Unraveling the causal genes and transcriptomic determinants of human telomere length.

Telomere length (TL) shortening is a pivotal indicator of biological aging and is associated with many human diseases. The genetic determinates of human TL have been widely investigated, however, most existing studies were conducted based on adult tissues which are heavily influenced by lifetime exposure. Based on the analyses of terminal restriction fragment (TRF) length of telomere, individual genotypes, and gene expressions on 166 healthy placental tissues, we systematically interrogate TL-modulated genes and their potential functions. We discover that the TL in the placenta is comparatively longer than in other adult tissues, but exhibiting an intra-tissue homogeneity. Trans-ancestral TL genome-wide association studies (GWASs) on 644,553 individuals identify 20 newly discovered genetic associations and provide increased polygenic determination of human TL. Next, we integrate the powerful TL GWAS with placental expression quantitative trait locus (eQTL) mapping to prioritize 23 likely causal genes, among which 4 are functionally validated, including MMUT, RRM1, KIAA1429, and YWHAZ. Finally, modeling transcriptomic signatures and TRF-based TL improve the prediction performance of human TL. This study deepens our understanding of causal genes and transcriptomic determinants of human TL, promoting the mechanistic research on fine-grained TL regulation.

The recombinant BMP-2 loaded silk fibroin microspheres improved the bone phenotype of mild osteogenesis imperfecta mice.

Osteogenesis imperfecta (OI) is an inherited congenital disorder, characterized primarily by decreased bone mass and increased bone fragility. Bone morphogenetic protein-2 (BMP-2) is a potent cytokine capable of stimulating bone formation, however, its rapid degradation and unanticipated in vivo effects restrict its application. The sustained release characteristic of silk fibroin (SF) microspheres may potentially address the aforementioned challenges, nevertheless they have not previously been tested in OI treatment. In the current investigation, recombinant BMP-2 (rBMP-2) loaded SF (rBMP-2/SF) microspheres-based release carriers were prepared by physical adsorption. The SF microparticles were characterized by scanning electron microscopy (SEM) and were investigated for their cytotoxicity behavior as well as the release profile of rBMP-2. The rBMP-2/SF microspheres were administered via femoral intramedullary injection to two genotypes of OI-modeled mice daily for two weeks. The femoral microstructure and histological performance of OI mice were evaluated 2 weeks later. The findings suggested that rBMP-2/SF spheres with a rough surface and excellent cytocompatibility demonstrated an initial rapid release within the first three days (22.15 ± 2.88% of the loaded factor), followed by a transition to a slower and more consistent release rate, that persisted until the 15th day in an in vitro setting. The factor released from rBMP-2/SF particles exhibited favorable osteoinductive activity. Infusion of rBMP-2/SF microspheres, as opposed to blank SF spheres or rBMP-2 monotherapy, resulted in a noteworthy enhancement of femoral microstructure and promoted bone formation in OI-modeled mice. This research may offer a new therapeutic approach and insight into the management of OI. However, further investigation is required to determine the systematic safety and efficacy of rBMP-2/SF microspheres therapy for OI.

miR-27a-3p alleviates lung transplantation-induced bronchiolitis obliterans syndrome (BOS) via suppressing Smad-mediated myofibroblast differentiation and TLR4-induced dendritic cells maturation.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS), induced by a chronic rejection, remains a significant obstacle for end-stage lung diseases after lung transplantation. We have previously determined that the small non-coding mRNA (miRNA) miR-27a-3p maintained the immature state of dendritic cells (DCs) via the interleukin 10 (IL-10)-dependent regulatory pathway. Such status helped in preventing rejection and alleviating BOS. The present study explored mechanisms how miR-27a-3p may suppress the fibrosis as well as the maturation of DCs, ultimately attenuating BOS in vitro and in vivo. METHODS/RESULTS: In our tracheal transplantation mouse model, the expression of Smad2, Smad4, and αSMA were significantly decreased in the miR-27a-3p-transfected DCs (p < 0.0001, p = 0.0006, and p = 0.0002 respectively). Moreover, the expression of fibrosis markers (α-SMA, collagen I, and Fn) were potently inhibited in the miR-27a-3p-transfected NIH-3 T3 cells (p < 0.0001, p = 0.00148, and p < 0.0001 respectively). At the same time, reversed results were observed in the inhibitor group (p = 0.0002, p < 0.0001, and p < 0.0001 respectively), indicating that miR-27a-3p could directly inhibit myofibroblast differentiation. Furthermore, in the tracheal transplanted mice, the population of Treg cells was significantly decreased (p < 0.0001). In contrast, Th17 cells were down-regulated in the miR-27a-3p-transfected DCs group (p < 0.0001), accompanied by the decreased IL-17 levels (p = 0.0007) and the induction of TGF-ß1 and IL-10 (p < 0.0001 and p = 0.0016 respectively). Further mechanistic studies indicated that miR-27a-3p altered the maturation of DCs by targeting TLR4 and IRAK (p < 0.0001 and p = 0.0002 respectively). CONCLUSIONS: Our study suggests that miR-27a-3p selectively blocked the TGF-ß1/Smad pathways to suppress the myofibroblast differentiation and targeted the TRL4/IRAK4 pathway to restrain DCs maturation, thus attenuating BOS. Our findings suggest that miR-27a-3p is a potential active molecule on BOS management after lung transplantation.

