GDF15 Regulates Malat-1 Circular RNA and Inactivates NFκB Signaling Leading to Immune Tolerogenic DCs for Preventing Alloimmune Rejection in Heart Transplantation

Recombinant human growth differentiation factor 15 (rhGDF15) affects dendritic cell (DC) maturation. However, whether GDF15 is expressed in DCs and its roles and signaling in DCs remain largely unknown. It is unclear whether GDF15-DCs can induce immune tolerance in heart transplantation (HT). This study aims to understand the impact of endogenous GDF15 on DC's development, function, underlying molecular mechanism including circular RNA (circRNA). This study will also explore GDF15-DC-mediated immune modulation in HT. Bone marrow (BM) derived DCs were cultured and treated to up- or down regulate GDF15 expression. Phenotype and function of DCs were detected. Expression of genes and circRNAs was determined by qRT-PCR. The signaling pathways activated by GDF15 were examined. The impact of GDF15 treated DCs on preventing allograft immune rejection was assessed in a MHC full mismatch mouse HT model. Our results showed that GDF15 was expressed in DCs. Knockout of GDF15 promoted DC maturation, enhanced immune responsive functions, up-regulated malat-1 circular RNA (circ_Malat 1), and activated the nuclear factor kappa B (NFκB) pathway. Overexpression of GDF15 in DCs increased immunosuppressive/inhibitory molecules, enhanced DCs to induce T cell exhaustion, and promoted Treg generation through IDO signaling. GDF15 utilized transforming growth factor (TGF) β receptors I and II, not GFAL. Administration of GDF15 treated DCs prevented allograft rejection and induced immune tolerance in transplantation. In conclusion, GDF15 induces tolerogenic DCs (Tol-DCs) through inhibition of circ_Malat-1 and the NFκB signaling pathway and up-regulation of IDO. GDF15-DCs can prevent alloimmune rejection in HT

Based on Atmospheric Physics and Ecological Principle to Assess the Accuracies of Field CO2 /H2O Measurements From Infrared Gas Analyzers in Closed-Path Eddy-Covariance Systems

Field CO2 /H2O measurements from infrared gas analyzers in closed-path eddy-covariance systems have wide applications in earth sciences. Knowledge about exactness of these measurements is required to assess measurement applicability. Although the analyzers are specified with uncertainty components (zero drift, gain drift, cross-sensitivities, and precision), exactness for individual measurements is unavailable due to an absence of methodology to comprehend the components as an overall uncertainty. Adopting an advanced definition of accuracy as a range of all measurement uncertainty sources, the specified components are composited into a model formulated for studying analyzers’ CO2 /H2O accuracy equations. Based on atmospheric physics and environmental parameters, the analyzers are evaluated using the equations for CO2 accuracy (±0.78 µmolCO2 mol−1, relatively ±0.18%) and H2O accuracy (±0.15 mmolH2 O mol−1). Evaluation shows that precision and cross-sensitivity are minor uncertainties while zero and gain drifts are major uncertainties. Both drifts need adjusting through zero/span procedures during field maintenance. The equations provide rationales to guide and assess the procedures. H2O span needs more attentions under humid conditions. Under freezing conditions while H2O span is impractical, this span is fortunately unnecessary. Under the same conditions, H2O zero drift dominates H2O measurement uncertainty. Therefore, automatic zero becomes a more applicable and necessary tactic. In general cases of atmospheric CO2 background, automatic CO2 zero/ span procedures can narrow CO2 accuracy by 36% (±0.74 to ± 0.47 µmolCO2 mol−1). Automatic/manual H2 O zero/span procedures can narrow H2O accuracy by 27% (±0.15 to ±0.11 mmolH2O mol−1). While ensuring system specifications, the procedures guided by equations improve measurement accuracies

Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA

<p>Abstract</p> <p>Background</p> <p>Proliferation and migration of vascular smooth muscle cells (VSMCs) play a key role in neointimal formation which leads to restenosis of vein graft in venous bypass. STAT-3 is a transcription factor associated with cell proliferation. We hypothesized that silencing of STAT-3 by siRNA will inhibit proliferation of VSMCs and attenuate intimal thickening.</p> <p>Methods</p> <p>Rat VSMCs were isolated and cultured in vitro by applying tissue piece inoculation methods. VSMCs were transfected with STAT 3 siRNA using lipofectamine 2000. In vitro proliferation of VSMC was quantified by the MTT assay, while in vivo assessment was performed in a venous transplantation model. In vivo delivery of STAT-3 siRNA plasmid or scramble plasmid was performed by admixing with liposomes 2000 and transfected into the vein graft by bioprotein gel applied onto the adventitia. Rat jugular vein-carotid artery bypass was performed. On day 3 and7 after grafting, the vein grafts were extracted, and analyzed morphologically by haematoxylin eosin (H&E), and assessed by immunohistochemistry for expression of Ki-67 and proliferating cell nuclear antigen (PCNA). Western-blot and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression in vivo and in vitro. Cell apoptosis in vein grafts was detected by TUNEL assay.</p> <p>Results</p> <p>MTT assay shows that the proliferation of VSMCs in the STAT-3 siRNA treated group was inhibited. On day 7 after operation, a reduced number of Ki-67 and PCNA positive cells were observed in the neointima of the vein graft in the STAT-3 siRNA treated group as compared to the scramble control. The PCNA index in the control group (31.3 ± 4.7) was higher than that in the STAT-3 siRNA treated group (23.3 ± 2.8) (P < 0.05) on 7d. The neointima in the experimental group(0.45 ± 0.04 μm) was thinner than that in the control group(0.86 ± 0.05 μm) (P < 0.05).Compared with the control group, the protein and mRNA levels in the experimental group in vivo and in vitro decreased significantly. Down regulation of STAT-3 with siRNA resulted in a reduced expression of Bcl-2 and cyclin D1. However, apoptotic cells were not obviously found in all grafts on day 3 and 7 post surgery.</p> <p>Conclusions</p> <p>The STAT-3 siRNA can inhibit the proliferation of VSMCs in vivo and in vitro and attenuate neointimal formation.</p

RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis

INTRODUCTION: We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40. METHODS: Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses. RESULTS: Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells. CONCLUSIONS: These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity

CD5 blockade enhances ex vivo CD8+ T cell activation and tumour cell cytotoxicity

CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti‐tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti‐tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti‐tumour response. Here, we show that ex vivo administration of a function‐blocking anti‐CD5 MAb to primary mouse CTLs of both tumour‐naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti‐CD3/anti‐CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN‐γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function‐blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1‐mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell‐mediated anti‐tumour immunity

Prevention of hyperglycemia-induced myocardial apoptosis by gene silencing of Toll-like receptor-4

<p>Abstract</p> <p>Background</p> <p>Apoptosis is an early event involved in cardiomyopathy associated with diabetes mellitus. Toll-like receptor (TLR) signaling triggers cell apoptosis through multiple mechanisms. Up-regulation of TLR4 expression has been shown in diabetic mice. This study aimed to delineate the role of TLR4 in myocardial apoptosis, and to block this process through gene silencing of TLR4 in the myocardia of diabetic mice.</p> <p>Methods</p> <p>Diabetes was induced in C57/BL6 mice by the injection of streptozotocin. Diabetic mice were treated with 50 μg of TLR4 siRNA or scrambled siRNA as control. Myocardial apoptosis was determined by TUNEL assay.</p> <p>Results</p> <p>After 7 days of hyperglycemia, the level of TLR4 mRNA in myocardial tissue was significantly elevated. Treatment of TLR4 siRNA knocked down gene expression as well as diminished its elevation in diabetic mice. Apoptosis was evident in cardiac tissues of diabetic mice as detected by a TUNEL assay. In contrast, treatment with TLR4 siRNA minimized apoptosis in myocardial tissues. Mechanistically, caspase-3 activation was significantly inhibited in mice that were treated with TLR4 siRNA, but not in mice treated with control siRNA. Additionally, gene silencing of TLR4 resulted in suppression of apoptotic cascades, such as Fas and caspase-3 gene expression. TLR4 deficiency resulted in inhibition of reactive oxygen species (ROS) production and NADPH oxidase activity, suggesting suppression of hyperglycemia-induced apoptosis by TLR4 is associated with attenuation of oxidative stress to the cardiomyocytes.</p> <p>Conclusions</p> <p>In summary, we present novel evidence that TLR4 plays a critical role in cardiac apoptosis. This is the first demonstration of the prevention of cardiac apoptosis in diabetic mice through silencing of the TLR4 gene.</p

Preventing autoimmune arthritis using antigen-specific immature dendritic cells: a novel tolerogenic vaccine

