[Specific amplification of plasma exosome miRNA in cancer patients for construction of cDNA library].

Plasma exosome microRNAs (miRNAs) are closely related with the occurrence, diagnosis, and treatment of cancers. However, the underlying molecular mechanisms remain unclear. We herein investigated the solution for tackling the unspecific amplification of plasma exosome microRNAs from cancer patients during the construction of its cDNA library. For the restriction enzyme digesting method, the primers were degraded by exonuclease T (EXOT) and phi29 DNA polymerase. For the magnetic bead separation method, the templates and primers were separated through the DNA binding beads. The separation effects of magnetic beads were detected by agarose gel electrophoresis and modified polyacrylamide gel electrophoresis. The levels of plasma exosome miRNAs from cancer patients and various primers were assayed by RT-qPCR. The results indicated that the unspecific amplification stemmed from USR5SR. EXOT and phi29 DNA polymerase could degrade USR5SR, but the templates were also degraded simultaneously. Regarding the magnetic bead separation method, the best effect was achieved via precipitation of primer fragments by 9% PEG and precipitation of templates by 15% PEG. In conclusion, the magnetic bead separation method efficiently circumvented the unspecific amplification during the construction of cDNA library, and therefore led to the successful construction of cDNA library from plasma exosome miRNA of cancer patients and 293T cells.

Natural lactucopicrin alleviates importin-α3-mediated NF-κB activation in inflammated endothelial cells and improves sepsis in mice.

Lactucopicrin, a bitter sesquiterpene lactone of leafy vegetables, such as chicory, curly escarole, and lettuce, possesses anti-malarial, anti-cancer and analgesic properties. However, it remains unknown whether lactucopicrin could inhibit vascular endothelial nuclear factor-κB (NF-κB) activation, a hallmark of vascular inflammatory diseases including sepsis. In tumor necrosis factor-α-stimulated human or mouse aortic endothelial cells, lactucopicrin dose-dependently inhibited NF-κB activation, and concomitantly repressed both vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1)-mediated monocyte adhesion. The lactucopicrin effect was not due to modulation of inhibitor of NF-κB kinases (IKK) α/ß/γ, inhibitor of NF-κB alpha (IκBα), and NF-κB/p65 DNA binding activity. Instead, lactucopicrin inhibited importin-α3 expression by destabilization of its mRNA, an effect mediating the lactucopicrin effect on NF-κB activity. More importantly, in lipopolysaccharide (LPS)-elicited septic mice, oral gavage with lactucopicrin decreased mortality by 30.5% as compared with the control treatment. This effect was associated with inhibited importin-α3 expression, suppressed NF-κB activation and VCAM-1/ICAM-1 expression, and inhibited leukocyte influx in the vascular endothelium of both lung and aorta. Collectively, our novel data suggest that dietary supplementation with lactucopicrin inhibits endothelial NF-κB activation by down-regulation of importin-α3 and thereby improves sepsis.

Lactucopicrin Inhibits Cytoplasmic Dynein-Mediated NF-κB Activation in Inflammated Macrophages and Alleviates Atherogenesis in Apolipoprotein E-Deficient Mice.

SCOPE: Nuclear factor-κB (NF-κB) activation in macrophages aggravates atherosclerosis. Dietary plant secondary metabolites including sesquiterpene lactone lactucopicrin target multiple organs. This study is focused on the impact of lactucopicrin on NF-κB activation in inflammed macrophages and atherogenesis in a mouse model of atherosclerosis. METHODS AND RESULTS: In LPS-stimulated mouse bone marrow-derived macrophages, lactucopicrin inhibits NF-κB activation, and concomitantly represses the expression of IL-1ß, IL-6, and tumor necrosis factor-alpha. This effect is not due to modulation of the inhibitor of NF-κB kinases (IKK) α/ß/γ and NF-κB inhibitor α, and NF-κB/p65 DNA binding activity. Instead, the lactucopicrin effect is reliant on the inhibition of cytoplasmic dynein-mediated p65 transportation, a prerequisite step for p65 nuclear translocation. In high-fat diet-fed apolipoprotein E-deficient mice, lactucopicrin consumption dose-dependently reduces plaque area, inhibits plaque macrophage accumulation, attenuates plaque macrophage NF-κB activation, and reduces both plaque and serum inflammatory burden. However, lactucopicrin consumption does not affect the levels of serum lipids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor beta). CONCLUSION: Dietary lactucopicrin inhibits atherogenesis in mice likely by its anti-inflammatory property. These findings suggest that dietary supplementation with lactucopicrin is a promising strategy to inhibit atherosclerotic cardiovascular disease.

