Immunomodulation of nutritional formula containing epigallocatechin-3-gallate, ginseng extract, and polydextrose on inflammation and macrophage polarization.

Single nutrient likes polyphenol or dietary fiber have been exhaustively investigated to validate their positive intervention in health or disease. Meanwhile, the common interaction of inner systems with the nutrient complex has not been well elucidated, which raises the scientific issue of the modulatory effect of the nutrient complex on immunity. The representative prebiotics of epigallocatechin-3-gallate (EGCG), ginseng extract, and polydextrose (PDX) were selected on behalf of the classification of polyphenol, flavone or polysaccharides, and dietary fiber to generally cover the daily food intake in this study to explore their intervention in inflammation and macrophage polarization. The intervention of selected nutrients on inflammation and macrophage polarization has been evaluated against macrophages to unveil their comprehensive effects. The synergistic effect of selected nutrients was demonstrated by inhibiting M1 macrophage polarization and the promotion of M2 macrophage polarization. Then, the nutrient formula was set up to verify the intervention effect, and the results revealed the significant inhibition of cell inflammation and the effect on cell proliferation through promoting the cell cycle in the G2 phase. The nutrient complex could inhibit M1 macrophage polarization to inhibit M1-mediated inflammation and promote M2 macrophages for anti-inflammatory effect and enhance cell phagocytosis. Moreover, the varied intervention effects of the nutrient complex with different formulas could be summarized. In general, the formula containing EGCG, ginseng extract, and PDX was demonstrated to possess an enhanced immunomodulatory effect on cell inflammation and macrophage polarization, which could potentially inspire the investigation of complex nutrients in health and diseases.

Gut Microbiota-Derived Indole Derivatives Alleviate Neurodegeneration in Aging through Activating GPR30/AMPK/SIRT1 Pathway.

SCOPE: Tryptophan (Trp) metabolites are closely related to neurological diseases, whereas, the underlying mechanism related to the alleviative effects of Trp metabolites on neurodegeneration in aging remains unclear. This study aims to evaluate the protective effects and mechanisms of Trp metabolites on neurodegeneration in aging process. METHODS AND RESULTS: The neuroprotective properties of Trp metabolites are evaluated in vitro and in vivo experimental model. Trp metabolites such as indole, indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-lactic acid (ILA), and indole-3-carboxyaldehyde (Icld) could significantly reduce oxidative stress, inflammation, and neuronal apoptosis induced by H2 O2 in HT-22 cells. Meanwhile, indoles could upregulate the expressions of G protein-coupled receptor 30 (GPR30)/5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK)/silent information regulator 1 (SIRT1) pathway in vitro. Furthermore, the neuroprotective effects of IAA and IPA are unveiled through activation of GPR30/AMPK/SIRT1 pathway in d-galactose induced aging mice. Finally, the regulatory effects of indoles on GPR30/AMPK/SIRT1 pathway are further confirmed by pretreating HT-22 and Neuro-2a with GPR30 antagonist of G15. In that case, indoles are furtherly proved with inhibitory effects on neurodegeneration by activating the GPR30/AMPK/SIRT1 pathway in aging process. CONCLUSIONS: The findings reveal that Trp metabolites significantly improve neurodegeneration via GPR30/AMPK/SIRT1 pathway in aging process. This study provides the potential novel intervention strategy and target to prevent the neurodegeneration.

Identification of Serum Ferritin-Specific Nanobodies and Development towards a Diagnostic Immunoassay.

Serum ferritin (SF) is an iron-rich protein tightly connected with iron homeostasis, and the variations are frequently observed in diseased states, including iron-deficiency anemia, inflammation, liver disease, and tumors, which renders SF level an indicator of potential malignancies in clinical practice. Nanobodies (Nbs) have been widely explored and developed into theranostic reagents. Surprisingly, no reports stated the identification of anti-SF Nbs, nor the potential of such Nbs as a diagnostic tool. In this study, we generated SF-specific Nbs and provided novel clinical diagnostic approaches to develop an immunoassay. An immune library was constructed after immunizing an alpaca with SF, and five Nbs specifically targeting human SF were retrieved. The obtained Nbs exhibited robust properties including high stability, affinity, and specificity. Then, an ELISA-based test using a heterologous Nb-pair was developed. The calibration curve demonstrated a linear range of SF between 9.0 to 1100 ng/mL, and a limit of detection (LOD) of 1.01 ng/mL. The detecting recovery and coefficient variation (CV) were determined by spiking different concentrations of SF into the serum sample, to verify the successful application of our selected Nbs for SF monitoring. In general, this study generated SF-specific Nbs and demonstrated their potential as diagnostic immunoassay tools.

