Number Of Cells In Peripheral Blood Research Articles

Screening of Translocation Ets-Leukemia-Acute-Myeloid-Leukemia-1 Fusion Gene and Expression Pattern of Multidrug Resistance Protein Protein in Children with Acute Lymphoblastic Leukemia

The study aimed to investigate the positive rate of TEL-AML 1 fusion gene in acute lymphoblastic leukemia (ALL) children and the clinical characteristics of ALL patients with TEL-AML 1 fusion gene positively expressed, as well as the expression level of MRP-1. 40 ALL children were selected, with their medical records collected. The TEL-AML 1 fusion gene was screened by nested RTPCR. Bone marrow specimens were taken for G-banded karyotype analysis and flow cytometry immunophenotyping of the marrow chromosome. A semi-quantitative RT-PCR method was used to study the mRNA expression level of MRP 1. The results showed that the positive rate of TEL-AML 1 fusion gene in ALL patients was 22.5% (9/40). The positive group exhibited lower gene expression level, the hepatosplenomegaly degree, the total number of peripheral white blood cells, the absolute count of naive cells, and the Hb level at the first visit, indicating that the tumor burden of children in the positive group was lower. The complete remission rate of the positive group was higher (P < 0.05). The mRNA expression level of MRP 1 gene positive group was lower. In conclusion, patients with positive TEL-AML 1 fusion gene were more sensitive to chemotherapeutic drugs, and their treatment responses and prognosis were better.

Novel Effects of Combination Therapy Through Inhibition of Caspase-1/Gasdermin D Induced-Pyroptosis in Lupus Nephritis.

ObjectivesCombination therapy with mycophenolate mofetil, tacrolimus and steroids are effective in achieving complete remission in lupus nephritis (LN). Combination therapy uniquely downregulated caspase-1 compared with monotherapies, which can cleave gasdermin D (GSDMD) and was recently identified as the pyroptosis executioner. We therefore investigated whether combination therapy enabled the suppression of caspase-1/GSDMD-mediated pyroptosis in LN.MethodsExpression and activation of GSDMD were detected in kidney specimens of the human and mouse with LN using immunohistochemical staining and immunoblotting. Primary podocytes isolated from MRL/lpr mice were incubated with LPS+ATP, and pretreated with monotherapy or combination therapy. Inhibition of caspase-1/GSDMD-induced pyroptosis by combination therapy were assessed in MRL/lpr mice and human specimens. Pyroptosis was examined using a FAM caspase-1 kit and flow cytometry. The correlation between pyroptosis in peripheral blood and the systemic lupus erythematosus disease activity index (SLEDAI) was analyzed.ResultsKidney tissue specimens from LN patients and mice exhibited greatly increased expression levels and cleavage of GSDMD. In cultured podocytes, combination treatment significantly suppressed the activation of NLRP3 and caspase-1 and reduced GSDMD N-terminal levels. Combination therapy repressed disease progression through inhibition of caspase-1/GSDMD-mediated pyroptosis in both humans and MRL/lpr mice. Caspase-1/PI positive cell numbers in peripheral blood were positively correlated with SLE-DAI. LN patients with complete remission and partial remission had remarkably reduced caspase-1/PI positive cell numbers compared to baseline. Ac-FLTD-CMK, a GSDMD-derived inhibitor, prevented the development of LN.ConclusionCombination therapy suppressed caspase-1/GSDMD-mediated pyroptosis in vitro and in vivo and reduced disease progression.

The IL-2/IL-15 Mimetic NL-201 Prevents Myeloma Relapse after ASCT By Expanding Highly Cytolytic T Cells in the Bone Marrow That Are Resistant to Exhaustion

