Deltex-1 is indispensible for the IL-6 and TGF-ß treatment-triggered differentiation of Th17 cells.

CSL(CBF1, Su(H) and LAG-1)-dependent Hes-1 signaling plays an important part in regulating Th17 cell differentiation. However, little is known about influence of CSL-independent Deltex-1 signaling on this subset. The current focus is on roles of the Deltex-1 signaling in the Th17 cell differentiation. IL-17-producing CD4+ T cell subpopulation could be induced in vitro by treatment of both IL-6 and TGF-ß. This could be reversed by knockdown of the deltex-1 gene, following the attenuation of retinoic acid-related orphan receptor γt (RORγt) and its DNA-binding activity in nuclei. Subsequently, Th17-associated cytokines generated by the treated cells were also diminished by the inhibition of Deltex-1 signaling, but the production of IL-10 was enhanced. Contrary to the alteration of RORγt, both zinc-finger transcription factor-3 (GATA3) and transcription factor Forkhead box P3 (Foxp3) were augmented at their mRNA and protein levels as well as DNA-binding activities with the emerging phenotypes of the corresponding cellular subpopulation and T-bet (encoded by TBX21) was not changed. These results reveal for the first time that Deltex-1 is indispensible for the IL-6 and TGF-ß treatment-triggered differentiation of Th17 cells, indicating that CSL-independent Deltex-1 signaling favors naïve CD4+ T cells to deviate into Th17 cells via the enhancement of RORγt/IL-17A.

Ionomycin inhibits Jurkat T cell behaviors in the presence of phorbol-12,13-dibutyrate.

Ionomycin in conjunction with phorbol-12,13-dibutyrate (PDBu) is conventionally used as a stimulator to activate cells, especially original T cells. But we accidently found it had an entirely opposite action on malignant tumor cells derived from T cells. Thus, influence of ionomycin on human leukemia Jurkat T cell behaviors and its preliminary mechanistic process were explored in the presence of PDBu. Ionomycin could remarkably inhibit colony formation of the cells, and inhibitory rate of the cell proliferation was increased with ionomycin treatment in a dose- or time-related relationship, following the reduction of ERK1/2 and phosphorylated-ERK1/2 levels. However, a high dose of ionomycin might moderately repress mid-stage activation of the cells. It also blocked the cell entry at S-phase and G2/M-phase with the attenuation of transforming growth factor-ß (TGF-ß) level in the cells, and promoted the cell apoptosis following the augment of caspase-3 and cleaved caspase-3 in the cells. The dramatic elevation of [Ca2(+)]i and intracellular pH (pHi) was simultaneously followed by the above alteration of the cell behaviors. These results indicate that ionomycin may strongly inhibit human acute T lymphocyte leukemia progress in the presence of PDBu through the inhibition of ERK1/2 signaling, the activation of caspase-3 and the attenuation of TGF-ß mediated by the [Ca2(+)]i and pHi enhancement, providing a novel insight into function and potential application of both ionomycin and PDBu.

Reduced stathmin-1 expression in natural killer cells associated with spontaneous abortion.

Female CBA/J mice impregnated by male DBA/2J mice (CBA/J×DBA/2J matings) are prone to spontaneous abortion, although the reason for this is unclear. In this study, the stathmin-1 expression pattern was evaluated in uterine natural killer (uNK) cells purified from CBA/J×DBA/2J matings. Results were compared with those in a CBA/J×BALB/c control group that yields successful pregnancies. The mean ± SD percentage of stathmin-1(+) cells in the CD49b(+) uNK cell population was lower in CBA/J×DBA/2J mice (0.7% ± 0.4%) than in control CBA/J×BALB/c mice (4.9% ± 1.5%, P < 0.01) using flow cytometry, and the intracellular stathmin-1 level in uNK cells was lower in CBA/J×DBA/2J mice than in control mice using Western blot analysis. Co-localization of lectin from Dolichos biflorus agglutinin (DBA-lectin) and stathmin-1 was confirmed using multivision immunohistochemical analysis. The frequency of stathmin-1(+)DBA-lectin(+) cells was lower in CBA/J×DBA/2J mice than in CBA/J×BALB/c mice. A similar trend in the frequency of stathmin-1(+)CD56(+) cells was seen in patients with unexplained spontaneous abortion compared with normal early pregnancy. A neutralizing antibody against stathmin-1 further increased the percentage of embryo loss in CBA/J×DBA/2J matings. These results provide evidence that stathmin-1 expression in uNK cells at the maternal-fetal interface may help modulate uNK cell function and may be beneficial for a successful pregnancy.

