Silicon-Containing Neurotensin Analogues as Radiopharmaceuticals for NTS1-Positive Tumors Imaging.

Several independent studies have demonstrated the overexpression of NTS1 in various malignancies, which make this receptor of interest for imaging and therapy. To date, radiolabeled neurotensin analogues suffer from low plasmatic stability and thus insufficient availability for high uptake in tumors. We report the development of 68Ga-radiolabeled neurotensin analogues with improved radiopharmaceutical properties through the introduction of the silicon-containing amino acid trimethylsilylalanine (TMSAla). Among the series of novel radiolabeled neurotensin analogues, [68Ga]Ga-JMV6659 exhibits high hydrophilicity (log D7.4 = -3.41 ± 0.14), affinity in the low nanomolar range toward NTS1 (Kd = 6.29 ± 1.37 nM), good selectivity (Kd NTS1/Kd NTS2 = 35.9), and high NTS1-mediated internalization. It has lower efflux and prolonged plasmatic half-life in human plasma as compared to the reference compound ([68Ga]Ga-JMV6661 bearing the minimum active fragment of neurotensin and the same linker and chelate as other analogues). In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV6659 uptake reached 7.8 ± 0.54 %ID/g 2 h post injection. Uptake was decreased to 1.38 ± 0.71 %ID/g with injection of excess of non-radioactive neurotensin. Radiation dose as extrapolated to human was estimated as 2.35 ± 0.6 mSv for a standard injected activity of 100MBq. [68Ga]Ga-JMV6659 was identified as a promising lead compound suitable for PET imaging of NTS1-expressing tumors.

Simple rules govern the diversity of bacterial nicotianamine-like metallophores.

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis, respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or α-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored the substrate specificity of CntM by combining bioinformatic and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity is mainly due to the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity toward α-ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity toward pyruvate, whereas an alanine leads to the consumption of both pyruvate and α-ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, the diversity of opine-type metallophore is governed by the absence/presence of a cntK gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Body mass index, fat free mass, uric acid, and renal function as blood pressure levels determinants in young adults.

AIM: We assessed some major determinants of blood pressure (BP) in young adulthood to plan a lifestyle changes policy METHODS: A cross sectional survey was held, involving 2373 high school people (age 18-21), measuring BP, body mass index (BMI), waist circumference (WCirc), fat free mass (FFM); alcohol and smoking habits were evaluated by a questionnaire. In a subset of this population (n = 60) uric acid (UA), estimated glomerular filtration rate (eGFR) were also evaluated. RESULTS: Smoking and not alcohol was correlated to systolic blood pressure (SBP) through quartiles (31.7%, 39.1%, 46.5%, 45.5%). Systolic BP was significantly correlated with FFM in the whole population (r = 0.51) as well as in SBP quartiles (r = 0.243, 0.138, 0.118, 0.204). FFM-SBP cluster analysis gave two centroids corresponding to sexes; females n = 998; coordinates (116.4 mmHg, 38.9 kg) and males n = 1068; coordinates (131.3 mmHg, 56.7 kg). In the n = 60 substudy a multiple linear regression model (multiple R = 0.741) with SBP as dependent variable and UA, FFM, BMI, eGFR as explicative ones, only UA (ß coefficent = 0.363, partial r = 0.240, P < 0.01) was the determinant of BP particularly in men. Moreover in the same group we found an inverse relationship between eGFR (albeit always in the normal range) and UA, as well as for women (r = -0.54, P < 0.01) and men (r = -0.43, P < 0.01) analyzed separately. CONCLUSIONS: A significant correlation exists between BP and FFM; UA has proven to be the most important SBP determinant. At variance with paediatric age UA was negatively correlated with renal function. Dietary intervention on UA and alcohol habits in young adults seems advisable to prevent hypertension.

Synthetic and natural small molecule TLR4 antagonists inhibit motoneuron death in cultures from ALS mouse model.

Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the involvement of both the innate and adaptive immune responses in ALS pathogenesis. In particular, abnormal TLR4 signaling in pro-inflammatory microglia cells has been related to motoneuron degeneration leading to ALS. In this study the effect of small molecule TLR4 antagonists on in vitro ALS models has been investigated. Two different types of synthetic glycolipids and the phenol fraction extracted from commercial extra-virgin olive oil (EVOO) were selected since they efficiently inhibit TLR4 stimulus in HEK cells by interacting with the TLR4·MD-2 complex and CD14 co-receptor. Here, TLR4 antagonists efficiently protected motoneurons from LPS-induced lethality in spinal cord cultures, and inhibited the interleukine-1ß production by LPS-stimulated microglia. In motoneurons/glia cocultures obtained from wild type or SOD1 G93A mice, motoneuron death induced by SOD1mut glia was counteracted by TLR4 antagonists. The release of nitric oxide by LPS treatment or SOD1mut glia was also inhibited by EVOO, suggesting that the action of this natural extract could be mainly related to the modulation of this inflammatory mediator.

Wastewater-based epidemiological evaluation of the effect of air pollution on short-acting beta-agonist consumption for acute asthma treatment.

Asthma, one of the most common chronic diseases in the world and a leading cause of hospitalization among children, has been associated with outdoor air pollution. We applied the wastewater-based epidemiology (WBE) approach to study the association between the use of salbutamol, a short-acting beta-agonist used to treat acute bronchospasm, and air pollution in the population of Milan, Italy. Composite 24-h samples of untreated wastewater were collected daily and analyzed for human metabolic residues of salbutamol by liquid chromatography tandem mass spectrometry. Corresponding daily outdoor concentrations of particular matter up to 10µm (PM10) and 2.5µm (PM2.5) in aerodynamic diameter, nitrogen dioxide, ozone, sulfur dioxide, and benzene were collected from the public air monitoring network. Associations at different lag times (0-10 days) were assessed by a log-linear Poisson regression model. We found significant direct associations between defined daily doses (DDD) of salbutamol and mean daily concentrations of PM10 and PM2.5 up to nine days of lag time. The highest rate ratio, and 95% confidence interval (CI), of DDD of salbutamol was 1.06 (95% CI: 1.02-1.10) and 1.07 (95% CI: 1.02-1.12) at seven days of lag time and for an increase of 10 µg/m(3) of PM10 and PM2.5, respectively. Reducing the mean daily PM10 concentration in Milan from 50 to 30µg/m(3) means that 852 (95% CI: 483-1504) daily doses of salbutamol per day would not be used. These results confirm the association between asthma and outdoor PM10 and PM2.5 and prove the potential of the WBE approach to quantitatively estimate the relation between environmental exposures and diseases.

Automated online solid-phase extraction-liquid chromatography mass spectrometric analysis of dithianon in water.

An automated online solid-phase extraction-liquid chromatography mass spectrometry (SPE-LC/MS) method was developed for the quantification of dithianon in surface water samples, using warfarin as internal standard. The method was developed on a liquid chromatography (LC) system with Flexible Cube interfaced to a quadrupole time-of-flight (Q-TOF) mass spectrometer. A small volume of acidified water (1 mL) was spiked with internal standard, pre-concentrated online on polymeric cartridges and analyzed by full-scan MS in high-resolution conditions. The quantitative data were obtained by [M]-• of dithianon and [M - H]- of warfarin, used as internal standard. The chromatographic separation was performed on a C18 column with a gradient mobile phase consisting of acetonitrile and water containing 0.05% acetic acid. The method was validated to measure concentrations of dithianon in the range of 0.010-4 µg L-1 in surface water samples. Twenty real water samples, collected from Torrente Novella, Val di Non (TN, Northern Italy), during fungicide treatments of large apple orchards, were analyzed. All samples were kept in glass bottles and stored in the lab at -20°C until analysis. It was found that in all samples dithianon was undetectable: if it is present, its concentration was lower than the limit of detection (LOD) (0.008 µg L-1.To investigate the stability of dithianon, a series of water samples were spiked at different concentrations and analyzed after different storage conditions. Results suggested that dithianon is not stable in water stored at -20°C at neutral or basic pH, but the addition of acetic acid to pH = 3.5 increases its stability to at least two weeks.

