Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.

Self-assembling supramolecular dendrimer nanosystem for PET imaging of tumors.

Bioimaging plays an important role in cancer diagnosis and treatment. However, imaging sensitivity and specificity still constitute key challenges. Nanotechnology-based imaging is particularly promising for overcoming these limitations because nanosized imaging agents can specifically home in on tumors via the "enhanced permeation and retention" (EPR) effect, thus resulting in enhanced imaging sensitivity and specificity. Here, we report an original nanosystem for positron emission tomography (PET) imaging based on an amphiphilic dendrimer, which bears multiple PET reporting units at the terminals. This dendrimer is able to self-assemble into small and uniform nanomicelles, which accumulate in tumors for effective PET imaging. Benefiting from the combined dendrimeric multivalence and EPR-mediated passive tumor targeting, this nanosystem demonstrates superior imaging sensitivity and specificity, with up to 14-fold increased PET signal ratios compared with the clinical gold reference 2-fluorodeoxyglucose ([18F]FDG). Most importantly, this dendrimer system can detect imaging-refractory low-glucose-uptake tumors that are otherwise undetectable using [18F]FDG. In addition, it is endowed with an excellent safety profile and favorable pharmacokinetics for PET imaging. Consequently, this dendrimer nanosystem constitutes an effective and promising approach for cancer imaging. Our study also demonstrates that nanotechnology based on self-assembling dendrimers provides a fresh perspective for biomedical imaging and cancer diagnosis.

Surface Charge of Supramolecular Nanosystems for In Vivo Biodistribution: A MicroSPECT/CT Imaging Study.

Bioimaging has revolutionized medicine by providing accurate information for disease diagnosis and treatment. Nanotechnology-based bioimaging is expected to further improve imaging sensitivity and specificity. In this context, supramolecular nanosystems based on self-assembly of amphiphilic dendrimers for single photon emission computed tomography (SPECT) bioimaging are developed. These dendrimers bear multiple In3+ radionuclides at their terminals as SPECT reporters. By replacing the macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid cage with the smaller 1,4,7-triazacyclononane-1,4,7-triacetic acid scaffold as the In3+ chelator, the corresponding dendrimer exhibits neutral In3+ -complex terminals in place of negatively charged In3+ -complex terminals. This negative-to-neutral surface charge alteration completely reverses the zeta-potential of the nanosystems from negative to positive. As a consequence, the resulting SPECT nanoprobe generates a highly sought-after biodistribution profile accompanied by a drastically reduced uptake in liver, leading to significantly improved tumor imaging. This finding contrasts with current literature reporting that positively charged nanoparticles have preferential accumulation in the liver. As such, this study provides new perspectives for improving the biodistribution of positively charged nanosystems for biomedical applications.

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants.

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction.

Dysfunction of the exocrine pancreas is observed in diabetes, but links between concurrent exocrine and endocrine pancreatic disease and contributing genetic factors are poorly characterized. We studied two families with diabetes and exocrine pancreatic dysfunction by genetic, physiological and in vitro functional studies. A genome-wide screen in Family 1 linked diabetes to chromosome 9q34 (maximal lod score 5.07). Using fecal elastase deficiency as a marker of exocrine pancreatic dysfunction refined the critical chromosomal region to 1.16 Mb (maximal lod score 11.6). Here, we identified a single-base deletion in the variable number of tandem repeats (VNTR)-containing exon 11 of the carboxyl ester lipase (CEL) gene, a major component of pancreatic juice and responsible for the duodenal hydrolysis of cholesterol esters. Screening subjects with maturity-onset diabetes of the young identified Family 2, with another single-base deletion in CEL and a similar phenotype with beta-cell failure and pancreatic exocrine disease. The in vitro catalytic activities of wild-type and mutant CEL protein were comparable. The mutant enzyme was, however, less stable and secreted at a lower rate. Furthermore, we found some evidence for an association between common insertions in the CEL VNTR and exocrine dysfunction in a group of 182 unrelated subjects with diabetes (odds ratio 4.2 (1.6, 11.5)). Our findings link diabetes to the disrupted function of a lipase in the pancreatic acinar cells.

Targeting a novel onco-glycoprotein antigen at tumoral pancreatic cell surface by mAb16D10 induces cell death.

