Trafficking between clonally related peripheral T-helper cells and tissue-resident T-helper cells in chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome. CXCR5-PD-1hi peripheral T-helper (Tph) cells have an important pathogenic role in autoimmune diseases, but the role of Tph cells in cGVHD remains unknown. We show that in patients with cGVHD, expansion of Tph cells among blood CD4+ T cells was associated with cGVHD severity. These cells augmented memory B-cell differentiation and production of immunoglobulin G via interleukin 21 (IL-21). Tph cell expansion was also observed in a murine model of cGVHD. This Tph cell expansion in the blood is associated with the expansion of pathogenic tissue-resident T-helper (Trh) cells that form lymphoid aggregates surrounded by collagen in graft-versus-host disease (GVHD) target tissues. Adoptive transfer experiments showed that Trh cells from GVHD target tissues give rise to Tph cells in the blood, and conversely, Tph cells from the blood give rise to Trh cells in GVHD target tissues. Tph cells in the blood and Trh cells in GVHD target tissues had highly overlapping T-cell receptor α and ß repertoires. Deficiency of IL-21R, B-cell lymphoma 6 (BCL6), or T-bet in donor T cells markedly reduced the proportions of Tph cells in the blood and Trh cells in GVHD target tissues and reduced T-B interaction in the lymphoid aggregates. These results indicate that clonally related pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues, and that IL-21R-BCL6 signaling and T-bet are required for the development and expansion of Tph and Trh cells in the pathogenesis of cGVHD.

LncRNA-Smad7 mediates cross-talk between Nodal/TGF-ß and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells.

Transforming growth factor ß (TGF-ß) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-ß and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-ß and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

Changes and bioactivities on volatile organic compounds of endophytic fungi Neurospora dictyophora 3ZF-02 in different ages.

The endophytic fungus Neurospora dictyophora 3ZF-02 with a special aroma was isolated from the arils of Torreya grandis. Analysis of volatile organic compounds was done by gas chromatography-mass spectrometry. A total of 46 compounds were identified in the volatile organic compounds by 3ZF-02. In the growth phase, esters, acids, along with some alcohols and ketones were the main components. During the recession period, a large amount of benzene and naphthalene compounds appeared, accompanied by the production of amines. Oleic acid, methyl abietate, terpinen-4-ol were also found. They were the same ingredients in essential oil of Torreya. The antioxidant activity and antifungal activity of all extracts were also evaluated. When cultured for 10 days, it exhibited the most significant antioxidant activity with IC50 of 1.44 and 0.95 g/L against diphenyl picryl hydrazinyl and hydroxyl radicals, respectively. In addition, the extracts cultured for 10 days exhibited the most significant antifungal activity against Candida albicans with minimum inhibitory concentration 1.56 mg/ml.

γδT cells drive myeloid-derived suppressor cell-mediated CD8+ T cell exhaustion in hepatitis B virus-induced immunotolerance.

The mechanisms of liver hepatitis B virus (HBV)-induced systemic immune tolerance are still elusive, and the role of γδT cells has not yet been described. We examined the function of γδT cells in HBV-carrier mice--immunocompetent mice with plasmid-mediated persistent HBV expression in the liver. In this study, we found that γδT cell deficiency led to a break in HBV-induced tolerance and subsequent recovery of hepatic HBV-specific CD8(+) T cells. Of interest, IL-17(-/-) mice phenocopied TCRδ(-/-) mice in terms of losing HBV persistence, and adoptive transfer of γδT cells restored HBV-persistent expression in TCRδ(-/-) mice. We further observed that hepatic CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) play a major role in this mechanism, as they were significantly reduced in both HBV-carrier TCRδ(-/-) and IL-17(-/-) mice. MDSC numbers also recovered after adoptive transfer of γδT cells, particularly Vγ4(+) T cells. Furthermore, anti-Gr1-mediated MDSC depletion in HBV-carrier mice accelerated HBV elimination from the host, whereas MDSCs transferred to γδT cell-deficient mice restored HBV-induced tolerance. Accordingly, inhibition of MDSCs by the arginase-1 inhibitor norNOHA enhanced the number of HBV-specific CD8(+) T cells and promoted HBV clearance. We also observed enhanced CD8(+) T cell number with a notable decline of MDSCs in TCRδ(-/-) mice compared with wild-type mice during the recombinant adeno-associated virus/HBV1.3 virus infection. Importantly, HBV-carrier TCRδ(-/-) mice not only exhibited increased anti-HBV CD8(+) T cells but also markedly reduced MDSCs. Overall, the current study reveals that γδT cells play a previously unrecognized regulatory role in liver tolerance by mobilizing MDSC infiltration to the liver, leading to MDSC-mediated CD8(+) T cell exhaustion.

