Peritoneal infection after colorectal cancer surgery induces substantial alterations in postoperative protein levels: an exploratory study.

PURPOSE: Peritoneal infection, due to anastomotic leakage, after resection for colorectal cancer have been shown to associate with increased cancer recurrence and mortality, as well as cardiovascsular morbidity. Alterations in circulating protein levels could help shed light on the underlying mechanisms, prompting this exploratory study of 64 patients operated for colorectal cancer with anastomosis. METHODS: Thirty-two cases who suffered a postoperative peritoneal infection were matched with 32 controls who had a complication-free postoperative stay. Proteins in serum samples at their first postoperative visit and at one year after surgery were analysed using proximity extension assays and enzyme-linked immunosorbent assays. Multivariate projection methods, adjusted for multiple testing, were used to compare levels between groups, and enrichment and network analyses were performed. RESULTS: Seventy-seven proteins, out of 270 tested, were differentially expressed at a median sampling time of 41 days postoperatively. These proteins were all normalised one year after surgery. Many of the differentially expressed top hub proteins have known involvement in cancer progression, survival, invasiveness and metastasis. Over-represented pathways were related to cardiomyopathy, cell-adhesion, extracellular matrix, phosphatidylinositol-3-kinase/Akt (PI3K-Akt) and transforming growth factor beta (TGF-ß) signaling. CONCLUSION: These affected proteins and pathways could provide clues as to why patients with peritoneal infection might suffer increased cancer recurrence, mortality and cardiovascular morbidity.

Multi-omics profiling to identify early plasma biomarkers in pre-diagnostic pancreatic ductal adenocarcinoma: a nested case-control study.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor survival. Novel biomarkers are urgently needed to improve the outcome through early detection. Here, we aimed to discover novel biomarkers for early PDAC detection using multi-omics profiling in pre-diagnostic plasma samples biobanked after routine health examinations. A nested case-control study within the Northern Sweden Health and Disease Study was designed. Pre-diagnostic plasma samples from 37 future PDAC patients collected within 2.3 years before diagnosis and 37 matched healthy controls were included. We analyzed metabolites using liquid chromatography mass spectrometry and gas chromatography mass spectrometry, microRNAs by HTG edgeseq, proteins by multiplex proximity extension assays, as well as three clinical biomarkers using milliplex technology. Supervised and unsupervised multi-omics integration were performed as well as univariate analyses for the different omics types and clinical biomarkers. Multiple hypothesis testing was corrected using Benjamini-Hochberg's method and a false discovery rate (FDR) below 0.1 was considered statistically significant. Carbohydrate antigen (CA) 19-9 was associated with PDAC risk (OR [95 % CI] = 3.09 [1.31-7.29], FDR = 0.03) and increased closer to PDAC diagnosis. Supervised multi-omics models resulted in poor discrimination between future PDAC cases and healthy controls with obtained accuracies between 0.429-0.500. No single metabolite, microRNA, or protein was differentially altered (FDR < 0.1) between future PDAC cases and healthy controls. CA 19-9 levels increase up to two years prior to PDAC diagnosis but extensive multi-omics analysis including metabolomics, microRNAomics and proteomics in this cohort did not identify novel early biomarkers for PDAC.

Metabolomics for early pancreatic cancer detection in plasma samples from a Swedish prospective population-based biobank.

Background: Pancreatic ductal adenocarcinoma (pancreatic cancer) is often detected at late stages resulting in poor overall survival. To improve survival, more patients need to be diagnosed early when curative surgery is feasible. We aimed to identify circulating metabolites that could be used as early pancreatic cancer biomarkers. Methods: We performed metabolomics by liquid and gas chromatography-mass spectrometry in plasma samples from 82 future pancreatic cancer patients and 82 matched healthy controls within the Northern Sweden Health and Disease Study (NSHDS). Logistic regression was used to assess univariate associations between metabolites and pancreatic cancer risk. Least absolute shrinkage and selection operator (LASSO) logistic regression was used to design a metabolite-based risk score. We used receiver operating characteristic (ROC) analyses to assess the discriminative performance of the metabolite-based risk score. Results: Among twelve risk-associated metabolites with a nominal P value <0.05, we defined a risk score of three metabolites [indoleacetate, 3-hydroxydecanoate (10:0-OH), and retention index (RI): 2,745.4] using LASSO. A logistic regression model containing these three metabolites, age, sex, body mass index (BMI), smoking status, sample date, fasting status, and carbohydrate antigen 19-9 (CA 19-9) yielded an internal area under curve (AUC) of 0.784 [95% confidence interval (CI): 0.714-0.854] compared to 0.681 (95% CI: 0.597-0.764) for a model without these metabolites (P value =0.007). Seventeen metabolites were significantly associated with pancreatic cancer survival [false discovery rate (FDR) <0.1]. Conclusions: Indoleacetate, 3-hydroxydecanoate (10:0-OH), and RI: 2,745.4 were identified as the top candidate biomarkers for early detection. However, continued efforts are warranted to determine the usefulness of these metabolites as early pancreatic cancer biomarkers.

