Expression of glucocorticoid receptor isoforms and associations with serine/arginine-rich protein 30c and 40 in patients with systemic lupus erythematosus.

OBJECTIVES: To investigate the expression of glucocorticoid receptor (GR) isoforms in patients with systemic lupus erythematosus (SLE), confirm the main GR isoforms involving in glucocorticoids (GC) resistance, and explore the associations of GR isoforms with serine/arginine-rich protein (SRp) 30c and SRp40. METHODS: Seventy patients with SLE and thirty-eight age- and sex-matched controls were recruited. All patients received prednisone (0.5-1 mg/kg/d) as their routine therapy. According to the therapeutic effect, patients were divided into glucocorticoid-resistant (GCR) and glucocorticoid-sensitive (GCS) groups. Transcript levels of GRα, GRß, GRγ, GR-P, SRp30c and SRp40 in peripheral blood mononuclear cells (PBMCs) were determined by real-time PCR. GRα and GRß proteins were detected by western blotting. Trial registration number is ChiCTR-RCH-12002808. RESULTS: Four GR transcripts in SLE patients showed the following trend: GRα (51.85%) > GR-P (23.78%) > GRγ (13.08%) >GRß (0.03%). GR-P transcript and ratio of GRα/GR-P in SLE patients were significantly higher than that in controls (p<0.05). GRα transcript and protein as well as SRp40 transcript in GCS group were significantly higher than that in the GCR group before GC treatment (p<0.05). In the GCS group, GRα transcript and SRp40 transcript were significantly higher after GC treatment than that before GC treatment (p<0.05). In the GCR group, GR-P transcript was significantly higher after GC treatment than that before GC treatment (p<0.05). Positive correlation between SRp40 and GRα transcript was found (p<0.05). Additionally, SLE Disease Activity Index scores were significantly negatively correlated with GRα transcript and protein expression (p<0.05). CONCLUSIONS: Our data demonstrated that the decreased expression of GRα might be the evidence of high disease activity and help to predict GC resistance. GR-P isoform might be implicated in the development of resistance. Additionally, the preliminary finding suggested that SRp40 might be associated with GRα transcripts in SLE patients.

Elevated serum leptin levels in patients with systemic lupus erythematosus.

Previous studies have indicated that leptin and the soluble leptin receptor (SLR) might influence inflammatory and immune processes in autoimmune diseases, but this remains unclear in systemic lupus erythematosus (SLE). The aim of our study was to assess if leptin and SLR are involved in the etiopathology of SLE and the possible mechanism of immune regulation. We studied 87 patients with SLE and 85 matched subjects. We assessed the levels of serum leptin and SLR, tested the long isoform leptin receptor (Ob-Rb) mRNA levels in SLE patients and a control group. Furthermore, we measured Th1 and Th2 percentage in SLE patients' lymphocytes and examined lymphocytes activation and proliferation assays with leptin stimulation in vitro. The study found a higher level of serum leptin in SLE patients, however, no difference was found in serum SLR levels or Ob-Rb mRNA levels between SLE patients and the control group. The percentage of Th1 cells decreased and Th2 cells increased after treatment with glucocorticoids in SLE patients. Leptin stimulated the proliferation of T cells in vitro, and differentiation to Th1 cells increased. The present study demonstrated that leptin may play an important role in the pathogenesis of SLE, inducing dysfunction of autoimmune processes.

Changes of glucocorticoid receptor isoforms expression in acute lymphoblastic leukemia correlate with glucocorticoid resistance.

