Zeta-associated protein 70 expression in chronic lymphocytic leukemia: Relevance in Indian context

Background: Chronic lymphocytic leukemia (CLL) is prognosticated using the Rai and the Binet's staging. In the past few years, new parameters have been considered for prognostication. One such marker that has been a subject of speculation and found useful by some western studies is zeta-associated protein 70 (ZAP-70). Aim: To investigate the prevalence of ZAP-70 and find out its association with other prognostic markers like Rai and Binet's stage and CD38 in Indian CLL patients. Materials and Methods: Twenty-nine newly diagnosed cases of CLL were selected over 1 year. Immunophenotyping was done and expression of CD38 and ZAP-70 was evaluated on gated CLL cells. Statistical Analysis: Qualitative data were expressed as frequency and percentage. Differences between groups were evaluated using Student's t-test for quantitative data and Chi-square test/Fisher's exact t-test for qualitative variables. A P value less than 0.05 was considered significant. Results and Conclusion: We found a lower prevalence rate of ZAP-70 (2/29, 6.89%) with no association with any of the conventional poor prognostic factors. A large number of our CLL patients fall into the good prognostic group (22/29, ZAP 70?/CD38?) with a least number in the poor prognostic group (2/29, ZAP-70 + CD38+). Also, no association was found between ZAP-70 and CD38. The findings of the present study suggest that the majority of CLL patients in India have a good prognosis, may not require treatment, and have good overall survival. Geographical variations, genetic makeup, and natural history of the CLL could be the cause of such differences from western literature.

78c can alleviate coliagen-induced arthritis and restore immune balance in rheumatoid arthritis / 中华风湿病学杂志

Objective:To explored the effect of 78c in treating collagen-induced arthritis (CIA) mice and to investigate its mechanism of effects.Methods:CIA mice model and CD38 +NK cells were treated with 78c. Cytokine concentrations and lymphocyte subtypes were measured in the mice peripheral blood and culture medium using flow cytometry. Mikenyi cell isolation kit was used to isolate CD4 + T cells and NK cells from peripheral blood mononuclear cells of healthy volunteers. CD38 + NK cells were enriched using the Miltenyi CD38 microbeads from the extracted NK cells. CD38 + NK cells with 78c pretreatment or not were cocultured with CD4 +T cells in transwells. The least significant difference (LSD) method was used for comparison between the two groups, and one-way analysis of variance was used for multi-group significance. Pearson correlation analysis was used for correlation analysis. Results:78c treatment significantly suppressed joint inflammation, inhibited the toe thickness of CIA mice, and reduced the number of while cell, neutrophils, platelets, and concentrations of IFN-γ, IL-6 and TNF-α ( t=6.10, P<0.001; t=4.00, P=0.002; t=3.09, P=0.012; t=2.31, P=0.043; t=3.58, P=0.005; t=2.68, P=0.002) in the CIA mice. The proportion of CD38 +NK cells decreased from (3.9±0.9)% to (2.4±0.3)% ( t=2.49, P=0.032), the proportion of regulatory T cell (Treg) increased from (0.81±0.33)% to (1.41±0.26)% ( t=2.74, P=0.021), and the concentration of IL-10 also increased from (99±37) pg/ml to (199±9) pg/ml( t=2.76 , P=0.020). The proportion of Treg in CD4 +T cells cocultured with 78c-pretreated CD38 +NK cells increased from (0.52±0.04)% to (0.69±0.08)% ( t=3.33, P=0.029) , the T helper cells (Th)17/Treg ratio decreased from (4.44±0.26) to (2.59±0.64) ( t=4.76 , P=0.009), and the Th1/Th2 ratio decreased from (14.8±1.6) to (8.1±1.3)( t=5.70 , P=0.005). Conclusion:78c can reduce the proportion of CD38 +NK cells, thereby reducing the inhibition of CD38 +NK cells on CD4 +T cell differentiation into Treg cells, leading to the restoration of immune balance. The results of this study suggest that 78c is a potential therapeutic agent for rheumatoid arthritis.

