Mesenchymal stromal cells and their small extracellular vesicles in allergic diseases: From immunomodulation to therapy.

Mesenchymal stromal cells (MSCs) have long been considered a potential tool for treatment of allergic inflammatory diseases, owing to their immunomodulatory characteristics. In recent decades, the medical utility of MSCs has been evaluated both in vitro and in vivo, providing a foundation for therapeutic applications. However, the existing limitations of MSC therapy indicate the necessity for novel therapies. Notably, small extracellular vesicles (sEV) derived from MSCs have emerged rapidly as candidates instead of their parental cells. The acquisition of abundant and scalable MSC-sEV is an obstacle for clinical applications. The potential application of MSC-sEV in allergic diseases has attracted increasing attention from researchers. By carrying biological microRNAs or active proteins, MSC-sEV can modulate the function of various innate and adaptive immune cells. In this review, we summarise the recent advances in the immunomodulatory properties of MSCs in allergic diseases, the cellular sources of MSC-sEV, and the methods for obtaining high-quality human MSC-sEV. In addition, we discuss the immunoregulatory capacity of MSCs and MSC-sEV for the treatment of asthma, atopic dermatitis, and allergic rhinitis, with a special emphasis on their immunoregulatory effects and the underlying mechanisms of immune cell modulation.

Mesenchymal stromal cells-derived small extracellular vesicles modulate DC function to suppress Th2 responses via IL-10 in patients with allergic rhinitis.

Mesenchymal stromal cells (MSCs) are well known for their immunoregulatory roles on allergic inflammation particularly by acting on T cells, B cells, and dendritic cells (DCs). MSC-derived small extracellular vesicles (MSC-sEV) are increasingly considered as one of the main factors for the effects of MSCs on immune responses. However, the effects of MSC-sEV on DCs in allergic diseases remain unclear. MSC-sEV were prepared from the induced pluripotent stem cells (iPSC)-MSCs by anion-exchange chromatography, and were characterized with the size, morphology, and specific markers. Human monocyte-derived DCs were generated and cultured in the presence of MSC-sEV to differentiate the so-called sEV-immature DCs (sEV-iDCs) and sEV-mature DCs (sEV-mDCs), respectively. The phenotypes and the phagocytic ability of sEV-iDCs were analyzed by flow cytometry. sEV-mDCs were co-cultured with isolated CD4+ T cells or peripheral blood mononuclear cells (PBMCs) from patients with allergic rhinitis. The levels of Th1 and Th2 cytokines produced by T cells were examined by ELISA and intracellular flow staining. And the following mechanisms were further investigated. We demonstrated that MSC-sEV inhibited the differentiation of human monocytes to iDCs with downregulation of the expression of CD40, CD80, CD86, and HLA-DR, but had no effects on mDCs with these markers. However, MSC-sEV treatment enhanced the phagocytic ability of mDCs. More importantly, using anti-IL-10 monoclonal antibody or IL-10Rα blocking antibody, we identified that sEV-mDCs suppressed the Th2 immune response by reducing the production of IL-4, IL-9, and IL-13 via IL-10. Furthermore, sEV-mDCs increased the level of Treg cells. Our study identified that mDCs treated with MSC-sEV inhibited the Th2 responses, providing novel evidence of the potential cell-free therapy acting on DCs in allergic airway diseases.

[Bladder wall neourethra technique improves early urinary continence after laparoscopic radical prostatectomy].

OBJECTIVE: To study the effect of the bladder wall neourethra (BWN) technique on early urinary continence after laparoscopic radical prostatectomy (LRP). METHODS: We prospectively selected 40 cases of LRP performed in our hospital from August 2020 to August 2021 and randomly divided them into a BWN group (n = 20) and a control group (n = 20). We recorded the urinary continence rate of the two groups of patients at 7, 30, 90 and 180 days, and measured the maximum urethral pressure (MUP), functional urethral length (FUL) and functional urethral area (UFA) and observed the shape of the neourethra closure by MRI at 1 month after catheter removal. RESULTS: The urinary continence rates were significantly higher in the BWN than in the control group at 7 days (90.0% vs 25.0%, P < 0.001), 30 days (95.0% vs 35.0%, P < 0.001), 90 days (100% vs 60.0%, P < 0.05) and 180 days (100% vs 90.0%, P > 0.05) after catheter removal. No statistically significant difference was observed in MUP between the two groups (P > 0.05). FUL and FUA were remarkably higher in the BWN than in the control group (P < 0.01). MRI showed tight closure of the neourethra in the BWN group in the urine storage period. CONCLUSION: The BWN technique can significantly prolong FUL and improve early urinary continence after LRP.

