TBC1D23 mediates Golgi-specific LKB1 signaling.

Liver kinase B1 (LKB1), an evolutionarily conserved serine/threonine kinase, is a master regulator of the AMPK subfamily and controls cellular events such as polarity, proliferation, and energy homeostasis. Functions and mechanisms of the LKB1-AMPK axis at specific subcellular compartments, such as lysosome and mitochondria, have been established. AMPK is known to be activated at the Golgi; however, functions and regulatory mechanisms of the LKB1-AMPK axis at the Golgi apparatus remain elusive. Here, we show that TBC1D23, a Golgi-localized protein that is frequently mutated in the neurodevelopment disorder pontocerebellar hypoplasia (PCH), is specifically required for the LKB1 signaling at the Golgi. TBC1D23 directly interacts with LKB1 and recruits LKB1 to Golgi, promoting Golgi-specific activation of AMPK upon energy stress. Notably, Golgi-targeted expression of LKB1 rescues TBC1D23 deficiency in zebrafish models. Furthermore, the loss of LKB1 causes neurodevelopmental abnormalities in zebrafish, which partially recapitulates defects in TBC1D23-deficient zebrafish, and LKB1 sustains normal neuronal development via TBC1D23 interaction. Our study uncovers a regulatory mechanism of the LKB1 signaling, and reveals that a disrupted Golgi-LKB1 signaling underlies the pathogenesis of PCH.

[Preparing Bifidobacteria for Quantitative Detections].

OBJECTIVES: To compare different preparation methods for quantitative real-time PCR (qPCR) detection of Bifidobacteria. METHODS: Standard strains of Bifidobacteria were prepared with concentration gradients using strain DNA, PCR product amplification and purification, and plasmid DNA methods. The concentrations of Bifidobacteria were determined with ultraviolet spectrophotometer and real-time quantitative PCR. RESULTS: Greater than 0.99 R 2 in values of standard curves were achieved by all three preparation methods. The plasmid DNA method obtained a higher level of concentration and purity of Bifidobacteria than the other two methods ( P<0.01). CONCLUSIONS: The plasmid DNA method produces high quality preparations and is more suitable for real-time quantitative PCR, which can provide a reference for the molecular biological detection of Bifidobacteria.

Increased serum levels of soluble vascular endothelial-cadherin in patients with systemic vasculitis.

Henoch-Schönlein purpura (HSP) is a commonest systemic vasculitis (SV) in childhood characterized by an inflammatory reaction directed at vessels. Endothelial damage and perivascular leukocyte infiltrates are vital in the development of HSP. Vascular endothelial (VE)-cadherin is an endothelial cell-specific adhesion molecule, which plays critical roles in angiogenesis and endothelial integrity. Herein, we investigated the serum levels of soluble VE-cadherin (sVE-cadherin) in patients with HSP and other forms of SV. The serum levels of sVE-cadherin in 30 patients with HSP, together with patients with urticarial vasculitis, allergic vasculitis, Behcet disease, psoriasis vulgaris (PV) and atopic dermatitis (AD) and 26 health controls were measured by enzyme-linked immunosorbent assay. Serum levels of sVE-cadherin were significantly increased in patients with HSP in acute stage and patients with other forms of SV but not in patients with PV or AD. Moreover, Serum sVE-cadherin levels in HSP patients were correlated with the severity of this disease and serum concentrations of IgA anticardiolipin antibodies and vascular endothelial growth factor. Taken together, we show firstly that serum sVE-cadherin is abnormally increased in HSP patients. Increased serum levels of sVE-cadherin might be a novel biomarker for evaluating the severity of HSP and useful for identifying the presence of SV in inflammatory skin conditions.

Paeoniflorin suppresses vascular damage and the expression of E-selectin and ICAM-1 in a mouse model of cutaneous Arthus reaction.

Paeoniflorin (PF) extracted from the root of Paeonia lactiflora pall, displays anti-inflammation properties in several animal models. Adhesion molecules are important for the recruitment of leucocyte to the vessel wall and involved in the pathogenesis of various autoimmune and inflammatory diseases. Herein, we investigate the effects of PF on adhesion molecule expression in a mouse model of cutaneous Arthus reaction and cultured human dermal microvascular endothelial cells (HDMECs). We showed that PF significantly ameliorated the immune complex (IC) induced vascular damage, leucocyte infiltrates and adhesion molecules expression. Furthermore, PF markedly blocked tumor necrosis factor-α (TNF-α)-induced E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression in HDMECs at both mRNA and protein levels. PF also suppressed TNF-α-induced adhesion of polymorphonuclear leucocytes (PMNs) to HDMECs. Finally, western blot data revealed that PF can inhibit the phosphorylation of p38, JNK in TNF-α-treated HDMECs. These data suggest that PF, as an anti-inflammatory agent, can downregulate adhesion molecules expression. PF may be a candidate medicine for the treatment of IC-induced inflammatory response.

