OBJECTIVETo explore the mechanism of 'erythroblast island (EI)' formation in the bone marrow of patients with immun-related hemocytopenia (IRP).

METHODSThe category of BM-auto antibody (au Ab) in 48 patients with IRP was detected with FCM. The BM-au Ab in the 'EI' of these cases were explored with immuonhistofluorescence (IF). Clinical and laboratory characteristics of these cases were also analyzed retrospectively.

RESULTSIgG could be detected in the 'EI' on the BM smear of 14 cases (29.17%), BM-au Ab mainly deposited at the edge/membranes between macrophage and erythroblasts rather than cyto plasm. Positive reaction were seen in all the cases with GlycoAIgG. The red blood cell count [(1.8 ± 0.5) × 10(12)/L] and hemoglobin level [(59.6 ± 16.2)g/L] were significantly lower than that in the IF(-) group [(2.5 ± 0.9) × 10(12)/L and (83.4 ± 25.0) g/L] (P < 0.05). The percentage of reticulocyte [(2.0 ± 0.8)%], serum level of IBIL [(9.4 ± 4.7) µmol/L], percentage of erythroblats in sternum BM (0.441 ± 0.139) and response rate to therapy (85.7%) in IF(+) group were significantly higher than that in IF(-)group [(1.3 ± 1.0)%, (6.6 ± 6.7)µmol/L, 0.298 ± 0.082, 61.3%, respectively] (P < 0.05).

CONCLUSIONMacrophage was connected with erythroblasts through autologous IgG in the 'EI's of some patients with IRP. 'EI' were the places where macrophages devoured and destroyed erythroblasts rather than erythroid development and differentiation. The pathogenetic mechanism of IRP might be associated with macrophages phagocytosing and destroying BM hematopoietic cells.

Blood Cell Count ; Bone Marrow ; Bone Marrow Cells ; immunology ; Coombs Test ; Erythroblasts ; Humans

OBJECTIVETo explore immunoregulatory mechanism of mesenchymal stem cells (MSCs) in H-2 haploidentical bone marrow cells transplantation mice.

METHODSBALB/c female mice irradiated with 8Gy 60Co gamma-rays were divided into two groups: MSCs group, infused cm-DiI labeled MSCs from female CB6F1 mice and monocytes from the bone marrow and spleen of male CB6F1; Control group, only infused monocytes from the bone marrow and spleen of male CB6F1. T-lymphocyte subpopulation of peripheral blood cells, T and B cells proliferation stimulated by ConA and LPS, mixed lymphocyte reaction between donor and recipient and third part, the sry-gene chimerism of bone marrow, spleen and thymus of the recipient, the distribution of MSCs in the recipient, the incidence rate of GVHD and survival were observed.

RESULTSThe CD3 at +90 d the percent of CD3+ CD4+ cells, and CD4/CD8 at +30 d in the MSCs group were higher than that in control post-transplantation, respectively (P < 0.05). The proliferation activity of B cells recovered more rapidly and that of T cells recovered comparably in MSCs group as compared with that in control group. The result of MLR between donor and recipient was lower in MSCs group than that in the control; and that between recipient and the third part had no difference. The sry-gene chimerism of bone marrow and spleen of the recipient was higher in MSCs group than in control at +30 d. The MSCs mainly distributed in intestine, thymus, bone marrow, liver, heart of the recipient after transplantation. The incidence of acute GVHD was higher and the survival rate was lower in MSCs group than that in control group (P < 0.05). Chronic GVHD occurred in the control group at +90 d, while in the MSCs group at +120 d.

CONCLUSIONSMSCs might improve stem cell engraftment, promote lymphocyte and humoral immunity recovery, decrease incidence of GVHD and increase survival by inducing specific immunologic tolerance and repairing organs injuries.

