Exchange transfusion combined with artesunate (ET-AS) as a safe and effective therapy in severe P. falciparum malaria: a case series.

BACKGROUND: the mortality associated with severe malaria due to Plasmodiun falciparum remains high despite improvements in malaria management. Case prensentation: this case series aims to describe the efficacy and safety of the exchange transfusion combined with artesunate (ET-AS) regimen in severe P. falciparum malaria. Eight patients diagnosed with severe P. falciparum malaria were included. All patients underwent ET using the COBE Spectra system. The aimed for a post-exchange hematocrit of 30%. Half the estimated blood volume was removed and replaced using fresh frozen plasma. The regimen was well-tolerated without complications. The parasite clearance time ranged from 1 ~ 5 days. Five patients with cerebral malaria exhibited full improved consciousness within 3 days, while patient2 with hemolysis improved on day 2. Liver function improved within 1 ~ 6 days, and patient 1 and patient 6 showed improvements renal function on days 18 and 19, respectively. The length of intensive care unit stay range from 2 ~ 10 days, and all patients treated with ET-AS remained in the hospital for 3 ~ 19 days. CONCLUSIONS: these preliminary results suggest that ET-AS regimens are a safe and effective therapy for severe P. falciparum malaria and can benefit patients in clinical settings.

Trimethylamine-N-oxide (TMAO) and basic fibroblast growth factor (bFGF) are possibly involved in corticosteroid resistance in adult patients with immune thrombocytopenia.

PURPOSE: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by accelerated platelet clearance. Gut dysbiosis was associated with its pathogenesis, but the underlying mechanisms have not been fully elucidated. Patients with ITP exhibit varying degrees of responsiveness to corticosteroid treatment. Therefore, prognostic indexes for corticosteroid responsiveness in ITP could offer valuable guidance for clinical practices. METHODS: The present study examined the signature of six types of gut-microbiota metabolites and forty-eight types of cytokines, chemokines, and growth factors and their clinical significance in patients with ITP. RESULTS: Both patients with good and poor corticosteroid responsiveness exhibited significantly elevated/suppressed secretion of twenty-two cyto(chemo)kins/growth factors in comparison to healthy controls. Additionally, patients with ITP demonstrated a significant decrease in plasma levels of trimethylamine-N-oxide (TMAO), which was found to be negatively correlated to circulating platelet counts, and positively correlated with Interleukin (IL)-1ß and IL-18. Notably, patients who exhibited poor response to corticosteroid treatment displayed elevated levels of TMAO and basic fibroblast growth factor (bFGF) in comparison to responders. Additionally, we found that the amalgamation of TMAO, bFGF and interleukin (IL)-13 could serve as a valuable prognostic tool for predicting CS responsiveness. CONCLUSION: Patients with ITP were characterized overall by an imbalanced secretion of cyto(cheo)kins/growth factors and inadequate levels of TMAO. The varying degrees of responsiveness to corticosteroid treatment can be attributed to different profiles of basic FGF and TMAO that might be related to overburdened oxidative stress and inflammasome overactivation, and ultimately mediate corticosteroid resistance.

Platelets fine-tune effector responses of naïve CD4+ T cells via platelet factor 4-regulated transforming growth factor ß signaling.

