Kuang-Den Chen

Impact of artificial sunlight therapy on the progress of non-alcoholic fatty liver disease in rats.

BACKGROUND & AIMS Non-alcoholic steatohepatitis (NASH) is recognized as the most severe form of non-alcoholic fatty liver disease, with likely progression to liver cirrhosis and hepatocellular carcinoma. However, there is no unified standard for diagnosis and therapeutics. This study aimed to characterize lipid transfer/metabolic proteins as non-invasive diagnostic markers, and to evaluate the therapeutic effects of phototherapy on the progression of NASH in rats. METHODS Lewis rats given a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet and Zucker fa/fa rats were used as a diet-induced and an obesity-related NASH models, respectively, with or without phototherapy. RESULTS Serum apolipoprotein E and low molecular weight-adiponectin levels were gradually reduced and reached the lowest level at fatty liver/NASH stage both in CDAA diet-induced NASH model and in genetically obese model. Total-adiponectin levels were dramatically elevated after NASH was established in CDAA diet-induced NASH model. Phototherapy ameliorated hepatocyte apoptosis, inflammation, fibrosis, and insulin/leptin resistance caused by CDAA diet with alteration of the levels of lipid transfer/metabolic proteins and elevation of the circulating active form of vitamin D(3). Vitamin D(3) supplementation ameliorated NASH progression in CDAA diet-induced NASH model. However, phototherapy failed to ameliorate the obesity and steatosis, suggesting that phototherapy may possess anti-inflammatory/fibrotic activity rather than anti-obesity/steatotic activity. CONCLUSIONS These results suggest that serum lipid transfer/metabolic proteins and vitamin D(3) status may be effective biomarkers for non-invasive diagnosis of NASH progression, and that phototherapy may be a good complementary therapy for NASH because of its regulation of lipid transfer/metabolic proteins and vitamin D(3).

Immunological and Regenerative Aspects of Hepatic Mast Cells in Liver Allograft Rejection and Tolerance

The precise roles of mast cells in liver allograft rejection and tolerance are still unknown. This study aimed to explore the roles of mast cells in immune regulation and liver regeneration for tolerance induction by using rat models of orthotopic liver transplantation (OLT). Stem cell factor (SCF) and its receptor c-Kit, which are critical to the migration and development of not only stem cells but also mast cells, significantly increased in the tolerogenic livers as compared with rejected livers. The significant elevation of mast cell tryptase, high-affinity IgE receptor, and histamine suggested the activation of mast cells in liver allografts at the tolerogenic phase after OLT. Immunohistochemical analysis using confocal microscope clearly showed colocalization of mast cells, Foxp3+ Tregs, γδ T cells, and recipient-derived hepatic progenitor cells with higher expression of SCF, IL-9, IL-10, TGF-β1, and IL-17 related to immunoregulation and liver regeneration in the donor grafts of a tolerogenic OLT model. Cross-talk among mast cells and other cells was evaluated by in vitro studies demonstrating that syngeneic bone marrow−derived mast cells (BMMCs) co-cultured with naïve splenocytes or primary hepatocytes significantly increased the population of splenic γδ T cells by mitogen stimulation or by mast cell degranulation, and also significantly induced the hepatocyte proliferation, respectively. Our results suggested that mast cells in the donor grafts may play important roles in the induction/maintenance of immune tolerance and liver regeneration resulting in the replacement of hepatic cells from donor to recipient.

