Genetic and Functional Analyses of the Novel KLF11 Pro193Thr Variant in a Three-Generation Family with MODY7.

KLF11 regulates insulin gene expression through binding to the insulin promoter and has been reported as a causative gene for maturity-onset diabetes of the young 7 (MODY7). Here, we report a novel KLF11 variant associated with a three-generation family with early childhood-onset diabetes and explore its clinical and functional characteristics. The three-generational pedigree contains five patients affected by diabetes. The pathogenic variant identified by whole-exome sequencing was further confirmed by Sanger sequencing and pedigree verification. Luciferase reporter assays and glucose-stimulated insulin secretion were used to examine whether the KLF11 variant binds to the insulin promoter and regulate insulin secretion in vitro. The proband, his son, and his uncle exhibited hyperglycemia at ages 32, 13 and 71 years, respectively. All three patients showed characteristics of metabolic syndrome (obesity, dyslipidemia, and diabetes), but the insulin secretion of islet ß-cells was impaired. A novel heterozygous missense variant, c.577 C>A (p.Pro193Thr) of the KLF11 gene was detected in all three patients. This variant co-segregates with the diabetes phenotype, consistent with an autosomal dominant disorder. The identified KLF11 p.Pro193Thr variant drastically decreased the transcriptional activity of KLF11, as demonstrated by luciferase reporter assay. Functional analyses revealed that the KLF11 Pro193Thr variant inhibited glucose-stimulated insulin secretion. We identified a novel KLF11 Pro193Thr variant in a three generation family with MODY7. These findings shed light on the molecular mechanisms underlying the pathogenesis of MODY7 and expand the genotype and clinical spectrum of MODY7.

Methylprednisolone pulse therapy promotes the differentiation of regulatory T cells by inducing the apoptosis of CD4+ T cells in patients with systemic lupus erythematosus.

OBJECTIVES: To investigate the differentiation of regulatory T cells (Tregs) induced by methylprednisolone (MP) pulse therapy in patients with Systemic Lupus Erythematosus (SLE). METHODS: We enrolled 30 patients with SLE and analyzed peripheral blood mononuclear cells (PBMCs) before and after MP pulse therapy. Peripheral Tregs, apoptosis of PBMCs subsets, and TGFß production by monocytes was quantified by flow cytometry. Proliferation and IFN-γ production of CD4+ T cells were measured. Furthermore, TGFß1 production by human monocyte-derived macrophages (HMDM) stimulated with MP-treated CD4+ T cells were quantified by ELISA. RESULTS: Peripheral Tregs was significantly increased after MP pulse therapy (6.76 ± 1.46% vs. 3.82 ± 1.02%, p < 0.01), with an expansion of Nrp1- induced Tregs (4.54 ± 0.46% vs. 1.75 ± 0.38%, p < 0.01). Proliferation and IFN-γ production of CD4+ T cells were significantly decreased after MP pulse therapy. MP pulse therapy induced CD4+ T cell apoptosis (early apoptosis, 26.34 ± 3.54% vs. 14.81 ± 2.89%, p < 0.01) and TGFß expression on monocytes (6.02% vs. 2.45%, p < 0.01). Furthermore, MP induced CD4+ T cell apoptosis in vitro, which stimulated HMDM to produce TGFß. Moreover, elevated TGFß level in supernatant from HMDM stimulated with MP-treated CD4+ T cells promoted Tregs differentiation. CONCLUSIONS: MP pulse therapy induces CD4+ T cell apoptosis, which promotes monocytes to produce TGFß and further facilitates Tregs differentiation. Newly-differentiated Tregs suppress proliferation and IFN-γ production of CD4+ T cells and contribute to immunoregulatory milieu after MP pulse therapy.

Cutaneous mucosa-associated lymphoid tissue lymphoma complicating Sjögren's syndrome: A case report and review of literature.