Changes of inflammatory cytokines and T lymphocyte activation in the peripheral blood of human immunodeficiency virus infected patients during anti-retroviral therapy / 中华传染病杂志

Objective:To explore the dynamic changes of inflammatory cytokines and T lymphocyte activation in the peripheral blood of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients during anti-retroviral therapy (ART).Methods:Two hundred and six HIV/AIDS patients with ART at clinic of the Department of Infectious Diseases in Second Xiangya Hospital, Central-South University between January 2017 and December 2019 were selected as HIV infection group. They were followed up regularly and the blood samples before treatment and at month 6, month 12, month 24 of treatment were collected. Meanwhile, 52 healthy cases were enrolled in the healthy control group and their blood samples were collected. Enzyme-linked immunosorbent assay was used to detect the plasma concentrations of interleukin (IL)-6, hypersensitive C-reactive protein (hsCRP) and tumor necrosis factor (TNF)-α. Flow cytometry was used to detect the CD3 + CD4 + T lymphocytes count and the percentage of CD4 + CD38 + T lymphocytes and CD8 + CD38 + T lymphocytes in the peripheral blood mononuclear cells. Plasma HIV RNA viral load was determined using a quantitative real-time polymerase chain reaction technique. Statistical methods used paired t test and Pearson correlation analysis. Results:The concentrations of IL-6, hsCRP and TNF-α in HIV infection group were (13.42±2.35) pg/mL, (4 012.46±1 012.35) μg/L and (51.78±11.32) μg/L, respectively, which were higher than those in healthy control group ((6.14±0.78) pg/mL, (707.21±305.76) μg/L and (19.01±6.48) μg/L, respectively). The differences were all statistically significant ( t=12.56, 16.79 and 13.45, respectively, all P<0.001). They decreased gradually after initiation of ART in HIV infection group, and returned to normal levels at month 24 of ART. CD3 + CD4 + T cells count was (256.00±65.32)/μL and HIV RNA viral load was (4.467±4.244) lg copies/mL before ART in HIV infection group, which were negatively correlated ( r=-0.625, P=0.041). The percentages of CD8 + CD38 + T lymphocytes before treatment and at month 12 or month 24 of treatment in HIV infection group were higher than those in healthy control group. The differences were all statistically significant ( t=3.85, 6.84 and 2.57, respectively, all P<0.050). The percentage of CD8 + CD38 + T lymphocytes was positively correlated with HIV RNA viral load before ART ( r=0.768, P=0.026). The percentages of CD4 + CD38 + T lymphocytes before treatment and at month 12 or month 24 of treatment in the HIV infection group were lower than those in the healthy control group, and the differences were all statistically significant ( t=6.80, 1.10, and 2.11, respectively, all P<0.050). Conclusions:HIV infection could not only cause insufficiency in immune system, but also abnormal activation of immune system, which could get better gradually with ART.

Aß promotes CD38 expression in senescent microglia in Alzheimer's disease

BACKGROUND: In Alzheimer's disease (AD), the neuroinflammatory response mediated by the activation of senescent microglia is closely related to energy dysmetabolism. However, the mechanism underlying the interaction between the energy metabolism of aging microglia and neuroinflammation remains unclear. METHODS: We used biochemical methods, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and western blot to determine the effects and mechanism of CD38 knockdown on energy metabolism and neuroinflammation in Aß1-40 injured BV2 cells. Using AD model mice, we detected CD38 enzyme activity, energy metabolism factors (ATP, NAD +, and NAD +/NADH), and neuroinflammatory factors (IL-1ß, IL-6, and TNF-α) following the addition of CD38 inhibitor. Using a combination of biochemical analysis and behavioral testing, we analyzed the effects of the CD38 inhibitor on energy metabolism disorder, the neuroinflammatory response, and the cognition of AD mice. RESULTS: Following Aß1-40 injury, SA-ß-Gal positive cells and senescence-related proteins P16 and P21 increased in BV2 cells, while energy-related molecules (ATP, NAD +, and NAD +/NADH) and mitochondrial function (mitochondrial ROS and MMP) decreased. Further studies showed that CD38 knockdown could improve Aß1-40-induced BV2 cells energy dysmetabolism and reduce the levels of IL-1ß, IL-6, and TNF-α. In vivo results showed an increase in senile plaque deposition and microglial activation in the hippocampus and cortex of 34-week-old APP/PS1 mice. Following treatment with the CD38 inhibitor, senile plaque deposition decreased, the number of Iba1 +BV2 cells increased, the energy metabolism disorder was improved, the proinflammatory cytokines were reduced, and the spatial learning ability was improved. CONCLUSIONS: Our results confirm that senescent microglia appeared in the brain of 34-week-old APP/PS1 mice, and that Aß1-40 can induce senescence of BV2 cells. The expression of CD38 increases in senescent BV2 cells, resulting in energy metabolism disorder. Therefore, reducing CD38 expression can effectively improve energy metabolism disorder and reduce proinflammatory cytokines. Following intervention with the CD38 inhibitor in APP/PS1 mice, the energy metabolism disorder was improved in the hippocampus and cortex, the level of proinflammatory cytokines was reduced, and cognitive impairment was improved.

