Characterization of miRNAs involved in response to poly(I:C) in porcine airway epithelial cells.

MicroRNAs (miRNA) have been implicated in a variety of pathological conditions including infectious diseases. Knowledge of the miRNAs affected by poly(I:C), a synthetic analog of viral double-stranded RNA, in porcine airway epithelial cells (PAECs) contributes to understanding the mechanisms of swine viral respiratory diseases, which bring enormous economic loss worldwide every year. In this study, we used high throughput sequencing to profile miRNA expression in PAECs treated with poly(I:C) as compared to the untreated control. This approach revealed 23 differentially expressed miRNAs (DEMs), five of which have not been implicated in viral infection before. Nineteen of the 23 miRNAs were down-regulated including members of the miR-17-92 cluster, a well-known polycistronic oncomir and extensively involved in viral infection in humans. Target genes of DEMs, predicted using bioinformatic methods and validated by luciferase reporter analysis on two representative DEMs, were significantly enriched in several pathways including transforming growth factor-ß signaling. A large quantity of sequence variations (isomiRs) were found including a substitution at position 5, which was verified to redirect miRNAs to a new spectrum of targets by luciferase reporter assay together with bioinformatics analysis. Twelve novel porcine miRNAs conserved in other species were identified by homology analysis together with cloning verification. Furthermore, the expression analysis revealed the potential importance of three novel miRNAs in porcine immune response to viruses. Overall, our data contribute to clarifying the mechanisms underlying the host immune response against respiratory viruses in pigs, and enriches the repertoire of porcine miRNAs.

Immunomodulatory effect of bone marrow mesenchymal stem cells on T lymphocytes in patients with decompensated liver cirrhosis.

We explored the immunomodulatory effects of bone marrow mesenchymal stem cells (BMSCs) on peripheral blood T lym-phocytes in patients with decompensation stage, hepatitis B-associated cirrhosis. MSCs from nine patients were analyzed by flow cytometry. Peripheral blood lymphocytes were isolated for fluorescent staining. Following stimulation by phytohemagglutinin (PHA), peripheral blood lymphocytes were co-cultured with BMSCs in serum and divided into four groups: (1) BMSC + lymphocyte + PHA contact culture group; (2) BMSC + lymphocyte + PHA non-contact culture group; (3) lym-phocyte + PHA positive control group; and (4) lymphocyte-only negative control group. Lymphocyte proliferation and frequencies of CD4(+)CD25(+)CD127(-) Tregs and CD4(+)CD8(-)IL-17(+) (Th17) cells were de-tected. Cell proliferation in groups 1 and 2 declined compared with group 3 (P < 0.01), and was notably higher than in group 4 (P < 0.01). CD4(+)CD25(+)CD127(-) Tregs frequencies in groups 1 and 2 were higher than in groups 3 and 4. In an intra-group comparison before and after culture, Th17 cell frequencies in groups 1 and 2 were higher than in group 4 (P < 0.01), but lower than in group 3 (P < 0.01). The Treg/Th17 ratio in groups 1 and 2 increased (P < 0.01), but did not change signifi-cantly in groups 3 and 4 (P > 0.05). In a comparison between groups after culture, the Treg/Th17 ratio in groups 1 and 2 increased more than in groups 3 and 4 (P < 0.01). BMSCs from cirrhotic patients can inhibit the proliferation of peripheral blood T lymphocytes, upregulate the ex-pression of CD4(+)CD25(+)CD127(-) Tregs, and improve Treg/Th17 imbal-ance. The mechanism by which this takes place may be associated with immunomodulatory effects induced by the secretion of soluble factors.

Association of cystatin C-based glomerular filtration rate with SYNTAX score in patients with diabetes.