DNA methyltransferases inhibitor azacitidine improves the skeletal phenotype of mild osteogenesis imperfecta by reversing the impaired osteogenesis and excessive osteoclastogenesis.

BACKGROUND: Osteogenesis imperfecta (OI), as a disease of congenital bone dysplasia, is often accompanied by the abnormal alteration of bone absorption and bone formation. DNA methyltransferases (Dnmts) can regulate the gene expression involved in osteogenesis and osteoclastogenesis. Dnmts changes and their effects on bone cells under OI is poorly understood. METHODS: The Dnmts expression in adipose derived mesenchymal stem cells (ADSCs), bone marrow derived pre-osteoclasts (pre-Ocs) and femurs of Col1a2oim/+ and Col1a1+/-365 mice, both modeling mild OI types, were determined. The effects of azacitidine (Aza) administration and Dnmt3a knockdown by ShRNA on the osteogenic differentiation of ADSCs together with osteoclasts (Ocs) production of pre-Ocs were studied in vitro. The synthesis and secretion of collagen fibers of OI derived ADSCs were examined. The therapeutic outcomes of intraperitoneal (i.p.) infused Aza (1 mg/kg/2d) for 30 days were evaluated in OI mice. RESULTS: Obviously elevated expression of Dnmts, especially Dnmt3a, existed in ADSCs, pre-Ocs, and femurs isolated from OI modeled mice. Much more collagen molecules of mutant ADSCs were secreted into the extracellular medium post Aza addition. Both Aza administration and Dnmt3a knockdown effectively enhanced the bone-forming capacity of affected ADSCs and reduced Ocs formation of OI mice in vitro. Aza treatment apparently improved the femora microstructure and biomechanical properties, increased bone formation and decreased the number of Ocs in mice with OI. CONCLUSION: Highly expressed Dnmt3a contributed to the impaired osteogenesis and enhanced osteoclastogenesis of collagen defect-related OI. Aza medication effectively improved the femora phenotype of the two types of OI modeled mice partly by Dnmts inhibition and modulating cell stress response. These findings facilitated understanding the role of Dnmts alteration in skeletal pathological development of mild OI and preliminary confirmed the therapeutic potential of Dnmts depressants in mild OI treatment. Still, further researches are needed to explore the specific function of Dnmts in OI bones and clarify the benefits of Aza administration in OI treatment.

Up-regulated IL-17 and Tnf signaling in bone marrow cells of young male osteogenesis imperfecta mice.

Osteogenesis imperfecta (OI) is a congenital bone dysplasia mainly caused by either defective production or assembly of type I collagen. The skeletal phenotypes especially fractures are often seen in OI adolescents. Studies have found that an increased number of osteoclasts and excessive bone resorption existed in collagen-related OI, which has not been well understood. Emerging evidence has suggested that inflammation may be associated with OI. We speculated that the bone marrow (BM) niche had similar inflammatory changes and performed RNA-sequencing (RNA-seq) in BM cells derived from young male mice to analyze the related differentially expressed genes (DEGs) and pathways. Data showed that there were 117 shared DEGs (Q ≤ 0.05, |log2FC| ≥ 1) in BM cells isolated from two types of OI murine models that respectively simulate different OI types. Gene Ontology (GO) (Q ≤ 0.05) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) (Q ≤ 0.05) analysis and real-time PCR validation indicated the dysregulated biology process of cellular response to interferon (Ifn) together with upregulated IL-17 signaling, tumor necrosis factor (Tnf) signaling and osteoclast differentiation in OI BM niche. Either defective collagen production or abnormal collagen assembly shared similar alterations in gene profiles and pathways involving inflammation and osteoclast activation. Data presented here not only contributed to understanding of the mechanism of the enhanced bone absorption in the bones of OI, but also provided more evidence to develop potential anti-inflammation therapies.

