Identification of a novel mutation in the factor XIII A subunit in a patient with inherited factor XIII deficiency.

Inherited factor XIII (FXIII) deficiency is an extremely rare and under-diagnosed autosomal recessive inherited coagulopathy, which is caused by genetic defects in the F13A1 or F13B gene. More than 200 genetic mutations have been identified since the first case of inherited FXIII deficiency was reported. This study aimed to identify underlying gene mutations in a patient with inherited FXIII deficiency who presented with recurrent intracerebral hemorrhage. Levels of plasma FXIII-A antigen were measured, F13A1 and F13B genes were sequenced, mutation information was analyzed, and the mutated protein structure was predicted using bioinformatics methods. Molecular genetic analysis identified four mutations of FXIII-related genes in the proband, including three previously reported mutations inherited from his parents (c.631G>A, p.Gly210Arg and c.1687G>A, p.Gly562Arg of F13A1 gene and c.344G>A, p.Arg115His of F13B gene) and a novel spontaneous mutation of F13A1 gene (c.2063C>G, p.Ser687Cys). Molecular structural modeling demonstrated that the novel Ser687Cys mutation may cause changes in the spatial structure of FXIII-A and increase its instability. In conclusion, we identified a novel and likely pathogenic mutation of the F13A1 gene, which enriched the gene mutation spectrum of inherited FXIII deficiency. The findings may provide promising targets for diagnosis and treatment of inherited FXIII deficiency.

Exploring the Potential Toxicological Mechanisms of Vine Tea on the Liver Based on Network Toxicology and Transcriptomics.

Objective: This study focuses on whether vine tea contains potentially toxic components that trigger hepatotoxicity as a mechanism of action, which further provides some reference for the consumption and guides future product development of vine tea. Methods: The chemical components of vine tea were collected from the reported literature and the toxicological information matched with the CTD database was collected, and the dataset of potential toxic components was established. The toxic components were submitted to the PharmMapper server to obtain potential targets. At the same time, the relevant targets were searched in the CTD database and GeneCards database with keywords such as "Hepatic Toxicity," "Liver Damage," and "Drug-induced liver injury." After intersection, the potential hepatotoxic targets of vine tea were obtained. The protein interactions of potential hepatotoxic targets of vine tea were analyzed by the STRING database. Protein-protein interaction (PPI) networks were constructed by Cytoscape3.6.1 software. The GO molecular function and KEGG pathway of hepatotoxic targets were enriched by the R package to screen the key targets. The role of the components and key targets was analyzed by the LEDOCK program. The data from GEO database were mined for the functional correlation characterized by cell transcriptional expression caused by vine tea as a disturbance factor. Results: This study has searched 34 potential toxic components and 57 potential hepatotoxic targets of vine tea, and the result showed that these targets were mainly involved in oxidative stress, cell metabolism, and apoptosis to affect the liver. Conclusion: Vine tea has the interrelationship of multi-components, multi-targets, and multi-pathways. At the cellular level, the toxic components of vine tea, mainly flavonoids, may promote oxidative stress, promote oxidation to produce free radicals, guide apoptosis, and affect cell metabolism and other cytotoxic mechanisms. However, this hepatotoxicity is related to the dose, duration of vine tea, and individual differences. This study revealed the potential hepatotoxic components of vine tea and provides a reference for further research and development of related functional products.

Proteomic analysis and microRNA expression profiling of plasma-derived exosomes in primary immune thrombocytopenia.