BACKGROUND AND AIM: Platelets are an able regulator of CD4+ T cell immunity. Herein, the mechanisms underlying platelet-regulated effector responses of naïve CD4+ T (Tn) cells were investigated. METHODS: Platelet-Tn cell co-cultures of human cells, genetically modified murine models, and high-throughput bioinformatic analyses were combined to elucidate molecular mechanisms of platelet-dependent regulation. RESULTS: Platelets exerted sophisticated regulation on effector responses of type 1, 2, and 17 T helper (Th1/Th2/Th17) and regulatory T (Treg) cells, in time-, concentration-, and organ-dependent manners and with close cooperation of transforming growth factor ß (TGFß) and platelet factor 4 (PF4). PF4 at low concentrations reinforced TGFß signaling by heteromerizing with type III TGFß receptor (TGFBRIII), and subsequently enhanced TGFBRII expression and TGFß signaling. High-concentration PF4 had, however, opposite effects by directly binding to TGFBRII, blocking TGFß-TGFBRII ligation, and thus inhibiting TGFß signaling. Furthermore, platelet depletion markedly hampered Treg and Th17 responses in the spleen but not in the lymph nodes, blockade of platelet-Tn cell contact diminished platelet effects, while spleen injection of PF4-immobilized microparticles in PF4-deficient mice mimicked platelet effects, suggesting the importance of direct platelet-Tn contact and platelet-bound PF4 for the optimal regulatory effects by platelets. CONCLUSION: Platelets exert context-dependent regulations on effector responses of Tn cells via PF4-TGFß duet, suggesting new possibilities of platelet-targeted interventions of T cell immunity.

VEGFR2 inhibition hampers breast cancer cell proliferation via enhanced mitochondrial biogenesis.

Objective: Vascular endothelial growth factor (VEGF), apart from its predominant roles in angiogenesis, can enhance cancer cell proliferation, but its mechanisms remain elusive. The purpose of the present study was therefore to identify how VEGF regulates cancer cell proliferation. Methods: VEGF effects on cancer cell proliferation were investigated with the VEGF receptor 2 inhibitor, Ki8751, and the breast cancer cell lines, MCF-7 and MDA-MB-231, using flow cytometry, mass spectrometry, immunoblotting, and confocal microscopy. Data were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test. Results: VEGF blockade by Ki8751 significantly reduced cancer cell proliferation, and enhanced breast cancer cell apoptosis. Mass spectrometric analyses revealed that Ki8751 treatment significantly upregulated the expression of mitochondrial proteins, suggesting the involvement of mitochondrial biogenesis. Confocal microscopy and flow cytometric analyses showed that Ki8751 treatment robustly increased the mitochondrial masses of both cancer cells, induced endomitosis, and arrested cancer cells in the high aneuploid phase. VEGFR2 knockdown by shRNAs showed similar effects to those of Ki8751, confirming the specificity of Ki8751 treatment. Enhanced mitochondrial biogenesis increased mitochondrial oxidative phosphorylation and stimulated reactive oxygen species (ROS) production, which induced cancer cell apoptosis. Furthermore, Ki8751 treatment downregulated the phosphorylation of Akt and PGC1α, and translocated PGC1α into the nucleus. The PGC1α alterations increased mitochondrial transcription factor A (TFAM) expression and subsequently increased mitochondrial biogenesis. Conclusions: VEGF enhances cancer cell proliferation by decreasing Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis, ROS production, and cell apoptosis. These findings suggested the anticancer potential of Ki8751 via increased mitochondrial biogenesis and ROS production.

Tumor Necrosis Factor-α Blockade Corrects Monocyte/Macrophage Imbalance in Primary Immune Thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder. Monocytes and macrophages are the major cells involved in autoantibody-mediated platelet clearance in ITP. In the present study, we found increased percentages of peripheral blood proinflammatory CD16+ monocytes and elevated frequencies of splenic tumor necrosis factor-α (TNF-α)-expressing macrophages in ITP patients compared with healthy controls. Concurrently, we observed elevated TNF-α secretion in plasma as well as higher TNF-α mRNA expression in total peripheral blood mononuclear cells and CD14+ monocytes of ITP patients. Of note, in vitro TNF-α blockade with neutralizing antibody remarkably reduced polarization to M1 macrophages by inhibiting the nuclear factor kappa B (NF-κB) signaling pathway. Moreover, TNF-α blockade dampened macrophage phagocytosis and T cell stimulatory capacity. Finally, in passive and active murine models of ITP, anti-TNF-α therapy reduced the number of nonclassical monocytes and M1 macrophages, ameliorated the retention of platelets in spleen and liver, and increased the platelet count of ITP mice. Taken together, TNF-α blockade decreased the number and function of proinflammatory subsets of monocytes and macrophages by inhibiting the NF-κB signaling pathway, leading to remarkable attenuation of antibody-mediated platelet destruction. Thus, TNF-α blockade may be a promising therapeutic strategy for the management of ITP.