BACKGROUND: The association of Sjögren's syndrome (SS) and lymphoma is similar. Mucosa-associated lymphoid tissue (MALT) or extranodal marginal zone B-cell lymphoma was the most common lymphomatous histology in SS patients. MALT in SS patients is frequently located in the parotid gland, while MALT lymphoma of the skin with SS is an exceedingly rare entity that needs to be recognized. CASE SUMMARY: A 60-year-old woman presented with a 3-year history of progressive dry mouth associated with a 1-year history of enlarging cutaneous nodules. Physical examination revealed two hard subcutaneous nodules on her right lower leg. The results of Schirmer's test were positive, despite the absence of dry eyes. Labial salivary gland biopsy revealed lymphocytic infiltration and chronic inflammation with a focus score of 2. The patient was diagnosed with SS. She underwent resection of one cutaneous nodule, and histopathological analysis identified the nodule as MALT lymphoma. Her dry mouth symptoms improved, and the nodules decreased after 6 mo of treatment with hydroxychloroquine sulfate and chemotherapy (thalidomide, cyclophosphamide, and dexamethasone). CONCLUSION: Lymphoma is a severe complication of SS, shown by the reported unique case of cutaneous MALT lymphoma with SS.

MicroRNA-139-5p upregulation is associated with diabetic endothelial cell dysfunction by targeting c-jun.

Dysfunction of endothelial cells (ECs) and their progenitor cells is an important feature of diabetic vascular disease. MicroRNA (miR)-139-5p is involved in inhibiting the metastasis and progression of diverse malignancies. However, the role of miR-139-5p in ECs still remains unclarified. Here we demonstrated that miR-139-5p expression was elevated in endothelial colony-forming cells (ECFCs) isolated from patients with diabetes, ECs derived from the aorta of diabetic rodents, and human umbilical vein endothelial cells (HUVECs) cultured in high glucose media. MiR-139-5p mimics inhibited tube formation, migration, proliferation, and down-regulated expression of c-jun, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF)-B, in ECFCs and HUVECs, respectively; moreover, miR-139-5p inhibitors reversed the tendency. Further, gain- and-loss function experiments and ChIP assay indicated that miR-139-5p regulate functions of ECFCs by targeting c-jun-VEGF/PDGF-B pathway. In vivo experiments (Matrigel plug assay and hindlimb ischemia model) showed that miR-139-5p downregulation further promoted ECFC-mediated angiogenesis and blood perfusion. In conclusion, diabetes-mediated high miR-139-5p expression inhibits the c-jun-VEGF/PDGF-B pathway, thus decreasing ECFCs migration, tube formation and proliferation, which subsequently reduces ECs survival. Therefore, miR-139-5p might be an important therapeutic target in the treatment of diabetic vasculopathy in the future.

Elevated EPSTI1 promote B cell hyperactivation through NF-κB signalling in patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by aberrant B cell hyperactivation, whose mechanism is partially understood. METHODS: We performed whole transcriptome sequencing of B cells from three pSS patients and three matched healthy controls (HC). Differentially expression genes (DEGs) were confirmed with B cells from 40 pSS patients and 40 HC by quantitative PCR and western blot. We measured the proliferation potential and immunoglobulins production of siRNA-transfected or plasmid-transfected B cells stimulated with cytosine-phosphate-guanine (CpG) or anti-IgM. We also explored Toll-like receptor 9 (TLR9) signalling to reveal the potential mechanism of B cell hyperactivation in pSS. RESULTS: We identified 77 upregulated and 32 downregulated DEGs in pSS B cells. We confirmed that epithelial stromal interaction (EPST1) expression in pSS B cells was significantly higher than that from HCs. EPSTI1-silencing B cells stimulated with CpG were less proliferated and produced lower level of IgG and IgM comparing with control B cells. EPSTI1-silencing B cells expressed lower level of p-p65 and higher level of IκBα, and B cells with overexpressed EPSTI1 showed higher level of p-p65 and lower level of IκBα. Finally, IκBα degradation inhibitor Dehydrocostus Lactone treatment attenuated p65 phosphorylation promoted by EPSTI1. CONCLUSION: Elevated EPSTI1 expression in pSS B cells promoted TLR9 signalling activation and contributed to the abnormal B cell activation, which was promoted by facilitating p65 phosphorylation and activation of NF-κB signalling via promoting IκBα degradation. EPSTI1 might be implicated in pSS pathogenesis and was a potential therapeutic target of pSS.