Exosomes are released into extracellular fluids and have emerged as vital biological mediators in autoimmune diseases. Plasma-derived exosomes have been reported to take part in the pathogenesis of primary immune thrombocytopenia (ITP), but the protein and miRNA cargoes have not been entirely elucidated. Via proteomic analysis and RNA sequencing on plasma-derived exosomes from ITP patients and healthy controls, we found one upregulated exosomal protein (apolipoprotein E, ApoE), six downregulated exosomal miRNAs (miR-584-5p, miR-4433a-5p, miR-4433b-3p, miR-6842-3p, miR-130b-5p and miR-222-3p), and 10 upregulated exosomal miRNAs (miR-29a-3p, miR-142-5p, miR-16-2-3p, miR-29b-3p, miR-501-3p, miR-144-5p, miR-192-5p, miR-182-5p, miR-363-3p and miR-96-5p) in ITP patients. The elevated exosomal protein candidate ApoE in the ITP cohort was further validated using western blot. Via quantitative real-time polymerase chain reaction assays, three differentially expressed miRNAs (miR-584-5p, miR-142-5p and miR-29b-3p) were identified. This study provides direct evidence for a restricted signature of exosomal protein and miRNAs which distinguishes ITP from healthy controls. The results require further validation in larger independent ITP cohorts, which will provide insights into the potential pathophysiological significance of circulating exosomes in ITP.

Dexamethasone plus oseltamivir versus dexamethasone in treatment-naive primary immune thrombocytopenia: a multicentre, randomised, open-label, phase 2 trial.

BACKGROUND: Primary immune thrombocytopenia is an autoimmune bleeding disorder. Preclinical reports suggest that the sialidase inhibitor oseltamivir induces a platelet response in the treatment of immune thrombocytopenia. This study investigated the activity and safety of dexamethasone plus oseltamivir versus dexamethasone alone as initial treatment in adult patients with primary immune thrombocytopenia. METHODS: This multicentre, randomised, open-label, parallel group, phase 2 trial was done in five tertiary medical hospitals in China. Eligible patients were aged 18 years or older with newly diagnosed, treatment-naive primary immune thrombocytopenia. Participants were randomly assigned (1:1), using block randomisation, to receive either dexamethasone (orally at 40 mg per day for 4 days) plus oseltamivir (orally at 75 mg twice a day for 10 days) or dexamethasone monotherapy (orally at 40 mg a day for 4 days). Patients who did not respond to treatment (platelet counts remained <30 × 109 cells per L or showed bleeding symptoms by day 10) were given an additional cycle of dexamethasone for 4 days in each group. Patients in the dexamethasone plus oseltamivir group who relapsed (platelet counts reduced again to <30 × 109 cells per L) after an initial response were allowed a supplemental course of oseltamivir (75 mg twice a day for 10 days). The coprimary endpoints were 14-day initial overall response and 6-month overall response. Complete response was defined as a platelet count at or above 100 × 109 cells per L and an absence of bleeding. Partial response was defined as a platelet count at or above 30 × 109 cells per L but less than 100 × 109 cells per L and at least a doubling of the baseline platelet count and an absence of bleeding. A response lasting for at least 6 months without any additional primary immune thrombocytopenia-specific intervention was defined as sustained response. All patients who were randomly assigned and received the allocated intervention were included in the modified intention-to-treat population analysis. This study has been completed and is registered with ClinicalTrials.gov, number NCT01965626. FINDINGS: From Feb 1, 2016, to May 1, 2019, 120 patients were screened for eligibility, of whom 24 were ineligible and excluded, 96 were enrolled and randomly assigned to receive dexamethasone plus oseltamivir (n=47) or dexamethasone (n=49), and 90 were included in the modified intention-to-treat analysis. Six patients did not receive the allocated intervention. Patients in the dexamethasone plus oseltamivir group had a significantly higher initial response rate (37 [86%] of 43 patients) than did those in the dexamethasone group (31 [66%] of 47 patients; odds ratio [OR] 3·18; 95 CI% 1·13-9·23; p=0·030) at day 14. The 6-month sustained response rate in the dexamethasone plus oseltamivir group was also significantly higher than that in the dexamethasone group (23 [53%] vs 14 [30%]; OR 2·17; 95 CI% 1·16-6·13; p=0·032). During the median follow-up of 8 months (IQR 5-14), two of 90 patients discontinued treatment due to serious adverse events (grade 3); one (2%) patient with general oedema in the dexamethasone plus oseltamivir group and one (2%) patient with fever in the dexamethasone group. The most frequently observed adverse events of any grade were fatigue (five [12%] of 43 in the dexamethasone plus oseltamivir group vs eight [17%] of 47 in the dexamethasone group), gastrointestinal reactions (eight [19%] vs three [6%]), insomnia (seven [16%] vs four [9%]), and anxiety (five [12%] vs three [6%]). There were no grade 4 or 5 adverse events and no treatment-related deaths. INTERPRETATION: Dexamethasone plus oseltamivir offers a readily available combination therapy in the management of newly diagnosed primary immune thrombocytopenia. The preliminary activity of this combination warrants further investigation. Multiple cycles of oseltamivir, as a modification of current first-line treatment, might be more effective in maintaining the platelet response. FUNDING: National Natural Science Foundation of China.

