Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1.

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells in vivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRß-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRß-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRß-sufficient Tfh cells. Loss of LXRß confers inactivation of GSK3ß induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/ß-catenin pathway, leading to elevated TCF-1 expression in CD4+ T cells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ T cells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3ß-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.

The Role of Carbamoyl Phosphate Synthetase 1 as a Prognostic Biomarker in Patients With Acetaminophen-induced Acute Liver Failure.

BACKGROUND & AIMS: Carbamoyl phosphate synthetase 1 (CPS1) is a highly abundant mitochondrial urea cycle enzyme that is expressed primarily in hepatocytes. CPS1 is constitutively and physiologically secreted into bile but is released into the bloodstream upon acute liver injury (ALI). Given its abundance and known short half-life, we tested the hypothesis that it may serve as a prognostic serum biomarker in the setting of acute liver failure (ALF). METHODS: CPS1 levels were determined using enzyme-linked immunosorbent assay and immunoblotting of sera collected by the ALF Study Group (ALFSG) from patients with ALI and ALF (103 patients with acetaminophen and 167 non-acetaminophen ALF etiologies). A total of 764 serum samples were examined. The inclusion of CPS1 was compared with the original ALFSG Prognostic Index by area under the receiver operating characteristic curve analysis. RESULTS: CPS1 values for acetaminophen-related patients were significantly higher than for non-acetaminophen patients (P < .0001). Acetaminophen-related patients who received a liver transplant or died within 21 days of hospitalization exhibited higher CPS1 levels than patients who spontaneously survived (P = .01). Logistic regression and area under the receiver operating characteristic analysis of CPS1 enzyme-linked immunosorbent assay values improved the accuracy of the ALFSG Prognostic Index, which performed better than the Model for End-Stage Liver Disease, in predicting 21-day transplant-free survival for acetaminophen- but not non-acetaminophen-related ALF. An increase of CPS1 but not alanine transaminase or aspartate transaminase, when comparing day 3 with day 1 levels was found in a higher percentage of acetaminophen transplanted/dead patients (P < .05). CONCLUSION: Serum CPS1 determination provides a new potential prognostic biomarker to assess patients with acetaminophen-induced ALF.

AAV-mediated skeletal muscle specific irisin expression does not contribute to weight loss in mice.

Obesity, a chronic disease, significantly increases the risk of various diseases, including diabetes, cardiovascular diseases, and cancers. Exercise is crucial for weight management not only through energy expenditure by muscle activity but also through stimulating the secretion of myokines, which affect various tissues. Irisin, derived from the proteolytic processing of fibronectin type III domain-containing protein 5 (Fndc5), is a well-studied myokine with beneficial effects on metabolism. This study explored the feasibility of adeno-associated virus (AAV)-mediated Fndc5 gene therapy to treat obesity in a mouse model using the AAV-DIO system to express Fndc5 specifically in skeletal muscle, and investigated its anti-obesity effect. Although Fndc5 was specifically expressed in the muscle, no significant impact on body weight under normal chow or high-fat diets was observed, and no change in thermogenic gene expression in inguinal white adipose tissue was detected. Notably, Fndc5 transduction did affect bone metabolism, consistent with previous reports. These findings suggest that AAV-mediated Fndc5 gene therapy may not be an efficient strategy for obesity, contrary to our expectations. Further research is needed to elucidate the complex mechanisms involved in irisin's role in obesity and related disorders.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

Timing of hormone therapy and its association with cardiovascular risk and metabolic parameters in 4-vinylcyclohexene diepoxide-induced primary ovarian insufficiency mouse model.