The mAb16D10 was raised against a pathological onco-glycoform of bile salt-dependent lipase isolated from the pancreatic juice of a patient suffering from a pancreatic adenocarcinoma. We previously showed that mAb16D10 specifically discriminates human pancreatic tumor tissues from other cancer and nontumor tissues. In this study, we report that mAb16D10 inhibited the proliferation of only human pancreatic tumor cells expressing 16D10 plasma membrane Ag. Interaction of mAb16D10 with its cognate surface Ag on pancreatic cells promoted cell death by activation of the p53- and caspase-dependent apoptotic pathway, and silencing of p53 decreased cell death. The decreased proliferation was also partly due to cell cycle arrest in G1/S phase, mAb16D10 triggering of glycogen synthase kinase-3ß (GSK-3ß) activation, degradation of ß-catenin, and decreased expression of cyclin D1. GSK-3ß positively affected p53 expression in pancreatic tumor cells after mAb16D10 binding. Inhibition of GSK-3ß activity reversed the effects induced by mAb16D10 in SOJ-6 cells, supporting the pivotal role of GSK-3ß signaling in the mechanisms of action induced by mAb16D10. Also, mAb16D10 cell treatment led to membrane overexpression of E-cadherin. Both E-cadherin and tumor Ag were localized in membrane lipid cholesterol-rich microdomains and are thought to belong to signaling platforms involved in the induction of cell cycle arrest and cell death. Overall, this study reveals that mAb16D10 holds great potential to prevent pancreatic tumor proliferation by apoptotic cell death, thus promising therapeutic prospects for treatment of pancreatic adenocarcinoma, a highly lethal disease.

A novel tumor-associated pancreatic glycoprotein is internalized by human dendritic cells and induces their maturation.

Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.

A glycosyltransferase gene signature to detect pancreatic ductal adenocarcinoma patients with poor prognosis.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an important heterogeneity, reflected by different clinical outcomes and chemoresistance. During carcinogenesis, tumor cells display aberrant glycosylated structures, synthetized by deregulated glycosyltransferases, supporting the tumor progression. In this study, we aimed to determine whether PDAC could be stratified through their glycosyltransferase expression profiles better than the current binary classification (basal-like and classical) in order to improve detection of patients with poor prognosis. METHODS: Bioinformatic analysis of 169 glycosyltransferase RNA sequencing data were performed for 74 patient-derived xenografts (PDX) of resected and unresectable tumors. The Australian cohort of International Cancer Genome Consortium and the microarray dataset from Puleo patient's cohort were used as independent validation datasets. FINDINGS: New PDAC stratification based on glycosyltransferase expression profile allowed to distinguish different groups of patients with distinct clinical outcome (p-value = 0.007). A combination of 19 glycosyltransferases differentially expressed in PDX defined a glyco-signature, whose prognostic value was validated on datasets including resected whole tumor tissues. The glyco-signature was able to discriminate three clusters of PDAC patients on the validation cohorts, two clusters displaying a short overall survival compared to one cluster having a better prognosis. Both poor prognostic clusters having different glyco-profiles in Puleo patient's cohort were correlated with stroma activated or desmoplastic subtypes corresponding to distinct microenvironment features (p-value < 0.0001). Besides, differential expression and enrichment analyses revealed deregulated functional pathways specific to different clusters. INTERPRETATION: This study identifies a glyco-signature relevant for a prognostic use, potentially applicable to resected and unresectable PDAC. Furthermore, it provides new potential therapeutic targets. FUNDING: This work was supported by INCa (Grants number 2018-078 and 2018-079), Fondation ARC (Grant number ARCPJA32020070002326), Cancéropôle PACA, DGOS (labelization SIRIC, Grant number 6038), Amidex Foundation and Ligue Nationale Contre le Cancer and by institutional fundings from INSERM and the Aix-Marseille Université.

In vitro and in vivo anticancer activity of a novel nano-sized formulation based on self-assembling polymers against pancreatic cancer.