Preclinical evaluation of 89Zr labeled Daratumumab for imaging diagnosis of multiple myeloma / 中华核医学与分子影像杂志

Objective:To prepare 89Zr labeled Daratumumab and evaluate its feasibility in the imaging diagnosis of multiple myeloma (MM). Methods:According to the principle of 89Y (p, n) 89Zr nuclear reaction, 89Zr was produced by cyclotron solid target system (30 μA, 1.5 h) and automatic purification module. The radionuclide purity, half-life and impurity metal ion concentration were detected. Desferrioxamine (DFO) was coupled with Daratumumab and then chelated with 89Zr to prepare 89Zr-DFO-Daratumumab. The quality control analyses of three consecutive batches were carried out. Pharmacokinetic evaluation and 89Zr-DFO-Daratumumab microPET/CT imaging were performed in normal rabbits and orthotopic myeloma mouse models, respectively. The SUV in situ myeloma and that in normal bone were compared by independent-sample t test. Results:About 560 MBq of 89Zr was obtained, and there were only two characteristic energy peaks of 89Zr (909 keV and 511 keV) by γ spectrometer. The half-life of 89Zr was 78.2 h, and the content of metal impurities was small. 89Zr-DFO-Daratumumab was prepared with pH of 7.2, radiochemical purity of more than 99%, good stability in vitro, and sterility and endotoxin tests were passed. Pharmacokinetic studies in rabbits showed that 89Zr-DFO-Daratumumab was gradually distributed from blood to liver, spleen, kidney and bone joints over time and metabolism. The results of microPET/CT imaging in orthotopic myeloma mouse models showed that the SUVs of 89Zr-DFO-daratumumab in situ myeloma were significantly higher than those in normal bone (2 h: 0.22±0.02 vs 0.06±0.00; 1 d: 0.38±0.01 vs 0.08±0.00; t values: 8.89, 21.90, both P=0.001). Conclusion:89Zr and 89Zr-DFO-daratumumab are successfully prepared, and relevant quality control and biological evaluation in vivo and in vitro are completed, which verify the feasibility of 89Zr-DFO-Daratumumab in the imaging diagnosis of MM, thus laying a foundation for clinical transformation.

A method for measuring the ADCP potency of anti-CD38 monoclonal antibody / 药学学报

A method to measure the antibody-dependent cell-mediated phagocytosis (ADCP) potency of anti-CD38 mAb was developed based on design of experiment (DoE) with a Jurkat/NFAT/CD32a-FcεRIγ transgenic cell line as the effector cell, the Daudi cell line as the target cells, and luciferase as the detection system. The DoE method was used for optimization of experimental parameters and methodological validation. The results show that anti-CD38 mAb exhibits a dose-response relationship with the following four-parameter equation: y = (A - D) / [1 + (x / C)B] + D. Several experimental parameters were optimized by statistical experimental design and determined as follows: the working concentration of anti-CD38 mAb was 800-20.81 ng·mL-1, the density of the target cells was 7.5×104 per well, and the density of effector cells was 2.5×104 per well, with an induction time of 6 h. The method showed good specificity. The recovery rate for samples from 5 different groups showed that the relative potencies of anti-CD38 mAb were (59.97 ± 4.74) %, (82.44 ± 5.15) %, (110.69 ± 11.71) %, (129.23 ± 5.22)% and (162.15 ± 3.66) %. The recoveries ranged from 103% to 120% and the RSDs of the above results were all less than 11%. The linear detection range was 50%-150%. Based on DoE design, this method for measuring ADCP potency of anti-CD38 mAb was optimized and validated with good specificity, repeatability and accuracy. This method can be used for evaluation of ADCP biological activity of anti-CD38 mAbs.