Plasma clusterin as a candidate pre-diagnosis marker of colorectal cancer risk in the Florence cohort of the European Prospective Investigation into Cancer and Nutrition: a pilot study.

BACKGROUND: Colorectal cancer is one of the major causes of cancer mortality world-wide. Prevention would improve if at-risk subjects could be identified. The aim of this study was to characterise plasma protein biomarkers associated with the risk of colorectal cancer in samples collected prospectively, before the disease diagnosis. METHODS: After an exploratory study on the comprehensive plasma proteome analysis by liquid chromatography-tandem mass spectrometry from ten colorectal cancer cases enrolled at diagnosis, and ten matched controls (Phase 1), a similar preliminary study was performed on prospective plasma samples from ten colorectal cancer cases, enrolled years before disease development, and ten matched controls identified in a nested case-control study within the Florence cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study (Phase 2); in Phase 3 the validation of the candidate biomarkers by targeted proteomics on 48 colorectal cancer cases and 48 matched controls from the Florence-EPIC cohort, and the evaluation of the disease risk were performed. RESULTS: Systems biology tools indicated that both in the Phase 1 and Phase 2 studies circulating protein levels differing in cases more than 1.5 times from controls, were involved in inflammation and/or immune response. Eight proteins including apolipoprotein C-II, complement C4-B, complement component C9, clusterin, alpha-2-HS-glycoprotein, mannan-binding lectin serine-protease, mannose-binding protein C, and N-acetylmuramoyl-L-alanine amidase were selected as promising candidate biomarkers. Targeted proteomics of the selected proteins in the EPIC samples showed significantly higher clusterin levels in cases than controls, but only in men (mean ± SD, 1.98 ± 0.46 and 1.61 ± 0.43 nmol/mL respectively, Mann-Whitney U, two-tailed P = 0.0173). The remaining proteins were unchanged. Using multivariate logistic models a significant positive association emerged for clusterin, with an 80% increase in the colorectal cancer risk with protein's unit increase, but only in men. CONCLUSIONS: The results show that plasma proteins can be altered years before colorectal cancer detection. The high circulating clusterin in pre-diagnostic samples suggests this biomarker can improve the identification of people at risk of colorectal cancer and might help in designing preventive interventions.

Stereoselective synthesis of unsaturated α-amino acids.

Stereoselective synthesis of unsaturated α-amino acids was performed by asymmetric alkylation. Two methods were investigated and their enantiomeric excess measured and compared. The first route consisted of an enantioselective approach induced by the Corey-Lygo catalyst under chiral phase transfer conditions while the second one involved the hydroxypinanone chiral auxiliary, both implicating Schiff bases as substrate. In all cases, the use of a prochiral Schiff base gave higher enantiomeric excess and yield in the final desired amino acid.

Computational design of novel peptidomimetic inhibitors of cadherin homophilic interactions.

We report a first set of peptidomimetic ligands mimicking the adhesive interface identified by recent crystallographic structures of E- and N-cadherin. Compounds 2 and 3 inhibit adhesion of epithelial ovarian cancer (EOC) cells with improved efficacy compared to the ADH-1 peptide, a N-cadherin antagonist that is in early clinical trials in EOC patients.

Determination of the binding epitope of RGD-peptidomimetics to αvß3 and α(IIb)ß3 integrin-rich intact cells by NMR and computational studies.

