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Context: Ewing sarcoma (ES) are malignant small round cell tumors (MSRCT) characterized by rearrangements of EWSR1 gene. Although gold standard for diagnosis is detection of specific fusion genes by molecular testing, these ancillary tests are costly and only available in limited number of settings. There is a persuasive evidence for reliability of NKX2.2 immunohistochemistry (IHC) as a surrogate marker for EWSR1 gene rearrangement in ES. Aims: The aim of this study is to correlate the NKX2.2 immuno-expression with genetically confirmed ES cases and also to assess the reliability and accuracy of NKX2.2 along with combined positivity of NXX2.2 and CD99 in diagnosing ES and differentiating it from other relevant histological mimics. Settings and Design: The present study is a retrospective study conducted over a period of 6-year duration in a tertiary cancer care center. Methods and Material: We evaluated NKX2.2 immunoexpression in 35 genetically confirmed cases of ES and also in pertaining differential entities (n = 58) of ES including rhabdomyosarcoma (n = 20), lymphoblastic lymphoma (n = 14), Wilms tumor (n = 10), poorly differentiated synovial sarcoma (n = 4), small-cell osteosarcoma (n = 4), neuroblastoma (n = 5), and mesenchymal chondrosarcoma (n = 1). CD99 was performed in the category of MSRCTs showing NKX2.2 positivity to evaluate combined specificity for the diagnosis of ES. Results: Of the 35 genetically confirmed cases of ES, 29 cases (83%) showed NKX2.2-positive expression (83% sensitivity). Compared to ES, NKX2.2 was positive in only 05% cases (3/58 cases) of non-ES MSRCT. Only two of five cases of neuroblastomas and one case of mesenchymal chondrosarcoma showed NKX2.2 positivity. CD99 positivity was seen in 100% of ES and in the single case of mesenchymal chondrosarcoma. All five cases (100%) of neuroblastoma were negative for CD99. Conclusions: The presented study, which is the first from an Indian oncology center, showed NKX2.2 IHC is quite reliable in diagnosis of ES in the right clinicopathological context. With remarkable sensitivity and specificity of NKX2.2 IHC for diagnosis of ES, we propose that combined positivity of CD99 and NKX2.2 IHC can obviate or minimize the need of EWSR1 gene rearrangement molecular testing for diagnosis of ES.
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@#[摘 要] 目的：设计并制备一种分别靶向B细胞表面抗原CD19和CD22的CAR-T细胞，检测其对肿瘤细胞的体内外杀伤效果。方法：将含有人源化 CD19 ScFv的二代CAR分子和带有CD3ε链作为共刺激结构域的CD22 ScFv CAR分子以P2A自剪切肽连接，序列连接于慢病毒载体pLTR-CMV-MCS中，以HEK-293T细胞包装相应的慢病毒载体，感染健康志愿者提供的T细胞制备CAR-19-22-T细胞，同时以相同二代结构分别构建单靶向CAR-T细胞作为参照。构建表达荧光素酶、CD19和/或CD22的前列腺癌3M细胞（靶细胞）。将各种CAR-T细胞与靶细胞共同培养，采用荧光素酶化学发光法和ELISA法检测其对靶细胞的杀伤能力和细胞因子的分泌水平。通过尾静脉注射Raji-Luc细胞构建NOD-SCID免疫缺陷小鼠白血病模型，分别注射各组CAR-T细胞进行治疗并评估其疗效。结果：培养7 d的CAR-19-22-T细胞的CAR-19表达率为13.7%，CAR-22表达率为14.3%。CAR-19-22-T细胞在10∶1效靶比时，对3M-CD19-Luc、3M-CD22-Luc和3M-CD19-CD22-Luc细胞的杀伤率均显著高于T细胞[（78.1±14.4）% vs （11.1±4.3）%、（46.7±10.7）% vs （12.4±2.7）%、（90.5±4.3）% vs （14.3±3.7）%，均P<0.01]；与3M-CD19-Luc、3M-CD22-Luc、3M-CD19-CD22-Luc靶细胞共培养后，CAR-19-22-T细胞IFN-γ、TNF-α和IL-2水平均显著低于CAR-19-T和CAR-22-T细胞（P<0.05或P<0.01）。CAR-19-22-T细胞对移植Raji-Luc细胞模型小鼠治疗效果明显，其生存期显著长于T细胞组（P<0.01），与CAR-19-T组和CAR-22-T组荷瘤小鼠比较差异均无统计学意义（均P>0.05）。结论：成功设计并制备了一种双靶点CAR-19-22-T细胞，其能够有效杀伤表达CD19和/或CD22抗原的肿瘤细胞，对Raji-Luc细胞的白血病模型小鼠有显著的治疗效果。
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t(8;21)(q22;q22) acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy with a high relapse rate in China. Two leukemic myeloblast populations (CD34
