[Equol and its enantiomers inhibited urethane-induced lung cancer in mice].

OBJECTIVE: To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice. METHODS: A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison. RESULTS: A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups. CONCLUSION: Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.

[Levels of human milk oligosaccharides in breast milk of mothers delivering preterm infants of different gestational ages and their effects on early growth and development].

Objective: To investigate the human milk oligosaccharides (HMOs) levels in breast milk of mothers delivering preterm infants and their effects on the early growth and development of infants. Methods: In this prospective cohort study, full-term and preterm newborns whose parents decided to breastfeed were recruited from Peking University Third Hospital between December 1, 2017 and November 30, 2018. The preterm infants were divided based on their gestational ages into extremely preterm (<28 weeks), very preterm (28-31+6 weeks) and moderate to late preterm (32-36+6 weeks) groups. Breast milk was collected from mothers at 7, 14, 28 and 120d postpartum. 368 breast milk samples were collected from 125 mothers in this study, including 54 mothers of full-term infants, 23 mothers of moderate to late preterm infants, 39 mothers of very preterm infants, and 9 mothers of extremely preterm infants. Ultra-performance liquid chromatography-mass spectrometer (UPLC-MS/MS) was used to determine the concentration of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), 3'-sialyllactose (3'SL), A-tetrasaccharide (P1), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose Ⅱ (LNFP-Ⅱ) and lacto-N-fucopentaose Ⅴ (LNFP-Ⅴ). Secretor status of mothers was defined as 2'-fucosyllactose (2'FL) concentration in colostrum and transitional milk greater than 200 µg/mL. Weight gain and the occurrence of allergic diseases of infants were collected at 120 d(4 months) postpartum. The chi-square test or the Fisher's exact test was used for the comparison of categorical data between groups; Kruskal-Wallis test and Wilcoxon rank sum test were used for comparison of continuous data between groups. Nemenyi test was used for multiple comparison. Results: 79.2% (99/125) of the mothers were secretor. There were no statistical differences between groups in the secretor status of mothers (χ²=1.31,P>0.05). The total concentration of HMOs peaked at 1-2 weeks postpartum. Compared to the preterm milk, the HMOs from the term milk was trending downwards at an earlier time. In the breast milk of secretor mothers on 28 d, total concentration of HMOs significant differed among the three groups of preterm milk and the term milk, with the median value of 4 587.09,4 615.25,5 277.44,5 476.03 µg/mL, respectively (Kruskal-Wallis χ²=8.1234,P=0.044). When analyzed by the median weight gain of the infants (low vs high weight gain) at 4 months postpartum, 2'FL was significantly lower in the high weight gain group at 7 d (1 818.04 µg/mL vs 2 181.67 µg/mL, W=1 386,P=0.018), while LNT & LNnT were significantly higher (1 182.36 µg/mL vs 1 053.62 µg/mL, W=816,P=0.044). The level of 3FL at 120 d was significantly affected by presence of allergic disease in infants, breast milk from mothers of infants with allergic disease had lower 3FL than those from mothers of infants without allergic disease (256.17 µg/mL vs 286.18 µg/mL, W=564,P=0.026). Conclusions: The overall profiles of HMOs in breast milk of mothers delivering preterm infants was basically the same as that of mothers delivering term infants; individual HMOs play a role in weight gain and the development of allergic diseases in preterm infants, but the mechanism is unclear and needs further study.

Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

miR-499A>G rs3746444 and miR-146aG>C expression and hepatocellular carcinoma risk in the Chinese population.