Alternative splicing of the glucocorticoid receptor (GR) gene results in several GR isoforms, we examined their expression (GRα, GRß, GRγ and GR-P) by real-time RT-PCR in glucocorticoid (GC) sensitive (CEM-C7), GC resistant (CEM-C1) cells and adult acute lymphoblastic leukemia (ALL) patients, to determine the association of GR isoform expression profiles and GC resistance in adult ALL patients. With GC treatment, GR levels in C1 cells showed no obvious changes. In C7 cells, the mRNA levels of GRα, GRß and GRγ first increased and then decreased, whereas GR-P mRNA had a continued rising trend. C7 cells had a higher GRα/GRγ, lower GRα/GR-P and GRγ/GR-P ratios than C1 cells (P < 0.01). In adult ALL patients, GRγ mRNA varied in different ALL stages (complete remission CR 15.82 vs. relapsed 8.21 vs. initial 1.93 P < 0.05). It also did in the ratios between GR isoforms that GRα/GRγ and GRα/GR-P in initial patients were higher than relapsed and CR (P < 0.05), while GRγ/GR-P in CR was higher than initial and relapsed patients (P < 0.05). GR-P mRNA in T-ALL patients was much higher than that in B-ALL patients (P < 0.05). Peripheral blood hemoglobin (HB) was positively correlated with GRα mRNA and GR-P mRNA (P < 0.05), while white blood cells (WBC) negatively correlated with GRγ mRNA (P < 0.05). The present study demonstrates that GR autoinduction is more important to GC sensitivity than its basal level expression. GC sensitivity is also significantly correlated with GRα mRNA and mildly associated with GRß mRNA expression. Both GRγ mRNA and the ratios between GR isoforms (GRα/GRγ, GRα/GR-P and GRγ/GR-P) are correlated with ALL stages. The changes of mRNA expression levels of GRα, GR-P and GRγ may provide valuable information for GC resistance. Peripheral blood HB and WBC affect GR isoform expression.

[The relationship between mRNA level of glucocorticoid receptor α, heat shock protein 90, protein level of macrophage migration inhibitory factor and glucocorticoid resistance in systemic lupus erythematosus].

OBJECTIVE: To investigate the mRNA level of glucocorticoid receptor α (GRα) and heat shock protein 90 (HSP90) in peripheral blood mononuclear cells (PBMCs) and the plasma protein level of macrophage migration inhibitory factor (MIF) in patients with systemic lupus erythematosus (SLE) and to analyze their association with glucocorticoid (GC) resistance. METHODS: One hundred and six patients with SLE and thirty-eight healthy controls were enrolled in this study. Transcription levels of GRα and HSP90 were determined by real-time polymerase chain reaction. Enzyme-linked immunosorbent assay was used to detect the protein level of plasma MIF. The association between these parameters and GC resistance was analyzed by Spearman correlation analysis. The multivariate logistic regression model was used to analyze the risk factors for GC resistance. RESULTS: The mRNA level of GRα and HSP90 in GC resistance group was significantly lower than that in GC sensitive group [10.18 (3.12, 17.20) vs 16.83 (12.01, 24.18), P=0.001; 18.46 (14.77, 26.45) vs 25.84 (17.97, 35.90), P= 0.005]. MIF protein level in GC resistance group was significantly higher than that in GC sensitive group [(23.21±7.98) µg/L vs (18.34±6.29) µg/L; P=0.013]. The mRNA level of HSP90 in the high MIF group was significantly lower than that in the low MIF group [23.67 (13.84, 28.32) vs 26.64 (23.61, 47.16); P=0.001], as well as HSP90/GRα ratio (P=0.008). Additionally, the plasma protein level of MIF was negatively correlated with HSP90 (r=-0.275, P=0.004) and HSP90/GRα ratio (r=-0.341, P<0.001). SLE activity index score in GC resistance group was significantly higher than that in GC sensitive group [(12.23±2.86) µg/L vs (9.63±3.48) µg/L; P=0.003]. Logistic regression model indicated that disease activity was an independent risk factor for GC resistance (OR=17.481, 95% CI 1.747-174.903, P=0.015). CONCLUSIONS: Our preliminary findings suggest that low mRNA level of GRα and HSP90 and high protein level of MIF are associated with GC resistance. Elevated MIF level in SLE patients may play an important role in the development of GC resistance through down-regulating HSP90 and destabilizing the balance of HSP90/Grα. Disease activity is the risk factor for GC resistance, which might be the viable evidence of therapy response.

The cytokines (IFN-gamma, IL-2, IL-4, IL-10, IL-17) and Treg cytokine (TGF-beta1) levels in adults with immune thrombocytopenia.