Multiple myeloma (MM) is a bone marrow (BM) resident hematological malignancy that is becoming increasingly recognized as one amenable to immunotherapy, although no therapies have yet provided durable, long-term disease control. Autologous stem cell transplantation (ASCT), the standard of care in eligible patients, provides a window for intervention with immunotherapy due to the induction of inflammation in the context of lymphodepletion at a time where there is also minimal residual disease and a disrupted tumor microenvironment (TME). We have previously established that the addition of T cells to BM grafts results in enhanced long-term myeloma control post-transplant in mice. Novel approaches aimed at improving and/or expanding the endogenous T cell response early post-ASCT may therefore prove highly effective with the benefit of avoiding ex vivo processing associated with other cellular therapies. To explore this, we utilized the IL-2/IL-15 mimetic NL-201: a de novo cytokine mimetic that signals via the beta and gamma subunits of the IL-2 receptor without engaging IL-2Rα (CD25). In pre-clinical studies, NL-201 has demonstrated the ability to signal to effector CD4 and CD8 T cells while avoiding the toxicity usually associated with IL-2 signaling via IL-2Rα.We hypothesized that NL-201 would enhance control of myeloma progression by stimulating T cell proliferation and activation early post-ASCT. We transplanted lethally irradiated Vk*MYC myeloma-bearing B6 recipients with BM and T cells graft from B6 donors and administered NL-201 from D+7 to week 6 (225 μg/kg weekly I.P). NL-201 promoted potent anti-myeloma immunity that was dependent on CD4 and CD8 T cells, but not NK cells (median survival was 68 days for control mice, unreached at >120 days for NL-201 alone or with NK depletion, 86 days for NL-201 with CD8 depletion, and 74 days with CD4 depletion; PBS vs NL-201 p<0.01; PBS vs NL-201 + αNK1.1 p<0.01; NL-201 vs NL-201 + αCD4 or αCD8 p<0.05). To further elucidate potential mechanisms of action we harvested BM from PBS and NL-201-treated mice 2 days after the last dose was administered and performed comprehensive immunophenotyping with high parameter flow cytometry. We grouped recipients based on whether they had controlled myeloma (MM-controlled) or had active disease progression at the time of harvest (MM-relapsed) to reveal immunological phenotypes that were dependent and independent of myeloma in the TME. In these experiments, all NL-201-treated recipients had controlled myeloma at time of harvest. Mechanistically, NL-201 significantly expanded the total number of CD8 T cells in the BM compared to PBS-treated mice with controlled or relapsed MM (PBS-treated mean CD8 T cell number was 1.0 x 10 5/femur vs 7.7 x 10 5/femur in NL-201-treated mice) but did not impact CD8 T cell number in peripheral blood. Memory CD8 T cells (CD44+CD62L+) were preferentially expanded, while the frequency of exhausted CD8 T cells (TOX +PD-1 +TIGIT +CD39 +; T EX) was reduced in NL-201-treated mice compared to both PBS-treated MM-relapsed and MM-controlled mice (75% T EX in PBS MM-relapsed, 15% PBS MM-controlled, 2% in NL-201; p<0.001). Surprisingly, >80% of the memory CD8 T cells in NL-201-treated mice produced granzyme B compared to <10% in PBS-treated mice. Granzyme B production was also observed in conventional CD4 T cells in response to NL-201 treatment, and the frequency of regulatory T cells was reduced by 50% after NL-201 compared to PBS MM-controlled and MM-relapsed mice (p<0.001).NL-201 expanded bone marrow resident cytotoxic memory CD8 and CD4 T cells that are resistant to exhaustion, whilst reducing the frequency of regulatory T cells in the BM TME. Together, these data highlight the promising therapeutic potential of NL-201 in multiple myeloma and support testing NL-201 in clinical trials for the treatment of hematological malignancies. DisclosuresHill: NapaJen Pharma: Consultancy; Roche: Research Funding; Syndax Pharmaceuticals: Research Funding; iTeos Therapeutics: Consultancy, Research Funding; Applied Molecular Transport: Research Funding; Compass Therapeutics: Research Funding; NeoLeukin Therapeutics: Consultancy; Generon Corporation: Consultancy.

Ex Vivo IL-2 Treatment Increases the Immunosuppressive Subset of T Lymphocytes to Suppress Allo-Immune Response

T lymphocytes are critical players in graft-versus-host disease (GVHD); however, their specific roles and mechanisms in GVHD have not been clearly defined. Co-inhibitory receptors including PD1, TIM3, TIGIT, and LAG3 play a key role in regulating T cell responses and maintaining immune homeostasis. TIM3 expression on lymphocytes was identified to induce immune tolerance in a mouse GVHD model; in addition, PD1 +TIM3 + double-positive T cells displayed more potent immunosuppression compared to PD1 + T cells. However, despite the strong immunosuppression exerted by PD1 +TIM3 + double-positive T cells, their clinical potential is greatly limited by their low cell number in peripheral blood. In this study, we introduce a novel method to isolate CD3 + cells from G-CSF mobilized peripheral blood stem cells (G-PBSCs), and culture CD3 +PD1 +TIM3 + lymphocytes to treat GVHD.Methods. The donors were subcutaneously injected with G-CSF (10μg/kg) for five days. G-PBSCs were collected from the donors using a COBE spectra cell separator. Then, the highly purified CD3 + cells were isolated by positive selection with magnetic-activated cell sorting (MACS) from G-PBSCs using CD3 Dynabeads™. Isolated CD3 + cells were cultured with a low concentration of IL-2 (50U/mL), and PD1, TIM3, LAG3, and TIGIT expressions were assessed using flow cytometry (FACS). CD3 +PD1 +TIM3 +, CD3 +PD1 +TIM3 +LAG3 +TIGIT - and CD3 +PD1 +TIM3 +LAG3 +TIGIT +cells are sorted and cultured with irradiated allo-MNCs for 4 days (Mixed Lymphocyte Reaction; MLR). We used 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) as an intracellular fluorescent dye in MLR (CFSE-MLR) to measure of T cells proliferation.Results. In normal, untreated G-PBSCs, very low percentages of cells are CD3 +PD1 +TIM3 +, CD3 +PD1 +LAG3 + and CD3 +PD1 +TIGIT + (0.6±0.4, 0.3±0.2 and 1.3±0.5% in G-PBSC, respectively). However, after treating G-PBSC cells with a low concentration of IL-2 (50 U/mL), we discovered the percentages of CD3 +PD1 +TIM3 +, CD3 +PD1 +LAG3 +, and CD3 +PD1 +TIGT + lymphocytes in G-PBSCs markedly increased by 19.6±5.9, 18.5±4.3, 17.7±6.5%, respectively. Of note, there was a very small change (~1.2 fold increase) in the total number of CD3 + lymphocytes, which indicates that the low dose IL-2 therapy alters subpopulations of T lymphocytes, rather than increasing the proliferation rate. Next, we tested the immunosuppressive capacity of the cultured CD3 +PD1 +TIM3 +cells. To do this, we used CSFE-MLR to measure T cell proliferation. CD3 +PD1 +TIM3 + lymphocytes were depleted in CD3 + G-PBSCs by flow cytometry-based cell sorting. Then we cultured the CD3 + G-PBSCs with allo-MNCs and discovered that the CD3 + G-PBSCs that lacked CD3 +PD1 +TIM3 + lymphocytes demonstrated a significantly increased level of T-cell proliferation compared to CD3 + G-PBSCs, and thereby confirming the immunosuppressive function of CD3 +PD1 +TIM3 + lymphocytes in inhibiting T cell proliferation (% of unstimulated T cells in CD3 + G-PBSCs vs. in CD3 +PD1 +TIM3 + depleted G-PBSC CD3 + cells; 33.5±7.5% vs. 6.3±4.2%).After confirming the immunosuppressive function of the IL-2 treated CD3 +PD1 +TIM3 + lymphocytes, we investigated expressions of other co-inhibitory surface markers such as LAG3 and TIGIT. Amongst the cultured CD3 +PD1 +TIM3 + lymphocytes, 96.2±3.3% of the CD3 +PD1 +TIM3 + lymphocytes were LAG3 +, 28.9±5.4% of the CD3 +PD1 +TIM3 + lymphocytes were TIGIT +, and 63.7±7.8% of the cells were TIGIT -. Interestingly, CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes demonstrated significantly enhanced levels of immunosuppression compared to CD3 +PD1 +TIM3 +LAG3 +TIGIT + lymphocytes (% of unstimulated T cells in CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes vs. in CD3 +PD1 +TIM3 +LAG3 +TIGIT + lymphocytes, 53.8±5.5% vs. 23.2±7.4%).Conclusion.We demonstrate that treating CD3 + G-PBSCs with low-dose IL-2 markedly increases the percentage of CD3 +PD1 +TIM3 + lymphocytes, which is known to exert strong immunosuppression. Also, we suggest that CD3 +PD1 +TIM3 +LAG3 +TIGIT - lymphocytes are the subpopulation within CD3 +PD1 +TIM3 + cells that demonstrate the most potent immunosuppression compared to other subpopulations of CD3 +PD1 +TIM3 + lymphocytes. Taken together, such findings suggest that the low-dose IL-2 therapy has therapeutic potential to treat patients with GVHD. DisclosuresNo relevant conflicts of interest to declare.