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.

Dexamethasone suppresses the Th17/1 cell polarization in the CD4+ T cells from patients with primary immune thrombocytopenia.

INTRODUCTION: Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease with increased Th17 cells in peripheral blood. Th17/1 cells, which were recently characterized as a new differentiated Th17 lineage secreting IL-17 and IFN-γ, play an important role in the pathogenesis of multiple autoimmune diseases. In this study, we investigated whether Th17/1 cells are involved in the pathogenesis of ITP. MATERIALS AND METHODS: Peripheral blood was obtained from 44 ITP patients and 50 healthy controls. The percentages of T cell subsets were evaluated. We also detected molecular signature of Th17/1 cells in CD4+ T cells. Besides, CD4+ T cells from ITP patients were treated with dexamethasone, the inhibitor of NF-κB, or rapamycin to evaluate the impact and mechanism of dexamethasone treatment on Th17/1 cells. RESULTS: We found an elevated percentage and an enhanced specific molecular signature of Th17/1 cells in CD4+ T cells in ITP patients. The percentage of Th17/1 cells was correlated positively with Th17 cells in ITP patients and healthy controls. The percentage of Th17/1 cells was correlated with corticosteroid resistance. Dexamethasone reversed the molecular signature of Th17/1 cells and decreased the percentage of Th17/1 cells in vitro. Treatment of dexamethasone and the inhibitor of NF-κB suppressed the phosphorylation of STAT3, while dexamethasone treatment also inhibited the phosphorylation of NF-κB p65. CONCLUSIONS: Our data suggested Th17/1 cells may contribute to the pathogenesis of ITP and dexamethasone could inhibit Th17/1 cells through NF-κB/STAT3 pathway. These results may provide a potential therapeutic strategy of correcting the Th17/1 cell deviation in ITP.

Interleukin-37 reduces inflammation and impairs phagocytosis of platelets in immune thrombocytopenia (ITP).

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet count with heterogeneous bleeding manifestations. Severe bleeding in ITP is not completely related with low platelet count. Fcγ receptor (FcγR)-mediated platelet destruction is one of the major mechanisms of ITP. Interleukin-37 (IL-37) is a fundamental natural suppressor of innate immunity and inflammatory responses in several autoimmune diseases. However, the role of IL-37 in the pathogenesis of ITP is unknown. In the present study, we identified that IL-37 expression was elevated in ITP patients, which was correlated with platelet count and the severity of bleeding in ITP, indicating that IL-37 could be a candidate in evaluating disease severity of ITP. In the in vitro study, IL-37 initiated an anti-inflammatory effect on monocytes/macrophages from ITP patients by down-regulating the phosphorylation of MAPK, AKT, and NF-κB signaling pathways. Moreover, IL-37 restored the balance of activating and inhibitory FcγRs and decreased antibody-mediated platelet phagocytosis by monocytes/macrophages. Our findings suggest that IL-37 may be a promising indicator of the disease severity and supplementation of IL-37 may be therapeutically beneficial for ITP patients.