PURPOSE: To evaluate the in vitro and in vivo pancreatic anticancer activity of a nano-sized formulation based on novel polyallylamine grafted with 5% mole cholesteryl pendant groups (CH(5)-PAA). METHODS: Insoluble novel anticancer drug, Bisnaphthalimidopropyldiaaminooctane (BNIPDaoct), was loaded into CH(5)-PAA polymeric self-assemblies by probe sonication. Hydrodynamic diameters and polydispersity index measurements were determined by photon correlation spectroscopy. The in vitro cytotoxicity evaluation of the formulation was carried out by the sulforhodamine B dye assay with human pancreatic adenocarcinoma BxPC-3 cells, while for the in vivo study, Xenograff mice were used. In vitro apoptotic cell death from the drug formulation was confirmed by flow cytometric analysis. RESULTS: The aqueous polymer-drug formulation had a mean hydrodynamic size of 183 nm. The drug aqueous solubility was increased from negligible concentration to 0.3 mg mL(-1). CH(5)-PAA polymer alone did not exhibit cytotoxicity, but the new polymer-drug formulation showed potent in vitro and in vivo anticancer activity. The mode of cell death in the in vitro study was confirmed to be apoptotic. The in vivo results revealed that the CH(5)-PAA alone did not have any anti-proliferative effect, but the CH(5)-PAA-drug formulation exhibited similar tumour reduction efficacy as the commercial drug, gemcitabine. CONCLUSIONS: The proposed formulation shows potential as pancreatic cancer therapeutics.

Monoclonal antibody 16D10 to the COOH-terminal domain of the feto-acinar pancreatic protein targets pancreatic neoplastic tissues.

We have shown that the 16D10 antigen located on the mucin-like COOH-terminal domain of the feto-acinar pancreatic protein (FAPP) is expressed at the surface of human pancreatic tumor cell lines such as SOJ-6 cell line. Furthermore, an in vivo study indicates that targeting this cell-membrane glycopeptide by the use of the monoclonal antibody (mAb) 16D10 inhibits the growth of SOJ-6 xenografts in nude mice. To validate the potential use of the mAb16D10 in immune therapy, this study examined the expression of 16D10 antigens at the surface of human pancreatic adenocarcinomas versus control tissues. We examined the reactivity of mAb16D10 and mAb8H8 with pancreatic ductal adenocarcinomas (PDAC) compared with controls by using immunohistochemistry and confocal laser scanning microscopy. mAb8H8 does react with control or nontumoral human pancreatic tissues. mAb16D10 has a strong and specific reactivity with PDAC and does not react with other cancers of epithelia or normal tissues tested. Notable, mAb16D10 mostly recognizes membrane of tumoral cells. Furthermore, mAb8H8 and mAb16D10 recognized a protein of 110 to 120 kDa in homogenates of nontumoral and tumoral human pancreatic tissues, respectively. This size correlates with that of FAPP or with that of the normal counterpart of FAPP, the so-called bile salt-dependent lipase. The results suggest that mAb16D10 presents a unique specificity against PDAC; consequently, it could be effective in immune therapy of this cancer. Furthermore, mAb16D10 and mAb8H8 pair might be useful for diagnosis purpose in discriminating tumoral from nontumoral human pancreatic tissues.

A squalamine derivative, NV669, as a novel PTP1B inhibitor: in vitro and in vivo effects on pancreatic and hepatic tumor growth.

NV669 is an aminosterol derived from squalamine found to possess strong anticancer effects. The aim of this study was to investigate NV669's beneficial effects on human pancreatic and hepatic cancer models and to decipher the cellular and molecular mechanisms involved in tumor growth decrease upon treatment with NV669. Pancreatic (BxPC3, MiaPaCa-2) and hepatic (HepG2, Huh7) cancer cells were treated with NV669, and the effects recorded on proliferation, cell cycle and death. Results showed that NV669 inhibited the viability of cancer cells, induced cell cycle arrest and subsequently promoted apoptosis. This was accompanied by a decrease in the expression of cyclin B1 and phosphorylated Cdk1 and by a cleavage of pro-apoptotic caspase-8 and PARP-1. Taken together, our studies showed that NV669 inhibits the proliferation of pancreatic and hepatic cancer cells through the regulation of G2/M phase transition via the cyclin B1-Cdk1 complex. In vitro NV669 inhibits PTP1B activity and FAK expression. NV669 impacts on the expression of adhesion molecules CDH-1, -2 and -3 in BxPC3 and Huh7 lines that form cell monolayers. Consecutively NV669 induces cell detachment. This suggests that NV669 by inhibiting PTP1B induces cell detachment and apoptosis. Subsequently, our in vivo results showed that NV669 inhibited the growth of pancreatic and hepatic tumor xenografts with a significant cell cycle arrest in pre-mitotic phase and an increase of tumor cell apoptosis. Therefore, NV669 may serve as an alternative anticancer agent, used alone or in association with other medications, for the treatment of pancreatic adenocarcinoma and hepatocellular carcinoma.

