Combined application of mesenchymal stem cells and different glucocorticoid dosing alleviates osteoporosis in SLE murine models.

OBJECTIVE: Bone mesenchymal stem cells (BMSCs) have been tentatively applied in the treatment of glucocorticoid-induced osteoporosis (GIOP) and systemic lupus erythematosus (SLE). However, the effects of BMSCs on osteoporosis within the context of glucocorticoid (GC) application in SLE remain unclear. Our aim was to explore the roles of BMSCs and different doses of GC interventions on osteoporosis in SLE murine models. METHODS: MRL/MpJ-Faslpr mice were divided into eight groups with BMSC treatment and different dose of GC intervention. Three-dimensional imaging analysis and hematoxylin and eosin (H&E) staining were performed to observe morphological changes. The concentrations of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in serum were measured by enzyme-linked immunosorbent assay (ELISA). The subpopulation of B cells and T cells in bone marrows and spleens were analyzed by flow cytometry. Serum cytokines and chemokines were assessed using Luminex magnetic bead technology. RESULTS: BMSCs ameliorated osteoporosis in murine SLE models by enhancing bone mass, improving bone structure, and promoting bone formation through increased bone mineral content and optimization of trabecular morphology. BMSC and GC treatments reduced the number of B cells in bone marrows, but the effect was not significant in spleens. BMSCs significantly promoted the expression of IL-10 while reducing IL-18. Moreover, BMSCs exert immunomodulatory effects by reducing Th17 expression and rectifying the Th17/Treg imbalance. CONCLUSION: BMSCs effectively alleviate osteoporosis induced by SLE itself, as well as osteoporosis resulting from SLE combined with various doses of GC therapy. The therapeutic effects of BMSCs appear to be mediated by their influence on bone marrow B cells, T cell subsets, and associated cytokines. High-dose GC treatment exerts a potent anti-inflammatory effect but may hinder the immunotherapeutic potential of BMSCs. Our research may offer valuable guidance to clinicians regarding the use of BMSC treatment in SLE and provide insights into the judicious use of GCs in clinical practice.

A novel enzyme-linked immunosorbent assay tool to evaluate plasma soluble CD226 in primary Sjögren's syndrome.

CD226 is an important receptor constitutively expressed on most immune cells, performing vital functions in immune responses. However, the levels of soluble CD226 (sCD226) and its roles in primary Sjögren syndrome (pSS) remain unclear. In this study, we developed two novel mouse anti-human CD226 monoclonal antibodies (mAbs) and established a novel sandwich enzyme-linked immunosorbent assay (ELISA) system, which proved to be highly effective in detecting human sCD226. We then analyzed the expression of sCD226 in the plasma of pSS patients. Our results showed that the levels of sCD226 were significantly lower in patients with pSS compared to healthy controls. The significant decline was also observed in active group and the patients with high levels of IgG or positive anti-SSB. Additionally, reduced sCD226 was found to be negatively correlated with the disease activity of pSS and several clinical manifestations, including arthralgia, fatigue, decayed tooth and interstitial lung disease (ILD). Furthermore, receiver operator characteristics (ROC) curve analysis showed that sCD226 displayed outstanding capacity in discriminating pSS and predicting the disease activity. Altogether, plasma sCD226 emerges as a promising candidate for diagnostic markers in the context of pSS.

Expression and clinical significance of PD-L1 and infiltrated immune cells in the gastric adenocarcinoma microenvironment.