TWEAK enhances E-selectin and ICAM-1 expression, and may contribute to the development of cutaneous vasculitis.

Our previous work indicated that TWEAK is associated with various types of cutaneous vasculitis (CV). Herein, we investigate the effects of TWEAK on vascular injury and adhesion molecule expression in CV mice. We showed that TWEAK priming in mice induced a local CV. Furthermore, TWEAK priming also increased the extravasation of FITC-BSA, myeloperoxidase activity and the expression of E-selectin and ICAM-1. Conversely, TWEAK blockade ameliorated the LPS-induced vascular damage, leukocyte infiltrates and adhesion molecules expression in LPS-induced CV. In addition, TWEAK treatment of HDMECs up-regulated E-selectin and ICAM-1 expression at both mRNA and protein levels. TWEAK also enhanced the adhesion of PMNs to HDMECs. Finally, western blot data revealed that TWEAK can induce phosphorylation of p38, JNK and ERK in HDMECs. These data suggest that TWEAK acted as an inducer of E-selectin and ICAM-1 expression in CV mice and HDMECs, may contribute to the development of CV.

Increased HMGB1 serum levels and altered HMGB1 expression in patients with psoriasis vulgaris.

High mobility group box-1 (HMGB1) has been implicated as a pro-inflammatory cytokine in the pathogenesis of various inflammatory and autoimmune diseases. However, information about HMGB1 in inflammatory skin diseases is unknown. Herein, we investigated the serum HMGB1 levels and tissue HMGB1 expression in patients with psoriasis vulgaris (PV) and atopic dermatitis (AD). Serum levels of HMGB1 in patients with PV and AD were detected by enzyme-linked immunosorbent assay (ELISA). The expression of HMGB1 in lesional skin was evaluated by immunohistochemistry and immunofluorescence. Protein levels of HMGB1 in the nuclear fraction and cytoplasmic fraction were determined by western blot. Serum levels of HMGB1 in patients with PV but not AD were significantly higher than those in nornal controls. Moreover, serum HMGB1 levels were correlated with the severity of PV according to PASI socres. Furthermore, by immunohistochemistry and immunofluorescence, we showed that the expression of HMGB1 in normal skin was almost completely restricted to the nucleus. However, abundant cytoplasmic expression of HMGB1 was observed in the epidermis in lesional skin of PV patients. In addition, western blot data indicated that HMGB1 expression was in the nucleus protein and was absent in the cytoplasm protein in control group. In contrast, HMGB1 expression in the cytoplasmic fraction was detectable in AD patients and more distinct in PV patients. Taken together, this study provides first observations on the association of HMGB1 with PV, and showed the elevated HMGB1 serum levels and altered HMGB1 distribution in lesional skin in patients with PV. We suggest that HMGB1 might be involved in the pathogenesis of PV.

Elevated serum interleukin-33 levels in patients with Henoch-Schönlein purpura.

Henoch-Schönlein purpura (HSP) is the most common systemic vasculitis and is known as an immunoglobulin (Ig) A related immune complex-mediated disease. However, the molecular mechanisms in the development of HSP are not yet fully understood. Herein, we investigated the serum levels of Interleukin (IL)-33 and soluble ST2 (sST2) in HSP patients and their association with disease severity and IgA autoantibodies production. The serum levels of IL-33 and sST2 were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) in the serum of 33 patients with HSP and 22 controls. Serum levels of IgA anti-endothelial cell antibodies (AECA) and IgA anticardiolipin antibodies (ACA) in HSP patients were detected by double antigen sandwich ELISA. Our results indicated that serum levels of IL-33 but not sST2 were significantly elevated in patients with HSP in acute stage and restored to normal levels in convalescent stage. Moreover, serum IL-33 levels were correlated with the severity of HSP and serum concentrations of AECA-IgA and ACA-IgA. Taken together, we show firstly that serum IL-33 is abnormally elevated in HSP patients. IL-33 might be associated with the IgA autoantibodies production in the pathogenesis of HSP.

Suppression of inflammation by tumor-derived exosomes: a kind of natural liposome packaged with multifunctional proteins.