We conducted a case-control study of a possible association of miR-499A>G rs3746444 and miR-146aG>C rs2910164 with risk of hepatocellular carcinoma. Samples from 172 hepatocellular carcinoma patients and 185 cancer-free controls were collected from October 2008 to December 2011. PCR-RFLP analysis was performed to determine the polymorphisms in each individual. The MAFs of miR-146aG>C and miR-499A>G in controls were similar to that known from the SNP database, and frequencies of genotypes in controls were in line with Hardy-Weinberg equilibrium. We found that miR-499 AG was significantly associated with decreased risk for hepatocellular carcinoma when compared with miR-499 AA genotype (adjusted odds ration = 0.74, 95% confidence interval = 0.24-0.96). However, subjects carrying miR-146a GG had a non-significant 0.62-fold decreased risk of hepatocellular carcinoma. We did not find a significant association of miR-146aG>C rs2910164 and miR-499A>G rs3746444 polymorphisms with hepatocellular carcinoma risk in the Chinese population. Further investigations are warranted to clarify the relationship between miRNA polymorphisms and susceptibility to hepatocellular carcinoma risk in various ethnic populations.

History of frequent gum chewing is associated with higher unstimulated salivary flow rate and lower caries severity in healthy Chinese adults.

This was a single-center, prospective, cross-sectional study stratified by age and gender with the objective of determining the relationship between gum chewing history, salivary flow, and dental caries severity in adults. We enrolled 191 subjects aged 18-65 years who underwent assessments for gum chewing history, unstimulated salivary flow rate, salivary pH, and caries severity. Unstimulated salivary flow rate tended to decline with increasing age (p = 0.04), and significant differences in unstimulated salivary flow rate were also found for males (0.58 ± 0.32 ml/min) versus females (0.48 ± 0.30 ml/min) (p = 0.02). Weekly gum chewing frequency was greater in younger subjects (p = 0.001) while no age group differences were noted in pieces per day or chewing duration. Gum chewing habits were similar in males and females. A multivariate model demonstrated that only days per week chewing gum (p < 0.001) and gender (p = 0.007) were predictive of unstimulated salivary flow rate (R(2) = 0.40). Mean caries severity scores, assessed via ICDAS II and DMFT, increased with age. In multivariate analysis, age was positively associated with ICDAS (p = 0.001) and days per week chewing gum was negatively associated with ICDAS (p = 0.004), indicating that caries severity increased with age, and that days of chewing provided an inverse effect, with increased days of chewing being associated with decreased severity of caries. Overall, a history of frequent gum chewing is associated with higher unstimulated salivary flow rate and lower caries severity.

Direct estimation and correction of residual aberrations in the reconstructed exit-wavefunction of a crystalline specimen.

Measuring residual aberrations up to second order using a crystalline specimen in transmission electron microscope is challenging. Here, we show by good examples of both experimental and simulated images that it is feasible to measure aberrations up to the second-order, using minimum amplitude contrast criterion for the exit wave function reconstructed. We propose a two-steps strategy for the task: (i) Firstly measuring defocus and two-fold astigmatism simultaneously to avoid error accumulation and to reduce the number of dimensions in parameters space. (ii) Then, with minimized misleading effects (or errors) in defocus and two-fold astigmatism, estimations of three-fold astigmatism and coma can be conducted more efficiently and effectively. Influences of other factors such as specimen structure, resolution and specimen thickness on the validity of the method are also discussed in detail. Our study provides a practical procedure for correcting residual aberrations in image wave using crystalline materials, which can then facilitate application of exit wave reconstruction technique to materials research.

BMSCs attenuate hepatic fibrosis in autoimmune hepatitis through regulation of LMO7-AP1-TGFß signaling pathway.

OBJECTIVE: In a previous study, we reported that transplantation of bone mesenchymal stem cells (BMSCs) significantly attenuated liver damage in a mouse autoimmune hepatitis (AIH) model. Moreover, expression of the LIM domain protein, LMO7, correlated positively with the invasive capacity of hepatoma cells. However, whether LMO7 plays a role in inflammation and fibrosis of AIH remains unknown. This investigation aimed to explore the effect of BMSC transplantation on LMO7 and the role of LMO7 in hepatic fibrosis. MATERIALS AND METHODS: S100-induced murine AIH and LPS-induced hepatocyte injury models were successfully established. Three doses of BMSCs were injected into AIH mice via the tail vein. LPS-treated AML12 cells were co-cultured with BMSCs in vitro. Small interfering (si) LMO7 RNA and T5224 (a specific inhibitor of AP-1) were used to demonstrate the relationship between LMO7-AP1-transforming growth factor (TGF)-ß. RESULTS: Pathological examination and serum alanine and aspartate aminotransferase levels indicated that liver damage was notably ameliorated in the BMSC-treated mice. LMO7 level was upregulated, while AP-1 and TGF-ß levels were downregulated upon intervention with BMSCs. AP-1 expression was upregulated in the siLMO7 group, whereas TGF-ß level was downregulated in the T5224 group when compared to those in the control group. CONCLUSIONS: BMSC transplantation significantly limits liver fibrosis and upregulates the expression of LMO7. LMO7 inhibits the TGF-ß pathway by inhibiting AP-1. This implies that BMSCs are a potential means of treating liver fibrosis. This approach has important implications for the treatment of AIH and other fibrotic diseases.

