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Background Immune checkpoint inhibitors (ICIs) have revolutionized the management of cancers. The risk factors and pathophysiological mechanisms of venous thromboembolic events (VTEs) of this new therapeutic class are still to be specified. Methods The included patients had to have cancer and should be treated with ICI. Data analyzed included demographic data, biological data, and immune-related adverse events (IRAEs). We studied the prevalence of VTEs and the factors associated with VTEs. Results Of 374 patients on ICI, over a median follow-up period of 15.2 months, the number of VTE was 50 (13.4%). The majority of patients were treated for metastatic melanoma or nonsmall cell lung cancer. There was no difference in prevalence or survival between cancer types. Patients with combined therapy composed of nivolumab and ipilimumab had higher 1-year cumulative VTE occurrence (29.3% [95% confidence interval [CI]: 9.7; 44.6]) than patients with pembrolizumab (14.9%, [95%CI: 2.5; 25.8], p = 0.03) or nivolumab (9.1%, [95% CI: 5.0; 12.9], p Conclusion The prevalence of VTE was 14.2% under ICIs. IRAE and combine treatment of nivolumab and ipilimumab were associated with VTE. The pathophysiological mechanisms are multiple and complex with a possible link to aberrant activation of the immune system.

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH) are well-known for their anticoagulant properties. There is also currently a growing interest in using LMWH in targeted cancer therapy. In particular, several types inhibit heparanase, a key enzyme overexpressed in the tumor microenvironment that promotes angiogenesis progression and metastasis spreading. Here, we propose iron oxide nanoparticles (HEP-IONP) coated with different heparins of distinct anticoagulant/anti-heparanase activity ratios and suitable for positive contrast in magnetic resonance imaging. As a proof of concept, magnetic resonance angiography (MRA) was conducted in mice up to 3 h after intravenous administration. This new IONP-based positive contrast appropriate for clinic together with the long vascular circulating times can enable innovative theranostic applications if combined with the various bioactivities of the heparins. Indeed, we showed, using advanced in vitro tests, how HEP-IONP anticoagulant or anti-heparanase activities were maintained depending on the heparin species used for the coating. Overall, the study allowed presenting an IONP coated with a commercial LMWH (Lovenox) suggested as a theranostic translational probe for MRA diagnostic and treatment of thrombosis, and an antitumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.

Strongly associated with tumor angiogenesis and metastasis, the enzyme heparanase is an endo-β-d-glucuronidase which is overexpressed in the tumor microenvironment. Its inhibition could be one of the most promising anti-angiogenic approaches to date. Although heparin is known as a good heparanase inhibitor, it also possesses major anticoagulant properties that may be incompatible with its use as an anti-angiogenic agent, hence the considerable interest for other sources of sulfated polysaccharides. Recent investigations point to λ-carrageenans, highly sulfated galactans with a tremendous potential that are found in red algae. This study describes the production of low-molecular-weight (LMW) heparins and λ-carrageenans, using a combination of glycol splitting and ultrasonically-assisted radical hydrolysis using hydrogen-peroxide. The structural characteristics, as well as the anticoagulant and antiheparanase activities of the resulting products were assessed. The best candidate was a LMW glycol-split λ-carrageenan that displayed major anti-heparanase properties, with an IC50 of 7.32ng/mL and a close-to-zero anticoagulant activity.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Extracellular products (ECPs) of the French V. tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1, 10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Even if taken together, our results let us wonder if this (these) zinc metalloprotease(s) could be involved in the impairment of hemocyte immunological functions, the direct implication of this (these) protein(s) in ECPs toxicity still has to be demonstrated.

To determine the angiotensin-I converting enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates, (N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-hippuryl-His-Leu hydrate salt (HHL)), and a natural one, angiotensin-I. The IC50 value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nanomolar range (1.79-15.1 nM) when synthetic substrates were used, whereas it exhibited IC50 of micromolar range (16.71 μM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC50 of micromolar range (17.13-146 μM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK), and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC50 displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK, and VIY show IC50 values over the IC50 value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW, and LKP exhibited IC50 value lower than the IC50 value of losartan for all substrates tested and were thus considered as good candidates for effectively decreasing hypertension. It appears that the comparison of IC50 is not consistent when IC50 values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references such as captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.