The challenging blood cross-matching and resolution for multiple myeloma patients in different disease stages / 中国输血杂志

【Objective】 To explore the challenging blood cross-matching and resolution for multiple myeloma (MM) patients in different disease stages. 【Methods】 For a patient who was first diagnosed as MM and scheduled for blood transfusion, his blood was cross matched with donors’ blood by microcolumn gel method and tube test. When the major side of cross-matching was agglutinated, the patient’s plasma was cross matched with donors’ red blood cell (RBC) by polybrene test, then plasma dilution cross matched with donors’ RBC by microcolumn gel method. For a patient who was diagnosed as recurrent refractory MM and scheduled for blood transfusion, his blood was cross matched with donors’ blood by microcolumn gel method. 【Results】 1) Case 1 was a first-visit outpatient. The major side of microcolumn cross-match test was agglutinated with the shape of fine line. The result of tube method also showed agglutination of major sides, and the rouleaux were detected by the microscopy. Then polybrene method and microcolumn gel method (after plasma diluted) were applied for cross-matching again with the above two donors’ blood and showed compatibility. 2) Case 2 was a recurrent refractory MM patient. The major and minor sides of microcolumn cross-match test were both agglutinated with the shape of granular. The patient was treated with anti-CD38 monoclonal antibody. The RBCs, after treated with dithiothreitol (DTT) was used to cross match with patient plasma by microcolumn test, and the result was compatible. 【Conclusion】 Polybrene method and microcolumn gel method after plasma diluted are suitable for blood cross-matching of newly diagnosed MM patients, also for those treated with CD38 monoclonal antibody, as the drug interference with cross-matching can be eliminated by DTT.

Removal of serological interference in the treatment of multiple myeloma with CD38 monoclonal antibody using polybrene method / 中国输血杂志

【Objective】 To investigate the clinical feasibility of polybrene method to remove serological interference in patients receiving anti-CD38 monoclonal antibody for the treatment of multiple myeloma, and its detection performance of alloantibodies. 【Methods】 For patients receiving anti-CD38 monoclonal antibody for the treatment of multiple myeloma, unexpected antibody screening and cross-matching blood test were performed by polybrene method. 【Results】 The polybrene method can remove the interference of anti-CD38 monoclonal antibodies on blood group serology; both methods can effectively remove anti-CD38 monoclonal antibody to detect anti-E, anti-D, anti-Fya and anti-S antibodies.The titers of anti-D, anti-E, anti-Fya and anti-S alloantibodies, yielded by enhanced polybrene method, were higher than those of the polybrene method.Seven patients received K-antigen-negative blood transfusion without any adverse reactions to blood transfusion. 【Conclusion】 For the treatment of multiple myeloma using CD38 monoclonal antibody, the polybrene method can quickly and effectively remove the interference of daratumumab with blood group serology.