NMR experiments (transferred NOE and Saturation Transfer Difference) were used to shed light on the binding epitope of RGD peptidomimetics 1-3 with integrins αvß3 and α(IIb)ß3, expressed on the membrane of ECV304 bladder cancer cells and human platelets, respectively. The NMR results were supported by docking calculations of 1-3 in the active sites of αvß3 and α(IIb)ß3 integrin receptors and were compared to the results of competitive αvß3 receptor binding assays and competitive ECV304 cell adhesion experiments. While cis RGD ligand 1 interacts mainly with the α integrin subunit through its basic guanidine group, trans RGD ligands 2 and 3 are able to interact with both the α and ß integrin subunits via an electrostatic clamp.

Post-dilution on line haemodiafiltration with citrate dialysate: first clinical experience in chronic dialysis patients.

BACKGROUND: Citrate has anticoagulative properties and favorable effects on inflammation, but it has the potential hazards of inducing hypocalcemia. Bicarbonate dialysate (BHD) replacing citrate for acetate is now used in chronic haemodialysis but has never been tested in postdilution online haemodiafiltration (OL-HDF). METHODS: Thirteen chronic stable dialysis patients were enrolled in a pilot, short-term study. Patients underwent one week (3 dialysis sessions) of BHD with 0.8 mmol/L citrate dialysate, followed by one week of postdilution high volume OL-HDF with standard bicarbonate dialysate, and one week of high volume OL-HDF with 0.8 mmol/L citrate dialysate. RESULTS: In citrate OL-HDF pretreatment plasma levels of C-reactive protein and ß 2-microglobulin were significantly reduced; intra-treatment plasma acetate levels increased in the former technique and decreased in the latter. During both citrate techniques (OL-HDF and HD) ionized calcium levels remained stable within the normal range. CONCLUSIONS: Should our promising results be confirmed in a long-term study on a wider population, then OL-HDF with citrate dialysate may represent a further step in improving dialysis biocompatibility.

Palm oil and palmitic acid: a review on cardiovascular effects and carcinogenicity.

We reviewed the scientific literature on the evidence of the relationship between palm oil and adverse effects on human health. Few studies have investigated the effects of palm oil per se, and the main reason why it has been associated with negative health effects is the relatively high content of saturated fatty acids (SFAs), particularly palmitic acid, which in turn have been associated with increased risk of coronary heart disease and some tumours. However, more recent investigations on the topic seem to have reconsidered the negative role of the dietary SFAs as a risk factor for cardiovascular diseases and show that not only the type of fat, but also that the triglyceride structure plays a role in cholesterolaemia. As regards to a role in cancer, specific studies on dietary palmitic acid or palm oil and the risk of cancer development are scanty, and the evidence is not convincing.

Proteomic analysis of mouse brain cortex identifies metabolic down-regulation as a general feature of ischemic pre-conditioning.

The molecular mechanisms that lead to ischemic pre-conditioning are not completely understood, and proteins are important players. We compared the mouse brain cortex proteome from different ischemia sets: transient (7 min) middle cerebral artery occlusion (7'MCAo, pre-conditioning stimulus), permanent MCAo (pMCAo, severe ischemia), and pMCAo 4 days after 7'MCAo (7'MCAo/pMCAo, pre-conditioned model). Proteins were analyzed by two-dimensional electrophoresis coupled to liquid chromatography-tandem mass spectrometry. Overall, 28 proteins were expressed differentially from sham controls, and identified. The ischemic pre-conditioning stimulus alone up-regulated the stress protein heat-shock protein 70 (HSP70), possibly activated by the androgen receptor. Western blotting confirmed the increased expression of HSP70 and showed that androgen receptor expression paralleled that of HSP70. In the ischemic-tolerant group (7'MCAo/pMCAo), a number of proteins over-expressed after pMCAo returned to sham levels, seven proteins remained up-regulated as in pMCAo, and five proteins mainly involved in energy metabolism and mitochondrial electron transport and unchanged in pMCAo were down-regulated only in ischemic tolerance, suggesting a role in brain pre-conditioning. Astrocytes participated in ischemic-tolerance induction, as shown by the down-regulation of glutamine synthetase in the 7'MCAo/pMCAo group. The results suggest that metabolic down-regulation was a general feature of ischemic pre-conditioning, playing a pivotal role in neuroprotection.