A Cross-Sectional and Longitudinal Analysis of Pre-Diagnostic Blood Plasma Biomarkers for Early Detection of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death that typically presents at an advanced stage. No reliable markers for early detection presently exist. The prominent tumor stroma represents a source of circulating biomarkers for use together with cancer cell-derived biomarkers for earlier PDAC diagnosis. CA19-9 and CEA (cancer cell-derived biomarkers), together with endostatin and collagen IV (stroma-derived) were examined alone, or together, by multivariable modelling, using pre-diagnostic plasma samples (n = 259 samples) from the Northern Sweden Health and Disease Study biobank. Serial samples were available for a subgroup of future patients. Marker efficacy for future PDAC case prediction (n = 154 future cases) was examined by both cross-sectional (ROC analysis) and longitudinal analyses. CA19-9 performed well at, and within, six months to diagnosis and multivariable modelling was not superior to CA19-9 alone in cross-sectional analysis. Within six months to diagnosis, CA19-9 (AUC = 0.92) outperformed the multivariable model (AUC = 0.81) at a cross-sectional level. At diagnosis, CA19-9 (AUC = 0.995) and the model (AUC = 0.977) performed similarly. Longitudinal analysis revealed increases in CA19-9 up to two years to diagnosis which indicates a window of opportunity for early detection of PDAC.

Type IV Collagen in Human Colorectal Liver Metastases-Cellular Origin and a Circulating Biomarker.

Circulating type IV collagen (cCOL IV) is a potential biomarker for patients with colorectal liver metastases (CLM) who present with elevated levels of COL IV in both CLM tissue and circulation. This study aimed to establish the cellular origin of elevated levels of COL IV and analyze circulating COL IV in CLM patients. The cellular source was established through in situ hybridization, immunohistochemical staining, and morphological evaluation. Cellular expression in vitro was assessed by immunofluorescence. Tissue expression of COL IV-degrading matrix metalloproteinases (MMPs)-2, -7, -9, and -13 was studied with immunohistochemical staining. Plasma levels of COL IV in CLM patients and healthy controls were analyzed with ELISA. This study shows that cancer-associated fibroblasts (CAFs) express COL IV in the stroma of CLM and that COL IV is expressed in vitro by fibroblasts but not by tumor cells. MMP-2, -7, -9, and -13 are expressed in CLM tissue, mainly by hepatocytes and immune cells, and circulating COL IV is significantly elevated in CLM patients compared with healthy controls. Our study shows that stromal cells, not tumor cells, produce COL IV in CLM, and that circulating COL IV is elevated in patients with CLM.

Preoperative biomarkers related to inflammation may identify high-risk anastomoses in colorectal cancer surgery: explorative study.

