Peripheral Blood White Blood Cell Research Articles

Efficient and Stable Makassar Base Editing in Nonhuman Primates

Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin chain. This nucleotide can be modified using adenine base editors (ABEs) to convert the defective SCD β-globin gene (HBB S) into the non-pathogenic Makassar β-globin (HBB M) variant. Proof-of-concept for this editing strategy in human and mouse hematopoietic stem cells (HSCs) has been previously demonstrated by transplantation after ex vivo modification as well as by in vivo adenoviral editing followed by selection. Here, we evaluated the feasibility, long-term efficiency, and safety of autologous ex vivo Makassar base editing in the preclinical nonhuman primate (NHP) large animal model to pave the way towards clinical applications in humans. As NHPs don't carry the sickle mutation (position A7 in the protospacer), bystander editing at position A9 was monitored as a surrogate. NHP CD34 + hematopoietic stem and progenitor cells (HSPCs) were enriched, CD34 +CD90 + HSCs FACS-purified as previously reported to increase the editing efficiency of short- and long-term engrafting cells, primed over-night, and ABE8e-NRCH base editor mRNA was delivered by electroporation. Base-edited HSCs were cultured overnight and then combined with unmodified CD34 +CD90 - cells and infused into animals after myeloablative total body irradiation. The infusion product was quality controlled by flow-cytometry, next generation sequencing (NGS), and assessing the mono-/bi-allelic base editing efficiency on a single cell level from colony-forming cell (CFC) assays. Animals were followed for up to 200 days analyzing the editing efficiency in peripheral blood (PB) white blood cells (WBCs), FACS-purified lineages from the PB, bone marrow (BM) WBCs, as well as FACS-purified BM HSPCs. Comprehensive bioinformatics analysis was performed on the generated NGS data to determine the frequency of editing over time as well as the on- and off-target editing within the protospacer at all four target locations. Finally, chromosomal integrity after base editing was confirmed performing Kromatid karyotyping assays on gene-edited BM-derived HSPCs. Ex vivo base editing of CD34 +CD90 + was highly reproducible at the Makassar locus reaching more than 60% efficiency within bulk HSCs. No impact of base editing on the myeloid and erythroid differentiation of HSCs was seen in CFC assays. Clonal analysis of colonies revealed that more than 90% of HSCs in the infusion product were edited with > 20% of cells showing biallelic editing. Gene-edited HSCs combined with unmodified progenitors rapidly engrafted in myeloablated animals without any noticeable delay in the recovery of neutrophils, red blood cells, as well as platelets and multilineage reconstitution was established within 3-6 months. Most importantly, stable editing of >20% was observed in PB WBCs and across all lineages throughout the entire follow-up. Similarly, the BM stem cell compartment fully recovered to baseline within 6-month post-transplant. Engrafted HSPCs demonstrated normal erythro-myeloid colony-formation potential. Assessment of editing in colonies showed persistence of HSCs with mono- and bi-allelic Makassar editing 6-month post-transplant. Closely matching the editing efficiency in the PB, all BM lineages, HSPC subsets, and colonies demonstrated >20% editing efficiency confirming successful editing of long-term persisting multipotent HSCs. Finally, comprehensive assessment of NGS data from all tissues in conjunction with the Kromatid assay revealed no unwanted off-target effects or chromosomal rearrangements. Editing was primarily observed at positions A9 and A12 confirming highly specific on-target editing. In summary, we demonstrate efficient, persistent, and safe base editing of NHP HSCs for the treatment of SCD. The editing efficiency of the bystander adenine in NHP HSCs is almost identical to the previously observed editing in human patient HSCs to generate the Makassar HBB. Base edited HSCs showed no impairment in homing, long-term engraftment, or multilineage differentiation in the NHP. These findings have major implications for the application of base editing in patients with hematological disease and disorders.

Administration of granulocyte-macrophage colony-stimulating factor enhanced chimeric antigen receptor T-cell expansion and cellular immunity recovery without inducing cytokine release syndrome.

Neutropenia and cytokine release syndrome (CRS) are two major toxicities of chimeric antigen receptor (CAR)-T cell therapy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an ideal candidate treatment for neutropenia except for its potential aggravation of CRS. We hypothesized that the optimal timing of supplemental with GM-CSF in a shortage of host immunity and CAR T-cell was chosen as avoidance of CRS. In the study we evaluated the safety and efficacy of GM-CSF intervention post-CAR T-cell therapy while circulating CAR T-cell declined. Nine patients received GM-CSF therapy who displayed moderate neutropenia with absolute neutrophil counts (ANC) < 1,500 cells/mm3 with concomitant declination of circulating CAR T-cell. The median duration of GM-CSF intervention was 15 days (4-30). CAR T-cell expansion was observed in peripheral blood (PB) of seven patients (7/9). The median baseline and peak CAR T cells count in PB of the seven patients with CAR T-cell expansion were 0.85 × 106/L (0-50.9) and 6.06 × 106/L (1.43-112.55). And the peaks of CAR T-cell levels in PB appeared in day 7 (2-11) following the initiation of GM-CSF administration with increases of 2.84 × 106/L (0.38-61.65). Also, increased white blood cells in PB were observed in all patients. The median onset and duration time of WBC recovery were 9 (1-14) and 17 (3-53) days. Moreover, the increment of WBC, neutrophil, lymphocyte and CD3-CD16 + CD56 + natural killer cell in PB was observed. In addition, no CRS or fatal infection occurred during GM-CSF treatment. This study provides evidence for the clinical feasibility of combining CAR T-cell therapy with the GM-CSF to treat neutropenia patients with concomitant declination of circulating CAR T-cell.

