MiR-181-5p protects mice from sepsis via repressing HMGB1 in an experimental model.

OBJECTIVE: Lentivirus-delivered microRNA (miR) has been reported to improve survival outcomes and organ dysfunction. The present study is aimed to explore whether sepsis-associated miR, miR-181-5p, could mitigate sepsis-induced inflammation and organ injury by the lentivirus-expressing system. MATERIALS AND METHODS: Cecal ligation and puncture (CLP)-operated mice were treated with lentivirus-expressing miR-181-5p (miR-agomir) 7 days before surgical operation by intravenous injection. Acute renal and hepatic injuries were assessed using specific biomarkers. Survival outcomes were evaluated following CLP operation within 72 hours. RESULTS: Lentivirus-delivered miR-181-5p improves survival outcomes of CLP-induced septic mice. The rescue of miR-181-5p expression by lentivirus expression vector protects against sepsis-induced renal and hepatic dysfunction. Sepsis-triggered inflammatory response and the release of HMGB1 level could be attenuated by miR-agomir administration. We also found that HMGB1 was a direct target of miR-181-5p, and that the overexpression of miR-81-5p led to a significant decrease in HMGB1 protein expression. CONCLUSIONS: miR-181-5p-mediated protective effects in septic mice were modulated, at least partially, through post-transcriptional repression of HMGB1 protein expression. The findings suggest that miR-181-5p may function as an HMGB1 antagonist for alleviating sepsis-induced systemic inflammatory diseases.

CalliSpheres® drug-eluting beads (DEB) transarterial chemoembolization (TACE) is equally efficient and safe in liver cancer patients with different times of previous conventional TACE treatments: a result from CTILC study.

PURPOSE: To assess the efficacy and safety of drug-eluting beads transarterial chemoembolization (DEB-TACE) in liver cancer patients with different times of previous conventional transarterial chemoembolization (cTACE) treatments. METHODS: 367 liver cancer patients about to receive DEB-TACE treatment were enrolled in this prospective cohort study. All patients were divided into no previous cTACE group (NPC group), 1-2 times previous cTACE group (PC group) and triple or above previous cTACE group (TPC group) according to the times of previous cTACE treatments. RESULTS: There was no difference in complete response (CR) (P = 0.671) and objective response rate (ORR) (P = 0.062) among three groups. Additionally, no difference in overall survival (OS) among groups (P = 0.899) was found. As to liver function, most liver function indexes were deteriorative at 1 week after DEB-TACE operation, but returned to baseline at 1-3 months after DEB-TACE operation in all three groups, while percentage of abnormal total bile acid (TBA) patients was higher in TPC group than NPC and PC groups at 1-3 month post-DEB-TACE (P = 0.018). As for safety profiles, the incidence of pain during DEB-TACE operation was lower in TPC group compared to NPC and PC groups (P = 0.005), while no difference of other adverse events was found during and 1 month post-DEB-TACE treatment among three groups. CONCLUSION: DEB-TACE treatment was equally efficient and tolerated in liver cancer patients with different times of previous cTACE treatments.

MiR-124a inhibits proliferation and invasion of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To investigate the impact of miR-124Aa on the proliferation, invasion and cytokine excretion of rheumatoid arthritis synovial fibroblasts (RASFs) in patients with rheumatoid arthritis (RA). PATIENTS AND METHODS: RASFs were separated for in-vitro culture, and transfected using lipidosome that connected with chemically synthesized miR-124a mimic or miR-124a inhibitor. Then, MTT, transwell chamber, and flow-cytometry were used to detect the impact on the proliferation, invasion, and apoptosis of RASFs; RT-PCR and Western-blotting were employed to measure the effect of miR-124a on the expressions of matrixmetalloproteinase3/13 (MMP3/13) and interleukin1ß (IL-1ß) of RASFs. RESULTS: miR-124a significantly suppresses the proliferation of RASFs, while inhibits the invasion of RASFs. The flow cytometry indicated that miR-124a showed no significant effect on the apoptosis of RASFs. Finally, miR-124a downregulates the expressions of MMP3/13 and IL-1ß. CONCLUSIONS: MiR-124a is of great significance for the onset of RA by inhibiting the proliferation and invasion of RASFs possibly through downregulating the expression of MMP3/13 and IL-1ß.

