The contribution of oxidative stress to platelet senescence during storage.

BACKGROUND: Platelets for transfusion become senescent and dysfunctional during storage, resulting in a markedly short shelf life (5 days). We hypothesized that oxidative stress might account for this decline. STUDY DESIGN AND METHODS: Human platelets were treated with or without antioxidants before storage, and samples were collected and analyzed at different time points. Platelet senescence was determined by senescence-associated ß-galactosidase assay, and senescence-related platelet qualities were also analyzed. RESULTS: Sign of senescence became evident after Day 3 and continued to increase over time. We also found that chemical induction of platelet activation did not affect senescence level, whereas apoptosis inducers showed a stimulative effect on platelet senescence. Moreover, this effect was not prevented by a pan-caspase inhibitor. Meanwhile, cellular and mitochondrial reactive oxygen species were found elevated during storage, and treatments with antioxidants successfully prevented this increase and also mitigated senescence levels of stored platelets. Finally, resveratrol, a natural antioxidant, was utilized as a novel storage additive to safely extend platelet shelf time. We showed that the addition of resveratrol efficiently postponed platelet senescence and ameliorated platelet storage lesion. CONCLUSIONS: Platelets during storage became senescent and dysfunctional over time, and we found that oxidative stress might account for this decline. The addition of antioxidants effectively postponed senescence and ameliorated platelet storage lesion, which might provide a valuable reference to future platelet storage methodologies.

Bcl-xL/Bak interaction and regulation by miRNA let-7b in the intrinsic apoptotic pathway of stored platelets.

Bcl-2 family proteins play key roles in the intrinsic apoptosis pathway in platelets, with both pro- and antiapoptotic protein expressions regulating survival during ex vivo storage. We detected a significant decrease in antiapoptotic Bcl-xL and increase in proapoptotic Bak expression on the third day of storage and as a result the ratio of Bak:Bcl-xL also decreased. Moreover, we identified an interaction between Bcl-xL and Bak. These shifts corresponded with activation of the apoptotic pathway, suggesting these proteins might play an important role in platelet survival. We then performed bioinformatic analysis to gain insight into protein expression regulation during storage. This identified a potential binding site of the microRNA (miRNA) let-7b in the 3'-UTR of the Bcl-xL gene, which we confirmed by a dual-luciferase reporter assay. We also determined that let-7b was upregulated during platelet storage, and let-7b transfection influenced Bcl-xL and Bak protein, but not mRNA, expression. Together, these data suggest that only posttranscriptional mechanisms are available for regulating gene expression in anucleate platelets.

Platelet mitochondrial dysfunction and the correlation with human diseases.

The platelet is considered as an accessible and valuable tool to study mitochondrial function, owing to its greater content of fully functional mitochondria compared with other metabolically active organelles. Different lines of studies have demonstrated that mitochondria in platelets have function far more than thrombogenesis regulation, and beyond hemostasis, platelet mitochondrial dysfunction has also been used for studying mitochondrial-related diseases. In this review, the interplay between platelet mitochondrial dysfunction and oxidative stress, mitochondrial DNA lesions, electron transfer chain impairments, mitochondrial apoptosis and mitophagy has been outlined. Meanwhile, considerable efforts have been made towards understanding the role of platelet mitochondrial dysfunction in human diseases, such as diabetes mellitus, sepsis and neurodegenerative disorders. Alongside this, we have also articulated our perspectives on the development of potential biomarkers of platelet mitochondrial dysfunction in mitochondrial-related diseases.

The Effect of Sphingosine 1-Phosphate/Sphingosine 1-Phosphate Receptor on Neutrophil Function and the Relevant Signaling Pathway.

BACKGROUND/AIMS: This study investigated the priming effect of sphingosine 1-phosphate (S1P) on formyl-Met-Leu-Phe-OH (fMLP)-activated neutrophils, by specific analysis of the neutrophil respiratory burst and the signaling pathway involved in S1P activity. METHODS: The neutrophil respiratory burst was indirectly detected by the cytochrome c reduction method and the dihydrorhodamine 123 staining method. The signal transduction pathways of neutrophil respiratory burst primed by S1P were detected by Western blotting. RESULTS: Our results showed that the S1P receptors (S1PRs) 1, 4 and 5 were the predominantly expressed neutrophil S1PRs at the cDNA level. After pretreatment with S1P, the fMLP-activated neutrophils released increased levels of superoxide anions. PI3K and Akt proteins were involved in the signaling pathway of the neutrophil respiratory burst primed by S1P. CONCLUSION: The results indicate that S1P is a new priming reagent for neutrophils and primes the respiratory burst of fMLP-activated neutrophils. S1P interacts with its receptors on neutrophils, resulting in NADPH oxidase activation by the PI3K-Akt cell signaling pathway and induction of the neutrophil respiratory burst.