Identification and Characterization of Specific Nanobodies against Trop-2 for Tumor Targeting.

Trophoblast cell-surface antigen 2 (Trop-2) is a tumor-associated antigen that is connected with the development of various tumors and has been identified as a promising target for tumor immunotherapy. To date, the immunotherapy against Trop-2 mainly relies on the specific targeting by monoclonal antibody in antibody-drug conjugate (ADC). Alternatively, the single domain antibodies of nanobodies (Nbs) possesses unique properties such as smaller size, better tissue penetration, etc., to make them good candidates for tumor targeting. Thus, it was proposed to develop anti-Trop-2 Nbs for tumor targeting in this study. Generally, three consecutive rounds of bio-panning were performed against immobilized recombinant Trop-2, and yielded three Nbs (Nb60, Nb65, and Nb108). The affinity of selected Nbs was determined in the nanomolar range, especially the good properties of Nb60 were verified as a promising candidate for tumor labeling. The binding to native Trop-2 was confirmed by flow cytometry against tumor cells. The inhibitory effects of the selected Nbs on tumor cell proliferation and migration were confirmed by wound healing and Transwell assay. The clear localization of the selected Nbs on the surface of tumor cells verified the potent labeling efficiency. In conclusion, this study provided several Nbs with the potential to be developed as targeting moiety of drug conjugates.

Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors.

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

Promotion Effect of EGCG on the Raised Expression of IL-23 through the Signaling of STAT3-BATF2-c-JUN/ATF2.

Tea polyphenol of epigallocatechin-3-gallate (EGCG) has been verified to possess multiple biological activities. Interleukin-23 (IL-23) is a heterodimeric cytokine consisting of two subunits of IL-23p19 and IL-12p40, with the functionality in regulating the production of cytokines under physiological or pathological conditions. By serendipity, the raised expression of IL-23 was observed after treating cells with EGCG, whereas the detailed mechanism remains poorly understood. This study was proposed to investigate the signaling related to EGCG-induced IL-23. The raised expression of IL-23 was confirmed primarily by intraperitoneally injecting with different concentrations of EGCG (0, 20, 50, 80 mg/kg) into BALB/c mice, and the raised expression was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results from enzyme-linked immunosorbent assay (ELISA) revealed the increase of IL-23 in serum from 116.09 to 153.90 pg/mL after treating with EGCG. The same results were also observed in RAW264.7 and peritoneal macrophages after treating with EGCG (0, 1, 5, 10, 25 µM) with the increased tendency of IL-23 in cultural medium (7.98 to 25.38 pg/mL for RAW264.7; 3.64 to 260.93 pg/mL for peritoneal macrophages). After preliminary exploration of the signaling related to the increased IL-23, the classical signaling pathways and key transcription factors, such as nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK) signaling pathways, and interferon regulatory factor 5 (IRF5), were demonstrated with no relevant contribution. A further study revealed the involvement of the key transcription factor of BATF2, which could antagonistically modulate the transcription and translation of IL-23. The signaling of STAT3-BATF2-c-JUN/ATF2-IL-23 has been further verified in RAW264.7 macrophages using the STAT3 inhibitor of AG490 and the activator of Colivelin TFA. The results indicated that EGCG inhibits the phosphorylation of STAT3 to facilitate the decreased level of BATF2, which contributed to the increased level of IL-23 by the enhancing heterodimerization of c-JUN and ATF2.

Ameliorative Effect of Dietary Tryptophan on Neurodegeneration and Inflammation in d-Galactose-Induced Aging Mice with the Potential Mechanism Relying on AMPK/SIRT1/PGC-1α Pathway and Gut Microbiota.

