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Diagnostic performance of serum miRNA panels.

Demographic, radiological and surgical characteristics of IPMN patients.

Purpose:Intraductal papillary mucinous neoplasm (IPMN) is a precursor of pancreatic ductal adenocarcinoma. Low-grade dysplasia has a relatively good prognosis, whereas high-grade dysplasia and IPMN invasive carcinoma require surgical intervention. However, diagnostic distinction is difficult. We aimed to identify biomarkers in peripheral blood for accurate discrimination.Experimental Design:Sera were obtained from 302 patients with IPMNs and 88 healthy donors. For protein biomarkers, serum samples were analyzed on microarrays made of 2,977 antibodies. A support vector machine (SVM) algorithm was applied to define classifiers, which were validated on a separate sample set. For microRNA biomarkers, a PCR-based screen was performed for discovery. Biomarker candidates confirmed by quantitative PCR were used to train SVM classifiers, followed by validation in a different sample set. Finally, a combined SVM classifier was established entirely independent of the earlier analyses, again using different samples for training and validation.Results:Panels of 26 proteins or seven microRNAs could distinguish high- and low-risk IPMN with an AUC value of 95% and 94%, respectively. Upon combination, a panel of five proteins and three miRNAs yielded an AUC of 97%. These values were much better than those obtained in the same patient cohort by using the guideline criteria for discrimination. In addition, accurate discrimination was achieved between other patient subgroups.Conclusions:Protein and microRNA biomarkers in blood allow precise diagnosis and risk stratification of IPMN cases, which should improve patient management and thus the prognosis of IPMN patients.See related commentary by Löhr and Pantel, p. 1387

Objective To propose a noninvasive diagnostic approach, which allows reliable distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMNs). Background IPMNs are identifiable precursor lesions of pancreatic cancer, of which surgical resection is warranted prior to the development of invasive carcinoma, but low-grade IPMNs should not be unnecessarily resected. However, diagnostic tools that preoperatively enable accurate risk stratification of IPMNs are missing. Methods This single-center, retrospective cohort study included 56 patients who underwent surgical resection for IPMN including 18 low-risk (low-grade) and 38 high-risk (high-grade/invasive carcinoma) IPMNs, from whom clinical features and serum samples were prospectively obtained. An antibody microarray platform was used to analyze the serum proteome. Based on serum markers and selected clinical characteristics support vector machine models were constructed to predict the risk of IPMN malignancy. Results A serum protein signature discriminating low- and high-risk IPMN patients was identified. Combinations of established clinical features and the newly identified serum biomarkers correctly distinguished low- and high-risk IPMNs in 93% on 1000-fold cross-validation. Conclusions This study highlights the synergistic predictive value of combining a novel serum protein signature with conventional clinical characteristics to risk-stratify IPMN patients. If these findings are supported by larger validation studies, they might enable more rational decision-making in clinical management of IPMN patients in conjunction with clinical guidelines.

BACKGROUND: Preclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor. METHODS: In this phase I trial, patients received either 1.5 × 10(6) MICs per kg BW on day –2 (n = 3, group A), or 1.5 × 10(8) MICs per kg BW on day –2 (n = 3, group B) or day –7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360. RESULTS: MIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 10(10) donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19(+)CD24(hi)CD38(hi) transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature. CONCLUSION: MIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs. TRIAL REGISTRATION: EudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220. FUNDING: Federal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany.