IL-12-based vaccination therapy reverses liver-induced systemic tolerance in a mouse model of hepatitis B virus carrier.

Liver-induced systemic immune tolerance that occurs during chronic hepadnavirus infection is the biggest obstacle for effective viral clearance. Immunotherapeutic reversal of this tolerance is a promising strategy in the clinic but remains to be explored. In this study, using a hepatitis B virus (HBV)-carrier mouse model, we report that IL-12-based vaccination therapy can efficiently reverse systemic tolerance toward HBV. HBV-carrier mice lost responsiveness to hepatitis B surface Ag (HBsAg) vaccination, and IL-12 alone could not reverse this liver-induced immune tolerance. However, after IL-12-based vaccination therapy, the majority of treated mice became HBsAg(-) in serum; hepatitis B core Ag was also undetectable in hepatocytes. HBV clearance was dependent on HBsAg vaccine-induced anti-HBV immunity. Further results showed that IL-12-based vaccination therapy strongly enhanced hepatic HBV-specific CD8(+) T cell responses, including proliferation and IFN-γ secretion. Systemic HBV-specific CD4(+) T cell responses were also restored in HBV-carrier mice, leading to the arousal of HBsAg-specific follicular Th-germinal center B cell responses and anti-hepatitis B surface Ag Ab production. Recovery of HBsAg-specific responses also correlated with both reduced CD4(+)Foxp3(+) regulatory T cell frequency and an enhanced capacity of effector T cells to overcome inhibition by regulatory T cells. In conclusion, IL-12-based vaccination therapy may reverse liver-induced immune tolerance toward HBV by restoring systemic HBV-specific CD4(+) T cell responses, eliciting robust hepatic HBV-specific CD8(+) T cell responses, and facilitating the generation of HBsAg-specific humoral immunity; thus, this therapy may become a viable approach to treating patients with chronic hepatitis B.

Lipocalin-2 induced LDHA expression promotes vascular remodelling in pulmonary hypertension.

Aerobic glycolysis is involved in the pathogenesis of pulmonary hypertension (PH). The mechanisms by which glycolysis is increased and how it contributes to pulmonary vascular remodelling are not yet fully understood. In this study, we demonstrated that elevated lipocalin-2 (LCN2) in PH significantly enhances aerobic glycolysis in human pulmonary artery smooth muscle cells (PASMCs) by up-regulating LDHA expression. Knockout of Lcn2 or having heterozygous LDHA deficiency in mice significantly inhibits the progression of hypoxic PH. Our study reveals that LCN2 stimulates LDHA expression by activating Akt-HIF-1α signalling pathway. Inhibition of Akt or HIF-1α reduces LDHA expression and proliferation of PASMCs. Both Akt and HIF-1α play critical roles in the development of PH and are suppressed in the pulmonary vessels of hypoxic PH mice lacking LCN2. These findings shed light on the LCN2-Akt-HIF1α-LDHA axis in aerobic glycolysis in PH.

Generation of two induced pluripotent stem cell lines from catecholaminergic polymorphic ventricular tachycardia patients carrying RYR2 mutations.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a congenital arrhythmic syndrome caused by the RYR2 gene encoded ryanodine receptor. Mutations on RYR2 are commonly associated with ventricular tachycardia after adrenergic stimulation, leading to lethal arrhythmias and sudden cardiac death. We generated two human induced pluripotent stem cell (iPSC) lines from CPVT affected patients carrying single missense heterozygote RYR2 mutations, c.1082 G > A and c.100 A > C. Pluripotency and differentiation capability into derivatives of three germ layers were evaluated along with karyotype stability in the report. The generated patient-specific iPSC lines provide a reliable tool to investigate the CPVT phenotype and understand underlaying mechanisms.

Generation of two induced pluripotent stem cell lines from spinal muscular atrophy type 1 patients carrying no functional copies of SMN1 gene.

Spinal muscular atrophy (SMA) is a severe neurodegenerative muscular disease caused by the homozygous loss of survival of motor neuron 1 (SMN1) genes. SMA patients exhibit marked skeletal muscle (SKM) loss, eventually leading to death. Here we generated two iPSC lines from two SMA type I patients with homozygous SMN1 mutations and validated the pluripotency and the ability to differentiate into three germ layers. The iPSC lines can be applied to generate skeletal muscles to model muscle atrophy of SMA that persists after treatment of motor neurons and will serve as a complementary platform for drug screening in vitro.