Programmed death-ligand 1 (PD-L1) is a crucial negative costimulatory molecule expressed on both tumor and immune cells. It binds to programmed death-1, facilitating tumor escape. Tumor-infiltrating immune cells play a vital role in this process. However, the clinical relationship between PD-L1 expression and tumor-infiltrating immune cells remains uncertain. Immunohistochemistry (IHC) was utilized to assess PD-L1 expression and TIIC markers (CD3, CD4, CD8, CD19, CD31, CD68, CD11c, CD56, and α-smooth muscle actin) in gastric adenocarcinoma tissues from 268 patients. The aim was to explore the prognostic significance of PD-L1 and the infiltration of different immune cell types. The study analyzed overall survival and the correlations between PD-L1 expression, immune cell infiltration, and clinicopathological characteristics. Among the 268 patients, 52 (19.40%) exhibited high PD-L1 expression on tumor cells (TPD-L1), while 167 (62.31%) displayed high PD-L1 expression on immune cells (IPD-L1). Patients with high IPD-L1 expression showed improved survival compared to those with low IPD-L1 expression (P = .028). High TPD-L1 expression associated with various clinicopathological features, such as larger tumor size, poorer differentiation, deeper invasion depth, and higher tumor stage. Conversely, patients with high IPD-L1 expression exhibited shallower tumor invasion and lower mortality rates. Univariate analysis indicated that superficial tumor infiltration, absence of lymph node and distant metastasis, low tumor stage, high IPD-L1 expression, and elevated CD8 and CD19 expression were associated with a reduced risk of tumor progression. Multivariate analysis revealed that patients with high IPD-L1 and CD8 expression or high TPD-L1 and low CD31 expression experienced significantly better overall survival than patients with other combinations. The findings indicate that patients with high PD-L1 expression in immune cells have a substantially improved prognosis. Additionally, the combination of PD-L1 with CD8 or CD31 expression status can serve as an indicator of prognosis in patients with gastric adenocarcinoma.

B7-H3 and CD47 co-expression in gastric cancer is a predictor of poor prognosis and potential targets for future dual-targeting immunotherapy.

BACKGROUND: Gastric cancer (GC) is one of the most prevalent types of cancer worldwide. B7-H3, an immune checkpoint molecule with promising potential, has been found to be overexpressed in various cancers. CD47 is an anti-phagocytic molecule that interacts with the signal regulatory protein alpha (SIRPα) to affect phagocytes. The relationship between the expression of B7-H3 and CD47, two potential therapeutic targets found in tumor cells, remains unknown. In this study, our objective is to investigate the clinical significance of co-expression of B7-H3 and CD47, as well as the potential therapeutic value of combination therapy in GC. METHODS: We utilized immunohistochemistry (IHC) to assess the expression of B7-H3, CD47, CD68, CD86 and CD163 in tissue microarrays obtained from 268 GC patients who underwent surgeries. Western blotting was employed to assess the protein level of B7-H3 and CD47 in GC tissues. The co-localization of B7-H3/CD47 and CD68 in GC tissues was determined using multiplex immunohistochemistry (m-IHC). We further verified the relationship between B7-H3/CD47 and macrophage infiltration via flow cytometry. To estimate the clinical outcomes of patients from different subgroups, we employed the Kaplan-Meier curve and the Cox model. RESULTS: Among the 268 GC cases, a total of 180 cases exhibited positive expression of B7-H3, while 122 cases showed positive expression of CD47. In fresh GC clinical tissues, B7-H3 and CD47 protein level was also higher in tumor tissue than in adjacent normal tissue. Remarkably, 91 cases demonstrated co-expression of B7-H3 and CD47. We observed a significant correlation between B7-H3 expression and tumor stage (P = 0.001), differentiation (P = 0.045), and depth (P = 0.003). Additionally, there was a significant association between B7-H3 and CD47 expression (P = 0.018). The percentage of B7-H3 and CD47 double positive cells in fresh GC tumor tissues were elevated compared with control adjacent tissues regardless of CD45- or CD45+ cells (P = 0.0029, P = 0.0012). Patients with high B7-H3 or CD47 expression had significantly lower overall survival (OS) rates compared to those with low expression levels (P = 0.0176 or P = 0.0042). Surprisingly, patients with combined high expression of B7-H3 and CD47 exhibited a considerably worse prognosis than others (P = 0.0007). Univariate analysis revealed that cases with high expression of B7-H3, CD47, or both had significantly higher hazard ratios (HR) than cases with low expression of these markers. Furthermore, the results of multivariate analysis indicated that B7-H3/CD47 co-expression and CD47 expression alone are independent prognostic factors for overall survival. Moreover, significant correlations were observed between B7-H3 and CD68 expression, CD47 and CD68 expression, as well as B7-H3/CD47 co-expression and CD68 expression in GC patients (P < 0.001, P = 0.003, and P < 0.001). Flow cytometry test showed that the percentage of CD68-positive cells but not CD86-positive cells among B7-H3-positive or CD47-positive immune cells in GC tumor tissue was elevated significantly compared with adjacent tissue. CONCLUSION: Our findings demonstrated a correlation between B7-H3 expression and CD47 expression in GC patient tissues. Co-expression of B7-H3 and CD47 can serve as an indicator of poor prognosis in GC patients. In GC tumor tissue, but not adjacent tissue, B7-H3 and CD47 expression was accompanied with macrophage infiltration.