Exosomes are small-membrane vesicles secreted by hematopoietic and malignant epithelial cells as well as trophoblasts. The composition of cancerous exosomes has been proven to play pivotal roles in the maintenance of the microenvironment that is beneficial for the progression of cancer, such as Fas-ligand-triggered lymphocyte apoptosis. We supposed that the immunosuppressive effect of cancerous exosomes might be helpful in the treatment of diseases characterized by overactivation of the immune system and subsequent tissue injury. The aim of this study was to evaluate the protective effect of tumor-derived exosomes in the mice model of lipopolysaccharide (LPS)-induced inflammation. Tetrazolium (MTT) and DNA electrophoresis were used to measure the cytotoxicity of exosomes on lymphocytes. Pathologic observation of tissue sections, serologic analysis of aspartate aminotransferase/alanine aminotransferase (AST/ALT), and urinary analysis of protein were used to assess the protection effect of exosomes in LPS-induced multiorgan damage. In vitro outcomes of MTT and DNA electrophoresis showed the cytotoxicity of exosomes on lymphocytes. Together with the alleviation of organ damages evaluated by urine protein, serum AST/ALT, and pathologic analysis, we confirmed the possibility that pretreatment of mice with exosomes, produced by H22 hepatic tumor cells, resulted in protection against LPS-induced tissue damage, which is caused by overactivation of the immune system and inflammation response. This therapeutic strategy will raise an interesting way to search new therapeutics in pairs of diseases with complementarities in etiology and pathology, namely, a strategy of taking advantage of the mutual complementarities between diseases.

CCL5, CXCL16, and CX3CL1 are associated with Henoch-Schonlein purpura.

Chemokines are involved in the pathogenesis of various vascular inflammations. However, information about chemokines in Henoch-Schonlein purpura (HSP) is limited. Herein, we investigated the serum CCL5, CXCL16, and CX3CL1 levels in HSP patients with controls and the ability of sera from HSP patients on chemokine production in human dermal microvascular endothelial cells. Enzyme-linked immunosorbent assay (ELISA) detected serum CCL5, CXCL16, and CX3CL1 levels in patients with HSP. Human dermal microvascular endothelial cell line (HMEC-1) was treated with sera from patients with HSP at different stages, patients with acute spontaneous urticaria, or controls. Serum levels of CCL5, CXCL16, and CX3CL1 were elevated in HSP patients at acute stage, which correlated with the severity of this disease. Sera from patients with active HSP markedly induced CCL5, CXCL16, and CX3CL1 production at both mRNA and protein levels. In addition, patients' sera-stimulated HMEC-1 supernatants enhanced HL-60 or THP-1 cells migration. Furthermore, patients' sera increased the phosphorylation of inhibitor of κB-α (IκBα) and phosphorylation of extracellular signal-regulated kinase (ERK)1/2 protein levels, upregulated the translocation of nuclear factor-κB (NF-κB) p65 to the nucleus. Taken together, we show firstly that CCL5, CXCL16, and CX3CL1 may be involved in the pathogenesis of HSP. Factors present in sera from patients with active HSP may act as an inducer of inflammatory response in HMEC-1 cells and contribute to chemokine production through NF-κB and ERK 1/2 pathways.

Peoniflorin suppresses tumor necrosis factor-α induced chemokine production in human dermal microvascular endothelial cells by blocking nuclear factor-κB and ERK pathway.

Peoniflorin (PF) extracted from the root of Paeonia lactiflora pall displays anti-inflammation and antioxidant properties in several animal models. Chemokines are vital for directing the movement of circulating leukocytes to the sites of inflammation and are involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated the effects and potential mechanisms of PF on tumor necrosis factor-α (TNF-α) induced chemokine production in human dermal microvascular endothelial cells. Human dermal microvascular endothelial cell line (HMEC-1) was treated by TNF-α with or without PF. PF markedly attenuated TNF-α-induced chemokines (including CCL2, CCL5, CCL20, CXCL8, CXCL16 and CX3CL1) mRNA expression in HMEC-1. PF also reduced the secretion of these chemokines in culture supernatants. In addition, endothelial activation in the presence of PF markedly blocked the chemotactic activities of TNF-α-stimulated HMEC-1 supernatant on promyelocytic leukemia cell line (HL-60) or the acute mature monocytic leukemia cell line (THP-1) cell migration. Furthermore, Western blot data revealed TNF-α upregulated phosphorylation of inhibitor of κB-α (IκBα) and phosphorylation of extracellular signal-regulated kinase (ERK)1/2, which was almost completely reversed by PF. Finally, PF inhibited nuclear factor-κB (NF-κB) nuclear translocation to the nucleus. Taken together, our data provide the first evidence that PF has an anti-inflammatory ability against TNF-α-induced chemokine production and leukocyte migration, which may be at least partly related to the inhibition of NF-κB and ERK pathway. PF may be a candidate medicine for the treatment of inflammatory skin diseases.