A Novel PiRNA Enhances CA19-9 Sensitivity for Pancreatic Cancer Identification by Liquid Biopsy.

Pancreatic cancer is one of the deadliest tumours worldwide, and its poor prognosis is due to an inability to detect the disease at the early stages, thereby creating an urgent need to develop non-invasive biomarkers. P-element-induced wimpy testis (PIWI) proteins work together with piwi-interacting RNAs (piRNAs) to perform epigenetic regulation and as such hold great potential as biomarkers for pancreatic cancer. PIWIL2 and PIWIL4 are associated with better prognosis, while PIWIL1 and PIWIL3 involvement appears to be associated with carcinogenesis. We aimed to discover PIWIL3- and PIWIL4-modulated piRNAs and determine their potential mechanisms in pancreatic cancer and the clinical implications. PIWIL3 or PIWIL4 was downregulated in pancreatic cancer-derived cell lines or in a non-tumour cell line. Differentially expressed piRNAs were analysed by next generation sequencing of small RNA. Nine fresh-frozen samples from solid human pancreases (three healthy pancreases, three intraductal papillary mucinous neoplasms, and three early-stage pancreatic cancers) were included in the sequencing analysis. Two piRNAs associated with PIWIL3 (piR-168112 and piR-162725) were identified in the neoplastic cells; in untransformed samples, we identified one piRNA associated with PIWIL4 (pir-366845). After validation in pancreatic cancer-derived cell lines and one untransformed pancreatic cell line, these piRNAs were evaluated in plasma samples from healthy donors (n = 27) or patients with pancreatic cancer (n = 45). Interestingly, piR-162725 expression identified pancreatic cancer patients versus healthy donors in liquid biopsies. Moreover, the potential of the serum carbohydrate antigen 19-9 (CA19-9) biomarker to identify pancreatic cancer patients was greatly enhanced when combined with piR-162725 detection. The enhanced diagnostic potential for the early detection of pancreatic cancer in liquid biopsies of these new small non-coding RNAs will likely improve the prognosis and management of this deadly cancer.

The Clinical Significance of PIWIL3 and PIWIL4 Expression in Pancreatic Cancer.

P-element-induced wimpy testis (PIWI) proteins have been described in several cancers. PIWIL1 and PIWIL2 have been recently evaluated in pancreatic cancer, and elevated expression of PIWIL2 conferred longer survival to patients. However, PIWIL3's and PIWIL4's role in carcinogenesis is rather controversial, and their clinical implication in pancreatic cancer has not yet been investigated. In the present study, we evaluated PIWIL1, PIWIL2, PIWIL3 and PIWIL4 expression in pancreatic cancer-derived cell lines and in one non-tumor cell line as healthy control. Here, we show a differential expression in tumor and non-tumor cell lines of PIWIL3 and PIWIL4. Subsequently, functional experiments with PIWIL3 and/or PIWIL4 knockdown revealed a decrease in the motility ratio of tumor and non-tumor cell lines through downregulation of mesenchymal factors in pro of epithelial factors. We also observed that PIWIL3 and/or PIWIL4 silencing impaired undifferentiated phenotype and enhanced drug toxicity in both tumor- and non-tumor-derived cell lines. Finally, PIWIL3 and PIWIL4 evaluation in human pancreatic cancer samples showed that patients with low levels of PIWIL4 protein expression presented poor prognosis. Therefore, PIWIL3 and PIWIL4 proteins may play crucial roles to keep pancreatic cell homeostasis not only in tumors but also in healthy tissues.

Clinical Implications of NRAS Overexpression in Resectable Pancreatic Adenocarcinoma Patients.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer, and its incidence is rising worldwide. Although survival can be improved by surgical resection, when detected at an early stage, this type of cancer is usually asymptomatic, and disease becomes only apparent after metastasis. Adjuvant treatment does not improve survival, thus after surgery there is a lack of predictive and prognosis biomarkers to predict treatment response and survival. The mitogen-activated protein-kinase and phosphoinositide 3-kinase signalling pathways play a crucial role in cancer development and progression. Especially, activated RAS proteins promote cell proliferation through constitutive stimulation of the downstream effectors RAF-MEK-ERK and PI3K-AKT. Mutational status of NRAS is required in several types of cancer like colorectal or cutaneous melanoma. However, mutations in this gene are very scarce in PDAC patients, and NRAS determination is not usually performed in clinical practice for this kind of tumor. In this study, we analyse the association between NRAS protein expression and progression-free survival and overall survival of an homogenous cohort of pancreatic ductal adenocarcinoma patients from a single-centre. Interestingly, we found that patients with high expression not only showed longer progression-free survival than those patients with low expression (22 versus 9 months, respectively) (P = 0.013), but also longer overall survival (43 versus 19 months, respectively) (P = 0.020). These results confirm NRAS expression could be used to differentiate patients according to their prognosis. Proportional hazard model revealed NRAS expression together with grade of differentiation as pathological variables to predict patient's outcome.

