British Society of Gastroenterology guidelines for the diagnosis and management of cholangiocarcinoma.

These guidelines for the diagnosis and management of cholangiocarcinoma (CCA) were commissioned by the British Society of Gastroenterology liver section. The guideline writing committee included a multidisciplinary team of experts from various specialties involved in the management of CCA, as well as patient/public representatives from AMMF (the Cholangiocarcinoma Charity) and PSC Support. Quality of evidence is presented using the Appraisal of Guidelines for Research and Evaluation (AGREE II) format. The recommendations arising are to be used as guidance rather than as a strict protocol-based reference, as the management of patients with CCA is often complex and always requires individual patient-centred considerations.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Translating Biomarkers of Cholangiocarcinoma for Theranosis: A Systematic Review.

Cholangiocarcinoma (CCA) is a rare disease with poor outcomes and limited research efforts into novel treatment options. A systematic review of CCA biomarkers was undertaken to identify promising biomarkers that may be used for theranosis (therapy and diagnosis). MEDLINE/EMBASE databases (1996-2019) were systematically searched using two strategies to identify biomarker studies of CCA. The PANTHER Go-Slim classification system and STRING network version 11.0 were used to interrogate the identified biomarkers. The TArget Selection Criteria for Theranosis (TASC-T) score was used to rank identified proteins as potential targetable biomarkers for theranosis. The following proteins scored the highest, CA9, CLDN18, TNC, MMP9, and EGFR, and they were evaluated in detail. None of these biomarkers had high sensitivity or specificity for CCA but have potential for theranosis. This review is unique in that it describes the process of selecting suitable markers for theranosis, which is also applicable to other diseases. This has highlighted existing validated markers of CCA that can be used for active tumor targeting for the future development of targeted theranostic delivery systems. It also emphasizes the relevance of bioinformatics in aiding the search for validated biomarkers that could be repurposed for theranosis.

Impact of blood transfusion on outcomes following resection for colorectal liver metastases in the modern era.

BACKGROUND: Evidence suggests that perioperative blood loss and blood transfusions are associated with poorer long-term outcomes in patients undergoing other oncological surgery. The aim of this study was to determine the long-term outcomes of patients requiring a blood transfusion post-hepatectomy for colorectal liver metastases (CRLM). METHODS: This is a retrospective review from 2005 to 2012. Overall survival (OS) and recurrence-free survival (RFS) were assessed using Kaplan-Meier curves. Red blood cell transfusion (RBCT) and other clinic-pathological parameters were handled as covariates for Cox regression analysis. RESULTS: Six hundred and ninety patients were included. Median follow-up was 33 months. Sixty-four (9.3%) patients required a perioperative RBCT. RBCT was a predictor for decreased OS (median 41 versus 49 months, P = 0.04). However, on multivariate regression analyses preoperative chemotherapy, post-operative complications and Clinical Risk Score were independently associated with reduced OS, though RBCT was not. There was no association between RBCT and RFS (median 15 versus 17 months, P = 0.28). CONCLUSIONS: RBCT is not independently associated with a poorer OS.

AZD1775 induces toxicity through double-stranded DNA breaks independently of chemotherapeutic agents in p53-mutated colorectal cancer cells.

AZD1775 is a small molecule WEE1 inhibitor used in combination with DNA-damaging agents to cause premature mitosis and cell death in p53-mutated cancer cells. Here we sought to determine the mechanism of action of AZD1775 in combination with chemotherapeutic agents in light of recent findings that AZD1775 can cause double-stranded DNA (DS-DNA) breaks. AZD1775 significantly improved the cytotoxicity of 5-FU in a p53-mutated colorectal cancer cell line (HT29 cells), decreasing the IC50 from 9.3 µM to 3.5 µM. Flow cytometry showed a significant increase in the mitotic marker pHH3 (3.4% vs. 56.2%) and DS-DNA break marker γH2AX (5.1% vs. 50.7%) for combination therapy compared with 5-FU alone. Combination therapy also increased the amount of caspase-3 dependent apoptosis compared with 5-FU alone (4% vs. 13%). The addition of exogenous nucleosides to combination therapy significantly rescued the increased DS-DNA breaks and caspase-3 dependent apoptosis almost to the levels of 5-FU monotherapy. In conclusion, AZD1775 enhances 5-FU cytotoxicity through increased DS-DNA breaks, not premature mitosis, in p53-mutated colorectal cancer cells. This finding is important for designers of future clinical trials when considering the optimal timing and duration of AZD1775 treatment.

