This study was aimed to investigate the effect of human umbilical vein endothelial cells (HUVEC) on dendritic cell (DC) development. First, HUVEC were isolated from human umbilical cord by collagenase digestion, and then the morphology, immunophenotypes and functions were identified. Furthermore, the HUVEC were cocultured with CD14(+) monocytes under the cytokine condition for detecting the influence of HUVEC on differentiation of CD14(+) cells to DC. The phenotype of dendritic cells derived from CD14(+) cells was analyzed by flow cytometry, the immunoregulatory function of DC was tested by mixed lymphocyte reaction (MLR). The change of IL-6 and VEGF as well as EPK and p38 signal pathway were analyzed by neutral antibody experiment and Western blot. The results showed that HUVEC isolated from human umbilical cord were characterized by spindle-shaped morphology, homogenous immunophenotypes (vWF(+)CD31(+)CD73(+)CD45(-)HLA-DR(-)CD86(-)CD34(low)), Dil-Ac-LDL incorporation ability and forming capillary-like structures. Following stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), HUVEC cocultures could inhibit the initial differentiation of CD14(+) monocyte to DC. Interestingly, IL-6 and VEGF enhanced the suppression effect of HUVEC on generation of DC via activation of the ERK or p38 mitogen activated protein kinase pathway. It is concluded that HUVEC are involved in DC development and can suppress the differentiation of monocyte to DC.

This study was aimed to investigate whether endothelium-specific deletion of PTEN can affect hemangioblast development in the AGM region of mouse embryos. Based on Cre/loxP system, the Tie2CrePten(loxp/loxp) and Tie2CrePten(loxp/wt) mouse embryos were obtained. The genotype was identified by PCR. After treated with type I collagenase, the AGM region was dispersed into single-cell suspension, and then was cultured in blast colony-forming cell (BL-CFC) media. The number of BL-CFC was counted 4 or 5 days later. The hematopoietic capacity of BL-CFC was detected in methylcellulose culture system and the endothelial potential was assessed by tube-like structure formation on Matrigel. The results showed that the number of BL-CFC in AGM region of Tie2CrePten(loxp/loxp) mouse embryo decreased as compared with Tie2CrePten(loxp/wt) embryo. Whereas the hematopoietic capacity of mutant BL-CFC was enhanced, the endothelial potential, as evaluated by tube-like structure formation in vitro, was significantly reduced. It is concluded that the endothelial PTEN is capable of exerting regulatory functions on both the numbers and the dual potential of hemangioblast in mouse AGM region.

This study was purposed to investigate the effect of RUNX1 on transcription activity of WNT5A promoter in mouse bone marrow derived mesenchymal stem cells (MSC), and to explore the mechanism by which bone marrow environments regulate MSC. RT-PCR was used to detect the expression of RUNX1 in MSC isolated from mouse bone marrow and cultured in vitro; the chromatin immunoprecipitation (ChIP) was used to investigate the direct in vivo interaction between the RUNX1 and WNT5A promoter; retrovirus system was utilized to introduce the RUNX1 gene into MSC to detect the regulation of RUNX1 on the transcription activity of WNT5A promoter. The results showed that mouse bone marrow derived MSC was positive for Oil Red O, van Kossa and toluidine blue staining respectively and RUNX1 expressed in MSC. WNT5A promoter could be bound by RUNX1, and the expression level of WNT5A was enhanced with the increase of RUNX1. It is concluded that RUNX1 expresses in mouse bone marrow derived MSC, WNT5A is a direct target gene of RUNX1 and its transcriptional activity is regulated by RUNX1.

This study was aimed to investigate the effect of RPMI 1640 and IMDM on the development of human peripheral blood monocyte-derived dendritic cells. Under the same cytokines and culture conditions, the different medium types were tested, and the morphology of mature and immature dendritic cells was observed by microscopy, the cell phenotype and endocytosis ability were detected by flow cytometry. Furthermore, the immunoregulatory function of various DC was analyzed by mixed lymphocyte reaction (MLR), the expression of cytokine in culture supernatant of MLR system was also analyzed by Bio-plex technology. The results showed that there were no difference in morphology, CD14, CD83 expression and endocytosis ability between IMDM-cultured DC and RPMI-1640 medium-cultured DC, but there was a lower expression of CD1a in IMDM-cultured DC. Moreover, DC cultured with IMDM displayed a significant reduction in stimulating T cell proliferation, and highly expressed IL-6, IL-8 and IL-10, but low expressed IL-12. It is concluded that the different cultural mediums can induce DC with different functions and DC cultured with IMDM may correlated with induction of immune tolerance. The results of this study will provide a new idea for DC clinical application.