Mesenchymal stem cells regulate type 2 innate lymphoid cells via regulatory T cells through ICOS-ICOSL interaction.

Group 2 innate lymphoid cells (ILC2s) are recognized as key controllers and effectors of type 2 inflammation. Mesenchymal stem cells (MSCs) have been shown to alleviate type 2 inflammation by modulating T lymphocyte subsets and decreasing TH 2 cytokine levels. However, the effects of MSCs on ILC2s have not been investigated. In this study, we investigated the potential immunomodulatory effects of MSCs on ILC2s in peripheral blood mononuclear cells (PBMCs) from allergic rhinitis patients and healthy subjects. We further investigated the mechanisms involved in the MSC modulation using isolated lineage negative (Lin- ) cells. PBMCs and Lin- cells were cocultured with induced pluripotent stem cell-derived MSCs (iPSC-MSCs) under the stimulation of epithelial cytokines IL-25 and IL-33. And the ILC2 levels and functions were examined and the possible mechanisms were investigated based on regulatory T (Treg) cells and ICOS-ICOSL pathway. iPSC-MSCs successfully decreased the high levels of IL-13, IL-9, and IL-5 in PBMCs in response to IL-25, IL-33, and the high percentages of IL-13+ ILC2s and IL-9+ ILC2s in response to epithelial cytokines were significantly reversed after the treatment of iPSC-MSCs. However, iPSC-MSCs were found directly to enhance ILC2 levels and functions via ICOS-ICOSL interaction in Lin- cells and pure ILC2s. iPSC-MSCs exerted their inhibitory effects on ILC2s via activating Treg cells through ICOS-ICOSL interaction. The MSC-induced Treg cells then suppressed ILC2s by secreting IL-10 in the coculture system. This study revealed that human MSCs suppressed ILC2s via Treg cells through ICOS-ICOSL interaction, which provides further insight to regulate ILC2s in inflammatory disorders.

Small extracellular vesicles derived from human MSCs prevent allergic airway inflammation via immunomodulation on pulmonary macrophages.

Allergic airway inflammation is a major public health disease that affects up to 300 million people in the world. However, its management remains largely unsatisfactory. The dysfunction of pulmonary macrophages contributes greatly to the development of allergic airway inflammation. It has been reported that small extracellular vesicles derived from mesenchymal stromal cells (MSC-sEV) were able to display extensive therapeutic effects in some immune diseases. This study aimed to investigate the effects of MSC-sEV on allergic airway inflammation, and the role of macrophages involved in it. We successfully isolated MSC-sEV by using anion exchange chromatography, which were morphologically intact and positive for the specific EV markers. MSC-sEV significantly reduced infiltration of inflammatory cells and number of epithelial goblet cells in lung tissues of mice with allergic airway inflammation. Levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid were also significantly decreased. Importantly, levels of monocytes-derived alveolar macrophages and M2 macrophages were significantly reduced by MSC-sEV. MSC-sEV were excreted through spleen and liver at 24 h post-administration in mice, and were able to be taken in by macrophages both in vivo and in vitro. In addition, proteomics analysis of MSC-sEV revealed that the indicated three types of MSC-sEV contained different quantities of proteins and shared 312 common proteins, which may be involved in the therapeutic effects of MSC-sEV. In total, our study demonstrated that MSC-sEV isolated by anion exchange chromatography were able to ameliorate Th2-dominant allergic airway inflammation through immunoregulation on pulmonary macrophages, suggesting that MSC-sEV were promising alternative therapy for allergic airway inflammation in the future.

