Panaxdiol saponins component promotes hematopoiesis by regulating GATA transcription factors of intracellular signaling pathway in mouse bone marrow.

Background: Our research team has identified a biological active component, panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng as a potential targeted drug for treating hemocytopenia. PDS-C possesses dual activities, namely that of promoting hematopoiesis and regulating immune function. Our study is to observe effects of PDS-C on promoting hematopoiesis in normal and aplastic anemia (AA) mice, furthermore, to explore its possible mechanism. Methods: Bone marrow nucleated cells were cultured for colony forming assay of CFU-GM, CFU-E and CFU-MK in the presence of PDS-C at different concentration. The proliferation and differentiation-related genes expression profile was analyzed with DNA membrane microarray. The mRNA expression levels and protein phosphorylated state of GATA-1, GATA-2 transcription factors and AKT-1, MAPK14 protein kinases were detected by RT-qPCR and Western blot, the DNA binding activity and components of GATA-DNA complex were analyzed by EMSA and antibody gel supershift assay. Results: In response to PDS-C at 10, 25 and 50 mg/L, the bone marrow colony numbers of CFU-GM, CFU-E and CFU-MK increased significantly by 25.7%±3.1% to 42.4%±4.5% respectively in normal mice, and 29.7%±3.7% to 53.2%±7.1% in AA mice. The gene microarray profile initiated by PDS-C provided the up-regulated genes by more than 3 times, which can be classified into 11 categories according to their functions, including GATA-1, GATA-2, and AKT-1, MAPK14. The mRNA expression levels of GATA-1, GATA-2 were consistent with their gene microarray profile in PDS-C treated erythroid and megakaryocytic hematopoietic cells. Meanwhile, PDS-C could not only up-regulate expression levels of GATA-1, GATA-2 proteins, but also enhance phosphorylated activity state. Furthermore, PDS-C obviously enhanced binding activity of GATA protein with DNA in erythroid and megakaryocytic cells, and the main composition of GATA-DNA complex was GATA-2 and GATA-1. Conclusions: PDS-C displays the role to promote proliferation and induce differentiation for hematopoietic cells. Its action mechanism may involve in GATA-1, GATA-2 transcription factors, including up-regulating mRNA and protein expression, enhancing DNA binding activity, phosphorylated functional activity and up-regulating AKT-1, MAPK14 protein kinases as the upstream signaling molecule for activation GATA-1, GATA-2 respectively in hematopoietic cells.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

Treatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

[Apoptosis of Endothelial Cells Induced by Anti-Platelet Integrin ß3 Antibody].

OBJECTIVE: To investigate the damaging of human umbilical vein endothelial cells (HUVEC) induced by antiplatelet integrin ß3 antibodies in vitro. METHODS: The serum from 36 chronic ITP patients were collected, flow cytometry and monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were used to collect antiplatelet integrin ß3 antibodies from the serum of the patients. After HUVEC were treated by ITP patient serum (PS) containing anti-integrin ß3 antibodies, the cell damage was detected by Lactate dehydrogenase (LDH) assay, cell apoptosis was detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR (RT-qPCR), and expression of Apoptosis-related signaling pathway protein Akt and related protein Bax were detected by Western blot. HUVEC were treated by PS combined with Akt activator SC79, the cells damage were detected by LDH assay, apoptosis of the cells were detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by RT-qPCR. RESULTS: Among 36 cases of serum from the chronic ITP patients, 5 patients' serum containing anti-integrin ß3 antibodies were collected. After HUVEC was treated by PS, the viability of LDH was significant increased（P<0.05）, so as for the apoptosis of the cells（P<0.05）, the expression of gene and protein of Bax was increased up-regulated（P<0.05）, the protein expression of pAkt was down-regulated（P<0.05）. Comparing with HUVEC cultured with PS alone, the viability of LDH of HUVEC treated by PS combined with SC79 was significantly reduced（P<0.05）, so as for the apoptosis of the cells（P<0.05）, and gene expression of Bax was significantly decreased（P<0.05）. CONCLUSION: Anti-integrin ß3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway，the apoptotic effects of anti-integrin ß3 antibodies to HUVEC was effectively reversed by SC79.

Functional MRI evaluation of the effect of carotid artery stenting: a case study demonstrating cognitive improvement.