Pharmacodynamic Analysis of CAR-T Cell Persistence in Patients with Hematologic Malignancies Treated with NKTR-255, an IL-15 Receptor Agonist That Enhances CD8 + T-Cells: Preliminary Results from a Phase 1 Study

Pharmacodynamic Analysis of CAR-T Cell Persistence in Patients with Hematologic Malignancies Treated with NKTR-255, an IL-15 Receptor Agonist That Enhances CD8 + T-Cells: Preliminary Results from a Phase 1 Study

Serum S100A8 and S100A9 as prognostic biomarkers in acute exacerbation of idiopathic pulmonary fibrosis.

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is a devastating and life-threatening condition during its clinical course. Biomarkers for precisely anticipating the prognosis of AE-IPF remain to be fully established. The objective of this study was to clarify whether S100A8 and S100A9, which are calcium-binding proteins mainly produced by activated neutrophils, are significant prognostic biomarkers in AE-IPF. Thirty-seven patients with AE-IPF who were diagnosed and treated at our hospital were retrospectively evaluated. The serum levels of S100A8 and S100A9 were measured using enzyme-linked immunosorbent assay, and the relationships between these levels and clinical parameters or prognosis were evaluated. The serum levels of S100A8 (median 386.5ng/mL) and S100A9 (median 60.2ng/mL) in patients with AE-IPF were significantly higher than those in age-matched healthy controls and in patients at IPF diagnosis (p<0.001 for all combinations). The serum levels of S100A8 negatively correlated with percent forced vital capacity (r=-0.356, p=0.049) and positively correlated with peripheral white blood cell number (r=0.509, p=0.002). Immunohistochemical staining of autopsy lung specimens showed that neutrophils, present mainly in the alveolar septum, were positive for S100A8 and S100A9. Patients with AE-IPF with higher levels of S100A8 or S100A9 showed significantly worse 3-month survival than those with lower levels (log-rank test, both p=0.028). Finally, in multivariate analysis, the serum levels of both S100A8 and S100A9 were significant prognostic factors (hazard ratio 4.032, p=0.023 and hazard ratio 4.327, p=0.012). The serum levels of S100A8 and S100A9 at AE were significant prognostic biomarkers in patients with AE-IPF.

The Influence of Low-Dose Occupational Radiation Exposure on Peripheral Blood Cell in a Cohort Chinese Medical Radiation Workers