Transforming growth factor beta 1 plays an important role in inducing CD4(+)CD25(+)forhead box P3(+) regulatory T cells by mast cells.

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (T(reg)) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow-derived mast cells (BMMCs) can induce T(regs) by expressing transforming growth factor beta 1 (TGF-ß1) in vitro, bone marrow cells obtained from C57BL/6 (H-2(b) ) mice were cultured with interleukin (IL)-3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co-cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti-CD3, anti-CD28 and IL-2 were administered into the co-culture system with (experiment groups) or without (control groups) TGF-ß1 neutralizing antibody. The percentages of CD4(+)CD25(+)forkhead box P3 (FoxP3)(+) T(regs) in the co-cultured system were analysed by flow cytometry on day 5. The T(reg) percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF-ß1 neutralizing antibody into the co-culture system. Our results indicated that the CD4(+) T cells can be induced into CD4(+)CD25(+)FoxP3(+) T cells by BMMCs via TGF-ß1.

Genetic Variants of Homocysteine Metabolism, Homocysteine, and Frailty - Rugao Longevity and Ageing Study.

OBJECTIVES: Recently, elevated homocysteine was reported to be associated with frailty in cross-sectional studies. However, whether homocysteine is causally associated with frailty is unknown. Here, we explore the inter-relationships between five non-synonymous genetic variants of homocysteine metabolic four genes, plasma homocysteine levels, and frailty. METHOD: Data of 1480 individuals aged 70-87 years from the ageing arm of Rugao Longevity and Ageing Study were used. Five variants of the four homocysteine metabolic enzyme genes were genotyped. Frailty was defined using Fried's phenotype criteria. RESULTS: The percentage of high homocysteine (>15µmol/L) is 33.3%. Two functional variants that decrease methylenetetrahydrofolate reductase (MTHFR) activities, C677T (Ala222Val, rs1801133) and A1298C (Glu429Ala, rs1801131), were significantly associated with increased homocysteine levels (ß=-1.16, p=0.01; and ß=1.46, p<0.001, respectively). In addition, homocysteine increase gradually from CC-CC, CC-AC, CT-AC, CT-AA, CC-AA, to TT-AA genotypes of the C677T-A1298C combinations. The five polymorphisms in the homocysteine metabolic gene was not associated with frailty. However, homocysteine was significantly associated with frailty with an OR of 2.27 (95% 1.36-3.78) for high homocysteine after adjusting for multiple confounding factors. CONCLUSION: Elevated homocysteine is not a causal factor but a biomarker that manifests greater possibility of frailty in high risk elderly individuals for prevention.

The liver mediates apoptotic cell-induced immune regulation.

