Biliary Strictures Are Associated With Both Early and Late Hepatic Artery Stenosis.

BACKGROUND: Hepatic artery stenosis (HAS) following liver transplantation results in hypoperfusion and ischemic damage to the biliary tree. This study aimed to investigate how vascular intervention, liver function test derangement, and time point of HAS onset influence biliary complications. METHODS: A single-center retrospective study of adult patients that underwent primary liver transplantation. Patients were grouped according to the presence or absence of HAS and then into early (≤90 d) or late (>90 d) subgroups. Biliary complications comprised anastomotic (AS) or non ASs (NASs). RESULTS: Computed tomography angiography confirmed HAS was present in 39 of 1232 patients (3.2%). This occurred at ≤90 and >90 days in 20 (1.6%) and 19 (1.5%), respectively. The incidence of biliary strictures (BSs) in the group with HAS was higher than the group without (13/39; 33% versus 85/1193; 7.1%, P = 0.01). BS occurred in 8/20 (40.0%) and 5/19 (26.3%) of the early and late groups, respectively. The need for biliary intervention increased if any liver function test result was ≥3× upper limit of normal (P = 0.019). CONCLUSIONS: BS occurs at a significantly higher rate in the presence of HAS. Onset of HAS at ≤90 or ≥90 days can both be associated with morbidity. Significant liver function test derangement at HAS diagnosis indicates a higher likelihood of biliary intervention for strictures.

Management of a large delayed esophageal perforation in a fresh liver transplant patient with endoscopic placement of a nasopleural drainage tube-a case report.

Esophageal perforation in liver transplant recipients is a rare phenomenon. We herein report a case of an esophageal perforation due to Sengstaken-Blakemore tube in a liver-transplant recipient diagnosed 6 weeks post-transplant. A 2.5-cm mid-esophageal perforation communicating with large complex fluid collection in the pleural space was found. During endoscopy, 16Fr Salem Sump nasopleural tube (NP) was placed traversing through esophageal perforation into inferior aspect of the collection. Over the following 4 weeks, NP decompressed the cavity, allowed its closure and the tube was slowly retracted. By the end of 4 weeks, NP was removed with follow-up esophagogram showing no extravasation of contrast and a healed perforation. Hence, the esophageal perforation was successfully treated via this unique nonoperative approach without the need for major surgery. In instances of chronic leak with a stable patient, this nonoperative strategy should be considered even in immunocompromised patients.

Large Riedel's lobe and atrophic left liver in a donor - Accept for transplant or call off?

BACKGROUND: In context of suboptimal liver utilisation, grafts with various risk factors are under consideration today. For example, impaired vascularity with severe arterial calcifications and modified liver shapes are no longer contraindications and their use depends on the centre policy and experience of the surgical team. Riedel liver lobes represent a tongue-like liver shape with inferior projection in the right liver lobe. Such development modifications were initially described when patients developed a lesion and subsequently presented with symptoms. We here present the first case report in the literature, where such livers with anatomical variations were used for transplantation. CASE SUMMARY: We describe here two cases of adult human liver transplantation, where we have accepted two donor livers with modified shape. The technical considerations for transplantation of such livers, found with enlarged right lobes, or Riedel shape, and hypo-trophic left lateral segment are highlighted. Both recipients experienced immediate liver function and overall good outcomes with a minimum follow up of 1 year. We also provide detailed pictures and outcome analysis in combination with a literature review. CONCLUSION: The utilisation of donor livers with modified shape, such as Riedel's Lobe appears safe and will increase the donor pool.

Neutrophil Extracellular Trap-Associated CEACAM1 as a Putative Therapeutic Target to Prevent Metastatic Progression of Colon Carcinoma.

Neutrophils promote tumor growth and metastasis at multiple stages of cancer progression. One mechanism through which this occurs is via release of neutrophil extracellular traps (NETs). We have previously shown that NETs trap tumor cells in both the liver and the lung, increasing their adhesion and metastasis following postoperative complications. Multiple studies have since shown that NETs play a role in tumor progression and metastasis. NETs are composed of nuclear DNA-derived web-like structures decorated with neutrophil-derived proteins. However, it is unknown which, if any, of these NET-affiliated proteins is responsible for inducing the metastatic phenotype. In this study, we identify the NET-associated carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) as an essential element for this interaction. Indeed, blocking CEACAM1 on NETs, or knocking it out in a murine model, leads to a significant decrease in colon carcinoma cell adhesion, migration and metastasis. Thus, this work identifies NET-associated CEACAM1 as a putative therapeutic target to prevent the metastatic progression of colon carcinoma.