Roles of monocyte chemotactic protein 1 and nuclear factor-kappaB in immune response to spinal tuberculosis in a New Zealand white rabbit model

This study aimed to explore the roles of monocyte chemotactic protein 1 (MCP-1) and nuclear factor kappa B (NF-κB) in immune response to spinal tuberculosis in a New Zealand white rabbit model. Forty-eight New Zealand white rabbits were collected and divided into four groups: experimental group (n=30, spinal tuberculosis model was established), the sham group (n=15, sham operation was performed) and the blank group (n=3). The qRT-PCR assay and western blotting were applied to detect the mRNA and protein expressions of MCP-1 and NF-κB in peripheral blood. ELISA was used to measure serum levels of MCP-1, NF-κB, IFN-γ, IL-2, IL-4, and IL-10. Flow cytometry was adopted to assess the distributions of CD4+, CD8+ lymphocytes and CD4+ CD25+ Foxp3 lymphocyte subsets. Compared with the sham and blank groups, the mRNA and protein expressions of MCP-1 and NF-κB in the experimental group were significantly increased. The experimental group had lower serum levels of IL-2 and IFN-γ and higher serum level of IL-10 than the sham and blank groups. In comparison to the sham and blank groups, CD4+ T lymphocyte subsets percentage, CD4+/CD8+ ratio and CD4+ CD25+ Foxp3+ Tregs subsets accounting for CD4+ lymphocyte in the experimental group were lower, while percentage of CD8+ T lymphocyte subsets was higher. Our study provided evidence that higher expression of MCP-1 and NF-κB may be associated with decreased immune function of spinal tuberculosis, which can provide a new treatment direction for spinal tuberculosis.

miR-143: a novel regulator of MyoD expression in fast and slow muscles of Siniperca chuatsi.

The fast and slow skeletal muscle fibers in fish are separated to a much greater degree than in mammals. MyoD is required for the maintenance of normal fiber type balance in muscles. So far, the upstream regulatory factors of MyoD in terms of controlling muscle phenotype are poorly understood. In the present study, we used Siniperca chuatsi as a model system and demonstrated that miR-143 expression was negatively correlated with MyoD expression in the fast and slow muscles of S. chuatsi. The luciferase reporter assay further verified the direct interaction between the miR-143 and MyoD. The miR-143 suppression also led to the significant increase in MyoD and fast myosin heavy chain gene expression in vivo. Taken together, our studies indicated that miRNA participates in controlling the performance of different muscle fiber types in vertebrates.

Decreased submandibular adiponectin is involved in the progression of autoimmune sialoadenitis in non-obese diabetic mice.

OBJECTIVE: To investigate a possible role of adiponectin in the pathogenesis of autoimmune sialoadenitis in non-obese diabetic (NOD) mouse model of Sjögren's syndrome. MATERIALS AND METHODS: Expression of adiponectin and its receptors (AdipoR1/2) was detected by PCR, immunoblotting, or immunofluorescence. The level of adiponectin was quantified by ELISA. Adiponectin-related signaling molecules and pro-inflammatory cytokines were examined by PCR or immunoblotting. Apoptosis was evaluated by TUNEL staining, flow cytometry, and caspase 3 activation. RESULTS: Adiponectin and AdipoR1/2 mRNA and protein were expressed in submandibular glands. Adiponectin immunostaining was widely diffused in the cytoplasm of acinar and ductal cells. AdipoR1 was mainly distributed in acinar cytoplasm, while AdipoR2 was predominantly located at acinar cell membrane. Submandibular adiponectin levels were reduced during the progression of autoimmune sialoadenitis in 7-, 14-, and 21-week-old NOD mice, while AdipoR1/2 levels were unchanged. The levels of phosphorylated adenosine monophosphate-activated protein kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase were decreased, while interferon (IFN)-γ and glandular apoptosis were temporally increased at all time points. Moreover, exogenous adiponectin supplement inhibited, whereas neutralizing endogenous adiponectin by its antibody promoted IFN-γ-induced apoptosis and caspase 3 activation in cultured submandibular acinar cells. CONCLUSIONS: Adiponectin plays a protective role on submandibular cells. Decreased adiponectin might promote glandular destruction in autoimmune sialoadenitis.

Distribution of immunoglobulin G subclasses of anti-thyroid peroxidase antibody in sera from patients with Hashimoto's thyroiditis with different thyroid functional status.