BACKGROUND: The narrowing of the carotid arteries with plaque formation represents a major risk factor for ischemic stroke and cognitive impairments. Carotid angioplasty and stenting is a standard clinical treatment to reduce stroke risk. The cognitive effect of carotid angioplasty and stenting remains largely unknown. PURPOSE: This study aims to provide direct evidence of possible effects of carotid angioplasty and stenting on cognition, using task-phase functional magnetic resonance imaging. MATERIAL AND METHODS: This study received harmonized institutional ethics board approval (Grant number REB ID =H18-02495/FHREB 2018-058). Two patients had MRI scans pre-carotid angioplasty and stenting and two-month post-carotid angioplasty and stenting. Case 1 had severe (>95%) flow-limiting stenosis in the right carotid artery. Case 2 had 70% non-flow limiting stenosis in the left carotid artery. At each scan, patients completed two functional magnetic resonance imaging sessions while performing a working memory task. Accuracy, reaction time, and brain activation were analyzed for each patient for possible pre-post carotid angioplasty and stenting changes. RESULTS: Case 1 showed increased activation in the right (treated-side) frontal and temporal lobes post-carotid angioplasty and stenting; associated with improvements in accuracy (from 58% to 74%) and task completion rate (from 17% to 72%). Case 2 completed the tasks pre- and post-carotid angioplasty and stenting with >90% accuracy, while decreased functional magnetic resonance imaging activation in the contralateral (untreated) hemisphere and mildly increased activation in the left (treated -side) anterior circulation territory were observed post-carotid angioplasty and stenting. CONCLUSION: These cases provided the first task-phase functional magnetic resonance imaging data demonstrating that carotid angioplasty and stenting improved cognitive function in the re-perfused vascular territory. The finding supports the role of carotid angioplasty and stenting in improving cognitive performance beyond reducing stroke risk.

SHP2 blockade enhances anti-tumor immunity via tumor cell intrinsic and extrinsic mechanisms.

SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor.

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

Differentiation of CD45­/CD31+ lung side population cells into endothelial and smooth muscle cells in vitro.

Side population (SP) cells are a small subpopulation of cells found in many mammalian tissues and organs, identified by their capacity to efflux Hoechst 33342 dye. They are enriched for stem/progenitor cell activity. SP cells isolated from the adult mouse lung can be separated into a CD45+ subset (bone marrow­derived) and a CD45­ subset that can be subdivided into CD31­ and CD31+ subpopulations. CD45­/CD31­ lung SP (LSP) cells are known to be mesenchymal stem cells. However, CD45­/CD31+ LSP cells are not fully characterized. In the present study, it was found that CD45­/CD31+ LSP cells were able to form colonies. Based on the expression of vascular endothelial growth factor receptor 2 (VEGFR2), these cells were separated into VEGFR2­ and VEGFR2+ cells. The CD45­/CD31+/VEGFR2­ LSP cells expressed genes characteristic of smooth muscle and endothelial progenitors, and were able to differentiate into smooth muscle and endothelial cells in vitro. The CD45­/CD31+/VEGFR2+ LSP cells expressed genes characteristic of endothelial progenitors and gave rise to endothelial cells, although not smooth muscle, in vitro. The data demonstrate that CD45­/CD31+/VEGFR2­ LSP cells differentiated into CD45­/CD31+/VEGFR2+ LSP cells and then endothelial cells, indicating that CD45­/CD31+/VEGFR2+ LSP cells are likely to be derived from CD45­/CD31+/VEGFR2­ LSP cells. Taken together, the results suggest that CD45­/CD31+ LSP cells can be separated into CD45­/CD31+/VEGFR2­ LSP cells, which may be progenitors of endothelial and smooth muscle, whereas CD45­/CD31+/VEGFR2+ LSP cells may serve as late commitment endothelial progenitors in the adult mouse lung.

The application of frequency-domain photoacoustics to temperature-dependent measurements of the Grüneisen parameter in lipids.

The Grüneisen parameter is an essential factor in biomedical photoacoustic (PA) diagnostics. In most PA imaging applications, the variation of the Grüneisen parameter with tissue type is insignificant. This is not the case for PA imaging and characterization of lipids, as they have a very distinct Grüneisen parameter compared with other tissue types. One example of PA applications involving lipids is the imaging and characterization of atherosclerotic plaques. Intravascular photoacoustic (IVPA) imaging is a promising diagnostic tool that can evaluate both plaque severity and composition. The literature for IVPA has mainly focused on using the difference in absorption coefficients between plaque components and healthy arterial tissues. However, the Grüneisen parameters for lipids and their behavior with temperature have not been well established in the literature. In this study we employ frequency-domain photoacoustic measurements to estimate the Grüneisen parameter by virtue of the ability of this modality to independently measure both the absorption coefficient and the Grüneisen parameter through the use of the phase channel. The values of the Grüneisen parameters of some lipids are calculated as functions of temperature in the range 25-45 °C.

Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability.

Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses. Here, we expanded upon these findings and mechanistically dissected the properties that contribute to the potency of this amphiphile-vaccine (amph-vaccine). We found that multiple linkage chemistries could be used to link peptides with DSPE-PEG, and further, that multiple albumin-binding moieties conjugated to peptide antigens enhanced LN accumulation and subsequent T-cell priming. In addition to enhancing lymphatic trafficking, DSPE-PEG conjugation increased the stability of peptides in serum. DSPE-PEG peptides trafficked beyond immediate draining LNs to reach distal nodes, with antigen presented for at least a week in vivo, whereas soluble peptide presentation quickly decayed. Responses to amph-vaccines were not altered in mice deficient in the albumin-binding neonatal Fc receptor (FcRn), but required Batf3-dependent dendritic cells (DCs). Amph-peptides were processed by human DCs equivalently to unmodified peptides. These data define design criteria for enhancing the immunogenicity of molecular vaccines to guide the design of next-generation peptide vaccines. Cancer Immunol Res; 6(9); 1025-38. ©2018 AACR.

The mouse passive-transfer model of MuSK myasthenia gravis: disrupted MuSK signaling causes synapse failure.

While the majority of myasthenia gravis patients express antibodies targeting the acetylcholine receptor, the second most common cohort instead displays autoantibodies against muscle-specific kinase (MuSK). MuSK is a transmembrane tyrosine kinase found in the postsynaptic membrane of the neuromuscular junction. During development, MuSK serves as a signaling hub, coordinating the alignment of the pre- and postsynaptic components of the synapse. Adult mice that received repeated daily injections of IgG from anti-MuSK+ myasthenia gravis patients developed muscle weakness, associated with neuromuscular transmission failure. MuSK autoantibodies are predominantly of the IgG4 type. They suppress the kinase activity of MuSK and the phosphorylation of target proteins in the postsynaptic membrane. Loss of postsynaptic acetylcholine receptors is the primary cause of neuromuscular transmission failure. MuSK autoantibodies also disrupt the capacity of the motor nerve terminal to adaptively increase acetylcholine release in response to the reduced postsynaptic responsiveness to acetylcholine. The passive IgG transfer model of MuSK myasthenia gravis has been used to test candidate treatments. Pyridostigmine, a first-line cholinesterase inhibitor drug, exacerbated the disease process, while 3,4-diaminopyridine and albuterol were found to be beneficial in this mouse model.

Modular Peptide Amphiphile Micelles Improving an Antibody-Mediated Immune Response to Group A Streptococcus.

Inducing a strong and specific immune response is the hallmark of a successful vaccine. Nanoparticles have emerged as promising vaccine delivery devices to discover and elicit immune responses. Fine-tuning a nanoparticle vaccine to create an immune response with specific antibody and other cellular responses is influenced by many factors such as shape, size, and composition. Peptide amphiphile micelles are a unique biomaterials platform that can function as a modular vaccine delivery system, enabling control over many of these important factors and delivering payloads more efficiently to draining lymph nodes. In this study, the modular properties of peptide amphiphile micelles are utilized to improve an immune response against a Group A Streptococcus B cell antigen (J8). The hydrophobic/hydrophilic interface of peptide amphiphile micelles enabled the precise entrapment of amphiphilic adjuvants which were found to not alter micelle formation or shape. These heterogeneous micelles significantly enhanced murine antibody responses when compared to animals vaccinated with nonadjuvanted micelles or soluble J8 peptide supplemented with a classical adjuvant. The heterogeneous micelle induced antibodies also showed cross-reactivity with wild-type Group A Streptococcus providing evidence that micelle-induced immune responses are capable of identifying their intended pathogenic targets.

[Differentiation of Cultivated CD45-/CD31+ Mouse Lung Side Population Cells into Vascular Smooth Muscle Cells in vitro].

OBJECTIVES: To investigate the characteristics of differentiation of lung side population cells (LSP cells)in vitro. METHODS: CD45-/CD31+ LSP cells sorted by flow cytometry were taken from mouse lung tissues and cultured for 14 d. The cultured LSP cells were observed with colony formation assay and flow cytometryin vitro. The mRNA expressions of ATP-binding cassette transporter G2 (ABCG2), smooth muscle actin (SMA) and α-smooth muscle tropomyosin (α-SMT) in both freshly isolated LSP cells and cultured LSP cells were examined. The expressions of ABCG2 and stem cell antigen 1 (Sca1) in LSP cells were detected using immunofluorescence. RT-PCR tests were performed to detect the expressions of ABCG2, SMA and α-SMT in LSP cells. RESULTS: The isolated CD45-/CD31+ lung side population cells expressed ABCG2, SMA and Sca1, but not α-SMT. A large number of LSP in aggregated state were observed after 14 d of culture. Before induction of differentiation, the CD45-/CD31+ LSP cells expressed ABCG2 and SMA, but not α-SMT. After induction of differentiation, the CD45-/CD31+ lung side population cells expressed α-SMT and SMA, but not ABCG2. CONCLUSIONS: CD45-/CD31+ LSP cells might be progenitor cells of vascular smooth muscle cells, possessing the characteristics of stem cell differentiations.