BACKGROUND: Colorectal anastomotic leakage can be considered a process of failed wound healing, for which related biomarkers might be a promising research area to decrease leak rates. METHODS: Patients who had elective surgery with a primary anastomosis for non-metastatic colorectal cancer, at two university hospitals between 1 January 2010 and 31 December 2015 were included. Patients with an anastomotic leak were identified and matched (1:1) to complication-free controls on the basis of sex, age, tumour stage, tumour location, and operating hospital. Preoperative blood samples were analysed by use of protein panels associated with systemic or enteric inflammation by proteomics, and enzyme-linked immunosorbent assays. Multivariable projection methods were used in the statistical analyses and adjusted for multiple comparisons to reduce false positivity. Rectal cancer tissue samples were evaluated with immunohistochemistry to determine local expression of biomarkers that differed significantly between cases and controls. RESULTS: Out of 726 patients undergoing resection, 41 patients with anastomotic leakage were matched to 41 controls. Patients with rectal cancer with leakage displayed significantly elevated serum levels of 15 proteins related to inflammation. After controlling for a false discovery rate, levels of C-X-C motif chemokine 6 (CXCL6) and C-C motif chemokine 11 (CCL11) remained significant. In patients with colonic cancer with leakage, levels of high-sensitivity C-reactive protein (hs-CRP) were increased before surgery. Local expression of CXCL6 and CCL11, and their receptors, were similar in rectal tissues between cases and controls. CONCLUSION: Patients with anastomotic leakage could have an upregulated inflammatory response before surgery, as expressed by elevated serological levels of CXCL6 and CCL11 for rectal cancer and hs-CRP levels in patients with colonic cancer respectively.

Circulating Tissue Polypeptide-Specific Antigen in Pre-Diagnostic Pancreatic Cancer Samples.

Early detection of pancreatic ductal adenocarcinoma (PDAC) is challenging, and late diagnosis partly explains the low 5-year survival. Novel and sensitive biomarkers are needed to enable early PDAC detection and improve patient outcomes. Tissue polypeptide specific antigen (TPS) has been studied as a biomarker in PDAC diagnostics, and it has previously been shown to reflect clinical status better than the 'golden standard' biomarker carbohydrate antigen 19-9 (CA 19-9) that is most widely used in the clinical setting. In this cross-sectional case-control study using pre-diagnostic plasma samples, we aim to evaluate the potential of TPS as a biomarker for early PDAC detection. Furthermore, in a subset of individuals with multiple samples available at different time points before diagnosis, a longitudinal analysis was used. We assessed plasma TPS levels using enzyme-linked immunosorbent assay (ELISA) in 267 pre-diagnostic PDAC plasma samples taken up to 18.8 years before clinical PDAC diagnosis and in 320 matched healthy controls. TPS levels were also assessed in 25 samples at PDAC diagnosis. Circulating TPS levels were low both in pre-diagnostic samples of future PDAC patients and in healthy controls, whereas TPS levels at PDAC diagnosis were significantly increased (odds ratio 1.03; 95% confidence interval: 1.01-1.05) in a logistic regression model adjusted for age. In conclusion, TPS levels increase late in PDAC progression and hold no potential as a biomarker for early detection.

Expression and Circulating Levels of Perlecan in Breast Cancer - Implications for Oestrogen Dependent Stromal Remodeling.

Localised breast cancer can be cured by surgery and adjuvant treatments, but mortality remains high as some tumours metastasize early. Perlecan is a basement membrane (BM) protein involved in tumour development and progression. Here, mRNA and protein expression of perlecan, and mRNA expression of matrix degrading enzymes were studied in normal breast and invasive breast cancer, and correlated to prognostic risk factors, in particular oestrogen status. Moreover, plasma levels of perlecan were measured in patients with breast cancer and compared with controls. mRNA data was extracted from the Cancer Genome Atlas database. Perlecan protein expression was visualized using immunofluorescence and plasma levels measured by ELISA assay. Perlecan mRNA levels were twice as high in normal breast compared with breast cancer tissue. A strong correlation was found between mRNA expression of perlecan and several matrix-degrading enzymes in oestrogen receptor positive (ER+) tumours. Perlecan protein was localized to both epithelial and vascular BMs, but absent in the stroma in normal breast. In breast cancer, the expression of perlecan in epithelial BM was fragmented or completely lost, with a marked upregulation of perlecan expression in the stroma. Significantly higher levels of perlecan were found in plasma of ER+ patients when compared with ER- patients. This study shows that perlecan expression and degradation in breast cancer may be linked to the ER status of the tumour.

Pharmacokinetics, pharmacodynamics and safety of the inverse retinoic acid-related orphan receptor γ agonist AZD0284.