Acid goat whey fermentation by a co-culture of Kluyveromyces marxianus and Lactobacillus rhamnosus yielded a hydrolysate inhibitory for the angiotensin-I-converting enzyme. The potential of this hydrolysate and its main lactokinin tryptophan against accumulation of lipid was investigated at different stages of immortalised human adipocytes development using Oil Red O staining and compared with that of captopril. A slight reduction of lipid content (8%) was observed when mature adipocytes were incubated with the whole hydrolysate at the concentration of 587 μg mL−1. Proliferation of pre-adipocytes was also reduced after incubation with this hydrolysate, which was not cytotoxic. Tryptophan induced a reduction of lipid content of 18% and was cytotoxic towards adipocytes at the concentration found in the hydrolysate: 25.7 μg mL−1. A decrease of the lipid content of mature adipocytes of 28% was observed with 2.9 ng mL−1 of captopril, which was also cytotoxic.

Unsupplemented acid goat whey containing 0·96% protein and 2·76% lactose was fermented aerobically with 32 microflora extracted from various raw milk cheeses and dairy products. These microflora were screened for their ability to hydrolyse whey proteins (α-lactalbumin and/or β-lactoglobulin) and to generate peptides inhibitors of Angiotensin I Converting Enzyme. Five microflora were able to degrade whey protein. The most efficient microflora was able to fully hydrolyse α-lactalbumin and to a lesser extend β-lactoglobulin. It was extracted from Bamalou des Pyrenées cheese. Micro-organisms involved consisted of yeast Kluyveromyces marxianus and lactobacillus Lactobacillus rhamnosus. Both were able to produce ACE inhibitory peptides after whey fermentation.

International audience; The microenvironment that surrounds tumor cells is characterized by hypoxic conditions and extracellular acidity. These hostile conditions induce crucial changes in cell behavior and can promote the secretion of many soluble factors such as growth factors, cytokines and enzymes. The lysosomal aspartyl-endopeptidase cathepsin D (CD) is a marker of poor prognosis in breast cancer and is associated with a metastatic risk. In this study, the transport of CD was investigated in a model of breast cancer cells line (MCF-7) cultivated under hypoxia and acidification of media. CD secretion was assessed using Western blot analysis and protease activity was measured in conditioned culture media. We demonstrate that cultured MCF-7 cells secrete an active 52 kDa pCD precursor and report that under hypoxia there was an increased amount of pCD secreted. More surprisingly, extracellular acidification (pH 6 and 5.6) induced the secretion of the fully-mature and active (34 kDa + 14 kDa) double chain CD. Our findings reflect the fact that chemical anomalies influence the secretion path of CD in a breast cancer cell model, resulting in altered trafficking of the mature form. This important result may provide new arguments in favor of the role of extracellular CD in the degradation of the matrix proteins that constitute the breast tumor microenvironment.

Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [α-lactalbumin (α-la) and β-lactoglobulin (β-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 °C using crude or pre-heated whey (10 min at 65, 75 or 85 °C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither β-lg hydrolysis nor peptide generation were observed. More than 50% of α-la was digested into a truncated form of α-la (±12 kDa) in crude whey and whey pre-heated at 65 °C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of α-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both α-la and β-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from α-la hydrolysis. The co-culture of both microorganisms was required for α-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for α-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.

Angiotensin-I-Converting Enzyme (ACE) inhibitors peptides were produced from unsupplemented acid goat whey fermented aerobically for 168 h byKluyveromyces marxianusandLactobacillus rhamnosus. This yeast-lactobacillus association is GRAS. Two novel lactokinins were identified: NYW and W with IC50of 20 and 0·86 μmrespectively. They both were resistant toward simulated gastrointestinal digestion. In addition, WLAHK was found in the hydrolysate. These three sequences belong tof(99–110) of α-la which seems to be a lactokinin cryptic zone. W was the major molecule released by the fermentation process. Considering that W is the precursor of serotonin, the hydrolysate produced could be of interest for the generation of functional health ingredient involved in regulation of affective disorders and hypertension.