Primary liver resection for patients with cirrhosis and hepatocellular carcinoma: the role of surgery in BCLC early (A) and intermediate stages (B).

AIM: This study aims to report the outcomes following primary liver resection (PLR) in patients with cirrhosis including stratification according to the Barcelona Clinic Liver Cancer (BCLC) classification. METHODS: Patients with cirrhosis and hepatocellular carcinoma (HCC) who had PLR between 2000 and 2013 were examined. Overall survival (OS), disease-free survival (DFS) and recurrence rate (RR) were analysed. Management after recurrence was reviewed as well as comparison to a series of 116 patients listed directly for liver transplant. RESULTS: Seventy-one patients underwent PLR. Median follow-up was 40 months. The 1-, 3- and 5-year OS and DFS for the series were 77, 69 and 61 % and 69, 39 and 28 % respectively. Overall recurrence was 59 % (44/71) and only 36 % (15/44) of those patients had a further potentially curative procedure. The 1-3-5-year OS and DFS in the BCLC-A (44 patients) were 86, 78 and 68 % and 78, 48 and 44 % respectively. The RR in BCLC-A was 45 % (20 patients) with half (11 patients) suitable for further treatment with curative intent. The 1-3-5-year OS and DFS in the BCLC-B (17 patients) were 81, 74 and 60 % and 58, 29 and 7 % respectively. The overall RR in BCLC-B was 76 % (13 patients). CONCLUSION: Recurrence following PLR for HCC in patients with cirrhosis is high with only a third of patients suitable for further potentially curative procedures. For patients with BCLC-A (or within Milan criteria), PLR provided a 68 % 5-year OS with 44 % of them free of disease. Surgery can offer satisfactory OS in carefully selected patients in the BCLC-B stage.

A cost-effective analysis of fibrin sealants versus no sealant following open right hemihepatectomy for colorectal liver metastases.

BACKGROUND: There is paucity of data regarding the cost-effectiveness of fibrin sealants during liver surgery. This study aimed to assess the cost-effectiveness of fibrin sealants following right hemihepatectomy for colorectal liver metastases. METHOD: A prospectively maintained database between 2004 and 2013 was reviewed to identify patients who underwent a right hemihepatectomy with and without fibrin sealant application. Perioperative and post-operative outcomes were analysed to assess its cost-effectiveness. RESULTS: One hundred and sixty-three right hemihepatectomies were performed, of which 79 were in the fibrin sealant treatment group and 84 were in the no sealant group. No difference was seen between fibrin sealant and no sealant with regard to bile leak (P = 0.366), intra-abdominal collections (P = 0.200) and overall post-operative complications (P = 0.480). Operating costs were significantly cheaper in the no sealant group (P = 0.010). There was no difference seen in median post-operative stay between fibrin sealant versus no treatment (8 versus 9 days, P = 0.327), median total bed cost (£3900 versus £4300, P = 0.400), mean transfusion cost per patient (P = 0.201) and overall cost (£6706.15 versus £6555.80, P = 0.792). CONCLUSION: Fibrin sealant application to cut surface during liver surgery confers no cost benefit and their routine use may not be recommended.

The Impact of Advancing Age on Recurrence and Survival Following Major Hepatectomy for Colorectal Liver Metastases.

INTRODUCTION: This study analysed the effect of age on survival in patients undergoing major hepatectomy (MH) for colorectal liver metastases (CRLM). The effect of adjuvant chemotherapy (AC) with age was also assessed. METHOD: Patients undergoing MH for CRLM between 1996 and 2011 were reviewed. Patients aged <75 or ≥75 were compared for disease-free (DFS) and overall survival (OS) as well as impact of AC on survival. RESULTS: Seven hundred twenty-seven patients underwent MH of which 105 (14 %) were aged ≥75. Morbidity was greater in the ≥75 group (25 versus 34 %, p = 0.048). No difference was noted in mortality. There was no difference in DFS between the two groups at 5 years (16.8 vs 18.9 months (p = 0.570). OS was longer in the <75 group (38.6 vs 32.0 months (p = 0.001). DFS was better in groups receiving AC than those not (<75 24.2 vs 12.2 months (p = <0.001) and ≥75 24 vs 12.1 months (p = 0.007)). OS in the ≥75 group was improved in the group receiving AC compared to the ≥75 group not (41.1 vs 16.6 months, p = 0.005). Age ≥75 was not an independent risk factor for reduced DFS on multivariate analysis. CONCLUSION: Well-selected patients aged ≥75 should be considered for MH followed by adjuvant chemotherapy.

Relationship between primary colorectal tumour and location of colorectal liver metastases.