AIMS: Retinoic acid-related orphan receptor γ (RORγ), a master regulator of T-helper 17 (Th17) cell function and differentiation, is an attractive target for treatment of Th17-driven diseases. This first-in-human study aimed to investigate the pharmacokinetics, pharmacodynamics, safety and tolerability of the inverse RORγ agonist AZD0284. METHODS: We conducted a phase I, randomized, single-blind, placebo-controlled, two-part, first-in-human study with healthy subjects receiving single (4-238 mg) or multiple (12-100 mg) oral doses of AZD0284 or placebo after overnight fasting. Subjects in the one single dose cohort additionally received a single dose of AZD0284 after a high-calorie meal. AZD0284 plasma concentrations, as well as inhibition of ex vivo-stimulated interleukin (IL)-17A release in whole blood, were frequently measured after both single and multiple dosing. RESULTS: Eighty-three men participated in the study. AZD0284 was absorbed rapidly into plasma after oral dosing and exhibited a terminal half-life of 13-16 hours. Both the area under the concentration-time curve (AUC) and maximum concentration (Cmax ) increased subproportionally with increasing dose (95% confidence intervals of slope parameter were 0.71-0.84 and 0.72-0.88 for AUC and Cmax , respectively). Food intake delayed the absorption of AZD0284 but did not affect the overall exposure or half-life. AZD0284 showed dose-dependent reduction of ex vivo-stimulated IL-17A release after both single and multiple doses. No significant safety concerns were identified in the study. CONCLUSIONS: AZD0284 was well tolerated, rapidly and dose-dependently absorbed, and reduced stimulated IL-17A release after single and multiple dosing. The results of this study support further clinical development of AZD0284.

Language interpretation conditions and boundaries in multilingual and multicultural emergency healthcare.

BACKGROUND: With an increasing migrant population globally the need to organize interpreting service arises in emergency healthcare to deliver equitable high-quality care. The aims of this study were to describe interpretation practices in multilingual emergency health service institutions and to explore the impact of the organizational and institutional context and possible consequences of different approaches to interpretation. No previous studies on these issues in multilingual emergency care have been found. METHODS: A qualitative descriptive study was used. Forty-six healthcare professionals were purposively recruited from different organizational levels in ambulance service and psychiatric and somatic emergency care units. Data were collected between December 2014 and April 2015 through focus-group and individual interviews, and analyzed by qualitative content analysis. RESULTS: Organization of interpreters was based on patients' health status, context of emergency care, and access to interpreter service. Differences existed between workplaces regarding the use of interpreters: in somatic emergency care bilingual healthcare staff and family members were used to a limited extent; in psychiatric emergency care the norm was to use professional interpreters on the spot; and in ambulance service persons available at the time, e.g. family and friends were used. Similarities were found in: procuring a professional interpreter, mainly based on informal workplace routines, sometimes on formal guidelines and national laws, but knowledge of existing laws was limited; the ideal was a linguistically competent interpreter with a professional attitude, and organizational aspects such as appropriate time, technical and social environment; and wishes for development of better procedures for prompt access to professional interpreters at the workplace, regardless of organizational context, and education of interpreters and users. CONCLUSION: Use of interpreters was determined by health professionals, based on the patients' health status, striving to deliver as fast and individualized care as possible based on humanistic values. Defects in organizational routines need to be rectified and transcultural awareness is needed to achieve the aim of person-centered and equal healthcare. Clear formal guidelines for the use of interpreters in emergency healthcare need to be developed and it is important to fulfill health professionals' wishes for future development of prompt access to interpreters and education of interpreters and users.