Unsupplemented caprine whey was fermented by 25 cheese microflora in order to produce peptides from α -lactalbumin ( α -la) and/or β -lactoglobulin ( β -lg) hydrolysis. Fourteen hydrolysates enriched in peptides mainly released from α -la were obtained. Angiotensin-I-converting enzyme (ACE) inhibitory activity of each hydrolysate was investigated. Six of them had high ACE inhibitory activities ranging from 31% to 56%. The highest ACE inhibitory activity was obtained after whey fermentation by the microflora from 18 months ripened Comte cheese. The microflora was identified as a co-culture of Candida parapsilosis and Lactobacillus paracasei . Hydrolysate activity remained stable after pepsin, trypsin and chymotrypsin treatments simulating an in vitro gastrointestinal digestion. This hydrolysate was further fractionated by RP-HPLC. The peptide exhibiting the highest ACE inhibitory activity was characterised as WLAHK ( α -la f (104–108): Trp–Leu–Ala–His–Lys). WLAHK was resistant toward pepsin and trypsin treatments but was digested by chymotrypsin.

The influence of pH (4·5–6·5), sodium chloride content (125–375 mM), calcium chloride content (10–30 mM), protein concentration (70–90 g/l) and lactose content on the gel hardness of goat whey protein concentrate (GWPC) in relation to the origin of the acid whey (raw or pasteurized milk) was studied using a factorial design. Gels were obtained after heat treatment (90 °C, 30 min). Gel hardness was measured using texture analyser. Only protein concentration and pH were found to have a statistically significant effect on the gel hardness. An increase in the protein concentration resulted in an increase in the gel hardness. GWPC containing 800 g/kg protein formed gels with a hardness maximum at the pHi, whereas GWPC containing 300 g/kg protein did not form true gels. Whey from pasteurized milk formed softer gels than whey from raw milk. A high lactose content (≈360 g/kg) also reduced the gelation performance of GWPC.

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This paper presents a novel contribution to the purification of goat β-lactoglobulin by using an ultrafiltration membrane enzymic reactor. The basis of the purification process was the enzymic hydrolysis of contaminating proteins, α-lactalbumin and traces of serum albumin, by pepsin at 40 °C and pH 2, conditions under which β-lactoglobulin is resistant to peptic digestion. Simultaneously, β-lactoglobulin and peptides were separated by ultrafiltration. β-Lactoglobulin was retained in the reactor while peptides generated by hydrolysis from α-lactalbumin and serum albumin permeated through the membrane. The process was made continuous by the addition of fresh whey to replace the lost permeate. Three mineral membranes with 10, 30 and 50 kDa molecular mass cut-off were tested and the 30 kDa membrane was selected for the continuous process. The simultaneous purification and concentration of β-lactoglobulin from clarified goats' whey was achieved in a single step. The ultrafiltration membrane enzymic reactor could treat eight reactor volumes of clarified whey. The recovery of β-lactoglobulin was 74%, its purity was 84% and its concentration 6·6-fold that in the initial clarified whey.

Goat whey was hydrolysed by pepsin in an ultrafiltration membrane enzymic reactor coupled with a 30 kDa mineral membrane. Peptides collected in the permeate were resolved using reversed-phase HPLC. Their sequences were determined by amino acid analysis, second order derivative spectra analysis and mass spectrometry. Owing to the resistance of β-lactoglobulin (β-lg) towards pepsin, the majority of peptides identified were derived from α-lactalbumin (α-la). Pepsin showed a broad specificity of hydrolysis sites and generated a wide range of products from dipeptides to very large peptides containing disulphide bridges. The molecular masses of peptides resulting from α-la degradation were between 150 and 6900 Da: 36% were < 600 Da, 24% were 600–2000 Da and 40% were > 2000 Da.

Bovine whey hydrolysate has been developed and applied to areas such as nutrition, culture media, and isolation of bioactive peptides. In order to produce such a type of hydrolysate, it is possible to use goat whey which constitutes also a food processing by-product. Enzymatic hydrolysis of goat whey by pepsin was carried out in a continuous ultrafiltration reactor. The permeate contained peptide hydrolysate that was resolved by RP-HPLC. Second order derivative spectroscopy, amino acid analysis, and mass spectrometry revealed the presence of a biologically active peptide called alpha-lactorphin. This constitutes preliminary information about goat whey enzymatic degradation for future applications.

The effect of RP-HPLC-purified fractions of fish waste hydrolysates issued from three fish industries was tested on guinea pig ileum in order to examine the presence of opioid molecules. The evaluation of anti-hypertensive activities of whole hydrolysates and fractions were also tested, monitoring the ability of the fraction to inhibit the activity of angiotensin I-converting enzyme involved in hypertension regulation. Sardine autolysate and cod head hydrolysate powder (50 µg) were able to inhibit near 30% of ACE activity, whereas 50 µg of shrimp hydrolysate allows the inhibition of 57% of ACE activity. HPLC fractionation of cod head hydrolysate and sardine autolysate was necessary to evidence biological activity, whereas HPLC separation of shrimp hydrolysate exhibited low biological activity fractions. Further studies are necessary to characterise bioactive molecules from cod head alcalase hydrolysate and from sardine autolysate.