OBJECTIVE: To evaluate the effects of various initiation time points and durations of hormone therapy (HT) on cardiovascular and metabolic parameters of premenarche, primary ovarian insufficiency (POI) mouse model, induced by 4-vinylcyclohexene diepoxide. METHODS: A total of 50 mice at 4 weeks of age were developed into POI mouse model, further randomly categorized into 5 groups: control group without any intervention; no HT group with only high-fat diet (NT); group 1 with delayed estradiol treatment (T1); group 2 with on-time, continuous estradiol treatment (T2); and group 3 with on-time estradiol treatment but early stop (T3). Cardiovascular risk and metabolic parameters were measured. RESULTS: Presenting with similar body weights, blood glucose levels of T1, T2, and T3 were all significantly lower than NT (p < .001). Serum total cholesterol and insulin were also significantly lower in all HT groups than in NT, especially in T2 (p < .001). For serum low-density lipoprotein-cholesterol, only T2 resulted in the statically lower level than those of NT, T1, and T3 (p < .001). Aortic thickness was significantly increased with aggravated fibrotic change of the intima in NT, and such consequence was significantly ameliorated in HT groups, mostly lowered in T2 (p < .05). Last, serum pro-inflammatory cytokines were significantly low in the HT groups than in NT, especially in T2 with the lowest level (p < .05). . CONCLUSIONS: On-time, continuous E2 treatment immediately after a biologic estrogen deprivation event significantly reduced metabolic and cardiovascular risks in young, pre-menarche female mouse models of POI, confirming decreased serum levels of pro-inflammatory cytokines.

Cold-induced adaptive thermogenesis is impaired by exposure of Asian sand dust in mice.

Desertification and desert sandstorms caused by the worsening global warming pose increasing risks to human health. In particular, Asian sand dust (ASD) exposure has been related to an increase in mortality and hospital admissions for respiratory diseases. In this study, we investigated the effects of ASD on metabolic tissues in comparison to diesel particulate matter (DPM) that is known to cause adverse health effects. We found that larger lipid droplets were accumulated in the brown adipose tissues (BAT) of ASD-administered but not DPM-administered mice. Thermogenic gene expression was decreased in these mice as well. When ASD-administered mice were exposed to the cold, they failed to maintain their body temperature, suggesting that the ASD administration had led to impairments in cold-induced adaptive thermogenesis. However, impaired thermogenesis was not observed in DPM-administered mice. Furthermore, mice fed a high-fat diet that were chronically administered ASD demonstrated unexplained weight loss, indicating that chronic administration of ASD could be lethal in obese mice. We further identified that ASD-induced lung inflammation was not exacerbated in uncoupling protein 1 knockout mice, whose thermogenic capacity is impaired. Collectively, ASD exposure can impair cold-induced adaptive thermogenic responses in mice and increase the risk of mortality in obese mice.

FK506 and Lactobacillus acidophilus ameliorate acute graft-versus-host disease by modulating the T helper 17/regulatory T-cell balance.

BACKGROUND: Graft-versus-host disease (GvHD) is a critical complication after allogeneic hematopoietic stem cell transplantation (HSCT). The immunosuppressants given to patients undergoing allogeneic HSCT disturb the microbiome and the host immune system, potentially leading to dysbiosis and inflammation, and may affect immune function and bone marrow transplantation. The intestinal microbiome is a target for the development of novel therapies for GvHD. Lactobacillus species are widely used supplements to induce production of antimicrobial and anti-inflammatory factors. METHODS: We determined the effect of the combination of Lactobacillus acidophilus and FK506 on GvHD following major histocompatibility complex-mismatched bone marrow transplantation. RESULTS: The combination treatment suppressed IFN-γ and IL-17-producing T cell differentiation, but increased Foxp3+Treg differentiation and IL-10 production. Also, the combination treatment and combination treated-induced Treg cells modulated the proliferation of murine alloreactive T cells in vitro. Additionally, the combination treatment upregulated Treg-related genes-Nt5e, Foxp3, Ikzf2, Nrp1 and Itgb8-in murine CD4+-T cells. The combination treatment also alleviated GvHD clinically and histopathologically by controlling the effector T cell and Treg balance in vivo. Moreover, the combination treatment decreased Th17 differentiation significantly and significantly upregulated Foxp3 and IL-10 expression in peripheral blood mononuclear cells from healthy controls and liver transplantation (LT) patients. CONCLUSIONS: Therefore, the combination of L. acidophilus and FK506 is effective and safe for patients undergoing allogeneic hematopoietic stem cell transplantation.

Constitutive release of CPS1 in bile and its role as a protective cytokine during acute liver injury.