BACKGROUND: There is conflicting evidence regarding whether streamlining of blood flow within the portal vein influences the anatomical distribution of colorectal liver metastases (CRLM). This study assesses the relationship between primary tumour location and metastases location. METHODS: Patients were identified using a prospectively maintained database, and those with known site of primary colorectal tumour and hemiliver involvement were included. Site of metastases and segments affected were confirmed via review of the radiology reports. The location of primary colonic tumour was confirmed via review of clinical correspondence letters. RESULTS: A total of 2364 metastases were identified in 891 patients. Of these, 379 metastases were in the right lobe and 156 in the left lobe, with 356 having bilobar disease. There was no significant relationship between the distribution of CRLM and the site of primary disease (left colon versus right colon) (P = 0.819). However, when the segmental location of the metastases was considered, there is a statistically significant difference between the number of right-sided CRLM compared with left-sided CRLM (P < 0.001). CONCLUSIONS: Right-sided CRLM is more likely regardless of the primary location. Portal streaming may have an effect, although the natural anatomical 'angulation', particularly of the left portal vein branch is more likely to play a role.

Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor.

OBJECTIVE: Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca(2+)) entry. Here, we sought understanding of a Synta66-resistant Ca(2+) entry pathway activated by VEGF. APPROACH AND RESULTS: Measurement of intracellular Ca(2+) in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca(2+) entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca(2+) entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca(2+) release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. CONCLUSIONS: VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca(2+) entry and downstream endothelial cell remodeling.

Anaesthetic and pharmacological techniques to decrease blood loss in liver surgery: a systematic review.

BACKGROUND: There is increasing evidence that perioperative blood loss and blood transfusions are associated with poorer short- and long-term outcomes in patients undergoing hepatectomy. The aim of this study was to systematically review the literature for non-surgical measures to decrease intraoperative blood loss during liver surgery. METHODS: The literature search was performed using PubMed, Embase, Cochrane Library, CINAHL and Google Scholar databases. The primary outcome measures were perioperative blood loss and transfusion requirements. A secondary outcome measure was development of ischaemia-reperfusion injury. RESULTS: Seventeen studies met the inclusion criteria and included 1573 patients. All were randomized controlled studies. In eight studies (n = 894), pharmacological methods, and in another nine studies (n = 679), anaesthetic methods to decrease blood loss were investigated. Anti-fibrinolytic drugs, acute normovolaemic haemodilution, autologous blood donation and use of inhalational anaesthetic agent may affect blood loss and post-operative hepatic function. CONCLUSIONS: There is potential for use of non-surgical techniques to decrease perioperative bleeding. However, on the basis of this review alone, due to heterogeneity of randomized trials conducted, no particular strategy can be recommended. Future studies should be conducted looking at pathways to decrease bleeding in liver surgery.

A systematic review and meta-analysis of portal vein ligation versus portal vein embolization for elective liver resection.

INTRODUCTION: This meta-analysis aimed to review the percentage increase in future liver remnant (FLR) and perioperative outcomes after portal vein ligation (PVL) and portal vein embolization (PVE) before liver resection. METHODS: An electronic search was performed of the MEDLINE, EMBASE, and PubMed databases using both subject headings (MeSH) and truncated word searches to identify all articles published that related to this topic. Pooled risk ratios were calculated for categorical outcomes and mean differences for secondary continuous outcomes using the fixed-effects and random-effects models for meta-analysis. RESULTS: Seven studies involving 218 patients met the inclusion criteria. There was no difference in the increase in FLR between the 2 groups 39% (PVE) versus 27% (PVL; mean difference [MD] 6.04; 95% CI, -0.23, 12.32; Z = 1.89; P = .06). Similarly, there was no difference in the morbidity (risk ratio [RR], 1.08; 95% CI, 0.55, 2.09; Z = 0.21; P = .83) and mortality (RR, 0.87; 95% CI, 0.19, 3.92; Z = 0.18; P = .85) in the 2 groups after liver resection. While awaiting liver resection after PVL and PVE, no difference was noted in the number of patients developing disease progression (RR, 0.93; 95% CI, 0.52, 1.66; Z = 0.24; P = .81). In a subset analysis comparing FLR with PVE and PVL as part of the procedure called an associating liver partition with PVL for staged hepatectomy (ALPPS), there was a significant increase in FLR in favor of ALPPS (MD, -17.09; 95% CI, -32.78, -1.40; Z = 2.14; P = .03). CONCLUSION: PVL and PVE result in comparable percentage increase in FLR with similar morbidity and mortality rates. The ALPPS procedure results in an improved percentage increase in FLR compared with PVE alone.