Insufficient peroxiredoxin-2 expression in uterine NK cells obtained from a murine model of abortion.

The CBA/J × DBA/2 mouse mating combination is prone to spontaneous embryo loss, in contrast to the MHC-identical CBA/J × BALB/c mating combination, which yields successful pregnancies. The underlying mechanisms for these observations are unclear. In this study, multi-vision immunohistochemical staining (IHC), flow cytometry and Western blot analysis were used to detect peroxiredoxin-2 (PRX-2) expression in the uterine natural killer (uNK) cells from CBA/J × DBA/2 and CBA/J × BALB/c mice. In IHC analysis, co-localization of PRX-2 and lectin from Dolichos biflorus agglutinin (DBA-lectin) was confirmed and the frequency of PRX-2(+) DBA-lectin(+) cells was significantly lower in CBA/J × DBA/2 than CBA/J × BALB/c. In flow cytometry and Western blotting, PRX-2 was found expressed at a significantly lower level in CBA/J × DBA/2 mice. PRX-2 inhibition with a neutralizing antibody significantly decreased PRX-2 expression, increased the cytotoxicity of uNK cells, and increased the percentage of embryo loss in CBA/J × DBA/2J mice. Our data suggest that PRX-2 may be involved in the modulation of maternal-fetal tolerance and that insufficient expression of this protein may correlate with increased embryo loss in CBA/J × DBA/2J mice.

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild-type and NOD mice.

Non-obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down-regulated. Western blot analysis confirmed that 8 proteins were up-regulated and 2 proteins were down-regulated in WT mice compared with NOD mice, consistent with the results of 2-DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up-regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3(+) and CD49b(+) cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early-stage development of embryos, and subsequently influence pregnancy outcome.

The immunosuppressive effect of gossypol in mice is mediated by inhibition of lymphocyte proliferation and by induction of cell apoptosis.

AIM: To investigate the immunosuppressive effect of gossypol in mice both in vitro and in vivo. METHODS: The in vitro effect of gossypol on the proliferation of lymphocytes isolated from lymph nodes of BALB/c mice was determined by CFSE staining and by an MTS assay. Lymphocyte activation and lymphoblastic transformation were evaluated with immunostaining. Cell apoptosis was detected by Annexin-V and Hoechst 33342 staining. The in vivo immunosuppressive effect of gossypol on the DTH reaction was evaluated using a mouse DTH model induced by 2,4-dinitro-1-fluorobenzene (DNFB). The thickness of the ears was measured, and the histological changes of the mouse auricles were observed after hematoxylin-eosin staining. The proliferation capacity of lymphocytes from DTH mice was also assayed. RESULTS: In vitro, gossypol could significantly inhibit the proliferation of mouse lymphocytes stimulated with phorbol ester plus ionomycin in a dose-dependent manner. Although the expression of the early activation antigen CD69 was not affected, the lymphoblastic transformation of both T and B lymphocyte subsets was significantly suppressed by gossypol. Moreover, gossypol could induce apoptosis of lymphocytes, and the effect was time- and dose-dependent. In vivo, the DTH reaction in mice was markedly alleviated by gossypol injected intraperitoneally. Lymphocytes from drug-treated DTH mice had a reduced proliferation capacity as compared with lymphocytes from untreated DTH mice. Gossypol treatment also markedly reduced the number of infiltrated lymphocytes in the auricles of DTH mice. CONCLUSION: Gossypol exhibited immunosuppressive effects in mice, probably by inhibition of lymphocyte proliferation and by induction of cell apoptosis.

Novel recombinant thrombolytic and antithrombotic staphylokinase variants with an RGD motif at their N-termini.