Carbamoyl phosphate synthetase-1 (CPS1) is the major mitochondrial urea cycle enzyme in hepatocytes. It is released into mouse and human blood during acute liver injury, where is has a short half-life. The function of CPS1 in blood and the reason for its short half-life in serum are unknown. We show that CPS1 is released normally into mouse and human bile, and pathologically into blood during acute liver injury. Other cytoplasmic and mitochondrial urea cycle enzymes are also found in normal mouse bile. Serum, bile, and purified CPS1 manifest sedimentation properties that overlap with extracellular vesicles, due to the propensity of CPS1 to aggregate despite being released primarily as a soluble protein. During liver injury, CPS1 in blood is rapidly sequestered by monocytes, leading to monocyte M2-polarization and homing to the liver independent of its enzyme activity. Recombinant CPS1 (rCPS1), but not control r-transferrin, increases hepatic macrophage numbers and phagocytic activity. Notably, rCPS1 does not activate hepatic macrophages directly; rather, it activates bone marrow and circulating monocytes that then home to the liver. rCPS1 administration prevents mouse liver damage induced by Fas ligand or acetaminophen, but this protection is absent in macrophage-deficient mice. Moreover, rCPS1 protects from acetaminophen-induced liver injury even when given therapeutically after injury induction. In summary, CPS1 is normally found in bile but is released by hepatocytes into blood upon liver damage. We demonstrate a nonenzymatic function of CPS1 as an antiinflammatory protective cytokine during acute liver injury.

Melatonin Regulates Iron Homeostasis by Inducing Hepcidin Expression in Hepatocytes.

The pineal hormone, melatonin, plays important roles in circadian rhythms and energy metabolism. The hepatic peptide hormone, hepcidin, regulates iron homeostasis by triggering the degradation of ferroportin (FPN), the protein that transfers cellular iron to the blood. However, the role of melatonin in the transcriptional regulation of hepcidin is largely unknown. Here, we showed that melatonin upregulates hepcidin gene expression by enhancing the melatonin receptor 1 (MT1)-mediated c-Jun N-terminal kinase (JNK) activation in hepatocytes. Interestingly, hepcidin gene expression was increased during the dark cycle in the liver of mice, whereas serum iron levels decreased following hepcidin expression. In addition, melatonin significantly induced hepcidin gene expression and secretion, as well as the subsequent FPN degradation in hepatocytes, which resulted in cellular iron accumulation. Melatonin-induced hepcidin expression was significantly decreased by the melatonin receptor antagonist, luzindole, and by the knockdown of MT1. Moreover, melatonin activated JNK signaling and upregulated hepcidin expression, both of which were significantly decreased by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation analysis showed that luzindole significantly blocked melatonin-induced c-Jun binding to the hepcidin promoter. Finally, melatonin induced hepcidin expression and secretion by activating the JNK-c-Jun pathway in mice, which were reversed by the luzindole treatment. These findings reveal a previously unrecognized role of melatonin in the circadian regulation of hepcidin expression and iron homeostasis.

Short-chain fatty acid butyrate induces IL-10-producing B cells by regulating circadian-clock-related genes to ameliorate Sjögren's syndrome.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune disease caused by inflammation of the exocrine gland. The pathological hallmark of SS is the infiltration of lymphocytes into the salivary glands. Increased infiltration of T and B cells into salivary glands exacerbates symptoms of SS. Several recent studies have identified the role of gut microbiota in SS. Butyrate, one of the metabolites of the gut microbiota, regulates T cells; however, its effects on B cells and SS remain unknown. This study determined the therapeutic effect of butyrate on regulating B cells in SS. METHODS: Various concentrations of butyrate were intraperitoneally injected three times per week in NOD/ShiLtJ (NOD) mice, the prototype animal model for SS, and observed for more than 10 weeks. Whole salivary flow rate and the histopathology of salivary glands were investigated. Human submandibular gland (HSG) cells and B cells in mouse spleen were used to confirm the anti-inflammatory and immunomodulatory effects of butyrate. RESULTS: Butyrate increased salivary flow rate in NOD mice and reduced inflammation of salivary gland tissues. It also regulated cell death and the expression of circadian-clock-related genes in HSG cells. Butyrate induced B cell regulation by increasing IL-10-producing B (B10) cells and decreasing IL-17-producing B cells, through the circadian clock genes RAR-related orphan receptor alpha and nuclear receptor subfamily 1 group D member 1. CONCLUSION: The findings of this study imply that butyrate may ameliorate SS via reciprocal regulation of IL-10- and IL-17-producing B cells.

