Gelatin methacryloyl microneedle loaded with 3D-MSC-Exosomes for the protection of ischemia-reperfusion.

Exosomes (Exo) generated from mesenchymal stem cells (MSCs) have great therapeutic potential in ischemia-reperfusion treatment. For best therapeutic effect, high quality Exo product and effective delivery system are indispensable. In this study, we developed a new strategy for ischemia-reperfusion recovery by combining MSCs 3D (3D-MSC) culturing technology to generate Exo (3D-MSC-Exo) and microneedle for topical delivery. Firstly, primary MSCs from neonatal mice were isolated and 3D cultured with gelatin methacryloyl (GelMA) hydrogel to prepare 3D-MSC-Exo. The 3D-MSC showed better viability and 3D-MSC-Exo exhibited more effective effects of reducing neuroinflammation, inhibiting glial scarring, and promoting angiogenesis. Subsequently, the biocompatible GelMA was used to construct microneedles for 3D-Exo delivery (GelMA-MN@3D-Exo). The results demonstrated GelMA microneedles had excellent 3D-Exo loading capacity and enabled continuous 3D-Exo release to maintain effective therapeutic concentrations. Furthermore, the rat middle cerebral artery occlusion (MCAO) model was established to evaluate the therapeutic effect of GelMA-MN@3D-Exo in ischemia-reperfusion in vivo. Animal experiments showed that the GelMA-MN@3D-Exo system could effectively reduce the local neuroinflammatory reaction, promote angiogenesis and minimize glial scar proliferation in ischemia-reperfusion. The underlying reasons for the stronger neuroprotective effect of 3D-Exo was further studied using mass spectrometry and transcriptome assays, verifying their effects on immune regulation and cell proliferation. Taken together, our findings demonstrated that GelMA-MN@3D-Exo microneedle can effectively attenuate ischemia-reperfusion cell damage in the MCAO model, which provides a promising therapeutic strategy for ischemia-reperfusion recovery.

Three-dimensional-cultured MSC-derived exosome with hydrogel for cerebral ischemia repair.

Microglia-mediated neuroinflammatory response, one of the most essential pathological processes of cerebral ischemia-reperfusion (I/R) injury, is acknowledged as the main factors leading to poor prognosis of cerebral ischemia. Exosome derived from mesenchymal stem cell (MSC-Exo) exhibits neuroprotective functions by reducing cerebral ischemia-induced neuroinflammatory response and promoting angiogenesis. However, MSC-Exo has disadvantages such as insufficient targeting capability and low production, which limits their clinical applications. Here, we fabricated gelatin methacryloyl (GelMA) hydrogel for three-dimensional (3D) culture of MSCs. It is indicated that 3D environment could simulate the biological niches of MSCs, thereby significantly increasing the cell stemness of MSCs and improving the yield of MSCs-derived exosomes (3D-Exo). In this study, we utilized the modified Longa method to induce middle cerebral artery occlusion (MCAO) model. Additionally, in vitro and in vivo studies were conducted to interrogate the mechanism of the stronger neuroprotective effect of 3D-Exo. Furthermore, the administration of 3D-Exo in MCAO model could promote neovascularization in infarct region and result in a significant suppression of inflammatory response. This study proposed an exosome-based targeting delivery system for cerebral ischemia and provided a promising strategy for efficient and large-scale production of MSC-Exo.

Establishment of a novel double-monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA): tool for human B7-H4 detection in autoimmune diseases.

B7-H4, one of the immunoregulatory proteins, plays an inhibitory role by inhibiting T cell proliferation and cytokine production. Nevertheless, the significance of soluble B7-H4 (sB7-H4) in autoimmune diseases is unclear. In our study, we developed two novel mouse anti-human B7-H4 monoclonal antibodies (mAbs) (clones 8D4 and 7E1) with utilities for flow cytometry, immunoblotting and immunofluorescence. We characterized 7E1 as a functional antibody with antagonistic activity, which could promote T cell proliferation and regulate cytokine production. Furthermore, based on the different epitope specificities, we established a novel enzyme-linked immunosorbent assay (ELISA) which could detect sB7-H4 sensitively and specifically. Using this ELISA kit, sB7-H4 was observed in a high proportion of autoimmune diseases patients. We found that the levels of sB7-H4 were significantly higher in patients with systemic lupus erythematosus (SLE), type I diabetes (T1D) and Graves' disease (GD). Together, sB7-H4 in human serum is regarded not only as a regulator of T cell activation but may also be a diagnostic marker of autoimmune diseases.