UNR/CDSE1 expression as prognosis biomarker in resectable pancreatic ductal adenocarcinoma patients: A proof-of-concept.

Pancreatic ductal adenocarcinoma is an aggressive form of pancreatic cancer and the fourth leading cause of cancer-related death. When possible, curative approaches are based on surgical resection, though not every patient is a candidate for surgery. There are clinical guidelines for the management of these patients that offer different treatment options depending on the clinical and pathologic characteristics. However, the survival rates seen in this kind of patients are still low. The CDSE1 gene is located upstream of NRAS and encodes an RNA-binding protein termed UNR. The aim of this study was to analyze UNR expression and its correlation with outcome in patients with resectable pancreatic ductal adenocarcinoma (PDAC). For this, samples from resectable PDAC patients who underwent duodenopancreatectomy were used to evaluate UNR protein expression by immunohistochemistry using a tissue microarray. Here, we observed that low UNR expression was significantly associated with shorter progression-free survival after surgery (P = 0.010). Moreover, this prognostic marker remained significant after Cox proportional hazards model (P = 0.036). We further studied the role of CDSE1 expression in patient's prognosis using data from public repositories (GEO and TGCA), confirming our results. Interestingly, CDSE1 expression correlated with that of genes characteristic of an immunogenic molecular subtype of pancreatic cancer. Based on these findings, UNR may be considered a potential prognostic biomarker for resectable PDAC and may serve to guide subsequent adjuvant treatment decisions.

Sonication of Abdominal Drains: Clinical Implications of Quantitative Cultures for the Diagnosis of Surgical Site Infection.

BACKGROUND: The use of drains in patients undergoing abdominal surgery has been a subject of debate for several decades. In this paper, the usefulness of quantitative cultures of sonicated abdominal drains for diagnosing surgical site of infection (SSI) and the association between culture results with patient outcome is evaluated. METHODS: Forty-five abdominal drainage tubes from 35 patients who underwent abdominal surgery were studied. Samples were sonicated for 5 min, the sonicate was centrifuged, and the sediment was cultured on different media. Total bacterial counts were adjusted to the actual surface of the drainage tubing. Clinical information of the patients was reviewed retrospectively. RESULTS: A relation was observed between SSI and the use of drains for more than 3 d (p = 0.0216). The presence of a suspected pathogen was related to the prevalence of SSI (p = 0.035), complications (p = 0.013), and greater leukocyte count (p = 0.048 Mann Whitney test), as well as to the use of drains for more than 3 d (p = 0.0386) and to the serous appearance of the exudates at the point of insertion of the drain (p = 0.0399). The sonication procedure showed a sensitivity of 50%, specificity of 84.2%, positive predictive value of 72.72%, and negative predictive value of 66.67% in the diagnosis of SSI. The most commonly isolated group of organisms was coagulase-negative staphylococci, being present in 18 patients (51.43%) who, however, were not associated with SSI. One or two organisms considered as pathogens were detected in 11 patients (31.43%). The more common pathogens detected were Enterobacteriae spp. (nine patients): Enterobacter aerogenes (2), Enterobacter cloacae (1), Escherichia coli (4), Klebsiella pneumoniae (1), Morganella morganii (1); and Pseudomonas aeruginosa (five patients). Candida spp. and Enterococcus spp. were detected in one patient each one. CONCLUSIONS: The detection and quantification of organisms not present in skin microbiota after drain sonication is helpful in the diagnosis of SSI and it is associated with a worse outcome in patients. Duration of use of drainage tubes is an independent risk factor for the development of SSI.

Biocompatibility reduces inflammation-induced apoptosis in mesothelial cells exposed to peritoneal dialysis fluid.

