme, Phosphofructokinase (PFK), reflected a sustained activation was over 1-4h in the HUW group only. In part B, groups were vascularly flushed as follows: Group 1, HUW solution (control); Group 2, Group 1+20mM glucose; and Group 3, Group 2+luminal flush. Elevated ATP and total adenylates over 2-10h in Group 3 compared to control were a direct consequence of improved glycolytic activity. This data supports the hypothesis that tissue energetics can be significantly improved during cold storage using a histidine-buffered UW solution supplemented with carbohydrate substrate.

An amino acid-based solution has been recently developed and has demonstrated significant protective effects during cold storage of small bowel (SB). This study was designed to examine the role of this novel solution in ameliorating intestinal injury in an in vivo model of ischemia-reperfusion (IR). The impact of luminal treatment with an amino acid-based (AA) solution was assessed throughout reperfusion after 60-min warm ischemia (WI) in rodent SB. Energetics (ATP and total adenylates) remained significantly elevated throughout 60-min reperfusion in AA-treated tissue compared with untreated controls. Increases in end-products (ammonia and alanine) and increases in alanine aminotransferase and glutaminase activity implicated greater amino acid metabolism in AA-treated tissues. After reperfusion, malondialdehyde levels were similar between all groups. Glutathione levels were consistently elevated in AA-treated tissues and by 60 min reperfusion values were sixfold greater than control. AA-mediated protection during IR resulted in reduced neutrophil infiltration suggesting a weaker inflammatory response. Barrier function and electrophysiology parameters exhibited a clear pattern of mucosal preservation in AA-treated tissues; histology supported these findings. This study raises the possibility of a role for a luminal nutrient-rich solution during ischemic storage of small bowel in the clinic.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 +/- 0.5 h after 11.1 +/- 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 +/- 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.