Construction of anti-CD38 CAR-T cells with targeted inhibition of CD38 by shRNA and preliminary identification of its functions / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨shRNA靶向抑制CD38的方法能否增强抗CD38 CAR-T细胞的抗癌功能。方法：构建shRNA靶向抑制CD38的抗CD38 CAR-T细胞的CAR分子，利用逆转录病毒载体包装成功后转导人原代T细胞，制备CAR-T细胞。实验分为shRNA1 CD38 CAR-T组、shRNA2 CD38 CAR-T组和对照组（shR-NC-CD38 CAR-T细胞）。采用qPCR法检测CAR-T细胞CD38 mRNA相对表达水平，计算CAR-T细胞培养0～14 d的增殖倍数，CFSE法检测CAR-T细胞与人Burkitt淋巴瘤细胞Raji-luc或人多发性骨髓瘤外周血B淋巴细胞RPMI-8226-luc共培养时的增殖情况，荧光素酶化学发光法检测CAR-T细胞在不同效靶比（1∶1、1∶2、1∶4、1∶8）时对Raji-luc和RPMI-8226-luc细胞的杀伤效率，ELISA法检测CAR-T细胞杀伤Raji-luc或RPMI-8226-luc细胞时上清液中IFN-γ水平，FCM检测CAR-T细胞表面耗竭T细胞生物标志物PD-1的表达水平。结果：shR-NC-CD38 CAR、shRNA1-CD38 CAR和shRNA2-CD38 CAR逆转录病毒载体的滴度均为1´107拷贝/mL，转导T细胞后，shR-NC-CD38 CAR-T、shRNA1-CD38 CAR-T和shRNA2-CD38 CAR-T细胞的转导效率（CAR的阳性率）分别为60.3%、67.0%和57.4%。与对照组比较，shRNA2-CD38 CAR-T组细胞中CD38 mRNA的表达水平显著降低（P<0.01），显示shRNA-CD38 CAR-T细胞构建成功。shRNA2-CD38 CAR-T组细胞在体外培养增殖能力更强（P<0.05），对2种CD38阳性的肿瘤细胞的杀伤效率更高（均P<0.05）、IFN-γ释放水平更高（均P<0.05）、细胞表面PD-1的表达水平更低（P<0.05）。结论：成功构建一种shRNA靶向抑制CD38的抗CD38 CAR-T细胞，其抗癌功能表现出明显的优势。

Interference of CD38 monoclonal antibody in blood compatibility testing and its countermeasures: A general consensus among experts / 中国输血杂志

With continuous discovery of tumor immune targets and continuous changes in antibody research and development technology, antibody drugs are becoming more and more widely used in clinical practice. However, some targets are not only expressed on tumor cells, but also on red blood cells. Therefore, the clinical application of antibodies against the corresponding targets may interfere with the detection of blood transfusion compatibility, resulting in difficulty in blood matching or delay of blood transfusion. This consensus summarizes the current solutions for the interference of CD38 monoclonal antibody (CD38 mAb) in transfusion compatibility testing. After analyzing the advantages and disadvantages of different methods, polybrene and sulfhydryl reducing agents [dithiothreitol (DTT) or 2-mercaptoethanol (2-Me)], as a solution for CD38 mAb interference in blood compatibility testing, are recommended for Chinese patients, so as to eliminate blood transfusion interference produce by CD38 mAb and further provide a pre-transfusion workflow for clinicians and technicians in Department of Blood Transfusion.

To remove monoclonal anti-CD38 interference from compatibility testing: A simple method / 中国输血杂志

【Objective】 To explore a simple method to remove the interference of monoclonal anti-CD38 from compatibility testing and evaluate its effectiveness and safety, in order to develop a reasonable clinical transfusion strategy. 【Methods】 Blood phenotype detection, direct antiglobulin testing(DAT) and antibody screening were carried out by standard methods. Antibody screening and cross-matching of serums after monoclonal anti-CD38 treatment were performed by anti-human globulin card with 0.2 mol/L or 0.04 mol/L dithiothreitol(DTT) treated red blood cells. 【Results】 The results showed that 0.04 mol/L DTT treated directly in the anti-human globulin card for 15 min can completely remove the interference of monoclonal anti-CD38 in antibody screening and cross-matching without compromising of the yielding of anti-K, anti-LW, anti-JMH and anti-Lub alloantibodies. However, the titer of IgM antibodies may decrease in different degrees, and antibody screening and cross-matching with saline methods are required to avoid the missed detection of IgM alloantibodies. All 12 patients had no acute or delayed haemolytic transfusion reactions and their routine blood tests showed that the red blood cells transfusion were effective. 【Conclusion】 Based on antibody screening and cross-matching plus saline method, the method of 0.04 mol/L DTT, treated directly in the anti-human globulin card, is safe, effective and simple, which can detect most alloantibodies.