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.

Human health risk in relation to air quality in two municipalities in an industrialized area of Northern Italy.

Air quality is one of the major environmental issues related to human health, and people and authorities are increasingly aware and concerned about it, asking to be involved in decisions whose fallout can have consequences on their health. The objectives of the present study were to provide quantitative data on the impact of air pollution on the health of people living in two small municipalities in a highly industrialized, densely populated area of Northern Italy. We applied the approach proposed by the World Health Organization (WHO) using the AirQ 2.2.3 software developed by the WHO European Centre for Environment and Health, Bilthoven Division. Daily concentrations of ozone, nitrogen dioxide, and particulate matter of aerodynamic diameter≤10 µm (PM10) and ≤2.5 µm (PM2.5) were used to assess human exposure and health effects in terms of attributable proportion of the health outcome, annual number of excess cases of mortality for all causes, and cardiovascular and respiratory diseases. Long-term effects were estimated for PM2.5 as years of life lost. Considering short-term effects, PM2.5 had the highest health impact on the 24,000 inhabitants of the two small towns, causing an excess of total mortality of 8 out of 177 in a year. Ozone and nitrogen dioxide each caused about three excess cases of total mortality. Results on long-term effects showed, respectively, 433, 180, and 72 years of life lost for mortality for all causes, cardiopulmonary diseases and lung cancer, in a year. These results are consistent with other reports of the impact of air quality on human health and the AirQ software seems an effective and easy tool, helpful in decision-making.

An integrated approach, based on mass spectrometry, for the assessment of imidacloprid metabolism and penetration into mouse brain and fetus after oral treatment.

Imidacloprid is an insecticide belonging to neonicotinoids, a class of agonists of the nicotinic acetylcholine receptors that shows higher affinities in insects compared to mammals. However, recent evidence show that neonicotinoids can bind to the mammalian receptors, leading to detrimental responses in cultured neurons. We developed an analytical strategy which uses mass spectrometry with multiple reaction monitoring (targeted approach) and high-resolution acquisitions (untargeted approach), which were applied to quantify imidacloprid and to identify its metabolites in biological tissues after oral treatments of mice. Mouse dams were treated with doses from 0.118 mg/kg bw day up to 41 mg/kg day between gestational days 6-9. Results showed quantifiable levels of imidacloprid in plasma (from 30.48 to 5705 ng/mL) and brain (from 20.48 to 5852 ng/g) of treated mice, proving the passage through the mammalian blood-brain barrier with a high correspondence between doses and measured concentrations. Untargeted analyses allowed the identification of eight metabolites including imidacloprid-olefin, hydroxy-imidacloprid dihydroxy-imidacloprid, imidacloprid-nitrosimine, desnitro-imidacloprid, 6-chloronicotinic acid, 5-(methylsulfanyl)pyridine-2-carboxylic acid and N-imidazolidin-2-ylidenenitramide in plasma and brain. Moreover, analysis of embryonic tissues after oral treatment of mouse dams showed detectable levels of imidacloprid (816.6 ng/g after a dose of 4.1 mg/Kg bw day and 5646 ng/g after a dose of 41 mg/Kg bw day) and its metabolites, proving the permeability of the placenta barrier. Although many studies have been reported on the neurotoxicity of neonicotinoids, our study paves the way for a risk assessment in neurodevelopmental toxicity, demostrating the capability of imidacloprid and its metabolites to pass the biological barriers.

Metabolically stable neurotensin analogs exert potent and long-acting analgesia without hypothermia.