Effects of coenzyme Q10 on ovarian surface epithelium-derived ovarian stem cells and ovarian function in a 4-vinylcyclohexene diepoxide-induced murine model of ovarian failure.

BACKGROUND: Several studies have shown that coenzyme Q10 (CoQ10) can rescue ovarian aging and that ovarian surface epithelium (OSE)-derived ovarian stem cells (OSCs) are useful for treating infertility due to ovarian aging. However, few studies have examined the effect of CoQ10 on OSCs. This study was aimed to investigate whether CoQ10 activates OSCs and recovers ovarian function in a 4-vinylcyclohexene diepoxide (VCD)-induced mouse model of ovarian failure. METHODS: Forty female C57BL/6 mice aged 6 weeks were randomly divided into four groups (n = 10/group): a control group administered saline orally, a CoQ10 group administered 150 mg/kg/day of CoQ10 orally in 1 mL of saline daily for 14 days, a VCD group administered 160 mg/kg/day of VCD i.p. in 2.5 mL of saline/kg for 5 days, and a VCD + CoQ10 group administered VCD i.p. for 5 days injection and CoQ10 (150 mg/kg/day) orally for 14 days. After treatment, follicle counts were evaluated by hematoxylin and eosin (H&E) staining, and ovarian mRNA expressions of Bmp-15, Gdf-9, and c-Kit were examined by quantitative real-time PCR. Serum FSH, AMH, and ROS levels were also measured. Oocyte-like structure counts and the expressions of Oct-4 and MVH were also evaluated after culturing OSE for 3 weeks. In a second experiment, 32 female mice were administered CoQ10 as described above, induced to superovulate using PMSG and hCG, and mated. Numbers of zygotes and embryo development rate were examined. RESULTS: Postcultured OSE showed significant increases in the numbers of oocyte-like structure and that the expression of Oct-4 and MVH were higher in the VCD + CoQ10 group than in the VCD group (p < 0.05). Numbers of surviving follicles from primordial to antral follicles, numbers of zygotes retrieved and embryo development rate to blastocyst were significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.01). Serum AMH level and ovarian expressions of Bmp-15, Gdf-9 and c-Kit were also significantly greater in the VCD + CoQ10 group than in the VCD group (p < 0.05). In contrast, serum ROS level was significantly lower in the VCD + CoQ10 group than in the VCD group (p < 0.05). CONCLUSION: This study shows that CoQ10 stimulates the differentiation of OSE-derived OSCs and confirms that CoQ10 can reduce ROS levels and improve ovarian function and oocyte quality in mice with VCD-induced ovarian failure.

Metformin Augments Anti-Inflammatory and Chondroprotective Properties of Mesenchymal Stem Cells in Experimental Osteoarthritis.

Mesenchymal stem cells (MSCs) can protect against cartilage breakdown in osteoarthritis (OA) via their immunomodulatory capacities. However, the optimization strategy for using MSCs remains challenging. This study's objective was to identify the in vivo effects of metformin-stimulated adipose tissue-derived human MSCs (Ad-hMSCs) in OA. An animal model of OA was established by intra-articular injection of monosodium iodoacetate into rats. OA rats were divided into a control group and two therapy groups (treated with Ad-hMSCs or metformin-stimulated Ad-hMSCs). Limb nociception was assessed by measuring the paw withdrawal latency and threshold. Our data show that metformin increased IL-10 and IDO expression in Ad-hMSCs and decreased high-mobility group box 1 protein, IL-1ß, and IL-6 expression. Metformin increased the migration capacity of Ad-hMSCs with upregulation of chemokine expression. In cocultures, metformin-stimulated Ad-hMSCs inhibited the mRNA expression of RUNX2, COL X, VEGF, MMP1, MMP3, and MMP13 in IL-1ß-stimulated OA chondrocytes and increased the expression of TIMP1 and TIMP3. The antinociceptive activity and chondroprotective effects were greater in OA rats treated with metformin-stimulated Ad-hMSCs than in those treated with unstimulated Ad-hMSCs. TGF-ß expression in subchondral bone of OA joints was attenuated more in OA rats treated with metformin-stimulated Ad-hMSCs. Our findings suggest that metformin offers a promising option for the clinical application of Ad-hMSCs as a cell therapy for OA.

Junctional Modulation of Round Window Membrane Enhances Dexamethasone Uptake into the Inner Ear and Recovery after NIHL.