// Shakhawan Mustafa 1, 2, * , Longqiang Pan 1, * , Aseel Marzoq 1 , Malak Fawaz 1 , Laureen Sander 1 , Felix Ruckert 3 , Andrea Schrenk 3 , Christina Hartl 1 , Rico Uhler 1 , Adem Yildirim 1 , Oliver Strobel 4 , Thilo Hackert 4 , Nathalia Giese 4 , Markus W. Buchler 4 , Jorg D. Hoheisel 1, * , Mohamed Saiel Saeed Alhamdani 1, * 1 Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany 2 Kurdistan Institution for Strategic Studies and Scientific Research, Kurdistan Region, Iraq 3 Chirurgische Klinik, Universitatsmedizin Mannheim, Theodor-Kutzer-Ufer 1-3, Mannheim, Germany 4 Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Mohamed Saiel Saeed Alhamdani, email: m.alhamdani@dkfz.de Keywords: secretome, pancreatic cancer, biomarkers, cell lines, antibody microarray Received: February 09, 2016 Accepted: December 25, 2016 Published: January 2, 2017 ABSTRACT Pancreatic cancer is the currently most lethal malignancy. Toward an accurate diagnosis of the disease in body liquids, we studied the protein composition of the secretomes of 16 primary and established cell lines of pancreatic ductal adenocarcinoma (PDAC). Compared to the secretome of non-tumorous cells, 112 proteins exhibited significantly different abundances. Functionally, the proteins were associated with PDAC features, such as decreased apoptosis, better cell survival and immune cell regulation. The result was compared to profiles obtained from 164 serum samples from two independent cohorts – a training and a test set – of patients with PDAC or chronic pancreatitis and healthy donors. Eight of the 112 secretome proteins exhibited similar variations in their abundance in the serum profile specific for PDAC patients, which was composed of altogether 189 proteins. The 8 markers shared by secretome and serum yielded a 95.1% accuracy of distinguishing PDAC from healthy in a Receiver Operating Characteristic curve analysis, while any number of serum-only markers produced substantially less accurate results. Utility of the identified markers was confirmed by classical enzyme linked immunosorbent assays (ELISAs). The study highlights the value of cell secretome analysis as a means of defining reliable serum biomarkers.

Platelet storage lesions (PSLs) occur during platelet concentrate (PC) storage. Adverse transfusion reactions (ATRs) have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Proteomic profiling of PC supernatants was thus performed to identify proteins associated with PSLs and ATRs. Twenty-four PCs were investigated daily from day 0 to day 9 for platelet pre-activation (PPA), platelet-derived extracellular vesicles (PEVs), and platelet function. Using antibody microarrays, 673 extracellular proteins were analysed in PC supernatants on days 0, 3, 5, 7, and 9. During 5 days of storage, PPA and PEVs continuously increased ( P P = 0.1751) and decreased thereafter. Comparison of all time points to day 0 revealed the identification of 136 proteins that were significantly changed in abundance during storage, of which 72 were expressed by platelets. Network analysis identified these proteins to be predominantly associated with exosomes ( P = 4.61 × 10 − 8 , n = 45 genes) and two clusters with distinct functions were found with one being associated with haemostasis and the other with RNA binding. These findings may provide an explanation for ATRs. Significance Changes in platelet concentrate (PC) supernatants during storage have been so far only poorly addressed and high abundant proteins burden the identification of quantitative changes in the secretome. We applied a high-throughput antibody microarray allowing for the sensitive quantification of 673 extracellular factors. PCs account for the highest number of adverse transfusion reactions (ATRs). ATRs have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Comprehensive interpretation of the changing proteins in the secretome during platelet storage under blood banking conditions may help to identify mechanisms leading to the occurrence of adverse transfusion reactions.

Pancreatic ductal adenocarcinoma (PDAC) exists in a complex desmoplastic microenvironment. As part of it, pancreatic stellate cells (PSCs) provide a fibrotic niche, stimulated by a dynamic communication between activated PSCs and tumour cells. Investigating how PSCs contribute to tumour development and for identifying proteins that the cells secrete during cancer progression, we studied by means of complex antibody microarrays the secretome of activated PSCs. A large number of secretome proteins were associated with cancer-related functions, such as cell apoptosis, cellular growth, proliferation and metastasis. Their effect on tumour cells could be confirmed by growing tumour cells in medium conditioned with activated PSC secretome. Analyses of the tumour cells’ proteome and mRNA revealed a strong inhibition of tumour cell apoptosis, but promotion of proliferation and migration. Many cellular proteins that exhibited variations were found to be under the regulatory control of eukaryotic translation initiation factor 4E (eIF4E), whose expression was triggered in tumour cells grown in the secretome of activated PSCs. Inhibition by an eIF4E siRNA blocked the effect, inhibiting tumour cell growth in vitro. Our findings show that activated PSCs acquire a pro-inflammatory phenotype and secret proteins that stimulate pancreatic cancer growth in an eIF4E-dependent manner, providing further insight into the role of stromal cells in pancreatic carcinogenesis and cancer progression.