Previous studies have indicated that autoimmune diseases might be caused by an imbalance of T helper cells (Th), cytokines, and regulatory T cells (Treg) cytokines. We measured the plasma concentrations of Th1-associated cytokines (IFN-gamma, IL-2), Th2 -associated cytokines (IL-4, IL-10), Th17-associated cytokine (IL-17) and Treg -associated cytokine (TGF-beta1) in adult patients with immune thrombocytopenia (ITP) and evaluated their clinical relevance. Plasma IFN-gamma, IL-2, IL-4, IL-10, IL-17 and TGF-beta1 concentrations of 52 ITP patients and 30 age- and sex-matched healthy controls were measured by enzyme-linked immunosorbent assay method (ELISA). Concentration of Th2 cytokines (IL-4 and IL-10) were significantly higher in ITP patients compared to controls (P < 0.05). However, concentrations of Th1 cytokines (IFN-gamma, IL-2), Th17 cytokine (IL-17) and Treg cytokine (TGF-beta1) were lower in ITP patients (P < 0.05). Concentration of IL-17 was significantly higher in chronic ITP patients compared to severe ITP patients (P < 0.05), and no significant difference of cytokine concentration among the other subgroups in ITP patients was found. Among the ITP patients, concentration of IFN-gamma correlated positively and significantly with PAIgG (r = 0.48, P = 0.02). A significant correlation was neither found between other cytokine levels and platelet count, nor between cytokine levels and megakaryocytes number, nor between cytokines levels and PAIgG or GPIIb/IIIa and/or GPIb/IX autoantibodies. The present study demonstrates that an imbalance of Th and Treg cytokines may mediate the pathogenesis of ITP.

Metagenomic next-generation sequencing on bronchoalveolar lavage fluid to contribute to diagnosis of subclinical pulmonary tuberculosis with scarce sputum and negative smear in a patient mimicking adult- onset still's disease: A case report.

Extremely high serum ferritin, which is regarded as a marker of adult-onset still's disease (AOSD), has been rarely observed in patients with TB. We report a case of TB diagnose by metagenomic next-generation sequencing(mNGS) who presented with clinical criteria of AOSD and extreme hyperferritinemia, which posed a diagnostic confusion. TB presenting with major clinical criteria of AOSD should be notable. Since TB remains a potentially curable disease, an awareness of its' protean manifestations is essential. A typical or even normal outcomes of clinical, microbiochemical, and radiologic evaluation should not be overlooked and dedicated diagnostic work-up should be performed for TB diagnosis. For equivocal cases, mNGS could be helpful.

A novel biological antibacterial polyvinyl alcohol/polyionic liquid hydrogel for wound dressing.

The release of antibiotics or anions by traditional bacteriostatic agents led to the development of bacterial drug resistance and environmental pollution. Ionic liquids (ILs) have become important choices for antibacterial agents because of their excellent physical, chemical and biological properties. In this paper, the bioactivities of 1-vinyl-3-butylimidazolium chloride ([VBIM]Cl, IL) and poly (1-vinyl-3-butylimidazolium chloride) (P[VBIM]Cl, PIL) were evaluated, and the potential antibacterial material was used to synthesize hydrogels. Using the colony formation assay and the Oxford cup method, antibacterial effect of IL and PIL were tested. Cell-Counting-Kit-8 (CCK-8) experiments were used to study the IC50 (half maximal inhibitory concentration) values of IL and showed 1.47 mg/mL, 0.35 mg/mL and 0.33 mg/mL at 24 h, 48 h and 72 h, respectively. The IC50 value of PIL were 12.15 µg/mL, 12.06 µg/mL and 11.76 µg/mL at 24 h, 48 h and 72 h, respectively. The PIL is further crosslinked with polyvinyl alcohol (PVA) to form a novel hydrogel through freeze-thaw cycles. The newly fabricated hydrogel exhibited a high water content, excellent water absorption properties and outstanding mechanical performance. Using the colony formation assay and the inhibition zone assay, the hydrogels exhibited favorable antibacterial effects (against E.coli and S.aureus) such that nearly 100% of the bacteria were killed in liquid medium while cultivating with H4 (synthesized by 0.5 g PIL and 1g PVA). In addition, the cytotoxicity of PIL was significantly reduced through hydrogen bond crosslinking. H4 showed the highest antibacterial activity and a good biocompatibility. The results indicated that the PVA&PIL hydrogels had great potential for wound dressing.