Association between methylation of DNA damage response-related genes and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride.

In the present study, we investigated the association between methylation of DNA damage response-related genes such as cyclin-dependent kinase inhibitor (CDKN)2A, Ras association (RalGDS/AF-6) domain family member (RASSF)1A, O6-methylguanine DNA methyltransferase (MGMT), Kirsten rat sarcoma viral oncogene homolog (KRAS), and spleen-associated tyrosine kinase (SYK) and DNA damage in hepatocytes of rats following subchronic exposure to vinyl chloride (VC). Sixty-four healthy rats were randomly divided into three VC exposure groups (5, 25, and 125 mg/kg) and an untreated negative control group (n = 16 each). VC was administered by intraperitoneal injection every other day for a total of three times a week. Eight randomly selected rats from each group were sacrificed at the end of 6 and 12 weeks, and liver tissue was harvested for the comet assay and for assessment of DNA methylation level and mRNA expression of related genes by PCR. Overall methylation levels in the genome of hepatocytes in VC-exposed rats were higher than those in the control group at 6 and 12 weeks (P < 0.05), although no differences were observed with regarding to dose (P > 0.05). After 12 weeks of exposure, differences in the methylation of RASSF1A and MGMT promoter regions were observed between the high-dose group and other groups (P < 0.05), whereas no differences were observed for the KRAS, SYK, and CDKN2A promoters (P > 0.05). These results suggest that DNA damage and increased genome-wide methylation are biomarkers for VC exposure and that RASSF1A and MGMT promoter methylation is related to the carcinogenic mechanism of VC.

Connexin 43-Mediated Mitochondrial Transfer of iPSC-MSCs Alleviates Asthma Inflammation.

We previously identified an immunomodulatory role of human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) in asthmatic inflammation. Mitochondrial transfer from bone marrow MSCs to epithelial cells can result in the attenuation of acute lung injury in mice. However, the effects of mitochondrial transfer from iPSC-MSCs to epithelial cells in asthma and the mechanisms underlying these effects are unclear. We found that iPSC-MSC transplantation significantly reduced T helper 2 cytokines, attenuated the mitochondrial dysfunction of epithelial cells, and alleviated asthma inflammation in mice. Tunneling nanotubes (TNTs) were formed between iPSC-MSCs and epithelial cells, and mitochondrial transfer from iPSC-MSCs to epithelial cells via TNTs was observed both in vitro and in mice. Overexpression or silencing of connexin 43 (CX43) in iPSC-MSCs demonstrated that CX43 plays a critical role in the regulation of TNT formation by mediating mitochondrial transfer between iPSC-MSCs and epithelial cells. This study provides a therapeutic strategy for targeting asthma inflammation.

Induced pluripotent stem cell-derived mesenchymal stem cells activate quiescent T cells and elevate regulatory T cell response via NF-κB in allergic rhinitis patients.

BACKGROUND: It has been demonstrated previously that induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSCs) have immunosuppressive effects on activated T cells. However, the effects of iPSC-MSCs on quiescent T cells are still unknown. The aim of this study was to identify the immunomodulatory role of iPSC-MSCs on resting peripheral blood mononuclear cells (PBMCs) from allergic rhinitis (AR) patients. METHODS: PBMCs were cocultured with iPSC-MSCs without any stimulation, following which lymphocyte proliferation, activation of T cells, TH1/TH2 and regulatory T (Treg) cell differentiation, and Treg cell function were analyzed. The roles of soluble factors and cell-cell contact were examined to investigate the mechanisms involved. RESULTS: iPSC-MSCs promoted the proliferation of resting lymphocytes, activated CD4+ and CD8+ T cells, and upregulated and activated Treg cells without any additional stimulation. In addition, iPSC-MSCs balanced biased TH1/TH2 cytokine levels. Cell-cell contact was confirmed to be a possible mechanism involved. NF-κB was identified to play an important role in the immunomodulatory effects of iPSC-MSCs on quiescent T cells. CONCLUSIONS: iPSC-MSCs activate quiescent T cells and elevate regulatory T-cell response in AR patients, suggesting different immunomodulatory functions of iPSC-MSCs according to the phases of diseases. Therefore, iPSC-MSCs are a potential therapeutic candidate for treating allergic airway inflammation.