Mitochondria-derived reactive oxygen species play an important role in Doxorubicin-induced platelet apoptosis.

Doxorubicin (DOX) is an effective chemotherapeutic agent; however; its use is limited by some side effects; such as cardiotoxicity and thrombocytopenia. DOX-induced cardiotoxicity has been intensively investigated; however; DOX-induced thrombocytopenia has not been clearly elucidated. Here we show that DOX-induced mitochondria-mediated intrinsic apoptosis and glycoprotein (GP)Ibα shedding in platelets. DOX did not induce platelet activation; whereas; DOX obviously reduced adenosine diphosphate (ADP)- and thrombin-induced platelet aggregation; and impaired platelet adhesion on the von Willebrand factor (vWF) surface. In addition; we also show that DOX induced intracellular reactive oxygen species (ROS) production and mitochondrial ROS generation in a dose-dependent manner. The mitochondria-targeted ROS scavenger Mito-TEMPO blocked intracellular ROS and mitochondrial ROS generation. Furthermore; Mito-TEMPO reduced DOX-induced platelet apoptosis and GPIbα shedding. These data indicate that DOX induces platelet apoptosis; and impairs platelet function. Mitochondrial ROS play a pivotal role in DOX-induced platelet apoptosis and GPIbα shedding. Therefore; DOX-induced platelet apoptosis might contribute to DOX-triggered thrombocytopenia; and mitochondria-targeted ROS scavenger would have potential clinical utility in platelet-associated disorders involving mitochondrial oxidative damage.

Regulatory effect of nicotine on collagen-induced arthritis and on the induction and function of in vitro-cultured Th17 cells.

OBJECTIVES: To determine the effect of nicotine stimulation on collagen-induced arthritis (CIA), especially on Th17 cells, and the influence of activated acetylcholine receptor signaling on the induction and function of in vitro-cultured Th17 cells. METHODS: Mice were divided into control and experimental (nicotine) group, and PBS or nicotine-PBS was orally administered from Day 21 to Day 28. Phenotypic changes in spleen CD4(+) cells were measured by flow cytometry. α7nAChR expression in Th17 cells was detected using flow cytometry, western blotting and real-time PCR. Purified Th17 cells were further stimulated with nicotine. The cytometric bead array (CBA) assay was employed to measure TNF-α levels in mice serum and IL-17A levels in the supernatants of nicotine-treated cell cultures. RESULTS: Compared with their counterparts, mice receiving oral nicotine showed a delayed progress of arthritis and more attenuated signs of histological changes. Moreover, serum TNFα levels were lower in the nicotine-treated group. Spleen IL-17 level of nicotine-treated mice was lower than that of the control group, and the mRNA expression of pro-inflammatory cytokines (IL-17A and IL-6) in splenocytes were also lower than that of the control group. α7nAChR expression was detected on in vitro-cultured IL-17A(+) cells. Cells treated with 10 (- 6) M nicotine expressed lower IL-17A levels. Similarly, supernatants from nicotine-treated cell cultures also showed lower IL-17A levels. CONCLUSIONS: Nicotine stimulation attenuated signs and severity of arthritis in mice. Activation of nicotine acetylcholine receptors on in vitro-cultured Th17 cells decreased their pro-inflammatory function, which may play a potential role in alleviating arthritis in mice.

Adverse effects of microparticles on transfusion of stored red blood cell concentrates.