Dahuang Mudan decoction repairs intestinal barrier in chronic colitic mice by regulating the function of ILC3

Ethnopharmacological relevanceDahuang Mudan decoction (DMD) is a classic prescription for treating intestinal carbuncle from Zhang Zhongjing's “Essentials of the Golden Chamber” in the Han Dynasty. Recent studies also prove that DMD has a therapeutic effect on ulcerative colitis (UC), but its mechanism is still unclear. Aim of studyIn this study, we aim to assess the therapeutic effect of DMD on DSS-induced chronic colitis in mice and deeply expound its underlying regulative mechanism. Materials and methodsThe efficacy of DMD on mice with 2% DSS-induced chronic colitis was examined by changes in mouse body weight, DAI score, colon length changes, peripheral blood white blood cells (WBC) and red blood cells (RBC) counts, and hemoglobin (HGB) content, using mesalazine as a positive control. A small animal imaging system observed the FITC-Dextran fluorescence distribution in mice, and the contents of IL-22 and IL-17A in colon tissue homogenate supernatant and LPS in peripheral blood were detected by ELISA. Fluorescence in situ molecular hybridization and bacterial culture were used to investigate bacterial infiltration in intestinal mucosa and bacterial translocation in mesenteric lymph nodes and spleen. Mice immune function was further evaluated by analyzing the changes in spleen index, thymus index, and the ratio of peripheral blood granulocytes, monocytes, and lymphocytes. Meanwhile, the proportion of NCR+ group 3 innate lymphoid cells (ILC3), NCR-ILC3, and IL-22+ILC3 in colonic lamina propria lymphocytes of mice was detected by flow cytometry. The contents of effectors IL-22, IL-17A, and GM-CSF were detected by RT-PCR. We use cell scratching to determine the effect of DMD conditioned medium on the migration of Caco-2 cells by establishing an in vitro model of MNK-3 conditioned medium (CM) intervening Caco-2 cells. RT-PCR and WB detect the expression of tight junction ZO-1, Occludin, and Claudin-1. ResultsDMD restored the body weight, colon length, peripheral blood RBC numbers, and HGB content of chronic colitis mice and reduced peripheral blood WBC and colon inflammatory cell infiltration. Moreover, DMD decreased LPS content in serum, bacterial infiltration of colonic mucosa, and bacterial translocation in spleen and mesenteric lymph nodes. Simultaneously, DMD intensified the expression of ZO-1, Occludin, and Claudin-1, the ratio of NCR+ILC3 and IL-22+ILC3, and decreased the proportion of NCR-ILC3. In vitro studies also confirmed that the conditioned medium of DMD promoted the migration of Caco-2 cells and the expression of tight junction proteins. ConclusionOur results confirm that DMD improves inflammation and restores intestinal epithelial function in mice with chronic colitis, and the mechanism may be related to regulating ILC3 function.

Reassessing accuracy of blood cell correction factor for traumatic lumbar puncture

ObjectiveThis study assessed the utility of correction formulas for procedural peripheral blood admixture with cerebrospinal fluid (CSF) in adult patients using in vitro addition of peripheral blood. MethodsParticipants from the Johns Hopkins CSF Disorders Center were recruited if greater than 18 years old, not acutely ill, and able to tolerate lumbar puncture (LP). Peripheral blood was added in vitro to acellular CSF and then cellular concentration and protein were quantified. ResultsCorrection formulas for white blood cell (WBC) count determined by linear regression for samples with more than 100,000 RBCs per uL were inaccurate. Linear correction formulas close to 1 WBC per 1000 red blood cells (RBCs) were more precise than the formula based on peripheral blood WBC to RBC ratio. Correction formulas for protein level derived by linear regression had minimal error. DiscussionThis study confirms in a controlled manner that CSF admixture with peripheral blood cannot always be reliably corrected for in a predictable manner. Use of correction formulas for protein level and for WBC count when CSF admixture with peripheral blood has RBCs less than 10,000 per microliter is possibly useful but will need to be assessed in larger studies that can confirm diagnostic utility.

Performance of digital morphology analyzer Vision Pro on white blood cell differentials.