Subject(s)
Humans , Gene Expression , Granulocyte Precursor Cells , Immunophenotyping , Leukemia, Myeloid, Acute/genetics , Membrane Glycoproteins , Prognosis , Proteins , Proto-Oncogene Proteins c-kit/geneticsABSTRACT
Objective: The aim of the present study was to determine the patterns of leukemia in Northern Saudi Arabia. Methodology: This was a retrospective descriptive study conducted in King Khalid hospital, Hail, Kingdom of Saudi Arabia (KSA) including records of leukemia from 2008 to 2016. Results: The overall Crude Incidence Rate (CIR) of leukemia was 7.45 per 100.000 person-year, including patients diagnosed with different patterns of leukemia in Northern Saudi Arabia. The mean age of patients was 45.4 years with a minimum of 5 years and a maximum of 107 years old. Around 43 (59%) were males and 30 (41%) were females. Conclusion: The incidence rates of leukemia are relatively higher in Northern Saudi Arabia, with an increase of all subtype.
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Objective To analyze the frequency of interleukin (IL)-22+CD161+CD4+ T cells in the peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) patients compared with healthy control subjects and investigate the relationship of IL-22+CD4+CD161+ T lymphocyte frequency changes with RA disease activity.In addition to explore the pathogenesis of RA,and to look for new treatment targets for RA.Methods Twenty-one RA cases were included in the Department of Rheumatology of Tangshan Gongren Hospital from 2017 to 2018.Fourteen patients were female and 7 were male with the age ranged from 36 to 74 years old.The average age of this group of patients was (55±10) years,the average disease course was (60±50) months.All patients fulfilled the classification criteria of American College of Rheumatology [American College of Rheumatology (ACR)].Twenty-one subjects were enrolled as the control group,all of them came to Tangshan Gongren Hospital for regular health check-up.Fifteen subjects in the control group were female and 6 were male.Their age ranged between 40-78 years old with the average age of (55±9) years.IL-22+CD4+CD161+ T cells in PBMCs were detected by flow cytometry.The frequency variation of different CD4+CD161 + T was compared between case and control groups.The correlation was studied between the frequency and RA disease activity score (DAS28),tender joints number,swollen joints number,red blood cell sedimentation rate,high sensitive C reactive protein and white blood cell counts,red blood cell counts,platelet counts,IgG,IgA,IgM,complement C3 level,complement C4 level.T-test or Mann-Whitney U test were used for single-factor analysis,Pearson's test was used for correlation analysis.Results The percentage of RA group secreted CD4+ T cells (0.33± 0.20)％ of INF-γand IL-22,CD4+ T cells (0.51±0.29)％ of IL-22,and CD4+CD161+ T cells of IL-22 simultaneously.The number (0.55 ±0.28)％ was.significantly higher than that of the healtby control group [(0.22±0.14)％,(0.25±0.18)％,(0.36±0.24)％],and the differences were statistically significant [P=0.002,P=-0.0.45,P=0.026].Conclusion The percentage of IL-22+CD4+CD161+ T lymphocytes in the peripheral blood monocytes in RA patients is significantly higher than that in the healthy controls.The results of this study suggest that IL-22+CD4+CD161+ T lymphocytes in RA patients maybe related to RA disease activity and joint lesions.