Animals ; Bone Marrow Cells ; immunology ; Bone Marrow Transplantation ; immunology ; Female ; Graft vs Host Disease ; immunology ; Male ; Mesenchymal Stromal Cells ; immunology ; Mice ; Mice, Inbred BALB C

This study was purposed to explore the immunoregulatory effects of human bone marrow mesenchymal stem cells (MSCs) on active T lymphocytes in vitro and the new strategy to prevent graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononuclear cells from human peripheral blood cells were isolated and cultured in the presence of phytohemagglutinin (PHA) (final concentration was 10 microg/ml) for different times. The ability of T lymphocyte proliferation and activation was measured by (3)H-Thyramine incorporation. The expressions of CD3(+)CD4(+), CD3(+)CD8(+), CD4(+)CD25(+) and CD4(+)CD152(+) on T cells were detected by FCM after coculture for 72 hours. Experiment was divided into 4 groups: A group as control (no added MSCs), B group (actived T cells + 2 x 10(4) MSCs), C group (actived T cells + 4 x 10(4) MSCs), D group (actived T cells + 8 x 10(4) MSCs). The results showed that the ability of T lymphocyte proliferation in the same PHA concentration increased with prolonging of time. ability of T lymphocyte proliferation was strongest when culturing for 48 hours (p < 0.01); the expressions of CD44, CD105, CD29 and FIK1 of MSCs were positive, expressions of CD33, CD34, CD45 and HLA-DR were negative. MSCs inhibited T lymphocyte proliferation and the inhibitory effect depended on the amount of MSCs. CD3(+)CD8(+), CD4(+)CD25(+) and CD4(+)CD152(+) T cells cocultured with MSCs increased obviously and CD3(+)CD4(+) expression significantly decreased, as compared with control group (p < 0.01). It is concluded that the MSCs inhibit T lymphocyte proliferation induced by mitogen (PHA), and perform their immunosuppressive function by up-regulation of CD3(+)CD8(+), CD4(+)CD25(+) and CD4(+)CD152(+) expressions and down-regulation of CD3(+)CD4(+) expression.
Bone Marrow Cells ; cytology ; Cell Separation ; Cells, Cultured ; Humans ; Lymphocyte Activation ; immunology ; Mesenchymal Stromal Cells ; cytology ; immunology ; T-Lymphocytes ; immunology

This study was purposed to clarify whether biology function of mesenchymal stem cells (MSCs) is changed by suppressing the development of dendritic cells (DC) derived from hematopoietic stem cells (HSCs). MSCs were cocultured with dendritic cells derived from CD34 positive hematopoietic stem cells (HSCs), and then the expression of cytokines and phenotypes of DCs/MSCs were detected by RT-PCR and flow cytometry respectively. Induced experiments were used to analyze the differentiation ability of MSCs. The results showed that DCs/MSCs were negative for the CD14, CD34, CD45, CD31, CD86, but positive for HLA-ABC, CD29, CD73, though the percentage decreased as MSCs vs DCs/MSCs (93.1% vs 13.44%, 98.3% vs 78.8%, 95.3% vs 75.9%). In addition, the expression of cytokines such as M-CSF, TGF-beta increased in DCs/MSCs. After differentiation induction, DCs/MSCs were deprived of the potential to differentiate into adipocytes, but maintained osteogenesis characteristics. It is concluded that the basic characteristics of MSCs are altered after coculture with DCs, and DCs/MSCs result in lower expression of mesenchymal phenotypes and decrease differentiation ability, but increase the expression of cytokines related to hematopoiesis and immunity.
Antigens, CD34 ; immunology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; Humans ; Mesenchymal Stromal Cells ; cytology

The purpose of this study was to detect the distribution of Treg and Th17 cells in bone marrow and to investigate the relationship of Treg/Th17 imbalance with the pathogenesis and progression of multiple myeloma (MM). The Bone marrow was collected from 37 MM patients and 12 healthy volunteers, the ratio of Treg and Th17 cells was detected by flow cytometry. The expression of Treg and Th17 cells simultaneously was examined in peripheral blood of 19 MM patients with same method. The results indicated that the frequency of Treg cells was higher in MM patients than that in control group (P < 0.05), there was a trend of increasing of Treg cell number in the ISS stage from I+II to III (P < 0.05). Furthermore, in the patients with MM, the Treg cell number in bone marrow was higher than that in peripheral blood (P < 0.05). Th17 cell rate was not statistically different between MM patients and control group (P > 0.05), and at different ISS stage (P > 0.05). Th17 cell number between bone marrow and peripheral blood was not significantly different (P > 0.05).The ratio of Treg/Th17 in patients with MM was higher than that in control group (P < 0.05), and increased gradually from ISS stage I+II to stage III (P < 0.05). It is concluded that the Treg/Th17 immune imbalance is presenced in bone marrow of patients with MM, this imbalance may promote the progression of MM.
Bone Marrow ; immunology ; Cell Count ; Disease Progression ; Flow Cytometry ; Humans ; Multiple Myeloma ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology

In an attempt to study the immunoregulatory effect of osteoblasts derived from mesenchymal stem cells (MSC), MSC was induced to differentiate into osteoblasts for one week. The growth pattern and the phenotype were evaluated by MTT and flow cytometry respectively. The immunoregulatory effect was tested by the inhibitory effect on T cell proliferation. The result showed that during the differentiation cells grew fast and there was no significant change in the phenotypes but keeping CD73, CD105, CD44, CD29 positive and CD34, CD45, HLA-DR, CD86 negative. Osteocyte derived from MSC also showed immunosuppressive effect on T cell proliferation in adose-dependent manner. It is concluded that osteoblasts derived from MSC also harbored immunoregulatory effect.
Bone Marrow Cells ; cytology ; immunology ; Cell Differentiation ; immunology ; Cell Lineage ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; immunology ; Osteoblasts ; cytology ; immunology ; T-Lymphocytes ; cytology ; immunology

Bone marrow hematopoietic microenvironment occupies the medullary cavities of bones throughout the skeleton and provides support for hematopoiesis and immune cells development. Bone-resorbing osteoclasts in bone marrow environment are specialized cells derived from the hematopoietic stem cells and play a pivotal role in process termed as bone remodeling that involves break down and build-up of bone. It is only recently that studies have provided a novel basis for understanding potential role of osteoclasts in homeostasis, stress-induced mobilization of hematopoietic progenitors and osteoimmunology. Further exploration on the interaction of osteoclasts with others in bone marrow hematopoietic microenvironment may contribute to future clinical treatments for hematopoietic and bone-related immunologic disorders including cancer. In this review the origin and identification of osteoclasts and regulation of mobilizing hematopoietic stem cells, as well as osteoclasts and osteoimmunity were mainly concerned.
Bone Marrow Cells ; physiology ; Bone Remodeling ; immunology ; Hematopoiesis ; physiology ; Hematopoietic Stem Cells ; cytology ; immunology ; Homeostasis ; immunology ; Humans ; Osteoclasts ; cytology ; immunology ; physiology

OBJECTIVETo explore the pathogenetic mechanism of cytopenia in the patients with systemic lupus erythematosus (SLE).

METHODSTwenty-one patients with SLE and 10 healthy controls were enrolled in this study. Bone marrow mononuclear cells (BMMNC) Coombs test was used to determine the autoantibodies and in vitro CFU-E, BFU-E and CFU-GM yields were used to determine the proliferation function of hematopoietic stem/progenitor cell (HSC).

RESULTSPositive results of BMMNC-Coombs test were observed in 12 patients with SLE (57.1%), among them, 10 with (58.2%) and 2 without blood cytopenia. The yields of CFU-GM (49 +/- 40), and CFU-E (49 +/- 35) in SLE patients without cytopenia were higher than that in normal controls, but lower than that in cytopenia SLE patients. The BFU-E yield in SLE patients (3 +/- 4) was significantly lower than that in normal controls (36 +/- 12).

CONCLUSIONThere were auto-antibodies on BMMNC in patients with SLE. The blood cytopenia in SLE patients maybe resulted from the destructions of bone marrow hematopoietic cells by the autoantibodies. The capacities of bone marrow HSC in SLE patients for proliferation compensation of hematopoietic cell destruction were not damaged.

Adolescent ; Adult ; Autoantibodies ; blood ; Bone Marrow ; immunology ; pathology ; Bone Marrow Cells ; immunology ; pathology ; Case-Control Studies ; Female ; Humans ; Lupus Erythematosus, Systemic ; immunology ; pathology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo explore the hematopoietic stem cell distribution and lymphocyte proliferation and differentiation in recipient mice after allogeneic bone marrow transplantation (allo-BMT).