BACKGROUND: Systemic and pulmonary coagulopathy and inflammation are important characteristics of transfusion-related acute lung injury (TRALI). Whether microparticles that accumulate in transfused red blood cell concentrates (RBCs) have proinflammatory and procoagulant potential and contribute to adverse reactions of RBC transfusions is unclear. AIM: To investigate the ability of microparticles in stored RBCs to promote thrombin generation and induce human pulmonary microvascular endothelial cell (HMVEC) activation and damage. METHODS: The number and size of microparticles were determined by flow cytometric and nanoparticle tracking analyses, respectively. Thrombin generation and the intrinsic coagulation pathway were assayed by a calibrated automated thrombogram and by measuring activated partial thromboplastin time (aPTT), respectively. The expression of ICAM-1 and the release of cytokines by endothelial cells were detected by flow cytometric analyses. HMVEC damage was assessed by incubating lipopolysaccharide-activated endothelial cells with MP-primed polymorphonuclear neutrophils (PMNs). RESULTS: The size of the microparticles in the RBC supernatant was approximately 100-300 nm. Microparticles promoted thrombin generation in a dose-dependent manner and the aPTT was shortened. Depleting microparticles from the supernatant of RBCs stored for 35 days by either filtration or centrifugation significantly decreased the promotion of thrombin generation. The expression of ICAM-1 on HMVECs was increased significantly by incubation with isolated microparticles. Furthermore, microparticles induced the release of interleukin-6 (IL-6) and interleukin-8 (IL-8) from HMVECs. Microparticles induced lipopolysaccharide-activated HMVEC damage by priming PMNs, but this effect was prevented by inhibiting the PMNs respiratory burst with apocynin. CONCLUSION: Microparticles in stored RBCs promote thrombin generation, HMVEC activation and damage which may be involved in TRALI development.

Comparative study of regulatory T cells expanded ex vivo from cord blood and adult peripheral blood.

In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers.

Amelioration of acute graft-versus-host disease by adoptive transfer of ex vivo expanded human cord blood CD4+CD25+ forkhead box protein 3+ regulatory T cells is associated with the polarization of Treg/Th17 balance in a mouse model.

BACKGROUND: Human cord blood (CB) is a superior source of regulatory T cells (Tregs) compared with peripheral blood. Initial studies have shown that CB-derived Tregs can be effectively expanded ex vivo. However, in vitro suppressor activity of expanded CB-Tregs and their efficacy in the prevention of acute graft-versus-host disease (aGVHD) in vivo are poorly understood. STUDY DESIGN AND METHODS: In vitro, human CB CD4+CD25+ T cells expanded with anti-CD3/CD28 beads plus interleukin (IL)-2 and the phenotypes, expression of cytokines, and suppression of expanded cells were analyzed after two cycles of stimulation. In vivo, the addition of human CB-Tregs was transferred in the major histocompatibility complex-mismatched aGVHD mouse model. Survival, body weight, GVHD scoring, histopathologic specimens, serum cytokines, and Th subsets were analyzed in CB-Treg-treated mice and untreated controls. RESULTS: After being expanded ex vivo, human CB-derived Tregs with potent suppressor function could meet clinical demands. Up to 85% of mice with CB-Tregs treatment survived beyond Day 63 after bone marrow transplantation; however, all aGVHD mice died within 18 days. In the serum of the CB-Treg-treated mice, the production of transforming growth factor-ß increased continuously, as opposed to IL-17, which decreased quickly. Consistent with the changes of cytokines, the percentage of mouse CD4+ forkhead box protein 3+ Tregs increased while that of Th17 cells decreased. CONCLUSION: Ex vivo expanded human CB-Tregs significantly prevented allogeneic aGVHD in vivo by modulating various cytokine secretion and polarizing the Treg/Th17 balance toward Treg, which suggests the potential use of expanded CB-Tregs as a therapeutic approach for GVHD.

MicroRNA-424-5p Alleviates Isoflurane Anesthesia-Induced Neurotoxicity in Human Embryonic Stem Cell-Derived Neurons by Targeting FASN.