The mRNA Expression and Regulation of Glucocorticoid Receptor Isoforms Associated with Response of Multiple Myeloma Treated with a Glucocorticoids-Dependent Regimen of Dexamethasone, Bortezomib and Thalidomide.

OBJECTIVE: Glucocorticoids (GCs) are the effective first-line drugs and indispensable in chemotherapy regimens to treat patients with multiple myeloma (MM). Previous studies in a variety of hematologic malignancies have shown that the biological action of GC is mediated through the expression and activation and of glucocorticoids receptor (GR) isoforms in vitro. GR and its regulation are crucial determinants of the efficacy of GC independent therapy. There is currently lack of research on patients with MM. METHODS: 132 patients with MM were divided into responders (78 cases) and nonresponders (54 cases) according to the efficacy evaluated after four cycles of GC-dependent regimen. 66 patients with iron-deficiency anemia were served as controls. Preparation of mononuclear bone marrow cells (MBMCs) was purified by Ficoll-Hypaque gradient centrifugation. The mRNA expression of GR α, ß, γ, P, SRp30, SRp40, HSP90, NF-κB and AP-1 were detected by real time RT-PCR. TRIAL REGISTRATION: CHiCTR-RCH-12002872. RESULTS: The expression of four GR isoforms exhibited the following trend in MM patients and controls: GRα>GR-P>GRγ>GRß. GRα and HSP90 expression in responders was significantly higher than that of the nonresponders (P<0.050). HSP90/GRα expression in MM patients exhibited significantly higher than that in controls (P<0.001). SRp30c and SRp40 mRNA expression both showed significant positive correlation with GRα transcript (P<0.001). Compared with controls, NF-kB and AP -1 expression in MM patients was higher. NF-kB and AP-1 expression of nonresponders were significantly higher than that of responders. The difference was not obvious statistically (P>0.050). CONCLUSION: Our findings raise the possibility that low expression of GRα and HSP90 plays important roles in nonresponders. Lack of HSP90 might affect GR structure and further take part in nonresponse. SRp30c and SRp40 mRNA expression both showed significant positive correlation with GRα. That might become new targets for treatment of nonresponders in MM patients, although further studies are needed for clarification.

Inhibition of autophagy overcomes glucocorticoid resistance in lymphoid malignant cells.

Glucocorticoid (GC) resistance remains a major obstacle to successful treatment of lymphoid malignancies. Till now, the precise mechanism of GC resistance remains unclear. In the present study, dexamethasone (Dex) inhibited cell proliferation, arrested cell cycle in G0/G1-phase, and induced apoptosis in Dex-sensitive acute lymphoblastic leukemia cells. However, Dex failed to cause cell death in Dex-resistant lymphoid malignant cells. Intriguingly, we found that autophagy was induced by Dex in resistant cells, as indicated by autophagosomes formation, LC3-I to LC3-II conversion, p62 degradation, and formation of acidic autophagic vacuoles. Moreover, the results showed that Dex reduced the activity of mTOR pathway, as determined by decreased phosphorylation levels of mTOR, Akt, P70S6K and 4E-BP1 in resistant cells. Inhibition of autophagy by either chloroquine (CQ) or 3-methyladenine (3-MA) overcame Dex-resistance in lymphoid malignant cells by increasing apoptotic cell death in vitro. Consistently, inhibition of autophagy by stably knockdown of Beclin1 sensitized Dex-resistant lymphoid malignant cells to induction of apoptosis in vivo. Thus, inhibition of autophagy has the potential to improve lymphoid malignancy treatment by overcoming GC resistance.