Histones of Neutrophil Extracellular Traps Directly Disrupt the Permeability and Integrity of the Intestinal Epithelial Barrier.

BACKGROUND: Increased neutrophil extracellular trap (NET) formation and abundant NET-associated proteins are frequently found in the inflamed colon of patients with inflammatory bowel disease. Peptidyl arginine deiminase 4 (PAD4) activation is essential for the generation of NET and NET-mediated pathogenesis. However, the role of PAD4-dependent NET formation in murine inflammatory bowel disease models and the molecular mechanisms responsible for the altered gut barrier function are unknown. METHODS: Wild-type and Pad4 knockout (Pad4-/-) mice were administrated 3% dextran sulfate sodium (DSS) in their drinking water. Caco-2 monolayers were used to test the effect of NETs on intestinal barrier function and cytotoxicity. Histones were intrarectally administrated to wild-type mice to determine their effects on intestinal barrier function and cytotoxicity in vivo. RESULTS: PAD4 deficiency reduced the severity of DSS-induced colitis with decreased intestinal NET formation and enhanced gut barrier function and integrity in mice. NETs disrupted the barrier function in intestinal epithelial Caco-2 monolayers through their protein, rather than DNA, components. Pretreatment of NETs with histone inhibitors abrogated the effects on epithelial permeability. Consistent with these observations, adding purified histone proteins to Caco-2 monolayers significantly damaged epithelial barrier function, which was associated with the abnormal distribution and integrity of tight junctions as well as with increased cell death. Furthermore, intrarectal administration of histones damaged the intestinal barrier integrity and induced cytotoxicity in the mouse colon epithelium. CONCLUSIONS: PAD4-mediated NET formation has a detrimental role in acute colitis. NET-associated histones directly inhibit intestinal barrier function, resulting in cytotoxicity in vitro and in vivo.

Peptidyl arginine deiminase 4­dependent neutrophil extracellular trap formation is detrimental to intestinal barrier function in acute colitis. Neutrophil extracellular trap­associated histones altered the integrity of tight junction and adherens junction proteins as well as induced intestinal epithelial cell death that resulted in increased gut epithelium permeability.

5-methoxytryptophan alleviates liver fibrosis by modulating FOXO3a/miR-21/ATG5 signaling pathway mediated autophagy.

Liver fibrosis is a critical health issue in the world due to its rapidly increasing prevalence. It is of great demand to develop effective drugs for the treatment of liver fibrosis. 5-methoxytryptophan (5-MTP) has been reported to play an important role in anti-inflammatory, anti-cancer, myocardial-protective effects. However, the anti-fibrotic effect of 5-MTP is never covered in liver. Here, we investigated anti-fibrotic effects of 5-MTP on liver fibrosis and its underlying mechanism. In vitro, 5-MTP treatment could inhibit TGF-ß1-induced elevated levels of collagen I, collagen III, fibronectin and α-smooth muscle actin (SMA) by stimulating autophagy process. Mechanically, the expression of FOXO3a was enhanced by 5-MTP and then repressed the level of miR-21, eventually leading to a restoration of autophagy-related gene ATG5. Furthermore, rescue experiments showed 5-MTP could activate autophagy process and suppress the activation of LX-2 cells by regulating FOXO3a/miR-21/ATG5 pathway. Consistently, 5-MTP significantly attenuated CCl4-induced hepatic fibrosis in rat model. In conclusion, our research discovered that 5-MTP effectively alleviated liver fibrosis in vitro and in vivo, which provided new insights into the application of 5-MTP for liver fibrosis.

The Role of IL-27 and its Receptor in the Pathogenesis of HIV/AIDS and Anti-viral Immune Response.

BACKGROUND: Cytokines have been widely demonstrated to involve in the pathogenesis of AIDS and the mechanisms of antiretroviral therapy. Interleukin 27 (IL-27) is a new member of the IL-12 cytokine family and has been shown to interfere HIV-1 virus replication with controversial findings. This study is to investigate the dynamic changes in plasma IL-27 level and cell surface IL-27 receptor expression in HIV/AIDS patients who underwent HAART. METHODS: Whole blood was collected from 34 HIV-positive/AIDS patients 0, 6, and 12 months after initiation of HAART and 27 healthy subjects. Plasma IL-27, IFN-γ, and IL-4 were measured by enzyme-linked immunosorbent assay, while peripheral blood CD3+CD4+ T cells count and the gp130 expressed CD3+CD4+cell were measured by flow cytometry. RESULTS: The plasma IL-27 concentration, IFN-γ concentration, and percentage of positive gp130 CD4 cells were significantly decreased in previously treatment-naive HIV/AIDS patients compared to healthy controls, but gradually increased 6 and 12 months after initiation of HAART. Conversely, IL-4 levels were significantly increased in treatment-naive HIV/AIDS patients compared to healthy controls, but gradually decreased 6 and 12 months after HAART. The concentrations of plasma IL-27 were positively correlated with the percentage of gp130 positive CD4 cells (r=0.438, p=0.016). Both plasma IL-27 concentration and gp130 positive cell percentage were positively associated with peripheral blood CD3+CD4+ T cell count (P<0.05 or P<0.01), but negatively associated with plasma HIV viral load (P<0.05 or P<0.01). CONCLUSION: IL-27 signaling (IL-27 and its receptor) may be involved in the pathogenesis of HIV infection and immune reconstitution in HIV/AIDS patients who underwent HAART. IL-27 may exert effects through regulating Th1 / Th2 ratio.