To develop a more potent thrombolytic agent, four Sak (staphylokinase) variants were constructed, in which RGD (Arg-Gly-Asp) sequences are introduced into diferent sites of the N-terminus of Sak. These variants were successfully expressed in Escherichia coli DH5alpha as soluble cytoplasmic proteins in a 5-litre fermentor and accounted for more than 40% of the total cellular protein. The expressed proteins were subsequently purified, employing a similar three-step chromatographic purification process. SDS/PAGE and HPLC-MS analyses indicated that the purified proteins were almost completely homogeneous, the purity of the variants exceeding 95%. Further investigations into the properties of the Sak variants showed that mutations at the N-terminus significantly affected N-terminal methionine excision, and serine residues at the N-terminus of Sak appeared to play an important role in the process. Kinetic analysis of r-Sak (recombinant Sak) and its variants using plasminogen as substrate indicated that the mutations affected the proteolysis. In addition, a significant inhibitory effect of the Sak variants at 2.0 muM was observed on the ADP-induced aggregation of platelets compared with that of r-Sak, whether N-terminally cleaved or not (P<0.05). Furthermore, the inhibitory activity of Sak variants after N-terminal proteolysis was higher than that of native Sak variants.

Preterm delivery induced by LPS in syngeneically impregnated BALB/c and NOD/SCID mice.

Strategies of lipopolysaccharide (LPS) stimulation with or without previous toll-like receptor 4 (TLR4) blocking were pursued to investigate the mechanism of LPS-induced preterm delivery in syngeneically impregnated BALB/c and non-obese diabetic (NOD)/LtSz-scid/scid (NOD/SCID [severe combined immunodeficiency] for short) mice. The LPS-stimulated mice were killed at the beginning of preterm labor and pooled placentas were collected in each mouse. Cell surface expression of TLR4, CD80, and intracellular TNF-alpha in placenta CD45(+) cell population was determined by flow cytometry. It displayed that preterm delivery could be induced by LPS in BALB/c, while the NOD/SCID seemed to be resistant to LPS induction. TLR4 expression was not changed in either BALB/c or NOD/SCID mice upon LPS-stimulation, but the CD45(+)CD80(+) cell percentage was elevated in both groups. The CD45(+)TNF-alpha(+) cell percentage was increased merely in BALB/c after the stimulation, while no such trend was observed in NOD/SCID mice. In BALB/c, the effect of LPS on CD80 and TNF-alpha expression could be abrogated by previous TLR4 blocking, subsequently prevent LPS-induced preterm delivery. In another design, NK cell blocking was performed at earlier stage of gestation by injections of anti-asialo GM1 antiserum (ASGM1). It appeared that LPS-induced preterm delivery could be partially prevented by this blocking in BALB/c mice. Such data, together with the diversity of sensitivity to LPS induction observed in BALB/c and NOD/SCID mice, imply that LPS interacts with TLR4, triggers the mobilization of CD45(+)CD80(+) cells, results in elevated production of inflammatory cytokines, and finally results in preterm delivery. In addition, NK cells may be involved in the signaling cascade, and the lack of functional NK cells in the NOD/SCID may be why these mice appeared to be less sensitive to LPS-induced premature labor.

Jagged-1 signaling suppresses the IL-6 and TGF-ß treatment-induced Th17 cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL-12rb1.

Jagged-1 signaling has recently been reported to be involved in the Th17 cell differentiation. However, little is known about its mechanisms. Soluble Jagged-1 was used to activate the Jagged-1-Notch signaling to interfere with the IL-6 and TGF-ß-induced Th17 cell skewing. Genes relevant to the autoimmunity or inflammation were screened for the first time in this system by qPCR array for the differential expressions. The 18 genes out of 84, including Clec7a, Il12b, Il12rb1, Il12rb2, Csf3, Il15, Il17a, Il17f, Il17rc, Il17rd, Il17re, Il23a, Myd88, Socs1, Stat4, Stat5a, Sykb and Tbx21, were downregulated, but only Cxcl2, Cxcl12 and Mmp3 were upregulated. The expressions of the genes, Rorγt, Il17a, Il17f, Il12rb1 and Il23a, induced by simultaneous IL-6 and TGF-ß treatment were significantly suppressed by Jagged-1, followed by the reduction of RORγt, IL-17A, and IL-17F. Consistent with the attenuation of RORγt, and the reduced production and secretion of IL-17A and IL-17F in the cell supernatant and the in situ stained cells, the number of CD4(+)IL-17(+) cells was also diminished. It is concluded that the Jagged-1-Notch signaling can suppress the IL-6 and TGF-ß treatment-induced Th17 cell skewing through the attenuation of RORγt and, hence by, the down-regulation of IL-17A, IL-17F, IL-23a, and IL-12rb1.