Vision Pro (West Medica, Perchtoldsdorf, Austria) is a recently developed digital morphology analyzer. We evaluated the performance of Vision Pro on white blood cell (WBC) differentials. In a total of 200 peripheral blood smear samples (100 normal and 100 abnormal samples), WBC preclassification and reclassification by Vision Pro were evaluated and compared with manual WBC count, according to the Clinical and Laboratory Standards Institute guidelines (H20-A2). The overall sensitivity was high for normal WBCs and nRBCs (80.1-98.0%). The overall specificity andoverall efficiency were high for all cell classes (98.1-100.0% and 97.7-99.9%, respectively). The absolute values of mean differences between Vision Pro and manual count ranged from 0.01 to 1.31. In leukopenic samples, those values ranged from 0.09 to 2.01. For normal WBCs, Vision Pro preclassification and manualcount showed moderate or high correlations (r=0.52-0.88) except for basophils (r=0.34); after reclassification, the correlation between Vision Pro and manual count was improved (r=0.36-0.90). This is the first study that evaluated the performance of Vision Pro on WBC differentials. Vision Pro showed reliable analytical performance on WBC differentials with improvement after reclassification. Vision Pro could help improve laboratory workflow.

Risk Factors for Relapse of Childhood B Cell Acute Lymphoblastic Leukemia.

BackgroundB cell acute lymphoblastic leukemia (B-ALL) is the most common type of ALL. This study aimed to explore risk factors for relapse of childhood B-ALL.Material/MethodsTotal of 102 pediatric B-ALL patients were included in this study. B-ALL patients were divided into a relapse group and a non-relapse group. Chemotherapy-induced agranulocytosis time, fusion gene, and minimal residual disease (MRD) were assessed. White blood cell (WBC) count in peripheral blood and risk stratification were evaluated in newly-diagnosed patients. Kaplan-Meier plots were used to evaluate the correlation between risk factors and relapse rates. Multivariate analysis was performed with Cox proportional hazard model to estimate relative risk (RR), 95% confidence interval (95% CI), and hazard ratio (HR). Finally, 99 cases of B-ALL were included in this study.ResultsThere were significant differences between the relapse group and the non-relapse group in age (p=0.004), chemotherapy-induced agranulocytopenia (p=0.001), WBC count in peripheral blood of newly diagnosed patients (p=0.016), risk stratification (p=0.000), and MRD at 12th week (p=0.007). Age over 10 years, high-risk stratification, long period of agranulocytopenia, higher WBC counts, and MRD more than 10−4 were correlated with higher B-ALL relapse rate (p<0.05). Multivariate analysis showed significantly higher relapse rates for age ≥10 years, high-risk stratification, and MRD at 12th week >10−4, with RR (95% CI) of 4.001 (1.005–15.930), 4.964 (1.050–23.456), and 4.646 (1.383–15.614), respectively.ConclusionsAgranulocytopenia ≤7 days, peripheral blood WBC >100×109/L, and MRD at 33rd day >10−4 were associated with B-ALL relapse. Age ≥10 years, high-risk stratification, and MRD at 12th week >10−4 were independent risk factors for relapse.

Antioxidant mechanism of diallyl sulfide in inhibiting leucopenia in peripheral blood induced by benzene in rats

Objective: To investigate the antioxidant mechanism of diallyl sulfide (DAS) in antagonizing the reduction in peripheral blood white blood cells (WBC) induced by benzene in rats. Methods: A total of 60 specific pathogen-free adult male Sprague-Dawley rats, with a body weight of 180-220 g, were selected, and after 5 days of adaptive feeding, they were randomly divided into blank control group, DAS control group, benzene model group, benzene+low-dose DAS group, benzene+middle-dose DAS group, and benzene+high-dose DAS group, with 10 rats in each group. The rats in the benzene+low-dose DAS group, the benzene+middle-dose DAS group, the benzene+high-dose DAS group, and the DAS control group were given DAS by gavage at a dose of 40, 80, 160, and 160 mg/kg·bw, respectively, and those in the blank control group and the benzene model group were given an equal volume of corn oil; 2 hours later, the rats in the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group were given a mixture of benzene (1.3 g/kg·bw) and corn oil (with a volume fraction of 50%), and those in the blank control group and the DAS control group were given an equal volume of corn oil. The above treatment was given once a day for 4 consecutive weeks. At 1 day before treatment, anticoagulated blood was collected from the jugular vein for peripheral blood cell counting. After anesthesia with intraperitoneally injected pentobarbital (50 mg/kg·bw), blood samples were collected from the abdominal aorta, serum was isolated, and the thymus, the spleen, and the femur were freed at a low temperature to measure oxidative and antioxidant indices. The femur at one side was freed for WBC counting in bone marrow. Results: Compared with the blank control group, the benzene model group had significant reductions in the volume, weight, and organ coefficient of the spleen and the thymus (P<0.05) ; compared with the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group had significant increases in the volume of the spleen and the thymus and the weight and organ coefficient of the spleen (P<0.05), and the benzene+middle-dose DAS group and the benzene+high-dose DAS group had significant increases in the weight and organ coefficient of the thymus (P<0.05). Compared with the blank control group, the benzene model group had a significant reduction in WBC count in peripheral blood and bone marrow (P<0.05), and compared with the benzene model group, the benzene+middle-dose DAS group and the benzene+high-dose DAS group had a significant increase in WBC count in peripheral blood and bone marrow (P<0.05). Compared with the blank control group, the benzene model group had a significant increase in the serum level of malondialdehyde (MDA) (P<0.05) and significant reductions in total superoxide dismutase (T-SOD) activity, reduced glutathione (GSH) level, GSH/oxidized glutathione (GSSG) ratio, total antioxidant capacity (T-AOC) (P<0.05) ; compared with the benzene model group, the benzene+high-dose DAS group had a significant reduction in the serum level of MDA and significant increases in T-SOD activity, GSH level, GSH/GSSG ratio, and T-AOC (P<0.05). Compared with the blank control group, the benzene model group had a significant increase in the level of MDA (P<0.05) and significant reductions in GSH level, GSH/GSSG ratio, and T-AOC (P<0.05) in the spleen; compared with the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group had a significant reduction in MDA level (P<0.05) and significant increases in GSH level and T-AOC (P<0.05), and the benzene+high-dose DAS group had significant increases in T-SOD activity and GSH/GSSG ratio (P<0.05). Compared with the blank control group, the benzene model group had a significant increase in the level of MDA in bone marrow cells (BMCs) and peripheral blood mononucleated cells (PBMCs) (P<0.05) and a significant reduction in T-AOC in PBMCs (P<0.05) ; compared with the benzene model group, the benzene+low-dose DAS group, the benzene+middle-dose DAS group, and the benzene+high-dose DAS group had a significant reduction in the level of MDA in BMCs and PBMCs (P<0.05), and the benzene+high-dose DAS group had significant increases in GSH level and GSH/GSSG ratio (P<0.05) . Conclusion: DAS can antagonize the benzene-induced reduction in peripheral blood WBC, possibly by exerting an anti-oxidative stress effect.