CtBP-a targetable dependency for tumor-initiating cell activity and metastasis in pancreatic adenocarcinoma.

Ctbp2 is a uniquely targetable oncogenic transcriptional coregulator, exhibiting overexpression in most common solid tumors, and critical to the tumor-initiating cell (TIC) transcriptional program. In the "CKP" mouse pancreatic ductal adenocarcinoma (PDAC) model driven by mutant K-Ras, Ctbp2 haploinsufficiency prolonged survival, abrogated peritoneal metastasis, and caused dramatic downregulation of c-Myc, a known critical dependency for TIC activity and tumor progression in PDAC. A small-molecule inhibitor of CtBP2, 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) phenocopied Ctbp2 deletion, decreasing tumor burden similarly to gemcitabine, and the combination of 4-Cl-HIPP and gemcitabine further synergistically suppressed tumor growth. Pharmacodynamic monitoring revealed that the 4-Cl-HIPP/gemcitabine combination induced robust and synergistic tumor apoptosis and marked downregulation of the TIC marker CD133 in CKP PDAC tumors. Collectively, our data demonstrate that targeting CtBP represents a fruitful avenue for development of highly active agents in PDAC that cooperate with standard therapy to limit both primary and metastatic tumor burden.

Correction to: Sepsis increases perioperative metastases in a murine model.

It has been highlighted that the original manuscript [1] contains a typesetting error in Fig. 1 and the Fig. 1c panel gas been inadvertently duplicated in panel Fig. 1d. This does not affect the results and conclusions of the article. The correct version of Fig. 1 is included with this Correction. The original article has been updated.

Sepsis increases perioperative metastases in a murine model.

BACKGROUND: Cancer surgery can promote tumour metastases and worsen prognosis, however, the effect of perioperative complications on metastatic disease remains unclear. In this study we sought to evaluate the effect of common perioperative complications including perioperative blood loss, hypothermia, and sepsis on tumour metastases in a murine model. METHODS: Prior to surgery, pulmonary metastases were established by intravenous challenge of CT26LacZ colon cancer cells in BALB/c mice. Surgical stress was generated through partial hepatectomy (PH) or left nephrectomy (LN). Sepsis was induced by puncturing the cecum to express stool into the abdomen. Hemorrhagic shock was induced by removal of 30% of total blood volume (i.e. stage 3 hemorrhage) via the saphenous vein. Hypothermia was induced by removing the heating apparatus during surgery and lowering core body temperatures to 30 °C. Lung tumour burden was quantified 3 days following surgery. RESULTS: Surgically stressed mice subjected to stage 3 hemorrhage or hypothermia did not show an additional increase in lung tumour burden. In contrast, surgically stressed mice subjected to intraoperative sepsis demonstrated an additional 2-fold increase in the number of tumour metastases. Furthermore, natural killer (NK) cell function, as assessed by YAC-1 tumour cell lysis, was significantly attenuated in surgically stressed mice subjected to intraoperative sepsis. Both NK cell-mediated cytotoxic function and lung tumour burden were improved with perioperative administration of polyI:C, which is a toll-like receptor (TLR)-3 ligand. CONCLUSIONS: Perioperative sepsis alone, but not hemorrhage or hypothermia, enhances the prometastatic effect of surgery in murine models of cancer. Understanding the cellular mechanisms underlying perioperative immune suppression will facilitate the development of immunomodulation strategies that can attenuate metastatic disease.

Surgical stress promotes the development of cancer metastases by a coagulation-dependent mechanism involving natural killer cells in a murine model.