The novel Nrf2 activator CDDO-EA attenuates cerebral ischemic injury by promoting microglia/macrophage polarization toward M2 phenotype in mice.

The aim of present study was to explore whether 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO)-ethylamide (CDDO-EA) attenuates cerebral ischemic injury and its possible mechanisms using a middle cerebral artery occlusion (MCAO) model in C57BL/6 mice. Our results showed that intraperitoneal injection (i.p.) of CDDO-EA (2 and 4 mg/kg) augmented NFE2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in ischemic cortex after MCAO. Moreover, CDDO-EA (2 mg/kg, i.p.) significantly enhanced Nrf2 nuclear accumulation, associated with increased cytosolic HO-1 expression, reduced neurological deficit and infarct volume as well as neural apoptosis, and shifted polarization of microglia/macrophages toward an antiinflammatory M2 phenotype in ischemic cortex after MCAO. Using an in vitro model, we confirmed that CDDO-EA (100 µg/mL) increased HO-1 expression and primed microglial polarization toward M2 phenotype under inflammatory stimulation in BV2 microglial cells. These findings suggest that a novel Nrf2 activator CDDO-EA confers neuroprotection against ischemic injury.

Determinants of multidrug-resistant tuberculosis in Henan province in China: a case control study.

BACKGROUND: Multi-drug resistance (MDR) has been a cause of concern for tuberculosis (TB) control in both developed and developing countries. This study described the characteristics and risk factors associated with MDR-TB among 287 cases and 291 controls in Henan province, China. METHODS: A hospital-based case-control study was conducted between June 2012 and December 2013. The study subjects were selected using multistage probability sampling. Multivariate conditional logistic regression models were used to determine the risk factors associated with MDR-TB. RESULTS: The following risk factors for MDR-TB were identified: previous TB treatment (AOR = 4.51, 95% CI: 3.55-5.56), male sex (AOR = 1.09, 95% CI: 0.24-1.88), high school or lower education degree (AOR = 1.87, 95% CI: 1.27-2.69), unemployment (AOR = 1.30, 95% CI: 0.78-2.52), long distance of residence from the health facility (AOR = 6.66,95% CI: 5.92-7.72), smoking (AOR = 2.07, 95% CI: 1.66-3.19), poor knowledge regarding MDR-TB (AOR = 2.06, 95% CI: 1.66-2.92), traveling by foot to reach the health facility (AOR = 1.85, 95% CI: 1.12-3.09), estimated amount of time to reach the health facility was greater than 3 h (AOR = 1.42, 95% CI: 0.51-2.35), social stigma (AOR = 1.17, 95% CI: 0.27-2.03), having an opportunistic infection (AOR = 1.45, 95% CI: 0.58-2.4), more than 3 TB foci in the lungs (AOR = 1.98, 95% CI: 1.49-3.25), total time of first treatment was more than 8 months (AOR = 1.39, 95% CI: 0.65-2.54), adverse effects of anti-TB medication (AOR = 2.39, 95% CI: 1.40-3.26), and more than 3 prior episodes of anti-TB treatment (AOR = 1.83, 95% CI: 1.26-2.80). CONCLUSION: The identified risk factors should be given priority in TB control programs. Additionally, there is a compelling need for better management and control of MDR-TB, particularly through increasing laboratory capacity, regular screening, enhancing drug sensitivity testing, novel MDR-TB drug regimens, and adherence to medication.

MicroRNA-223 Is Upregulated in Active Tuberculosis Patients and Inhibits Apoptosis of Macrophages by Targeting FOXO3.