Inducible costimulator ligand (ICOSL) on CD19+ B cells is involved in immunopathological damage of rheumatoid arthritis (RA).

Inducible costimulator (ICOS) and its ligand (ICOSL) are critical to regulate the immune response in autoimmune diseases. The participation of B lymphocytes exhibits pathogenic potential in the disease process of rheumatoid arthritis (RA). However, the precise role of ICOSL in RA remains unclear. In this study, we aimed to explore the regulatory effects of CD19+ICOSL+ B cells in the pathogenesis of RA. We demonstrated the increased expression of ICOS and ICOSL in patients with RA and collagen-induced arthritis (CIA) mice. The population of CD19+ICOSL+ B-cell subset was significantly correlated with clinicopathological characteristics of RA patients and CIA mice. Adoptive transfer of CD19+ICOSL+ B cells aggravated arthritic progression in CIA mice. Moreover, microarray analysis revealed that CD19+ICOSL+ cells could exert pivotal effect in pathological process of RA. Further blocking of ICOSL significantly inhibited proinflammatory responses and ameliorated arthritic progression. Therefore, CD19+ICOSL+ B-cell subset could be defined as a specific pathogenic cell subpopulation involved in immunopathological damage of RA. Blockade of ICOSL is promising to be a potential new approach for RA therapy.

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.

Decreased expression of programmed death-1 on CD8+ effector memory T lymphocytes correlates with the pathogenesis of type 1 diabetes.

AIMS: Chronic inflammation of autoimmune diseases, including type 1 diabetes (T1D), is mainly mediated by memory T(Tm) cells, predominantly effector memory T (Tem) cells. The roles of the programmed death-1 (PD-1) receptor on lymphocytes have been well studied in tumor and other infection models. However, little is known about the relationship between the expression of PD-1 on CD8+ Tem cells and the pathogenesis of T1D. METHODS: A total of 52 patients diagnosed with T1D and 39 gender-, age-, and ethnically matched health control individuals were enrolled in this study. Peripheral blood mononuclear cells from these individuals were isolated and analyzed by flow cytometry. We evaluated the frequencies of PD-1+ CD8+ memory T cell subsets from patients' peripheral blood with T1D and the spleen cells of nonobese diabetic (NOD) mice in the present study. We also investigated the effects of blocking PD-1/PD-L1 pathway on islet's inflammation in NOD mice. RESULTS: Frequencies of PD-1+ CD8+ Tem cells were decreased significantly in PBMC of patients with T1D (40.73 ± 12.72 vs 47.43 ± 15.56, *p < 0.05). The frequencies of PD-1+ CD8+ Tem cells were decreased in patients with T1D who were positive for two or more autoantibodies compared with the patients with one autoantibody (13.46% vs 46.95 ± 12.72%, *p < 0.05). Meanwhile, the frequencies of PD-1+ CD8+ central memory T (Tcm) cells were also significantly decreased in patients with two or more autoantibodies compared with other groups (≥ 2AAb vs HC 33.1 ± 8.92% vs 43.71 ± 11.78%, *p < 0.05; ≥ 2AAb vs AAb-33.1 ± 8.92% vs 41.65 ± 11.2%, *p < 0.05; ≥ 2AAb vs 1AAb 33.1 ± 8.92% vs 48.09 ± 10.58%, ***p < 0.001). The frequencies of PD-1+CD8+ Tem cells were positively correlated with fasting serum C-peptide levels (r = 0.4308, *p < 0.05) and C-peptide levels 2 h after meal in T1D patients (r = 0.5723, **p < 0.01). The frequencies of PD-1+CD8+ Tcm cells were only negatively correlated with the levels of HbA1c (r = - 0.2992, *p < 0.05). Similarly, the frequencies of PD-1+CD8+ Tem were significantly decreased in intervention group (anti-mouse PD-1 mAb) compared with the control group (14.22 ± 6.455% vs 27.69 ± 9.837%, *p < 0.05). Pathologically, CD8, PD-1 and PD-L1 were strongly expressed in the islets of diabetic mice after PD-1 blockade. CONCLUSIONS: It is the first report of the expression of PD-1 on CD8+ Tem cells in T1D in the present study. Our observations suggest that the PD-1/PD-L1 signal pathway on CD8+ Tem cells of T1D subjects might identify a new pathway for delaying the occurrence and development by inhibiting autoimmunity.