The study aims to assess the change of peripheral blood cell number following protracted low-dose radiation exposure among medical radiation workers.A cohort of 185 Chinese radiation workers which divided into 4 groups by different occupational categories which include Interventional radiologists group (IRG); nuclear medicine doctors group (NMD); medical diagnostic radiographers group (MDR); radiologists group (RG) were followed by 5 years (2015-2019). This study observed and recorded the changes in the cumulative dose and peripheral blood cell counts consecutively for five years. While exploring the changes in blood cells and cumulative dose, the correlation between dose and the quantity of peripheral blood cells was also discussed. Individual doses were recorded by dose monitoring system. The counts of peripheral blood cell were based on the physical examination data of the workers of the annual physical examination in the hospitality-test, SD-T test and Variance analysis were used to analyze the radiation dose-response changes of the peripheral blood cell. Correlation analysis was used to analyze the correlation between the number of peripheral blood cells and the cumulative dose.The results of this study showed that compared with the control group, the number of white blood cells (WBCs), the number of hemoglobin (Hb) and the ratio of eosinophils (EO%) in the study group decreased (P < 0.05). Correlation analysis between the average cumulative dose in different years and the number of peripheral blood cells in different groups showed that the average cumulative dose of nuclear medicine group in 2016 was negatively correlated with WBC (r = -0.602, P < 0.05) and NE% (r = -0.596, P < 0.05). There was a significant positive correlation between the average cumulative dose of the radiological diagnosis group in 2015 and MO% (r = 0.530, P < 0.01). For the radiologists' group, there was a significant negative correlation between the average cumulative dose and the number of WBCs in 2016 (r = -3.37, P < 0.05), and there was a significant negative correlation between the average cumulative dose and RBC in 2017 (r = -4.12, P < 0.05).The results of the study suggest that the blood cell levels of workers exposed to low-dose ionizing radiation for a long time can show dynamic changes, and relevant workers should take appropriate protection measures and regularly monitor peripheral blood cell indicators.

There is no gene for CVID - novel monogenetic causes for primary antibody deficiency.

'There is no gene for fate' (citation from the movie 'GATTACA') - and there is no gene for CVID. Common Variable ImmunoDeficiency (CVID) is the most prevalent primary immunodeficiency in humans. CVID is characterized by an increased susceptibility to infections, hypogammaglobulinemia, reduced switched memory B cell numbers in peripheral blood and a defective response to vaccination, often complicated by autoimmune and autoinflammatory conditions. However, as soon as a genetic diagnosis has been made in a patient with CVID, the diagnosis must be changed to the respective genetic cause (www.esid.org). Therefore, there are genetic causes for primary antibody deficiencies, but not for CVID. Primary antibody deficiencies (PADs) are a heterogeneous group of disorders. Several attempts have been made to gain further insights into the pathogenesis of PAD, using unbiased approaches such as whole exome or genome sequencing. Today, in just about 35% of cases with PAD, monogenic mutations (including those in the gene TNFRSF13B) can be identified in a set of 68 genes [1•]. These mutations occur either sporadically or are inherited and do explain an often complex phenotype. In our review, we not only discuss gene defects identified in PAD patients previously diagnosed with CVID and/or CVID-like disorders such as IKZF1, CTNNBL1, TNFSF13 and BACH2, but also genetic defects which were initially described in non-CVID patients but have later also been observed in patients with PAD such as PLCG2, PIK3CG, PMS2, RNF31, KMT2D, STAT3. We also included interesting genetic defects in which the pathophysiology suggests a close relation to other known defects of the adaptive immune response, such as DEF6, SAMD9 and SAMD9L, and hence a CVID-like phenotype may be observed in the future. However, alternative mechanisms most likely add to the development of an antibody-deficient phenotype, such as polygenic origins, epigenetic changes, and/or environmental factors.

Comparative study of peripheral blood cells in two marine fishes (Synechogobius hasta and Sebastes schlegelii): Morphological and cytochemical characterization

The morphology, cell size and relative number of peripheral blood cells in two teleosts, Synechogobius hasta and Sebastes schlegelii, were compared using different staining methods. The results showed significant differences in cell size. The percentage of monocytes in S. hasta was greater than that in S. schlegelii (P < 0.01); however, the opposite results were obtained in the percentage of lymphocytes and thrombocytes. The two fishes shared common cytochemical-staining results, which showed that both erythrocytes were negative for all cytochemical staining; monocytes were strongly positive for PAS and positive for SBB and NAE; lymphocytes were negative for SBB, POX and NAE; neutrophils were positive for SBB and NAE; and thrombocytes were negative for SBB, ALP, POX and NAE. However, species specificity existed in the cytochemical properties. For S. hasta, monocytes were strongly positive for ALP and ACP; lymphocytes were strongly positive for ALP and weakly positive for ACP; neutrophils were strongly positive for ACP and POX; and thrombocytes were weakly positive for PAS and positive for ACP. Unlike S. hasta, monocytes were strongly positive for ACP and positive for ALP in S. schlegelii; lymphocytes were positive for ALP and partially positive for PAS; neutrophils were positive for ACP and POX; and thrombocytes were negative for PAS and ACP. The POX activity of monocytes in S. hasta was greater than that in S. schlegelii (P < 0.05), while the POX activity of neutrophils and the NAE activity of monocytes in S. hasta were significantly greater than those in S. schlegelii (P < 0.01). The results of this study can be used as a reference for the construction of haematological parameters in S. hasta and S. schlegelii for the assessment of fish health and can provide a research basis for fish diseases and immunity.