The mechanism of disease progression in Hashimoto's thyroiditis (HT) is still unclear. Anti-thyroid peroxidase antibody (TPOAb), a diagnostic hallmark of HT, is principally of the immunoglobulin G (IgG) isotype, and it appears to be a response to thyroid injury. The aim of our study was to evaluate the distribution of IgG subclasses of TPOAb in sera from patients with HT with different thyroid functional status. Sera from 168 patients with newly diagnosed HT were collected and divided into three groups according to thyroid function: patients with hypothyroidism (n = 66), subclinical hypothyroidism (n = 60) and euthyroidism (n = 42). Antigen-specific enzyme-linked immunosorbent assay was used to detect the distribution of TPOAb IgG subclasses. The prevalence of TPOAb IgG subclasses in all patients' sera with HT was IgG1 70.2%, IgG2 35.1%, IgG3 19.6% and IgG4 66.1% respectively. The prevalence of IgG2 in sera from patients with hypothyroidism (51.5%) was significantly higher than that of subclinical hypothyroidism (33.3%) (P < 0.05), and the latter was also significantly higher than that of euthyroidism (11.9%) (P < 0.05). The positive percentage of IgG2 subclass in sera from patients with hypothyroidism and subclinical hypothyroidism was significantly higher than that of euthyroidism (P < 0.05), the prevalence and positive percentage of IgG4 subclass in sera from patients with hypothyroidism and subclinical hypothyroidism was significantly higher than that of euthyroidism respectively (P < 0.05). The predominant TPOAb IgG subclasses in sera from patients with HT were IgG1 and IgG4. Patients with high levels of TPOAb IgG2, IgG4 subclasses might be at high risk of developing overt hypothyroidism.

Long-term outcomes of patients with propylthiouracil-induced anti-neutrophil cytoplasmic auto-antibody-associated vasculitis.

OBJECTIVE: It was well known that propylthiouracil (PTU) could induce ANCA-associated vasculitis (AAV) and clinical evident vasculitis could resolve after cessation of PTU with or without immunosuppressive therapy. However, the treatment strategy for patients with PTU-induced AAV remained inconclusive and their long-term outcomes were lacking. The aim of our study was to summarize these data. METHODS: Fifteen patients with PTU-induced AAV, receiving immunosuppressive agents for <12 months and following over 24 months, were selected in the current study. The clinical and pathological data, including treatment protocols and outcomes, were retrospectively investigated. RESULTS: All the patients were followed for a mean of 55.0 (25-98) months. PTU was discontinued upon diagnosis of PTU-induced AAV. Immunosuppressive therapy was administrated only for patients with vital organ involvements, such as lung and kidney, and lasted only 7.9 +/- 3.3 (0.27-12) months. No relapse of vasculitis occurred during follow-up, even after withdrawal of immunosuppressive therapy. Twelve (80%) patients remained in complete remission and one patient remained in partial remission at the latest follow-up. Two patients were treatment resistant due to late referral and late withdrawal of PTU, both of them progressed to end-stage renal disease. For uncontrolled hyperthyroidism on presentation, six patients switched to methimazole and none of them experienced relapse of vasculitis. CONCLUSIONS: The long-term outcomes of patients with PTU-induced AAV were relatively good. PTU should be discontinued immediately after diagnosis. Immunosuppressive therapy may be only used in patients with vital organ involvements, and a long-term maintenance therapy may not be necessary.

Association of TNF-alpha promoter polymorphisms with the outcomes of hepatitis B virus infection in Chinese Han population.

Host genetic factors and environment factors including hepatitis B virus (HBV) genotypes are widely studied for the different outcomes of HBV infection. Recent studies suggest that tumour necrosis factor-alpha (TNF-alpha) plays a pivotal role in the viral clearance and host immune response to HBV, and the capacity for TNF-alpha production in individuals is influenced by a major genetic component. In this study, we aimed to explore whether the single-nucleotide polymorphisms (SNPs) of TNF-alpha promoter are associated with the outcomes of HBV infection in the Chinese Han population. One hundred and forty-three spontaneously recovered HBV subjects and 196 chronic hepatitis B patients were recruited in this case-control study in the Beijing area of China. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) and sequence-specific primer-PCR (SSP-PCR) were used to detect the SNPs of five sites in the TNF-alpha promoter (-238G/A, -308G/A, -857C/T, -863C/A, -1031T/C). The frequency distributions of genotypes and haplotypes in two groups were analysed by EPI and EH programs. The presence of the -238GG genotype was significantly correlated with persistence of HBV infection (OR = 4.08, P = 0.02), and -857TT genotype appeared in relation to the spontaneous clearance of HBV (OR = 0.47, P = 0.03). Frequency of haplotype GGCCT (-238/-308/-857/-863/-1031) in the chronic HB group was significantly lower than that in spontaneously recovered group (P = 0.03), and frequencies of haplotypes GGCAT and GGTAT in the chronic HB group were significantly higher than those in the spontaneously recovered group (P = 0.0001, P = 0.0004). In conclusion, TNF-alpha promoter polymorphisms are independently associated with different outcomes of HBV infection.