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@#AIM:To investigate the changes of Notch receptors and interleukin(IL)-22 expression in patients with Vogt-Koyanagi-Harada(VKH)syndrome, and to assess the regulatory activity of Notch signaling to IL-22 production by CD4+ T cells in patients with VKH syndrome.<p>METHODS: Thirty-five patients with VKH syndrome(including fifteen active VKH and twenty inactive VKH)and twelve healthy controls were enrolled. Plasma was isolated, and CD4+T cells were purified. Notch receptors were investigated by qRT-PCR and Western blot. Plasma IL-22 expression was measured by ELISA. The percentage of Th17 and Th22 cells was investigated by flow cytometry. CD4+T cells, which were purified from active VKH patients, were stimulated with Notch signaling inhibitor DAPT. mRNA expression of transcription factor in CD4+T cells as well as IL-22 secretion by CD4+T cells was investigated.<p>RESULTS: Notch1-Notch3 in CD4+T cells from active VKH syndrome patients was significantly elevated in comparison with inactive VKH and healthy controls. Plasma IL-22 expression and percentage of Th17 and Th22 was notably increased in active VKH syndrome in comparison with inactive VKH and controls. DAPT stimulation inhibited Notch signaling pathway in CD4+T cells, leading to the down-regulation of AhR mRNA and IL-22 secretion.<p>CONCLUSION:Notch-AhR-IL-22 signaling pathway might take part in the pathogenesis of VKH syndrome.
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Objective To investigate the immunophenotypic characteristics of potential leukemia cells transfected with CD19 antigen receptor( CAR) during CAR-T cell preparation. Methods Morphological chan-ges in CD19 CAR-transfected cells were observed under inverted microscope. The transfection rate and immuno-phenotype of transfected Nalm-6 cells were analyzed by flow cytometry. Secretion of cytokines in the culture sys-tem was detected by chemiluminescence. Results The transfection rate of Nalm-6 cells by CD19 CAR was (46. 50±3. 78) ％ and that of KG1a cells was (15. 70±1. 22) ％. CD19 CAR-transfected Nalm-6 cells prolifer-ated more rapidly than Nalm-6 cells ( P values on 0 d, 4 d, 7 d and 12 d were 6. 339, 3. 447, 0. 012 and 0. 009). In the culture of CD19 CAR-transfected Nalm-6 cells, cell aggregation and adhesion were observed and they gradually gathered into a group. The rate of CD19 expression was only 1. 19％ in the CD19 CAR-transfect-ed Nalm-6 cell culture system with the transfection rate of (46. 50±3. 78) ％. After increasing the proportion of Nalm-6 cells in the culture system, CD19 expression was gradually increased, while the expression of CD22 re-mained stable. CD19 expressed by Nalm-6 cells cultured in the supernatant of CD19 CAR-transfected Nalm-6 cell culture system was decreased gradually. The levels of IL-10 and TNF-αsecreted by CD19 CAR-transfected Nalm-6 cells were higher than those by Nalm-6 cells. Conclusions Results of the immunophenotypic analysis of CD19 CAR-transfected leukemia cells suggested that CD22 CAR-T cell therapy could be used as a rescue or combination therapy for CD19 CAR transfection into leukemia cells.
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Objective To investigate the cell origin of interleukin (IL)-22-secreting cell of mice infected with Trichinella spiralis (T.spiralis) at the early encapsulated stage.Methods Twelve Balb/c mice were divided into the infected group and the control group according to body weight by random number table.The infected mice were intragastrically administrated with 300 muscle larvae of T.spiralis,and the control mice were given the same amount of normal saline.The IL-22-secreting cell subsets in mouse splenic lymphocytes were detected by flow cytometry at the fourth week after infection.Results The proportion of IL-22-secreting cells in splenic lymphocytes of T.spiralis infected mice was increased when compared with control group [(0.88 ± 0.25)％ vs (0.28 ±0.17)％,t =-4.899,P ＜ 0.05].There was no significant difference between the proportion of CD3+IL-22+ cells and CD3-IL-22+ cells in the splenic lymphocytes of the infected group [(0.29 ± 0.17)％ vs (0.51 ± 0.17)％,t =-2.195,P ＞ 0.05],and the percentage of CD3-IL-22+ cells were similar between the infected group and the control group [(0.51 ± 0.17)％ vs (0.44 ± 0.22)％,t =-0.600,P ＞ 0.05].The proportion of CD3+IL-22+ cells in the infected group was significantly higher than that in the control group [(0.29 ± 0.17)％ vs (0.07 ± 0.06)％,t =-3.068,P ＜ 0.05],and the percentage of CD4+IL-22+ T cells and γδTCR+IL-22+ T cells were obviously increased in CD3+ lymphocytes [(1.28 ± 0.54)％ vs (0.16 ± 0.07)％,(0.33 ± 0.22)％ vs (0.02 ± 0.00)％,t =-4.997,-3.342,P ＜ 0.05].Conclusions The proportion of IL-22-secreting splenic lymphocytes is increased in mice infected with T.spiralis at the early encapsulated stage.The rise is caused by increased numbers of IL-22-secreting CD3 + lymphocytes,especially CD4+ T cells and γδT cells.
