NLRP3 regulates CIITA/MHC II axis and interferon-γ-inducible chemokines in Malassezia globosa-infected keratinocytes.

CIITA, a member of NOD-like receptor (NLR) family, is the major MHC II trans-activator and mediator of Th1 immunity, but its function and interaction with NLRP3 have been little studied. We found activation of NLRP3 inflammasome, increased expression of CIITA, CBP, pSTAT1, STAT1, MHC II, IFN-γ and IFN-γ-inducible chemokines (CCL1 and CXCL8), and colocalisation of NLRP3 with CIITA in Malassezia folliculitis lesions, Malassezia globosa-infected HaCaT cells and mouse skin. CoIP with anti-CIITA or anti-NLRP3 antibody pulled down NLRP3 or both CIITA and ASC. NLRP3 silencing or knockout caused CIITA downexpression and their colocalisation disappearance in HaCaT cells and mouse skin of Nlrp3-/- mice, while CIITA knockdown had no effect on NLRP3, ASC, IL-1ß and IL-18 expression. NLRP3 inflammasome inhibitors and knockdown significantly suppressed IFN-γ, CCL1, CXCL8 and CXCL10 levels in M. globosa-infected HaCaT cells. CCL1 and CXCL8 expression was elevated in Malassezia folliculitis lesions and reduced in Nlrp3-/- mice. These results demonstrate that M. globosa can activate NLRP3 inflammasome, CIITA/MHC II signalling and IFN-γ-inducible chemokines in human keratinocytes and mouse skin. NLRP3 may regulate CIITA by their binding and trigger Th1 immunity by secreting CCL1 and CXCL8/IL-8, contributing to the pathogenesis of Malassezia-associated skin diseases.

Five miRNAs as novel diagnostic biomarker candidates for primary nasopharyngeal carcinoma.

MicroRNAs (miRNAs) play an essential role in the development and progression of nasopharyngeal carcinomas (NPC). Despite advances in the field of cancer molecular biology and biomarker discovery, the development of clinically validated biomarkers for primary NPC has remained elusive. In this study, we investigated the expression and clinical significance of miRNAs as novel primary NPC diagnostic biomarkers. We used an array containing 2, 500 miRNAs to identify 22 significant miRNAs, and these candidate miRNAs were validated using 67 fresh NPC and 25 normal control tissues via quantitative real-time PCR (qRT-PCR). Expression and correlation analyses were performed with various statistical approaches, in addition to logistic regression and receiver operating characteristic curve analyses to evaluate diagnostic efficacy. qRT-PCR revealed five differentially expressed miRNAs (miR-93-5p, miR-135b-5p, miR-205-5p and miR-183-5p) in NPC tissue samples relative to control samples (p<0.05), with miR-135b-5p and miR-205-5p being of significant diagnostic value (p<0.01). Moreover, comparison of NPC patient clinicopathologic data revealed a negative correlation between miR-93-5p and miR- 183-5p expression levels and lymph node status (p<0.05). These findings display an altered expression of many miRNAs in NPC tissues, thus providing information pertinent to pathophysiological and diagnostic research. Ultimately, miR-135b-5p and miR-205-5p may be implicated as novel NPC candidate biomarkers, while miR- 93-5p, miR-650 and miR-183-5p may find application as relevant clinical pathology and diagnostic candidate biomarkers.

Identification of steroid biosynthetic defects in genotype-proven heterozygous individuals for 17alpha-hydroxylase/17,20-lyase deficiency.

OBJECTIVE: P450c17 deficiency (17alpha-hydroxylase/17,20-lyase deficiency, 17OHD) is a rare form of congenital adrenal hyperplasia caused by CYP17A1 gene mutations. The D487_F489 deletion in exon 8 and Y329fs in exon 6 are relatively frequent mutations of the CYP17A1 gene in China that completely abolish the enzyme activity of P450c17. However, little remains known about steroid biosynthetic functions in carriers with these mutations in a single allele of the CYP17A1 gene, who are assumed to have 50% P450c17 activity. We investigated adrenal steroidogenic function in genotype-proven heterozygotes carrying such mutations in the CYP17A1 gene in vivo. PATIENTS AND DESIGN: Eight patients and fourteen family members from five families with 17OHD were recruited. The mutations of the CYP17A1 gene in these individuals were screened by sequencing. The hormonal response to cosyntropin (ACTH) was evaluated in the 14 genotype-proven carriers and 45 age- and gender-matched normal controls. RESULTS: Fourteen carriers of the CYP17A1 mutation - seven with the D487_F489 deletion, six with Y329fs and one with H373L - were identified from the five families with 17OHD. Compared with normal controls, carriers showed lower basal and ACTH-stimulated cortisol levels but higher ACTH-stimulated corticosterone levels. The ratios of corticosterone to cortisol in the genotype-proven heterozygotes were higher than those of the normal controls at the baseline and after cosyntropin stimulation. Similarly, the progesterone levels and the ratios of progesterone to 17-hydroxyprogesterone in the male heterozygotes were also higher than those of the normal controls, both before and after ACTH stimulation. CONCLUSION: Genotype-proven carriers of the CYP17A1 mutation who lack apparent clinical symptoms exhibit decreased adrenal 17alpha-hydroxylase activity and altered adrenal gland reserve for steroid biosynthesis.