Isoflurane (ISO) is a type of anesthetic that might cause neurotoxicity in children. Although miR-424-5p is considerably downregulated in ISO-treated rat brain samples, its physiological role in ISO-induced neuronal injury in human embryonic stem cell-derived neurons remains unknown (hESC-derived neurons). miR-424-5p expression and fatty acid synthase (FASN) in ISO-treated hESC-derived neurons were tested via qRT-PCR. The amount of protein for Bax, Cleaved-caspase-8, Bcl-2, and FASN was investigated through western blot analysis. The viability and apoptosis of hESC-derived neurons were estimated through cell counting kit-8 assessment and TUNEL assay, accordingly. Superoxide dismutase, glutathione, and malondialdehyde levels were discovered via corresponding kits. The contents of inflammatory factors including interleukin-6 and tumor necrosis factor-α were examined by enzyme-linked immunosorbent assays. The combination between FASN and miR-424-5p was resolute via dual-luciferase reporter assessment. After exposure to ISO, induced neurotoxicity and a decreased miR-424-5p production were identified in hESC-derived neurons. Upregulation of miR-424-5p repressed ISO-induced apoptosis and mitigated ISO-induced inflammatory response and oxidative stress in vitro. FASN expression levels were reduced by elevation of miR-424-5p and upregulated after ISO treatment. Mechanically, FASN was directly targeted by miR-424-5p in hESC-derived neurons. Of note, the miR-424-5p elevation-suppressed neuronal apoptosis, inflammatory response, and oxidative stress were countered by upregulation of FASN.

Significant Shifts in Microbial Communities Associated with Scleractinian Corals in Response to Algae Overgrowth.

Microbes play a key role in reef dynamics, mediating the competition between scleractinian corals and benthic algae; however, major shifts in bacterial communities among coral species in response to increases in the abundance of algae are not well understood. We investigated the taxonomic composition of coral-associated microbial communities under algae-overgrowth conditions using 16S rRNA gene sequencing. The results showed that non-algal (i.e., healthy) tissue (HH) had lower bacterial abundance and diversity than tissue collected from the coral-algae interface boundary (HA) and areas of algae growth (AA). Specifically, the HA and AA samples had higher relative abundances of Saprospiraceae, Rhodobacteraceae, and Alteromonadaceae. Compared with Platygyra sp. and Montipora sp., the physiological response of Pocillopora sp. was more intense under algae-induced stress based on microbial gene function prediction. Our results indicate that algal pressure can significantly alter the microbial community structure and function of coral ecosystems. Our data thus provide new insight into the relationship between corals and their microbiome under environmental stress.

Exosomal-like vesicles with immune-modulatory features are present in human plasma and can induce CD4+ T-cell apoptosis in vitro.

BACKGROUND: Exosomes are small membrane vesicles that are secreted from many cell types into various body fluids. These vesicles are thought to play a role in cell-cell interactions. STUDY DESIGN AND METHODS: Vesicles were isolated from human plasma of healthy donors by differential ultracentrifugation and ultrafiltration. The vesicles were identified by transmission electron microscopy, and their biochemical characteristics were analyzed by Western blot and flow cytometry. The immune-modulatory ability of exosomal-like vesicles was examined by incubating them with CD4+ T cells for CD4+ T-cell proliferation and apoptosis assays in vitro. RESULTS: Vesicles purified from human plasma displayed shapes and sizes similar to those of previously described exosomes and contained exosomes marker proteins CD63 and CD81. They also expressed molecules such as MHC Class II molecules, CD80, CD86, and the cell signal transduction molecules Wnt3a, Wnt5a, and FasL. Furthermore, functional analysis showed that allogeneic plasma exosomes restrained the survival of CD4+ T cells. Plasma exosomes can induce dose-dependent suppression of proliferation of activated CD4+ T cells, with the strongest responses induced by 500 µg/mL exosomes in vitro. Antibodies against exosomes FasL can block the activity of exosomes on CD4+ T-cell apoptosis. Moreover, three different concentrations of CD4+ T cells were inhibited by plasma exosomes and the suppressive function was not dependent on interleukin-2. CONCLUSION: Exosomes present in human plasma contain immunity-associated molecules and can induce CD4+ T-cell apoptosis in vitro. Plasma exosomes have the capacity to influence immune responses.

MicroLet-7b Regulates Neutrophil Function and Dampens Neutrophilic Inflammation by Suppressing the Canonical TLR4/NF-κB Pathway.

