Pharmacodynamic Evaluation and PK/PD-Based Dose Prediction of Tulathromycin: A Potential New Indication for Streptococcus suis Infection.

Tulathromycin is the first member of the triamilide antimicrobial drugs that has been registered in more than 30 countries. The goal of this study is to provide a potential new indication of tulathromycin for Streptococcus suis infections. We investigated the pharmacokinetic and ex vivo pharmacodynamics of tulathromycin against experimental S. suis infection in piglets. Tulathromycin demonstrated a relatively long elimination half-life (74.1 h) and a mean residence time of 97.6 h after a single intramuscular administration. The minimal inhibitory concentration (MIC) and bactericidal concentration in serum were markedly lower than those in broth culture, with Mueller-Hinton broth/serum ratios of 40.3 and 11.4, respectively. The post-antibiotic effects were at 1.27 h (1× MIC) and 2.03 h (4× MIC) and the post-antibiotic sub-MIC effect values ranged from 2.47 to 3.10 h. The ratio of the area under the concentration-time curve divided by the MIC (AUC/MIC) correlated well with the ex vivo antimicrobial effectiveness of tulathromycin (R2 = 0.9711). The calculated AUC12h/MIC ratios in serum required to produce the net bacterial stasis, 1-log10 and 2-log10 killing activities were 9.62, 18.9, and 32.7, respectively. Based on the results of Monte Carlo simulation, a dosage regimen of 3.56 mg/kg tulathromycin was estimated to be effective, achieving for a bacteriostatic activity against S. suis infection over 5 days period. Tulathromycin may become a potential option for the treatment of S. suis infections.

LincRNA-Gm4419 knockdown ameliorates NF-κB/NLRP3 inflammasome-mediated inflammation in diabetic nephropathy.

Diabetic nephropathy (DN) as the primary cause of end-stage kidney disease is a common complication of diabetes. Recent researches have shown the activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) and NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome are associated with inflammation in the progression of DN, but the exact mechanism is unclear. Long noncoding RNAs (lncRNAs) have roles in the development of many diseases including DN. However, the relationship between lncRNAs and inflammation in DN remains largely unknown. Our previous study has revealed that 14 lncRNAs are abnormally expressed in DN by RNA sequencing and real-time quantitative PCR (qRT-PCR) in the renal tissues of db/db DN mice. In this study, these lncRNAs were verified their expressions by qRT-PCR in mesangial cells (MCs) cultured under high- and low-glucose conditions. Twelve lncRNAs displayed the same expressional tendencies in both renal tissues and MCs. In particular, long intergenic noncoding RNA (lincRNA)-Gm4419 was the only one associating with NF-κB among these 12 lncRNAs by bioinformatics methods. Moreover, Gm4419 knockdown could obviously inhibit the expressions of pro-inflammatory cytokines and renal fibrosis biomarkers, and reduce cell proliferation in MCs under high-glucose condition, whereas overexpression of Gm4419 could increase the inflammation, fibrosis and cell proliferation in MCs under low-glucose condition. Interestingly, our results showed that Gm4419 could activate the NF-κB pathway by directly interacting with p50, the subunit of NF-κB. In addition, we found that p50 could interact with NLRP3 inflammasome in MCs. In conclusion, our findings suggest lincRNA-Gm4419 may participate in the inflammation, fibrosis and proliferation in MCs under high-glucose condition through NF-κB/NLRP3 inflammasome signaling pathway, and may provide new insights into the regulation of Gm4419 during the progression of DN.

Tumor-derived chemokine CCL5 enhances TGF-ß-mediated killing of CD8(+) T cells in colon cancer by T-regulatory cells.