Circulating Vascular Basement Membrane Fragments are Associated with the Diameter of the Abdominal Aorta and Their Expression Pattern is Altered in AAA Tissue.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is characterised by enhanced proteolytic activity, and extracellular matrix (ECM) remodelling in the vascular wall. Type IV and XVIII collagen/endostatin are structural proteins in vascular basement membrane (VBM), a specialised ECM structure. Here the association between plasma levels of these collagens with the aortic diameter and expansion rate is studied, and their expression in aortic tissue characterised. METHODS: This was a retrospective population based cohort study. Type IV and XVIII collagen/endostatin were analysed in plasma by ELISA assay in 615 men, divided into three groups based on the aortic diameter: 1) normal aorta ≤ 25 mm, 2) sub-aneurysmal aorta (SAA) 26-29 mm, and 3) AAA ≥ 30 mm. Follow up data were available for 159 men. The association between collagen levels and aortic diameter at baseline, and with the expansion rate at follow up were analysed in ordinal logistic regression and linear regression models, controlling for common confounding factors. Tissue expression of the collagens was analysed in normal aorta (n = 6) and AAA (n = 6) by immunofluorescence. RESULTS: Plasma levels of type XVIII collagen/endostatin (136 ng/mL [SD 29] in individuals with a normal aorta diameter, 154 ng/ml [SD 45] in SAA, and 162 ng/ml [SD 46] in AAA; p = .001) and type IV collagen (105 ng/mL [SD 42] normal aorta, 124 ng/ml [SD 46] SAA, and 127 ng/ml [SD 47] AAA; p = .037) were associated with a larger aortic diameter. A significant association was found between the baseline levels of type XVIII/endostatin and the aortic expansion rate (p = .035), but in the multivariable model, only the initial aortic diameter remained significantly associated with expansion (p = .005). Altered expression patterns of both collagens were observed in AAA tissue. CONCLUSION: Plasma levels of circulating type IV and XVIII collagen/endostatin increase with AAA diameter. The expression pattern of VBM proteins is altered in the aneurysm wall.

Plasma Micro-RNA Alterations Appear Late in Pancreatic Cancer.

OBJECTIVES: The aim of this research was to study whether plasma microRNAs (miRNA) can be used for early detection of pancreatic cancer (PC) by analyzing prediagnostic plasma samples collected before a PC diagnosis. BACKGROUND: PC has a poor prognosis due to late presenting symptoms and early metastasis. Circulating miRNAs are altered in PC at diagnosis but have not been evaluated in a prediagnostic setting. METHODS: We first performed an initial screen using a panel of 372 miRNAs in a retrospective case-control cohort that included early-stage PC patients and healthy controls. Significantly altered miRNAs at diagnosis were then measured in an early detection case-control cohort wherein plasma samples in the cases are collected before a PC diagnosis. Carbohydrate antigen 19-9 (Ca 19-9) levels were measured in all samples for comparison. RESULTS: Our initial screen, including 23 stage I-II PC cases and 22 controls, revealed 15 candidate miRNAs that were differentially expressed in plasma samples at PC diagnosis. We combined all 15 miRNAs into a multivariate statistical model, which outperformed Ca 19-9 in receiver-operating characteristics analysis. However, none of the candidate miRNAs, individually or in combination, were significantly altered in prediagnostic plasma samples from 67 future PC patients compared with 132 matched controls. In comparison, Ca 19-9 levels were significantly higher in the cases at <5 years before diagnosis. CONCLUSION: Plasma miRNAs are altered in PC patients at diagnosis, but the candidate miRNAs found in this study appear late in the course of the disease and cannot be used for early detection of the disease.

Prolonged mounted patrolling is a risk factor for developing knee pain in Danish military personnel deployed to the Helmand Province.

INTRODUCTION: Non-battle injuries have been the leading cause of medical evacuation in the recent wars in Afghanistan and Iraq. This study investigates the hypothesis, that the occurrence of knee problems could be associated with mounted patrolling in armoured vehicles independent of other risk factors. METHOD: Retrospective questionnaire-based cohort study of Danish soldiers deployed to Afghanistan during 1 February-31 July 2013. RESULTS: 307 soldiers included. Response rate 70%. 33% reported knee pain. MAIN FINDING: Significant association between knee pain and time spent weekly on mounted patrols (OR 1.23, CI 1.07 to 1.41, p=0.003). Controlled for confounders age, body mass index and duration of military employment (OR 1.22, CI 1.06 to 1.41, p=0.006). Adjusted for confounders and all other risk factors (OR 1.25, CI 1.07 to 1.48, p=0.007). The main finding in a subset of the 33% with knee pain: Significant association between more severe knee problems with Knee injury and Osteoarthritis Outcome Score below 400 and time spent weekly on mounted patrols (OR 1.49, CI 1.17 to 1.56, p=0.002). CONCLUSIONS: A major concern regarding knee problems among Danish deployed military personnel is identified. The risk of suffering from knee problems and the severity of symptoms increase with the amount of time spent inside a vehicle on mounted patrols.

Boundaries and conditions of interpretation in multilingual and multicultural elderly healthcare.