Indirubin modulates CD4+ T-cell homeostasis via PD1/PTEN/AKT signalling pathway in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by an immune mediated decrease in platelet number. Disturbance of CD4+ T-cell homeostasis with simultaneous decrease of CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4+ effector T cells underpin the pathophysiology of ITP. Indirubin is an active ingredient of a traditional Chinese herb called Indigofera tinctoria L. which is clinically used for the treatment of ITP patients. Whether indirubin targets the Tregs/effector T cell-axis to restore platelet number is unknown. In our in vitro studies, Indirubin could significantly enhance the number and function of Tregs and meanwhile dampen the activation of effector T cells in a dose-dependent manner. Indirubin was observed to restore the expression of programmed cell-death 1 (PD1) and phosphatase and tensin homolog (PTEN) on the CD4+ T cells of ITP patients, leading to the subsequent attenuation of the AKT/mTOR pathway. Furthermore, these observations were recapitulated in an active murine model of ITP with a prominent platelet response. Thus, our results identified a potentially novel mechanism of the therapeutic action of indirubin in the treatment of ITP through regulating the homeostasis of CD4+ T cells in a PD1/PTEN/AKT signalling pathway.

Platelet desialylation is a novel mechanism and a therapeutic target in thrombocytopenia during sepsis: an open-label, multicenter, randomized controlled trial.

BACKGROUND: Studies in murine models suggested that platelet desialylation was an important mechanism of thrombocytopenia during sepsis. METHODS: First, we performed a prospective, multicenter, observational study that enrolled septic patients with or without thrombocytopenia to determine the association between platelet desialylation and thrombocytopenia in patients with sepsis, severe sepsis, and septic shock. Gender- and age-matched healthy adults were selected as normal controls in analysis of the platelet desialylation levels (study I). Next, we conducted an open-label randomized controlled trial (RCT) in which the patients who had severe sepsis with thrombocytopenia (platelet counts ≤50 × 109/L) were randomly assigned to receive antimicrobial therapy alone (control group) or antimicrobial therapy plus oseltamivir (oseltamivir group) in a 1:1 ratio (study II). The primary outcomes were platelet desialylation level at study entry, overall platelet response rate within 14 days post-randomization, and all-cause mortality within 28 days post-randomization. Secondary outcomes included platelet recovery time, the occurrence of bleeding events, and the amount of platelets transfused within 14 days post-randomization. RESULTS: The platelet desialylation levels increased significantly in the 127 septic patients with thrombocytopenia compared to the 134 patients without thrombocytopenia. A platelet response was achieved in 45 of the 54 patients in the oseltamivir group (83.3%) compared with 34 of the 52 patients in the control group (65.4%; P = 0.045). The median platelet recovery time was 5 days (interquartile range 4-6) in the oseltamivir group compared with 7 days (interquartile range 5-10) in the control group (P = 0.003). The amount of platelets transfused decreased significantly in the oseltamivir group compared to the control group (P = 0.044). There was no difference in the overall 28-day mortality regardless of whether oseltamivir was used. The Sequential Organ Failure Assessment score and platelet recovery time were independent indicators of oseltamivir therapy. The main reason for all of the mortalities was multiple-organ failure. CONCLUSIONS: Thrombocytopenia was associated with increased platelet desialylation in septic patients. The addition of oseltamivir could significantly increase the platelet response rate, shorten platelet recovery time, and reduce platelet transfusion. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-IPR-16008542 .

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.

Thrombopoietin: a potential diagnostic indicator of immune thrombocytopenia in pregnancy.