A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism.

Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537-3 C>A). Computational analysis of the missense mutation in the three-dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice-acceptor mutation (c. 537-3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene.

[The rhythmicity of adiponectin and its relation with glucocorticoids, insulin and leptin].

OBJECTIVE: To investigate the circadian rhythmicity of adiponectin in patients with Cushing's syndrome and obesity, as well as in normal subjects. The effects of glucocorticoids, insulin and leptin on the secretion of adiponectin were also explored. METHODS: 15 patients with Cushing's syndrome and 10 with obesity as well as 9 normal subjects were recruited and their serum adiponectin, glucocorticoids, insulin and leptin levels were determined 6 times a day (8:00, 12:00, 16:00, 20:00, 24:00, 3:00). RESULTS: Circadian rhythmicity of adiponectin was not observed, but the adiponectin levels in patients with obesity were lower than those in the normal subjects at all 6 time spots. The area under curve (AUC) of serum adiponectin concentrations were significantly negatively correlated with body mass index (BMI, r = 0.46, P < 0.01), waist circumference (r = -0.33, P < 0.05) and insulin AUC (r = -0.36, P < 0.05). A multiple linear regression analysis revealed that BMI and waist circumference were two significant independent parameters of the plasma adiponectin level. The adiponectin levels did not manifest any change after short term dexamethasone administration with both high and low doses, but were obviously decreased after surgery. CONCLUSIONS: Neither long-term endogenous hyperglucocorticoidism nor short-term dexamethasone administration affects the serum adiponectin levels and the serum adiponectin concentrations remain unchanged even with elevated postprandial insulin levels.

[Effect of berberine on the differentiation of adipocyte].

OBJECTIVE: To observe the effect of berberine on the differentiation of 3T3-L1 preadipocytes into adipocytes and to elucidate its mechanism. METHODS: 3T3-L1 preadipocytes were cultured and then divided into 7 groups into whose media were added berberine of the concentrations of 0, 0.1, 1, 10, and 100 micro mol/L, 100 nmol/Linsulin, and 10 micro mol/L berberine + 100 nmol/L insulin. The proliferation of 3T3-L1 preadipocytes was detected by MTT method. The accumulation of lipid in the cytoplasm of differentiated adipocytes was observed by oil red O staining. The peroxisome proliferation activated receptor gamma2 (PPARgamma2) mRNA and protein were detected by RT-PCR and Western blotting respectively. RESULTS: MTT method showed that the absorbance at 570 nm of 3T3-L1 preadipocytes increased by 17% (P < 0.01), 36% (P < 0.001), and 22% (P < 0.05) in the groups of 1, 10, and 100 micro mol/L berberine, by 53% (P < 0.0001)in the group of 100 nmol/L insulin, and by 66% in the group of 10 micro mol/L berberine + 100 nmol/L insulin. There were less and smaller lipid droplets in the 3T3-L1 adipocytes treated with berberine as compared with the untreated control cells and only 10% - 20% of the treated cells displayed big lipid drops. RT-PCR showed that berberine significantly reduced the expression of PPARgamma2 mRNA by 48% (P < 0.01) in the course of 3T3-L1 adipocyte differentiation. Western blotting showed that berberine inhibited the expression of PPARgamma2 protein. CONCLUSION: Berberine promotes the proliferation of 3T3-L1 preadipocytes, decreases the accumulation of lipid drops therein, and inhibits the terminal differentiation of adipocyte, which may be associated with its effect on decreasing the expression of PPARgamma2 mRNA and protein, suggesting that berberine has advantages in the treatment of obesity patients with type 2 diabetes.