MicroRNA-21 Mediates the Protective Effects of Mesenchymal Stem Cells Derived from iPSCs to Human Bronchial Epithelial Cell Injury Under Hypoxia.

Airway epithelial cell injury is a key triggering event to activate allergic airway inflammation, such as asthma. We previously reported that administration of mesenchymal stem cells (MSCs) significantly alleviated allergic inflammation in a mouse model of asthma, and the mmu-miR-21/ACVR2A axis may be involved. However, whether MSCs protect against bronchial epithelial cell injury induced by hypoxia, and the underlying mechanism, remain unknown. In our study, the human bronchial epithelial cell line BEAS-2B was induced to undergo apoptosis with a hypoxia mimic of cobalt chloride (CoCl2) damage. Treatment of MSCs derived from induced pluripotent stem cells (iPSCs) significantly decreased apoptosis of BEAS-2B cells. There was high miR-21 expression in injured BEAS-2B cells after MSC treatment. Transfection of the miR-21 mimic significantly decreased apoptosis of BEAS-2B, and transfection of a miR-21 inhibitor significantly increased apoptosis. More importantly, the protective effects of MSCs on injured BEAS-2B were reversed by transfection of the miR-21 inhibitor. Binding sites of human miR-21 were identified in the 3'UTR of human ACVR2A. We further determined that CoCl2 stimulation increased ACVR2A expression at both the mRNA and protein levels. Moreover, transfection of the miR-21 mimic further up-regulated ACVR2A expression induced by CoCl2, whereas transfection of the miR-21 inhibitor down-regulated ACVR2A expression. In addition, MSCs increased ACVR2A expression in BEAS-2B cells; however, this effect was reversed after transfection of the miR-21 inhibitor. Our data suggested that MSCs protect bronchial epithelial cells from hypoxic injury via miR-21, which may represent an important target. These findings suggest the potentially wide application of MSCs for epithelial cell injury during hypoxia.

Rb deficiency accelerates progression of carcinoma of the urinary bladder in vivo and in vitro through inhibiting autophagy and apoptosis.

Urinary bladder cancer is known as a common cancer diagnosed across the world and results in significant mortality and morbidity rates among patients. The retinoblastoma (Rb) protein, as a main tumor suppressor, controls cellular responses to potentially oncogenic stimulation. Rb phosphorylation could disrupt E2F complex formation, resulting in diverse transcription factor dysfunction. In our study, we investigated how Rb is involved in controlling urinary bladder cancer progression. The results indicate that Rb expression is reduced in mice with urinary bladder tumor, and its suppression leads to urinary bladder cancer progression in vivo and in vitro. Rb mutation directly results in tumor size with lower survival rate in vivo. Rb knockdown in vitro promoted bladder tumor cell proliferation, migration and invasion. Interestingly, Rb knockout and knockdown result in autophagy and apoptosis inhibition via suppressing p53 and caspase-3 signaling pathways, enhancing bladder cancer development in vitro and in vivo. These findings reveal that Rb deficiency accelerated urinary bladder cancer progression, exposing an important role of Rb in suppressing urinary bladder cancer for treatment in the future.

Rb knockdown accelerates bladder cancer progression through E2F3 activation.

Bladder cancer is one of the most common cancers diagnosed in the world and leads to significant mortality and morbidity among affected patients. The retinoblastoma (Rb) protein is a main tumor suppressor, controlling cellular responses to potentially oncogenic stimulation. E2F3 was invariably disrupted in different human cancers for its central role in the control of cellular proliferation. Here, we investigated how Rb is integrated to control bladder cancer progression through E2F3 and p53 regulation. The results exhibit that Rb expression is lower in patients with bladder tumor, while E2F3 level is high. Rb knockdown enhanced bladder tumor cell proliferation and migration, aggravated with p53 silence. Interestingly, Rb silence results in E2F3, Myc and mTOR signaling pathway activation, contributing to bladder cancer cell proliferation and apoptosis suppression mainly through caspase-3 inhibition in vitro and in vivo. Immunohistochemical analysis revealed that Rb is highly expressed in normal bladder cells, but was repressed in tumor tissues of the bladder completely, suggesting a possible role of Rb as a tumor suppressor.