Allogeneic apoptotic cells have been demonstrated to induce allograft tolerance, but the mechanisms for this remain unclear. The study presented here investigates organs in which the tolerogenic immune responses may occur. Distribution of live or apoptotic CFSE(+) splenocytes in recipients' organs and phagocytosis by liver antigen-presenting cells (APC) were investigated by fluorescence microscopy and flow cytometry, and cytokine expression was analysed by Multiplex and ELISA. It was found that allogeneic or autogenic apoptotic cells preferentially accumulated in the liver within 30 min, peaked at 60 min, and disappeared at 12 h after infusion, whereas these cells scarcely appeared in the spleen. The accumulation in the liver was apoptotic cell-specific as both allogeneic and autogenic live splenocytes were completely deposited in the spleen. Liver phagocytes, including Kupffer cells, liver sinusoidal endothelial cells and dendritic cells, all efficiently phagocytized apoptotic cells in vitro and in vivo. Although a Th1 cytokine profile found both in the spleen and liver in the recipients of allogeneic apoptotic cells, a rapid and consistent Th2 cytokine profile specifically was initiated in the liver. From this, we conclude that liver APC phagocytize donor apoptotic cells and induce liver-specific Th2 cytokines, which may contribute to the mechanisms of allograft tolerance induced by donor apoptotic cells.

A combination of donor specific transfusion and rapamycin prolongs cardiac allograft survival in mice.

BACKGROUND: We sought to investigate the effects of donor-specific transfusion (DST) combined with rapamycin (RPM) on engraftment and demonstrate the mechanisms. METHODS: B6 was treated with DST (on day -8) combined with RPM (from day -7 to -1, 1.5 microL/g per day). On day 0, B6 splenocytes were harvested to coculture with Balb/c splenocytes pretreated with mitomycin C in 1-way mixed lymphocyte cultures (MLC). After 48 hours, the proliferation, we measured apoptosis and the CD4(+)CD25(+) T-cell ratio of cultured cells. The heterotopic cardiac transplantation model was performed from Balb/c to B6 using the recipients pretreated with this protocol. On day 0, RPM was withdrawn and the transplantations performed. The mean survival time (MST) of the grafts evaluated and in vitro assays measured. RESULTS: Both in vitro and in vivo, the protocol suppressed the proliferation of splenocytes as well as enhanced apoptosis and the CD4(+)CD25(+) T-cell ratio. The MST of the grafts was 35 +/- 14.4 days. CONCLUSIONS: Our study showed that DST combined with RPM prolonged cardiac allograft survival even if we withdrew immunosuppressants after transplantation. This result may be associated with inhibition of T-cell proliferation. We also demonstrated that RPM expanded CD4(+)CD25(+) T cells and increased splenocyte apoptosis.

An improved iterative wave function reconstruction algorithm in high-resolution transmission electron microscopy.

Exit wavefunction reconstruction is a powerful image processing technique to enhance the resolution and the signal-to-noise ratio for atomic-resolution imaging in both aberration uncorrected and corrected transmission electron microscopes. The present study aims to improve the performance of the iterative wavefunction reconstruction algorithm in comparison not only with its conventional form but also with the popular commercial Trueimage software for exit wavefunction reconstruction. It is shown that by implementing a wave propagation procedure for refining its image alignment, the iterative wavefunction reconstruction algorithm can be greatly improved in accurately retrieving the wavefunctions while keeping its original advantages, which allow the reconstruction be performed with less images and a larger defocus step in the data set of through-focus image series. In addition, calculations of this algorithm can be accelerated drastically by the graphic processing unit (GPU) hardware programming using the popular computer unified device architecture language, whose computing speed can be 25-38 times as fast as a central processing unit (CPU) program.

Accumulation of High Levels of Monocytic Myeloid-Derived Suppressor Cells Enhances Graft Survival in Almost-Tolerant Kidney Transplant Recipients.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that suppress immune responses during organ transplantation and participate in mediating long-term graft survival and immune tolerance in animal transplant models. However, their role in regulating transplant tolerance in human subjects is not well understood. In the present study, we investigated the role of MDSCs in mediating long-term graft survival in almost-tolerant kidney transplant recipients (ATKTRs) and the mechanism(s) responsible for increasing MDSC numbers in these recipients. METHODS: Peripheral blood mononuclear cells (PBMCs) from whole blood samples were collected from 30 ATKTRs (graft survival, > 10 years after kidney transplant [KTx]) treated with low doses of immunosuppressive drugs and with stable kidney function, 10 short-term graft survival kidney transplant recipients (STKTRs; graft survival, ∼1-3 years post-KTx) with stable kidney function, and 10 healthy donors (HDs). MDSC and regulatory T cell (Tregs) levels were analyzed using multicolor flow cytometry in PBMCs. RESULTS: ATKTRs had significantly higher levels of monocytic MDSCs (P < .001) and CD4+CD25+FoxP3+ Tregs than STKTRs and HDs. Furthermore, the M-MDSC levels correlated positively with the survival rates, estimated glomerular filtration rates (eGFRs) of grafts, and the levels of CD4+CD25+FoxP3+ Tregs in ATKTRs. CONCLUSIONS: Accumulation of high levels of MDSCs was observed in ATKTRs. Changes in MDSC levels may play important roles in mediating transplant tolerance and regulating Tregs. Therefore, we propose that MDSCs may be potentially used for recognizing tolerant transplant recipients and guiding dosage reduction for immunosuppressive drugs for KTx.