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Objective To determine the frequency of T helper type 22 (Th22) cells and expression level of interleukin-22 (IL-22) in peripheral blood of patients with psoriasis vulgaris,and to investigate their relationship with disease severity and clinical course.Methods Peripheral blood samples were obtained from 40 patients with psoriasis vulgaris and 30 healthy human controls.Five-color flow cytometry was performed to determine the percentage of Th22 cells in peripheral blood,and enzyme-linked immunosorbent assay (ELISA) to measure the expression of serum IL-22.Statistical analysis was carried out by t test and Pearson correlation analysis.Results Both the percentage of Th22 cells and serum level of IL-22 in peripheral blood were significantly higher in patients with psoriasis vulgaris than in healthy human controls (Th22 cells:0.65％ ± 0.48％ vs.0.33％ ± 0.15％,t =3.89,P ＜ 0.01; IL-22:(67.96 ± 14.32) vs.(40.59 ± 9.91) ng/L,t =9.45,P ＜ 0.01).Further more,Th22 cell percentage and IL-22 serum level in peripheral blood were both positively correlated with psoriasis area and severity index (PASI) in these patients (r =0.38,0.94,P ＜ 0.05 and 0.01,respectively),but neither of them correlated with clinical course of psoriasis vulgaris (r =0.20,0.10,respectively,both P ＞ 0.05).Conclusions The percentage of Th22 cells and level of IL-22 are increased in peripheral blood of patients with psoriasis vulgaris,and both of them are correlated with disease severity.
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Objective To detect the quantity of Th17 cells and expressions of related cytokines including interleukin (IL)-17 and IL-22, in peripheral blood and skin lesions of patients with psoriasis vulgaris, and to analyze their correlation with disease severity and clinical course. Methods Peripheral blood was obtained from 44 patients with progressive psoriasis vulgaris and 28 normal human controls. Three-color flow cytometry was carried out to detect the quantity of Th17 cells, and ELISA to examine the levels of serum IL-17 and -22.Skin samples were obtained from 20 patients with psoriasis vulgaris and 8 normal human controls, and a quantum dot-based double labled immumofluorescence method was used to determine the quantity of Th17 cells.Results The percentage of peripheral blood Th17 cells was higher in patients with psoriasis vulgaris than in normal human controls (4.71% ± 2.55% vs. 0.55% ± 0.39%, P ＜ 0.01 ). Elevated expressions of IL-17 and IL-22 were noted in the patients compared with the normal human controls (24.02 ± 12.31 ng/L vs. 7.16 ±4.04 ng/L, P ＜ 0.05; 18.32 ± 8.14 ng/L vs. 6.52 ± 4.15 ng/L, P ＜ 0.01 ). The percentage of peripheral blood Th17 cells and serum levels of IL-17 and IL-22 were positively correlated with psoriasis area and severity index (r= 0.53, 0.47, 0.53, respectively, all P ＜ 0.01 ), but unrelated to the clinical course of psoriasis (r = 0.09,0.03, 0.19, respectively, all P ＞ 0.05). There was an infiltrate of Th17 cells in psoriatic lesions, which was mainly distributed around the blood vessels in superficial dermis, whereas there were only a small number of CD4+ T cells in the normal control skin with the absence of Th17 cells. Conclusions Th17 cells are involved in the development of psoriasis, and Th17 cell-secreted cytokines, such as IL-17 and IL-22, may serve as a new therapeutic target for psoriasis.