Actein induces autophagy and apoptosis in human bladder cancer by potentiating ROS/JNK and inhibiting AKT pathways.

Human bladder cancer is a common genitourinary malignant cancer worldwide. However, new therapeutic strategies are required to overcome its stagnated survival rate. Triterpene glycoside Actein (ACT), extracted from the herb black cohosh, suppresses the growth of human breast cancer cells. Our study attempted to explore the role of ACT in human bladder cancer cell growth and to reveal the underlying molecular mechanisms. We found that ACT significantly impeded the bladder cancer cell proliferation via induction of G2/M cycle arrest. Additionally, ACT administration triggered autophagy and apoptosis in bladder cancer cells, proved by the autophagosome formation, LC3B-II accumulation, improved cleavage of Caspases/poly (ADP-ribose) polymerase (PARP). Furthermore, reduction of reactive oxygen species (ROS) and p-c-Jun N-terminal kinase (JNK) could markedly reverse ACT-induced autophagy and apoptosis. In contrast, AKT and mammalian target of rapamycin (mTOR) were greatly de-phosphorylated by ACT, while suppressing AKT and mTOR activity could enhance the effects of ACT on apoptosis and autophagy induction. In vivo, ACT reduced the tumor growth with little toxicity. Taken together, our findings indicated that ACT suppressed cell proliferation, induced autophagy and apoptosis through promoting ROS/JNK activation, and blunting AKT pathway in human bladder cancer, which indicated that ACT might be an effective candidate against human bladder cancer in future.

MicroRNAs Involved in Asthma After Mesenchymal Stem Cells Treatment.

Administration of human bone marrow-derived mesenchymal stem cells (BM-MSCs) significantly alleviates allergic airway inflammation. There are no studies that refer to the role of microRNAs (miRNAs) after the BM-MSCs treatment in airway allergic inflammation. We induced a mouse model of asthma and performed the transplantation of BM-MSCs. We analyzed aberrant miRNAs and key immune regulators using both miRNA and messenger RNA (mRNA) polymerase chain reaction (PCR) arrays. We identified that 296 miRNAs were differently expressed after the induction of asthma and/or the treatment of BM-MSCs, in which 14 miRNAs presented the reverse variation tendency between asthma induction and BM-MSCs transplantation. Mmu-miR-21a-3p, mmu-miR-449c-5p, and mmu-miR-496a-3p were further confirmed to be differently expressed with additional samples and quantitative real-time PCR. With an mRNA PCR array, we identified 19 genes to be involved in the allergy induction and the administration of BM-MSCs. Further target genes analysis revealed that mmu-miR-21a-3p was significantly correlated with the immune regulator activin A receptor, Type IIA (Acvr2a). Mmu-miR-21a-3p had opposite expression with Acvr2a after asthma and BM-MSCs treatment. Acvr2a had binding sites for miR-21a for both mice and human, suggesting that miR-21/Acvr2a axis is conserved between human and mice. Dual-luciferase reporter assay showed that mmu-miR-21a-3p negatively regulated the transcript of Acvr2a. In addition, has-miR-21a inhibitor significantly increased the expression of Acvr2a mRNA in BEAS-2B cells under lipopolysaccharide stimulation. Our results suggest that there were different miRNA and mRNA profiles after asthma induction and BM-MSCs treatment, and the miR-21/Acvr2a axis is an important mechanism for the induction of asthmatic inflammation.

Computed tomography and magnetic resonance imaging findings of peripheral primitive neuroectodermal tumors of the head and neck.