International audience; Antiproliferative activity of 18 fish protein hydrolysates was measured on 2 human breast cancer cell lines grown in vitro. Three blue whiting, three cod, three plaice and one salmon hydrolysates were identified as significant growth inhibitors on the two cancer cell lines. Preliminary analysis of hydrolysates composition evidenced they contained a complex mixture of free amino acids, peptides with various sizes ranging up to 7 kDa and in a lower proportion, lipids and sodium chloride. RP-HPLC fractionation of fish hydrolysates is currently undertaken to purify anticancer peptides, lipids or other bioactive trace compounds responsible for this antiproliferative activity.

The development of obesity and breast cancer is influenced by exposure to environmental pollutants. These pollutants interfere with the normal body function causing changes in cells behavior. They can act each alone or as a cocktail. In this context, we have chosen three pollutants in our project : bisphenol A (endocrin disruptor), glyphosate (herbicide), okadaic acid (marin toxine). We have studied their effect of alone or in combination in cellular models : human breast cancer cells MCF7 and MDA-MB 231, human mammary epithelial cells HME1 and human preadipocytes HWP. In the first part, we have studied the effect of pollutants on cells viability. 2D electrophoresis coupled to MS/MALDI-TOF, measurement of proteins phosphatase activity were conducted to understand pollutants action mechanism on cells viability. In the following part we detected and quantified pollutants in culture medium to determine their pathway of action : extra or intra cellular. Finally, we studied the effect of pollutants on adipocytes life cycle and lipogenesis. Also, effect of secretome from HWP (+/- pollutants) on cancer cells viability was studied.; L’apparition de l’obésité et du cancer du sein est influencée par l’exposition aux polluants environnementaux. Ces polluants interfèrent avec le fonctionnement normal de l’organisme causant des modifications dans le comportement cellulaire. Ils peuvent agir chacun seul ou sous forme de cocktail. Dans ce contexte, nous avons choisi trois polluants dans notre projet : le bisphénol A (perturbateur endocrinien), le glyphosate (désherbant) et l’acide okadaïque (toxine marine). Nous avons étudié leur effet seul ou en cocktail sur les cellules de cancer du sein MCF7 et MDA-MB231, les cellules épithéliale mammaire humaine HME1 et les préadipocytes humain HWP. Dans une première partie, nous avons étudié leur effet sur la viabilité cellulaire. L’électrophorèse 2D couplée à MS/MALDI-TOF, le dosage de l’activité des protéines phosphatases ont été menés pour comprendre le mécanisme d’action des polluants sur la viabilité cellulaire. La partie suivante a porté sur la détection et la quantification des polluants dans le milieu de culture afin de déterminer leur voie d’action : extra ou intra cellulaire. Finalement, nous avons étudié l’effet des polluants sur le cycle de vie des adipocytes et la lipogenèse. Également, l’effet de sécrétome des HWP (+/-polluants) sur la viabilité cellulaire des cellules cancéreuses a été étudié.

Marine products represent an important source of active substances, in particular bioactive peptides called cryptides. Cryptides are hidden within the sequence of a parent protein and are released during digestion or industrial proteolytic processes. These cryptides could provide physiological benefit or protection against diseases such as those of metabolic syndrome. In this context, we investigated the action of some marine cryptides on hypertension, diabetes and obesity. We demonstrated that some cryptides can target in vitro several factors associated with the development of metabolic syndrome.; Les ressources marines constituent un réservoir considérable de substances actives, en particulier, de peptides bioactifs appelés cryptides. Les cryptides, qui sont initialement dissimulés au cœur des protéines, sont libérés lors de la digestion ou lors de procédés protéolytiques industriels. Ces cryptides pourraient procurer des bienfaits physiologiques ou assurer une protection contre des pathologies telles que celles du syndrome métabolique. Dans ce contexte, nous nous sommes intéressés à l’action de certains cryptides marins sur des cibles impliquées dans l’hypertension, le diabète et l’obésité. Nous avons pu mettre en évidence que certains cryptides pouvaient cibler in vitro plusieurs facteurs de risques associés au développement des anomalies du syndrome métabolique.