OBJECTIVE: To determine whether the postoperative hypercoagulable state is responsible for the increase in metastases observed after surgery. BACKGROUND: Surgery precipitates a hypercoagulable state and increases the formation of cancer metastases in animal models. Coagulation promotes metastases by facilitating the formation of microthrombi around tumor cell emboli (TCE), thereby inhibiting natural killer (NK) cell-mediated destruction. METHODS: Mice underwent surgery preceded by tumor cell inoculation to establish pulmonary metastases in the presence or absence of various perioperative anticoagulants. Pulmonary TCE were quantified and characterized using fluorescently labeled fibrinogen and platelets. The role of NK cells was evaluated by repeating these experiments after antibody depletion in a genetically deficient strain and by adoptively transferring NK cells into NK-deficient mice. RESULTS: Surgery resulted in a consistent and significant increase in metastases while a number of different anticoagulants and platelet depletion attenuated this effect. Impaired clearance of TCE from the lungs associated with an increase in peritumoral fibrin and platelet clot formation was observed in surgically stressed mice, but not in control mice or mice that received perioperative anticoagulation. The increase in TCE survival conferred by surgery and inhibited by perioperative anticoagulation was eliminated by the immunological or genetic depletion of NK cells. Adoptive transfer experiment confirms that surgery impairs NK cell function. CONCLUSIONS: Surgery promotes the formation of fibrin and platelet clots around TCE, thereby impairing NK cell-mediated tumor cell clearance, whereas perioperative anticoagulation attenuates this effect. Therapeutic interventions aimed at reducing peritumoral clot formation and enhancing NK cell function in the perioperative period will have important clinical implications in attenuating metastatic disease after cancer surgery.

The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer.

AIMS: If stratified medicine is to be applied in the neoadjuvant setting, predictive testing will have to be undertaken on preoperative diagnostic biopsy specimens. The aim of this study was to evaluate whether a diagnostic biopsy was adequately representative of the main tumour in colorectal cancer. METHODS AND RESULTS: Thirty cases of paired biopsy and subsequent resection specimens were randomly selected. Samples were screened for mutation in KRAS (codons 12/13, 61, and 146), BRAF (codon 600 and exon 11), PIK3CA (exons 1, 9, and 20), TP53 (exons 5-8), and microsatellite instability, using the quick multiplex consensus or standard polymerase chain reaction (PCR) protocols followed by high-resolution melting analysis. A total of 570 paired PCR tests were performed for mutation detection, and identical results were obtained in both biopsy and resection specimens in 569 tests (>99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens. CONCLUSIONS: This is the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer.

MLH1 function is context dependent in colorectal cancers.

BACKGROUND AND AIMS: Loss of mismatch repair (MMR) function in sporadic colorectal cancer occurs most commonly because of inactivation of MLH1, and it causes an increase in mutation rate. However, it is uncertain whether loss of MMR alters any other cellular function. The aim of this study was to investigate the role of MMR in regulating cell numbers and apoptosis. METHODS: MLH1 protein levels were manipulated by (a) cloning and forcibly expressing MLH1 in HCT116 (a cell line with MLH1 mutation) and RKO (a cell line with MLH1 silencing), and (b) knockdown of MLH1 in SW480 (a cell line with normal MMR function). Cell number and apoptotic bodies were measured in standard and 'high stress' (ie, after staurosporine exposure) conditions. RESULTS: Restoration of MLH1 function in HCT116 and RKO resulted in increased cell number (p<0.001 for both cell lines) and decreased numbers of floating apoptotic bodies (p<0.01 in HCT116) in standard culture conditions. However, on induction of apoptotic stress, restoration of MLH1 resulted in reduced cell numbers (p<0.005). Knockdown of MLH1 in SW480 had no effect on cell numbers or apoptosis. CONCLUSIONS: MLH1 function may be context dependent: in 'low stress' conditions it may act to inhibit apoptosis, while in 'high stress' conditions it may induce apoptosis. However, within the context of chromosomal instability, the effect of MLH1 on cell numbers is limited.

The stem cell marker CD133 associates with enhanced colony formation and cell motility in colorectal cancer.

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133-expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause "off-target" effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133- populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133- populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133- population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133- population of SW480. Prolonged culture of a pure CD133- population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features.

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue.