Identification of miR-27b as a Novel Signature from the mRNA Profiles of Adipose-Derived Mesenchymal Stem Cells Involved in the Tolerogenic Response

Adipose-derived mesenchymal stem cells (adipose-derived MSCs, ASCs) possess the ability to differentiate into multiple tissue types and have immune-modulatory properties similar to those of MSCs from other origins. However, the regulation of the MSC-elicited immune-modulatory activity by specific microRNA (miRNA) mechanisms remains unexplored. Gene expression profiling with knowledge-based functional enrichment analysis is an appropriate approach for unraveling these mechanisms. This tool can be used to examine the transcripts and miRNA regulators that differentiate the rat tolerogenic orthotopic liver transplantation (OLT; DA liver into PVG) and rejection OLT (DA liver into LEW) models. In both models, the rejection reaction was observed on postoperative day 7∼14 (rejection phase) but was overcome only by the PVG recipients. Thus, the global gene expression patterns of ASCs from spontaneously tolerant (PVG) and acute rejecting (LEW) rats in response to LPS activation were compared. In this study, we performed miRNA enrichment analysis based on the analysis of pathway, gene ontology (GO) terms and transcription factor binding site (TFBS) motif annotations. We found that the top candidate, miR-27, was specifically enriched and had the highest predicted frequency. We also identified a greater than 3-fold increase of miR-27b expression in the ASCs of tolerant recipients (DA to PVG) compared to those of rejecting recipients (DA to LEW) during the rejection phase in the rat OLT model. Furthermore, our data showed that miR-27b knockdown has a positive influence on the allosuppressive activity that inhibits T-cell proliferation. We found that miR-27 knockdown significantly induced the expression of CXCL12 in cultured ASCs and the expression of CXCL12 was responsible for the miR-27b antagomir-mediated inhibition of T-cell proliferation. These results, which through a series of comprehensive miRNA enrichment analyses, might be relevant for stem cell-based therapeutic applications in immunosuppressive function using ASCs.

Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype.

STUDY OBJECTIVES We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). METHODS Illuminas (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). RESULTS Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (-114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (-531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (-608/-618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (-194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. CONCLUSIONS IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. COMMENTARY A commentary on this article appears in this issue on page 723.

Peripheral Immune Cell Gene Expression Changes in Advanced Non-Small Cell Lung Cancer Patients Treated with First Line Combination Chemotherapy

Introduction Increasing evidence has shown that immune surveillance is compromised in a tumor-promoting microenvironment for patients with non-small cell lung cancer (NSCLC), and can be restored by appropriate chemotherapy. Methods To test this hypothesis, we analyzed microarray gene expression profiles of peripheral blood mononuclear cells from 30 patients with newly-diagnosed advanced stage NSCLC, and 20 age-, sex-, and co-morbidity-matched healthy controls. All the patients received a median of four courses of chemotherapy with cisplatin and gemcitabine for a 28-day cycle as first line treatment. Results Sixty-nine differentially expressed genes between the patients and controls, and 59 differentially expressed genes before and after chemotherapy were identified. The IL4 pathway was significantly enriched in both tumor progression and chemotherapy signatures. CXCR4 and IL2RG were down-regulated, while DOK2 and S100A15 were up-regulated in the patients, and expressions of all four genes were partially or totally reversed after chemotherapy. Real-time quantitative RT-PCR for the four up-regulated (S100A15, DOK2) and down-regulated (TLR7, TOP1MT) genes in the patients, and the six up-regulated (TLR7, CRISP3, TOP1MT) and down-regulated (S100A15, DOK2, IL2RG) genes after chemotherapy confirmed the validity of the microarray results. Further immunohistochemical analysis of the paraffin-embedded lung cancer tissues identified strong S100A15 nuclear staining not only in stage IV NSCLC as compared to stage IIIB NSCLC (p = 0.005), but also in patients with stable or progressive disease as compared to those with a partial response (p = 0.032). A high percentage of S100A15 nuclear stained cells (HR 1.028, p = 0.01) was the only independent factor associated with three-year overall mortality. Conclusions Our results suggest a potential role of the IL4 pathway in immune surveillance of advanced stage NSCLC, and immune potentiation of combination chemotherapy. S100A15 may serve as a potential biomarker for tumor staging, and a predictor of poor prognosis in NSCLC.