About 70% of newly diagnosed cases of bladder cancer are low-stage, low-grade, non muscle-invasive. Standard treatment is transurethral resection. About 60% of the tumors will recur, however, and in part progress to become invasive. Therefore, surveillance cystoscopy is performed after resection. However, in the USA and Europe alone, about 54 000 new patients per year undergo repeated cystoscopies over several years, who do not experience recurrence. Analysing in a pilot study resected tumors from patients with (n = 19) and without local recurrence (n = 6) after a period of 5 years by means of an antibody microarray that targeted 724 cancer-related proteins, we identified 255 proteins with significantly differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. A multivariate classifier was constructed based on 20 proteins. It facilitates the prediction of recurrence with a sensitivity of 80% and a specificity of 100%. As a measure of overall accuracy, the area under the curve value was found to be 91%. After validation in additional sample cohorts with a similarly long follow-up, such a signature could support decision making about the stringency of surveillance or even different treatment options.

Exchange of molecules via exosomes is a means of eukaryotic intercellular communication, especially within tumour microenvironments. However, no data are available on alterations of exosomal molecular cargo by environmental cues (eg, pharmacological treatments). To approach this issue, we compared the abundance of 754 miRNAs and 741 cancer-related proteins in exosomes secreted by Caco-2 (Cetuximab-responsive) and HCT- 116 (Cetuximab-resistant) CRC cells, before and after Cetuximab treatment, with that in their source cells. Cetuximab significantly altered the cargo of Caco-2 exosomes: it increased abundance of miRNAs and proteins activating proliferation and inflammation and reduced miRNAs and proteins related to immune suppression. These alterations did not precisely mirror those in source cells, suggesting a Cetuximab-linked effect. Analogous alterations were detected in HCT-116. Transfection of exosomes from Cetuximab-treated Caco-2 into HCT-116 significantly increased HCT-116 viability; conversely, no viability alteration was detected in Caco-2 transfected with exosomes from Cetuximab-treated HCT-116. Analysis of networks, comprising targets of differentially expressed (DE) exosomal miRNAs and DE exosomal proteins, demonstrates a significant involvement of processes related to proliferation, inflammation, immune response, apoptosis. Our data extend existing knowledge on molecular mechanisms of eukaryotic intercellular communication, especially in oncological processes. Their translation to clinical settings may add new weapons to existing therapeutic repertoires against cancer.

Based on about a decade of technical developments in analysing the human proteome with antibody microarrays and experience in performing such analyses, now there are the means at hand for detailed and simultaneously global investigations of this kind. Many technical aspects have been dealt with of both the microarray format itself - such as overcoming kinetic and mass transport limitations and thus achieving accurate measurements - and ancillary processes - such as extraction procedures that provide good protein solubilisation, produce reproducible yields and preserve the native protein conformation as much as possible. The overall analysis process is robust and reproducible, highly sensitive down to the level of single-molecule detection and permits an analysis of several parameters on many molecules at a time. While the study of body liquids is widely applied, analyses of tissue proteomes are still scarce. However, conditions do exist to perform the latter at a quality level that meets the standards for clinical applications. This review highlights methodological aspects relevant for a biomedically useful analysis of cellular samples and discusses the potential of such studies, in particular, in view of personalised medicine approaches.

The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While antibody microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.