Myeloid neoplasms with eosinophilia and abnormal PDGFRA/B: report of three cases and review of literature

Objective To investigate clinical and hematological features of myeloid neoplasms with eosinophilia and abnormal PDGFRA/B and the effect of imatinib. Methods The data of three eosinophilia patients with abnormal PDGFRA/B fusion gene in Changhai Hospital, the Second Military Medical University and 22 Chinese cases reported in Chinese medical journals were analyzed. Thirty-one cases of idiopathic hypereosinophilic syndrome from Changhai Hospital, the Second Military Medical University were used as the controls. Results Compared with idiopathic hypereosinophilic syndrome, no differences were found in age, percentage of bone marrow eosinophils and counts of platelets in peripheral blood in myeloid neoplasms with eosinophilia and abnormal PDGFRA/B (all P > 0.05), but statistical differences were found in gender (χ 2= 5.080, P= 0.016), peripheral blood white blood cell count (t= 4.908, P= 0.001), eosinophilic granulocyte absolute value (χ 2= 17.230, P= 0.001) and hemoglobin concentration (t= 2.770, P= 0.013). The median follow-up time was 17 months (3-108 months) in 24 myeloid neoplasms patients with eosinophilia and abnormal PDGFRA/B from Chinese report. Complete hematopoietic remission (CHR) rate was 91.7% (22/24) after the treatment of imatinib. The total complete molecular remission (CMR) rate was 75.0% (18/24). The median time of remission was 3 months (1-8 months). CMR in patients with PDGFRA and with PDGFRB was 76.5% (13/17) and 85.7% (6/7), respectively. Only one patient (4.2%) died of disease relapse. Conclusion Imatinib has a favorable effect on myeloid neoplasms with eosinophilia and abnormal PDGFRA/B featured by distinct hematologic and clinical manifestations. Key words: Eosinophilia; Gene fusion; Myeloid neoplasms; Remission

Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient's own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model.The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion.We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing.These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases.Figure 1:Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A). DisclosuresNegre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair:RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

Clinical Effectiveness of Traditional Herbal Medicine in the Treatment of Chemotherapy-induced Leukopenia: A Systematic Review and Meta-analysis of Randomized Clinical Trials

Objectives We performed a systematic review and meta-analysis to explore the recent clinical research trends regarding the effects of traditional herbal medicine (THM) on chemotherapy-induced leukopenia. Methods Randomized controlled trials that verified effects of herbal medicine treating chemotherapy induced leukopenia were included in the study. A literature search was performed in the English, Chinese, and Korean databases for papers published from January 1, 2007, to September 1, 2017. The selected literature was assessed by Cochrane’s Risk of Bias (RoB). Results Forty-two of 232 randomized controlled trials met the inclusion criteria. The most commonly used herbal prescriptions called ‘升白湯 (shengbai decoction)’ which means elevating the numbers of White blood cells (WBCs). And the mostly included herbs such as Astragali Radix, Angelicae Gigantis Radix, Atractylodis Rhizoma Alba, Codonopsis Pilosulae, Glycyrrhizae Radix, Ligustri Lucidi Fructus are commonly used to elevate qi, blood and yin. The count of WBC in peripheral blood, the level of leukopenia presented in WHO (World Health Organization), and clinical symptoms were used to evaluate the treatments. The effective rate was 68.6-98.18% and the effectiveness was significantly higher in the intervention group than in the control group in 38 articles (p<0.05). No serious adverse events were reported. Only five articles (5/42, 11.9%) were rated as having adequate methodological quality with a low level of bias. Conclusion Some traditional herbal medicines may be effective as therapeutic treatments for chemotherapy-induced leukopenia, but the majority of reviewed studies were of poor quality. The present findings need to be confirmed by rigorously conducted high-quality trials, including pharmacokinetic studies, to confirm the absence of interactions between traditional herbal medicine agents and chemotherapy. Keywords: chemotherapy, leukopenia, herbal medicine, systemic review, meta-analysis