Overexpression of B7-H4 is associated with infiltrating immune cells and poor prognosis in metastatic colorectal cancer.

Metastasis commonly occurs in colorectal cancer (CRC) patients and confers a poor prognosis. B7-H4, an immune checkpoint molecule, has been found to be expressed in numerous tumor tissues and play critical roles in tumor progression. However, B7-H4 expression and its prognostic significance in different metastases from CRC remain unclear. In the present study, we screened a novel mouse anti-human B7-H4 monoclonal antibody (mAb) which exhibited a higher degree of recognition and sensitivity than the commercial reagent in immunohistochemistry (IHC). Using this antibody, overall 110 metastatic and paired primary lesions of CRC were analyzed for their expression of B7-H4, CD8 and CD68. Our results showed that expression of B7-H4 and CD68 in metastastic lesions was significantly higher than that in matched primary lesions (P = 0.0016, P < 0.0001). We also found a significant increase of CD68-positive immune cell infiltration in the B7-H4 high expressing metastases (P = 0.041). Moreover, upregulated B7-H4 in metastatic lesions was correlated with poor prognosis of patients (P = 0.014), while in primary lesions, B7-H4 combined with CD8 was associated with the overall survival (OS) (P = 0.043). Further, B7-H4 expression in metastatic lesions was significantly correlated with hazard ratio (HR) both in univariate and multivariate analysis. Altogether, B7-H4 in metastatic lesions is promising to be a potential prognostic indicator of CRC, and may promote tumor progression and metastasis of this cancer.

BMSC-derived exosomes from congenital polydactyly tissue alleviate osteoarthritis by promoting chondrocyte proliferation.

In the past decade, mesenchymal stem cells (MSCs) have been widely used for the treatment of osteoarthritis (OA), and exosomes may play a major role. Here, we acquired a special kind of MSCs from the bone marrow of surgically resected tissue from the hand of a patient with polydactyly. Experiments were focused on the role of polydactyly bone marrow-derived MSCs (pBMSCs) in osteoarthritis. The results showed that the pBMSCs had a greater ability than the BMSCs to differentiate into chondrocytes. Mechanistically, the expression of BMP4 was significantly higher in the pBMSCs than it was in the BMSCs. Furthermore, we showed that the migration and proliferation of chondrocytes were stimulated by exosomes secreted by pBMSC (pBMSC-EXOs). Notably, the downregulation of BMP4 in pBMSCs by siRNA inhibited both the chondrogenic differentiation potential of the MSCs and the function of the chondrocytes. In addition, the injection of pBMSC-EXOs and BMSC-EXOs attenuated OA in an OA mouse model, but the pBMSC-EXOs had a superior therapeutic effect compared with that of the BMSC-EXOs. Taken together, the data indicate that pBMSCs have greater ability to differentiate into chondrocytes and regulate chondrocyte formation through BMP4 signaling. Therefore, pBMSC-EXOs may represent a novel treatment for OA.