Platelet factor 4 enhances CD4+ T effector memory cell responses via Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis.

BACKGROUND: Cell metabolism drives T cell functions, while platelets regulate overall CD4+ T cell immune responses. OBJECTIVE: To investigate if platelets influence cell metabolism and thus regulate CD4+ T effector memory cell (Tem) responses. METHODS: Human CD4+ Tem cells were activated with αCD3/αCD28 and cultured without or with platelets or platelet-derived mediators. RESULTS: Polyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4+ Tem cells. Platelet co-culture enhanced Th1 response transiently, while it persistently enhanced Treg cell activation of Tem cells, with an enhancement that plateaued by day 3. Platelet factor 4 (PF4) was the key platelet-derived mediator regulating CD4+ Tem cell responses, which involved cellular metabolisms as indicated by mass spectrometric analyses. PF4 exerted its effects via its receptor CXCR3, attenuated Akt activity, and reduced PGC1α phosphorylation, and resulted in elevations of PGC1α function and mitochondrial transcription factor A (TFAM) synthesis. The latter increased mitochondrial biogenesis, and subsequently enhanced Th1 and Treg responses. Consistent with these observations, inhibition of mitochondrial function by rotenone counteracted the enhancements by recombinant PF4, and TFAM overexpression by TFAM-adenovirus infection mimicked PF4 effects. Furthermore, increased mitochondrial mass elevated oxygen consumption, and enhanced adenosine triphosphate and reactive oxygen species production, which, in turn, stimulated Th1 (T-bet) and Treg (FoxP3) transcription factor expression and corresponding CD4+ T effector cell responses. CONCLUSIONS: Platelets enhance CD4+ T cell responses of Tem cells through PF4-dependent and Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet-regulated immune responses.

PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis.

The binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases. In this study, we detected PD-1 and PD-L expression on T cells and dendritic cells (DCs) in immune thrombocytopenia (ITP) patients with active disease by flow cytometry. The effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells and on secretion of interferon-γ (IFN-γ) and interleukin-2 (IL-2) were detected by flow cytometry and enzyme-linked immunosorbent assay, respectively. Compared with healthy controls, PD-1 expression was significantly increased in CD4+ T cells and CD8+ T cells from patients with active ITP. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls. In vitro assays revealed that PD-L1-Fc increased T cell apoptosis, inhibited activation and proliferation of CD4+ T cells and CD8+ T cells and decreased IFN-γ and IL-2 secretion in patients with active ITP. These results suggest that the aberrant PD-1/PD-L negative co-stimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP patients by enhancing T cell apoptosis, inhibiting T cell activation and proliferation and reducing secretion of inflammatory factors.

CD8(+) T cells induce platelet clearance in the liver via platelet desialylation in immune thrombocytopenia.

In addition to antiplatelet autoantibodies, CD8(+) cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8(+) T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8(+) T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8(+) T cells than those without cytotoxicity and controls. In vitro, CD8(+) T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8(+) splenocytes were used. Platelets co-cultured with these CD8(+) splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8(+) splenocytes. These findings suggest that CD8(+) T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP.

Low-dose decitabine promotes megakaryocyte maturation and platelet production in healthy controls and immune thrombocytopenia.