Background/Aims: Several studies have implicated reactive carbonyl compounds (RCOs), especially those derived from lipid peroxidation, in the development of complications frequently associated with hemodialysis (HD) treatment. However, there is still much unknown regarding the nature and concentration of RCOs in HD patients. This study was designed to evaluate the level of toxic aldehydes in the plasma of HD patients and to determine the extent to which these aldehydes contribute to RCO toxicity among these patients. Methods: 15 aldehydes of the alkanal, alkenal and 4-HO-alkenal type were measured in the plasma of 17 HD patients and 20 healthy controls. In addition, protein modification markers such as carbonyl content (CO), free thiol (SH) and residual free amino groups, as well as amyloid fibrils were also determined. Results: 11 of the 15 aldehydes were significantly elevated in the HD group when compared with the controls. Correlation studies in the HD group revealed high relationships between total alkenals plus total 4-HO-alkenals versus CO, total alkanals versus NH2, total aldehydes versus SH, and total 4-HO-alkenals versus fibril. Conclusion: The increased levels of alkanals, alkenals and 4-HO-alkenals of lipid peroxidation in the plasma of HD patients may greatly contribute to the toxicity of RCOs. The pattern of modification of plasma protein by each group of aldehydes may provide new evidence on the in vivo mechanisms of toxicity triggered by these aldehydes on their target molecules.

We are analyzing in detail the molecular communication between the different cell types in the microenvironment of pancreatic ductal adenocarcinomas (PDAC). Also, its regulation is being investigated, looking at aspects such as the kind and mixture of molecules needed for triggering a particular regulative function, and unraveling the inter- and intracellular processes utilized to transmit a molecular message. Although a rare tumor entity, adding up to only about 3% of all cancer cases in the Western world, PDAC is the fourth most frequent cause of cancer-related death; mortality is nearly identical to incidence. There is no real remedy apart from surgery, which can be applied to only 10 to 20% of patients. Even then, basically all patients will experience tumor recurrence. Our study is based on a large basic set of data at the levels of DNA, RNA and protein that was generated from some 1000 pancreatic tumor samples [1], supplemented with information from studies that aim at an elucidation of functional molecules and comparative analyses in other tumors. This has already led to the creation of basic functional knowledge about metastasis [e.g., 2], the establishment of means for diagnosis [3, 4], an accurate disease prognosis [1], the characterization of communication processes [5] and the identification of new therapeutic avenues [6]. At the conference, we will report about the status of mapping particularly the protein-mediated communication between the different cell types in the tumor microenvironment. Also, we will show results about the effect that PDAC tissue has on the wider peritumoral environment and vice versa. The peritumoral tissue is exhibiting a field defect that does not seem to be dependent on DNA-methylation. A comparison of PDAC and cystic pancreatic tumors revealed that differences in communicating with the peritumoral tissue could actually be more important to the known pathological difference between the two cancer entities than the molecular variations in the tumor cells. 1. Bauer et al. (2017), work submitted for publication 2. Botla et al. (2016) Cancer Res. 76, 4149-4159 3. Keller et al. (2014) BMC Med. 12, 224 4. Moskalev et al. (2015) Oncotarget 6, 4418-4427 5. Dror et al. (2016) Nature Cell Biol. 18, 1006-1017 6. Jandaghi et al. (2016) Gastroenterology, in press (doi: 10.1053/j.gastro.2016.08.040) www.dkfz.de/funct_genome/ Note: This abstract was not presented at the meeting. Citation Format: Mohamed S. Alhamdani, Andrea S. Bauer, Jörg D. Hoheisel. Mapping the molecular communication within the tumor microenvironment of pancreatic cancer as well as between tumor and peritumoral tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5925. doi:10.1158/1538-7445.AM2017-5925

After the establishment of DNA/RNA sequencing as a means of clinical diagnosis, the analysis of the proteome is next in line. As a matter of fact, proteome-based diagnostics is bound to be even more informative, since proteins are directly involved in the actual cellular processes that are responsible for disease. However, the structural variation and the biochemical differences between proteins, the much wider range in concentration and their spatial distribution as well as the fact that protein activity frequently relies on interaction increase the methodological complexity enormously, particularly if an accuracy and robustness is required that is sufficient for clinical utility. Here, we discuss the contribution that protein microarray formats could play towards proteome-based diagnostics.