PURPOSE: We aimed to analyze the computed tomography (CT) and magnetic resonance imaging (MRI) findings of peripheral primitive neuroectodermal tumor (pPNET) of the head and neck. METHODS: Eight patients with pPNET of the head and neck confirmed by histopathological examination were analyzed retrospectively. RESULTS: The average patient age was 8 years. The tumor location in the 8 patients was as follows: maxillofacial region (right, 2; left, 1), left maxillary sinus (1), right masticator space (1), left carotid space (1), right infratemporal fossa (1), and left parotid gland (1). All 5 patients who underwent CT demonstrated ill-defined soft masses and no calcification. Three patients with tumors in the maxillofacial region showed homogeneous small masses and a mild enhancement. The patient with left maxillary sinus tumor showed a heterogeneous mass with patchy, necrotic foci and mild heterogeneous enhancement. The patient with right masticator space tumor showed a heterogeneous mass, and marked heterogeneous enhancement. The T1-weighted images of the patients with right infratemporal fossa, left carotid space, and left parotid gland tumors were isointense. The T2-weighted images were heterogeneous and mildly hyperintense in 2 patients and hyperintense in 1 patient. Heterogeneous intermediate enhancement was demonstrated in 2 patients and mild ring enhancement in 1 patient. CONCLUSION: The imaging features of pPNET of the head and neck are non-specific. An ill-defined, aggressive mass and variable enhancement on CT and MR images may suggest the diagnosis of pPNET. Peripheral PNET should be included in the differential diagnosis of children and adolescents' regional tumors.

[The value of endoscopic mucosal resection for dysplasia and early-stage cancer of the esophagus and gastric cardia].

OBJECTIVE: To evaluate the long-term effect and clinical value of endoscopic mucosal resection (EMR) with transparent cap for dysplasia and early-stage cancer of the esophagus and gastric cardia. METHODS: From September 1996 to June 2007, 154 lesions in the esophagus or gastric cardia of 147 patients were treated using EMR with transparent cap. Among the lesions, there were 69 early-stage squamous-cell carcinomas in 64 patients and 47 squamous cell precancerous lesions of the esophagus in 45 patients, with an average lesion size of (14.8 +/- 6.1) mm (range, 3-40 mm), furthermore, there were 23 early-stage adenocarcinomas in 23 patients and 15 precancerous lesions in the gastric cardia in 15 patients, with an average lesion size of (8.2 +/- 4.3) mm (rang, 5-25 mm). All lesions were finally confirmed histopathologically. RESULTS: Of the 154 lesions, 139 (90.3%) were resected completely through EMR procedure. A close relationship between the complete resection rate and the lesion size was observed. The bigger the lesion size, the lower the complete resection rate. Endoscopic follow-up was carried out in 7 patients for more than 10 years, in 43 for 5 - 10 years, in 31 for 3 - 5 years and in 66 for less than 3 years. Of 11 dead patients during following-up, 10 died of other diseases, only 1 of recurrence. The 5-year survival rate was 96.2% for early-stage esophageal cancer, and 100% for early cancer of the gastric cardia. Perioperative complications included oozing bleeding in 5 patients (3.4%) and stricture in 1 (0.7%), no perforation occurred in this series. CONCLUSION: Endoscopic mucosal resection is suitable to treat precancerous lesions or early-stage esophageal cancers without invasion into submucosa. Compared with conventional resection through open thoracotomy, similar long-term survival and curative effect can be achieved by this EMR treatment, preserving a good quality of life.

Molecular simulation of interaction between estrogen receptor and selective estrogen receptor modulators.

AIM: To study the mechanism of interaction between a series of potent racemic selective estrogen receptor modulators (SERM) and estrogen receptors (ER). METHODS: Active conformations of these conformationally restricted raloxifene analogues in binding pocket were determined by molecular mechanics. The interactive energies between ligand and receptor were calculated by docking program. RESULTS: Both R and S configurations of these SERM were accommodated by the binding pocket of ER. The hydroxy group of compounds forms hydrogen bonds with amino acid residues of ER and the phenolic group mimics the A ring of estradiol. The most potential compounds were those with two hydroxy groups and accommodated by binding pocket in S configuration with phenolic group at C(16) imitating A ring of estradiol. CONCLUSION: Chiral center conferred little effect on the binding affinity of these conformationally restricted raloxifene analogues. The hydroxy group(s) play(s) a critical role to the orientation of compounds in active pocket of ER and the binding between ligand and receptor.