Efficacy and safety of abatacept for interstitial lung disease associated with antisynthetase syndrome: a case series.

OBJECTIVES: This study investigated the efficacy and safety of abatacept (ABA) in interstitial lung disease (ILD) associated with antisynthetase syndrome (ASS). METHODS: Eight patients were identified through retrospective analysis of the medical records of our centre. All patients fulfilled the Solomon criteria and had a disease complicated with ILD. Lung function, imaging, serum markers, clinical evaluation indicators of ILD, peripheral blood cell classification, cytokines, and prednisone doses were analysed. RESULTS: Seven of the eight patients were female. The mean age was 54.4 (standard deviation [SD] 6.0) years. Antibodies against Jo-1, PL-12, and PL-7 were present in three, three, and two patients respectively. At baseline, the mean diffusing lung capacity for carbon monoxide (DLCO) was 53.8% (SD 9.2%), the mean score of King's Brief Interstitial Lung Disease (KBILD) was 40.6 (SD 13.8), the median Krebs Von den Lungen-6 (KL-6) was 1612.5 (interquartile range [IQR] 1180.5-2431.5) U/ml. All patients experienced symptom alleviation after ABA therapy. The mean and median changes in DLCO percentage, KBILD, and KL-6 were 12.3% (p<0.05), 21.4 (p<0.01), and 174.5U/ml (p<0.01), respectively. No obvious adverse events related to ABA were observed during the treatment. CONCLUSIONS: Our study offers preliminary, but encouraging, clinical evidence in favour of ABA as a therapy for ASS-ILD. ABA demonstrated favourable effects on ILD and was well-tolerated. Well-designed randomised controlled studies are required to confirm the efficacy and safety of this strategy.

Development of biocompatible DES/NADES as co-solvents for efficient biosynthesis of chiral alcohols.

The novel deep eutectic solvents (DESs) and natural deep eutectic solvents (NADESs) were designed and synthesized by cell protective components, in which the compounds were derived from natural alternative sources. The performances of designed DESs/NADESs as co-solvent were investigated in asymmetric reduction catalyzed by microbial cells. The DESs/NADESs synthesized by three different types of hydrogen bond receptor (betaine, L-proline and L-carnitine) conferred an advantage over conventional choline chloride-based DESs/NADESs and aqueous buffer system, with regard to efficient bioproduction of (R)-1-[4-(trifluoromethyl)phenyl]ethanol by recombinant Escherichia coli cells. TEM images exhibited that the cell membrane integrity during exposure to the developed NADESs was better than that after treatment with choline chloride-based NADES, which accounted for enhanced catalytic efficiency. This bioprocess was also feasible at 500 mL preparation scale with 92.4% yield under 400 mM substrate loading. To broaden the applicability of three types of DES/NADESs that increased catalytic efficiency in the process of E. coli-mediated reduction, the production of various chiral alcohols in developed reaction media were further examined, with some positive results. It was also found that lysine-based NADES could even reverse the enantioselectivity of biocatalyst at high water content in the reaction medium. These findings may aid in the development of novel DESs/NADESs for biocatalysis.

Production of anti-Trichophyton rubrum egg yolk immunoglobulin and its therapeutic potential for treating dermatophytosis.

The aim of this study was to estimate the therapeutic potential of specific egg yolk immunoglobulin (IgY) on dermatophytosis caused by Trichophyton rubrum. The IgY was produced by immunizing hens with cell wall proteins of T. rubrum, extracted from eggs by PEG precipitation and then purified by ammonium sulfate precipitation. The cross-reactivity (CR) with other fungi, growth inhibition on T. rubrum in vitro and therapeutic effect on T. rubrum infection in BALB/C mice of the specific IgY were then evaluated. Anti- T. rubrum cell wall proteins IgY (anti-trCWP IgY) presented a certain degree of cross-reactivity with different fungi. In the in vitro and in vivo activity researches, Anti-trCWP IgY showed a significant dose-dependent growth inhibitory effect on T. rubrum in vitro and a significant dose-dependent therapeutic effect on T. rubrum infection in BALB/C mice.

Development of a double-antibody sandwich ELISA for rapid detection to C-peptide in human urine.

C-peptide level is recognized as an important indicator of diabetes diagnosis. A sensitive and specific double-antibody sandwich enzyme-linked immunosorbent assay for the detection of C-peptide based on double antibody sandwich method was studied in this paper. The rabbit and hen were innunized with PLL-C-peptide and BSA-C-peptide respectively to obtain specific Yolk antibody (IgY) and polyclonal antibody used to construct the sandwich ELISA for the measurement of C-peptide. The limit of detection was 0.51 µg/mL and the half maximal inhibitory concentration (IC50) was 3.26 µg/mL. The method developed in the study showed no evident cross-reactivity with other similar analogs. The detection standard curve of C-peptide exhibited a good linearity (R2 = 0.9896, n = 15). 17 types of the urine of diabetes patients on c-peptide levels compared with the hospital type of diabetes information, with a conclusion of a high consistent rate. Therefore, the methods could be selectively used for rapid screening of C-peptide in human urine, and the type of diabetes has some referential significance.