Murine CD45+CD86+ cells isolated from para-aortic lymph nodes in an abortion-prone model.

The present study aims to address whether the analysis of CD45+CD86+ cells isolated from para-aortic lymph nodes (pLNs) is valuable in assessment of the status of local immunity at the murine feto-maternal interface. CBA/J x DBA/2 mice, virgin CBA/J mice, and CBA/J x BALB/c mice were used as an abortion-prone model (group A), nonpregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45+CD86+ cells in the CD45+ cell group (CD45+CD86+ percentage for short) and the absolute number of these cells were determined by means of flow cytometry (FCM), using mononuclear cells isolated from pLNs collected 5.5, 9.5, and 13.5 days post-coitum (dpc), respectively, and mononuclear cells isolated from placentas 13.5 dpc. To clarify the identity of these CD86+ cells, FCM was also performed with CD3, CD19, and DX5 as specific markers for murine T-cells, B-cells, and NK cells, respectively. Both resorption rate and absolute number of resorptions were significantly higher in group A (29.3%, 1.8+/-1.0) than in group F (4.8%, 0.3+/-0.5, P<0.001, respectively). Similarly, both cell percentage and absolute number of CD45+CD86+ cells in pLNs collected 13.5 dpc were significantly higher in group A than in group F (27.5+/-14.0% versus 12.3+/-7.1%, and 1362+/-687 versus 615+/-353, P=0.001, respectively). The CD45+CD86+ percentage was around 7.5% in nonpregnant CBA/J mice, similar to the 10.6% in CBA/JxDBA/2 mice 5.5 dpc, but had increased dramatically, to 23.9%, by 9.5 dpc (P<0.001 versus nonpregnant mice and P=0.002 versus CBA/JxDBA/2 mice 5.5 dpc), and remained at a higher level (27.5%) until 13.5 dpc. However, this trend was not observed in group F during pregnancy. The increased CD45+CD86+ percentage at day 9.5 of gestation, when resorption begins, may support the assumption that CD45+CD86+ cells play a role in the course of embryo resorption. Lymphocyte phenotypic analysis in the lymph nodes that drain the pregnant uterus may be helpful to assess the status of local immunity at the feto-maternal interface.

In vitro and in vivo evaluation of anisomycin against Ehrlich ascites carcinoma.

Anisomycin eminently inhibits cell proliferation in vitro. The aim of this study was to explore the potential of anisomycin to treat tumors in vivo and its mechanism(s) of action. The results showed that peritumoral administration of anisomycin significantly suppressed Ehrlich ascites carcinoma (EAC) growth resulting in the survival of approximately 60% of the mice 90 days after EAC inoculation. Enhancement of infiltrating lymphocytes was noted in the tumor tissue, which was dramatically superior to adriamycin. The growth inhibitory rate of EAC cells was enhanced with increasing concentrations of anisomycin, following an enhanced apoptotic rate. The total apoptotic rate induced by 160 ng/ml of anisomycin was higher when compared to that induced by 500 ng/ml of adriamycin. DNA breakage and nanostructure changes were also noted in the EAC cells. The levels of caspase-3 mRNA, caspase-3 and cleaved-caspase-3 proteins in the anisomycin­treated EAC cells were augmented in a dose- and time-dependent manner, following the activation of caspase-8 and caspase-9, which finally triggered PARP cleavage. The cleaved-caspase-3, cleaved-caspase-8 and cleaved-caspase-9 proteins were mainly localized in the nuclei of the cells. These results indicate that anisomycin efficaciously represses in vitro and in vivo growth of EAC cells through caspase signaling, significantly superior to the effects of adriamycin. This suggests the potential of anisomycin for the treatment of breast cancer.

Anisomycin suppresses Jurkat T cell growth by the cell cycle-regulating proteins.