BACKGROUND: Mutation detection in tumours will become increasingly important in pathological diagnosis as 'predictive' mutations are identified. A cheap and reliable test that works on formalin-fixed paraffin-embedded (FFPE) tissue is required. METHODS: The quick-multiplex-consensus (QMC)-PCR protocol was developed to be used with high-resolution melting (HRM) analysis. The assay was compared with Sanger sequencing. Robustness of the assay was tested in DNA from FFPE tissue. RESULTS: QMC-PCR with HRM could detect a minimum of 2.5% of mutant alleles (compared with 20% detectable for Sanger sequencing). Ten mutation hotspots in KRAS, BRAF, PIK3CA and CDC4 were screened in 29 cell lines with 100% sensitivity and specificity. Forty-three FFPE colorectal tumours were sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. There was 100% sensitivity, although, of 21 mutations detected by QMC-PCR, 16 were confirmed by sequencing (71% specificity, positive predictive value 76%). All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (positive predictive value 84%). As our technique is more sensitive than direct sequencing, the remaining seven tests are probably sequencing false-negatives. Precision tests showed that there was little intra-assay and interassay variation. CONCLUSIONS: QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues.

Comparative analysis of pyrosequencing and QMC-PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection.

Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.

CD24 is upregulated in inflammatory bowel disease and stimulates cell motility and colony formation.

BACKGROUND: We investigated whether CD24 (reportedly a stem cell marker and adhesion molecule) was expressed in regenerative mucosa in inflammatory bowel disease (IBD) and whether it could be functionally relevant. METHODS: CD24 expression was examined in 10 cases of IBD and the relationship of CD24 with Wnt signaling was tested using dominant negative (DN)-TCF4 expression. For functional evaluation, CD24 was 1) cloned and forcibly expressed in HCT116 (which expresses very low levels of CD24) and 2) knocked-down by RNA interference in HT29 (which expresses high levels of CD24). The effect of altered CD24 expression on proliferation/apoptosis, staurosporine-induced apoptosis, colony formation in soft agar, migration, and invasion was examined. RESULTS: CD24 was not expressed in normal tissue, while 10/10 cases of IBD showed CD24 upregulation. Inhibition of Wnt signaling with DN-TCF4 caused CD24 downregulation. Forced expression of CD24 did not influence cell proliferation, apoptosis, or staurosporine-induced apoptosis but it did significantly enhance colony forming efficiency (P < 0.01). Furthermore, there was increased transwell migration (P < 0.001) and invasion (P < 0.03) and there was increased cell migration in wounding assays. Conversely, knockdown of CD24 reduced transwell migration (P < 0.01) and invasion (P < 0.01) and reduced cell motility in wounding assays. CD24 knockdown did not influence proliferation, apoptosis resistance, or staurosporine-induced apoptosis. CONCLUSIONS: This is the first study to report upregulation of CD24 in regenerating tissue in IBD. This may be regulated by Wnt signaling and can confer enhanced colony forming ability and enhanced cell motility-features that may be important in tissue healing in the colon.

C-terminal Tensin-like (CTEN) is an oncogene which alters cell motility possibly through repression of E-cadherin in colorectal cancer.

The Tensin gene family encodes proteins thought to modulate integrin function. C-terminal Tensin-like (CTEN) is a member of the Tensin gene family which lacks the N-terminus actin-binding domain. Cten is reported to have both oncogenic and tumour-suppressor functions. We investigated the role that Cten may play in colorectal cancer (CRC). By quantitative RT-PCR CTEN is up-regulated (i.e. > two-fold increase) in 62% of cell lines and 69% of tumours compared with normal mucosa, consistent with CTEN being a possible oncogene. Stable transfection of HCT116 and SW480 (CRC cell lines with low endogenous Cten expression) with a Cten expression vector gave identical results in both cell lines. Forced Cten expression did not cause change in cell numbers, although it did confer resistance to staurosporine-induced apoptosis (p < 0.005). Cten also induced epithelial-mesenchymal transition (EMT) in tumour cells accompanied by a significant increase in both cell migration (transwell migration and cell wounding assays, p < 0.001 and p < 0.05, respectively) and cell invasion (invasion through Matrigel, p < 0.001). Given the observed EMT, we investigated the levels of E-cadherin. Cten induction was associated with a reduction in E-cadherin protein expression but not levels of E-cadherin mRNA. These data suggest that CTEN is an oncogene in CRC which stimulates EMT, cell migration and invasion and may therefore have a role in tumour invasion/spread. Furthermore, Cten induction is associated with post-transcriptional repression of E-cadherin.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

BACKGROUND: Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. METHODS: Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. RESULTS: Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P < 0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P < 0.05). There were no significant differences in AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. CONCLUSIONS: In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer.

Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Acute lung injury and cell death: how many ways can cells die?

