Colorectal cancer-derived small extracellular vesicles induce TGFß1-mediated epithelial to mesenchymal transition of hepatocytes.

BACKGROUND: Metastatic disease is the major cause of cancer-related deaths. Increasing evidence shows that primary tumor cells can promote metastasis by preparing the local microenvironment of distant organs, inducing the formation of the so-called "pre-metastatic niche". In recent years, several studies have highlighted that among the tumor-derived molecular components active in pre-metastatic niche formation, small extracellular vesicles (sEVs) play a crucial role. Regarding liver metastasis, the ability of tumor-derived sEVs to affect the activities of non-parenchymal cells such as Kupffer cells and hepatic stellate cells is well described, while the effects on hepatocytes, the most conspicuous and functionally relevant hepatic cellular component, remain unknown. METHODS: sEVs isolated from SW480 and SW620 CRC cells and from clinical samples of CRC patients and healthy subjects were used to treat human healthy hepatocytes (THLE-2 cells). RT-qPCR, Western blot and confocal microscopy were applied to investigate the effects of this treatment. RESULTS: Our study shows for the first time that TGFß1-carrying CRC_sEVs impair the morphological and functional properties of healthy human hepatocytes by triggering their TGFß1/SMAD-dependent EMT. These abilities of CRC_sEVs were further confirmed by evaluating the effects elicited on hepatocytes by sEVs isolated from plasma and biopsies from CRC patients. CONCLUSIONS: Since it is known that EMT of hepatocytes leads to the formation of a fibrotic environment, a well-known driver of metastasis, these results suggest that CRC_sEV-educated hepatocytes could have an active and until now neglected role during liver metastasis formation.

Late-Onset Pompe Disease with Normal Creatine Kinase Levels: The Importance of Rheumatological Suspicion.

Pompe disease (PD), also defined as acid maltase deficiency, is a rare autosomal recessive disease that causes glycogen accumulation due to a deficiency of the lysosomal enzyme acid α-glucosidase. An excessive amount of undisposed glycogen causes progressive muscle weakness throughout the body. It particularly affects skeletal muscles and the nervous system, especially in the late-onset phase. Here, we present a clinical case of late-onset PD (LOPD) with normal CK (creatinine kinase) values treated after a misdiagnosis of demyelinating motor polyneuropathy and chronic inflammatory neuropathy. The suspicion of possible fibromyalgia induced the patient to seek a rheumatology consultation, and the investigations performed led to the diagnosis of PD. The patient was investigated for genetic and enzymatic studies. PD was diagnosed using the α-glucosidase assay on DBS. In LOPD, clinical manifestations, such as muscle weakness, exercise intolerance, myalgia, or even high hyperCKemia, often appear as nonspecific and may mimic a wide variety of other muscle disorders, such as limb muscle dystrophies, congenital, metabolic, or inflammatory myopathies. In our case, the patient had CK values in the normal range but with continued complaints typical of PD. An analysis of enzyme activity revealed a pathologic value, and genetic analysis identified the c.-32-13T>G mutation in homozygosis. The association of the pathological enzyme value and mutation in homozygosity with LOPD led to a familial segregation study. Our results contribute to the characterization of PD in Italy and support the importance of rheumatologic attention. This suggests further studies are needed to define the broad clinical and pathological spectrum observed in this disease.

Tumor-Derived Small Extracellular Vesicles Induce Pro-Inflammatory Cytokine Expression and PD-L1 Regulation in M0 Macrophages via IL-6/STAT3 and TLR4 Signaling Pathways.

Tumor-associated macrophages play a key role in promoting tumor progression by exerting an immunosuppressive phenotype associated with the expression of programmed cell death ligand 1 (PD-L1). It is well known that tumor-derived small extracellular vesicles (SEVs) affect the tumor microenvironment, influencing TAM behavior. The present study aimed to examine the effect of SEVs derived from colon cancer and multiple myeloma cells on macrophage functions. Non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured with SEVs derived from a colorectal cancer (CRC) cell line, SW480, and a multiple myeloma (MM) cell line, MM1.S. The expression of PD-L1, interleukin-6 (IL-6), and other inflammatory cytokines as well as of the underlying molecular mechanisms were evaluated. Our results indicate that SEVs can significantly upregulate the expressions of PD-L1 and IL-6 at both the mRNA and protein levels and can activate the STAT3 signaling pathway. Furthermore, we identified the TLR4/NF-kB pathway as a convergent mechanism for SEV-mediated PD-L1 expression. Overall, these preliminary data suggest that SEVs contribute to the formation of an immunosuppressive microenvironment.

The influence of the wooden equipment employed for cheese manufacture on the characteristics of a traditional stretched cheese during ripening.