BACKGROUND: Recent studies have shown that anisomycin significantly inhibits mammalian cell proliferation, but its mechanism remains unclear. In this study, Jurkat T cells were used to first explore a relationship between effect of anisomycin on them and alteration of cell cycle-regulating proteins. METHODS: Cell colony formation, CCK-8 assay, flow cytometry, RT-PCR and western blot were employed to evaluate correlation of ten cell cycle-regulating proteins with suppression of the cell proliferation and arrest of the cell cycle by anisomycin. RESULTS: Our data showed that anisomycin inhibited the colony-formation and proliferation of Jurkat T cells in a dose-dependent manner, and arrested the cells into S and G2/M phases with the production of sub-diploid cells. The levels of P21, P-P27 and P53/P-P53 reached their peaks 4 h after anisomycin treatment, presenting a positive correlation with anisomycin concentration, and P16, P-P21, P27, P57, P73/P-P73 and P-Rb changed little with the prolonged exposure time or increased concentrations of anisomycin. But the level of Rb protein was increased at 24 h after the treatment of anisomycin. The expression of an inverted CCAAT box binding protein (ICBP90) in Jurkat T cells came to decrease 12 h after the treatment of anisomycin, presenting a negative correlation with anisomycin concentration. Subsequently, the expression of P-CDK2 was also decreased at 24 h, presenting an obviously negative correlation, whereas P-CDK1 showed no differences among the differently treated Jurkat T cells. Furthermore, the level of P21 and P53 mRNA was increased with the enhanced concentrations of anisomycin. CONCLUSION: The results indicate that anisomycin may activate the P53/P21/P27 signaling to decrease the expression of ICBP90, inhibit expression of P-CDK2 to block the cells into S and G2/M phases, and finally result in proliferation inhibition of Jurkat T cells.

In vivo toxicological evaluation of Anisomycin.

Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. Recent studies have shown that Anisomycin as a novel immunosuppressive agent is superior to Cyclosporine A (J. Immunother. 31, 858-870, 2008). In order to make toxicological evaluation of Anisomycin, acute and four-week continuously intravenous toxicity studies were performed in mice. IC(50) value tested on peripheral lymphocytes was 25.44 ng/ml. The calculated LD(50) for Anisomycin was 119.64 mg/kg. The mice were intravenously injected through mouse tail vein with a total dose of 5, 15, 30 and 60 mg/kg/mice of Anisomycin every other day for 4 weeks. Just in the high-dose mice, death of three mice happened and body weight of the mice was significantly decreased. Statistically significant changes in organ index included increases in ratios of the spleen, liver, lung and brain to the body weight, and decrease in ratio of the thymus to the body weight. Changes in clinical biochemistry parameters included increases in the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and decreases in the glucose (GLU) activity. The distinct inflammation appeared in the lung, liver and kidney, and the number and size of megakaryocytes in the spleen were significantly increased. Anisomycin did not induce formation of the peripheral blood micronucleus, but increased the number of micronucleated polychromatic erythrocytes in bone marrow and sperm aberrations. However, the above aberrant changes occurred only in the mice treated with the high-dose Anisomycin. These results indicate that although Anisomycin has no significant side effects at effectively therapeutic doses, its over-dosage may lead to toxicity, particularly pulmo-, nephro- and hepato-toxicity.

The FGF2-binding peptide P7 inhibits melanoma growth in vitro and in vivo.

PURPOSE: Melanoma is a malignant tumor and causes majority of deaths related to skin cancer. Fibroblast growth factor 2 (FGF2) greatly contributes to melanoma growth and progress. In this paper, we attempt to evaluate the therapeutic potential of FGF2-binding peptide (named P7) using as a potent FGF2 antagonist via exploration of its antitumor effect on melanoma in vitro and in vivo. METHODS: Cell viability was measured by WST-1. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Western blotting was carried out to detect the activation of Erk1/2, P38, Akt, and MEK, and the expression of apoptosis-associated proteins. The influence of P7 on FGF2 internalization was assessed by separation of nuclear and cytoplasmic protein fractions followed by Western blotting. Female C57BL/6 mice bearing xenograft melanoma were established and used to evaluate the antitumor effect of P7 in vivo. RESULTS: In this study, we first proved that P7 peptides significantly inhibited proliferation of FGF2-induced melanoma cell line B16-F10. Further investigations revealed that the mechanisms of P7 peptides inhibiting cell proliferation of melanoma cells stimulated with FGF2 in vitro involved cell cycle arrest at the G0/G1 phase, blockade of the activation of Erk1/2, P38, and Akt cascades, and inhibition of FGF2 internalization. Finally, treatment of P7 peptides in a murine melanoma model resulted in significant inhibition of tumor growth and angiogenesis in vivo, which was associated with blockade of mitogen-activated protein kinase signal activation, and suppression of the expressions of anti-apoptotic Bcl-2 protein and angiogenic factor in the melanoma tumors. CONCLUSIONS: The FGF2-binding peptide with potent antiproliferation and anti-angiogenic activity may have therapeutic potential in melanoma.