A self-assembling amphiphilic dendrimer nanotracer for SPECT imaging.

Bioimaging has revolutionized modern medicine, and nanotechnology can offer further specific and sensitive imaging. We report here an amphiphilic dendrimer able to self-assemble into supramolecular nanomicelles for effective tumor detection using SPECT radioimaging. This highlights the promising potential of supramolecular dendrimer platforms for biomedical imaging.

Deciphering the Crosstalk Between Myeloid-Derived Suppressor Cells and Regulatory T Cells in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with rising incidence and a remarkable resistance to current therapies. The reasons for this therapeutic failure include the tumor's extensive infiltration by immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). By using light sheet fluorescent microscopy, we identified here direct interactions between these major immunoregulatory cells in PDAC. The in vivo depletion of MDSCs led to a significant reduction in Tregs in the pancreatic tumors. Through videomicroscopy and ex vivo functional assays we have shown that (i) MDSCs are able to induce Treg cells in a cell-cell dependent manner; (ii) Treg cells affect the survival and/or the proliferation of MDSCs. Furthermore, we have observed contacts between MDSCs and Treg cells at different stages of human cancer. Overall our findings suggest that interactions between MDSCs and Treg cells contribute to PDAC immunosuppressive environment.

High histone deacetylase 7 (HDAC7) expression is significantly associated with adenocarcinomas of the pancreas.

BACKGROUND: Alterations in HDACs gene expression have been reported in a number of human cancers. No information is available concerning the status of HDACs in pancreatic cancer tumors. The aim of the present study was to evaluate the expression levels of members of class I (HDAC1, 2,, 3), class II (HDAC4, 5, 6, and 7), and class III (SIRT1, 2, 3, 4, 5, and 6) in a set of surgically resected pancreatic tissues. METHODS: Total RNA was isolated from 11 pancreatic adenocarcinomas (PA): stage 0 (n = 1), IB (n = 1), IIB (n = 6), III (n = 1), IV (n = 2), one serous cystadenoma (SC), one intraductal papillary mucinous tumor of the pancreas (IMPN), one complicating chronic pancreatitis (CP), and normal pancreas (NP) obtained during donor liver transplantation. Moreover, six other control pancreatic were included. HDACs gene expression was conducted using quantitative real-time polymerase chain reaction (qPCR). Protein expression levels were analyzed by Western blot and their localization by immunohistochemistry analyses of cancer tissues sections. RESULTS: Remarkably, 9 of the 11 PA (approximately 81%) showed significant increase of HDAC7 mRNA levels. In contrast to PA samples, message for HDAC7 was reduced in CP, SC, and IMPN specimens. The Western blot analysis showed increased expression of HDAC7 protein in 9 out of 11 PA samples, in agreement with the qPCR data. Most of the PA tissue sections examined showed intense labeling in the cytoplasm when reacted against antibodies to HDAC7. CONCLUSION: The data showed alteration of HDACs gene expression in pancreatic cancer. Increased expression of HDAC7 discriminates PA from other pancreatic tumors.

Pancreatic adenocarcinoma, chronic pancreatitis, and MODY-8 diabetes: is bile salt-dependent lipase (or carboxyl ester lipase) at the crossroads of pancreatic pathologies?