Adenovirus-mediated heme oxygenase transfer inhibits graft arteriosclerosis in rat aortic transplants.

PURPOSE: Heme oxygenase-1 (HO-1) has been implicated in graft protection because its induction is associated with long-surviving allografts. The aim of this study was to analyze the efficacy of adenovirus-mediated HO-1 overexpression to inhibit graft arteriosclerosis and estimate whether the protective role correlated with nuclear factor kappaB (NF=kappaB) expression and grafts apoptosis. METHODS: Aortic transplantation was performed using inbred Brown-Norway (BN) rats (RT1n male) as donors and Lewis rats (RT1l male) as recipients. The experiments were divided into four groups: group A, Lewis-Lewis (n=6), no treatment; group B, BN-Lewis (n=6), no treatment; group C, BN-Lewis (n=6), donor aorta perfused with buffer containing 4x10(10) pfu Ad-Null (the noncoding adenoviral vector); and group D, BN-Lewis (n=6), donor aorta perfused with buffer containing 4x10(10) pfu Ad-HO-1 (adenoviral vector coding for HO-1). All grafts were harvested at 60 days after transplantation. Reverse transcriptase polymerase chain reaction was performed to detect the expression of HO-1 gene. Hematoxylin and eosin staining was used to detect the morphology and cytology of the transplanted vessels. Intimal thickening was detected by Masson staining. Immunohistochemistry was used to detect the localization and expression of HO-1 and NF-kappaB and TUNEL assays to detect the apoptosis of the grafts. RESULTS: Gene transfer of HO-1 grafts using an adenoviral vector resulted in the expression of HO-1 protein in endothelium and adventitia. We observed that the intimal thickness in Ad-HO-1-treated aortas (11.11+/-0.92 microm) was significantly thinner compared with untreated (85.20+/-6.90 microm) or Ad-null treated (87.20+/-6.20 microm) aortas (P<.01). Immunohistology showed that treatment with Ad-HO-1 resulted in a significant reduction in leukocyte infiltration and a decreased number of vascular smooth muscle cells in the intima, compared with Ad-null-treated aortas. The levels of NF-kappaB and the number of apoptotic cells in Ad-HO-1-treated aortas showed significantly lower compared with Ad-null-treated arotas (P<.05). CONCLUSION: HO-1 prevented the development of graft arteriosclerosis in the rat aortic transplant model. The protective role of HO-1 in chronic rejection lesions seemed to correlate with downregulation of the expression of NF-kappaB and inhibition of apoptosis in the grafts.

Heme oxygenase-1 protects INF-gamma primed endothelial cells from Jurkat T-cell adhesion.