Advances in immunotherapy of extranodal NK/T cell lymphoma / 中华耳鼻咽喉头颈外科杂志

Extranodal NK/T-cell lymphoma (ENKTCL) is a relatively rare group of highly aggressive non-Hodgkin′s lymphoma (NHL). The disease has rapid clinical progress, high degree of malignancy and poor prognosis. Traditional chemoradiotherapy regimens have not shown good efficacy. In recent years, the immunotherapy of tumors has developed rapidly. At present, it has shown strong therapeutic activity in the treatment of various solid tumors such as non-small cell lung cancer, prostate cancer, melanoma and kidney cancer. Multiple tumor immunotherapy drugs have been approved by the US Food and Drug Administration (FDA) for clinical use. This article reviews recent novel immunotherapeutic regimens of ENKTCL, hoping to change the treatment modality of this malignant disease.

Characteristic and prognostic significance of leukemia stem cells associated antigens expressions in t (8;21) acute myeloid leukemia / 中华血液学杂志

Objective@#To investigate the characteristic and prognostic significance of leukemia stem cells associated antigens expressions including CD34, CD38, CD123, CD96 and TIM-3 in t (8;21) AML.@*Methods@#Bone marrow samples of 47 t (8;21) AML patients were collected at diagnosis from October 2015 to April 2018 in Peking University Peoples’ Hospital, then flow cytometry method was performed to detect the expression frequencies of CD34, CD38, CD123, CD96 and TIM-3 to analyze the relationship between leukemia stem cells associated antigens expressions and relapse.@*Results@#Of 47 t (8;21) AML patients tested, the median percentages of CD34+CD38-, CD34+ CD38-CD123+, CD34+CD38- CD96+ and CD34+ CD38- TIM-3+ cells among nucleated cells were 2.37%, 0.24%, 0.27% and 0.06%, respectively. All the frequencies of CD34+CD38-, CD34+CD38-CD123+, CD34+CD38-CD96+ and CD34+ CD38-TIM-3+ cells had no impact on the achievement of CR after the first course of induction. All higher frequencies of CD34+CD38-, CD34+CD38-CD123+, CD34+CD38-CD96+ cells were related to higher 2-year CIR rate. Whereas, the frequency of CD34+ CD38- TIM-3+ cells had no impact on CIR rate. Both high frequency of CD34+ CD38- cells and the high level of minimal residual diseases (patients with <3-log reduction in the RUNX1-RUNX1T1 transcript level after the second consolidation therapy) were independent poor prognostic factors of CIR[P=0.025, HR=6.9 (95%CI 1.3-37.4) ; P=0.031, HR=11.1 (95%CI 1.2-99.2) ].@*Conclusion@#Different leukemia stem cells associated antigens had distinct prognostic significance in t (8;21) AML. High frequencies of CD34+ CD38-, CD34+ CD38- CD123+ and CD34+CD38-CD96+ cells at diagnosis predicted relapse in patients with t (8;21) AML.

Ginsenoside Rg_1 induces leukemia stem cell senescence via SIRT1/TSC_2 signal axis / 中国中药杂志

The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 μmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated β-Galactosidase(SA-β-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-β-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.

Transfusion management for patients taking an anti-CD38 monoclonal antibody

Abstract Introduction: Pre-transfusion tests, essential for the release of blood components, may be affected by drugs. Monoclonal antibodies represent a class of medications increasingly used in the clinical practice, with anti-CD38 monoclonal antibodies (daratumumab) being a promising resource in the treatment of refractory myeloma. This monoclonal antibody recognizes CD38 in myeloma cells and interferes with pre-transfusion tests by causing panreactivity in indirect antiglobulin tests thereby clinically masking alloantibodies. Dithiothreitol is a reagent that breaks disulfide bonds and effectively destroys antigenic sites for CD38 on red blood cells. This study reports the immunohematological findings of pre-transfusion tests of patients with multiple myeloma receiving daratumumab and on solutions to prevent the interference of this monoclonal antibody. Methods: Serum samples from five patients on anti-CD38 monoclonal antibody treatment were evaluated. Tests performed included ABO/RhD typing, indirect antiglobulin test, direct antiglobulin test and eluate test. A daily evaluation was performed to determine the shelf life of dithiothreitol-treated red blood cells when stored in Alsever's solution. Results: No interference in the ABO/RhD typing results was noted but in all samples, a panreactivity was observed in indirect antiglobulin tests. Regarding the direct antiglobulin test, two samples presented positive results but negative eluates. In all samples, treatment of reagent red blood cells with 0.2 M dithiothreitol offset interference by anti-CD38 monoclonal antibodies. Dithiothreitol-treated red blood cells stored in Alsever's solution were stable for up to 15 days. Conclusion: Treatment of reagent red blood cells with dithiothreitol can be efficient and accessible to offset the interference of the anti-CD38 drug in pre-transfusion tests. The number of costly serological workups can be reduced by having stored dithiothreitol red blood cells with this proving to be a useful reagent for investigating anti-CD38.