FTY720-induced conversion of conventional Foxp3- CD4+ T cells to Foxp3+ regulatory T cells in NOD mice.

PROBLEM: FTY720 is known as an agonist of sphingosine-1-phosphate (S1P) receptor, but little is known about the possibility that FTY720 induces the conversion of conventional Foxp3(-) CD4(+) T cells to Foxp3(+) regulatory T cells in non-obese diabetic (NOD) mice. METHOD OF STUDY: FTY720 treatment was performed using Foxp3(-) CD4(+) T cells purified from NOD mice. RESULTS: FTY720 caused an increase in Foxp3(+) Treg cells in lymphoid organs in NOD mice. FTY720 effectively induced Foxp3 expression in Foxp3(-) CD4(+) T cells both in vitro and in vivo, an effect that was inhibited by a TGF-ß-neutralizing antibody or the proinflammatory cytokine IL-6. T-cell-mediated embryo rejection in NOD mice was prevented upon FTY720 treatment. CONCLUSIONS: The use of FTY720 along with Ag administration may represent a useful therapeutic strategy to selectively expand Ag-specific Foxp3(+) Tregs to intervene autoimmune and infectious diseases.

[Protective effects and mechanism of N-acetylcysteine on cardiac injury induced by ischemia/reperfusion in diabetic rats].

AIM: To study the protective effects and mechanism of N-acetylcysteine on cardiac injury induced by ischemia/reperfusion in diabetic rats. METHODS: Diabetes mellitus was induced by intraperitoneal injection of streptozotocin(STZ). The animals were randomly reassigned into sham-operated group, I/R group and I/R+treated with NAC group. The content of GSH and GSSG, and the activity of Caspase-3 were measured. The apoptosis index (AI) by TUNEL staining was calculated. In addition, the apoptosis of Cardiomyocyte was also confirmed by DNA Ladder. RESULTS: Treatment with NAC decreased the activity of Caspase-3, the content of GSSG, the values of AI but increased the content of GSH in both non-diabetic and diabetic rats (P<0.05), but there were still significant difference about the values of above parameters between diabetic rats and non-diabetic rats (P<0.05). CONCLUSION: NAC can attenuated cardiomyocyte apoptosis by decreasing the activity of Caspase-3 and increasing the content of GSH, which has protective effect on ischemic/reperfused myocardium injury in both non-diabetic and diabetic rats, but the cardioprotective effect is less effective in diabetic rats than that in non-diabetic rats.

[Protective effect of N-acetylcysteine on liver and lung in mice after ischemia-reperfusion injury].

AIM: To investigate protective effect of N-acetylcysteine (NAC) on liver and lung in mice after hepatic ischemia/reperfusion injury. METHODS: BALB/s mice were used in a model of partial hepatic ischemia/reperfusion (I/R) injury. They are divided randomly to sham-operated control group (SH), hepatic I/R group or NAC pretreated in hepatic I/R group (I/R-NAC). The level of TNF-alpha in portal vein and plasma ALT were measured at 1 hour and 3 hour, respectively after reperfusion. Lung tissue wet-to-dry (W/D) weight ratio compared. RESULTS: Lung tissue W/D ratio showed significant difference between two groups; The expressions of TLR2/4 mRNA in liver and lung increased obviously after hepatic I/R injury. Histological evaluation showed several changes in lung tissue in I/R group. The level of TNF-alpha and ALT declined significantly in the group pretreated by NAC. CONCLUSION: N-acetylcysteine can inhibit the activation of TLR2/4 and reduce TNF-alpha secretion resulted from I/R injury it might abate liver and lung injury following partial hepatic ischemia-reperfusion in mice.

Effect of TLR3 and TLR7 activation in uterine NK cells from non-obese diabetic (NOD) mice.