Pathogenic and diagnostic analysis of bacterial meningitis children in the Midwest area

Objective To investigate the diagnostic analysis and pathogenic characteristics of bacterial meningitis (BM) in children in Midwest area in China. Methods Clinical and pathogenic data of inpatients with BM were analyzed retrospectively at Xiangya Hospital, Central South University and Children′s Hospital of Chongqing Medical University, including age, gender, index of peripheral inflammation, cerebrospinal fluid routine, biochemistry, culture, drug sensitivity and image change. Results In total, 446 cases with BM were analyzed in this study.The male-to-female ratios was 1.671.00.Peripheral blood detected abnormal ratios of white blood cell (WBC), C reactive protein(CRP) and procalcitonin (PCT) which were 67.7%(302/446 cases), 64.9%(268/413 cases) and 71.7%(279/389 cases), respectively.Cerebrospinal fluid detected abnormal ratios of WBC count, glucose and protein, which were 76.5%(332/434 cases), 71.2%(306/430 cases) and 91.7%(397/433 cases), respectively; 79.4% of the patients revealed abnormal head CT/MRI image.Bacterial culture positive rate was 33.6%(150/446 cases), and the positive rates of abnormality in peripheral blood WBC, CRP, PCT or cerebrospinal fluid WBC, glucose and protein were 34.9% (116/332 cases), 34.8%(138/397 cases), 36.6%(112/306 cases), respectively, which were higher than the normal patients, who were 29.4%(30/102 cases), 25.0%(9/36 cases), 21%(26/124 cases), respectively.The most common pathogenic bacteria were Streptococcus pneumoniae, Escherichia coli and coagulase negative staphylococcus.Infants within one year presented dominantly with infection of Escherichia coli, accounting for 26.6%(29/109 cases). Children above 1-year-old presented dominantly with Streptococcus pneumoniae, accounting for 61.0%(25/41 cases). A drug-resistance phenomenon was common among pathogenic bacteria. Conclusion Abnormality in peripheral blood WBC, CRP, PCT or cerebrospinal fluid WBC, glucose and protein, and subdural effusion/empyema can help the early diagnosis and differential diagnosis of BM.The most common pathogenic bacteria in Midwest area of China are Streptococcus pneumoniae, Escherichia coli and coagulase negative staphylococcus, which suggests recommend the application of Streptococcus pneumoniae vaccine. Drug-resistance phenomenon to pathogens is very common.For the treatment strategy, it should be made and adjusted timely according to the drug sensitivity and common hospital antimicrobial spectrum. Key words: Bacterial meningitis; Child; Pathogenic analysis; Diagnostic analysis

Clinical and laboratory characteristics of chronic neutrophilic leukemia: analyses of five cases

Objective To investigate the clinical characteristics, cell morphology, genetics, gene mutations of the patients with chronic neutrophilic leukemia (CNL). Methods Five CNL patients from the Second Hospital of Shanxi Medical University between May 2011 and May 2017 who conformed to 2016 World Health Organization (WHO) diagnostic criteria were retrospectively analyzed from clinical characteristics, laboratory features and treatment methods. Results The peripheral blood white blood cell count (WBC) of 5 CNL patients was significantly increased, and the average WBC was 81.26×109/L [(29-217)×109/L]. Morphological analysis of peripheral blood cell showed a sustained increasing number of matured neutrophilia (0.80-0.85). Neutrophil alkaline phosphatase (NAP) activity was increased (144-266). Bone marrow cell morphology typically showed granulocyte proliferation without obvious dysplasia. Gene detection showed 3 patients with CSF3R T618I mutation and 2 patients with JAK2 V617F mutation in 5 WHO-defined CNL patients. Bone marrow biopsy with reticular staining showed that marrow fibrosis (MF) degree in patients with JAK2 V617F mutation (MF≥2) was higher than that in patients with CSF3R T618I mutation(MF<2). Conclusions CNL is a rare type of chronic leukemia, and CSF3R T618I mutation is a specific diagnostic index for CNL. JAK2 V617F mutations alone may be related to myelofibrosis, which remains to be further studied. Key words: Leukemia, myeloid, chronic-phase; Cellular morphology; Genes; Receptors, colony-stimulating factor; Janus kinase 2

Multiple mechanisms underlying neuroprotection by secretory phospholipase A2 preconditioning in a surgically induced brain injury rat model