Pancreatic adenocarcinomas and diabetes mellitus are responsible for the deaths of around two million people each year worldwide. Patients with chronic pancreatitis do not die directly of this disease, except where the pathology is hereditary. Much current literature supports the involvement of bile salt-dependent lipase (BSDL), also known as carboxyl ester lipase (CEL), in the pathophysiology of these pancreatic diseases. The purpose of this review is to shed light on connections between chronic pancreatitis, diabetes, and pancreatic adenocarcinomas by gaining an insight into BSDL and its variants. This enzyme is normally secreted by the exocrine pancreas, and is diverted within the intestinal lumen to participate in the hydrolysis of dietary lipids. However, BSDL is also expressed by other cells and tissues, where it participates in lipid homeostasis. Variants of BSDL resulting from germline and/or somatic mutations (nucleotide insertion/deletion or nonallelic homologous recombination) are expressed in the pancreas of patients with pancreatic pathologies such as chronic pancreatitis, MODY-8, and pancreatic adenocarcinomas. We discuss the possible link between the expression of BSDL variants and these dramatic pancreatic pathologies, putting forward the suggestion that BSDL and its variants are implicated in the cell lipid metabolism/reprogramming that leads to the dyslipidemia observed in chronic pancreatitis, MODY-8, and pancreatic adenocarcinomas. We also propose potential strategies for translation to therapeutic applications.

Docetaxel-trastuzumab stealth immunoliposome: development and in vitro proof of concept studies in breast cancer.

BACKGROUND: Trastuzumab plus docetaxel is a mainstay to treat HER2-positive breast cancers. However, developing nanoparticles could help to improve the efficacy/toxicity balance of this doublet by improving drug trafficking and delivery to tumors. This project aimed to develop an immunoliposome in breast cancer, combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab, and comparing its performances on human breast cancer cell lines with standard combination of docetaxel plus trastuzumab. METHODS: Several strategies to engraft trastuzumab to pegylated liposomes were tested. Immunoliposomes made of natural (antibody nanoconjugate-1 [ANC-1]) and synthetic lipids (ANC-2) were synthesized using standard thin film method and compared in size, morphology, docetaxel encapsulation, trastuzumab engraftment rates and stability. Antiproliferative activity was tested on human breast cancer models ranging from almost negative (MDA-MB-231), positive (MDA-MB-453) to overexpressing (SKBR3) HER2. Finally, cell uptake of ANC-1 was studied by electronic microscopy. RESULTS: ANC-1 showed a greater docetaxel encapsulation rate (73%±6% vs 53%±4%) and longer stability (up to 1 week) as compared with ANC-2. Both ANC presented particle size ≤150 nm and showed similar or higher in vitro antiproliferative activities than standard treatment, ANC-1 performing better than ANC-2. The IC50s for docetaxel combined to free trastuzumab were 8.7±4, 2±0.7 and 6±2 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. The IC50s for ANC-1 were 2.5±1, 1.8±0.6 and 3.4±0.8 nM and for ANC-2 were 1.8±0.3 nM, 2.8±0.8 nM and 6.8±1.8 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. Cellular uptake appeared to depend on HER2 expression, the higher the expression, the higher the uptake. CONCLUSION: In vitro results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models using nanoparticles.

Dendritic cell-based vaccination: powerful resources of immature dendritic cells against pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAC) has a poor prognosis. One treatment approach, investigated here, is to reinforce antitumor immunity. Dendritic cells (DCs) are essential for the development and regulation of adaptive host immune responses against tumors. A major role for DCs may be as innate tumoricidal effector cells. We explored the efficacy of vaccination with immature (i)DCs, after selecting optimal conditions for generating immunostimulatory iDCs. We used two models, C57BL/6Jrj mice with ectopic tumors induced by the PAC cell line, Panc02, and genetically engineered (KIC) mice developing PAC. Therapeutic iDC-vaccination resulted in a significant reduction in tumor growth in C57BL/6Jrj mice and prolonged survival in KIC mice. Prophylactic iDC-vaccination prevented subcutaneous tumor development. These protective effects were long-lasting in Panc02-induced tumor development, but not in melanoma. iDC-vaccination impacted the immune status of the hosts by greatly increasing the percentage of CD8+ T-cells, and natural killer (NK)1.1+ cells, that express granzyme B associated with Lamp-1 and IFN-γ. Efficacy of iDC-vaccination was CD8+ T-cell-dependent but NK1.1+ cell-independent. We demonstrated the ability of DCs to produce peroxynitrites and to kill tumor cells; this killing activity involved peroxynitrites. Altogether, these findings make killer DCs the pivotal actors in the beneficial clinical outcome that accompanies antitumor immune responses. We asked whether efficacy can be improved by combining DC-vaccination with the FOLFIRINOX regimen. Combined treatment significantly increased the lifespan of KIC mice with PAC. Prolonged treatment with FOLFIRINOX clearly augmented this beneficial effect. Combining iDC-vaccination with FOLFIRINOX may therefore represent a promising therapeutic option for patients with PAC.