BACKGROUND/AIMS: Peritonitis is a major complication that arises out of peritoneal dialysis (PD), leading to death and loss of mesothelium and peritoneal injury, which may impede PD. We studied the combined impact of inflammatory mediators and PD fluids on mesothelial cell death. METHODS: Cultured human mesothelial cells. RESULTS: Inflammatory cytokines (TNF-α and interferon-γ) cooperate with bioincompatible PD fluids containing high glucose degradation product (GDP) concentrations to promote mesothelial cell death. Thus, the inflammatory cytokine cocktail induced a higher rate of death in cells cultured in high GDP PD fluid than in low GDP PD fluid or cell culture medium (cell death expressed as % hypodiploid cells: TNF-α and interferon-γ in RPMI: 14.15 ± 1.68, TNF-α and interferon-γ in 4.25% low GDP PD fluid 13.16 ± 3.29, TNF-α and interferon-γ in 4.25% high GDP PD fluid 25.88 ± 2.18%, p < 0.05 vs. the other two groups). BclxL BH4 peptides, Apaf-1 inhibition or caspase inhibition failed to protect from apoptosis induced by the combination of inflammatory cytokines and bioincompatible PD fluids, although they protected from other forms of mesothelial cell apoptosis. CONCLUSION: Inflammation cooperates with high GDP PD fluids to promote mesothelial cell death, which is resistant to several therapeutic approaches. This information provides a framework for selection of PD fluid during peritonitis.

Bacterial adherence to different meshes used in abdominal surgery.

BACKGROUND: We studied the influence of morphology and type of material of abdominal wall prostheses in the avoidance of bacterial adhesion in acute and chronic mesh infections. METHODS: Three different types of prostheses were compared: 1) High-density polypropylene monofilament mesh (PMM); 2) low-density PMM; and 3) prostheses composed of low-density polypropylene and a non-porous hydrophilic film (composite prostheses). Microbial adhesion tests were performed using reference strains of Staphylococcus aureus 15981, Staphylococcus epidermidis ATCC 35984, Mycobacterium abscessus DSM 44196, and Mycobacterium fortuitum ATCC 13756 using a protocol described previously. RESULTS: Both Staphylococcus spp. and M. fortuitum strains showed lower adherence to PMM. Mycobacterium abscessus also exhibited lower adherence to composite prostheses. Both Mycobacterium spp. strains had lower adherence than Staphylococcus spp. strains for all materials except for low-density PMM. Mycobacterium fortuitum showed higher adherence to composite prostheses than M. abscessus, whereas the latter species had higher adherence to high-density PMM than M. fortuitum. CONCLUSION: Depending on the type of bacteria, collagen-coated hydrophilic prostheses with a large surface increased bacterial adherence significantly. These differences should be taken into consideration when choosing a mesh graft, which limits infection in abdominal wall reconstruction.

A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury.

BACKGROUND: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization. METHODOLOGY: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice. CONCLUSION: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

The open mesh repair of Spigelian hernia.

BACKGROUND: Spigelian hernias are rare defects of the abdominal wall usually appearing between the abdominal muscles, lateral to the rectus abdominis and through a debilitated Spigelian aponeurosis. Recently, mesh repair has been introduced for the treatment of these types of hernias and different approaches have been proposed. METHODS: Nine patients with Spigelian hernia were prospectively treated by placing a mesh prosthesis between the external oblique and the internal oblique muscles, based on principles of mesh repair established by the Lichtenstein group. RESULTS: Five women and 4 men, mean age 75.7 years, were operated on. In 1 patient the Spigelian hernia was an incidental finding during an inguinal hernia repair. Two patients were operated on an emergency basis. Elective operations were undertaken in ambulatory facilities in 3. Two patients had postoperative ecchymosis with no associated morbidity. No recurrences have been observed during follow-up. CONCLUSIONS: Open mesh repair of Spigelian hernias placing the mesh between the external and internal oblique muscles is a simple and safe approach that can prevent morbidity related to other techniques.

The role of antibiotic prophylaxis on wound infection after mesh hernia repair under local anesthesia on an ambulatory basis.

Mesh prosthesis, local anesthesia, and ambulatory care have been widely introduced in recent decades in the treatment of inguinal hernia. The use of antibiotic prophylaxis during open inguinal hernia repair has been controversial. No prospective trial has been conducted to assess the role of antibiotic prophylaxis in patients operated on for inguinal hernia under the above-mentioned conditions. A prospective, randomized, double-blinded trial was initiated to assess the efficacy of antibiotic prophylaxis in the prevention of wound infection during open mesh inguinal hernia repair under local anesthesia on an ambulatory basis. Ninety-nine consecutive hernia repairs were randomized to receive 1 g of parenteral Cefazolin preoperatively or a placebo. No wound infections existed in the therapeutic group (0/50). Four infections appeared in the control group (4/49), and the study was suspended for ethical reasons when differences reached values close to statistical significance ( P=0.059). We conclude that a single dose of intravenous Cefazolin decreases the risk of wound infection during open mesh inguinal hernia repair under local anesthesia on an ambulatory basis.

Sevoflurane reduces endothelium-dependent vasorelaxation: role of superoxide anion and endothelin.