Impaired proliferation of growth plate chondrocytes in a model of osteogenesis imperfecta.

Short stature is the second conspicuous characteristic of osteogenesis imperfecta (OI), but the etiological mechanism is unclear. The proliferation of growth plate chondrocytes (GPCs) plays an essential role in longitudinal bone growth, and chondrocyte division deficiency can cause shortened limbs. However, few studies have reported the abnormal changes of growth plate and GPCs in OI. In this study, the cell proliferative performance of GPCs in heterozygous Col1a2oim/+ mice were studied and the underlying mechanism was explored by RNA-Sequencing. The results indicated that chondrocytes of Col1a2oim/+ background displayed impaired cell division when compared with cells of wild-type littermates. A group of differentially expressed genes involving chondrocyte proliferation related pathways including cell cycle, TGF-ß signaling pathway and Hedgehog signaling pathway were identified. These dysregulated genes and pathways in GPCs of Col1a2oim/+ mice are likely to play an important role in their shortened long bones. Further investigations to reveal the effect of these genes on bone elongation not only facilitate the understanding of OI short stature, but also contribute to developing new treatments.

Large chromosomal deletions and impaired homologous recombination repairing in HEK293T cells exposed to polychlorinated biphenyl 153.

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent pollutants with carcinogenesis and mutagenesis effects which have been closely associated with PCBs-induced DNA damage. However, the detailed DNA damage events and corresponding pathway alterations under PCBs poisoning is still not well understood. METHODS: Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) were used to explore genome wide variations and related pathway changes in HEK293T cells that challenged by 15 µM PCB153 for 96 h in vitro. Double strand breaks (DSBs) were measured by 53BP1 foci detection, altered pathways were confirmed by quantitative real-time PCR (qPCR). RESULTS: The results indicated that abundant copy number variations (CNVs), including four duplications and 30 deletions, occurred in PCB153-exposed HEK293T cells. Multiple large fragment deletions (>1 Mb) involving up to 245 Mb regions on many chromosomes. Missense mutations were found in six tumor susceptibility genes, two of which are key members participating in homologous recombination (HR) repair response, BRCA1 and BRCA2. RNA-seq data showed that PCB153 poisoning apparently suppressedHR repairing genes. Besides, 15 µM PCB153 exposure significantly increased 53BP1 foci formation and effectively reduced BRCA1, RAD51B and RAD51C expression, indicating an elevated DSBs and impaired HR repairing. CONCLUSION: This study firstly reported multiple large chromosomal deletions and impaired HR repairing in PCB153-exposed HEK293T cells, which provided a new insight into the understanding of early response and the mechanism underlying PCB153 genotoxicity. The chromosomal instabilities might be related to the impaired HR repairing that induced by PCB153; however, further investigations, especially on actual toxic effects of human body, are needed to confirm such speculation.

Modification of COL1A1 in Autologous Adipose Tissue-Derived Progenitor Cells Rescues the Bone Phenotype in a Mouse Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a congenital genetic disorder mainly manifested as bone fragility and recurrent fracture. Mutation of COL1A1/COL1A2 genes encoding the type I collagen are most responsible for the clinical patients. Allogenic mesenchymal stem cells (MSCs) provide the potential to treat OI through differentiation into osteoblasts. Autologous defective MSCs have not been utilized in OI treatment mainly because of their impaired osteogenesis, but the latent mechanism has not been well understood. Here, the relative signaling abnormality of adipose-derived mesenchymal stem cells (ADSCs) isolated from OI type I mice (Col1a1+/-365 mice) was explored. Autologous ADSCs transfected by retrovirus carrying human COL1A1 gene was first utilized in OI therapy. The results showed that decreased activity of Yes-associated protein (YAP) due to hyperactive upstream Hippo kinases greatly contributed to the weakened bone-forming capacity of defective ADSCs. Recovered collagen synthesis of autologous ADSCs by COL1A1 gene modification normalized Hippo/YAP signaling and effectively rescued YAP-mediated osteogenesis. And the COL1A1 gene engineered autologous ADSCs efficaciously improved the microstructure, enhanced the mechanical properties and promoted bone formation of Col1a1+/-365 mice after femoral bone marrow cavity delivery and might serve as an alternative source of stem cells in OI treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

Mir-27a-3p attenuates bronchiolitis obliterans in vivo via the regulation of dendritic cells' maturation and the suppression of myofibroblasts' differentiation.