The quest for a better understanding of organisms, and the human body in particular, at a comprehensive level has stimulated the development of techniques and processes that permit the analysis and assessment of biomedical information at high throughput. This has had substantial impact, not only by merely gathering knowledge but also by making scientists aware of the necessity to view organisms as complex biological systems rather than an assembly of individual biochemical pathways. Proteomics is still lagging behind genomics in this holistic analysis, however, because of the much higher degree of complexity that needs to be dealt with. Protein arrays, among other techniques, offer the prospect of advancing global protein analysis, similar to the impact of arrays in genomics. In basic research, protein arrays already contributed substantially, permitting the identification of many protein interactions and providing information on expression variations and structural changes, for example. Beside the fact of high throughput, arrays require relatively small reaction volumes, which is critical in view of the lack of means for in vitro protein amplification and beneficial for good assay sensitivity. Applications comprise many facets, such as the search for drug targets, analysis of host-parasite interactions and, above all, biomarker discovery. Despite the achievements and promises of the technology, it is still far from being a standard approach and many technical developments are ongoing. In this review, we look at the state of protein array technology and discuss future perspectives.

Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.

Antibody microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-microarrays, the implementation of a dual-colour assay adds to assay robustness and reproducibility and was therefore a focus of our technical implementation. In order to perform antibody microarray experiments for large sets of samples and analytes in a robust manner, it was essential to optimise the experimental layout, the protein extraction, labelling and incubation as well as data processing steps. Here, we present our current protocol, which is used for the simultaneous analysis of the abundance of more than 800 proteins in plasma, urine, and tissue samples.

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.

Background/Aim:Toxicity with advanced glycation end products (AGEs) is a major problem in uremic patients. Treatment with peritoneal dialysis (PD) exacerbates AGE formation as a result of bioincompatibility of the conventional peritoneal dialysis fluid (PDF). The presence of glucose degradation products (GDPs) in PDF is the main cause of its bioincompatibility. Carnosine is an endogenous dipeptide with a powerful antiglycation/antioxidant activity. In an attempt to improve PDF biocompatibility, we evaluated the effect of carnosine in human peritoneal mesothelial cells (HPMC) incubated with PDF or GDPs in vitro. Methods: HPMC were incubated for short or prolonged time with PDF in the presence or absence of carnosine. Similarly, HPMC were incubated in the same condition but with a combination of GDPs. Following the incubation, cells were tested for their viability, protein carbonyl content and reactive oxygen species (ROS) production. Results: Results demonstrated a significant protective effect of carnosine to HPMC in both acute and chronic conditions with PDF or GDPs as judged by the enhancement of cell viability, preserved protein from modification and decreased ROS production. Conclusion: Carnosine enhanced HPMC viability against the toxic effect of GDPs probably through protection of cellular protein from modification and from ROS-mediated oxidative damage. The salutary effect of carnosine may render it a desirable candidate for improving PDF biocompatibility and reducing AGE complications in PD patients.

Background Formation of advanced glycation end-products (AGEs) is a major problem in uremic patients treated with peritoneal dialysis (PD). Application of additives with known anti-glycosylation properties to PD fluid may be beneficial in minimizing the formation of AGEs. This study aimed to evaluate the effect of carnosine and its related peptides homocarnosine and anserine against the formation of AGEs in PD fluid. Methods PD solutions (1.5% dextrose) were incubated with human serum albumin (HSA) or collagen (type IV) with or without 10 mmol/L of each of carnosine, anserine, homo-carnosine, histidine, and aminoguanidine. The formation of AGEs was followed by fluorescence spectrophotometry at weekly intervals for 7 weeks. For the determination of the acute effect of carnosine and related compounds, HSA and collagen were incubated with 4.25% dextrose PD solutions for 24 hours, followed by incubation with 20 mmol/L of carnosine and related compounds for another 24 hours. The rate of AGE formation was monitored by fluorescence spectrophotometry. Results Carnosine and related compounds showed effective regression in AGE formation in both types of proteins in both long- and short-term exposure to PD fluids at a rate of effectiveness of the order of carnosine > homocarnosine > anserine, aminoguanidine > histidine in long-term exposure, and homocarnosine > carnosine > aminoguanidine > anserine > histidine in short-term exposure. Conclusion Carnosine and related peptides could suppress the formation of AGEs initiated by PD fluid. This observation may provide a new therapeutic approach for the prevention and treatment of the AGE-related complications in PD patients.