Delivery of substances into the inner ear via local routes is increasingly being used in clinical treatment. Studies have focused on methods to increase permeability through the round window membrane (RWM) and enhance drug diffusion into the inner ear. However, the clinical applications of those methods have been unclear and few studies have investigated the efficacy of methods in an inner ear injury model. Here, we employed the medium chain fatty acid caprate, a biologically safe, clinically applicable substance, to modulate tight junctions of the RWM. Intratympanic treatment of sodium caprate (SC) induced transient, but wider, gaps in intercellular spaces of the RWM epithelial layer and enhanced the perilymph and cochlear concentrations/uptake of dexamethasone. Importantly, dexamethasone co-administered with SC led to significantly more rapid recovery from noise-induced hearing loss at 4 and 8 kHz, compared with the dexamethasone-only group. Taken together, our data indicate that junctional modulation of the RWM by SC enhances dexamethasone uptake into the inner ear, thereby hastening the recovery of hearing sensitivity after noise trauma.

Hearing Impairment in a Mouse Model of Diabetes Is Associated with Mitochondrial Dysfunction, Synaptopathy, and Activation of the Intrinsic Apoptosis Pathway.

Although previous studies continuously report an increased risk of hearing loss in diabetes patients, the impact of the disease on the inner ear remains unexplored. Herein, we examine the pathophysiology of diabetes-associated hearing impairment and cochlear synaptopathy in a mouse model of diabetes. Male B6.BKS(D)-Leprdb/J (db/db, diabetes) and heterozygote (db/+, control) mice were assigned into each experimental group (control vs. diabetes) based on the genotype and tested for hearing sensitivity every week from 6 weeks of age. Each cochlea was collected for histological and biological assays at 14 weeks of age. The diabetic mice exerted impaired hearing and a reduction in cochlear blood flow and C-terminal-binding protein 2 (CtBP2, a presynaptic ribbon marker) expression. Ultrastructural images revealed severely damaged mitochondria from diabetic cochlea accompanied by a reduction in Cytochrome c oxidase subunit 4 (COX4) and CR6-interacting factor 1 (CRIF1). The diabetic mice presented significantly decreased levels of platelet endothelial cell adhesion molecule (PECAM-1), B-cell lymphoma 2 (BCL-2), and procaspase-9, but not procaspase-8. Importantly, significant changes were not found in necroptotic programmed cell death markers (receptor-interacting serine/threonine-protein kinase 1, RIPK1; RIPK3; and mixed lineage kinase domain-like pseudokinase, MLKL) between the groups. Taken together, diabetic hearing loss is accompanied by synaptopathy, microangiopathy, damage to the mitochondrial structure/function, and activation of the intrinsic apoptosis pathway. Our results imply that mitochondrial dysfunction is deeply involved in diabetic hearing loss, and further suggests the potential benefits of therapeutic strategies targeting mitochondria.

Protein arginine methyltransferase-1 stimulates dopaminergic neuronal cell death in a Parkinson's disease model.

Recent studies have revealed that protein arginine methyltransferases (PRMTs) are responsible for diverse neurodegenerative diseases. However, their pathophysiological role in dopaminergic neuronal death in Parkinson's disease (PD) has not been evaluated. In this study, we demonstrated that 1-Methyl-4-phenylpyridinium iodide (MPP+), rotenone and paraquat, which cause dopaminergic neuronal cell death, increased PRMT1 expression in dopaminergic cell line. Dopaminergic neuronal cell death was increased by PRMT1 overexpression. MPP+-induced cell death was attenuated by PRMT1 knockdown. Poly (ADP-ribose) polymerase-1 (PARP1) expression and activity, poly-ADP-ribosylation (PARylation), were elevated by MPP+. Moreover, we found that PRMT1 positively regulates nuclear translocation of apoptosis-inducing factor (AIF). Elevated PRMT1 expression was observed in the substantia nigra pars compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected mice. Furthermore, MPTP-induced dopaminergic neuronal death was reduced in PRMT1 haploinsufficient (prmt1+/-) mice. These data suggest that PRMT1 is implicated in PARP1/AIF-mediated dopaminergic neuronal cell death, which might be involved in the pathology of PD. Therefore, our results propose PRMT1 as a new target to develop a potential treatment of PD.