Bronchiolitis obliterans (BO), is a chronic rejection phenotype characterized by chronic small airway fibrous obliteration, hinders the patients who suffer from lung transplanting for surviving longer. Cell-based therapies using dendritic cells (DCs) and T regulatory cells (Tregs) have been developed to regulate allograft rejection, and to induce and maintain immune tolerance. In the present study, the effects of mir-27a-3p on regulating DCs as well as resulting effects on BO attenuation have been investigated. According to our reporter assays, the potential targets of mir-27a-3p were Smad2, sprouty2, and Smad4, respectively. Furthermore, sprouty2 inhibition by mir-27-3p indirectly activated extracellular regulated protein kinases (ERK) and increased IL-10 production in DCs. This led to a positive feedback loop that maintained the immature state of DCs via IL-10/JAK/STAT3 pathway, and caused an increase in Foxp3+ CD4+ T cells amount as well as TGF-ß level. Furthermore, mir-27a-3p regulated TGF-ß function, inhibited TGF-ß/Smad pathway, and suppressed myofibroblast differentiation through influencing the function of Smad2 and Smad4. In short, the study indicated the effect of mir-27a-3p on suppressing DC maturation, which implicated the potential clinical application in treating postlung transplant BO.

The synergistic effect of NELL1 and adipose-derived stem cells on promoting bone formation in osteogenesis imperfecta treatment.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by bone fragility and deformity. Mesenchymal stem cells (MSCs) infusion can improve bone performance mainly due to their differentiation into osteoblasts in OI therapy. The osteoinductive activity of NELL1 have benefited various bone defect and osteoporotic models by promoting bone formation. The present study investigated the efficacy of combined use of NELL1 and adipose-derived mesenchymal stem cells (ADSCs) in OI treatment. METHODS: Lentiviral vector carrying mouse Nell1 gene was constructed and lentivirus were used to infect ADSCs. The osteogenic capacity of MC3T3-E1 and ADSCs stimulated by recombinant mouse NELL1 protein (rmNELL1) and Nell1 gene genetically modified ADSCs (lenti-Nell1-ADSCs) were estimated by real-time quantitative PCR. Thirty adult male OI type I mice with single Col1a1 gene knockout were randomly divided into five groups and received intravenously injected PBS, rmNELL1 (1.25 mg/Kg), ADSCs (2 × 105 cells per mice), rmNELL1 (1.25 mg/Kg) combined with ADSCs (2 × 105 cells per mice), or lenti-Nell1-ADSCs (2 × 105 cells per mice) respectively. Six wildtype (WT) mice served as positive control. Bone formation was examined after 4 weeks using micro-CT, histological and immunohistochemical methods. RESULTS: Three osteoblast related genes of MC3T3-E1 and ADSCs were significantly up-regulated by rmNELL1 in vitro. Lenti-Nell1-ADSCs showed greatly enhanced osteogenic differentiation capacity. The infused lenti-Nell1-ADSCs could migrate to femur and differentiate into ALPL-positive cells. Systemic administration of rmNELL1 combined with ADSCs or lenti-Nell1-ADSCs markedly improved the femoral microstructure and promoted bone formation through increasing the ALPL and osteocalcin (OCN) expression, much better than mice that received single rmNELL1 or ADSCs. And Nell1 gene engineered ADSCs achieved slightly better outcomes than that of combinative use of rmNELL1 and ADSCs. CONCLUSIONS: NELL1 and ADSCs exhibited synergistic effect on stimulating bone formation of OI mice, which might provide an alternative strategy in OI treatment. Compared with dose escalation or multiple administration of rmNELL1, lentivirus-mediated long term expression of NELL1 might be more feasible and convenient. However, further studies are needed to confirm the safety and optimize the therapeutic regime.

Pretreatment with interleukin 35-engineered mesenchymal stem cells protected against lipopolysaccharide-induced acute lung injury via pulmonary inflammation suppression.