PURPOSE: The heme oxygenase-1 (HO-1) system is associated with the rate-limiting step of conversion of heme, one of the most critical roles in cytoprotective mechanisms. Our study investigated its potential role in protection of endothelial cells from T cells. METHODS: The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells. Indirect fluorescent staining was used to examine the expression of HO-1 protein. Then endothelial cells primed by INF-gamma were mixed in culture with Jurkat T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The number of adhesive Jurkat T cells was determined using FACS to evaluate the adhesion effect. After being cultured with endothelial cells, the cell cycle of Jurkat T cells was detected using FACS. RESULTS: Expression of HO-1 on endothelial cells conferred significant protection against Jurkat T-cell-mediated adhesion. The rate of Jurkat T-cell adhesions was reduced to 19.06%, in contrast with 31.42% in the control group (P<.05). After using ZnPP, an inhibitor of HO-1, the rate of Jurkat T-cell adhesion recovered to 29.08%. The binding activities between endothelial cells and Jurkat T cells was blocked by HO-1 expression. The proliferation of Jurkat T cells was inhibited after culture with endothelial cells, which had been transfected with HO-1, which blocked cell cycle entry of T cells. More than 60% of Jurkat T cells remained in G0/G1 compared with 40% among the control group. CONCLUSION: HO-1 directly protected endothelial cells primed by INF-gamma from Jurkat T cells and down-regulated the expression of HLA-DR on the surface of endothelial cells. These results indicated that transgenic expression of HO-1 may be useful to prevent lymphocytes from responding to endothelial cells.

Expression of neuropilin-1 in kidney graft biopsies: what is the significance?

OBJECTIVE: Recent work has suggested that neuropilin-1 (NRP-1) is a surface marker of regulatory T cells (Treg). However, no further relative evidence has been provided to confirm this finding. Since Treg should decline during rejection, the expression of NRP-1 on lymphocytes should decline in a rejected graft. To test this proposal, we examined NRP-1 expression in kidney graft biopsies. MATERIALS AND METHODS: Tissue samples were obtained from 20 kidney graft biopsies with pathologically confirmed acute rejection and from 10 without rejections. We performed immunohistochemistry assays using an anti-NRP-1 monoclonal antibody. The positive cells were counted and the ratios among lymphocytes analyzed. RESULTS: Compared with samples from nonrejected graft biopsies (18.71 +/- 20.60), the number of positive cells among lymphocytes in the rejected samples showed a lower percentage (3.16 +/- 1.72; P < .05). CONCLUSIONS: NRP-1 has an important role in directing the growth of nervous synapses and immune synapses. We found in rejected grafts that the percentage of NRP-1 positive cells among lymphocytes decreased significantly. Therefore, NRP-1 may have a previously unrecognized role to predict the immune state of the graft as a potential marker for Treg.

Induction of accommodation model by combined RNA interference targeting 1,3-galactosyltransferase gene and low-dose GS-IB4 lectin in vitro.

OBJECTIVE: This study sought to mimic the interaction of xenograft endothelial cells and human serum in vitro after successfully silencing the expression of porcine alpha1,3-galactosyltransferase (alpha1,3GT) gene by RNA interference (RNAi), and to investigate the possibility of inducing accommodation in vitro by stimulation of alpha-Gal-specific binding lectin, Griffonia simplicifolia isolectin B4 (GS-IB4) and RNAi. MATERIALS AND METHODS: Various alpha-Gal expression patterns on a pig endothelial cell immortalized line (PED) was achieved by serial doses of small interfering RNA (siRNA) targeting porcinc alpha1,3GT gene. alpha1,3GT-siRNA transfected PEDs were exposed to increasing doses of GS-IB4 lectin (0.5, 2, and 8 microg/mL) for 4 hours before incubation with normal human serum (NHS). Accommodation phenomenon of PEDs in NHS was observed by 51Cr release and antibody/complement binding assays. RESULTS: With combined RNAi and low-dose GS-IB4 stimulation, PEDs remarkably inhibited complement-mediated cytotoxicity, which showed a better protective effect than using RNAi alone. At a concentration of 2 mug/mL, GS-IB4 exhibited the maximum protective effect. The expression of E-selectin on alpha1,3GT-siRNA transfected PEDs did not differ from that on parental PEDs with heat-inactivated NHS (HINHS) stimulation. Combined with GS-IB4 stimulation, however, it inhibited expression of E-selectin, which was GS-IB4 dose dependent, resulting in mean fluorescence intensity values of 98.5, 42.0, and 36.3 at 0.5, 2, and 8 microg/mL. The mRNA expression of the protective gene HO-1 was significantly up-regulated after treatment with RNAi and low-dose of GS-IB4. CONCLUSIONS: Combined RNAi and low-dose GS-IB4 induced pig endothelial cell accommodation in vitro. The level of alpha-Gal expression played an important role in the induction of accommodation.