Altered HSC Programming Drives Aberrant Blood Output Underlying RA Pathogenesis

Rheumatoid arthritis (RA) is a debilitating autoimmune disorder characterized by autoantibody production towards collagen type-II in the synovial joints of the skeletal system, leading to progressive joint damage. RA is accompanied by chronic systemic production of pro-inflammatory cytokines including IL-6, TNF, and IL-1. Importantly, RA patients often suffer from significant hematological complications including production of tissue-damaging macrophages, chronic anemia, impaired lymphopoiesis and suppressed bone marrow function. As blood-forming hematopoietic stem cells (HSC) constitute the root of the immune system, identifying mechanisms by which altered HSC function may contribute to disease could provide key insights into RA pathogenesis and therapeutic intervention.In this study, we used a mouse model of collagen-induced arthritis (CIA), which faithfully recapitulates many features of human autoimmune arthritis, to identify the impact of disease on the hematopoietic compartment, and identify mechanisms underlying alteration in blood lineage output and function. Strikingly, bone marrow hematopoietic populations were significantly altered in CIA mice. This was typified by decreased numbers of peripheral red blood cells (RBC) and bone marrow erythroid progenitors, alongside decreases in bone marrow B cells and common lymphoid progenitors (CLP). On the other hand, we observed increased numbers of platelets, bone marrow granulocytes, and myeloid-biased multipotent progenitors (MPP3). Strikingly, we also observed reductions in the stromal cells comprising the bone marrow niche, with decreases in epithelial, mesenchymal, and osteoblastic cell types, suggesting that the inflammatory effects of RA influence the bone marrow compartment at multiple levels. We functionally validated our phenotypic analyses using colony forming unit (CFU) assays, and observed increased myeloid CFU, with concomitant decreases in erythroid and lymphoid colony forming activity in unfractionated bone marrow cells from CIA mice. Despite these changes, transplantation assays using purified HSCs from CIA and control mice showed that long-term reconstitution potential of HSCs was largely unaltered, suggesting that HSC self-renewal capacity is not significantly impaired. Taken together, these observations suggest that autoimmune arthritis induces substantial remodeling of the bone marrow, resulting in overproduction of myeloid cells at the expense of other lineages.In line with our phenotypic and functional analyses, RNA-seq analysis of HSCs from CIA versus control mice uncovered precocious activation of myeloid-specific gene programs that likely underlie shifts in lineage output. Moreover, in line with our transplantation data, we did not observe significant shifts in the expression of core genes governing HSC self-renewal. Collectively, these data show that the inflammatory environment of RA causes changes to hematopoietic output, leading to an increase in damaging myeloid cell types, further fueling the inflammatory environment and disease progression.We are currently identifying the mechanism underlying myeloid overproduction in the bone marrow, with focus on systemic inflammatory factors and/or shifts in the gastrointestinal microbiota, as potential underwriters of deregulated hematopoiesis and disease development. We are also assessing the functionality of myeloid cells generated by HSCs during RA to determine whether they exhibit a stronger pro-inflammatory phenotype. Ultimately, these studies will increase our understanding of the crosstalk between RA and the hematopoietic system, and potentially opens new avenues for therapeutic intervention to re-establish the hematopoietic system and even rescue RA progression. DisclosuresNo relevant conflicts of interest to declare.

Platelet-Targeted Gene Therapy of Hemophilia a Using Hematopoietic Stem Cells Derived from Genome-Edited Induced Pluripotent Stem Cells

Hemophilia A (HA), an X-linked recessive hereditary bleeding disorder, is caused by mutations in the F8 gene, which encodes the coagulation factor VIII (FVIII). Replacement therapy is the mainstay in clinic, but it can also cause inhibitory antibodies and is therefore restricted in wide use. Effective therapy of HA with inhibitors needs to be considered as a matter of priority. Evidence has been obtained that functional FVIII can be ectopically expressed and stored in platelets after transplantation of F8 gene modified-hematopoietic stem cells (HSCs), and the stored-FVIII can be released during the activation of platelets to exert therapeutic effect on HA with inhibitors. This unique strategy, however, requires enough sources of HSCs and a safe and effective way of HSCs modification. Induced pluripotent stem cells (iPSCs) are capable of self-renewing, large-scale expansion and differentiating into almost all types of cells and tissues. These features make iPSCs a promising path in the field of regenerative medicine and cell therapy. The recently developed techniques of genome editing, especially the CRISPR/Cas9 system, provide a powerful tool for gene therapy, owing to significantly reduced risk of insertional mutagenesis as compared to the random integration in retroviral-mediated gene therapy. Hence, we seek to explore the possibility of utilizing iPSCs and CRISPR/Cas9 in the platelet-targeted gene therapy of HA.Here, we established a HA mouse iPSCs (HA-iPSCs) derived from mouse-tail tip fibroblasts, and verified the pluripotency of these cells. On the other hand, we synthesized a codon optimized B domain-deleted FVIII construct (opBDD-F8) in order to enhance the expression efficiency of FVIII. Indeed, our in vitro expression data showed that opBDD-F8 achieved a nearly 4-fold increase of FVIII level as compared to BDD-F8, either under control of the platelet specific GPαIIb promoter or CMV promoter. This increased FVIII expression was further confirmed by hydrodynamic tail vein injection. Then, we inserted the GPαIIb promoter controlled opBDD-F8 (2bopF8) cassette into the Rosa26 locus of HA-iPSCs via CRISPR/Cas9-mediated homologous recombination, and obtained 2bopF8-modified HA-iPSCs (2bopF8-HAiPSCs). No obvious off-target mutation was found in the clones. To generate functional HSCs for transplantation, we performed an in vivo differentiation strategy. We injected 2bopF8-HAiPSCs with hHoxB4 retrovirus transduction into immunodeficient mice to create teratoma, from which we isolated HSCs. The cells were subsequently transplanted into lethally irradiated HA recipient mice. Hematopoiesis was recovered in the recipients, and normal peripheral blood cell numbers sustained for more than 40 weeks. FVIII was detected in platelets of the recipients, suggesting that the cells were originated from 2bopF8-HAiPSCs and FVIII could be expressed during thrombopoiesis. Bleeding phenotype was corrected in the recipients, demonstrating that FVIII released during platelet activation was functional in ensuring coagulation cascade. Furthermore, we did secondary HSC transplantation by using the recipients as the donors. FVIII activity could be detected in the platelets of the new recipients, and bleeding phenotype was corrected in these animals, indicating that long-term HSCs could be generated from 2bopF8-HAiPSCs. During the whole period of research, the recipients were all in good health without teratoma formation or other sickness. Furthermore, we successfully produced chimeric mice with the 2bopF8-HAiPSCs. FVIII expression was observed in the platelets and HSCs were isolated from the mice. Transplantation of these HSCs into lethally irradiated HA mice reconstituted hematopoiesis and generated platelets with FVIII activity. In these mice, coagulation deficiency was also corrected.Taken together, our proof of principle studies suggest that the genome-edited 2bopF8-HAiPSCs could differentiate towards HSCs with physiological functions, and the functional platelets could be derived from these HSCs. Meanwhile, FVIII could be expressed in platelets and abrogate bleeding diathesis of HA mice. Thus, iPSC-derived HSCs together with genome editing tools represent a potential route for platelet-targeted gene therapy that is worth further investigation. DisclosuresNo relevant conflicts of interest to declare.