KPNBeta1 promotes palmitate-induced insulin resistance via NF-kappaB signaling in hepatocytes

It has been intensively studied that inflammation contributes to the insulin resistance development in obesity-induced type 2 diabetes mellitus (T2DM). In this study, we assessed the effect of karyopherin Beta1 (KPNBeta1) in hepatic insulin resistance and the underlying mechanisms using high-fat diet (HFD) fed mice and palmitate (PA)-stimulated hepatocytes (HepG2). KPNBeta1 expression is increased in the HFD fed mice liver. PA upregulated KPNBeta1 expression in HepG2 cells in a time-dependent manner. PA also increased pro-inflammatory cytokines expression, including tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and interleukin 1Beta (IL-1Beta). KPNBeta1 knockdown reversed PA-induced pro-inflammatory cytokines expression and insulin-stimulated glucose uptake in HepG2 cells. In addition, KPNBeta1 knockdown reduced intracellular lipid accumulation. Mechanistically, KPNBeta1 transports nuclear factor kB (NF-kappaB) p65 from the cytoplasm to the nucleus to increase pro-inflammatory genes expression. In summary, KPNBeta1 acts as a positive regulator in the NF-kappaB pathway to enhance palmitate-induced inflammation response and insulin resistance in HepG2 cells

TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway.

High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3ß was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3ß in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3ß. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway.

KPNß1 promotes palmitate-induced insulin resistance via NF-κB signaling in hepatocytes.

It has been intensively studied that inflammation contributes to the insulin resistance development in obesity-induced type 2 diabetes mellitus (T2DM). In this study, we assessed the effect of karyopherin ß1 (KPNß1) in hepatic insulin resistance and the underlying mechanisms using high-fat diet (HFD) fed mice and palmitate (PA)-stimulated hepatocytes (HepG2). KPNß1 expression is increased in the HFD fed mice liver. PA upregulated KPNß1 expression in HepG2 cells in a time-dependent manner. PA also increased pro-inflammatory cytokines expression, including tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1ß (IL-1ß). KPNß1 knockdown reversed PA-induced pro-inflammatory cytokines expression and insulin-stimulated glucose uptake in HepG2 cells. In addition, KPNß1 knockdown reduced intracellular lipid accumulation. Mechanistically, KPNß1 transports nuclear factor kB (NF-κB) p65 from the cytoplasm to the nucleus to increase pro-inflammatory genes expression. In summary, KPNß1 acts as a positive regulator in the NF-κB pathway to enhance palmitate-induced inflammation response and insulin resistance in HepG2 cells.

The Role of PTP1B O-GlcNAcylation in Hepatic Insulin Resistance.

Protein tyrosine phosphatase 1B (PTP1B), which can directly dephosphorylate both the insulin receptor and insulin receptor substrate 1 (IRS-1), thereby terminating insulin signaling, reportedly plays an important role in insulin resistance. Accumulating evidence has demonstrated that O-GlcNAc modification regulates functions of several important components of insulin signal pathway. In this study, we identified that PTP1B is modified by O-GlcNAcylation at three O-GlcNAc sites (Ser104, Ser201, and Ser386). Palmitate acid (PA) impaired the insulin signaling, indicated by decreased phosphorylation of both serine/threonine-protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3ß) following insulin administration, and upregulated PTP1B O-GlcNAcylation in HepG2 cells. Compared with the wild-type, intervention PTP1B O-GlcNAcylation by site-directed gene mutation inhibited PTP1B phosphatase activity, resulted in a higher level of phosphorylated Akt and GSK3ß, recovered insulin sensitivity, and improved lipid deposition in HepG2 cells. Taken together, our research showed that O-GlcNAcylation of PTP1B can influence insulin signal transduction by modulating its own phosphatase activity, which participates in the process of hepatic insulin resistance.

TAB3 involves in hepatic insulin resistance through activation of MAPK pathway.

Insulin resistance is often accompanied by chronic inflammatory responses. The mitogen-activated protein kinase (MAPK) pathway is rapidly activated in response to many inflammatory cytokines. But the functional role of MAPKs in palmitate-induced insulin resistance has yet to be clarified. In this study, we found that transforming growth factor ß-activated kinase binding protein-3 (TAB3) was up-regulated in insulin resistance. Considering the relationship between transforming growth factor ß-activated kinase (TAK1) and MAPK pathway, we assumed TAB3 involved in insulin resistance through activation of MAPK pathway. To certify this hypothesis, we knocked down TAB3 in palmitate treated HepG2 cells and detected subsequent biological responses. Importantly, TAB3 siRNA directly reversed insulin sensitivity by improving insulin signal transduction. Moreover, silencing of TAB3 could facilitate hepatic glucose uptake, reverse gluconeogenesis and improve ectopic fat accumulation. Meanwhile, we found that the positive effect of knocking down TAB3 was more significant when insulin resistance occurred. All these results indicate that TAB3 acts as a negative regulator in insulin resistance through activation of MAPK pathway.