High affinity monoclonal antibody targeting Siglec-15 for cancer immunotherapy.

BACKGROUND AND AIM: Recently, Siglec-15 has been proved as a novel immune suppressor and a potential target for normalization cancer immunotherapy, which is non-redundant to the well-known PD-L1/PD-1 pathway. Herein, anti-Siglec-15 mAb, a monoclonal antibody (mAb) with a high affinity against Siglec-15, was prepared. METHODS: The engineered CHO-K1 Siglec-15 cell line was constructed to heterologously expressed Siglec-15 for the affinity test with the mAb. Antigens Siglec-15-mIgG and Siglec-15-his were recombinantly expressed by 293F cells and purified by high-performance liquid chromatography (HPLC). Hybridoma cell line against Siglec-15 was prepared and validated by enzyme-linked immunoabsorbant assay (ELISA) and fluorescent-activated cell sorting (FACS). Finally, the anti-Siglec-15 mAb was produced, purified, and confirmed by SDS-PAGE, ELISA, and FACS. RESULTS: The EC50 of the anti-Siglec-15 mAb with Siglec-15 is 76.65 ng/mL, lower than that of the positive control 5G12 (90.7 ng/mL), indicating a high affinity of the anti-Siglec-15 mAb. In vitro and in vivo studies verified that the anti-Siglec-15 mAb blocks the Siglec-15-mediated suppression of T cell and moderately prevents the tumor growth. CONCLUSIONS: The anti-Siglec-15 mAb can be considered as an effective immunotherapy for tumor suppression. RELEVANCE FOR PATIENTS: The anti-Siglec-15 mAb prepared in this study is useful as an immune checkpoint inhibitor against Siglec-15 for normalization cancer immunotherapy. This immunotherapy provides an alternative treatment for cancer patients who are refractory to the well-known PD-L1/PD-1-targeting therapies.

Low Oral Bioavailability and Partial Gut Microbiotic and Phase II Metabolism of Brussels/Witloof Chicory Sesquiterpene Lactones in Healthy Humans.

Free and glycosylated sesquiterpene lactones (SLs), which are abundant in leafy vegetables including Brussels/witloof chicory, possess health-promoting effects in vivo. However, the pharmacokinetics of dietary source of SLs remain largely unknown. In this open-label and single-dose trial, sixteen healthy volunteers consumed 150 g of Brussels/witloof chicory juice containing 48.77 µmol SLs in 5 min. Blood, urine, and fecal samples were collected before and after chicory consumption in 24 h. No SLs were detected in the serum, urine, and fecal samples before chicory consumption in all of the participants. Chicory consumption increased lactucin, 11ß,13-dihydrolactucin, and their glucuronide/sulfate conjugates, rather than lactucopicrin and 11ß,13-dihydrolactucopicrin, as well as glycosylated SLs in biological samples. The peak concentration of total SLs in serum reached 284.46 nmol/L at 1 h, while, in urine, this peak was 220.3 nmol between 2 and 6 h. The recovery of total SLs in blood, urine, and feces was 7.03%, 1.13%, and 43.76% of the ingested dose, respectively. Human fecal suspensions with intestinal microbiota degraded glycosylated SLs in chicory, and converted lactucopicrin and 11ß,13-dihydrolactucopicrin to lactucin and 11ß,13-dihydrolactucin, respectively. Collectively, Brussels/witloof chicory SLs are poorly bioavailable and they undergo partial gut microbial and phase II metabolism in humans.

Protocatechuic Acid Inhibits Vulnerable Atherosclerotic Lesion Progression in Older Apoe-/- Mice.