Shengxuening Extracted from Silkworm Excrement Mitigates Myelosuppression via SCF-Mediated JAK2/STAT3 Signaling.

Shengxuening (SXN) is a Chinese patent medicine with main ingredients (including chlorophyll derivatives and sodium iron chlorophyllin) extracted from silkworm excrement. SXN exhibited efficacy in clinical trials of renal anemia and iron deficiency anemia; however, the specific mechanisms remain unclear. This study found that SXN increased the number of peripheral blood cells and improved the bone marrow morphology in myelosuppressed mouse model, reversed the reduction in body weight and spleen indices, and increased the serum levels of erythropoietin and granulocyte-macrophage colony-stimulating factor. Quantitative real-time PCR array and Western blot analysis showed the enhanced expression of stem cell factor (SCF), JAK2, and STAT3 in the liver. These results suggested that SXN promoted the recovery of hemopoietic function in myelosuppressed models by increasing the secretion of hematopoietic factors and activating the JAK2/STAT3 pathway. Therefore, this medicine may be applied as therapeutic pharmaceutical drug to mitigate myelosuppression.

Screening of biomarkers in exhaled breath of mice exposed to benzene

Objective: To screen the biomarkers in the exhaled breath of mice exposed to benzene by using exhaled breath online analysis system. Methods: Thirty 8-week-old male C57BL/6 mice were randomly divided into six groups (0, 3, 32, 324, 648, and 1 296 mg/m3) and treated with benzene vapour for 28 days. At the end of the exposure, the peripheral blood cell counts and blood glutathione (GSH) were detected. The content of malondialdehyde (MDA) in HL60 cells treated by mice plasma was examined. Exhaled breath data from mice were collected by Secondary electrospray ionization source high resolution mass spectrometry (SESI-HRMS). Targeted analysis underlying benzene metabolites and oxidative stress metabolites was performed to screen the biomarkers in exhaled breath. Results: After benzene exposure, the number of peripheral blood cells was decreased in different degrees, particularly in the white blood cells (WBC) number. The WBC in 32 and 324 mg/m3 groups was declined by 27.76% and 52.87%, respectively compared to that in control group (P<0.05). Meanwhile, compared with the control group, the GSH content of peripheral blood cells from 324 mg/m3 group decreased by 13.16% (P<0.05). In addition, MDA content was increased by 18.11% in HL60 cells treated with plasma from 324 mg/m3 group mice (P<0.05). The phenol, hydroquinone/catechol, benzenetriol and trans, trans-Muconic acid (t,t-MA) in the exhaled gas of mice could be used as biomarkers for benzene exposure (R2>0.8, P<0.001). The peak intensity of five small molecular metabolites related to oxidative stress (ω-carboxylic fatty acid C5H10O3, ω-carboxylic fatty acid C6H12O3, glutamate, cysteine and MDA) increased with the increase of benzene concentration (P<0.05), which was negatively correlated with WBC decline (P<0.001), suggesting that these molecules mignt be used as biomarkers of benzene-induced toxicity. Conclusions: Phenol, hydroquinone/catechol, benzenetriol and trans, trans-Muconic acid (t,t-MA) in exhaled breath of mice could be used as biomarkers for benzene exposure; ω-carboxylic fatty acid C5H10O3, ω-carboxylic fatty acid C6H12O3, glutamate, cysteine and MDA might be used as markers of benzene-induced toxicity.

Radioprotective Effect of Whey Hydrolysate Peptides against γ-Radiation-Induced Oxidative Stress in BALB/c Mice.

Radiation therapy is widely used in the treatment of tumor diseases, but it can also cause serious damage to the body, so it is necessary to find effective nutritional supplements. The main purpose of this study is to evaluate the protective effect of whey hydrolysate peptides (WHPs) against 60Coγ radiation damage in mice and explore the mechanism. BALB/c mice were given WHPs by oral gavage administration for 14 days. Then, some mice underwent a 30-day survival test after 8 Gy radiation, and other mice received 3.5 Gy radiation to analyze the changes in body weight, hematology and bone marrow DNA after three and 14 days. In addition, through further analysis of the level of oxidative stress and intestinal barrier function, the possible mechanism of the radioprotective effect of WHPs was explored. The study found WHPs can prolong survival time, restore body weight, and increase the number of peripheral blood white blood cells and bone marrow DNA content in irradiated mice. In addition, WHPs can significantly improve the antioxidant capacity, inhibit pro-inflammatory cytokines and protect the intestinal barrier. These results indicate that WHPs have a certain radioprotective effect in mice, and the main mechanism is related to reducing oxidative damage.