To evaluate whether the serum thrombopoietin levels in pregnancy-associated immune thrombocytopenia (ITP) differ from those in gestational thrombocytopenia, and reveal the possibility of thrombopoietin serving as a marker for differential diagnosis. Serum thrombopoietin concentration was determined in ITP in pregnancy (n = 35), gestational thrombocytopenia (n = 31), healthy pregnancy (n = 32), age-matched nonpregnant ITP (n = 32) and nonpregnant healthy controls (n = 35) by ELISA. The serum thrombopoietin level of ITP in pregnancy (1283 ± 646 pg/mL) was significantly higher than gestational thrombocytopenia (187 ± 64 pg/mL) (P < 0.01), although the platelet counts of these two disorders may overlap. Twenty-nine of 35 patients with ITP in pregnancy had thrombopoietin values >500 pg/mL, whereas none of the gestational thrombocytopenia patients' thrombopoietin levels exceeded 500 pg/mL. In addition, ITP in pregnancy presented a markedly higher thrombopoietin level than nonpregnant ITP (88 ± 41 pg/mL) (P < 0.01), indicating that the pathogenesis of pregnant and nonpregnant ITP was different. Our findings suggest that measurement of serum thrombopoietin concentration provides valuable diagnostic information for differentiating ITP in pregnancy from gestational thrombocytopenia. Thrombopoietin represents a reliable marker for ITP in pregnancy.

High-dose dexamethasone corrects impaired myeloid-derived suppressor cell function via Ets1 in immune thrombocytopenia.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. In this study, the MDSC population was evaluated in adult patients with primary immune thrombocytopenia (ITP), where cell-mediated immune mechanisms are involved in platelet destruction. Our data demonstrated that both the numbers and suppressive functions of MDSCs were impaired in the peripheral blood and spleens of patients with ITP compared with healthy control patients. High-dose dexamethasone (HD-DXM) treatment rescued MDSC numbers in patients with ITP. And DXM modulation promoted the suppressive function of MDSCs induced in vitro. Moreover, the expression of interleukin 10 and transforming growth factor ß was significantly upregulated in DXM-modulated MDSCs compared with the unmodulated cultures. DXM-modulated MDSCs inhibited autologous CD4(+)T-cell proliferation and significantly attenuated cytotoxic T lymphocyte-mediated platelet lysis, further indicating enhanced control over T-cell responses. Elevated expression of the transcription factor Ets1 was identified in DXM-modulated MDSCs. Transfection of Ets-1 small interfering RNA efficiently blocked regulatory effects of MDSCs, which almost offset the augmentation of MDSC function by DXM. Meanwhile, splenocytes from CD61 knockout mice immunized with CD61(+)platelets were transferred into severe combined immunodeficient (SCID) mouse recipients (C57/B6 background) to induce a murine model of severe ITP. We passively transferred the DXM-modulated MDSCs induced from bone marrow of wild-type C57/B6 mice into the SCID mouse recipients, which significantly increased platelet counts in vivo compared with those receiving splenocyte engraftment alone. These findings suggested that impaired MDSCs are involved in the pathogenesis of ITP, and that HD-DXM corrected MDSC functions via a mechanism underlying glucocorticoid action and Ets1.

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.

Interleukin 27 inhibits cytotoxic T-lymphocyte-mediated platelet destruction in primary immune thrombocytopenia.

Cytotoxic T-lymphocyte (CTL)-mediated platelet destruction and aberrant cytokine profiles play important roles in the pathogenesis of primary immune thrombocytopenia (ITP). Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the effect of IL-27 on CTL activity in ITP has not been reported. In the present study, platelets from ITP patients were cultured with autologous CTLs in the presence of IL-27. We found that IL-27 could inhibit CTL-mediated platelet destruction. In these IL-27-treated CTLs, granzyme B and T-bet expression decreased significantly, whereas granzyme A, perforin, and eomesodermin were not affected. To further investigate the role of granzyme B in CTL-mediated platelet destruction, granzyme B inhibitor was added and platelet apoptosis was significantly inhibited. These results suggest that IL-27 negatively regulates CTL cytotoxicity toward platelets in ITP by decreasing granzyme B expression, which is associated with reduced T-bet expression. IL-27 may have a therapeutic role in treating ITP patients.