BACKGROUND: Elderly migrants who do not speak the official language of their host country have increased due to extensive international migration, and will further increase in the future. This entails major challenges to ensure good communication and avoid communication barriers that can be overcome by the use of adequate interpreter services. To our knowledge, there are no previous investigations on interpreting practices in multilingual elderly healthcare from different healthcare professionals' perspectives. This study examines issues concerning communication and healthcare through a particular focus on interpretation between health professionals and patients of different ethnic and linguistic backgrounds. The central aim of the project is to explore interpretation practices in multilingual elderly healthcare. METHODS: A purposive sample of 33 healthcare professionals with experience of using interpreters in community multilingual elderly healthcare. Data were collected between October 2013 and March 2014 by 18 individual and four focus group interviews and analysed with qualitative content analysis. RESULTS: The main results showed that interpreting practice in multilingual elderly healthcare was closely linked to institutional, interpersonal and individual levels. On the organizational level, however, guidelines for arranging the use of interpreters at workplaces were lacking. Professional interpreters were used on predictable occasions planned long in advance, and bilingual healthcare staff and family members acting as interpreters were used at short notice in everyday caring situations on unpredictable occasions. The professional interpreter was perceived as a person who should interpret spoken language word-for-word and who should translate written information. Furthermore, the use of a professional interpreter was not adapted to the context of multilingual elderly healthcare. CONCLUSION: This study found that interpreter practice in multilingual elderly healthcare is embedded in the organizational environment and closely related to the individual's language skills, cultural beliefs and socio-economic factors. In order to formulate interpreter practice in the context of multilingual elderly healthcare it is important to consider organizational framework and cultural competence, cultural health knowledge, beliefs and customs.

Combining conventional and stroma-derived tumour markers in pancreatic ductal adenocarcinoma.

BACKGROUND: A lack of disease-specific symptoms and good tumour markers makes early detection and diagnosis of pancreatic ductal adenocarcinoma (PDAC) challenging. OBJECTIVE: To analyse the tissue expression and circulating levels of four stroma-derived substances (type IV collagen, endostatin/type XVIII collagen, osteopontin and tenascin C) and four conventional tumour markers (CA 19-9, TPS, CEA and Ca 125) in a PDAC cohort. METHODS: Tissue expression of markers in normal pancreas and PDAC tissue was analysed with immunofluorescence. Plasma concentrations of markers were measured before and after surgery. Patients with non-malignant disorders served as controls. RESULTS: The conventional and stromal substances were expressed in the cancer cell compartment and the stroma, respectively. Although most patients had increased levels of many markers before surgery, 2/12 (17%) of patients had normal levels of Ca 19-9 at this stage. High preoperative endostatin/type XVIII collagen, and postoperative type IV collagen was associated with short survival. Neither the pre- nor postoperative levels of TPS, Ca 125 or CA 19-9 were associated to survival. CONCLUSIONS: PDAC is characterized by an abundant stroma. These initial observations indicate that the stroma can be a source of PDAC tumour markers that are found in different compartments of the cancer, thus reflecting different aspects of tumour biology.

Interaction between the anticancer drug Cisplatin and the copper chaperone Atox1 in human melanoma cells.

Cisplatin (CisPt) is one of the most common anticancer drugs used against many severe forms of cancers. However, treatment with this drug causes many side effects and often, it results in the development of cell resistance. A majority of side effects as well as cell resistance are thought to develop due to CisPt interactions with proteins prior to reaching the nucleus and the DNA target. The copper (Cu) transport proteins Ctr1 and ATP7A/B have been implicated in cellular resistance of CisPt, possibly exporting the drug out of the cell. Recent in vitro work demonstrated that CisPt also interacts with the cytoplasmic Cu-chaperone Atox1, binding in or near the Cu-binding site, without expulsion of bound Cu. Whereas Ctr1 and ATP7B interactions with CisPt have been shown in vivo or ex vivo, there is no such information for Atox1-CisPt interactions. To address this, we developed a method to probe if CisPt interacts with Atox1 in human melanoma cells. Atox1-specific antibodies were linked to magnetic beads and used to immune-precipitate Atox1 from melanoma cells that had been pre-exposed to CisPt. Analysis of extracted Atox1 with inductively coupled plasma mass spectrometry demonstrated the presence of Pt in the protein fraction. Thus, CisPt-exposed human melanoma cells contain Atox1 molecules that bind some derivative of CisPt. This study gives the first indication for the intracellular presence of Atox1-CisPt complexes ex vivo.