An imperative need to change organ donation and transplant curriculum results of a nationwide United kingdom junior doctor survey.

BACKGROUND: Awareness among the medical students and junior doctors about organ donation and transplantation (ODT) may play an important role in increasing organ donor pool. This study surveys the knowledge, perceptions, and attitudes of ODT among the U.K. junior doctors and attempts to identify their educational needs. To our knowledge, this is first such study in the United Kingdom. METHODS: A cross-sectional online survey was conducted among 1,696 junior doctors (809 foundation and 887 core trainees). A 36-point questionnaire explored the junior doctor's knowledge, perceptions, and attitudes toward ODT. RESULTS: There were 523 respondents (30.8%). Majority were foundation trainees (58.9%). Only 29.6% had previous exposure to transplantation, which reflected in their poor knowledge on the basics of ODT. Only 47.0% of the respondents were aware that consent from next of kin or family is sought for all deceased organ donation. Those registered as organ donor (69.8%) had better knowledge, perceptions, and attitudes in comparison to those not registered. Majority (84.1%) felt that they were inadequately exposed to ODT, and 96.8% stated that ODT should be part of undergraduate curriculum. CONCLUSION: Junior doctors in the United Kingdom have limited knowledge about ODT. Although subjected to investigators bias, the results demonstrate that junior doctors' perceptions and attitudes toward ODT were favorable. Majority felt that their ODT knowledge was inadequate and suggested the need for a change in undergraduate ODT curriculum. Increasing knowledge and awareness among junior doctors may help to improve the continuing organ shortage for transplantation.

A cost effective analysis of a laparoscopic versus an open left lateral sectionectomy in a liver transplant unit.

INTRODUCTION: This study aimed to assess the cost effectiveness of a laparoscopic left lateral sectionectomy (LLLS) compared with an open (OLLS) procedure and its role as a training operation as well as the learning curve associated with a laparoscopic approach. METHOD: Between 2004 and 2013, a prospectively maintained database was reviewed. LLLS were compared with age- and sex-matched OLLS. In addition, the outcomes of LLLS with a consultant as the primary surgeon were compared with those performed by trainees. RESULTS: Forty-three LLLS were performed during the study period. LLLS was a significantly cheaper operation compared with OLLS (P = 0.001, £3594.14 versus £5593.41). The median hospital stay was shorter in the laparoscopic group (P = 0.002, 3 versus 7 days). No difference was found in outcomes between a LLLS performed by a trainee or consultant (operating time, morbidity or R1 resection rate). The procedure length was significantly shorter during the later half of the study period [120 versus 129 min (P = 0.045)]. CONCLUSION: LLLS is a significantly cost effective operation compared with an open approach with a reduction in hospital stay. In addition, it is suitable to use as a training operation.

Epidural versus local anaesthetic infiltration via wound catheters in open liver resection: a meta-analysis.

BACKGROUND: This meta-analysis was designed to systematically analyse all published studies comparing local anaesthetic infiltration with wound catheters and epidural catheters in open liver resection. METHODS: A literature search was performed using the Cochrane Colorectal Cancer Group Controlled Trials Register, the Cochrane Central Register of Controlled Trials in the Cochrane Library, MEDLINE, Embase and Science Citation Index Expanded. Randomized trials, and prospective and retrospective studies comparing wound catheters with epidural catheters were included. Statistical analysis was performed using Review Manager Version 5.2 software. The primary outcome measures were pain scores in the post-operative period operation. Secondary outcome measures were hospital stay, time to opening bowels, overall complications and analgesia-specific complications. RESULTS: Four studies including 705 patients were included in the analysis. The pain scores were significantly lower in those patients with epidural on the first post-operative day (POD) (mean difference of -0.90 [-1.29, -0.52], Z = 4.61) (P < 0.00001) with comparable pain scores on PODs 2 and 3. There was no significant difference in the time to opening bowels, opioid use and hospital stay between the techniques. The post-operative complication rate was higher in the epidural group (risk ratio 1.40 [1.07, 1.83]; χ(2) = 0.60, df = 1) (P = 0.44); I(2) = 0%; Z = 2.42 (P = 0.02). CONCLUSION: Local anaesthetic infiltration via wound catheters combined with patient-controlled opiate analgesia provides comparable pain relief to epidural catheters except for the first POD. Both techniques are associated with similar hospital stay and opioid use with wound catheters associated with lower complication rate.