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (T(reg)). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T(reg) infiltration and CD8(+) T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T(reg) against CD8(+) T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T(reg) cell infiltration and CD8(+) T-cell apoptosis was noted. TGF-ß signaling blockade diminished apoptosis of CD8(+) T cells, implicating TGF-ß as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-ß by CCR5-deficient T(reg) or to enhance their cytotoxic effects against CD8(+) T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of T(reg) in inhibiting the antitumor responses of CD8(+) T cells, in the same way as CCL5 signaling blockade. Together, our findings establish that CCL5/CCR5 signaling recruits T(reg) to tumors and enhances their ability to kill antitumor CD8(+) T cells, thereby defining a novel mechanism of immune escape in colorectal cancer.

[Construction of let-7a expression plasmid and its inhibitory effect on k-Ras protein in A549 lung cancer cells].

OBJECTIVE: To elucidate the role of let-7a-mediated gene regulation in the pathogenesis of lung cancer. METHODS: Two template DNA sequences were designed based on hsa-let-7a sequence in miRBase database. The let-7a expression construct and a control plasmid, namely pGenesil-let-7a and pGenesil-control, respectively, were generated by cloning the annealed oligonucleotides into pGenesil-1 and then transfected into A549 cells, which were selected by G418 to establish the lung cancer cell lines stably expressing let-7a-GFP and control-GFP. The living cells were counted by MTT assay and cell growth curves were drawn to analyze the cell proliferation. The k-Ras mRNA level was assessed by semi-quantitative RT-PCR, and the expression of k-Ras protein was determined by Western blotting and immunocytochemistry. RESULTS: The recombinant vectors were verified by sequencing. The cell growth curves indicated that the proliferation of the cells transfected with pGenesil- let-7a were inhibited significantly compared with that of cells transfected with pGenesil-control and A549 cells. Semi- quantitative RT-PCR analysis showed that the levels of k-Ras mRNA almost remained unchanged in cells with or without the treatments. Western blotting and immunocytochemistry demonstrated a significant decrease of k-Ras protein levels in cells transfected with pGenesil-let-7a, but not in cells transfected with pGenesil-control, when compared to A549 cells. CONCLUSION: let-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.

[Kinetic analysis of laccase catalyze phenolic and aniline compounds and detecting catechol in wastewater].

Phenolic or aniline compounds were important pollutants in the industrial wastewaters to seriously polluted water environment. This research developed a detecting method of phenolic and aniline compounds based on the kinetic parameters of the substrates of laccase. Catalytic reaction between laccase and phenolic and aniline compounds was characterized using spectrophotometic method, which resulted 0-10 mg/L substrate reaction rate and calibration curve of substrate concentration and reaction rate. And then the non-volatile phenols in three kinds of coking wastewater were screened and the contents were detected. The result showed that polyhydric phenol, multi-amine and aminophenol were the main substrates of laccase. The optimum pH of phenols was around 7.0 and anilines 4.5-5.0, K(m) values of each substrates was 0.4-10 mmol/L. The calibration curve performed good first order kinetics linear relationship except benzidine with correlation coefficients above 0.96. Using laccase method, the contents of catechol in three kinds of coking wastewater were respectively detected to be 190.5, 265.8 and 155.3 mg/L with recoveries ranged from 89.9% to 115.8%.

The let-7a microRNA protects from growth of lung carcinoma by suppression of k-Ras and c-Myc in nude mice.