Circulating T Cells Exhibit Different TIM3/Galectin-9 Expression in Patients with Obesity and Obesity-Related Diabetes.

AIMS: Obesity is highly associated with type 2 diabetes mellitus (T2DM). The TIM3/galectin-9 pathway plays an important role in immune tolerance. Herein, we aimed to investigate the expression of TIM3 and galectin-9 in peripheral blood and to evaluate their clinical significance in patients with obesity and obesity-related T2DM. METHODS: We performed flow cytometry on peripheral blood samples from healthy donors (HC), patients with simple obesity (OB), and patients with obesity comorbid T2DM (OD). The expression of TIM3 on CD3+, CD4+, and CD8+ T cells was determined. The level of galectin-9 in plasma was detected by ELISA. RESULTS: We demonstrated the enhancement of TIM3 on CD3+, CD4+, and CD8+ T cells in the OB group when compared with healthy controls, while it was decreased significantly in the OD group. The TIM3+CD8+ T cells of the OB group were positively correlated with risk factors including BMI, body fat rate, and hipline. The concentration of galectin-9 of the OD group in plasma was significantly higher than that of healthy donors and the OB group. Moreover, the level of galectin-9 of the OD group was positively correlated with fasting insulin and C-peptide, which were two clinical features that represented pancreatic islet function in T2DM. CONCLUSIONS: Our results suggested that TIM3 and galectin-9 may be potential biomarkers related to the pathogenesis of obesity-related T2DM.

Evaluation of the role of B7-H3 haplotype in association with impaired B7-H3 expression and protection against type 1 diabetes in Chinese Han population.

BACKGROUND: Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disorder caused by the destruction of insulin-secreting cells. B7-H3 (CD276) plays a vital role in T cell response. However, B7-H3 expression and its clinical significance in T1D remain unclear. The aim of this study was to investigate the correlations between the expression of B7-H3 and clinical parameters in T1D patients. The possible role of B7-H3 gene variants with T1D was also discussed. METHODS: Four B7-H3 single nucleotide polymorphisms (SNPs) were genotyped in 121 T1D patients and 120 healthy controls by polymerase chain reaction (PCR) direct sequencing. Expression of membrane B7-H3 (mB7-H3) in peripheral blood lymphocytes was determined by flow cytometry. Levels of soluble B7-H3 (sB7-H3) in serum were analyzed by enzyme linked immunosorbent assay (ELISA). RESULTS: The B7-H3 haplotype T-A-C-T was less frequently observed in T1D patients compared to the controls (OR: 0.31, 95% CI: 0.16-0.61). B7-H3 expression on monocytes showed significant upregulation in T1D patients and was positively correlated with several clinical features including ALT, fast C-peptide 120 min, HbAlc, IFN-γ, IL-6 and TNF-α (P < 0.05). The concentration of sB7-H3 in serum increased in T1D patients (P < 0.0001). We also observed that B7-H3-T-A-C-T was associated with the decreased release of sB7-H3 but not the membrane form. CONCLUSIONS: B7-H3 may act as a potential biomarker related to the pathogenesis of T1D. The B7-H3-T-A-C-T polymorphism variant is associated with the low risk of T1D as well as less release of sB7-H3.

Enhancement of the Soluble Form of OX40 and OX40L Costimulatory Molecules but Reduction of the Membrane Form in Type 1 Diabetes (T1D).

This study analyzed the expression of membrane OX40 and OX40L (mOX40 and mOX40L) and levels of soluble OX40 and OX40L (sOX40 and sOX40L) in T1D patients to determine their clinical significance. Peripheral blood (PB) was collected from patients with T1D and healthy control participants. Expression of mOX40 and mOX40L on immune cells was detected by flow cytometry. Levels of sOX40 and sOX40L in sera were measured by ELISA. We demonstrated for the first time enhanced sOX40 and sOX40L expression and reduced mOX40 and mOX40L levels in T1D patients which correlated with the clinical characteristics and inflammatory factors. These results suggest that OX40/OX40L signal may be promising biomarkers and associated with the pathogenesis of T1D.

A novel mutation in INS gene linked to permanent neonatal diabetes mellitus.