BackgroundIntra-operative bleeding, post-operative brain edema and neuroinflammation are major complications in patients with surgical brain injury (SBI). Phospholipase A2 (PLA2) is the upstream enzyme which initiates the PLA2, 5-lipoxygenase (5-LOX) and leukotriene B4 (LTB4) inflammatory pathway. We hypothesized PLA2preconditioning (PPC) prior to SBI can activate endogenous anti-inflammatory responses to protect against SBI. This study evaluated if PPC can ameliorate neurosurgical complications and elucidated PPC-mediated possible protective mechanisms in a rat SBI model. MethodsTotal 105 adult male Sprague Dawley rats were used for this study. SBI was induced by partial resection of the right frontal lobe. PLA2 or 0.9% NaCl was injected via rats' tail vein for 3 consecutive days prior to SBI. For mechanism study, a selective PLA2 inhibitor, Manoalide and 5-LOX inhibitor, Zileuton were injected intravenously with PPC to elucidate the role of PLA2 and 5-LOX in PPC-mediated anti-inflammatory effects. Brain water content (BWC) and lung water content, neurological tests, ELISA, western blot, immunohistochemistry, white blood cells (WBC) count, and spectrophotometric assay for intra-operative hemorrhage volume were evaluated. ResultsFirst, PPC reduced brain water content, intra-operative bleeding, and improved neurological function after SBI. Second, PPC decreased 5-LOX expression and brain leukocyte infiltration, while increasing glial fibrillary acidic protein (GFAP) expression in the peri-resection brain tissue after SBI. Third, PPC induced peripheral inflammation represented by mild pulmonary inflammation and increased peripheral blood WBC count and LTB4 level. Lastly, PPC increased blood glucose concentration and glucocorticoid levels after SBI. In addition, PPC mediated above-mentioned changes were partially reversed by administration of PLA2 inhibitor, Manoalide and 5-LOX inhibitor, Zileuton. ConclusionsPPC conferred neuroprotection against SBI via multi-target involvement induced anti-inflammatory mechanisms.

Clinical significance of peripheral blood leukocytosis in patients with advanced malignant tumors

Objective To explore the capability of leukocytosis in predicting the survival time of patients with advanced malignant tumors, in order to provide evidences for clinicians to make the best therapy regimen. Methods The clinical data of 103 dead patients with advanced malignant tumors treated from September 2015 to September 2016 were collected. The relationship between peripheral blood white blood cell count (WBC) and survival time of patients was analyzed retrospectively. The survival analysis was performed by Kaplan-Meier and Log-rank test,and Cox regression was used for multivariate analysis. Results An elevated peripheral blood WBC was found in 61.1% (63/103) dead patients with advanced malignant tumors, and the elevated WBC was negatively correlated with the life span (r=-0.569, P= 0.000). Conclusion Elevated WBC in some advanced malignancy patients predicts they are in the end-stage, which can guide clinicians adjust treatment regimens in time, and they may consider palliative care for those patients. Key words: Neoplasms; Leukocyte count; Prognosis

Optimizing Progenitor Cell Mobilization in Patients with Myeloma: Effect of a Pre-Emptive Day 4 Plerixafor-Based Mobilization Algorithm

BACKGROUND: Peripheral blood progenitor cell (PBPC) mobilization strategies vary considerably. Optimization of this critical aspect of autologous hematopoietic stem cell transplantation (ASCT) requires efficient use of resources including apheresis kits, machine run time, nursing and cell processing time and consumables. Equally important is the patient experience, which is strongly influenced by collection days and caregiver time that reduces their economic productivity and raises their expenses. We developed a mobilization algorithm designed to collect the target number of cells on day 1 of collection. The algorithm utilizes pre-emptive day 4 plerixafor to maximize collection day peripheral blood (PB) CD34+ cell numbers. METHOD: We analyzed data on all patients with multiple myeloma undergoing PBPC mobilization between March 2014 and July 2016. All patients were mobilized with the intent of collecting sufficient numbers of progenitor cells to permit two ASCT procedures. Patients received filgrastim 10 mcg/kg daily x 4 days. Patients in whom the PB white blood cell (WBC) count was RESULTS: We identified 105 patients with MM. Median age was 61 years (range, 25 - 76) and 57% were female. Disease status at mobilization included: 7 CR, 14 stringent CR, 5 unconfirmed CR, 52 very good PR (VGPR), and 27 PR. The median day 4 PBCD34+ cell number was 19.9/uL (range, 0.8 - 118.7/uL), and median day 5 PBCD34+ count was 107.8/uL (range, 15.6 - 307.8/uL). Ninety percent of patients required plerixafor of which 16% (n = 17) received the 12 mg fixed dose. The median increase in day 4 to day 5 PBCD34+ cell count for patients receiving plerixafor was 6.6 fold (range, 1.5 - 56.4 fold). In patients not receiving plerixafor, the median increase was 1.8 fold (range, 1.5 - 2.6 fold). The median collection yield was 10.95 million CD34+ cells/Kg (range, 2.9 - 22.5 million CD34+ cells/Kg) no significant difference between patients who received standard dose or fixed dose plerixafor. By the criteria outlined above 96.2% of patients had an optimal mobilization i.e. ≥ 6 million CD34+ cells/Kg sufficient for 2 ASCT procedures with 94.2% achieving this with only 1 day of collection. Per our institutional criteria, 71.4% achieved a successful collection (> 8 million CD34+ cells/Kg) in 1 day. There was no significant difference in days of collection among patients receiving 4 or less cycles of lenalidomide versus those receiving more than 4 cycles (p value = 0.104). The median duration of collection was 399 minutes (range, 180 - 495 minutes) with a median collection efficiency (CE2) of 42.3%. The mean number of days of collection was 1.23 days (range, 1 - 2 days). The median transplanted CD34+ cell dose was 5.48 million CD34+ cells/Kg. All patients had hematopoietic recovery with median neutrophil and platelet engraftment of 11 days and 18 days, respectively. CONCLUSION : The pre-emptive use of plerixafor on day 4 is effective and results in a high percentage of optimal day 1 collections. The cost of a more liberal plerixafor algorithm is offset by the savings incurred from reduced collection day numbers with the majority of patients requiring only 1 day of collection. Cost savings include limiting days away from work and family for the patient and caregiver and decreasing expenses for travel and overnight stays. Reducing collection day numbers also reduces the impact on quality of life for patients, caregivers and family members. Collectively, these data demonstrate utility of the pre-emptive day 4 plerixafor-based mobilization algorithm described here. Disclosures Usmani: Amgen: Consultancy, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity9s Board of Directors or advisory committees, Research Funding; BioPharma: Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Research Funding; Britsol-Myers Squibb: Consultancy, Research Funding; Skyline: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Array: Research Funding; Millenium: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau. Jacobs: Magellan Health: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau. Bhutani: Onyx, an Amgen subsidiary: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Takeda Oncology: Research Funding, Speakers Bureau; Prothena: Research Funding. Avalos: Seattle Genetics: Membership on an entity9s Board of Directors or advisory committees.

