ZCH-2B8a (IgG2a) is a novel monoclonal antibody (McAb) generated in laboratory of Children Hospital of Medical College, Zhejiang University recently using human myeloblastic leukemia cell line KG1a as immunogen. This antibody has been submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. The aim of this study was to investigate the reactivity of this antibody on normal blood cells and malignant cell lines and to explore its possible application in clinical practice. The multi-parameter flow cytometry was used to analyze the expression pattern of 2B8a antigen in triplicate on normal blood components including T cells, B cells, natural killers (NK), neutrophils, monocytes, dendritic cells (DC), red blood cells (RBC), platelets (Plt), hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34(+) cells and malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines. The amount of positive cells > or = 20% was considered as positivity. The results showed that 2B8a antibody reacted to 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive to neutrophils (23.72%) and negative for T cells, NK, DC, RBC and Plt. The antibody reacted to all 3 marrow CD34(+) cells with an average positive rate of 39.33% while it was negative for G-CSF-mobilized CD34(+) peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody notably reacted to three out of 4 cell lines (Raji, SMS-SB, Nalm-6 and Nall-1) with the positive rates of 98.78%, 98.61%, 94.93% respectively and weakly to one of them with 5.68% in B lineage cell lines and monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including Meg01 (33.40%), HL-60 (29.70%), K562 (28.19%), KG1a (16.23%) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia, 5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KCNR, BE, SK-N-SH, SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). It is concluded that 2B8a antibody primarily reacts to B lineage and monocytic lineage cells which may bear the diagnostic and therapeutic applications among different types of hematopoietic malignancies.
Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; Antigen-Antibody Reactions ; Antigens, Neoplasm ; analysis ; immunology ; Antigens, Surface ; immunology ; B-Lymphocytes ; cytology ; immunology ; HLA Antigens ; immunology ; Hematopoietic Stem Cells ; cytology ; immunology ; Hematopoietic System ; cytology ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; Tumor Cells, Cultured

The purpose of this review is to provide an overview of the effect of (lymph)angiogenic cytokines on hematopoietic cells involved in acute myeloid leukemia (AML). Like angiogenesis, lymphangiogenesis occurs in pathophysiological conditions but not in healthy adults. AML is closely associated with the vasculature system, and the interplay between lymphangiogenic cytokines maintains leukemic blast survival in the bone marrow (BM). Once AML is induced, proangiogenic cytokines function as angiogenic or lymphangiogenic factors and affect hematopoietic cells, including BM-derived immune cells. Simultaneously, the representative cytokines, VEGFs and their receptors are expressed on AML blasts in vascular and osteoblast niches in both the BM and the peripheral circulation. After exposure to (lymph)angiogenic cytokines in leukemogenesis and infiltration, immune cell phenotypes and functions are affected. These dynamic behaviors in the BM reflect the clinical features of AML. In this review, we note the importance of lymphangiogenic factors and their receptors in hematopoietic cells in AML. Understanding the functional characterization of (lymph)angiogenic factors in the BM niche in AML will also be helpful in interrupting the engraftment of leukemic stem cells and for enhancing immune cell function by modulating the tumor microenvironment.
Animals ; Cytokines/*immunology ; Hematopoietic Stem Cells/immunology/*pathology ; Humans ; Immunity, Cellular ; Leukemia, Myeloid, Acute/immunology/*physiopathology ; *Lymphangiogenesis ; Lymphatic Vessels/immunology/*physiopathology ; Vascular Endothelial Growth Factor A/immunology

OBJECTIVESTo investigate the stroma-independent growth ability, multilineage differentiation and expansion of the single hematopoietic stem/progenitor cell from patients with paroxysmal nocturnal hematoglobinuria (PNH).

METHODThe CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) CD(59)(+) cells from normal volunteers were sorted as each single cell into a well of 96 well culture plates containing culture medium supplemented with SCF, IL-3, Epo, GM-CSF, G-CSF, IL-6, Tpo and Flt-3 ligand.

RESULTS(1) Single PNH CD(34)(+) CD(59)(-) cell had a higher capacities for plating efficiency, colony (>/= 50 cells) formation and cell expansion than that of the PNH CD(34)(+) CD(59)(+) cells (P < 0.05); (2) Both the single CD(34)(+) CD(59)(-) cells from PNH patients and the single CD(34)(+) CD(59)(+) cells from normal controls had similar capacities for cell plating efficiency and colony and large colony formation. The PNH CD(34)(+) CD(59)(-) cells had a lower average cell production and cell expansion capacity. (3) The single CD(34)(+) CD(59)(+) cells from both PNH patients and normal controls showed the same capacities for cell plating efficiency and colony formation. The PNH CD(34)(+) CD(59)(+) cells exhibited much lower capacity for large colony formation, average cell production and total cell expansion. (4) A diminished secondary colony formation ability was also observed in the PNH CD(34)(+) CD(59)(+) and CD(34)(-) CD(59)(-) clones.