METHODSBALB/c (H-2(d)) mice were total body irradiated 5.5 Gy x 2 by (137)Cs and then transplanted with bone marrow cells from GFP transgenic C57BL/6J (H-2(b)) mice. The femur, spleen, Peyer patches, thymus, liver and peripheral blood of the host were collected on days 3, 7, 21, 35 and 70 post transplantation, and their sections were observed by fluorescent microscopy. The green fluorescent cells were counted with FACS. The phycoerythrin (PE) labeled antibodies to CD4, CD8 and B220 were used for sorting T and B lymphocytes.

RESULTS(1) On day 3 and day 7 after allo-BMT, there were (1.06 +/- 0.02)% and (76.60 +/- 1.80)% of donor's green bone marrow cells in host's spleen respectively, whereas only (0.37 +/- 0.06)% and (39.70 +/- 5.38)% in the bone marrow, respectively. (2) In bone marrow and other organs of 21 day-old chimerism mice, over 60% cells were of donor origin. (3) There were (0.36 +/- 0.04)% donor's bone marrow cells lodging at host's Peyer patches, similar to that in bone marrow.

CONCLUSION(1) The engrafted allogeneic hematopoietic stem cell can move into spleen, bone marrow, Peyer patches and thymus. The spleen is the main lodging place of the engrafted cells early after all-BMT. (2) The majority of cells in chimerism mice immunologic organs were of donor origin. (3) Peyer patches is another lodging place early after allo-BMT.

Animals ; Bone Marrow Cells ; immunology ; Bone Marrow Transplantation ; immunology ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Tissue Donors

OBJECTIVEMesenchymal stem cells (MSCs) were adult stem cells which contribute to the regeneration of mesenchymal tissues such as bone cartilage, muscle, ligament, tendon, adipose and stroma. Due to the multipotential ability and self-renewal capacity, the mesenchymal stem cells can be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy. To enhance the clinical use of MSCs, the investigators studied the isolation and expansion of MSCs from adult bone marrow, fetal bone marrow and human umbilical cord blood, and investigated their biological identities.

METHODSTwo kinds of incubation systems containing L-DMEM or MSC special culture medium were used to purify and expand MSCs. The growth, purification and proliferative abilities of 3 kinds of MSCs were observed and their immunophenotypes were determined by flow-cytometry.

RESULTS(1) The shapes of 3 kinds of cells were same. There was no difference in number and size. The colonies formed early in adult bone marrow MSCs. (2) There was no difference in the expansion speed of the 3 kinds of MSCs, but after the colonies confluenced there had no touching constrain in MSCs from umbilical cord blood and fetal bone marrow. When the colonies confluenced, the cells also had proliferation ability. But in adult bone marrow, the touching constrain was significant. (3) MSCs had strong self-renewal capacity. After primary culture approximately 5 - 6 x 10(5) MSCs were obtained from 8 x 10(6) MNC of bone marrow and 25 x 10(6) MNC of umbilical cord blood. After passage 3, passage 5 and passage 10, the investigators could get 10(7), 10(8) and 10(10) MSCs, respectively. (4) Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased, but they maintained good puripotentiality. After passage 2, passage 3 and passage 5, the purity of MSCs was 90%, 95% and 99%, respectively. (5) Three kinds of MSCs were all positive for CD(29), CD(44), CD(59), CD(90), CD(105), CD(166) and all negative for the markers of hematopoietic cells such as CD(11a), CD(14), CD(33), CD(34), CD(28), CD(45). All the important GVHD correlation markers were negative, such as HLA-DR, B7-1 (CD(80)), B7-2 (CD(86)), CD(40) and CD(40L). There were no differences in the phenotype among the 3 kinds of MSCs cells. (6) The 2 kinds of culture mediums used did not markedly affect isolation and expansion of MSCs, and the biological properties of MSCs.

CONCLUSIONS(1) Human MSCs could be isolated from many kinds of human tissues, and they had no difference in their origin; (2) Human MSCs maintained good puripotentiality and self-renewal capacity. Therefore, they could meet with the need of clinical tissue engineering. (3) The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier but had broad clinical use.

Bone Marrow Cells ; cytology ; immunology ; Cell Culture Techniques ; Cell Differentiation ; immunology ; Cell Division ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; immunology ; Time Factors