Type IV collagen stimulates pancreatic cancer cell proliferation, migration, and inhibits apoptosis through an autocrine loop.

BACKGROUND: Pancreatic cancer shows a highly aggressive and infiltrative growth pattern and is characterized by an abundant tumor stroma known to interact with the cancer cells, and to influence tumor growth and drug resistance. Cancer cells actively take part in the production of extracellular matrix proteins, which then become deposited into the tumor stroma. Type IV collagen, an important component of the basement membrane, is highly expressed by pancreatic cancer cells both in vivo and in vitro. In this study, the cellular effects of type IV collagen produced by the cancer cells were characterized. METHODS: The expression of type IV collagen and its integrin receptors were examined in vivo in human pancreatic cancer tissue. The cellular effects of type IV collagen were studied in pancreatic cancer cell lines by reducing type IV collagen expression through RNA interference and by functional receptor blocking of integrins and their binding-sites on the type IV collagen molecule. RESULTS: We show that type IV collagen is expressed close to the cancer cells in vivo, forming basement membrane like structures on the cancer cell surface that colocalize with the integrin receptors. Furthermore, the interaction between type IV collagen produced by the cancer cell, and integrins on the surface of the cancer cells, are important for continuous cancer cell growth, maintenance of a migratory phenotype, and for avoiding apoptosis. CONCLUSION: We show that type IV collagen provides essential cell survival signals to the pancreatic cancer cells through an autocrine loop.

Risk of post-traumatic stress disorder among Danish junior medical officers deployed to Afghanistan is not increased.

INTRODUCTION: Since August 2006, the Danish Armed Forces have deployed junior medical officers (JMOs) to the Helmand Province in Afghanistan. Research has shown an increased incidence of post-traumatic stress disorder (PTSD) in deployed military personnel throughout the history of modern warfare. No investigation of the mental health of Danish military medical personnel has been performed. We wanted to investigate the extent of potentially traumatizing events experienced by Danish JMOs and the prevalence of PTSD among them. MATERIAL AND METHODS: We included all JMOs deployed for one or more tours of duty in Afghanistan from January 2006 to August 2010. Potential participants received a questionnaire to their home address including the PTSD Checklist - Civilian Version (PCL-C). RESULTS: A total of 72 JMOs were included in the survey. The completion rate was 65%. We found that 98% of the respondents had experienced a potentially traumatizing event and that 47% had experienced feeling fear, horror or helplessness in the context of such an event. The prevalence of PTSD was 0%. CONCLUSION: Danish JMOs do not seem to have an increased risk of PTSD after deployment to Afghanistan. However, further research on the mental health of this personnel group is needed. FUNDING: This study was partly funded by The Danish Armed Forces Health Services. TRIAL REGISTRATION: This study was registered with the Danish Data Protection Agency.

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro.

Cisplatin (cisPt), Pt(NH(3))(2)Cl(2), is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

Evaluation of cell lysis methods for platinum metallomic studies of human malignant cells.

Three cell lysis methods-freeze-thaw, osmosis, and a chemical detergent-based method-were evaluated as sample treatment procedures for platinum metallomic studies of in vitro grown human malignant cells exposed to cisplatin. The lysis methods are relatively mild, resemble those commonly used in proteomic studies, and were selected because of the proven reactivity of platinum drug metabolites and indications that platinum in exposed cells and plasma is mainly associated with proteins. The chemical method gave an absolute lysis efficiency of greater than 80%, whereas the freeze-thaw and osmosis methods gave approximately 30% lower efficiency. The within- and between-batch lysis reproducibilities were, for all methods, better than 20 and 24% relative standard deviations, respectively. Total platinum concentration normalized to lysate protein content was statistically the same for all lysis methods. Reagents in the chemical lysis buffer did, however, react with platinum analyte compounds, making this method unsuitable for analysis of reactive compounds or for metallome profiling encompassing analytes with unknown reactivity. Of the lysis methods evaluated here, osmosis gave the highest cisplatin recovery, likely because this protocol is chemically inert and can be carried out at a constant low temperature. Therefore, it is the recommended cell lysis method for the determination of reactive and unknown intracellular platinum compounds.