Dynamic changes and influencing factors of leukocyte and platelet count in preterm infants

Objective To investigate the dynamic changes and influencing factors of peripheral blood white blood cells (WBC), differential counts (DCs) and platelet (PLT) count in preterm infants to understand the changing characteristics of these blood parameters in preterm infants of different postnatal age, gestational age, and birth weight. Methods Totally 2 849 preterm infants admitted to the Department of Neonatology of Northwest Women's and Children's Hospital from November 30, 2011 to November 30, 2014 were retrospectively analyzed except for those diagnosed with infectious diseases, hematological system diseases, or immunologic diseases. All of the subjects were divided into seven groups based on their postnatal age, three groups based on gestational age and three groups based on birth weight, or male and female groups, respectively. Peripheral blood samples were obtained for determination of WBC, DCs and PLT. Statistical analysis was performed with one-way analysis of variance, t-test and Spearman linear correlation analysis. Results WBC, neutrophil (Ne), lymphocyte (Ly), monocyte (Mo), eosinophil (Eo), basophil (Ba) and PLT counts were significantly different among the seven groups of preterm babies of different postnatal age (F=172.00, 364.90, 34.88, 14.22, 80.82, 168.10 and 86.64, respectively, all P 2- ≤5 [(10.62±4.68)×109/L], further gradual decrease thereafter, and then being stable in day >14-≤21 and >21-≤30 [(10.54±3.09)×109/L and (10.27±3.70)×109/L, respectively]. PLT counts showed no significant change within one day after birth and in day >1-≤2 [(240.56±63.54)×109/L and (240.85±71.47)×109/L, respectively], then began to increase in day >2-≤5 [(249.21±80.55)×109/L], peaked in day >7-≤14 [(339.11±121.84)×109/L], and decreased gently and became stable finally. The changing trends of Ne and Ly were cross and inverted in day > 5-≤7. WBC, Ne, Ly, Mo, Eo, Ba and PLT counts of the preterm infants were all correlated with the postnatal age shown by Spearman linear correlation analysis (r=-0.46, -0.60, 0.18, -0.07, 0.33, -0.47 and 0.29, respectively, all P<0.01). With the increase of gestational age, WBC, Ne, Mo, and PLT counts increased, but Ly and Eo counts decreased. And all of the above showed significant difference (F=81.00, 124.49, 13.34, 18.35, 5.35 and 4.11, respectively, all P<0.05). While, the WBC, Ne, Mo, Ba and PLT counts showed positive relationship with the increase of birth weight (F=122.12, 133.09, 39.38, 13.77 and 21.24, respectively, all P<0.05). WBC, Ne and PLT counts of female infants were higher than those of male babies (t=16.35, 16.72 and 13.19, respectively, all P<0.05). Conclusions The peripheral WBC, DCs and PLT counts of preterm infants change dynamically with postnatal age with the remarkable variations on day >2-≤5 after birth and stable after 14 days of age. WBC, DCs and PLT counts might all be influenced by gestational age, birth weight and gender to some extend. Key words: Leukocyte count; Platelet count; Infant, premature