Imbalance of T-helper 1/T-helper 2 cytokines and impaired glucose tolerance among patient with acute coronary syndrome.

PURPOSE: The balance between T helper (Th) cells Th1- and Th2-related cytokines plays a key role in the clinical process of acute coronary syndrome (ACS) and type 2 diabetes mellitus (T2DM) or impaired glucose tolerance (IGT). The objective of this study was to assess the status of Th1/Th2 cytokines in patients with ACS and T2D or IGT. METHODS: A total of 201 ACS patients were enrolled in the study. All ACS patients were divided into three groups: Group I-patients with normal glucose tolerance (NGT), Group II-patients with IGT and Group III-patients with T2D. We measured circulating Th1/Th2-type cytokines (interleukin [IL]-4, IL-13, interferon-gamma [IFN-γ], and tumor-necrosis factor-alpha [TNF-α]) using enzyme-linked immunosorbent assay and calculated the ratio of Th1/Th2. RESULTS: Significant elevations in serum levels of IL-4, IL-13, IFN-γ, and TNF-α were found in ACS-T2D and ACS-IGT groups compared to that in both ACS-NGT group and healthy individuals. Higher serum levels of IL-4, IL-13, and TNF-α were found in ACS-NGT group than that in the control group. Furthermore, IL-4 and IFN-γ concentrations were significantly higher in ACS-T2D patients than in ACS-IGT patients. IFN-γ/IL-4, IFN-γ/IL-13, and TNF-α/IL-4 ratios as markers of Th1/Th2 ratio were significantly higher for the ACS-T2D group and ACS-IGT group as compared to that in the ACS-NGT group and control group (P < 0.05). CONCLUSION: Shifts in the balance of Th1/Th2 toward a predominance of Th1 may represent more severe inflammatory status in ACS patients with type T2D or IGT.

Adiponectin exerts its negative effect on bone metabolism via OPG/RANKL pathway: an in vivo study.

To explore the effects of adiponectin on the bone metabolism in vivo. Bone mineral density (BMD), bone microstructure, serum adiponectin levels, and biochemical markers of the bone turnover were measured in 12-week-old male Adipo-/- and WT mice. In addition, the osteoclast formation, osteoprotegerin (OPG), and the receptor activator of nuclear factor-κB ligand (RANKL) expression were examined. The serum adiponectin levels were normal in the WT mice while undetectable in the Adipo-/- mice. Compared with the WT mice, the Adipo-/- mice had higher BMD, more trabecular bone, greater bone volume fraction, and trabecular thickness in the left femur. On the contrary, fewer osteoclasts were observed in the Adipo-/- mice when compared with the WT mice. Meanwhile, the Adipo-/- mice had a significantly decreased serum carboxyl-terminal telopeptide of type 1 collagen (CTX)/osteocalcin (OC) ratio. Interestingly, both the adiponectin and RANKL would cause a significant increase of CTX/OC ratio in the co-culture of the CD14+ peripheral blood mononuclear cells and the osteoblasts from Adipo-/- mice. Further, immunohistochemistry assays in tibias and both the RT-PCR and immunoblot analyses in the cultured osteoblasts showed the Adipo-/- mice expressed lower levels of RANKL but higher levels of OPG. Adiponectin had a negative effect on the bone metabolism, and this negative effect might be mediated, at least in part, by the OPG/RANKL pathway.

Analysis of conformational motions and residue fluctuations for Escherichia coli ribose-binding protein revealed with elastic network models.

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The open (ligand-free) and closed (ligand-bound) forms of RBP have been found. Here we investigate the conformational motions and residue fluctuations of the RBP by analyzing the modes of motion with two coarse-grained elastic network models, the Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). The calculated B-factors in both the calculated models are in good agreement with the experimentally determined B-factors in X-ray crystal structures. The slowest mode analysis by GNM shows that both forms have the same motion hinge axes around residues Ser103, Gln235, Asp264 and the two domains of both structures have similar fluctuation range. The superposition of the first three dominant modes of ANM, consisting of the rotating, bending and twisting motions of the two forms, accounts for large rearrangement of domains from the ligand-free (open) to ligand-bound (closed) conformation and thus constitutes a critical component of the RBP's functions. By analyzing cross-correlations between residue fluctuation and the difference-distance plot, it is revealed that the conformational change can be described as a rigid rotation of the two domains with respect to each other, whereas the internal structure of the two domains remains largely intact. The results directly indicate that the dominant dynamic characteristics of protein structures can be captured from their static native state using coarse-grained models.