Sepsis is a heterogeneous syndrome caused by a dysregulated host response during the process of infection. Neutrophils are involved in the development of sepsis due to their essential role in host defense. COVID-19 is a viral sepsis. Disfunction of neutrophils in sepsis has been described in previous studies, however, little is known about the role of microRNA-let-7b (miR-let-7b), toll-like receptor 4 (TLR4), and nuclear factor kappa B (NF-κB) activity in neutrophils and how they participate in the development of sepsis. In this study, we investigated the regulatory pathway of miR-let-7b/TLR4/NF-κB in neutrophils. We also explored the downstream cytokines released by neutrophils following miR-let-7b treatment and its therapeutic effects in cecal ligation and puncture (CLP)-induced septic mice. Six-to-eight-week-old male C57BL/6 mice underwent CLP following treatment with miR-let-7b agomir. Survival (n=10), changes in liver and lungs histopathology (n=4), circulating neutrophil counts (n=4), the liver-body weight ratio (n=4-7), and the lung wet-to-dry ratio (n=5-6) were recorded. We found that overexpression of miR-let-7b could significantly down-regulate the expression of human-derived neutrophilic TLR4 at a post-transcriptional level, a decreased level of proinflammatory factors including interleukin-6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), and an upregulation of anti-inflammatory factor IL-10 in vitro. After miR-let-7b agomir treatment in vivo, neutrophil recruitment was inhibited and thus the injuries of liver and lungs in CLP-induced septic mice were alleviated (p=0.01 and p=0.04, respectively), less weight loss was reduced, and survival in septic mice was also significantly improved (p=0.013). Our study suggested that miR-let-7b could be a potential target of sepsis.

Adoptive therapy by transfusing expanded donor murine natural killer T cells can suppress acute graft-versus-host disease in allogeneic bone marrow transplantation.

BACKGROUND: Invariant natural killer T cells (iNKT cells) may suppress graft-versus-host disease (GVHD) after allogeneic transplantation. The purpose of this study was to investigate the therapeutic potential of iNKT cells from major histocompatibility complex (MHC)-mismatched donors for preventing GVHD after allogeneic bone marrow transplantation (BMT). STUDY DESIGN AND METHODS: In vitro, mouse iNKT cells were expanded with alpha-galactosylceramide and interleukin (IL)-2 treatment. In the NKT-treated group, lethally irradiated DBA/2(H-2K(d)) mice were adoptively transferred with expanded iNKT, bone marrow (BM), and spleen cells (SCs) from C57BL/6 (H-2K(b)) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and serum cytokine analysis. RESULTS: In the iNKT-treated group, 80% of mice survived past Day 60 after BMT, but all died within 38 days in the control group. The mice treated with iNKT did not exhibit signs of GVHD after Day 42 except for a change in fur color. There were higher IL-4 levels by Day 7 in serum of mice that received iNKT compared to those without iNKT treatment, while the interferon-gamma levels showed no significant difference between two groups. Levels of IL-2 and IL-5 increased by Day 21 only in iNKT-treated mice. CONCLUSION: The results suggest that donor iNKT cells could alleviate GVHD symptoms and prolong survival after MHC-mismatched allogeneic BMT, which may be associated with the maintenance in IL-4 levels. These findings indicate that the therapy based on iNKT cells from MHC-mismatched donors has great potential in protection against GVHD after allogeneic hematopoietic stem cell transplantation.

14-3-3ζ Regulates the Platelet Apoptosis During Storage.

There is an increasing interest in the possibility of storing platelet concentrates below standard temperatures. The role of 14-3-3 proteins has been demonstrated in numerous cellular functions, including both its positive and negative roles in apoptosis. The 14-3-3ζ protein has a potential role in regulation of storage induced apoptosis in platelets. Apheresis platelets were collected and stored under either at room temperature (RT, 20-24 °C) or cold temperature (CT, 2-6 °C) conditions (n = 7 in each group). Flow cytometry was used to assess changes in phosphatidylserine and mitochondrial membrane potential in washed platelets. Proteomic changes were analyzed using Western blot and coimmunoprecipitation. During RT storage conditions used in this study, we found that both Annexin V and JC-1 exhibited significant increases at Day 3 compared to Day 1. In comparison to RT storage, a 3-day cold storage exhibited higher positive rates of Annexin V and lower positive rates of JC-1. The release of cytochrome c and caspase 3 and 9 cleavage were only observed in platelets maintained under RT storage conditions for 3 days. The anti-apoptosis protein Bcl-xL was downregulated and the pro-apoptosis protein Bak was upregulated under RT storage conditions. However, both Bcl-xL and Bak of CT-D3 exhibited no significant changes in comparison to either RT-D1 or CT-D1. Expression levels of 14-3-3ζ and GPIbα decreased significantly in RT-D3 compared to those in RT-D1, while the expression levels of CT-D3 were found to be significantly higher than those in RT-D3. Expression levels of Bad protein in CT-D3 were significantly lower than those in RT-D3. A comparative analysis of RT-D3 and CT-D3 demonstrated that both the ratios of Bad/14-3-3ζ and GPIbα/14-3-3ζ increased significantly following cold storage. The ratio was significantly larger following a 3-day cold storage in comparison to that at RT. During cold storage of platelets, the enhanced association between 14-3-3ζ and GPIbα was demonstrated to improve exposure of phosphatidylserine, and the enhanced association between 14-3-3ζ and Bad was shown to delay the depolarization of the mitochondrial membrane potential.