Role of beta(1)-integrins and their associated tetraspanin molecules in fibronectin-enhanced megakaryopoiesis.

BACKGROUND: We have shown previously that fibronectin (FN) together with megakaryocyte (Mk) growth and development factor (MGDF) enhanced generation of Mk progenitors determined by colony-forming unit (CFU)-Mk assay. MGDF can activate beta(1)-integrins on progenitor cells and increase binding to FN. We studied the role of beta(1)-integrin-tetraspanin complexes by which FN may enhance megakaryopoiesis. METHODS: Cord blood CD34(+) cells were cultured for up to 8 days in serum-free medium containing IL-3, IL-6 and SCF with or without MGDF in the presence or absence of FN. Immunofluorescence flow cytometry was used to monitor changes in beta(1)-integrin-tetraspanin complexes. CFU-Mk assay was used to assess Mk commitment. RESULTS: The cocktail of cytokines irrespective of the presence of MGDF altered the percentage expression of beta(1)-integrins CD49d and CD49e on CD34(+) cells. CD49d expression fell initially (98% to 15%) and then rose to 75%, whereas CD49e progressively increased over the 8 days of culture, from 5.4% to 22%. However, with the addition of FN, similar changes in the expression of beta(1)-integrins were observed but the expression was maintained at a higher level. MGDF and FN increased expression of tetraspanin molecules, CD63 and CD151, as well as their co-expression with the beta(1)-integrins on both the CD34(+) and CD34(-) cells (e.g. and increase from 0% to 80% co-expression of CD49d and CD151 on CD34(+)). Blocking of beta(1)-integrins or the tetraspanin CD151 with the respective MAb reduced Mk progenitor generation in a stromal cell model. DISCUSSION: FN enhanced Mk progenitor generation through modulation of beta(1)-integrin-tetraspanin complexes, such as CD151/CD49d.

Anti-myeloperoxidase antibodies in sera from patients with propylthiouracil-induced vasculitis might recognize restricted epitopes on myeloperoxidase molecule.

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic antibodies (ANCA) in primary systemic vasculitis. It is known that propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The production of anti-MPO antibodies in patients with PTU-induced vasculitis may be different from that in patients with primary microscopic polyangiitis (MPA). One possible reason for this may be differences in epitope recognition. The aim of this study is to compare the epitopes of antibodies to MPO in sera from patients with PTU-induced vasculitis (n = 10) and MPA (n = 10). The sera were collected and used to inhibit monoclonal antibodies against human MPO (3D8 and 6B9) and affinity purified, horseradish peroxidase conjugated human anti-MPO antibodies (Pab1-HRP, Pab2-HRP) in a competitive inhibition enzyme-linked immunosorbent assay (ELISA) system using soluble human MPO as solid phase ligand. The Pab1-HRP and Pab2-HRP were affinity purified from plasma exchanges of a patient with PTU-induced vasculitis and a patient with MPA, respectively. The inhibition rates were evaluated and compared between the PTU and primary MPA groups. In the PTU group all 10 sera could inhibit 3D8: the average inhibition rate was 44.7% +/- 5.0%; 9/10 sera could inhibit 6B9: the average inhibition rate was 35.6% +/- 6.0%. However, in the MPA group all 10 sera could inhibit 3D8 and 6B9; the average inhibition rates were 68.4% +/- 16.1% (P < 0.01) and 62.2% +/- 17.2% (P < 0.01), respectively. Sera in both the PTU and MPA groups could inhibit Pab1-HRP and the inhibition rates were 81.4% +/- 9.4%versus 86.6% +/- 17.2% (P > 0.05). However, the average inhibition rate for Pab2-HRP in the MPA group was significantly higher than that in the PTU group (76.3% +/- 7.8%versus 58.9% +/- 15.5%, P < 0.01). We conclude that anti-MPO antibodies from patients with PTU-induced vasculitis and from patients with primary MPA could recognize more than one epitope on the native MPO molecule. Although the epitopes overlapped between the two groups, the epitopes of anti-MPO antibodies from patients with PTU-induced vasculitis might be more restricted.

Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.