The research of inflammatory cytokines and T cell activation in peripheral blood of opportunistic infections and death in HIV / 中国医师杂志

Objective The plasma levels of interleukin 6 (IL-6),high sensitive C reactive protein (hs-CRP) and the level of T cell activation were detected in the peripheral blood inflammatory factors of acquired immunodeficiency syndrome (AIDS) patients,and the relationship with opportunistic infection and prognosis was analyzed.Methods 79 human immunodeficiency virus (HIV)-positive/aids cases from May 2014 to January 2015 in first hospital of Changsha were enrolled in the study.They were divided into three groups:HIV-infected without opportunistic infections group (n =20),HIV-infected with infections group (n =43,including HIV-infected with tuberculosis group,HIV-infected with merge fungus group.HIV combined hepatitis C),death group (n =16).Serum IL-6 and the concentration of hs-CRP were detected by enzyme-linked immunosorbent assay (ELISA).Flow cytometry was used to test the CD3 + CD4 + cell count,the percentage of CD4 + CD38 + cell and CD8 + CD38 + cell in peripheral blood mononuclear cell (PBMC).Compare the differences among the three groups.Results The results showed that:the concentration of hsCRP and IL-6 in peripheral blood of HIV death group was significantly higher than that in other three groups (P ＜ 0.05).The concentration of hs-CRP and IL-6 in the peripheral blood of the HIV-infected with tuberculosis group and HIV-infected with merge fungus group were significantly higher than that in the HIV-infected without opportunistic infections group (P ＜ 0.05),and the hs-CRP in the peripheral blood of the HIV combined with the hepatitis C group was higher than that in the HIV-infected without opportunistic infections group (P ＜ 0.05).The number of CD3,CD4T lymph nodes in the death group and the combined opportunistic infection group was significantly lower than that in the HIV-infected without opportunistic infections group (P ＜ 0.05).The HIV-RNA expression in peripheral blood of the death group and the combined opportunistic infection group was significantly lower than that in the HIV-infected without opportunistic infections group (P ＜ 0.05).The expression of CD8+CD38+ on PBMC in the HIV-infected without opportunistic infections group was significantly lower than that in the death group,the tuberculosis group,the fungus group and the hepatitis C group (P ＜ 0.05).The expression of CD4+ CD38 + on PBMC in the HIV-infected without opportunistic infections group was higher than that in the death group (P ＜ 0,05).Conclusions The concentration of inflammatory cytokines (IL-6,hs-CRP) and the expression level of T cell surface CD8 + CD38 + related to immune activation were associated with opportunistic infection and prognosis of AIDS.

Expressions of CD34, CD123 and CD38 in acute myelogenous leukemia and their clinical significances / 白血病·淋巴瘤

Objective To investigate the expressions of CD34, CD123 and CD38 in acute myelogenous leukemia (AML) and their clinical significances. Methods A total of 164 patients with AML in Xiangya Hospital of Central South University from February 2014 to July 2015 were enrolled. Cellular immunophenotyping was performed by flow cytometry. According to the expressions of CD34, CD38 and CD123, 164 patients were divided into positive group and negative group, and the clinical data and immature cells ratio of two groups were compared. Results In 164 patients with AML, 102 cases (62.2 ％) were CD34 positive, 126 cases (76.8％) were CD123 positive, and 144 cases (88.3％) were CD38 positive. There were no significant differences in age and sex between the positive and negative groups (P> 0.05). But there were significant differences in the proportion of immature cells, white blood cell count and hemoglobin between the two groups (all P< 0.05). The expression rates of CD34, CD38 and CD123 were correlated with minimal residual disease and complete remission rate (all P< 0.05). Conclusions CD34, CD123 and CD38 are effective markers for AML detection. The expressions of CD34, CD123 and CD38 can be used as the judgment marker of cell maturity, which is conducive to the determination of the condition and prognosis of AML patients.