PURPOSE: Neonatal diabetes mellitus (NDM) is caused by mutations in the genes responsible for pancreatic ß cell mass or function. This study aimed to screen the mutations in the KCNJ11, ABCC8, and INS genes in a Chinese patient with clinical features of NDM. METHODS: The entire coding sequence and exon/intron boundaries of KCNJ11, ABCC8, and INS genes were detected by Sanger sequencing. The pathogenicity of the mutation was determined by using online prediction programs SIFT and Mutation Taser. The conformational alterations which contribute to the change of protein function were analyzed at the structural level. RESULTS: A novel mutation L35Q (B11) of the INS gene was discovered in the patient. As L35 residue contributes to its hydrophobic core of the protein, the L35Q substitution is predicated to affect B19-A20 disulfide bond and therefore disrupt the folding of the proinsulin, which ultimately results in beta cell apoptosis by inducing ER stress. CONCLUSIONS: This case could help us understand the role of the INS mutation in the development of diabetes.

Preparation, characterization and application of anti-human OX40 ligand (OX40L) monoclonal antibodies and establishment of a sandwich ELISA for autoimmune diseases detection.

OX40L (CD252, TNFSF4), a type II transmembrane protein which like other tumor necrosis factor ligands, involved in the costimulation and differentiation of T cells, functions as a positive signal in immune response. To investigate the biological function of soluble OX40L (sOX40L), three functional anti-OX40L monoclonal antibodies (mAbs) 3D2, 3F7 and 2H3 were obtained by hybridoma technology. Besides, specificity of the mAbs was further demonstrated by ELISA, Western blot and Immunofluorescence experiments. We also developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human OX40L antibodies 3D2 and 3F7 with different epitopes. Using the ELISA system, we found that sOX40L in the sera of healthy donors increases in an age-dependent manner and that enhanced sOX40L expression in some autoimmune diseases especially in rheumatoid arthritis (RA) patients, suggesting the potential diagnostic significance of sOX40L in the autoimmune diseases. Together, these data demonstrate that the existence of circulating sOX40L in human sera might play an important role in immunoregulation.

Soluble B7-H3 (sB7-H3) is over-expressed in the serum of type 1 diabetes patients.

Type 1 Diabetes (T1D) is an autoimmune disease resulting from insulin-secreting ß-cells mediated by autoreactive T cells. We demonstrated increased level of sB7-H3 in T1D patients than in healthy control group. This result suggests that B7-H3 may be may be a promising biomarker associated with the pathogenesis of T1D.

Amniotic fluid stem cells provide considerable advantages in epidermal regeneration: B7H4 creates a moderate inflammation microenvironment to promote wound repair.

The current treatments for severe skin injury all involve skin grafting. However, there is a worldwide shortage of donor skin tissue. In this study, we examined the advantages of using human amniotic fluid stem (hAFS) cells in skin wound healing. In vitro, hAFS cells differentiate into keratinocytes (termed hAFS-K). Like keratinocytes, hAFS-K cells express the markers K5, K14, K10 and involucrin; display typical cellular structure, including a tonofibril-rich cytoplasm; and construct a completely pluristratified epithelium in 3D culture. In vivo, in a mouse excisional wound model, GFP-positive hAFS cells participate in wound repair. Co-localization of GFP/K14 and GFP/K10 in the repaired epidermis demonstrated that hAFS cells can differentiate into keratinocytes. Real-time PCR results confirmed that hAFS cells can initiate and promote early-stage repair of skin damage. During wound repair, hAFS cells did not directly secrete repair-related factors, such as bFGF, VEGF, CXCL12, TGF-ß1 and KGF, and provided a moderate inflammation reaction with lower expression of IL-1ß, IL-6, TNF-α, Cox2 and Mac3. In hAFS cells, the negative co-stimulatory molecule B7H4 regulates low immunogenicity, which can provide a modest inflammatory reaction microenvironment for wound repair. Furthermore, with their uniquely high proliferation rate, hAFS cells offer a promising alternative for epidermal regeneration.