Generation of two induced pluripotent stem cell lines from Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that leads to death in early adulthood. Patients with DMD have null mutations leading to loss of functional dystrophin protein. Here we generated two DMD induced pluripotent stem cell (iPSC) lines, one with deletion of exon 51 and the other with a single nucleotide nonsense mutation (c.10171C > T). Both lines expressed high levels of pluripotency markers, had the capability of differentiating into derivatives of the three germ layers, and possessed normal karyotypes. These iPSC lines can serve as powerful tools to model DMD in vitro and as a platform for therapeutic development.

Donor T cell STAT3 deficiency enables tissue PD-L1-dependent prevention of graft-versus-host disease while preserving graft-versus-leukemia activity.

STAT3 deficiency (STAT3-/-) in donor T cells prevents graft-versus-host disease (GVHD), but the impact on graft-versus-leukemia (GVL) activity and mechanisms of GVHD prevention remains unclear. Here, using murine models of GVHD, we show that STAT3-/- donor T cells induced only mild reversible acute GVHD while preserving GVL effects against nonsusceptible acute lymphoblastic leukemia (ALL) cells in a donor T cell dose-dependent manner. GVHD prevention depended on programmed death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) signaling. In GVHD target tissues, STAT3 deficiency amplified PD-L1/PD-1 inhibition of glutathione (GSH)/Myc pathways that regulate metabolic reprogramming in activated T cells, with decreased glycolytic and mitochondrial ATP production and increased mitochondrial ROS production and dysfunction, leading to tissue-specific deletion of host-reactive T cells and prevention of GVHD. Mitochondrial STAT3 deficiency alone did not reduce GSH expression or prevent GVHD. In lymphoid tissues, the lack of host-tissue PD-L1 interaction with PD-1 reduced the inhibition of the GSH/Myc pathway despite reduced GSH production caused by STAT3 deficiency and allowed donor T cell functions that mediate GVL activity. Therefore, STAT3 deficiency in donor T cells augments PD-1 signaling-mediated inhibition of GSH/Myc pathways and augments dysfunction of T cells in GVHD target tissues while sparing T cells in lymphoid tissues, leading to prevention of GVHD while preserving GVL effects.

Investigation on bioactive metabolites produced by an endophytic fungus Trichoderma citrinoviride from the arils of Torreya grandis.

An endophytic fungus Trichoderma citrinoviride capable of producing active substances was isolated from the arils of Torreya grandis. Seven compounds were separated from the ethyl acetate extract of fermentation broth and mycelium by chromatography, respectively identified as trichomerol (1), bisorbicillinolide (2), sohirnone A (3), emodin (4), stigmasterol (5), ergosterol (6), daidzein (7). This study is the first to report of the isolation of the endophytic fungus T. citrinoviride from the arils of T. grandis with complete assignments of 1-7. Compound 1 and 2 exhibited significant antioxidant activity of diphenyl picryl hydrazinyl with IC50 38.92 and 3.91 µg/mL, respectively. Compound 1, 2, 4 and 7 significantly inhibited the growth of Staphylococcus aureus with MIC 0.78; 0.39; 0.20 and 0.20 mg/mL, respectively. Current study indicated the presence of endophytic fungus in the arils of Torreya grandis that could be responsible for the bioactive metabolite synthesis.

A Nodal enhanced micropeptide NEMEP regulates glucose uptake during mesendoderm differentiation of embryonic stem cells.

TGF-ß family proteins including Nodal are known as central regulators of early development in metazoans, yet our understanding of the scope of Nodal signaling's downstream targets and associated physiological mechanisms in specifying developmentally appropriate cell fates is far from complete. Here, we identified a highly conserved, transmembrane micropeptide-NEMEP-as a direct target of Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells (mESCs), and this micropeptide is essential for mesendoderm differentiation. We showed that NEMEP interacts with the glucose transporters GLUT1/GLUT3 and promotes glucose uptake likely through these interactions. Thus, beyond expanding the scope of known Nodal signaling targets in early development and showing that this target micropeptide augments the glucose uptake during mesendoderm differentiation, our study provides a clear example for the direct functional impact of altered glucose metabolism on cell fate determination.

An enzyme linked aptamer photoelectrochemical biosensor for Tau-381 protein using AuNPs/MoSe2 as sensing material.

