Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.

The combination of chemotherapy and immunotherapy motivates a potent immune system by triggering immunogenic cell death (ICD), showing great potential in inhibiting tumor growth and improving the immunosuppressive tumor microenvironment (ITM). However, the therapeutic effectiveness has been restricted by inferior drug bioavailability. Herein, we reported a universal bioresponsive doxorubicin (DOX)-based nanogel to achieve tumor-specific co-delivery of drugs. DOX-based mannose nanogels (DM NGs) was designed and choosed as an example to elucidate the mechanism of combined chemo-immunotherapy. As expected, the DM NGs exhibited prominent micellar stability, selective drug release and prolonged survival time, benefited from the enhanced tumor permeability and prolonged blood circulation. We discovered that the DOX delivered by DM NGs could induce powerful anti-tumor immune response facilitated by promoting ICD. Meanwhile, the released mannose from DM NGs was proved as a powerful and synergetic treatment for breast cancer in vitro and in vivo, via damaging the glucose metabolism in glycolysis and the tricarboxylic acid cycle. Overall, the regulation of tumor microenvironment with DOX-based nanogel is expected to be an effectual candidate strategy to overcome the current limitations of ICD-based immunotherapy, offering a paradigm for the exploitation of immunomodulatory nanomedicines.

BACKGROUND: Freshly isolated uncultured adipose tissue-derived stromal cells (u-ADSCs), containing miscellaneous cells like the relatively abundant mesenchymal stem cells, are attractive for repair and regenerative therapy. However, the detailed characteristics and therapeutic efficacy of u-ADSCs obtained from disease-affected hosts are unknown. We compared the properties of u-ADSCs obtained from wild-type mice and from a mouse model of non-alcoholic steatohepatitis (NASH).
METHODS: The NASH model was established by feeding C57BL/6J mice an atherogenic high-fat diet for 4 (NASH (4w)) or 12 weeks (NASH (12w)), followed by the isolation and characterization of u-ADSCs. Wild-type u-ADSCs or NASH-derived u-ADSCs were administered to mice with NASH cirrhosis, followed by analyses of hepatic inflammatory cells, antigen profiles, fibrosis, and gene expression.
RESULTS: Wild-type u-ADSCs and NASH-derived u-ADSCs did not show marked differences in surface antigen profiles. In NASH (4w) u-ADSCs, but not NASH (12w) u-ADSCs, the frequencies of the leukocyte markers CD11b, CD45, and CD44 were elevated; furthermore, we observed an increase in the M1/M2 macrophage ratio only in NASH (12w) u-ADSCs. Only in NASH-4w u-ADSCs, the expression levels cell cycle-related genes were higher than those in u-ADSCs. Wild-type u-ADSCs administered to mice with NASH-related cirrhosis decreased the infiltration of CD11b+, F4/80+, and Gr-1+ inflammatory cells, ameliorated fibrosis, and had a restorative effect on liver tissues, as determined by gene expression profiles and the NAFLD activity score. The therapeutic effects of NASH (4w) u-ADSCs and NASH (12w) u-ADSCs on NASH-related cirrhosis were highly similar to the effect of wild-type u-ADSCs, including reductions in inflammation and fibrosis.
CONCLUSIONS: NASH-derived u-ADSCs, similar to wild-type u-ADSCs, are applicable for reparative and regenerative therapy in mice with NASH.

Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial-mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative in vitro approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor β1 (TGF-β1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-β1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-β1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-β1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.

Hepatocellular carcinoma (HCC) has been known as the second common leading cancer worldwide, as it responds poorly to both chemotherapy and medication. Triptolide (TP), a diterpenoid triepoxide, is a promising treatment agent for its effective anticancer effect on multiple cancers including HCC. However, its clinical application has been limited owing to its severe systemic toxicities, low solubility, and fast elimination in the body. Therefore, to overcome the above obstacles, photo-activatable liposomes (LP) integrated with both photosensitizer Ce6 and chemotherapeutic drug TP (TP/Ce6-LP) was designed in the pursuit of controlled drug release and synergetic photodynamic therapy in HCC therapy. The TP encapsulated in liposomes accumulated to the tumor site due to the enhanced permeability and retention (EPR) effect. Under laser irradiation, the photosensitizer Ce6 generated reactive oxygen species (ROS) and further oxidized the unsaturated phospholipids. In this way, the liposomes were destroyed to release TP. TP/Ce6-LP with NIR laser irradiation (TP/Ce6-LP+L) showed the best anti-tumor effect both in vitro and in vivo on a patient derived tumor xenograft of HCC (PDXHCC). TP/Ce6-LP significantly reduced the side effects of TP. Furthermore, TP/Ce6-LP+L induced apoptosis through a caspase-3/PARP signaling pathway. Overall, TP/Ce6-LP+L is a novel potential treatment option in halting HCC progression with attenuated toxicity.

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 μmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

Although trastuzumab has succeeded in breast cancer treatment, acquired resistance is one of the prime obstacles for breast cancer therapies. There is an urgent need to develop novel HER2 antibodies against trastuzumab resistance. Here, we first rational designed avidity-imporved trastuzumab and pertuzumab variants, and explored the correlation between the binding avidity improvement and their antitumor activities. After characterization of a pertuzumab variant L56TY with potent antitumor activities, a bispecific immunoglobulin G-like CrossMab (Tras-Permut CrossMab) was generated from trastuzumab and binding avidity-improved pertuzumab variant L56TY. Although, the antitumor efficacy of trastuzumab was not enhanced by improving its binding avidity, binding avidity improvement could significantly increase the anti-proliferative and antibody-dependent cellular cytotoxicity (ADCC) activities of pertuzumab. Further studies showed that Tras-Permut CrossMab exhibited exceptional high efficiency to inhibit the progression of trastuzumab-resistant breast cancer. Notably, we found that calreticulin (CRT) exposure induced by Tras-Permut CrossMab was essential for induction of tumor-specific T cell immunity against tumor recurrence. These data indicated that simultaneous blockade of HER2 protein by Tras-Permut CrossMab could trigger CRT exposure and subsequently induce potent tumor-specific T cell immunity, suggesting it could be a promising therapeutic strategy against trastuzumab resistance.

Various components of mulberry leaves, such as iminosugars, flavonoids and polysaccharides, have been reported to exert anti-diabetic activity. The purpose of our present study was to examine the modulating effect of mulberry leaf polysaccharides (MLPs) on murine bone-marrow-derived dendritic cells (BMDCs). The ultrastructure, phenotype and functional maturation of BMDCs were studied using transmission electron microscopy (TEM), flow cytometry (FCM), and tested for phagocytosis, acid phosphatase (ACP) activity using an enzyme linked immunosorbent assay (ELISA). Our results demonstrated that MLPs could markedly induce BMDC maturation by up-regulating the expression of membrane phenotypic markers, such as CD80, CD86, CD83,CD40, and MHC II, down-regulating phagocytosis and ACP activity, and by enhancing the production of interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) secreted by BMDCs. We therefore concluded that MLPs can positively modulate BMDCs.