The endogenous tridecapeptide neurotensin (NT) has emerged as an important inhibitory modulator of pain transmission, exerting its analgesic action through the activation of the G protein-coupled receptors, NTS1 and NTS2. Whereas both NT receptors mediate the analgesic effects of NT, NTS1 activation also produces hypotension and hypothermia, which may represent obstacles for the development of new pain medications. In the present study, we implemented various chemical strategies to improve the metabolic stability of the biologically active fragment NT(8-13) and assessed their NTS1/NTS2 relative binding affinities. We then determined their ability to reduce the nociceptive behaviors in acute, tonic, and chronic pain models and to modulate blood pressure and body temperature. To this end, we synthesized a series of NT(8-13) analogs carrying a reduced amide bond at Lys8-Lys9 and harboring site-selective modifications with unnatural amino acids, such as silaproline (Sip) and trimethylsilylalanine (TMSAla). Incorporation of Sip and TMSAla respectively in positions 10 and 13 of NT(8-13) combined with the Lys8-Lys9 reduced amine bond (JMV5296) greatly prolonged the plasma half-life time over 20 h. These modifications also led to a 25-fold peptide selectivity toward NTS2. More importantly, central delivery of JMV5296 was able to induce a strong antinociceptive effect in acute (tail-flick), tonic (formalin), and chronic inflammatory (CFA) pain models without inducing hypothermia. Altogether, these results demonstrate that the chemically-modified NT(8-13) analog JMV5296 exhibits a better therapeutic profile and may thus represent a promising avenue to guide the development of new stable NT agonists and improve pain management.

Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks.

The cancer secretome is a rich repository in which to mine useful information for both cancer biology and clinical oncology. To help understand the mechanisms underlying the progression of pancreatic cancer, we characterized the secretomes of four human pancreatic ductal adenocarcinoma (PDAC) cell lines versus a normal counterpart. To this end, we used a proteomic workflow based on high-confidence protein identification by mass spectrometry, semiquantitation by a label-free approach, and network enrichment analysis by a system biology tool. Functional networks significantly enriched with PDAC-dysregulated proteins included not only expected alterations within key mechanisms known to be relevant for tumor progression (e.g., cell-cell/cell-matrix adhesion, extracellular matrix remodeling, and cytoskeleton rearrangement), but also other extensive, coordinated perturbations never observed in pancreatic cancer. In particular, we highlighted perturbations possibly favoring tumor progression through immune escape (i.e., inhibition of the complement system, deficiency of selected proteasome components within the antigen-presentation machinery, and inhibition of T cell cytoxicity), and a defective protein folding machinery. Among the proteins found concordantly oversecreted in all of our PDAC cell lines, many are reportedly overexpressed in pancreatic cancer (e.g., CD9 and Vimentin), while others (PLOD3, SH3L3, PCBP1, and SFRS1) represent novel PDAC-secreted proteins that may be worth investigating.

Proteasomal processing of albumin by renal dendritic cells generates antigenic peptides.

The role of dendritic cells (DC) that accumulate in the renal parenchyma of non-immune-mediated proteinuric nephropathies is not well understood. Under certain circumstances, DC capture immunologically ignored antigens, including self-antigens, and present them within MHC class I, initiating an autoimmune response. We studied whether DC could generate antigenic peptides from the self-protein albumin. Exposure of rat proximal tubular cells to autologous albumin resulted in its proteolytic cleavage to form an N-terminal 24-amino acid peptide (ALB1-24). This peptide was further processed by the DC proteasome into antigenic peptides that had binding motifs for MHC class I and were capable of activating syngeneic CD8+ T cells. In vivo, the rat five-sixths nephrectomy model allowed the localization and activation of renal DC. Accumulation of DC in the renal parenchyma peaked 1 wk after surgery and decreased at 4 wk, concomitant with their appearance in the renal draining lymph nodes. DC from renal lymph nodes, loaded with ALB1-24, activated syngeneic CD8+ T cells in primary culture. The response of CD8+ T cells of five-sixths nephrectomized rats was amplified with secondary stimulation. In contrast, DC from renal lymph nodes of five-sixths nephrectomized rats treated with the proteasomal inhibitor bortezomib lost their capacity to stimulate CD8+ T cells in primary and secondary cultures. These data suggest that albumin can be a source of potentially antigenic peptides upon renal injury and that renal DC play a role in processing self-proteins through a proteasome-dependent pathway.