Dietary tryptophan affects intestinal homeostasis and neurogenesis, whereas the underlying mechanism and the reciprocal interaction between tryptophan and gut microbiota in aging are unclear. This investigation was performed to determine the effect and mechanism of tryptophan on intestinal- and neuro- health in aging. In present study, the 0.4% tryptophan diet significantly ameliorated the oxidative stress and inflammation in the aging mice, potentially through the regulation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) and nuclear factor κB (NF-κB) pathways. The 0.4% tryptophan diet increased the levels of indoles in colon contents, which indicated the potential contribution of tryptophan metabolites. Microbiome analysis revealed that the 0.4% tryptophan diet raised the relative abundance of Akkermansia in aging. The ameliorated effect of 0.4% tryptophan on neurodegeneration and neuroinflammation was summarized to potentially rely on the brain-derived neurotrophic factor- (BDNF) and NF-κB-related pathways. These findings provide the research evidence for the beneficial effect of tryptophan on aging.

The lipoprotein NlpD in Cronobacter sakazakii responds to acid stress and regulates macrophage resistance and virulence by maintaining membrane integrity.

Cronobacter sakazakii, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in C. sakazakii virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that nlpD encodes an acid-resistance factor in C. sakazakii.  Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.

Tailor-Made Cell-Based Biomimetic Nanoprobes for Fluorescence Imaging Guided Colorectal Cancer Chemo-immunotherapy.

Colorectal cancer has become one of the malignant tumors with a high rate of morbidity and mortality. Therefore, how to effectively treat colorectal cancer is crucial. Although nanodelivery system has been applied to the therapy of colorectal cancer, the majority of existing nanodelivery systems still have drawbacks such as low biocompatibility and poor targeting ability. In this work, tailor-made cell-based biomimetic nanoplatform was prepared to enhance the targeting and therapeutic effect for colorectal cancer chemo-immunotherapy. First, hollow long persistence luminescence nanomaterials were synthesized with superior background-free bioimaging effect and high drug-loading content. After loaded with cisplatin, the nanoplatform was camouflaged with tailor-made erythrocyte and programmed cell death receptor 1 (PD-1) expressed hybrid cell membrane. In vivo animal imaging experiment showed that the nanoplatform camouflaged with hybrid cell membrane not only had excellent immune escapability but also had excellent tumor active targeting ability. In vivo anticancer experiments showed that combined chemotherapy and immunotherapy of the nanoplatform could significantly inhibit tumor growth in tumor-bearing mice. In summary, the tailor-made cell-based membrane camouflage produced excellent immune escapability and cancer active targeting ability, providing a modality for biomimetic nanodelivery systems.

Green tea leaf powder prevents dyslipidemia in high-fat diet-fed mice by modulating gut microbiota.

BACKGROUND: In the past, most researchers paid more attention to the biological activity of tea infusion and tea polyphenols; however, the prebiotic role of tea leaf powder is still unknown. Green tea leaf powder is rich in dietary fiber and is suggested to be beneficial for human health. Only limited studies have looked at the effects of tea leaf powder (which mainly contains tea dietary fiber) on gut microbiota and health. OBJECTIVE: The purpose of our study was to determine the effects of green tea leaf powder in preventing hyperlipidemia and to understand its potential lipid-lowering mechanism. DESIGN: Mice in three treatment groups were fed high-fat diets (HFDs) by administering either 0.5, 1.0, or 2.0 g/kg•d dietary fiber-enriched green tea leaf powder of low, medium, or high, respectively, for 12 weeks. Serum biochemical analyses and mRNA gene expression levels of related energy and lipid metabolism biomarkers from the liver were investigated. In addition, 16S rRNA cecal microbiota and fecal short chain fatty acids (SCFAs) were tested. RESULTS: Green tea leaf powder reduced body weight and total cholesterol of HFD-fed mice in a dose-dependent manner. Green tea leaf powder also increased satiety hormone secretion and reduced systemic inflammation of HFD-fed mice. Real-time polymerase chain reaction (PCR) analyses reconfirmed that green tea leaf powder prevented dyslipidemia by enhancing hepatic mRNA expression levels of peroxisome proliferator-activated receptor alpha, cholesterol 7α-hydroxylase, and Adenosine triphosphate (ATP)-binding cassette transporter A1 and decreasing the expression of fatty acid synthase, sterol regulatory element-binding protein 1c, and liver X receptor. Green tea leaf powder promoted the growth of Blautia, Oscillibacter, Ruminiclostridium, Alloprevotella, and Butyrivibrio and inhibited the growth of Erysipelatoclostridium, Desulfovibrio, and Candidatus_Saccharimonas in the cecum of HFD-fed mice. CONCLUSION: In summary, our results indicate that green tea leaf powder improves lipid metabolism of HFD-fed mice in a dose-dependent manner. The potential mechanism involves a synergistic role in reprogramming gut microbiota, increasing satiety hormone secretion, and reducing systemic inflammation.