Alzheimer's disease is a worldwide health problem and it has attracted extensive attention. Tau protein is an important biomarker in the pathogenesis of Alzheimer's disease. Herein, we devise a in situ enzyme catalysis generating electron donor photoelectrochemical (PEC) biosensor for Tau-381 protein. Tau-381 protein aptamer is immobilized onto the surface of AuNPs/MoSe2 nanosheets modified electrode. In the presence of Tau-381 protein, an aptamer-protein duplex is formed. Meanwhile, the Tau-381 antibody and the protein G/AP (protein G labeled with alkaline phosphatase) are captured with the affinity interaction between Tau-381 protein and Tau-381 antibody, Tau-381 antibody and protein G/AP. The electron donor, ascorbic acid, is in situ produced by the catalyzing of ascorbic acid 2-phosphate in the PEC detection solution. As a result, low blank noise and strong photocurrent response are engendered. The photocurrent response is related to the concentration of Tau-381 protein. The detection range of Tau-381 protein is from 0.5 fM to 1.0 nM with detection limit of 0.3 fM. This in situ generating electron donor PEC biosensor can detect various targets by simply alternating antibody, antigen, or aptamer commercially. Thus, this work represents a simple and general sensing protocol.

Murine Models Provide New Insights Into Pathogenesis of Chronic Graft-Versus-Host Disease in Humans.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for hematologic malignancies, but its success is complicated by graft-versus-host disease (GVHD). GVHD can be divided into acute and chronic types. Acute GVHD represents an acute alloimmune inflammatory response initiated by donor T cells that recognize recipient alloantigens. Chronic GVHD has a more complex pathophysiology involving donor-derived T cells that recognize recipient-specific antigens, donor-specific antigens, and antigens shared by the recipient and donor. Antibodies produced by donor B cells contribute to the pathogenesis of chronic GVHD but not acute GVHD. Acute GVHD can often be effectively controlled by treatment with corticosteroids or other immunosuppressant for a period of weeks, but successful control of chronic GVHD requires much longer treatment. Therefore, chronic GVHD remains the major cause of long-term morbidity and mortality after allo-HCT. Murine models of allo-HCT have made great contributions to our understanding pathogenesis of acute and chronic GVHD. In this review, we summarize new mechanistic findings from murine models of chronic GVHD, and we discuss the relevance of these insights to chronic GVHD pathogenesis in humans and their potential impact on clinical prevention and treatment.

Tissue-resident PSGL1loCD4+ T cells promote B cell differentiation and chronic graft-versus-host disease-associated autoimmunity.

CD4+ T cell interactions with B cells play a critical role in the pathogenesis of systemic autoimmune diseases such as systemic lupus and chronic graft-versus-host disease (cGVHD). Extrafollicular CD44hiCD62LloPSGL1loCD4+ T cells (PSGL1loCD4+ T cells) are associated with the pathogenesis of lupus and cGVHD, but their causal role has not been established. With murine and humanized MHC-/-HLA-A2+DR4+ murine models of cGVHD, we showed that murine and human PSGL1loCD4+ T cells from GVHD target tissues have features of B cell helpers with upregulated expression of programmed cell death protein 1 (PD1) and inducible T cell costimulator (ICOS) and production of IL-21. They reside in nonlymphoid tissues without circulating in the blood and have features of tissue-resident memory T cells with upregulated expression of CD69. Murine PSGL1loCD4+ T cells from GVHD target tissues augmented B cell differentiation into plasma cells and production of autoantibodies via their PD1 interaction with PD-L2 on B cells. Human PSGL1loCD4+ T cells were apposed with memory B cells in the liver tissues of humanized mice and cGVHD patients. Human PSGL1loCD4+ T cells from humanized GVHD target tissues also augmented autologous memory B cell differentiation into plasma cells and antibody production in a PD1/PD-L2-dependent manner. Further preclinical studies targeting tissue-resident T cells to treat antibody-mediated features of autoimmune diseases are warranted.

Dendritic carbon architectures formed by nanotube core-directed diffusion-limited aggregation of nanoparticles.

A regular array of fractal patterns with macroscopic dendritic carbon architecture was prepared by catalytic chemical vapor deposition (CVD). The dendritic carbon architectures have micrometre-sized stems and hyperbranches evolved by lateral growth, and they are formed by diffusion-limited aggregation of carbon-encapsulated iron nanoparticle building blocks generated from catalytic pyrolysis of toluene, which is directed by carbon nanotube cores, and followed by subsequent restructuring from surface to bulk. Incorporation of such proposed processes in Monte Carlo simulations generates dendritic architectures similar to the morphologies observed from the experiments. The findings provide direct information to the time resolved evolution of the morphology and microstructure of the dendritic carbon architecture, which mimic the nature behavior as snowflake attaching on the tree branches. Those will be important to understand the growth of vapor grown carbon fibers and carbon filamentous structures, and further possibility to control branching out of vapor grown carbon fibers.