Development of arterial calcification in adiponectin-deficient mice: adiponectin regulates arterial calcification.

Arterial calcification is common, but the mechanisms remain unclear. This study was undertaken to investigate the arterial calcification in adiponectin-deficient mice in vivo and the effects of adiponectin on cultured vascular smooth muscle cells in vitro. Alizarin red S staining was used to detect arterial calcification of adiponectin(-/-) mice. Alkaline phosphatase activity, osteocalcin secretion, and Runx2 protein expression were examined in cultured calcifying vascular smooth muscle cells (CVSMCs). The involved signal pathway was studied using a mitogen-activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. Adiponectin(-/-) mice developed slight arterial calcification after being fed with normal chow diet for 30 wk. Adenovirus-mediated supplement of adiponectin attenuated arterial calcification in these mice. On cultured CVSMCs, adiponectin inhibited ALP activity, osteocalcin secretion, Runx2 protein expression, and the formation of mineralized nodules. Adiponectin receptor 1 (AdipoR1) protein was detected in CVSMCs, and adiponectin activated p38 mitogen-activated protein kinase. Furthermore, inhibition of AdipoR1 expression or p38 activation reversed the effects of adiponectin on ALP activity. These results showed that adiponectin inhibited osteoblastic differentiation of CVSMCs through the AdipoR1/p38 signaling pathway. Our findings showed that adiponectin(-/-) mice developed arterial calcification, and this could be attributed to the loss of inhibitory action of adiponectin on osteoblastic differentiation of CVSMCs. It suggested that adiponectin plays a protective role against arterial calcification.

[Bone mass change and its possible mechanisms in murine knockout apolipoprotein E(ApoE-/-)].

OBJECTIVE: To study bone mass changes and its mechanisms in murine knockout apolipoprotein E (ApoE-/-). METHODS: Both 28-week-old male ApoE-/- mice (n = 6) and wild-type (WT) mice (n = 10) were euthanized. The trabecular and cortical bone microarchitecture were assessed by micro-CT (microCT) in right distal femur. The total body BMD (bone mineral density) of left femur was determined by dual-energy X-ray absorptiometry (DXA). The serum concentrations of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) were analyzed using enzyme-linked immunosorbent assay (ELISA). The left tibias were dissected and fixed in 4% paraformaldehyde after decalcified in 0.15M EDTA buffer for 30 days. And the expressions of OPG and RANKL were detected by immunohistochemical staining. RESULTS: Compared with WT mice, the ApoE-/- mice showed an increased volumetric BMD (294 +/- 38) mg/mm(3), tissue BMD (627 +/- 23) mg/mm(3), BMC (bone mineral content) (0.57 +/- 0.08) mg, bone volume fraction (20.38 +/- 4.74)%, trabecular number (6.67 +/- 0.78) mm(-1), trabecular thickness (0.03 +/- 0.01) mm with an decreased bone surface fraction (69 +/- 18) mm(-1), trabecular separation (0.12 +/- 0.01) and structure mode index (1.73 +/- 0.24). The total body BMD was higher in ApoE-/- mice than WT mice [(0.063 +/- 0.004) g/cm(2) vs (0.056 +/- 0.004) g/cm(2)]. The serum level of OPG was significantly higher in ApoE-/- mice than WT mice [(4.98 +/- 0.97) ng/ml vs (3.54 +/- 0.65) ng/ml]. The results of immunohistochemical staining showed that the OPG expression was at a higher level in bone cell of ApoE-/- mice and RANKL showed no obvious change in either sera or bone cells. CONCLUSION: A decreased bone resorption causes an increased bone mass in ApoE-/- mice and leads to an imbalanced ratio of OPG/ RANKL.