PRDX1 is involved in palmitate induced insulin resistance via regulating the activity of p38MAPK in HepG2 cells.

Studies have identified that type 2 diabetes mellitus (T2DM) patients displayed higher levels of plasma peroxiredoxin1(PRDX1) than non-diabetics. However, the impact of PRDX1 on insulin resistance and the underlying mechanism remains totally unknown. Here, we investigated the influence of PRDX1 on hepatic insulin resistance. We showed that the protein and mRNA levels of PRDX1 were significantly elevated under insulin-resistant conditions. In addition, we showed that interference of PRDX1 ameliorated palmitate-induced insulin resistance in HepG2 cells, which was indicated by elevated phosphorylation of protein kinase B (AKT) and of glycogen synthase kinase-3 (GSK3ß). Furthermore, the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were down-regulated following PRDX1 depletion. Accordingly, glucose uptake was suppressed in PRDX1-interferred HepG2 cells. In addition, Over-expression of PRDX1 enhanced PA-induced insulin resistance in HepG2 cells. Moreover, we found that knocking down PRDX1 improves insulin sensitivity and decreased the activation of p38 mitogen-activated protein kinase (p38MAPK). Our results demonstrate that PRDX1 can induce hepatic insulin resistance by activating p38MAPK signaling and identifies potential targets for new treatments.

PCBP2 regulates hepatic insulin sensitivity via HIF-1α and STAT3 pathway in HepG2 cells.

Elevated free fatty acids (FFAs) are fundamental to the pathogenesis of hepatic insulin resistance. However, the molecular mechanisms of insulin resistance remain not completely understood. Transcriptional dysregulation, post-transcriptional modifications and protein degradation contribute to the pathogenesis of insulin resistance. Poly(C) binding proteins (PCBPs) are RNA-binding proteins that are involved in post-transcriptional control pathways. However, there are little studies about the roles of PCBPs in insulin resistance. PCBP2 is the member of the RNA-binding proteins and is thought to participate in regulating hypoxia inducible factor-1 (HIF-1α) and signal transducers and activators of transcription (STAT) pathway which are involved in regulating insulin signaling pathway. Here, we investigated the influence of PCBP2 on hepatic insulin resistance. We showed that the protein and mRNA levels of PCBP2 were down-regulated under insulin-resistant conditions. In addition, we showed that over-expression of PCBP2 ameliorates palmitate (PA)-induced insulin resistance, which was indicated by elevated phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3ß (GSK3ß). We also found that over-expression of PCBP2 inhibits HIF1α and STAT3 pathway. Furthermore, glucose uptake was found to display a similar tendency with the phosphorylation of Akt. The expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, were down-regulated following Over-expression of PCBP2. Accordingly, PA-induced intracellular lipid accumulation was suppressed in over-expression of PCBP2 HepG2 cells. In addition, we found that over-expression of PCBP2 inhibits HIF1α and STAT3 pathway. Our results demonstrate that PCBP2 was involved in hepatic insulin sensitivity might via HIF-1α and STAT3 pathway in HepG2 cells.

TRAM1 protect HepG2 cells from palmitate induced insulin resistance through ER stress-JNK pathway.

Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. With high-fat feeding (HFD), FFAs can activate chronic endoplasmic reticulum (ER) stress in target tissues, initiating negative crosstalk between FFAs and insulin signaling. However, the molecular link between insulin resistance and ER stress remains to be identified. We here reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, was involved in the onset of insulin resistance in hepatocytes. TRAM1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAM1 led to hyperactivation of CHOP and GRP78, and the activation of downstream JNK pathway. Given the fact that the activation of ER stress played a facilitating role in insulin resistance, the phosphorylation of Akt and GSK-3ß was also analyzed. We found that depletion of TRAM1 markedly attenuated the phosphorylation of Akt and GSK-3ß in the cells. Moreover, application with JNK inhibitor SP600125 reversed the effect of TRAM1 interference on Akt phosphorylation. The accumulation of lipid droplets and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt. Glucose uptake assay indicated that knocking down TRAM1 augmented PA-induced down-regulation of glucose uptake, and inhibition of JNK using SP600125 could block the effect of TRAM1 on glucose uptake. These data implicated that TRAM1 might protect HepG2 cells against PA-induced insulin resistance through alleviating ER stress.