Proteomic profiling of HIV-1 infection of human CD4+ T cells identifies PSGL-1 as an HIV restriction factor.

Human immunodeficiency virus (HIV) actively modulates the protein stability of host cells to optimize viral replication. To systematically examine this modulation in HIV infection, we used isobaric tag-based mass spectrometry to quantify changes in the abundance of over 14,000 proteins during HIV-1 infection of human primary CD4+ T cells. We identified P-selectin glycoprotein ligand 1 (PSGL-1) as an HIV-1 restriction factor downregulated by HIV-1 Vpu, which binds to PSGL-1 and induces its ubiquitination and degradation through the ubiquitin ligase SCFß-TrCP2. PSGL-1 is induced by interferon-γ in activated CD4+ T cells to inhibit HIV-1 reverse transcription and potently block viral infectivity by incorporating in progeny virions. This infectivity block is antagonized by Vpu via PSGL-1 degradation. We further show that PSGL-1 knockdown can significantly abolish the anti-HIV activity of interferon-γ in primary CD4+ T cells. Our study identifies an HIV restriction factor and a key mediator of interferon-γ's anti-HIV activity.

WU and KI polyomavirus present in the respiratory tract of children, but not in immunocompetent adults.

BACKGROUND: Recently, two new polyomaviruses (PyV), termed WUPyV and KIPyV, were identified in respiratory tract specimens from children with acute respiratory tract infections (ARTIs). However, their roles in the disease have not been determined. OBJECTIVES: To determine the prevalence of WUPyV and KIPyV in the Chinese population suffering from ARTIs in Beijing, China, and to examine their possible role in causing disease. STUDY DESIGN: Nasopharyngeal aspirates, nasal swabs and throat swabs were collected from 415 children and 297 immunocompetent adults with lower ARTIs (LARTIs). The specimens were screened by polymerase chain reaction for the presence of WUPyV, KIPyV, and other common respiratory pathogens. RESULTS: Although none of the adults sampled were positive for either virus, WUPyV in 10 (2.4%) children and KIPyV was detected in 2 (0.5%) of the children sampled, respectively. Eleven of the positive cases were co-detected with either rhinovirus (6/11), respiratory syncytial virus (4/11), parainfluenzavirus virus (3/11) or Mycoplasma pneumoniae (2/11). Phylogenetic analysis of the WUPyV and KIPyV isolates showed that the nucleotide sequences were homologous to those of previously reported strains. CONCLUSIONS: The presence of WUPyV and KIPyV in samples from children but not from immunocompetent adults suffering from LARTIs suggests that these viruses primarily infect the young population. Co-detection of additional respiratory pathogens in most of the specimens containing either WUPyV or KIPyV suggests that these viruses do not cause disease independently.

Flow cytometric analyses of the viability, surface marker expression and function of lymphocytes from children following cryopreservation.

Flow cytometric analysis is important for the investigation and clinical preparation of lymphocytes from children. However, the strict requirement of cell freshness and inter­assay variability limits the application of this methodology for pediatric investigations. Therefore, it is necessary to identify a reliable cryopreservative method capable of maintaining high cell viability and proper cell function in lymphocytes from children. In the present study, eight commonly­used cell cyropreservative methods were used, and their effects on cell viability, surface marker expression and cell function were examined. In addition, how these methods affect the distribution of T­cell receptor Vß subfamilies were also determined. The results of the present study provided valuable experimental evidence, based on which the optimal method for the cryopreservation of lymphocytes from children in pediatric investigations and clinical applications can be selected.

Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility.

Skin integrity is essential for protection from external stress and trauma. Defects in structural proteins such as keratins cause skin fragility, epitomized by epidermolysis bullosa (EB), a life-threatening disorder. Here we show that dominant mutations of KLHL24, encoding a cullin 3-RBX1 ubiquitin ligase substrate receptor, cause EB. We have identified start-codon mutations in the KLHL24 gene in five patients with EB. These mutations lead to truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-ΔN28). KLHL24-ΔN28 is more stable than its wild-type counterpart owing to abolished autoubiquitination. We have further identified keratin 14 (KRT14) as a KLHL24 substrate and found that KLHL24-ΔN28 induces excessive ubiquitination and degradation of KRT14. Using a knock-in mouse model, we have confirmed that the Klhl24 mutations lead to stabilized Klhl24-ΔN28 and cause Krt14 degradation. Our findings identify a new disease-causing mechanism due to dysregulation of autoubiquitination and open new avenues for the treatment of related disorders.