Increased morbidity in overweight and obese liver transplant recipients: a single-center experience of 1325 patients from the United Kingdom.

Obesity levels in the United Kingdom have risen over the years. Studies from the United States and elsewhere have reported variable outcomes for obese liver transplant recipients in terms of post-liver transplant morbidity, mortality, and graft survival. This study was designed to analyze the impact of the body mass index (BMI) on outcomes following adult liver transplantation. Data from 1994 to 2009 were retrieved from a prospectively maintained database. Patients were stratified into 5 World Health Organization BMI categories: underweight (<18.5 kg/m(2)), normal weight (18.5-24.9 kg/m(2)), overweight (25.0-29.9 kg/m(2)), obese (30.0-34.9 kg/m(2)), and morbidly obese (≥35.0 kg/m(2)). The primary outcome was an evaluation of graft and patient survival, and the secondary outcome was an assessment of postoperative morbidity. Bonferroni correction was applied with statistical significance set at P < 0.012. Kaplan-Meier curves were used to study the effects of BMI on graft and patient survival. A total of 1325 patients were included in the study: underweight (n = 47 or 3.5%), normal-weight (n = 643 or 48.5%), overweight (n = 417 or 31.5%), obese (n = 145 or 10.9%), and morbidly obese patients (n = 73 or 5.5%). The rate of postoperative infective complications was significantly higher in the overweight (60.7%, P < 0.01) and obese recipients (65.5%, P < 0.01) versus the normal-weight recipients (50.4%). The morbidly obese patients had a longer mean intensive care unit (ICU) stay than the normal-weight patients (4.7 versus 3.2 days, P = 0.03). The mean hospital stay was longer for the overweight (22.4 days, P < 0.001), obese (21.3 days, P = 0.04), and morbidly obese recipients (22.4 days, P = 0.047) versus the normal-weight recipients (18.0 days). There was no difference in death-censored graft survival or patient survival between the groups. In conclusion, this is the largest and only reported UK series on BMI and outcomes following liver transplantation. Overweight and obese patients have significantly increased morbidity in terms of infective complications after liver transplantation and, consequently, longer ICU and hospital stays.

Treatment and surveillance of polypoid lesions of the gallbladder in the United Kingdom.

OBJECTIVES: The increase in the routine use of abdominal imaging has led to a parallel surge in the identification of polypoid lesions in the gallbladder. True gallbladder polyps (GBP) have malignant potential and surgery can prevent or treat early gallbladder cancer. In an era of constraint on health care resources, it is important to ensure that surgery is offered only to patients who have appropriate indications. The aim of this study was to assess treatment and surveillance policies for GBP among hepatobiliary and upper gastrointestinal tract surgeons in the UK in the light of published evidence. METHODS: A questionnaire on the management of GBP was devised and sent to consultant surgeon members of the Association of Upper Gastrointestinal Surgeons (AUGIS) of Great Britain and Ireland with the approval of the AUGIS Committee. It included eight questions on indications for laparoscopic cholecystectomy and surveillance based on GBP (size, number, growth rate) and patient (age, comorbidities, ethnicity) characteristics. RESULTS: A total of 79 completed questionnaires were returned. The vast majority of surgeons (>75%) stated that they would perform surgery when a single GBP reached 10 mm in size. However, there was a lack of uniformity in the management of multiple polyps and polyp growth rate, with different surveillance protocols for patients treated conservatively. CONCLUSIONS: Gallbladder polyps are a relatively common finding on abdominal ultrasound scans. The survey showed considerable heterogeneity among surgeons regarding treatment and surveillance protocols. Although no randomized controlled trials exist, national guidelines would facilitate standardization, the formulation of an appropriate algorithm and appropriate use of resources.

An in vitro model of warm hypoxia-reoxygenation injury in human liver endothelial cells.

BACKGROUND: Ischemia-reperfusion or hypoxia-reoxygenation (H-R) injury adversely affects hepatic function following transplantation and major resection; the death of human sinusoidal endothelial cells (SECs) by apoptosis may play a central role in this process. Caspase-3 is an important intracellular protease in the intrinsic and extrinsic pathways of apoptosis. MATERIALS AND METHODS: SECs and EAhy926 cells were exposed to warm hypoxia at 37°C, followed by reoxygenation at 37°C. Activity of caspase-3 was quantified using Western blotting and colorimetric kinase assays. RESULTS: H-R caused a significant increase in caspase-3 activity compared with controls in both cell types. CONCLUSIONS: Warm H-R injury causes apoptotic cell death of SECs and immortalized cells, but with differing patterns of caspase activity.