Apoptosis has been considered as an underlying mechanism in acute lung injury/acute respiratory distress syndrome and multiorgan dysfunction syndrome. Recently, several alternative pathways for cell death (such as caspase-independent cell death, oncosis, and autophagy) have been discovered. Evidence of these pathways in the pathogenesis of acute lung injury has also come into light. In this article, we briefly introduce cell death pathways and then focus on studies related to lung injury. The different types of cell death that occur and the underlying mechanisms utilized depend on both experimental and clinical conditions. Lipopolysaccharide-induced acute lung injury is associated with apoptosis via Fas/Fas ligand mechanisms. Hyperoxia and ischemia-reperfusion injury generate reactive oxidative species, which induce complex cell death patterns composed of apoptosis, oncosis, and necrosis. Prolonged overexpression of inflammatory mediators results in increased production and activation of proteases, especially cathepsins. Activation and resistance to death of neutrophils also plays an important role in promoting parenchymal cell death. Knowledge of the coexisting multiple cell death pathways and awareness of the pharmacological inhibitors targeting different proteases critical to cell death may lead to the development of novel therapies for acute lung injury.

Intestinal ischemia-reperfusion-induced acute lung injury and oncotic cell death in multiple organs.

Most acute respiratory distress syndrome studies have been focused on the lung injury. Little is known about other organs during the development of acute respiratory distress syndrome. Herein, we investigated the injury and cell death in multiple organs after intestinal ischemia-reperfusion (IIR) in C57BL/6 mice. Terminal transferase dUTP nick end labeling staining was used as a marker of cell death. Caspase 3 and cathepsin B activation as markers of caspase-dependent and caspase-independent apoptosis, respectively, and electron microscopy for ultimate characterization of cell death were used. In comparison with control and sham-operated mice, the IIR group showed interstitial inflammatory infiltrates in the lung and significant increases of lung injury parameters and plasma lactate dehydrogenase and aspartate aminotransferase levels. Terminal transferase dUTP nick end labeling-positive cells and immunostaining for hemeoxygenase 1, an enzyme induced by inflammatory stimuli, were increased in the lung, heart, and kidney, but not in the liver. The number of hemeoxygenase 1-positive cells positively and significantly correlated to the number of terminal transferase dUTP nick end labeling-positive cells. Cell death was not associated with caspase 3 or cathepsin B activation. Electron microscopy showed morphological features compatible with oncotic rather than apoptotic cell death or necrosis, including mitochondrial swelling and cytoplasm disorganization in pulmonary and renal epithelial cells, lung and cardiac endothelial cells, and myocytes. These results indicate that, although lung injury is the most significant manifestation after IIR, oncotic cell death occurs in the lung, heart, and kidney, which may be related to ischemia and inflammation.

Identification of a novel prostate cancer-associated tumor antigen.

BACKGROUND: The identification of antigens that distinguish cancer cells from normal cells is of major importance for the definition of therapeutic targets in human malignancies. Using sera from cancer patients, we have previously reported on the identification of immunologically recognized proteins that belong to the family of cancer testis antigens (CTAs). METHODS: A normal testicular cDNA library was screened with pooled allogeneic sera from patients with prostate cancer using a modified SEREX approach. Subsequently we have identified and characterized a novel antigen, T21, with an expression pattern similar to that of CTAs. mRNA expression of T21 was determined using a panel of whole tissues and prostate cell lines using Q-RT-PCR. For laser microdissection, fresh prostate cancer and benign tissue was obtained using our novel validated harvesting technique. Protein expression and cellular localization of T21 were assessed in prostate cell lines using Western blotting, confocal microscopy and flow cytometry. RESULTS: T21 showed tissue-restricted mRNA expression in gastric, kidney and prostate cancers, and in normal testis and prostate tissues. Following laser microdissection, T21 was significantly over-expressed in malignant compared to benign prostatic epithelium. We have demonstrated expression of T21 at the protein level and confocal microscopy on PC3 cells probed with a T21-monospecific antibody revealed cytoplasmic localization of T21 protein. CONCLUSIONS: The highly restricted expression pattern of T21 makes it an attractive vaccine target for prostate cancer. Several CTAs reportedly induce cytotoxic T-lymphocyte responses, therefore it is reasonable to assume that T21 will be a valuable target for cancer immunotherapy.