Dietary Supplementation of Foxtail Millet Ameliorates Colitis-Associated Colorectal Cancer in Mice via Activation of Gut Receptors and Suppression of the STAT3 Pathway.

Coarse cereal intake has been reported to be associated with reduced risk of colorectal cancer. However, evidence from intervention studies is absent and the molecular basis of this phenomenon remains largely unexplored. This study sought to investigate the effects of foxtail millet and rice, two common staple grains in Asia, on the progression of colitis-associated colorectal cancer (CAC) and define the mechanism involved. In total, 40 BALB/c mice were randomized into four groups. The Normal and azoxymethane/dextran sodium sulfate (AOM/DSS) groups were supplied with an AIN-93G diet, while the millet- and rice-treated groups were supplied with a modified AIN-93G diet. Compared to the AOM/DSS-induced CAC mice supplemented with rice, an increased survival rate, suppressed tumor burden, and reduced disease activity index were observed in the millet-treated group. The levels of IL-6 and IL-17 were decreased in the millet-treated group compared to both the AOM/DSS and AOM/DSS + rice groups. Millet treatment inhibited the phosphorylation of STAT3 and the related signaling proteins involved in cell proliferation, survival and angiogenesis. These beneficial effects were mediated by the activation of gut receptors AHR and GPCRs via the microbial metabolites (indole derivates and short-chain fatty acids) of foxtail millet. Moreover, millet-treatment increased the abundance of Bifidobacterium and Bacteroidales_S24-7 compared to the rice-treated mice. This study could help researchers to develop better dietary patterns that work against inflammatory bowel disease (IBD) and for CAC patients.

Correlation Analysis of Intestinal Redox State with the Gut Microbiota Reveals the Positive Intervention of Tea Polyphenols on Hyperlipidemia in High Fat Diet Fed Mice.

Tea polyphenols (TP) possess the ability to regulate dyslipidemia and gut microbiota dysbiosis. However, the underlying mechanism is still elusive. The present study explored the intervention of TP on high fat diet induced metabolic disorders, gut microbiota dysbiosis in mice, and the underlying intestinal mechanism. As a result, TP significantly ameliorated hyperlipidemia, improved the expression levels of hepatic lipid metabolism genes, and modulated gut microbiota. The underlying mechanism was supposed to rely on the maintaining of intestinal redox state by TP. Intestinal redox related indicators were significantly correlated with the distribution of gut microbiota. An unidentified genus of Lachnospiraceae, Bacteroides, Alistipes, and Faecalibaculum were identified as the biomarkers for intestinal redox state. Importantly, different dosages of TP modulated intestinal redox state and gut microbiota in varied patterns, and an overdose intake attenuated the beneficial effects on gut health. Our findings offered novel insights into the mechanism of TP on intestinal homeostasis.

Antler extracts stimulate chondrocyte proliferation and possess potent anti-oxidative, anti-inflammatory, and immune-modulatory properties.

The Sika deer antler is well known for its unique ability to regenerate repeatedly and grow rapidly. Furthermore, it is a precious traditional Chinese medicine and has been widely used for more than 20 centuries. The major bioactive components within the antlers are water-soluble proteins, polypeptides, and free amino acids. Many studies have shown that water-soluble antler extracts play pivotal roles in wound healing, immune system modulation, anti-oxidation, and anti-inflammation. However, the exact effects on chondrocytes are still largely unknown. In this study, we prepared fresh, aqueous extracts from growing deer antlers in a rapid growth stage. We isolated the chondrocytes from neonatal mouse rib cartilage and investigated the effects of antler extracts on chondrocyte viability. We also used the RNA-Seq method to analyze the gene expression pattern under antler extract treatment. We demonstrated that fresh extracts from Sika deer antlers in a rapid growth stage significantly promoted chondrocyte viability and kept chondrocytes proliferating continuously, while blocking maturation and further differentiation. Additionally, our results indicated that antler extracts might serve as a potent anti-oxidant, anti-inflammatory agent, and immune modulator to boost the abilities of chondrocytes against oxidative, inflammatory, and immune stresses. Thus, this study has greatly deepened our current knowledge of the molecular control of antler extracts on chondrocytes. It has also shed light on possible new strategies to further prevent and treat diseases of cartilage and other related diseases.

Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy.

The development of innovative targeted therapeutic approaches are expected to surpass the efficacy of current forms of treatments and cause less damage to healthy cells surrounding the tumor site. Since the first development of targeting agents from hybridoma's, monoclonal antibodies (mAbs) have been employed to inhibit tumor growth and proliferation directly or to deliver effector molecules to tumor cells. However, the full potential of such a delivery strategy is hampered by the size of mAbs, which will obstruct the targeted delivery system to access the tumor tissue. By serendipity, a new kind of functional homodimeric antibody format was discovered in camelidae, known as heavy-chain antibodies (HCAbs). The cloning of the variable domain of HCAbs produces an attractive minimal-sized alternative for mAbs, referred to as VHH or nanobodies (Nbs). Apart from their dimensions in the single digit nanometer range, the unique characteristics of Nbs combine a high stability and solubility, low immunogenicity and excellent affinity and specificity against all possible targets including tumor markers. This stimulated the development of tumor-targeted therapeutic strategies. Some autonomous Nbs have been shown to act as antagonistic drugs, but more importantly, the targeting capacity of Nbs has been exploited to create drug delivery systems. Obviously, Nb-based targeted cancer therapy is mainly focused toward extracellular tumor markers, since the membrane barrier prevents antibodies to reach the most promising intracellular tumor markers. Potential strategies, such as lentiviral vectors and bacterial type 3 secretion system, are proposed to deliver target-specific Nbs into tumor cells and to block tumor markers intracellularly. Simultaneously, Nbs have also been employed for in vivo molecular imaging to diagnose diseased tissues and to monitor the treatment effects. Here, we review the state of the art and focus on recent developments with Nbs as targeting moieties for drug delivery systems in cancer therapy and cancer imaging.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated.

Selection by phage display of nanobodies directed against hypoxia inducible factor-1α (HIF-1α).

Hypoxia, which promotes tumor invasion and metastasis, is a common phenomenon in solid tumors. Hypoxia generally leads to a higher expression level of hypoxia inducible factor-1 (HIF-1) in tumors (cells) relative to normal tissues (cells). Given the unique expression of HIF-1α in human cancers and its vital importance in mediating hypoxic adaptation, we have identified 20 different HIF-1α-specific nanobodies by using a llama-derived nonimmune phage display library. PAS-B domain of HIF-1α (HIF-1α-PAS-B) has been used as an antigen. Nanobody (VHH16) was selected from these 20 nanobodies by phage enzyme-linked immunosorbent assay. The preliminary analysis of biological activity demonstrates that VHH16 can specifically bind to HIF-1α with high affinity. VHH16 is the first nanobody that specifically binds to HIF-1α-PAS-B as well. We suggest here that VHH16 is useful in disease diagnosis and also has potential in medical applications.

Recent agents targeting HIF-1α for cancer therapy.

The discovery of hypoxia-inducible factor-1 (HIF-1) has led to an increasing understanding of the mechanism of tumor hypoxia in the past two decades. As a key transcriptional regulator, HIF-1 plays a central role in the adaptation of tumor cells to hypoxia by activating the transcription of targeting genes, which regulate several biological processes including angiogenesis, cell proliferation, survival, glucose metabolism and migration. The inhibitors of HIF-1 in cancer have provided us a new clue for the targeting cancer therapy. This review will introduce the general knowledge of the biology characteristic of HIF-1 and mechanism of O(2)-dependent regulation. Moreover, a number of chemical inhibitors plus protein and nucleic acid inhibitors are included and classified mainly based on their different mechanism of inhibiting action. We also prefer to discuss the advantages of protein and nucleic acid inhibitors compared with chemical inhibitors.