BACKGROUND: Macrophage apoptosis is a host innate defense mechanism against tuberculosis (TB). AIM: In this study, we aimed to investigate the role of microRNA-223 (miR-223) in macrophage apoptosis of TB. METHODS: We analyzed apoptosis in peripheral blood macrophages of active TB patients, infected human macrophages (TDMs and MDMs) with the Mycobacterium tuberculosis (Mtb) strain H37Rv, and observed the expression of miR-223 to investigate the relationship between miR-223 and macrophage apoptosis induced by Mtb. RESULTS: The apoptosis rate of peripheral blood macrophages decreased in active TB patients compared with healthy controls, and miR-223 expression increased significantly in macrophages after H37Rv infection. Transfection of human macrophages (TDMs and MDMs) with miR-223 inhibited macrophage apoptosis. We also demonstrated that miR-223 directly suppressed forkhead box O3 (FOXO3), and FOXO3 played a critical role as a mediator of the biological effects of miR-223 in macrophage apoptosis. The overexpression of FOXO3 remarkably reversed the apoptosis inhibitory effect of miR-223. CONCLUSION: Our data provide new clues for the essential role of miR-223 in the regulation of anti-Mtb-directed immune responses, which relies on the regulation of FOXO3 expression.

MiR-155 is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO3.

The aim of the present study was to investigate the association between microRNA (miR)-155 and apoptosis of monocytes infected by Mycobacterium tuberculosis, to examine the effect of forkhead box O3 (FOXO3) on miR­155. The present study analysed the apoptosis of CD14+ in the peripheral blood of patients with active tuberculosis, disposed the THP­1 human monocytic cell line by BCG and examined the expression of miR­155. Furthermore, the expression of FOXO3 in THP­1 cells was determined, and wild- and mutant-type luciferase reporter plasmids containing FOXO3 3'­untranslated regions (UTRs) were constructed to analyse the expression of luciferase. Finally, an over­expression plasmid was constructed, and THP-1 cells were transfected with control miRNA, miR­155 and the plasmid, which revealed that miR­155 inhibited the apoptosis of THP­1 cells. miR­155 in the THP­1 cells infected by BCG was upregulated and apoptosis also increased. However, the apoptosis declined when the cells were transfected with the control miRNA and miR­155. Folllowing transfection with miR­155, the expression of FOXO3 decreased. Transfection with miR­155 and the FOXO3 3'-UTRs significantly reduced luciferase, and overexpression of FOXO3 reversed the inhibitory role of miR­155. From these results, it was concluded that mycobacteria can improve the level of miR­155, while BCG can induce apoptosis in THP­1 cells. The results suggested FOXO3 is a downstream target gene of miR­155, which combines 3'-UTRs to inhibit the expression of FOXO3.

The association between serum miR-155 and natural killer cells from tuberculosis patients.

Tuberculosis (TB) is still an infectious disease that greatly threatens human health, and is always refractory to the current therapeutic modalities. Accumulated evidence revealed that microRNAs (miRNAs) are closely with various pathologies, such as TB. The possibilities of miRNAs as diagnostic biomarkers and therapeutic targets have been proved. However, it is still unknown if miRNA is implicated in the TB-associated immunity. This study revealed that miR-155, which has been shown to suppress the activation of natural killer (NK) cells associated with tumors, was downregulated in serum samples of TB patients (n=90), compared with healthy controls (n=31). Cytotoxicity assays indicated that NK cells, which have been demonstrated to promote TB progression, exhibited lower cytotoxicity in high serum miR-155 TB patients (n=37). There is an inverse relationship between serum miR-155 abundance and NK cell cytotoxicity (R=-0.659, P=0.000). Further studies demonstrated that miR-155 level is inversely associated with the concentration of TNFα secreted by NK cells from TB patients (n=37, R=-0.694, P=0.000). Collectively, serum miR-155 level was shown to be negatively associated with the TB-suppressing activity of NK cells, and this miRNA can be used as a potential therapeutic agent for TB treatment.