Impaired megakaryocyte maturation and insufficient platelet production have been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Our previous study demonstrated that low expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in megakaryocytes contributed to impaired platelet production in ITP. Decitabine (DAC), a demethylating agent, is known to promote cell differentiation and maturation at low doses. However, whether decitabine is potential in promoting megakaryocyte maturation and platelet release in ITP is unclear. In this study, we evaluated the effect of DAC on megakaryocyte maturation and platelet release in the presence of ITP plasma that has been shown to cause impaired megakaryocyte maturation and platelet production. We observed that low-dose DAC (10 nM) could significantly increase the number of mature polyploid (≥ 4N) megakaryocytes in cultures with plasma from healthy controls and more than one-half of ITP patients in vitro. Furthermore, the number of platelets released from these megakaryocytes significantly increased compared with those untreated with DAC. In these megakaryocytes, DAC significantly enhanced TRAIL expression via decreasing its promoter methylation status. These findings demonstrate that low-dose DAC can promote megakaryocyte maturation and platelet production and enhance TRAIL expression in megakaryocytes in healthy controls and ITP. The potential therapeutic role of low-dose DAC may be beneficial for thrombocytopenic disorders.

Thalidomide corrects impaired mesenchymal stem cell function in inducing tolerogenic DCs in patients with immune thrombocytopenia.

Thalidomide (THD) is an immunomodulatory agent used to treat immune-mediated diseases. Immune thrombocytopenia (ITP) is an autoimmune disorder in which impaired mesenchymal stem cells (MSCs) are potentially involved. We demonstrated that MSCs in ITP patients had reduced proliferative capacity and lost their immunosuppressive function, which could be corrected with THD treatment. According to the gene profile, the downregulation of caspase-8 and caspase-10, and upregulation of oct3/4 and tgf-ß1, may be associated with THD modulation. Dendritic cells (DCs) played an important role in mediating the inhibitory activity of MSCs. To study the functional alteration of DCs elicited by MSCs, we sorted DCs after incubation with MSCs and performed T-lymphocyte reaction assays. The THD-modulated MSCs from ITP patients induced mature DCs to become tolerogenic DCs, whereas unmodulated MSCs had no effect. The induction of tolerogenicity in DCs by MSCs was dependent on the expression of TIEG1 in DCs. The study reveals the inability of MSCs from ITP patients to induce tolerogenic ability in DCs. THD could restore the regulatory effect of MSCs on DCs. These findings will help us understand the pathogenesis of ITP, and with appropriate safeguards, THD may benefit patients with ITP.

Decreased IDO activity and increased TTS expression break immune tolerance in patients with immune thrombocytopenia.

INTRODUCTION: Indoleamine 2,3-dioxygenase (IDO) can promote peripheral immune tolerance and control autoimmune responses through tryptophan catabolism. Tryptophanyl-tRNA synthetase (TTS) can protect T cells from IDO-mediated cell injury. Impaired IDO-mediated tryptophan catabolism has been observed in some autoimmune diseases. MATERIALS AND METHODS: The concentrations of plasma kynurenine and tryptophan were detected by high-pressure liquid chromatography. The expressions of IDO and TTS were analyzed by real-time quantitative polymerase chain reaction and flow cytometry. RESULTS: Compared with healthy controls, the PBMCs of patients with immune thrombocytopenia (ITP) had significantly increased expressions of IDO and TTS, especially IDO. However, the plasma tryptophan concentration was significantly elevated, and kynurenine concentration was significantly reduced in ITP patients. In CD4(+) and CD8(+) T cells of the ITP patients, IDO expressions were significantly lower than those in healthy controls, but in CD19(+) and CD14(+) cells, IDO expression significantly increased. Conversely, TTS expressions in CD4(+) and CD8(+) T cells of the ITP patients were significantly higher than those in healthy controls, but there was no difference either in CD19(+) or CD14(+) cells. CONCLUSION: These results suggest that the activity of IDO enzyme is insufficient in ITP patients. Increased TTS expressions from CD4(+) and CD8(+) T cells might link to a pathogenic mechanism involved in increasing survival of autoreactive T cells in ITP patients.