Toll-like receptor (TLR)-TLR cross talk is thought to be important in TLR signaling. Herein, we investigated the effect of specific TLR3 and TLR7 agonists, poly (I:C) and R837, individually and in combination, on uterine immune cell function and their subsequent effects on pregnancy outcome. Allogeneic pregnancies in the non-obese diabetic (NOD) mousexC57BL/6 and wild-type BALB/cxC57BL/6 model were used. An additive increase in embryo resorption was observed after induction with both poly (I:C) and R837, and was associated with elevated numbers of both TNF-alpha- and IFN-gamma-producing CD45(+) cells in the uterus. Further examination showed that while cytokine expression was detected in both CD3(+) cells and CD49b(+) cells in BALB/c mice, NOD mouse cells behaved differently. In NOD mice, elevated cytokine expression was attributed to CD3(+) T cells, with no response detected in the CD49b(+) NK cells. The additive effect of combined agonists was partially inhibited by the Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) inhibitor SP600125 and almost completely abrogated by the extracellular signal-regulated kinase (ERK) MAPK inhibitor PD98059. These results suggest that increased TLR3 and TLR7 signals are transmitted via Th1-type T cells, rather than NK cells, in NOD mice. Furthermore, the ERK MAPK pathway may be critical in TLR3 and TLR7 signaling.

Comparison of murine thymic stromal lymphopoietin- and polyinosinic polycytidylic acid-mediated placental dendritic cell activation.

We confirmed previously the existence of thymic stromal lymphopoietin (TSLP)-positive cells in murine placenta by flow cytometry. To compare the characteristics of Toll-like receptor 3 (TLR3)- and TSLP-mediated placental dendritic cell (DC) activation, pregnant BALB/c mouse mated with C57BL/6 male were used as a model of allogenic gestation. Placental CD11c(+) DCs were potently activated by the TLR3-agonist polyinosinic polycytidylic acid [poly (I:C)], subsequently causing increased expression of co-stimulatory molecules. Accordingly, increased intracellular production of interleukin (IL)-12 and interferon (IFN)-gamma, but not IL-4 or IL-10, were detected after stimulation by poly (I:C). In the case of TSLP-stimulation, although increased expression of co-stimulatory molecules was also detected, there was no substantial increase of intracellular production of IL-12, IFN-gamma, IL-4 or IL-10. In contrast, the expression of the Th2 cell-attracting chemokine, the thymus and activation-regulated chemokine (TARC) or CCL17, was significantly boosted in response to TSLP induction, whereas no significant increase of CCL17 was observed when triggering TLR3 with its specific agonist poly (I:C). The data were further supported by a CD4(+)IL-10(+) cell migratory assay. These results suggest that TSLP-TSLP receptor interaction may result in a Th2-type microenvironment at the feto-maternal interface by inducing the production of Th2 cell-attracting chemokine and modulating the immigration of Th2-type cells.

CXCL12 enhances exogenous CD4+CD25+ T cell migration and prevents embryo loss in non-obese diabetic mice.

OBJECTIVE: To investigate the possible role of CXCL12 in the migration of regulatory T (Treg) cells. DESIGN: Animal model-based study. SETTING: Academic. ANIMAL(S): Pregnant non-obese diabetic (NOD) mice were compared with non-immunodeficient mice. INTERVENTION(S): In vivo and in vitro CXCL12 induction. MAIN OUTCOME MEASURE(S): Flow cytometric analysis and Treg cell migratory assay. RESULT(S): A significantly high percentage of spontaneous embryo resorption was observed in both syngeneic and allogeneic pregnant NOD mice. The percentage of embryo loss in allogeneic pregnant NOD mice was significantly decreased by treatment with Treg cells and CXCL12 injection; however, no such effect was observed in syngeneic pregnant NOD mice. In addition, the migration of Treg cells induced by CXCL12 was confirmed by both in vitro and in vivo migratory assays. CXCR4, the specific receptor for CXCL12, was expressed more intensively on Treg cells than on non-Treg CD3(+) T cells, whereas CXCL12 was dominantly expressed in cytokeratin 7(+) trophoblast cells at an early stage of gestation, and its expression reduced gradually during pregnancy. CONCLUSION(S): The higher level of embryo loss in allogeneic pregnant NOD mice may be due to the lack of Treg cells. CXCL12 can cause CXCR4(+) Treg cells to migrate into the pregnant uterus and establish a beneficial microenvironment for the fetus.