Danggui Buxue Decoction enhances the anticancer activity of gemcitabine and alleviates gemcitabine-induced myelosuppression

Ethnopharmacological relevanceDanggui Buxue Decoction (DBD) as a traditional Chinese medicine (TCM) has been widely used to treat blood deficiency. With the immune regulation and hematopoietic effect, DBD improved the quality of life in non-small-cell lung cancer (NSCLC) patients. We previously reported that DBD sensitized the response of NSCLC to Gemcitabine (Gem); however, the synergism and attenuation mechanism on the combination of Gem and DBD has not yet been elucidated. Aim of the studyTo investigate the mechanisms of DBD in enhancing the anticancer activity of Gem and alleviating Gem-induced myelosuppression. Materials and methodsA549 nude mice model was established to study the effect on the combination of Gem and DBD. The organ indices, peripheral blood cells and the hematopoiesis-related cytokines were analyzed in Gem-induced myelosuppressive mice. Then we studied the whole process from Gem-induced bone marrow suppression to self-healing, and the mechanism of DBD's attenuation by the experiments of bone marrow nucleated cells (BMNCs). ResultsThere were an enhanced anticancer effect and an improvement of hematopoietic function by combining of Gem and DBD in A549 nude mice model. DBD regulated Hu antigen R (HuR), deoxycytidine kinase (dCK) and nuclear factor erythroid 2-related factor (Nrf2), increased the expression of thrombopoietin (TPO) and granulocyte-macrophage colony stimulating factor (GM-CSF). For Gem-induced myelosuppressive mice, DBD improved the number of peripheral blood cells and the levels of hematopoiesis-related cytokines. Moreover, DBD was observed to reduce deoxyribonucleic acid (DNA) content at the G1 phase, promoted BMNCs proliferation and up-regulated cycle-related proteins. ConclusionsThe results indicated that DBD not only improved the sensitivity of Gem but also alleviated Gem-induced myelosuppression. This study may provide a pharmacological basis for the combination of DBD and Gem in clinical application.

Benign Prostatik Patolojilerde Regülatuvar T Hücrelerinin (Treg) Değerlendirilmesi: Pilot Çalışma

Objective: We aimed to evaluate the number and function of regulatory T (Treg) cells in peripheral blood and prostate tissues of patients with histopathologically diagnosed benign prostate hyperplasia (BPH) and asymptomatic chronic prostatitis.&#x0D; Material and Methods: Blood and histopathological data of 19 patients (BPH=10, ACP=9) that underwent transurethral prostate resection were evaluated. Treg cell count in peripheral blood and prostatic tissue with flowcytometry, Forkhead box P3 (FOXP3) expression in prostatic tissue by reverse transcription polymerase chain reaction (PCR), and IL-17 measurement in blood samples with ELISA were performed. &#x0D; Results: Flowcytometric analyses showed that mean CD4+T cell count and mean FOXP3 levels in both peripheral blood (CD4+T, p= 0.752; FOXP3, p= 1.000) and prostate tissue (CD4+T, p= 0.458; FOXP3, p= 0.590) were higher in the BPH group compared to the chronic prostatitis group. However this difference was not statistically significant. Similarly, the mean blood IL-17 levels were also higher in BPH groups, but the difference was not statistically significant (p= 0.870). The PCR analyses showed that mean FOXP3 gene expression in the tissue was higher in the chronic prostate group, but again there was no statistically significant difference between the groups (p= 0.116).&#x0D; Conclusion: Since no statistically significant difference was found between BPH and chronic prostatitis in terms of Treg cell number and function in peripheral blood and prostatic tissue, our study supports the thesis that both these pathologies could be autoimmune inflammatory diseases.

Immunophenotype of Gastric Tumors Unveils a Pleiotropic Role of Regulatory T Cells in Tumor Development.

Simple SummaryThe role of regulatory T cells (Tregs) in gastric cancer (GC) is still controversial and poorly understood. GC patients have increased numbers of Tregs in peripheral blood and among tumor infiltrating lymphocytes; however, their prognostic value depends on specific tumor features (e.g., tumor location and/or microsatellite instability status). We found that Tregs might induce membrane expression of IL2Rα in intestinal-type GC cells, which associates with MAPK signaling pathway activation and spheroid growth. Moreover, Tregs accumulate at early steps of intestinal-type GCs progression, when tumors are starting to grow through the stomach wall, and do not present vascular and perineural invasion. Our findings suggest a novel non-immunosuppressive role of Treg cells in intestinal-type GC, which may unlock novel therapeutic immuno-oncology strategies for intestinal-type GC or other tumors with similar immune context.Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context.