Patients with diabetes mellitus are likely to develop certain complication such as retinopathy, nephropathy and neuropathy as a result of oxidative stress and overwhelming free radicals. Treatment of diabetic patients with antioxidant may be of advantage in attenuating these complications. Oleuropein, the active constituent of olive leaf (Olea europaea), has been endowed with many beneficial and health promoting properties mostly linked to its antioxidant activity. This study aimed to evaluate the significance of supplementation of oleuropein in reducing oxidative stress and hyperglycemia in alloxan-induced diabetic rabbits. After induction of diabetes, a significant rise in plasma and erythrocyte malondialdehyde (MDA) and blood glucose as well as alteration in enzymatic and non-enzymatic antioxidants was observed in all diabetic animals. During 16 weeks of treatment of diabetic rabbits with 20 mg/kg body weight of oleuropein the levels of MDA along with blood glucose and most of the enzymatic and non-enzymatic antioxidants were significantly restored to establish values that were not different from normal control rabbits. Untreated diabetic rabbits on the other hand demonstrated persistent alterations in the oxidative stress marker MDA, blood glucose and the antioxidant parameters. These results demonstrate that oleuropein may be of advantage in inhibiting hyperglycemia and oxidative stress induced by diabetes and suggest that administration of oleuropein may be helpful in the prevention of diabetic complications associated with oxidative stress.

Background. Glutathione (GSH), the predominant intracellular antioxidant, reportedly has been shown to be decreased in chronic renal failure patients, which renders these patients more susceptible to oxidative damage by free radicals. To our knowledge, the ability of erythrocytes to normalize the GSH level by de novo synthesis in uraemic and dialysis patients has not been studied previously. The main goal of the present study was to measure the activities of the enzymes that are responsible for de novo GSH generation, namely g-glutamylcysteine synthetase (g-GCS) and glutathione synthetase (GSH-S), in erythrocytes from uraemic and dialysis patients. Methods. Erythrocyte total GSH level and g-GCS and GSH-S activities as well as plasma malondialdehyde (MDA) levels were measured in 19 non-dialysis patients (ND), 34 haemodialysis patients (HD), 22 continuous ambulatory peritoneal dialysis patients (CAPD) and 21 normal healthy controls. The effect of a single haemodialysis session was determined in 16 HD patients. Results. Significant decreases in GSH levels and g-GCS activity but not GSH-S were observed in ND, HD and CAPD patients compared with controls. However, GSH levels as well as g-GCS and GSH-S activities were not different among the ND, HD and CAPD patients. The decrease in GSH was strongly and positively correlated with the decrease in g-GCS in ND, HD and CAPD patients (r ¼ 0.717, P

Following the advances in human genome sequencing, attention has shifted in part toward the elucidation of the encoded biological functions. Since proteins are the driving forces behind very many biological activities, large-scale examinations of their expression variations, their functional roles and regulation have moved to the central stage. A significant fraction of the human proteome consists of secreted proteins. Exploring this set of molecules offers unique opportunities for understanding molecular interactions between cells and fosters biomarker discovery that could advance the detection and monitoring of diseases. Antibody microarrays are among the relatively new proteomic methodologies that may advance the field significantly because of their relative simplicity, robust performance and high sensitivity down to single-molecule detection. In addition, several aspects such as variations in amount, structure and activity can be assayed at a time. Antibody microarrays are therefore likely to improve the analytical capabilities in proteomics and consequently permit the production of even more informative and reliable data. This review looks at recent applications of this novel platform technology in secretome analysis and reflects on the future.

The incidence of cancer and its associated mortality are increasing globally, indicating an urgent need to develop even more effective and sensitive sets of biomarkers that could help in early diagnosis and consequent intervention. Given that many cellular processes are carried out by proteins, cancer research has recently shifted toward an exploration of the full proteome for such discovery. Among the advanced methodologies that are being developed for analyzing the proteome, antibody microarrays have become a prominent tool for gathering the information required for a better understanding of disease biology, early detection, discrimination of tumors and monitoring of disease progression. Here, we review the technical aspects and challenges in the development and use of antibody microarray assays and examine recently reported applications in oncoproteomics.