Proliferation, differentiation and migration of SCA1-/CD31- cardiac side population cells in vitro and in vivo.

BACKGROUND: Side-population (SP) cells, identified by their capacity to efflux Hoechst dye, are highly enriched for stem/progenitor cell activity. They are found in many mammalian tissues, including mouse heart. Studies suggest that cardiac SP (CSP) cells can be divided into SCA1+/CD31-, SCA1+/CD31+ and SCA1-/CD31- CSP subpopulations. SCA1+/CD31- were shown to be cardiac and endothelial stem/progenitors while SCA1+/CD31+ CSP cells are endothelial progenitors. SCA1-/CD31- CSP cells remain to be fully characterized. In this study, we characterized SCA1-/CD31- CSP cells in the adult mouse heart, and investigated their abilities to proliferate, differentiate and migrate in vitro and in vivo. METHODS AND RESULTS: Using fluorescence-activated cell sorting, reverse transcriptase/polymerase chain reaction, assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction we show that SCA1-/CD31- CSP cells are located in the heart mesenchyme and express genes characteristic of stem cells and endothelial progenitors. These cells were capable of proliferation, differentiation, migration and vascularization in vitro and in vivo. Following experimental myocardial infarction, the SCA1-/CD31- CSP cells migrated from non-infarcted areas to the infarcted region within the myocardium where they differentiated into endothelial cells forming vascular (tube-like) structures. We further demonstrated that the SDF-1α/CXCR4 pathway may play an important role in migration of these cells after myocardial infarction. CONCLUSIONS: Based on their gene expression profile, localization and ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we conclude that SCA1-/CD31- CSP cells may serve as endothelial progenitor cells in the adult mouse heart.

Forced expression of muscle specific kinase slows postsynaptic acetylcholine receptor loss in a mouse model of MuSK myasthenia gravis.

We investigated the influence of postsynaptic tyrosine kinase signaling in a mouse model of muscle-specific kinase (MuSK) myasthenia gravis (MG). Mice administered repeated daily injections of IgG from MuSK MG patients developed impaired neuromuscular transmission due to progressive loss of acetylcholine receptor (AChR) from the postsynaptic membrane of the neuromuscular junction. In this model, anti-MuSK-positive IgG caused a reduction in motor endplate immunolabeling for phosphorylated Src-Y418 and AChR ß-subunit-Y390 before any detectable loss of MuSK or AChR from the endplate. Adeno-associated viral vector (rAAV) encoding MuSK fused to enhanced green fluorescent protein (MuSK-EGFP) was injected into the tibialis anterior muscle to increase MuSK synthesis. When mice were subsequently challenged with 11 daily injections of IgG from MuSK MG patients, endplates expressing MuSK-EGFP retained more MuSK and AChR than endplates of contralateral muscles administered empty vector. Recordings of compound muscle action potentials from myasthenic mice revealed less impairment of neuromuscular transmission in muscles that had been injected with rAAV-MuSK-EGFP than contralateral muscles (empty rAAV controls). In contrast to the effects of MuSK-EGFP, forced expression of rapsyn-EGFP provided no such protection to endplate AChR when mice were subsequently challenged with MuSK MG IgG. In summary, the immediate in vivo effect of MuSK autoantibodies was to suppress MuSK-dependent tyrosine phosphorylation of proteins in the postsynaptic membrane, while increased MuSK synthesis protected endplates against AChR loss. These results support the hypothesis that reduced MuSK kinase signaling initiates the progressive disassembly of the postsynaptic membrane scaffold in this mouse model of MuSK MG.

Peptide amphiphile micelles self-adjuvant group A streptococcal vaccination.