Cytochrome P450 in living donor liver transplantation

Cytochrome P450 metabolizes many drugs in the liver. Three genotypes of CYP2C19 with extensive, intermediate, and poor metabolizing activity, respectively, have been identified in peripheral blood of transplant recipients and new liver grafts in living donor liver transplantation (LDLT). The expression of the final genotype in liver graft biopsies depends on the donor, whereas the expression in peripheral blood mononuclear cells depends on the recipient. The metabolizing isoenzyme of the major anti-rejection agents passes through CYP3A4, CYP3A5 and MDR1, which have also been identified to have similar biological characteristics as genotype of CYP2C19 in liver tissue. Recently, pyrosequencing has been used to investigate the expressions of different genotypes in liver grafts in LDLT. This review focuses on recent findings regarding the biological expressions of the CYP2C19, CYP3A4, CYP3A5 and MRD1 genotypes in liver grafts before and after LDLT. The application of pyrosequencing may be beneficial in further research on liver transplantation. Laser capture microdissection of hepatocytes in liver grafts may be a direction for future research.

Pyrosequencing to Identify Homogeneous Phenomenon When Using Recipients/Donors with Different CYP3A5*3 Genotypes in Living Donor Liver Transplantation

This study used pyrosequencing to determine the proportional distribution of CYP3A5*3 genotypes to further confirm the homogeneous phenomenon that is observed when recipients and donors in living donor liver transplantation (LDLT) have a different single nucleotide polymorphism (SNP) genotype. We enrolled 42 recipient/living donor pairs and the SNPs of CYP3A5*3 were identified by polymerase chain reaction-restriction fragment length polymorphism. We performed 120 liver graft biopsies as part of clinical investigations after LDLT. Pyrosequencing of the CYP3A5*3 SNPs revealed that among the 16 recipients with the G/G genotype, 94.68% had the G and 5.32% the A allele. Among the 14 recipients with the A/G genotype, 78.08% had the G and 21.92% the A allele, and among the 12 recipients with the A/A genotype, 18.45% had the G and 81.55% the A allele. Among the 12 donors with the G/G genotype, 93.85% had the G and 6.14% the A allele. Among the 26 donors with the A/G genotype, 75.73% had the G and 24.27% the A allele, and among the 4 donors with the A/A genotype, 11.09% had the G and 88.91% the A allele. There were a total of 120 liver graft biopsy samples; among the 37 recipients with the G/G genotype, 89.74% had the G and 10.26% the A allele, among the 70 recipients with the A/G genotype, 71.57% had the G and 28.43% the A allele, and among the 13 recipients with the A/A genotype, 48.25% had the G and 51.75% the A allele. The proportional distribution of G and A alleles of the CYP3A5*3 SNP between recipients/donors and liver grafts after LDLT was significantly different (p<0.001). Pyrosequencing was useful in identifying detailed proportional changes of the CYP3A5*3 SNP allele distribution, and to confirm the homogeneous phenomenon when recipients and donors in LDLT have a different genotype.

Biological interactions of CYP2C19 genotypes with CYP3A4*18, CYP3A5*3, and MDR1-3435 in living donor liver transplantation recipients