Myeloid-derived suppressor cells therapy enhance immunoregulatory properties in acute graft versus host disease with combination of regulatory T cells.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) play a critical role in modulating the immune response and promoting immune tolerance in models of autoimmunity and transplantation. Regulatory T cells (Tregs) exert therapeutic potential due to their immunomodulatory properties, which have been demonstrated both in vitro and in clinical trials. Cell-based therapy for acute graft-versus-host disease (aGVHD) may enable induction of donor-specific tolerance in the preclinical setting. METHODS: We investigated whether the immunoregulatory activity of the combination of MDSCs and Tregs on T cell and B cell subset and alloreactive T cell response. We evaluated the therapeutic effects of combined cell therapy for a murine aGVHD model following MHC-mismatched bone marrow transplantation. We compared histologic analysis from the target tissues of each groups were and immune cell population by flow cytometric analysis. RESULTS: We report a novel approach to inducing immune tolerance using a combination of donor-derived MDSCs and Tregs. The combined cell-therapy modulated in vitro the proliferation of alloreactive T cells and the Treg/Th17 balance in mice and human system. Systemic infusion of MDSCs and Tregs ameliorated serverity and inflammation of aGVHD mouse model by reducing the populations of proinflammatory Th1/Th17 cells and the expression of proinflammatory cytokines in target tissue. The combined therapy promoted the differentiation of allogeneic T cells toward Foxp3 + Tregs and IL-10-producing regulatory B cells. The combination treatment control also activated human T and B cell subset. CONCLUSIONS: Therefore, the combination of MDSCs and Tregs has immunomodulatory activity and induces immune tolerance to prevent of aGVHD severity. This could lead to the development of new clinical approaches to the prevent aGVHD.

FTY720 ameliorates GvHD by blocking T lymphocyte migration to target organs and by skin fibrosis inhibition.

BACKGROUND: Fibrosis is the formation of excess connective tissue in an organ or tissue during a reparative or reactive process. Graft-versus-host disease (GvHD) is a medical complication of allogeneic tissue transplantation with transplanted donor T cell-mediated inflammatory response; it is characterized by a severe immune response with fibrosis in the final stage of the inflammatory process. T helper 17 cells play a critical role in the pathogenesis of GvHD. Fingolimod (FTY720), an analogue of sphingosine-1-phosphate (S1P), is an effective immunosuppressive agent in experimental transplantation models. METHODS: In this study, we evaluated the effects of FTY720 as a treatment for an animal GvHD model with inflammation and fibrosis. The splenocytes, lymph nodes, blood, tissues from Syngeneic mice and GvHD-induced mice treated vehicle or FTY720 were compared using flow cytometry, hematological analyses, histologic analyses. RESULTS: FTY720 reduced clinical scores based on the following five clinical parameters: weight loss, posture, activity, fur texture, and skin integrity. FACS data showed that T lymphocyte numbers increased in mesenteric lymph nodes and decreased in splenocytes of FTY720-treated mice. Tissue analysis showed that FTY720 reduced skin, intestinal inflammation, and fibrotic markers. FTY720 dramatically decreased α-smooth muscle actin, connective tissue growth factor, and fibronectin protein levels in keloid skin fibroblasts. CONCLUSIONS: Thus, FTY720 suppressed migration of pathogenic T cells to target organs, reducing inflammation. FTY720 also inhibited fibrogenesis marker expression in vitro and in vivo. Together, these results suggest that FTY720 prevents GvHD progression via immunosuppression of TH17 and simultaneously acts an anti-fibrotic agent.

Mitochondrial Damage and Necroptosis in Aging Cochlea.

Age-related hearing loss (ARHL) is an irreversible, progressive neurodegenerative disorder and is presently untreatable. Previous studies using animal models have suggested mitochondrial damage and programmed cell death to be involved with ARHL. Thus, we further investigated the pathophysiologic role of mitochondria and necroptosis in aged C57BL/6J male mice. Aged mice (20 months old) exhibited a significant loss of hearing, number of hair cells, neuronal fibers, and synaptic ribbons compared to young mice. Ultrastructural analysis of aged cochleae revealed damaged mitochondria with absent or disorganized cristae. Aged mice also showed significant decrease in cochlear blood flow, and exhibited increase in gene expression of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α), receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) and the pseudokinase mixed-lineage kinase domain-like (MLKL). Immunofluorescence (IF) assays of cytochrome C oxidase I (COX1) confirmed mitochondrial dysfunction in aged cochleae, which correlated with the degree of mitochondrial morphological damage. IF assays also revealed localization and increased expression of RIPK3 in sensorineural tissues that underwent significant necroptosis (inner and outer hair cells and stria vascularis). Together, our data shows that the aging cochlea exhibits damaged mitochondria, enhanced synthesis of proinflammatory cytokines, and provides new evidence of necroptosis in the aging cochlea in in vivo.