Conventional treatments for autoimmune diseases have relied heavily on nonspecific immune suppressants, which possess a variety of adverse effects without inhibiting the autoimmune process in a specific manner. In the present study we demonstrate the effectiveness of antigen-specific, maturation-resistant, tolerogenic dendritic cells (DC) in suppressing collagen-induced arthritis, a murine model of rheumatoid arthritis. Treatment of DC progenitors with the NF-κB inhibiting agent LF 15-0195 (LF) resulted in a population of tolerogenic DC that are characterized by low expression of MHC class II, CD40, and CD86 molecules, as well as by poor allostimulatory capacity in a mixed leukocyte reaction. Administering LF-treated DC pulsed with keyhole limpet hemocyanin antigen to naïve mice resulted hyporesponsiveness specific for this antigen. Furthermore, administration of LF-treated DC to mice with collagen-induced arthritis resulted in an improved clinical score, in an inhibited antigen-specific T-cell response, and in reduced antibody response to the collagen. The efficacy of LF-treated DC in preventing arthritis was substantiated by histological examination, which revealed a significant decrease in inflammatory cell infiltration in the joints. In conclusion, we demonstrate that in vitro-generated antigen-specific immature DC may have important potential as a tolerogenic vaccine for the treatment of autoimmune arthritis

Gene silencing of IL-12 in dendritic cells inhibits autoimmune arthritis

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that immune modulation can be accomplished by administration of gene silenced dendritic cells (DC) using siRNA. In this study, we demonstrate the therapeutic utilization of shRNA-modified DC as an antigen-specific tolerogenic vaccine strategy for autoimmune arthritis.</p> <p>Methods</p> <p>A shRNA that specifically targets IL-12 p35 was designed and cloned into a plasmid vectors (IL-12 shRNA). Bone marrow-derived DC from DBA/1 mice were transfected with the IL-12 shRNA construct in vitro. Mice with collagen II (CII)-induced arthritis (CIA) were treated with the modified DCs expressing the shRNA. Recall response and disease progression were assessed.</p> <p>Results</p> <p>After gene silencing of IL-12 in DC, DC were shown to selectively inhibit T cell proliferation on recall responses and in an MLR. In murine CIA, we demonstrated that administration of IL-12 shRNA-expressing DC that were pulsed with CII inhibited progression of arthritis. The therapeutic effects were evidenced by decreased clinical scores, inhibition of inflammatory cell infiltration in the joint, and suppression of T cell and B cell responses to CII.</p> <p>Conclusion</p> <p>We demonstrate a novel tolerance-inducing protocol for the treatment of autoimmune inflammatory joint disease in which the target antigen is known, utilizing DNA-directed RNA interference.</p

Exosomes as a tumor immune escape mechanism: possible therapeutic implications

Advances in cancer therapy have been substantial in terms of molecular understanding of disease mechanisms, however these advances have not translated into increased survival in the majority of cancer types. One unsolved problem in current cancer therapeutics is the substantial immune suppression seen in patients. Conventionally, investigations in this area have focused on antigen-nonspecific immune suppressive molecules such as cytokines and T cell apoptosis inducing molecules such as Fas ligand. More recently, studies have demonstrated nanovesicle particles termed exosomes are involved not only in stimulation but also inhibition of immunity in physiological conditions. Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules such as PD-1L and FasL. Concentrations of exosomes from plasma of cancer patients have been associated with spontaneous T cell apoptosis, which is associated in some situations with shortened survival. In this paper we place the "exosome-immune suppression" concept in perspective of other tumor immune evasion mechanisms. We conclude by discussing a novel therapeutic approach to cancer immune suppression by extracorporeal removal of exosomes using hollow fiber filtration technolog

Predicting the rupture status of small middle cerebral artery aneurysms using random forest modeling

ObjectiveSmall intracranial aneurysms are increasingly being detected; however, a prediction model for their rupture is rare. Random forest modeling was used to predict the rupture status of small middle cerebral artery (MCA) aneurysms with morphological features.MethodsFrom January 2009 to June 2020, we retrospectively reviewed patients with small MCA aneurysms (&lt;7 mm). The aneurysms were randomly split into training (70%) and internal validation (30%) cohorts. Additional independent datasets were used for the external validation of 78 small MCA aneurysms from another four hospitals. Aneurysm morphology was determined using computed tomography angiography (CTA). Prediction models were developed using the random forest and multivariate logistic regression.ResultsA total of 426 consecutive patients with 454 small MCA aneurysms (&lt;7 mm) were included. A multivariate logistic regression analysis showed that size ratio (SR), aspect ratio (AR), and daughter dome were associated with aneurysm rupture, whereas aneurysm angle and multiplicity were inversely associated with aneurysm rupture. The areas under the receiver operating characteristic (ROC) curves (AUCs) of random forest models using the five independent risk factors in the training, internal validation, and external validation cohorts were 0.922, 0.889, and 0.92, respectively. The random forest model outperformed the logistic regression model (p = 0.048). A nomogram was developed to assess the rupture of small MCA aneurysms.ConclusionRandom forest modeling is a good tool for evaluating the rupture status of small MCA aneurysms and may be considered for the management of small aneurysms