PURPOSE: Down-regulation of let-7 microRNA (miRNA) plays an important role in the pathogenesis of lung cancer. k-Ras and c-Myc, two key oncogenes in lung cancer, have been found to be targeted by let-7 in vitro. However, the in vivo relevance of these findings is unknown. The aim of the present study is to determine the effect of let-7a, a member of let-7 family, on the growth of lung cancer in vivo and to investigate whether let-7-induced suppression of k-Ras and c-Myc is involved in lung cancer. METHODS: A549-let-7a cell line and A549-control cell line, two stable transfected cell lines over-expressing let-7a and the control miRNA, were established and preserved in our lab. A549, A549-control, and A549-let-7a cells were injected subcutaneously into nude mice, respectively. After 30 days, the mice were killed; the xenografts were excised and weighed. The expression of let-7a in tumor xenografts was assessed by real-time reverse transcription-PCR (RT-PCR). The expression of k-Ras and c-Myc in xenografts were determined by western blot and immunohistochemistry detection. RESULTS: Real-time RT-PCR showed the expression of let-7a was increased significantly in A549-let-7a cells-injected group, compared with A549-control cells-injected group and A549 cells-injected group (P < 0.01). In the xenografts of A549-let-7a cells-injected group, a significant depression in tumor weight (P < 0.05) and significant decrease of k-Ras and c-Myc protein were observed (P < 0.01), compared to A549 cells-injected group and A549-control cells-injected group. CONCLUSION: Overexpression of let-7a can inhibit the growth of lung cancer transplanted subcutaneously in nude mice by suppression of k-Ras and c-Myc.

Effector/memory but not naive regulatory T cells are responsible for the loss of concomitant tumor immunity.

The phenomenon of concomitant tumor immunity involves a tumor-bearing host rejecting another similar tumor at a distant site and suggests the existence of tumor-specific immunity. Loss of this immunity may contribute to tumor metastasis. However, mechanisms underlying the loss of concomitant immunity are largely unknown. We set up a concomitant tumor immunity model in which this immunity is gradually lost as the primary tumor progresses. We found that CD8(+) T cells, especially tumor-infiltrating CD8(+) T cells, from mice that lost concomitant tumor immunity, possessed potent antitumor properties and strongly expressed effector molecules. Furthermore, effector/memory regulatory T cells (Treg cells, CD103(+)CD4(+)Foxp3(+) T cells) increased as the primary tumor progressed. They initially accumulated around the tumor and in the spleen at later points. Not only did these cells more greatly express killing molecules, they also suppressed the functions of tumor-bearing CD8(+) T cells in vitro and in vivo. Finally, we show that these effector/memory Treg cells inhibit concomitant tumor immunity in vivo. Taken together, data suggest that effector/memory Treg cells are responsible for the loss of concomitant tumor immunity associated with tumor progression.

[Inducement effect of ginsenoside Rg3 on apoptosis of human bladder transitional cell carcinoma cell line EJ].

OBJECTIVE: To explore the effect of Rg3 on inhibiting and inducing apoptosis of bladder cancer cells. METHOD: The bladder cancer cell line EJ was treated with Rg3 of various concentrations. Cell proliferation was measured by MTT assay. Morphological changes of cells were observed by fluorescent staining of Hoechst 33258. Cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). The expression of caspase-3 in cells was detected by immunocytochemistry. DNA ladder was showed by agarose gel electrophoresis. RESULT: Rg3 inhibited proliferation of EJ cells in a manner of concentration-dependent relationship, IC50 of Rg3 in 48 h treatment was 125.5 mg x L(-1) to EJ cells. When treated with 150 mg x L(-1) of Rg3 for 24 h and 48 h, the cells showed apoptotic morphological characteristics including the condensed chromatin, the nuclear fragmentation, the apoptotic body and bright fluorescent granules as well as a higher caspase-3 expression. FCM assay indicated that Rg3 regulated cell cycle and induced apoptosis of EJ cells. When treated for 24 h and 48 h with 75 mg x L(-1) of Rg3 as well as for 48 h with 150 mg x L(-1) of Rg3, the percentages of cells in S phase and G2/M phase were increased, whereas the percentage of cells in G0-G1 was decreased. The apoptosis rates were increased from (1.05 +/- 0.17)% in control group cells to (8.41 +/- 0.98)%, (18.57 +/- 2.20)% and (33.98 +/- 1.64)%, respectively. Remarkable DNA ladders were revealed. The effects showed a manner in dose and time dependent of Rg3. CONCLUSION: The results suggest that ginsenoside Rg3 exerts an inhibiting effect on proliferation of EJ cells by inducing apoptosis.