Effect of ginsenoside Rg1 on senescence characteristics of leukemic stem cells / 局解手术学杂志

Objective To explore the effect of ginsenoside Rg1 on leukemia stem cells through comparing the biological senescence characteristics of HSCs in the patients with leukemia and healthy people,and provide new ideas and methods for leukemia prevention and treatment.Methods Fifteen cases of normal bone marrow in normal group and sixteen cases of chronic myeloid leukemia in leukemia group were divided into control group and Rg1 group,respectively.The control group used the conventional culture.The Rg1 group used the culture system with 10 μg/mL ginsenoside Rg1,other conditions were the same as control group.The bone marrow mononuclear cell of all groups were extracted after 2 days,and the CD34 +/CD38-cells population was isolated and purified by immunomagnetic adsorption cell sorting(MACS).The purity of the cells and cell cycles phase were detected by flow cytometry.Cell viability was detected by trypan blue staining.The percentage of positive cells was detected by SA-β-gal staining.CCK-8 detected the CD34 +/CD38-proliferation ability of each group.Results The ratio of CD34 +/CD38-cell population was (1.76 ± 0.34) ％ in every 1 × 106 BMNCs before sorting;the proportion of CD34 +/CD38-cell population per 1 × 106 cells after immunomagnetic sorting was (91.15 ± 2.41)％.The positive rate of SA-β-gal staining in human bone marrow CD34 +/CD38-cells of leukemia Rg1 group was significantly higher than that in leukemia control group,the difference was not significant (P ＞ 0.05);meanwhile there was no significant difference between normal control group and normal Rg1 group,but higher than that in leukemia control group,the difference was significant(P ＜ 0.05).CCK-8 results showed that the proliferation of CD34 +/CD38-cells was significantly increased in leukemia control group than those in the other groups.The survival rate of CD34 +/CD38-cells in human bone marrow was 99.1％ in all groups.Cell cycle phase results showed that the G1 arrest of CD34 +/CD38-cells in leukemia control group was significantly lower than those in the other three groups.Conclusion CD34 +/CD38-cells in chronic myeloid leukemia patients may be caused by some chronic myeloid leukemia.Ginsenoside Rg1 can effectively delay the process of aging.

Effects of CD38 gene knockout on inflammatory cytokine production in murine spleen B-cells / 第二军医大学学报

Objective To analyze the number of spleen B-cells and the expression of inflammatory factors and sirtuin 1 (SIRT1) in spleen B-cells of CD38-/- mice, so as to explore the effects of CD38 gene knockout on inflammatory factors in B-cells and its potential mechanism. Methods The DNA levels of CD38 and Neo gene in mouse tail tissues were detected by polymerase chain reaction (PCR). Spleen B-cells from wide-type (WT) C57BL/6 and CD38-/-mice were sorted by magnetic activated cell sorting (MACS), and the purity of sorting B-cells were identified by flow cytometry. The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and CD38 gene were detected by real-time PCR, the protein expressions of CD38 and SIRT1 were detected by Western blotting analysis. Results We confirmed the successful establishment of CD38-/-mice and sorted spleen B-cells from WT and CD38-/-mice (purity>95%). Compared with WT mice, the development of spleen was hampered in the CD38-/-mice, the number of spleen cells and spleen B-cells were significantly reduced (P<0. 01), the mRNA levels of TNF-α and IL-1β were significantly decreased (P<0. 01), and the expression level of SIRT1 was significantly increased in CD38-/-mice (P<0. 05). Conclusion CD38 gene knockout can reduce the number of B-cells in the spleen; and it can inhibit the expression of inflammatory factors (TNF-α and IL-1β) in spleen B-cells by activating SIRT1 pathway.

Recent progress in multiple myeloma / 白血病·淋巴瘤

The treatments of multiple myeloma (MM) have been made remarkable progress in recent years,and especially in 2015,FDA approved a number of new drugs for treatment of relapsed and refractory MM.At the 21th European Hematology Association Annual Meeting,the issue of MM has received a lot of attention.The recent progress of MM in this conference will be briefly introduced in this review.