Acute lung injury (ALI)-triggered pulmonary injury has been associated with high mortality, despite advances in drug treatment and supportive therapy. Remarkable progress has been made in attenuating the inflammatory injury associated with ALI using mesenchymal stem cells (MSCs)-based cell and gene therapy. However, to date, the benefits of interleukin-35 (IL-35)-modified MSCs in ALI intervention have not been investigated. In the present study, adult male C57BL/6 mice randomly received intravenous infusion of adipose-derived mesenchymal stem cells (ADSCs) constitutively expressing IL-35 (IL-35-GFP-ADSCs) or GFP (GFP-ADSCs) via retrovirus-mediated transduction (8 × 105 cells per mice) or isotonic saline 7 days before ALI modeling to investigate the effect and related mechanism. ALI was performed by lipopolysaccharide (LPS) inhalation for 24 h. Normal mice served as the sham group. The results indicated that compared with GFP-ADSCs, IL-35-modified ADSCs significantly increased cellular and pulmonary IL-10 and IL-35 production. Pretreatment with IL-35-ADSCs markedly reduced body weight loss, pulmonary wet/dry weight ratio and pathological injury. The PO2 was rescued to normal levels in mice that received IL-35-ADSCs. IL-35-ADSCs infusion apparently inhibited IL-6 release, protein leakage and MPO activity but greatly elevated IL-35 level in the bronchoalveolar lavage fluid (BALF). Splenic regulatory T cells in IL-35-ADSCs-pretreated mice got effective increase. Moreover, IL-35-ADSCs pretreatment remarkably inhibited neutrophil and macrophage infiltration and greatly decreased IL-6, tumor necrosis factor α (TNF-α) and Toll-like receptor 4 (TLR4) expression. In conclusion, pretreatment with IL-35-engineered ADSCs provided effective protection against LPS-induced ALI through suppression of pulmonary inflammation and, thus, might be a promising strategy to improve outcomes after ALI. The enhanced paracrine and immunosuppressive capacity of IL-35-ADSCs might contribute to their beneficial effects. However, further studies are needed to illuminate the detailed mechanism.

A novel transgenic murine model with persistently brittle bones simulating osteogenesis imperfecta type I.

Osteogenesis imperfecta (OI) type I caused by the null allele of COL1A1 gene is in the majority in clinical OI cases. Currently, heterozygous Mov-13 mice generated by virus insertion in the first intron of col1a1 is the exclusive model to modulate OI type I, in spite of the gradually recovered bone mineral and mechanical properties. A newly designed heterozygous col1a1±365 OI mouse was produced in the present study by partial exons knockout (exon 2-exon 5, 365 nt of mRNA) using CRISPR/Cas9 system. The deletion resulted in generally large decrease in type I collagen synthesis due to frameshift mutation and premature chain termination, closely mimicking the pathogenic mechanism in affected individuals. And the strain possessed significantly sparse mineral scaffolds, bone loss, lowered mechanical strength and broken bone metabolism by 8 and 20 weeks compared to their littermates, suggesting a sustained skeletal weakness. Notably, the remarkable down-regulation of Yes-associated protein (YAP), one of the key coactivator in Hippo signaling pathway, was first found both in the femur and adipose derived mesenchymal stem cells (ADSCs) under osteogenic differentiation of col1a1±365 mice, which might be responsible for the reduced osteogenic potential and brittle bones. Still, further research was needed in order to illuminate the underlying mechanism.

iTRAQ-based quantitative proteomic analysis reveals potential early diagnostic markers in serum of acute cellular rejection after liver transplantation.

Liver transplantation (LT) is the most effective treatment method for advanced stage liver disease but acute cellular rejection (ACR) seriously affects the prognosis of LT. To discover novel diagnostic biomarkers of ACR after LT, Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-based mass spectrometry was performed to characterize alterations of serum proteins among patients validated to be pathologically ACR or pathologically no-ACR after LT and healthy controls. As a result, 10 differentially expressed proteins were found out between the ACR group and the No-ACR group; 88 differentially expressed proteins were found out between the ACR group and the Healthy Control group; 39 differentially expressed proteins were found out between No-ACR group and Healthy Control group. After analysis and ELISA validation, the results showed that CFHR1, CFHR5 and CFH could be candidate protein biomarkers for the early diagnosis of ACR after LT.

Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice.