Antisense ERK1/2 oligodeoxynucleotide gene therapy attenuates graft arteriosclerosis of aortic transplant in a rat model.

UNLABELLED: Chronic rejection is a major cause of transplant loss that is effected by the extracellular signal-regulated kinases (ERK) pathway. This study investigated the effects of antisense ERK1/2 oligodeoxynucleotide(ODN) gene therapy on chronic rejection. METHODS: Lewis (RT1(1)) rats served as recipients of Brown-Norway (BN, RT1n) grafts. The BN rat abdominal aortas were harvested and orthotopically grafted into Lewis rats. The recipients were divided into three groups: (1) control group (n = 9), (2) random ODN transfer group (n = 10), and (3) antisense ODN transfer group (n = 10). At day 60 after transplantation, the recipients were sacrificed; the grafted aortas were evaluated histologically and immunohistochemically. ERK1/2 protein expression in the grafts was determined using Western Blot assays. Serum levels of slCAM-1 were detected by ELISA. RESULTS: In the control group and random ODN transfer group, we observed a remarkable degree of intimal hyperplasia and inflammatory cell infiltration, including macrophages and T cells. Compared with the control group, antisense ERK1/2 ODN gene therapy resulted in a significant reduction in neointimal proliferation (P < .01), inhibition of ERK1/2 protein expression (P < .01), decreased graft infiltration with CD4+ T lymphocytes (P < .01), CD8+ T lymphocytes(P < .05), and ED-1 macrophages (P < .01) with decreased serum levels of sICAM-1 (P < .05). We obtained a negative correlation between ERK1/2 expression and immune cell infiltration or ICAM-1 level. CONCLUSIONS: Antisense ERK1/2 gene therapy can attenuate graft arteriosclerosis so as to protect aortic allografts. The protection seemed to correlate with inhibition of inflammatory infiltration, implying that the ERK1/2 signal transduction pathway plays an important role in the process of chronic vascular rejection.

Magnesium supplementation prevents chronic cyclosporine nephrotoxicity via adjusting nitric oxide synthase activity.

INTRODUCTION: Nitric oxide synthase (NOS) is a protective factor for chronic cyclosporine nephrotoxicity by virtue of adjusting the production of nitric oxide (NO). The aim of this study was to explore the role of NOS in the effect of magnesium supplementation to prevent chronic cyclosporine nephrotoxicity. METHODS: Rats maintained on a low-salt diet were divided into three groups: normal controls, cyclosporine group (CsA 15 mg x kg(-1) x d(-1) subcutaneously) and CsA + Mg2+ group (CsA subcutaneously and dietary supplementation with 0.6% Mg enriched by MgCl2). On day 28, plasma Mg2+, plasma creatinine, NOS activity, and NO content in renal tissue were examined. The renal expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in kidneys was determined by an immunohistochemistry technique. The lesions of chronic cyclosporine nephrotoxicity were identified by HE and PAS stains as well as electron microscope. RESULTS: After 28 days of CsA administration, characteristic histological lesions of chronic cyclosporine nephotoxicity were observed, including arteriolopathy, tubular atrophy and interstitial fibrosis. Giant mitochondria and microcalcifications were observed by electron microscopy. Simultaneously, constitutive nitric oxide synthase (cNOS) activity in kidneys was increased, but NO content did not increase correspondingly (P < .05) compared with normal controls. Dietary supplementation with Mg2+ ameliorated the CsA-induced histological lesions. cNOS activity was decreased to normal levels and NOS was increased (P < .05) compared with animals that only received CsA. CsA and magnesium supplementation did not change iNOS activity. CONCLUSIONS: Dietary supplementation with Mg2+ seems to improve renal function and almost abolish CsA-induced histological lesions via altering the abnormal activation of cNOS in this model.