PURPOSE: There are several reports suggesting that volatile anesthetics alter vascular endothelial function. We analyzed the effect of sevoflurane, a fluorinated volatile anesthetic, on nitric oxide (NO)-dependent relaxation, evaluating the role of the endothelium-derived vasoconstrictor endothelin-1 (ET-1). METHODS: The experiments were performed in rat isolated aortic segments aerated in the absence and in the presence of sevoflurane (2%). RESULTS: Acetylcholine-induced relaxation was reduced in aortic segments aerated with sevoflurane. Sevoflurane failed to modify relaxation in response to an exogenous NO donor, sodium nitroprusside. Superoxide dismutase, a scavenger of superoxide anion, partially restored the impaired vasorelaxation induced by sevoflurane, an effect that was associated with the release of superoxide anion. The presence of BQ-123, an antagonist of endothelin ETA-type receptors, normalized the vasorelaxing response to acetylcholine in the presence of sevoflurane. In addition, BQ-123 also reduced the ability of the sevoflurane-incubated vascular wall to release superoxide anion. CONCLUSIONS: Our results suggest that sevoflurane impairs the endothelium-dependent vasorelaxation but that the endothelium-independent response remains intact. ET-1 and superoxide anion are involved in the endothelial dysfunction induced by sevoflurane. Further studies are needed to associate the endothelial dysfunction related to sevoflurane shown herein and its reported preconditioning properties on the myocardium.

Aspirin prevents Escherichia coli lipopolysaccharide- and Staphylococcus aureus-induced downregulation of endothelial nitric oxide synthase expression in guinea pig pericardial tissue.

The aim was to analyze whether pericardial tissue expresses endothelial NO synthase (eNOS) protein and to determine the presence of cytosolic proteins that bind to eNOS mRNA. The effect of aspirin on the above-mentioned parameters was also analyzed. eNOS protein was expressed in pericardial tissue from male guinea pigs. Escherichia coli lipopolysaccharide (LPS, 10 microgram/mL) and Staphylococcus aureus endotoxin (SA, 10 microgram/mL) reduced eNOS protein expression and shortened the half-life of the eNOS messenger. Under basal conditions, cytosolic extracts from pericardial samples bound to the 3'-untranslated region (3'-UTR) of eNOS mRNA, which was enhanced by LPS and SA. Proteinase K fully prevented the binding of cytosolic pericardial extracts to 3'-UTR of eNOS mRNA, suggesting the involvement of proteins that were further characterized as 60- and 51-kDa proteins. Aspirin (1 to 10 mmol/L) restored eNOS expression in either LPS- and SA-stimulated pericardial samples and reduced the binding activity of the pericardial cytosolic proteins to 3'-UTR of eNOS mRNA. Indomethacin also reduced the downregulation of eNOS by LPS and diminished the binding activity of the cytosolic proteins, although higher doses of indomethacin than of aspirin were needed to improve these parameters. In conclusion, eNOS protein is expressed in guinea pig pericardial tissue. LPS and SA stimulate the binding activity of pericardial cytosolic proteins to 3'-UTR of eNOS mRNA and reduce eNOS protein expression. High doses of aspirin and indomethacin protect eNOS protein expression and reduce the binding activity of the cytosolic proteins to 3'-UTR of eNOS mRNA, suggesting an inverse association between the presence of these cytosolic proteins and eNOS expression.

Expression of endothelial nitric oxide synthase in human peritoneal tissue: regulation by Escherichia coli lipopolysaccharide.

Changes in the expression of endothelial nitric oxide synthase (eNOS) in the peritoneum could be involved in the peritoneal dysfunction associated with peritoneal inflammation. Demonstrated recently in bovine endothelial cells was the existence of cytosolic proteins that bind to the 3'-untranslated region (3'-UTR) of eNOS mRNA and could be implicated in eNOS mRNA stabilization. The present work demonstrates that eNOS protein is expressed in human endothelial and mesothelial peritoneal cells. Escherichia coli lipopolysaccharide shortened the half-life of eNOS message, reducing eNOS protein expression in peritoneal mesothelial and endothelial cells. Moreover, under basal conditions, human peritoneal samples expressed cytosolic proteins that bind to the 3'-UTR of eNOS mRNA. The cytosolic proteins that directly bind to 3'-UTR were identified as a 60-kD protein. After incubation of human peritoneal samples with lipopolysaccharide, the binding activity of the cytosolic 60-kD protein increased in a time-dependent manner. Studies are now necessary to determine the involvement of this 60-kD protein in the regulation of eNOS expression in peritoneal cells and particularly its involvement in the peritoneal dysfunction associated with inflammatory reactions.