[Cellular HIV DNA quantitative testing and its significance in Chinese AIDS patients during 48 weeks' highly-active antiretroviral treatment].

OBJECTIVE: To observe the dynamic changes of peripheral blood T lymphocytes and monocytes, which serve as HIV-1 viral reservoirs, in Chinese HIV-infected patients receiving highly-active antiretroviral treatment (HAART) for 48 weeks and its clinical significance. METHODS: A total of 35 chronic HIV-1 infected adults initial received HAART. The peripheral blood T lymphocyte subsets counts were determined by flux cytometry at week 0, 24 and 48. Magnetic activated cell sorting was used to extract cellular DNA from monocytes and T lymphocytes purified from peripheral blood mononuclear cells. Real-time fluorescent quantitative PCR was used to detect the serum HIV RNA and HIV DNA of monocytes and T lymphocytes. SPSS 18.0 software was used to analyze the collected data. RESULTS: At week 0, 24, and 48 after initiation of HAART, HIV RNA levels of peripheral blood were (4.12 ± 1.41), ≤ 1.69, and ≤ 1.69 lg copies/ml, respectively; CD(4)(+) T cells were (196 ± 101), (321.90 ± 112) and (392 ± 127) cells/µl, respectively; HIV DNA level in T lymphocytes were (4.03 ± 0.53), (2.74 ± 1.16) and (2.45 ± 0.41) lg copies/10(6) cells respectively; while in monocytes, HIV DNA levels were (2.51 ± 0.68), (2.16 ± 0.34)and (2.03 ± 0.25)lg copies/10(6) cells. Statistical analysis revealed that HIV RNA level was negatively correlated with the CD(4)(+) T cell count through the whole trail, while positively correlated with the HIV DNA level in blood T lymphocytes and monocytes. HIV DNA level in T lymphocytes decreased more slowly than HIV DNA in monocytes. Moreover, peripheral blood CD(4)(+) T cell count was negatively associated with the HIV DNA capacity from T lymphocytes. CONCLUSIONS: Both T lymphocyte and monocyte may serve as viral reservoirs, and T lymphocyte might play a more important role as HIV reservoirs. The blood HIV RNA is correlated positively with the cellular HIV DNA, whereas, CD(4)(+) T cell count is correlated negatively with HIV DNA from lymphocytes, which suggests that HIV DNA levels in T lymphocyte might be one of indicators of AIDS progress during HAART.

Inhibition of microtubule polymerization by 3-bromopropionylamino benzoylurea (JIMB01), a new cancericidal tubulin ligand.

3-Bromopropionylamino benzoylurea (JIMB01) is a small molecular weight compound (MW 313) that has been synthesized in our laboratory. This compound showed antiproliferative activities in a panel of thirteen human tumor cell lines with IC(50) values in the range of 0.25 to 0.51 micro M for leukemia and lymphoma cell lines and 0.33 to 9.26 micro M for solid tumor cell lines. The primary action of JIMB01 is to inhibit microtubule polymerization but not depolymerization. A 4 micro M concentration of the compound caused a complete inhibition of microtubule assembly in a cell-free reaction. An increase in the number of human hepatocarcinoma cells blocked in the M-phase was detected 12hr after exposure to JIMB01. The kinase activity of cyclin B1, which is responsible for the G(2)/M transition, was increased accordingly. Bcl-2 phosphorylation became visible, in a western blot, within 6hr in hepatocarcinoma cells treated with JIMB01 at 0.8 micro M or higher. JIMB01-induced apoptosis in liver cancer cells was confirmed by morphological methods, flow cytometry, as well as DNA gel electrophoresis, which clearly demonstrated DNA degradation in the form of a multiple-unit DNA ladder. Furthermore, in vivo experiments using nude mice showed that intraperitoneal injection of JIMB01 at 15mg/kg (with seven injections at 4-day intervals) significantly inhibited the growth of a human hepatocarcinoma (BEL-7402) by 66% in tumor volume (P=0.01), at least compatible to the inhibition by vincristine (43% inhibition), indicating good bioavailability of the compound in the circulation. Side-effects of the compound were not observed, and the body weight of the treated mice remained stable during the 4-week treatment. Since JIMB01 is a small compound, targets a specific molecule in tumor cells, and has promising activity against human hepatocarcinoma in vivo, we believe JIMB01 merits consideration for further investigation.