Baicalin inhibits IgG production by regulating Treg/Th17 axis in a mouse model of red blood cell transfusion.

This study was conducted to evaluate whether baicalin inhibits red blood cell (RBC) immunization and elucidate the underlying mechanism. We used human RBCs with adjuvant lipopolysaccharide (LPS) and transfused mice to induce antibodies as an experimental system for studying the effect of baicalin on RBC immunization. Mice were divided into a human RBC transfused positive control group administered with human RBC and LPS intravenously once or weekly for 4 weeks, control group administered dexamethasone (DEX) intraperitoneally daily for 4 weeks, and treatment group administered baicalin intraperitoneally daily for 4 weeks. Assessment of human RBC immunization was performed by measuring serum immunoglobulin G (IgG) and immunoglobulin M (IgM) against human RBC weekly. Lymphocyte changes in spleen were monitored by flow cytometry. We found that baicalin treatment significantly decreased serum IgG but not IgM production in a time and does dependent manner, with a concomitant reduction in Th17 cells and increase in CD4 regulatory T cells in the spleen. The percentage of CD4-positive cells in the spleen was not decreased in the baicalin-treated group but was decreased in the dexamethasone-treated group. In conclusion, baicalin inhibited RBC immunization, particularly IgG production by regulating the Treg/Th17 axis without damaging spleen function.

Efficient Therapeutic Function and Mechanisms of Human Polyclonal CD8+CD103+Foxp3+ Regulatory T Cells on Collagen-Induced Arthritis in Mice.

OBJECTIVE: To investigate the potential therapeutic effect in a rheumatoid arthritis model of stable human CD8+ regulatory T cells (hCD8+Tregs) induced by TGF-ß1 and rapamycin (RAPA) in vitro. METHODS: Human CD8+T cells were isolated from human peripheral blood mononuclear cells and induced/expanded with TGF-ß1 and RAPA along with anti-CD3/28 beads and IL-2 in vitro and harvested as hCD8+Tregs. The phenotypes, suppressive characteristics, and stability of the hCD8+Tregs in an inflammatory microenvironment were examined in vitro. Human CD8+Tregs were transfused into an acollagen-induced arthritis (CIA) mouse model, and their therapeutic effects and related mechanisms were investigated. RESULTS: Human CD8+Tregs induced by TGF-ß1/RAPA showed high expression of Foxp3 and CD103, exhibited vigorous suppression ability, and were stable in inflammatory microenvironments. In CIA mice, the clinical scores, levels of anti-collagen IgG antibody, and cartilage destruction were significantly reduced after adoptive transfusion with hCD8+Tregs. Moreover, hCD8+Treg treatment significantly reduced the number of Th17 cells, increased the number of CD4+IFN-γ +T cells, and produced self CD4+Foxp3+Tregs in vivo. In an in vitro cell coculture assay, hCD8+Tregs significantly inhibited mouse CD4+ effector T cell proliferation, induced mouse CD4+Foxp3+Treg and CD4+IFN-γ +Th1 cell production, reduced Th17 cell development, and downregulated CD80/86 expression on mature DCs (mDCs). CONCLUSION: TGF-ß1/RAPA can induce hCD8+Tregs with stable suppressive characteristics, which could significantly alleviate the severity of CIA based on their stable suppressive ability in an inflammatory microenvironment and further influence the function of other downstream cell subtypes. Human CD8+Tregs might be a therapeutic strategy for rheumatoid arthritis.