Evaluation of the effectiveness and safety of N-Chromosome Royal Jelly on the number of peripheral white blood cells in patients with acute lymphoblastic leukemia

Objectives: This study to investigate the immune response blood cells following N-Chromosome RJ administration in patients with childhood ALL.&#x0D; Methods: This single center before and after 12-weeks clinical trial was done in Ali-Asghar Children Hospital on twenty-six patients diagnosed with ALL during maintenance phase of chemotherapy. After collecting their demographics, patients received a starter dose of 2-gram processed new natural N-chromosome RJ before breakfast and were followed up each 2 weeks, counting their peripheral WBC, ANC and ALC by a blood cell count and differential analysis.&#x0D; Results: A total of Twenty-six patients were enrolled in this study (16 males and 10 females). Mean Peripheral WBC count of total patients were raised significantly after administering N-RJ Being 2510 ±1192 cells per cubic millimeter at the beginning and then raised to 4549±1500 cells per cubic millimeter at the end of trial. (P&lt;0.005) None of patients suffered from any adverse reaction during the trial.&#x0D; There was a positive statistical relationship between total peripheral WBC, ANC and ALC count and N-Chromosome Royal jelly increased dosage, Being more prominent at the beginning of trial (p&lt;0.001) and the last two weeks of follow-ups. (p&lt;0.0005)&#x0D; Conclusion: This study has successfully demonstrated that N-chromosome RJ can be a promising immune- enhancing supplement in patients diagnosed with acute lymphoblastic leukemia in their complete remission and maintenance therapy time. In addition, it is a natural alternative for drugs like G-CSF, but without those long-term adverse effects, we can see in G-CSF drugs.

Effect of Indirubin and Sheng-Xue-Xiao-Ban Capsule (SXXBC) on Promoting Peripheral Platelet in ITP Model Mice

To compare the effect of Sheng-Xue-Xiao-Ban Capsule (SXXBC) and indirubin to the peripheral platelets of the Idiopathic thrombocytopenic purpura (ITP) model mouse. The ITP mouse model was established by the method of passive immunization. SXXBC and indirubin were used for intervention treatment. Then the hemorrhagic phenomena of ITP mice were observed and the numbers of peripheral platelets, hemoglobin and white blood cells, bone marrow megakaryocytes and their classification and coagulation function were detected and compared. The improvement rate of hemorrhage in SXXBC group was 40% for small dose, 60% for medium dose and 80% for high dose, while the improvement rate of hemorrhage in indirubin group was 30% for small dose, 50% for medium dose and 60% for high dose. There was no statistically significant difference in the improvement rate of hemorrhage between the two groups (P＞0.05). Compared with the model control group, PLT and Hb increased in different doses of SXXBC and indirubin group 4th-8th day after drug intervention (P＜0.05, 0.01). However, there was no significant difference between the different doses of SXXBC group and indirubin group (P＞0.05). Compared with the model control group, the WBC in each group was significantly lower (P＜0.05, 0.01) on the 4th-8th day after drug intervention; However, there was no statistical significance between the two groups of SXXBC and indirubin (P＞0.05). Compared with the model control group, the total number of megakaryocytes in each treatment group were decreased (P＜0.05, P＜0.01), in which the number of primary megakaryocytes in the large and medium dose groups of SXXBC and indirubin were decreased (P＜0.05, 0.01), and the number of juvenile megakaryocytes in the large dose group of SXXBC and indirubin were also decreased (P＜0.05). The number of granular megakaryocytes were decreased in each intervention groups (P＜0.05, 0.01), and the number of thromocytogenic megakaryocyte was increased in the high and medium dose groups of SXXBC and indirubin (P＜0.01). The time of prothrombin was shortened in the high and medium dose groups of SXXBC and indirubin (P＜0.05), and the fibrinogen (FIB) content in the high and medium dose groups of SXXBC was close to that of the normal control group. Both of the SXXBC and the indirubin standard all show good hemostatic effects. Indirubin shows a positive effect on increasing the peripheral platelet and hemoglobin in ITP model mice, regulating the immune response, reducing the total number of bone marrow megakaryocytes, increasing the thromocytogenic megakaryocyte, and increasing coagulation function.

Elucidation of the molecular mechanism of Sanguisorba Officinalis L. against leukopenia based on network pharmacology

Leukopenia is the most common hallmark of hematopoietic diseases in clinic. Sanguisorba Officinalis L., a traditional Chinese medicine, has long been used for alleviating leukopenia. However, its associated mechanism still remains unknown. In this study, a network pharmacology approach was used to elucidate the underlying mechanisms of Sanguisorba Officinalis L. against leukopenia. Firstly, 12 active compounds of Sanguisorba Officinalis L. were identified through TCMSP database with absorption, distribution, metabolism, excretion (ADME) screening, and UHPLC-MS analysis. Then, 258 leukopenia related targets of the identified active compounds were predicted via the Swiss Target Prediction database, GeneCards database and DisGeNET database, respectively. After taking the intersection of two related targets, 72 common targets were selected. Among them, 8 core targets (VEGFA, HSP90AA1, EGFR, PTGS2, MTOR, ESR1, ERBB2, MDM2) of Sanguisorba Officinalis L. against leukopenia were obtained through the topological analysis. Meanwhile, both the GO and KEGG pathway analysis reveal that the core targets are mainly enriched in PI3K-Akt, HIF-1, VEGF and estrogen signaling pathways. In addition, molecular docking simulation was performed to explore the binding ability between the 12 active compounds of Sanguisorba Officinalis L. with 8 core targets. Furthermore, a myelosuppressive mice model was established to evaluate the protective effect of Sanguisorba Officinalis L. against leukopenia. The results showed that the ethanol extract of Sanguisorba Officinalis L. significantly raised the number of peripheral white blood cells. Overall, this study provides an insight into the underlying mechanisms of Sanguisorba Officinalis L. against leukopenia, which lays a theoretical foundation for the further experimental verification and clinical application.