BackgroundPolymorphisms in CYP2C19 are related to the metabolic oxidation of drugs to varying degrees. The CYP3A4*18, CYP3A5*3, and MDR1-3435 variant alleles are very important, particularly in tacrolimus metabolism in organ transplant rejection.AimThe aim of this study is o explore possible interactions among different CYP2C19 genotypes, namely, between homozygous extensive metabolizers (HomEM), heterozygous extensive metabolizers (HetEM), and poor metabolizers (PM), and the CYP3A4*18, CYP3A5*3, and MDR1-3435 variants in living donors and patients who received a living donor liver transplant (LDLT).MethodsThis prospective study enrolled 133 living donors and 133 corresponding recipients. On the basis of the HomEM, HetEM, and PM CYP2C19 genotypes, the distributions of CYP3A4*18 (exon 10; T878C), CYP3A5*3 (intron 3; A6986G), and MDR1-3435 (exon 26; C3435T) genotypes were analyzed for single nucleotide polymorphisms among donors and recipients.ResultsAmong 102 HomEM genotypes, including 56 donors and 46 recipients, 91.2% of individuals harbored the T/T genotype of CYP3A4*18; 53.9% possessed G/G, and 34.3% had A/G genotypes of CYP3A5*3; and 38.2% had C/C and 50.0% had C/T genotypes at MDR1-3435. Among 130 HetEM genotypes, including 58 donors and 72 recipients, 97.7% of individuals possessed T/T genotype at CYP3A4*18; 50.0% harbored G/G and 41.5% had A/G genotypes at CYP3A5*3; and 40.0% had C/C and 49.2% had C/T genotypes at MDR1-3435. In 34 PMs, including 19 donors and 15 recipients, 88.2% had T/T genotypes at CYP3A4*18; 41.2% had G/G and 58.8% had A/G genotypes at CYP3A5*3; and 47.1% possessed C/C and 47.1% had C/T genotypes at MDR1-3435. On the basis of the CYP2C19 genotypes, no statistically significant distribution of genotypes were observed between donors and recipients for all genotypes of CYP3A4*18, CYP3A5*3, and MDR1-3435 (P >0.05).ConclusionsIn conclusion, the CYP2C19 genotypes do not affect the expression of CYP3A4*18, CYP3A5*3, or MDR1-3435 variants, which are independently distributed among donors and recipients during LDLT.

Low-dose LBH589 increases the sensitivity of cisplatin to cisplatin-resistant ovarian cancer cells

OBJECTIVE There is a need to develop alternative therapeutic strategies to overcome cisplatin-associated resistance in patients with ovarian cancer. Histone deacetylation (HDAC) associated with inactivation of genes has been implicated in the epigenetic silencing of tumor suppressor genes affecting critical biological activities in cancer cells and may be an important factor in acquired cisplatin-associated resistance. In this report, we tested a combination of cisplatin and LBH589 (histone deacetylation inhibitor) in cisplatin-resistant ovarian cancer cells to explore the reversal effect of cisplatin resistance and changes of gene expression. MATERIALS AND METHODS To detect the synergistic effects of antiproliferation between cisplatin and LBH589 in ovarian cancer cells, we performed a cell viability assay and a clonogenic assay. To investigate the differences of gene expression between cells treated by cisplatin alone and cotreated with cisplatin and LBH589, a microarray mRNA analysis was performed. RESULTS In the presence of low-dose LBH589, the inhibition concentration value of cisplatin for A2780-cp70 cells was much lower than with cisplatin treatment alone. Gene expression profiles identified that a total of 354 genes had been significantly upregulated and a total of 63 genes been downregulated with LBH589 cotreatment. CONCLUSION We hypothesized that combination of cisplatin and LBH589 can override cisplatin-associated resistance in ovarian cancer cells. These results provide initial evidence for testing this combination in clinical use.

Effects of Different Operating Room Temperatures on the Body Temperature Undergoing Live Liver Donor Hepatectomy

OBJECTIVE We sought to compare the effects of operation room temperature (ORT) at typical ambient environment (19-21 degrees C) and ORT at 24 degrees C on the core temperature of patients undergoing living donor hepatectomy. METHODS AND PATIENTS Sixty-two patients undergoing living donor hepatectomy were divided into 2 groups. In group I (n = 31), surgery was performed at typical ambient ORT, and in group II (n = 31) in ORT at 24 degrees C. Anesthesia and measures to prevent heat loss, except ORT, were all the same. Nasopharyngeal temperature (NT) was recorded after anesthesia induction, then hourly until completion of the operation. Changes in NTs were analyzed as well as patient age, weight, anesthetic duration, blood loss, intravenous fluids, total urine output, and pre- and postoperative hemoglobin and hematocrit values. The Mann-Whitney U test was used for comparisons between groups. RESULTS The patients characteristics between groups were not statistically different. However, a significantly higher core temperature was noted in group II compared with group I. Increased ORT from 19 to 21 degrees C to 24 degrees C resulted in an increased core temperature of at least 0.5 degrees C during living donor hepatectomy.