HIF1-alpha Regulates Acinar Cell Function and Response to Injury in Mouse Pancreas.

We investigated whether intrapancreatic coagulation, with deposition of the fibrinogen-γ dimer (Fib-γD) and hypoxia, affect the severity of acute pancreatitis (AP) in mice. Pancreata of mice with AP induced by administration of cerulein or by L-arginine, or from patients with pancreatitis, had increased deposition of Fib-γD compared with control pancreata. Heparin administration protected mice from cerulein-induced AP and prevented Fib-γD formation. Cerulein administration resulted in activation and stabilization of hypoxia-inducible factor-1α (HIF1α) in pancreata of oxygen-dependent degradation domain-luciferase HIF1α reporter mice. Cerulein also led to induction of genes regulated by HIF1α, including Vegfa and Ero1a, before evidence of Fib-γD deposition or histologic features of AP. Expression of tissue factor, which is regulated by vascular endothelial growth factor, also increased following cerulein administration. Mice with acinar cell-specific disruption of Hif1a (Hif1aAc-/-) developed spontaneous endoplasmic reticulum stress and less severe AP, but did not accumulate Fib-γD following administration of cerulein. Feeding mice increased pancreatic expression of HIF1α, indicating a physiologic role in the exocrine pancreas. Therefore, HIF1α has bifunctional roles, in exocrine pancreas homeostasis and progression of AP that is promoted by intrapancreatic coagulation.

Contrast-enhanced US with Perfluorobutane for Hepatocellular Carcinoma Surveillance: A Multicenter Diagnostic Trial (SCAN).

Background US has served as a standard surveillance tool for hepatocellular carcinoma (HCC); however, the detection rate and false referral rate with this modality are suboptimal. Purpose To evaluate the added value of perfluorobutane-enhanced US when combined with conventional B-mode US as an HCC surveillance tool in participants with liver cirrhosis. Materials and Methods This prospective multi-institution diagnostic trial (https://ClinicalTrials.gov, NCT02188901) used an intraindividual comparison design in a single arm of study participants and was conducted at five referral hospitals. Eligible participants who had liver cirrhosis related to viral hepatitis and were undergoing US for HCC surveillance were enrolled from October 2014 to August 2016. Immediately after completion of B-mode US but before performance of perfluorobutane-enhanced US, operating radiologists entered the results of B-mode US. After completion of subsequent perfluorobutane-enhanced US (Kupffer phase with or without vascular-phase US), the radiologists recorded the results. The presence of HCC was confirmed either with pathologic analysis or radiologically by using dynamic contrast material-enhanced CT or gadoxetic acid-enhanced MRI. The primary end points were the detection rate of early-stage HCC (Barcelona Clinic Liver Cancer staging system stage 0 or A) and false referral rate. The primary end points were compared in a per-participant manner by using the McNemar test. Results A total of 524 participants (mean age, 54 years ± 9 [standard deviation]) were included. Of these, 493 (94.1%) had liver cirrhosis related to the hepatitis B virus. Ten HCCs were confirmed in eight participants. The detection rate of early-stage HCC was not significantly improved by adding perfluorobutane-enhanced US to conventional B-mode US (difference, 0.4% [95% confidence interval: -0.3%, 1.1%]; P = .16). The false referral rate was significantly reduced (difference, -3.2% [95% confidence interval: -5.0%, -1.4%]; P < .001). Conclusion The addition of perfluorobutane-enhanced US to conventional B-mode US reduced the false referral rate without a significant improvement in the detection rate of early-stage hepatocellular carcinoma for surveillance in a population in which the hepatitis B virus predominated. © RSNA, 2019 Online supplemental material is available for this article.