BACKGROUND: Normalization of arterial inflammation inhibits atherosclerosis. The preventive role for protocatechuic acid (PCA) in early-stage atherosclerosis is well recognized; however, its therapeutic role in late-stage atherosclerosis remains unexplored. OBJECTIVE: We investigated whether PCA inhibits vulnerable atherosclerosis progression by normalizing arterial inflammation. METHODS: Thirty-wk-old male apolipoprotein E-deficient (Apoe-/-) mice with vulnerable atherosclerotic lesions in the brachiocephalic artery were fed the AIN-93G diet alone (control) or supplemented with 0.003% PCA (wt:wt) for 20 wk. Lesion size and composition, IL-1ß, and NF-κB in the brachiocephalic arteries, and serum lipid profiles, oxidative status, and proinflammatory cytokines (e.g., IL-1ß, monocyte chemoattractant protein-1, and serum amyloid A) were measured. Moreover, the effect of PCA on the inflammation response was evaluated in efferocytic macrophages from C57BL/6J mice. RESULTS: Compared with the control treatment, dietary PCA supplementation significantly reduced lesion size (27.5%; P < 0.05) and also improved lesion stability (P < 0.05) as evidenced by increased thin fibrous cap thickness (31.7%) and collagen accumulation (58.3%), reduced necrotic core size (37.6%) and cellular apoptosis (73.9%), reduced macrophage accumulation (45.1%), and increased vascular smooth muscle cell accumulation (51.5%). Moreover, PCA supplementation inhibited IL-1ß expression (53.7%) and NF-κB activation (64.4%) in lesions. However, PCA supplementation did not change serum lipid profiles, total antioxidant capacity, and inflammatory cytokines. In efferocytic macrophages, PCA at 0.5 and 1 µmol/L inhibited Il1b/IL-1ß mRNA (27.2-46.5%) and protein (29.2-49.6%) expression and NF-κB activation (67.0-80.3%) by upregulation of MER proto-oncogene tyrosine kinase (MERTK) and inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1). Strikingly, the similar pattern of the MERTK and MAPK3/1 changes in lesional macrophages of mice after PCA intervention in vivo was recapitulated. CONCLUSION: PCA inhibits vulnerable lesion progression in mice, which might partially be caused by normalization of arterial inflammation by upregulation of MERTK and inhibition of MAPK3/1 in lesional macrophages.

Protocatechuic acid from chicory is bioavailable and undergoes partial glucuronidation and sulfation in healthy humans.

Protocatechuic acid exerts multiple health-promoting effects such as anticancer, anti-atherosclerosis, and neuroprotection in animal models. While protocatechuic acid produced in the lower gastrointestinal tract by microbial catabolism of several flavonoids is bioavailable, the pharmacokinetics of protocatechuic acid has not been evaluated so far in humans following its oral consumption. In this open-label and single-dose pharmacokinetic trial, 16 healthy adults followed a low-phytochemical diet for three days. Next, after overnight fasting, participants consumed 150 g of chicory containing 248 µmol of protocatechuic acid. Blood, urine, and fecal samples were collected before and up to 24 hr after chicory consumption. Protocatechuic acid in the free and glucuronide/sulfate-conjugated forms was almost undetectable in serum, urine, and fecal samples before chicory consumption. Chicory consumption increased the levels of protocatechuic acid and its glucuronide/sulfate conjugates in biological samples. The maximum serum concentrations of protocatechuic acid in the free-, glucuronide-, and sulfate-conjugated forms were 3,273, 519, and 340 nmol/L, respectively. The recovery of total protocatechuic acid in blood circulation, urine, and feces was 23.79%, 12.17%, and 12.79% of the ingested dose, respectively. Moreover, glucuronide and sulfate conjugates of protocatechuic acid made up 34.79%, 60.15%, and 72.70% of its total recovery in blood circulation, urine, and feces, respectively. Collectively, protocatechuic acid from chicory is bioavailable and undergoes partial glucuronidation and sulfation in human adults, and its regular consumption may exert health-promoting effects.