CONCLUSIONThe single PNH CD(34)(+) CD(59)(-) cells had growth advantage over the single PNH CD(34)(+) CD(59)(+) cells to some extent, but they both had impaired growth abilities as compared with CD(34)(+) cells from normal volunteers.

Antigens, CD34 ; immunology ; CD59 Antigens ; immunology ; Cell Culture Techniques ; Cell Division ; physiology ; Colony-Forming Units Assay ; Hematopoietic Stem Cells ; immunology ; pathology ; Hemoglobinuria, Paroxysmal ; physiopathology ; Humans

This study was purposed to investigate the change of Th cell subsets in the patients with acute graft-versus host disease (aGVHD) and to explore its role in the pathogenesis of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT). 23 patients underwent allo-HSCT were selected for analysis. The aGVHD in patients was diagnosed according to clinical features, and was confirmed by skin biopsy in some patients. The peripheral blood from 23 patients was collected before and after allo-HSCT. The quantitative chenges of Th1 and Th2 cells in peripheral blood samples were detected by using flow cytometry. The results showed that out of 23 patieats the aGVHD occured in 8 patients including 1 case of grade I, 2 case of grade II, 3 cases of grade III; no aGVHD occured in 15 patients. The flow cytometry analysis revealed that the amount of Th1 cells in patients with aGVHD was much higher than that in patients without aGVHD (p < 0.01), the IFN-gamma expression of Th1 cells in patients with aGVHD of grad II - III significantly was higher than that in patients without aGVHD (p < 0.01), meanwhie the IL-4 expression of Th2 cells in patients with aGVHD of grade II - III was significantly lower than that in patients without aGVHD (p < 0.05). Dynamical detection indicated that the Th1 obviously increased before occurrence of aGVHD and before treatment of aGVHD, while the Th1 cells obviously decreased after treatment of aGVHD. The Th1 cells not changed significantly in patients without aGVHD before and after allo-HSCT. It is concluded that Th1 cells obviously increase in patients with aGVHD, this increase is related to aGVHD pathogenesis. Detecting the changes of Th cell subsets in the early stage after allo-HSCT may be contributed to early diagnosis and therapy of aGVHD.
Graft vs Host Disease ; blood ; immunology ; Hematologic Neoplasms ; immunology ; therapy ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Th1 Cells ; immunology ; pathology

To investigate the effects of sera on the growth of single CD34(+) cells from patients with paroxysmal nocturnal hematoglobinuria (PNH), sera from both PNH patients and normal individuals were added separately to the single cell culture system and semi-solid colony formation system. The growth of the normal CD34(+) and PNH CD34(+) CD59(+) and CD34(+) CD59(-) cells was evaluated. No growth difference was found for growth of the normal CD34(+) and PNH CD34(+) CD59(+) cells when PNH or normal sera were added to the culture media in either single cell culture or colony formation culture. While no difference was detected for single PNH CD34(+) CD59(-) cells growth when PNH or normal sera were added, more colonies were observed in semi-solid culture with PNH sera. A conclusion was reached that compared with those from normal controls, the sera from PNH patients had no significant influence on single hematopoietic stem/progenitor cells derived from normal subjects and from PNH patients, but the PNH sera might promote the colony formation of the CD34(+) CD59(+) cells in semi-solid culture
Antigens, CD34 ; analysis ; Blood Proteins ; pharmacology ; CD59 Antigens ; analysis ; Cell Division ; drug effects ; Cells, Cultured ; Hematopoietic Stem Cells ; drug effects ; pathology ; Hemoglobinuria, Paroxysmal ; immunology ; pathology ; Humans