Delivery system design and adjuvant development are crucially important areas of research for improving vaccines. Peptide amphiphile micelles are a class of biomaterials that have the unique potential to function as both vaccine delivery vehicles and self-adjuvants. In this study, peptide amphiphiles comprised of a group A streptococcus B cell antigen (J8) and a dialkyl hydrophobic moiety (diC16) were synthesized and organized into self-assembled micelles, driven by hydrophobic interactions among the alkyl tails. J8-diC16 formed cylindrical micelles with highly α-helical peptide presented on their surfaces. Both the micelle length and secondary structure were shown to be enhanced by annealing. When injected into mice, J8-diC16 micelles induced a strong IgG1 antibody response that was comparable to soluble J8 peptide supplemented with two classical adjuvants. It was discovered that micelle adjuvanticity requires the antigen be a part of the micelle since separation of J8 and the micelle was insufficient to induce an immune response. Additionally, the diC16 tail appears to be non-immunogenic since it does not stimulate a pathogen recognition receptor whose agonist (Pam3Cys) possesses a very similar chemical structure. The research presented in this paper demonstrates the promise peptide amphiphile micelles have in improving the field of vaccine engineering.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR ß-subunit-Y390 was reduced at endplates. In contrast, endplate staining for ß-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold.

Migration of resident cardiac stem cells in myocardial infarction.

Ischemic heart disease is a major cause of morbidity and mortality worldwide. Stem cell-based therapy, which aims to restore cardiac structure and function by regeneration of functional myocardium, has recently been proposed as a novel alternative treatment modality. Resident cardiac stem cells (CSCs) in adult hearts are a key cell type under investigation. CSCs have been shown to be able to repair damaged myocardium and improve myocardial function in both human and animal studies. This approach relies not only on the proliferation of the CSCs, but also upon their migration to the site of injury within the heart. Here, we briefly review reported CSC populations and discuss signaling factors and pathways required for the migration of CSCs.

Polysaccharides from the root of Angelica sinensis promotes hematopoiesis and thrombopoiesis through the PI3K/AKT pathway.

BACKGROUND: Dozens of Traditional Chinese Medicine (TCM) formulas have been used for promotion of "blood production" for centuries, and we are interested in developing novel thrombopoietic medicines from these TCMs. Our previous studies have demonstrated the hematopoietic effects of DangGui BuXue Tong (DBT), a formula composed of Radix Angelicae Sinensis and Radix Astragali in animal and cellular models. As a step further to identify and characterize the active chemical components of DBT, we tested the hematopoietic and particularly, thrombopoietic effects of polysaccharide-enriched fractions from the root of Radix Angelicae Sinensis (APS) in this study. METHODS: A myelosuppression mouse model was treated with APS (10 mg/kg/day). Peripheral blood cells from APS, thrombopoietin and vehicle-treated samples were then counted at different time-points. Using the colony-forming unit (CFU) assays, we determined the effects of APS on the proliferation and differentiation of hematopoietic stem/progenitor cells and megakaryocytic lineages. Using a megakaryocytic cell line M-07e as model, we analyzed the cellular apoptosis progression with and without APS treatment by Annexin V, Mitochondrial Membrane Potential and Caspase 3 assays. Last, the anti-apoptotic effect of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor (PI3K) was also tested. RESULTS: In animal models, APS significantly enhanced not only the recovery of platelets, other blood cells and their progenitor cells, but also the formation of Colony Forming Unit (CFU). In M-07e cells, we observed the anti-apoptotic effect of APS. Treatment by Ly294002 alone increased the percentage of cells undergoing apoptosis. However, addition of APS to Ly294002-treated cells significantly reduced the percentage of cells undergoing apoptosis. CONCLUSIONS: APS promotes hematopoiesis and thrombopoiesis in the mouse model. This effect likely resulted from the anti-apoptosis activity of APS and is likely to involve the PI3K/AKT pathway.

Drug-induced thrombocytopenia: development of a novel NOD/SCID mouse model to evaluate clearance of circulating platelets by drug-dependent antibodies and the efficacy of IVIG.

Drug-induced immune thrombocytopenia (DITP) is an adverse drug effect mediated by drug-dependent antibodies. Intravenous immunoglobulin (IVIG) is frequently used to treat DITP and primary immune thrombocytopenia (ITP). Despite IVIG's proven beneficial effects in ITP, its efficacy in DITP is unclear. We have established a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of DITP in which human platelets survive for more than 24 hours, allowing platelet clearance by DITP/ITP antibodies to be studied. Rapid human platelet clearance was uniformly observed with all quinine-induced thrombocytopenia (QITP) patient sera studied (mean platelet lifespans: QITP 1.5 ± 0.3 hours vs controls 16.5 ± 4.3 hours), consistent with the clinical presentation of DITP. In contrast, clearance rates with ITP antibodies were more variable. IVIG treatment partially prevented platelet clearance by DITP and ITP antibodies. Our results suggest that the NOD/SCID mouse model is useful for investigating the efficacy of current and future DITP therapies, an area in which there is little experimental evidence to guide treatment.