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BACKGROUND: CD22 is a glycoprotein expressed on the surface of normal mature B cells and in the cytoplasm of normal B cell precursors. Cytoplasmic CD22 (cCD22) has been proposed as a immunologic marker for the diagnosis of B-lineage acute lymphoblastic leukemia (ALL) while membrane CD22 (mCD22) has been used as the marker for chronic lymphocytic leukemia, B-lineage lymphoma, and hairly cell leukemia, and mCD22 has not been routinely used for the diagnosis and subgrouping of ALL. The purpose of this study was to examine the expression of mCD22 in B-lineage ALL and its clinical significance. METHODS: From 1992 to April, 1998, the leukemic cells of 64 patients newly diagnosed as B-lineage ALL by immunophenotyping were analyzed by the direct immunofluorescence method using monoclonal antibodies including mCD22. RESULTS: mCD22 was positive in 53% (34/64) of all patients, 50% (21/42) of children and 59% (13/22) of adults. According to the immunologic classification, mCD22 was positive in 44% (4/9) of group II, 53% (19/36) of group III, 69% (11/16) of group IV, but negative in 3 cases of group V and VI. The complete remission rate of the mCD22 negative group in group III was significantly higher than that of the mCD22 positive group (P=0.008). There were significant differences in survival rates between the mCD22 positive group and the mCD22 negative group in group II, III and IV (P=0.046) and the above observed significant difference was seen when group III was separately tested (P=0.014). CONCLUSIONS: Our study demonstrated that the expression of mCD22 may be a poor prognostic factor in B-lineage ALL and that mCD22 shall be clinically used as a prognostic marker especially in group III, which is most common among the subgroups of B-lineage ALL.
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Adult , Child , Humans , Antibodies, Monoclonal , B-Lymphocytes , Biomarkers , Classification , Cytoplasm , Diagnosis , Fluorescent Antibody Technique, Direct , Glycoproteins , Immunophenotyping , Leukemia , Leukemia, Lymphocytic, Chronic, B-Cell , Lymphoma , Membranes , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Prognosis , Survival RateABSTRACT
BACKGROUND: Detection of bcr/abl fusion mRNA using reverse transcription polymerase chain reaction has been used for diagnosis of chronic myelogenous leukemia (CML) and monitoring after treatment. However, this conventional method is not quantitative. Therefore, new quantitative marker for CML is necessary for the follow-up of the patients after treatment including bone marrow transplantation. Whether the lymphocytes are involved in CML clone or not is still a moot question. If the lymphocytes are involved in CML clone, there could be an abnormal surface antigen expressed on these cells. We tried to find out abnormal surface antigen expression on the lymphocytes of CML. METHODS: We analyzed the immunophenotypic distribution of the bone marrow lymphocytes using flow cytometry in 22 cases of CML and 20 normal persons, and searched for characteristic abnormal immunophenotype of CML. Both peripheral blood and bone marrow samples were analyzed using dual color immunophenotying for CD19 and CD22 expression in 14 cases of CML. RESULTS: The proportion of lymphocytes with T cell antigen expression and CD10, CD19, CD20 expression were lower in CML than in normal control (P<0.05). In contrast to these antigens, the proportion of CD22 positive lymphocytes was higher in CML than in normal control (P=0.0434). And loss of correlation between CD19 and CD22 expression was observed in CML. The proportion of CD19-/CD22+ lymphocytes in the bone marrow of 14 cases of CML was higher than that of normal control (P=0.0001). The proportion of CD19-/CD22+ lymphocytes in the peripheral blood of CML was 45.0+/-22.6%, and higher than 1.0+/-0.3% of normal control (P= 0.0000). The proportion of CD19-/CD22+ lymphocytes in the peripheral blood was higher than that of the bone marrow (P=0.0018). CD22 was not coexpressed with T cell antigens (CD2, CD3) or myeloid antigen (CD14) in CML or normal control. CONCLUSION: We demonstrated that the cells, representing unusual immunophenotype of CD19-/ CD22+, are characteristically increased in the bone marrow and peripheral blood of CML. This immunophenotype could be a valuable marker for the diagnosis of CML and monitoring after treatment.