Bile-salt-stimulated lipase and mucins from milk of 'secretor' mothers inhibit the binding of Norwalk virus capsids to their carbohydrate ligands.

Breast-feeding-associated protection against calicivirus diarrhoea is associated with the presence of high levels of 2-linked oligosaccharides in mother's milk, and human calicivirus strains including the NV (Norwalk virus) use gut 2-linked fucosylated glycans as receptors, suggesting the presence of decoy receptors in milk. Our aim was to analyse the ability of human milk to inhibit the attachment of rNV VLPs (recombinant NV-like particles) to their carbohydrate ligands and to characterize potential inhibitors found in milk. Milk from women with the secretor phenotype was strongly inhibitory, unlike milk from women that are non-secretors, which is devoid of 2-linked fucosylated structures. At least two fractions in human milk acted as inhibitors for the NV capsid attachment. The first fraction corresponded to BSSL (bile-salt-stimulated lipase) and the second to associated mucins MUC1 and MUC4. These proteins present tandem repeat O-glycosylated sequences that should act as decoy receptors for the NV, depending on the combined mother/child secretor status.

Expression of truncated bile salt-dependent lipase variant in pancreatic pre-neoplastic lesions.

Pancreatic adenocarcinoma (PDAC) is a dismal disease. The lack of specific symptoms still leads to a delay in diagnosis followed by death within months for most patients. Exon 11 of the bile salt-dependent lipase (BSDL) gene encoding variable number of tandem repeated (VNTR) sequences has been involved in pancreatic pathologies. We hypothesized that BSDL VNTR sequences may be mutated in PDAC. The amplification of BSDL VNTR from RNA extracted from pancreatic SOJ-6 cells allowed us to identify a BSDL amplicon in which a cytosine residue is inserted in a VNTR sequence. This insertion gives rise to a premature stop codon, resulting in a truncated protein and to a modification of the C-terminal amino-acid sequence; that is PRAAHG instead of PAVIRF. We produced antibodies directed against these sequences and examined pancreatic tissues from patients with PDAC and PanIN. Albeit all tissues were positive to anti-PAVIRF antibodies, 72.2% of patient tissues gave positive reaction with anti-PRAAHG antibodies, particularly in dysplastic areas of the tumor. Neoplastic cells with ductal differentiation were not reactive to anti-PRAAHG antibodies. Some 70% of PanIN tissues were also reactive to anti-PRAAHG antibodies, suggesting that the C insertion occurs early during pancreatic carcinogenesis. Data suggest that anti-PRAAHG antibodies were uniquely reactive with a short isoform of BSDL specifically expressed in pre-neoplastic lesions of the pancreas. The detection of truncated BSDL reactive to antibodies against the PRAAHG C-terminal sequence in pancreatic juice or in pancreatic biopsies may be a new tool in the early diagnosis of PDAC.

Pancreatic bile salt-dependent lipase induces smooth muscle cells proliferation.

BACKGROUND: Because bile salt-dependent lipase (BSDL), an enzyme secreted by the pancreatic acinar cells and associated with LDL in circulating blood, also locates with smooth muscle cells (SMCs) in atherosclerotic lesions, we aimed to investigate its effects on SMCs. METHODS AND RESULTS: Immunohistochemical experiments allowed us to detect an expression of BSDL in atherosclerotic lesions from hypercholesterolemic monkeys and from human arteries. BSDL was found to be associated with SMCs but not with macrophages. BSDL was significantly mitogenic for cultured SMCs. This effect was inhibited by heparin and anti-BSDL antibodies, whereas heat-denaturated and diisopropylfluorophosphate-treated BSDL were inefficient. The mitogenic effect of BSDL was associated with an activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway, which was inhibited by heparin, and involved several mechanisms, among them diacylglycerol and oleic acid production as well as a rapid basic fibroblast growth factor release. CONCLUSIONS: Circulating BSDL is associated with SMCs within the intimal arteria and may trigger SMC proliferation, which could contribute to the development of atherosclerotic lesions.