Microparticles in red cell concentrates prime polymorphonuclear neutrophils and cause acute lung injury in a two-event mouse model.

Red cell-derived microparticles (RMPs) are potential mediators of transfusion-related acute lung injury (TRALI). The aim of this study was to investigate the effects of microparticles present in red cell concentrates (RCC) on polymorphonuclear neutrophil (PMN) respiratory burst and acute lung injury (ALI) in mice. Microparticles (MPs) in RCC supernatant were quantified using flow cytometry. The priming activity of either isolated MPs or RCC supernatant toward human PMN was measured in vitro. Mice were injected with lipopolysaccharide (LPS), followed by an infusion of either isolated MPs or heat-treated RCC supernatant. The lungs were harvested to assess myeloperoxidase (MPO) activity, histology and pulmonary edema. Protein content in bronchoalveolar lavage fluid (BALF) was measured. The number of RMPs increased significantly during storage. Both isolated MPs and the supernatants from RCCs that had been stored for 28 and 35days effectively primed the PMN respiratory burst. The infusion of isolated MPs or supernatants that had been stored for >28days into LPS-treated mice caused ALI. The filtered supernatant resulted in significantly ameliorated mouse ALI. MPs that accumulate during RCC storage prime the PMN respiratory burst and cause ALI in a two-event mouse model.

[The effect of two leukocyte depletion in-line filters on the efficiency of whole blood filtration].

The aim of this study was to observe the difference in respect to the leukocyte reduction efficiency and quality of fresh-frozen plasma (FFP) from filtered whole blood between two types of in-line filters wherein only filter materials were surface modified by the two methods respectively. Whole blood was kept in refrigerator and filtered within 6 h of collection at ambient temperature. Samples were taken pre- and post filtration for analysis of WBC numbers, coagulation factors and complement activation (n = 8 for each type of filter). All filtered units contained < 2. 5 x 10(6) residual leucocytes. RBCs recovery was over 93%. No significant difference between group A and B was seen. But group B appeared to take longer time for filtration than did group A (9'29" vs. 8'01"). Neither group A nor group B showed statistically significant losses of total protein, album, IgG, IgM, fibrin, factors VIII, IX, vWF and C3 (P > 0.05). Factor V, XI and AT-III decreased significantly in two group filters. Group B showed more significant losses of IgA content and factor V activity than did group A, which appeared to be related to the difference in surface character between group A and group B filters. These two types of filters could remove leukocytes effectively, and no significant changes were observed in the quality of FFP from the filtered whole blood. It is presumed that the filter material with better bio-compatibility will give a high recovery of plasma protein and coagulation factors after filtration.

Adoptive Cell Therapy of Induced Regulatory T Cells Expanded by Tolerogenic Dendritic Cells on Murine Autoimmune Arthritis.

OBJECTIVE: Tolerogenic dendritic cells (tDCs) can expand TGF-ß-induced regulatory T cells (iTregs); however, the therapeutic utility of these expanded iTregs in autoimmune diseases remains unknown. We sought to determine the properties of iTregs expanded by mature tolerogenic dendritic cells (iTregmtDC) in vitro and explore their potential to ameliorate collagen-induced arthritis (CIA) in a mouse model. METHODS: After induction by TGF-ß and expansion by mature tDCs (mtDCs), the phenotype and proliferation of iTregmtDC were assessed by flow cytometry. The ability of iTregs and iTregmtDC to inhibit CD4+ T cell proliferation and suppress Th17 cell differentiation was compared. Following adoptive transfer of iTregs and iTregmtDC to mice with CIA, the clinical and histopathologic scores, serum levels of IFN-γ, TNF-α, IL-17, IL-6, IL-10, TGF-ß and anti-CII antibodies, and the distribution of the CD4+ Th subset were assessed. RESULTS: Compared with iTregs, iTregmtDC expressed higher levels of Foxp3 and suppressed CD4+ T cell proliferation and Th17 cell differentiation to a greater extent. In vivo, iTregmtDC reduced the severity and progression of CIA more significantly than iTregs, which was associated with a modulated inflammatory cytokine profile, reduced anti-CII IgG levels, and polarized Treg/Th17 balance. CONCLUSION: This study highlights the potential therapeutic utility of iTregmtDC in autoimmune arthritis and should facilitate the future design of iTreg immunotherapeutic strategies.