AIM: Serum cystatin C has been proposed as a better marker of glomerular filtration rate than serum creatinine. SYNTAX score (SXscore) can accurately reflect the severity of coronary artery disease (CAD). However, the association between Cystatin C-based glomerular filtration rate (eGFRcys) and SXscore in patients with diabetes has never been reported. METHODS: We prospectively included 656 consecutive patients with diabetes who were angiographically diagnosed with CAD from January 2010 to December 2011. Renal function was assessed by eGFRcys. SXscore was calculated using SXscore algorithm. Ordinal logistic regression and Pearson correlation were used to analyze the association between eGFRcys and SXscore. RESULTS: Patients with renal dysfunction were older, more often female, more likely to have a history of hypertension and less tobacco use when compared with those patients with normal renal function. Age, sex, SBP, DBP, fasting glucose, HbA1c, TC, LDL, HDL, TG, BMI and CRP were not different among SXscore tertile groups. Incidence of hypertension, hyperlipidemia, family history and tobacco use were similar among these groups. Correlation analysis suggested that eGFRcys was negatively correlated with SXscore (R=-0.255, P<0.001). Ordinal logistic regression showed that eGFRcys was an independent predictor of SXscore (ß=-0.027, P<0.001). CONCLUSIONS: eGFRcys was an independent predictor of SXscore in patients with diabetes. This might help explain the increased risk of CVD events and mortality in patients with renal dysfunction. Further prospectively multiple centre studies are required to better quantify this finding.

Expression patterns of specific promyelocytic/retinoic acid receptor-alpha transcripts in patients with acute promyelocytic leukemia.

Several additional promyelocytic/retinoic acid receptor-alpha (PML/RARalpha) transcripts besides bcr1, bcr2, and bcr3 have been identified in patients with acute promyelocytic leukemia (APL). However, the expression levels of these specific isoforms and their clinical relevance have not been studied to date. The real-time quantitative polymerase chain reaction was established to detect each specific isoform of PML/RARalpha transcripts (bcr1/2, P46R3, P4R3, bcr3, and P2R3) in 46 APL patients. Whereas P46R3 and P4R3 isoforms were concurrently expressed in both bcr1- and bcr2-positive patients, P2R3 isoform was expressed only in bcr3-positive patients. A total of 13 patients had lower expression of bcr1/2 (median 11.60%, 0.86-108.51%) than that of P46R3 (median 14.26%, 6.03-222.91%; P = 0.001). The expression level of P4R3 (median 19.10%, 0.71-266.19%) was lower than the sum of bcr1/2 and P46R3 (median 37.94%, 9.62-403.51%) in all cases (P < 0.001). All 16 cases with bcr3 had concurrent low expression of P2R3 (P < 0.001). Structural analysis revealed that both P4R3 and P2R3 splicing resulting in the generation of a premature termination codon, which was recognized by nonsense-mediated decay (NMD). We suggest that alternative splicing of PML/RARalpha transcripts might be involved in NMD and each isoform should be quantified to further understand the pathogenesis of APL, stratify the risk of relapse, and monitor minimal residual disease.

Methylation of DAPK1 promoter: frequent but not an adverse prognostic factor in myelodysplastic syndrome.

Promoter hypermethylation plays an important role in the inactivation of cancer-related genes. This abnormality occurs early in leukemogenesis and seems to be associated with poor prognosis in myelodsplastic syndrome (MDS). The identification of more inactivated tumor suppressor genes contributing to the development of MDS may lead to further elucidation of the biology of this disease and help to identify novel targets for therapy. In this study, the methylation status of death-associated protein kinase 1 (DAPK1) gene promoter was analyzed by using methylation-specific polymerase chain reaction in bone marrow (BM) samples from 59 patients with different stages of MDS. The abnormal methylation of the DAPK1 gene was found in 37 of 59 (62.7%) MDS cases. The correlation was significant between the sex and the methylation status of DAPK1 promoter in MDS patients (R=0.332, P=0.010). Furthermore, methylation status of DAPK1 promoter was associated with the percentage of BM blasts (R=0.346, P=0.010) and International Prognosis Scoring System (IPSS) groups (R=0.278, P=0.034). The estimated 50% survival time of the methylated DAPK1 group and unmethylated group was 20 and 33 months, respectively. There was no significant difference between these two groups (chi2=0.652, P=0.419). Multivariate analysis identified the age older than 50 years, the Int-2/high-risk categories of IPSS and the advanced stage MDS (RAEB-1/RAEB-2) in WHO classification as negative prognostic factors (P<0.05). Aberrant methylation of DAPK1 gene promoter had no influence on the prognosis of MDS despite of its increasing occurrence during disease progression.