This study was purposed to investigate the expansion and hematopoietic reconstitution capability of CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) by using BALB/c nude mice so as to provide experimental basis for clinical anto-BMT or auto-PBHSCT in patients with PNH. CD34(+)CD59(+) cells were selected from the bone marrow mononuclear cells in normal persons and PNH patients by immunomagnetic positive double sorting and were engrafted sublethally irradiated BALB/c nude mice. The human CD45(+) cells in bone marrow, spleen and peripheral blood of recipient mice were detected by flow cytometry and DNA assay. The results showed that the CD34(+)CD59(+) cells in PNH patient group and normal person group could expanded ex vivo, but ex vivo expansion capability of CD34(+)CD59(+) cells in PNH patient group at day 7 seemed inferior to that in normal control. While CD34(+)CD59(+) cells of PNH patients and normal persons were transfused into recipient mice, the human CD45(+) cells could be detected in bone marrow, spleen and peripheral blood at 6 weeks after transfusion, but there was no statistical difference in counts of CD45 cells between 2 groups. It is concluded that CD34(+)CD59(+) cells from PNH patients may keep characteristics of normal hematopoietic stem cells, and possess ability to expand ex vivo and support hemopoiesis.
Animals ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Female ; Hematopoiesis ; physiology ; Hematopoietic Stem Cell Transplantation ; Hemoglobinuria, Paroxysmal ; pathology ; Humans ; Immunomagnetic Separation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Transplantation, Heterologous ; Whole-Body Irradiation

Ex vivo expanded human bone marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) were transplanted into BALB/c mice in order to investigate their proliferation ability and reconstruction of hemopoiesis, and to lay the groundwork for clinical ABMT/APBSCT in PNH patients. CD34(+)CD59(+) cells were selected from the bone marrow mononuclear cells in PNH patients by using immunomagnetic positive double sorting. Sublethally irradiated BALB/c mice were transplanted with CD34(+)CD59(+) cells enriched from bone narrow of PNH patients. The results showed that human CD45(+) cells were detected in the bone marrow, spleen and peripheral blood of the nude mice by flow cytometry and DNA analysis at 6 weeks post-transplant. Blood routine indicators of nude mice were found to recover to some extent, but did not fully recover. It is concluded that ex vivo expanded bone marrow CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria could keep their biological characteristics and ability to reconstruct hemopoiesis in irradiated BALB/c mice.
Animals ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Female ; Hematopoietic Stem Cell Transplantation ; methods ; Hemoglobinuria, Paroxysmal ; pathology ; therapy ; Humans ; Immunomagnetic Separation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Transplantation, Heterologous

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.
Acute Disease ; Adolescent ; Adult ; Angiogenesis Inducing Agents ; immunology ; metabolism ; pharmacology ; Angiopoietin-1 ; genetics ; immunology ; pharmacology ; Angiopoietin-2 ; genetics ; immunology ; pharmacology ; Antineoplastic Agents ; therapeutic use ; Female ; Gene Expression Regulation, Neoplastic ; Graft vs Host Disease ; genetics ; immunology ; pathology ; Hematopoietic Stem Cell Transplantation ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; immunology ; Humans ; Leukemia, Myeloid ; genetics ; immunology ; pathology ; therapy ; Lymphoma, Non-Hodgkin ; genetics ; immunology ; pathology ; therapy ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; pathology ; therapy ; Retrospective Studies ; Signal Transduction ; Transplantation, Homologous ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; immunology

OBJECTIVETo investigate the leukogenic function of Shuanghuang Shengbai (SHSB) granule and the related mechanisms.

METHODMouse leukopenic models were induced by radiation. Mice were divided into normal control group, model control group, positive control group-Li kejun tablet group and three different dose (high, middle, low-dose) groups of SHSB granule. The peripheral hemogram, thymus index (TI), spleen index (SI), bone marrow nucleated cell (BMNC) and colony forming unit-spleen (CFU-S) were evaluated. The proliferation of bone marrow cells was determined. The in vitro cultured colony forming unit granulocyte macrophage (CFU-GM) was estimated. The index of CD34+ cell in BMNC were determined by flow cytometry. The ultra-micro structure of bone marrow were observed by electromicroscope.

RESULT(1)SHSB rranule could increase the WBC of model mice; (2)SHSB granule could increase BMNC and promote the proliferation of bone marrow cell; (3)SHSB granule could increase CFU-S, CFU-GM and CD34+ cell index in BMNC of model mice significantly; (4)SHSB Granule could also protect the bone marrow hemotopoietic microenvironment from the harm of radiation; (5)SHSB granule could increase the SI of model mice, indicating the enhancement of immunological function.

CONCLUSIONSHSB granule has apparent leukogenic function. The mechanism may be related to enhancing the proliferation of hematopoietic cells and protecting the bone marrow hemotopoietic microenvironment.

Animals ; Antigens, CD34 ; metabolism ; Bone Marrow Cells ; drug effects ; pathology ; Cell Proliferation ; drug effects ; Colony-Forming Units Assay ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; Leukocyte Count ; Leukopenia ; immunology ; pathology ; Male ; Mice ; Mice, Inbred ICR ; Plants, Medicinal ; chemistry