OBJECTIVE: The benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice. METHODS: A plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR. RESULTS: Western blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results. CONCLUSION: The results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.

Long-term Persistent Organic Pollutants Exposure Induced Telomere Dysfunction and Senescence-Associated Secretary Phenotype.

Environmentally persistent organic pollutant (POP) is the general term for refractory organic compounds that show long-range atmospheric transport, environmental persistence, and bioaccumulation. It has been reported that the accumulation of POPs could lead to cellular DNA damage and adverse effects of on metabolic health. To better understand the mechanism of the health risks associated with POPs, we conducted an evidence-based cohort investigation (n = 5,955) at the Jinghai e-waste disposal center in China from 2009 to 2016, where people endure serious POP exposure. And high levels of aging-related diseases, including hypertension, diabetes, autoimmune diseases, and reproductive disorders were identified associated with the POP exposure. In the subsequent molecular level study, an increased telomere dysfunction including telomere multiple telomere signals, telomere signal-free ends, telomere shortening and activation of alternative lengthening of telomeres were observed, which might result from the hypomethylated DNA modification induced telomeric repeat-containing RNA overexpression. Moreover, dysfunctional telomere-leaded senescence-associated secretory phenotype was confirmed, as the proinflammatory cytokines and immunosenescence hallmarks including interleukin-6, P16INK4a, and P14ARF were stimulated. Thus, we proposed that the dysfunctional telomere and elevated systemic chronic inflammation contribute to the aging-associated diseases, which were highly developed among the POP exposure individuals.

The correlation between the expression of genes involved in drug metabolism and the blood level of tacrolimus in liver transplant receipts.

Immunosuppressive medications, such as tacrolimus and mycophenolate mofetil, are commonly used for reducing the risk of organ rejection in receipts of allogeneic organ transplant. The optimal dosages of these drugs are required for preventing rejection and avoiding toxicity to receipts. This study aimed to identify the correlation between the expression profiling of genes involved in drug metabolism and the blood level of tacrolimus in liver transplant receipts. Sixty-four liver transplant receipts were enrolled in this retrospective study. Receipts were divided into low (2-5.9 ng/ml) and high (6-15 ng/ml) tacrolimus groups. Clinical assessment showed that the blood level of tacrolimus was inversely correlated with the liver function evaluated by blood levels of total bilirubin and creatinine. Compared to the high tacrolimus group, expression levels of six cytochrome P450 enzymes, CYP1A1, CYP2B6, CYP3A5, CYP4A11, CYP19A1, and CYP17A1 were significantly higher in the low tacrolimus group. The expression levels of these genes were negatively correlated with the tacrolimus blood level. Enzyme assays showed that CYP3A5 and CYP17A1 exerted direct metabolic effects on tacrolimus and mycophenolate mofetil, respectively. These results support clinical application of this expression profiling of genes in drug metabolism for selection of immunosuppressive medications and optimal dosages for organ transplant receipts.

The Effect of Traditional Chinese Formula Danchaiheji on the Differentiation of Regulatory Dendritic Cells.

Recently, regulatory dendritic cells (DCregs), a newly described dendritic cell subset with potent immunomodulatory function, have attracted increased attention for their utility in treating immune response-related diseases, such as graft-versus-host disease, hypersensitivity, and autoimmune diseases. Danchaiheji (DCHJ) is a traditional Chinese formula that has been used for many years in the clinic. However, whether DCHJ can program dendritic cells towards a regulatory phenotype and the underlying mechanism behind this process remain unknown. Herein, we investigate the effects of traditional Chinese DCHJ on DCregs differentiation and a mouse model of skin transplantation. The current study demonstrates that DCHJ can induce dendritic cells to differentiate into DCregs, which are represented by high CD11b and low CD86 and HLA-DR expression as well as the secretion of IL-10 and TGF-ß. In addition, DCHJ inhibited DC migration and T cell proliferation, which correlated with increased IDO expression. Furthermore, DCHJ significantly prolonged skin graft survival time in a mouse model of skin transplantation without any liver or kidney toxicity. The traditional Chinese formula DCHJ has the potential to be a potent immunosuppressive agent with high efficiency and nontoxicity.

Generation of regulatory dendritic cells after treatment with paeoniflorin.