The influence of the wooden equipment used for the traditional cheese manufacturing from raw milk was evaluated on the variations of chemico-physical characteristics and microbial populations during the ripening of Caciocavallo Palermitano cheese. Milk from two farms (A, extensive; B, intensive) was processed in traditional and standard conditions. Chemical and physical traits of cheeses were affected by the farming system and the cheese making technology, and changed during ripening. Content in NaCl and N soluble was lower, and paste consistency higher in cheese from the extensive farm and traditional technology, whereas ripening increased the N soluble and the paste yellow and consistency. The ripening time decreased the number of all lactic acid bacteria (LAB) groups, except enterococci detected at approximately constant levels (10(4) and 10(5) cfu g(-1) for standard and traditional cheeses, respectively), till 120 d of ripening. In all productions, at each ripening time, the levels detected for enterococci were lower than those for the other LAB groups. The canonical discriminant analysis of chemical, physical and microbiological data was able to separate cheeses from different productions and ripening time. The dominant LAB were isolated, phenotypically characterised and grouped, genetically differentiated at strain level and identified. Ten species of LAB were found and the strains detected at the highest levels were Pediococcus acidilactici and Lactobacillus casei. Ten strains, mainly belonging to Lactobacillus rhamnosus and Lactobacillus fermentum showed an antibacterial activity. The comparison of the polymorphic profiles of the LAB strains isolated from the wooden vat with those of the strains collected during maturation, showed the persistence of three enterococci in traditional cheeses, with Enterococcus faecalis found at dominant levels over the Enterococcus population till 120 d; the absence of these strains in the standard productions evidenced the contribution of vat LAB during Caciocavallo Palermitano cheese ripening.

Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

Anti-inflammatory properties of an aldehydes-enriched fraction of grapefruit essential oil.

Chronic inflammation is linked to the development of numerous diseases and is accompanied by increased cytokine secretion. Macrophages provide a first line of defense against pathogens that under inflammatory stimuli release pro-inflammatory cytokines. The essential oil (EO) fractions obtained from Citrus spp. rich in different compounds have gained the attention of both researchers and users during the last decades. In particular, grapefruit (Citrus paradisi) peel is rich in phenolics and flavonoids with several health benefits, including anti-inflammatory actions. Additionally, its EO consists of a large number of compounds such as monoterpenes, sesquiterpenes, alcohols, aldehydes, esters, and oxides. Among the methods for encapsulating EOs, spray-drying is the main one. In the present study, we aimed to determine the in vitro anti-inflammatory activity of EO from C. paradisi (grapefruit essential oil [GEO]) (whole and fractions) in a lipopolysaccharide (LPS)-induced inflammation model. Results indicate that Fr-GEO and Fr-GEO_SD exert protective effects against LPS-induced inflammation by decreasing gene expression and levels of pro-inflammatory cytokines as IL-6 and TNF-α. Monoterpenes as the most common components, as well as aldehydes and sesquiterpenes, might be responsible for such effects, although a synergistic action is not excluded. Furthermore, a higher percent of aldehydes is linked to improved olfactory properties. Our findings support the anti-inflammatory effects of selected Fr-GEO with a great potential for the development of new nutraceuticals and/or functional food for the treatment of inflammatory-associated diseases. PRACTICAL APPLICATION: The findings of this study support the anti-inflammatory effects of selected Fr-GEO with a great potential for the development of new nutraceuticals and/or functional food for the treatment of inflammatory-associated diseases.

Citral-Enriched Fraction of Lemon Essential Oil Mitigates LPS-Induced Hepatocyte Injuries.

Lemon essential oil (LEO) is known for its aromatic and healthy properties; however, less consideration is given to the biological properties of the fractions obtained from LEO. This study aims to evaluate the ability of a citral-enriched fraction obtained from LEO (Cfr-LEO) to counteract lipopolysaccharide (LPS)-mediated inflammation, oxidative stress, and epithelial-mesenchymal transition (EMT) in healthy human hepatocytes. Human immortalized hepatocytes (THLE-2 cell line) were pretreated with Cfr-LEO and subsequently exposed to LPS at various time points. We report that the pretreatment with Cfr-LEO counteracts LPS-mediated effects by inhibiting inflammation, oxidative stress, and epithelial-mesenchymal transition in THLE-2. In particular, we found that pretreatment with Cfr-LEO reduced NF-κB activation and the subsequent proinflammatory cytokines release, ROS production, and NRF2 and p53 expression. Furthermore, the pretreatment with Cfr-LEO showed its beneficial effect in counteracting LPS-induced EMT. Taken together, these results support Cfr-LEO application in the nutraceutical research field not only for its organoleptic properties, conferred by citral enrichment, but also for its biological activity. Our study could lay the basis for the development of foods/drinks enriched with Cfr-LEO, aimed at preventing or alleviating chronic conditions associated with liver dysfunction.

Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum.

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

Biological Properties of a Citral-Enriched Fraction of Citrus limon Essential Oil.

Lemon essential oil (LEO) is a well-known flavoring agent with versatile biological activities. In the present study, we have isolated and characterized four citral-enriched fractions of winter LEO. We reported that in murine and human macrophages the pre-treatment with a mix of these fractions (Cfr-LEO) reduces the expression of the pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 induced by LPS. In addition, Cfr-LEO counteracts LPS-induced oxidative stress, as shown by the increase in the GSH/GSSG ratio in comparison to cells treated with LPS alone. Overall, the results reported here encourage the application of EO fractions, enriched in citral, in the nutraceutical industry, not only for its organoleptic properties but also for its protective action against inflammation and oxidative stress.

Nutraceutical Value of Pantelleria Capers (Capparis spinosa L.).

Unopened flower buds of Capparis spinosa L. (capers), generally used in the Mediterranean area as food flavoring, are known to be a good source of bioactive compounds. The aim of this work was to evaluate the nutraceutical value of salt-fermented capers collected from different areas of Pantelleria Island (Italy), testing their methylglyoxal and glyoxal trapping capacity and antioxidant activity by 2,2-diphenyl-1-picryl hydrazyl (DPPH), [2,2-azinobis(3-ethylben- zothiazoline-6-sulfonic acid)] diammonium salt (ABTS), and oxygen radical absorbance capacity (ORAC) assays. Hydrophilic extracts were also characterized by high-performance liquid chromatography-electrospray ionization/mass spectrometry. Among 24 detected compounds, several flavonol derivatives and glucosinolates were identified. The levels of kaempferol and quercetin derivatives varied considerably among the five accessions considered (6.46 to 267.93 and 22.39 to 367.14 mg kaempferol and quercetin equivalent /g fresh weight, respectively), with kaempferol derivatives more representative than quercetin ones. Person's coefficient indicated a high correlation between total phenolic content and anti-DPPH radical capacity (R2 = 0.665), as well as between total flavonoid content and antioxidant capacity (by ORAC assay; R2 = 0.888) and between total flavonoid content and glyoxal and methylglyoxal trapping capacity (R2 = 0.918). Results indicate that capers from Pantelleria Island represent a rich source of bioactive compounds with potential nutraceutical relevance. PRACTICAL APPLICATION: The findings of this study highlight the health benefits of Pantelleria capers consumption due to their composition in antioxidants and their biological properties (antiradical and alpha-dicarbonyls trapping) correlated with the development of a high number of chronic-degenerative diseases. These results are also important for the agricultural and commercial sectors involved in the production of capers from Pantelleria, which received the Protected Geographical Indications recognition.

The individual contribution of starter and non-starter lactic acid bacteria to the volatile organic compound composition of Caciocavallo Palermitano cheese.

The contribution of two starter (Lactobacillus delbrueckii and Streptococcus thermophilus) and nine non-starter (Enterococcus casselliflavus, Enterococcus faecalis, Enterococcus durans, Enterococcus gallinarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Pediococcus acidilactici and Pediococcus pentosaceus) species of lactic acid bacteria (LAB) to the volatile organic compounds (VOCs) of Caciocavallo Palermitano cheese was investigated. The strains used in this study were isolated during the production/ripening of the stretched cheese and tested in a cheese-based medium (CBM). The fermented substrates were analyzed for the growth of the single strains and subjected to the head space solid phase micro-extraction (HS-SPME) and gas chromatography - mass spectrometry (GC-MS). The 11 strains tested were all able to increase their numbers in CBM, even though the development of the starter LAB was quite limited. GC-MS analysis registered 43 compounds including seven chemical classes. A lower diversity of VOCs was registered for the unfermented curd based medium (CuBM) analyzed for comparison. The class of ketones represented a consistent percentage of the VOCs for almost all LAB, followed by alcohols and esters. The volatile profile of Pediococcus acidilactici and Lactobacillus delbrueckii was mainly characterized by 2-butanol, butanoic acid and hexanoic acid and their esters, while that of Lactobacillus casei and Lactobacillus rhamnosus was characterized by 2,3-butanedione and 2-butanone, 3-hydroxy. In order to correlate the VOCs produced by Caciocavallo Palermitano cheeses with those generated by individual LAB, the 4-month ripened cheeses resulting from the dairy process monitored during the isolation of LAB were also analyzed for the volatile chemical fraction and the compounds in common were subjected to a multivariate statistical analysis. The canonical analysis indicated that the VOCs of the ripened cheeses were mainly influenced by E. gallinarum, L. paracasei, L. delbrueckii, L. rhamnosus and L. casei and that 1-hexanol, o-xylene and m-xylene were the cheese VOCs highly correlated with LAB.