Serum carbohydrate antigen-125 levels in patients with bronchiectasis

To evaluate the level of serum carbohydrate antigen-125(CA125) and its related factors in patients with bronchiectasis. The clinical data of 504 patients with bronchiectasis in Zhejiang Putuo People's Hospital from June 2009 to June 2014 were collected in the study.The patients were divided into CA125 elevated group and CA125 normal group according to serum CA125 level,and the differences of serum CA125,age,gender, white blood cell(WBC),C-reactive protein(CRP), blood glucose and other test indicators were compared between two groups. There were 276 patients including 117 male and 159 female with elevated serum CA125.Their mean age was(66.3±13.1)years and the mean level of CA125 was(83.70±43.87) U/mL. There were 228 patients including 84 male and 144 female with normal CA125 levels. Their mean age was(67.5±10.5) years and the mean level of CA125 was(20.68±9.67)U/mL.The peripheral blood WBC in patients with CA125 elevated group[(10.08±5.68)×10(9)/L] was significantly higher than that in CA125 normal group[7.73±3.46)×10(9)/L], the difference was statistically significant(P<0.05).The medium of CRP level in patients with CA125 elevated group[22.98(3.18~196.88)mg/L] was significantly higher than that in CA125 normal group[6.34(0.50~97.66)mg/L](P<0.05). Correlation analysis showed that CA125 was positively correlated with WBC and CRP(P<0.05). Stepwise regression analysis showed that CRP was the only independent prognostic factors of CA125. Paired t test showed the presence of CA125 serum in patients with bronchiectasis had a significant difference between before and after anti-infection therapy(P<0.05). The serum levels of CA125 rise in patients with bronchiectasis,while it decrease after anti-infection therapy.CRP is an independent associated factor of serum CA125 level.

Radioprotective effects of Sipunculus nudus L. polysaccharide combined with WR-2721, rhIL-11 and rhG-CSF on radiation-injured mice.

This study investigated the radioprotective effect of Sipunculus nudus L. polysaccharide (SNP) in combination with WR-2721, rhIL-11 and rhG-CSF on irradiated mice. A total of 70 Imprinting Control Region (ICR) mice were divided into seven groups: the control group, the model group and five administration groups. All groups, except the control group, were exposed to a 5 Gy 60Co γ-ray beam. Blood parameters [including white blood cell (WBC), red blood cell (RBC) and platelet counts and hemoglobin level] were assessed three days before irradiation, and the on the 3rd, 7th and 14th days after irradiation. Spleen, thymus and testicular indices, DNA contents of bone marrow cells, bone marrow nucleated cells, sperm counts, superoxide dismutase (SOD), malondialdehyde (MDA), testosterone and estradiol levels in the serum were assessed on the 14th day after irradiation. The combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF exerted synergistic recovery effects on peripheral blood WBC, RBC and platelet counts and hemoglobin levels in irradiated mice, and synergistic promotion effects on spleen, thymus, testicle, bone marrow nucleated cells and sperm counts in irradiated mice. The synergistic administration increased the serum SOD activities and serum testosterone content of irradiated mice, but synergy decreased the content of serum MDA and estradiol in irradiated mice. These results suggest that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF should increase the efficacy of these drugs for acute radiation sickness, protect immunity, hematopoiesis and the reproductive organs of irradiated-damaged mice, and improve oxidation resistance in the body.

Chinese herbal medicine for myelosuppression induced by chemotherapy or radiotherapy: a systematic review of randomized controlled trials.

Background. Myelosuppression is one of the major side effects of chemo- and radiotherapy in cancer patients and there are no effective interventions to prevent it currently. Chinese herbal medicine (CHM) may be helpful due to its multidrug targets. Objectives. This study was designed to evaluate effectiveness of CHM on preventing patients from experiencing myelosuppression by chemo- or radiotherapy. Search Methods. Randomized controlled trials (RCTs) were retrieved from seven different databases from the date of database creation to April 2014. We assessed all included studies using Cochrane Handbook for Systematic Reviews of Interventions 5.1.0 and performed statistical analysis using RevMan 5.2.1. Results. Eight RCTs were included (818 patients). Pooled data showed that increase of white blood cells (WBCs) is higher with CHM plus chemotherapy/radiotherapy than with chemotherapy/radiotherapy only. Both CHM compared to placebo and CHM combined with chemotherapy/radiotherapy compared to chemotherapy/radiotherapy lacked significant differences in the peripheral platelets, red blood cells (RBCs), and hemoglobin changes. Conclusions. Our results demonstrated that CHM significantly protected peripheral blood WBCs from a decrease caused by chemotherapy or radiotherapy. There were no significant protective effects on peripheral RBCs, hemoglobin, or platelets, which may be related to low quality and small sample of included studies.

The alteration and clinical significance of Th22/Th17/Th1 cells in patients with chronic myeloid leukemia.

T helper- (Th-) cell immunodeficiency plays important roles in tumor development and their effects in chronic myeloid leukemia (CML) remain unclear. In the present study, we mainly investigated the role of Th22, Th17, and Th1 cell and their related cytokines (IL-22, IL-17, and IFN-r) in the pathophysiology of CML. Bone marrow (BM) and peripheral blood (PB) were extracted from newly diagnosed (ND), chronic phase- (CP-) CML patients, and controls. Th subsets were examined by flow cytometry. Plasma IL-22, IL-17, and IFN-r concentrations were measured by ELISA. AHR and RORC mRNA expressions were examined by RT-PCR. The frequencies of Th22, Th17, and Th1 cells, along with the expression of specific transcription factors RORC and AHR, were significantly decreased in ND patients compared with healthy controls, while all these abnormality recovered in CP patients. In addition, there existed a significantly positive relationship between Th22 and Th17 cells in PB or BM. A significantly negative relationship was found between Th cells (Th22, Th17, or Th1) and BCR-ABL (%) IS or the number of PB white blood cells. All these results demonstrated that Th22, Th17, and Th1 cells might be important therapeutic targets in CML and could facilitate a better outcome for tumor immunotherapy.