Regulatory dendritic cells are a potential therapeutic tool for assessing a variety of immune overreaction diseases. Paeoniflorin, a bioactive glucoside extracted from the Chinese herb white paeony root, has been shown to be effective at inhibiting the maturation and immunostimulatory function of murine bone marrow-derived dendritic cells. However, whether paeoniflorin can program conventional dendritic cells toward regulatory dendritic cells and the underlying mechanism remain unknown. Here, our study demonstrates that paeoniflorin can induce the production of regulatory dendritic cells from human peripheral blood monocyte-derived immature dendritic cells in the absence or presence of lipopolysaccharide (LPS) but not from mature dendritic cells, thereby demonstrating the potential of paeoniflorin as a specific immunosuppressive drug with fewer complications and side effects. These regulatory dendritic cells treated with paeoniflorin exhibited high CD11b/c and low CD80, CD86 and CD40 expression levels as well as enhanced abilities to capture antigen and promote the proliferation of CD4(+)CD25(+) T cells and reduced abilities to migrate and promote the proliferation of CD4(+) T cells, which is associated with the upregulation of endogenous transforming growth factor (TGF)-ß-mediated indoleamine 2,3-dioxygenase (IDO) expression. Collectively, paeoniflorin could program immature dendritic cells (imDCs) and imDCs stimulated with LPS toward a regulatory DC fate by upregulating the endogenous TGF-ß-mediated IDO expression level, thereby demonstrating its potential as a specific immunosuppressive drug.

Protein-DNA array-based identification of transcription factor activities differentially regulated in obliterative bronchiolitis.

Lung transplantation has already become the preferred treatment option for a variety of end-stage pulmonary failure. However the long-term results of lung transplantation are still not compelling and the major death reason is commonly due to obliterative bronchiolitis (OB) which is considered as chronic rejection presenting manifests physiologically as a progressive decline in FEV1. Transcription factors (TFs) play a key role in regulating gene expression and in providing an interconnecting regulatory between related pathway elements. Although the transcription factors are required for expression of the proinflammatory cytokines and immune proteins which are involved in obliterative bronchiolitis following lung transplantation, the alterations of the transcription factors in OB have not yet been revealed. Therefore, to investigate the alteration pattern of the transcription factors in OB, we used protein/DNA arrays. Mice orthotopic tracheal transplantation model was used in this studying. In this study, we explored the activity profiles of TFs in Protein/DNA array data of tracheal tissue in 14 and 28 day after transplanted. From a total of 345 screened TFs, we identified 42 TFs that showed associated with OB progression. Our data indicate that TFs may be potentially involved in the pathogenesis of OB, and can prevent, diagnose and treat OB after lung transplantation. In development of OB, some of the TFs may have ability to modulate the transcription of inflammatory proteins such cytokines, inflammatory enzymes and so on.

Effects of CYP3A5 genotypes, ABCB1 C3435T and G2677T/A polymorphism on pharmacokinetics of Tacrolimus in Chinese adult liver transplant patients.

1. The purpose of this study was to establish a population pharmacokinetic (PK) model of tacrolimus and evaluate the influence of clinical covariates, including the genetic polymorphisms of the cytochrome P450 3A5 gene (CYP3A5) and gene-encoding P-glycoprotein (ABCB1), on the PK parameters in Chinese adult liver transplant recipients. 2. Details of drug dose, sampling times and concentrations were collected retrospectively from routine therapeutic drug monitoring data early after liver transplant. Tacrolimus PKs was studied by a non-linear mixed-effect modeling (NONMEM) method. CYP3A5 genotypes, ABCB1 C3435T and G2677T/A polymorphism and a number of clinical covariates were tested for their influence on TAC PKs. 3. A one-compartment model with first-order absorption and elimination adequately described the data. Apparent clearance (CL/F) and apparent volumes of distribution (V/F) in final population model were 17.6 L/h and 225 L, respectively. The absorption rate constant (Ka) was fixed at 4.48 h(-1). The inter-individual variability in CL/F and V/F was 53.9 and 68%, respectively. In the final model, CYP3A5 genotype, post-operative day, alanine aminotransferase, total bilirubin, hematocrit and blood urea nitrogen were found to significantly influence the CL/F, whereas POD and HB influence V/F. 4. Population PK analysis of tacrolimus in Chinese adult liver transplant patients resulted in identification of the CYP3A5 genotype, POD, BUN, ALP, HCT, TBIL and HB as significant covariates on the PK parameters of tacrolimus.