Bone remodeling involves bone resorption by osteoclasts and synthesis by osteoblasts and is tightly regulated by the receptor activator of the NF-κB ligand (RANKL)/receptor activator of the NF-κB (RANK)/osteoprotegerin molecular triad. RANKL, a member of the TNF superfamily, induces osteoclast differentiation, activation and survival upon interaction with its receptor RANK. The decoy receptor osteoprotegerin inhibits osteoclast formation by binding to RANKL. Imbalance in this molecular triad can result in diseases, including osteoporosis and rheumatoid arthritis. In this study, we report the crystal structures of unliganded RANK and its complex with RANKL and elucidation of critical residues for the function of the receptor pair. RANK represents the longest TNFR with four full cysteine-rich domains (CRDs) in which the CRD4 is stabilized by a sodium ion and a rigid linkage with CRD3. On association, RANK moves via a hinge region between the CRD2 and CRD3 to make close contact with RANKL; a significant structural change previously unseen in the engagement of TNFR superfamily 1A with its ligand. The high-affinity interaction between RANK and RANKL, maintained by continuous contact between the pair rather than the patched interaction commonly observed, is necessary for the function because a slightly reduced affinity induced by mutation produces significant disruption of osteoclast formation. The structures of RANK and RANKL–RANK complex and the biological data presented in the paper are essential for not only our understanding of the specific nature of the signaling mechanism and of disease-related mutations found in patients but also structure based drug design.

This study was purposed to investigate the influence of inflammatory microenvironment on mesenchymal stem cells (MSCs) and regulatory effect of MSCs on osteoblast formation. The MSCs were isolated from synovial fluid of patients with rheumatoid arthritis (RASF-MSCs) and were cultured, the immunotypes of RASF-MSCs were detected by flow cytometry, the ability to differentiate RASF-MSCs into osteoblasts and adipocytes was determined by means of osteogenic and adipogenic induction, the regulatory effect of RASF-MSCs on osteoblast formation was assayed by co-culturing RASF-MSCs whth CD14(+) monocytes and in situ tartrate-resistant acid phosphatase staining. The results showed that RASF-MSCs highly expressed CD105, CD73, CD29, CD44, CD166 and HLA-ABC. Meanwhile, they lowly expressed CD34, CD45, CD31, HLA-DR, CD80 and CD86. However, RASF-MSCs decreased multi-differentiation capability as compared with BM-MSCs. More interestingly, RASF-MSC significantly promoted osteoclasts formation (p < 0.05) when co-cultured with monocytes. It is concluded that MSCs from rheumatoid arthritis synovial fluid exert typical MSC phenotypes but displayed decline of multi-differentiation capability. RASF-MSCs especially show promoting effect on osteoclastogenesis. The findings of this study may contribute to the understanding biological behavior of MSCs in pathological microenvironment.

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.

Mesenchymal stem cells (MSCs) are characterized by their hematopoiesis-supporting and immunosuppressive capacity, while osteoclasts are main cell components in the endosteal hematopoietic stem cell niche and pivotal players in osteoimmunology. To clarify the association of these 2 kinds of cells, mouse CD11b(+) monocytes were cultured onto MSC layers in the presence or absence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). The results showed that MSCs independently supported osteoclast development and this effect was enhanced by M-CSF and RANKL. Interestingly, tumor necrosis factor-alpha (TNF-alpha)-stimulated MSCs turned to inhibit osteoclast formation and protect tusk slices from osteoclastic resorption. Real-time PCR and ELISA assays demonstrated that osteoprotegerin expression at both mRNA and protein levels in TNF-alpha-stimulated MSCs was up-regulated, at least partially by activating the mitogen-activated protein kinase pathway. Furthermore, TNF-alpha-stimulated MSCs maintained their immunophenotypic, multipotential differentiation and immunosuppressive characteristics. Moreover, MSCs treated with synovial fluid from rheumatoid arthritis patients modulated osteoclast generation in close relation with the TNF-alpha levels. This study suggests that MSCs exhibit dual modulatory function on osteoclasts and the result might shed light on understanding the involvement of MSCs in the inflammatory diseases.

We have reported a new approach to fabricate mesoporous silica composite nanosheets by milling the corresponding hollow spheres. The mesoporous nanosheets are amphiphilic, and can be well dispersible both in water and oil, serving as particulate emulsifiers in o/w or w/o systems. The mesoporous silica can assist other functional materials, for example metal and carbon to be dispersible. An example is given to demonstrate the support of catalysts for a heterogeneous catalytic aerobic oxidation of benzyl alcohols by Pt/silica composite nanosheets.

This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.

word counts: 200 Scientific heading: Transplantation Blood First Edition Paper, prepublished online February 3, 2005; DOI 10.1182/blood-2004-02-0586 Copyright © 2005 American Society of Hematology For personal use only. on November 16, 2017. by guest www.bloodjournal.org From

OBJECTIVE:E7 is regarded as one of the main oncoproteins of high-risk human papillomaviruses (HPVs). It may affect the transforming growth factor beta 1 (TGF-beta1) signaling pathway. In this study, the relationship between HPV-16 infection and the functions of three critical factors of the TGF-beta1/Smads pathway was explored to assess the possible role of E7 in the development of cervical cancer. METHODS:The expression of E7, TGF-beta1, TbetaR-II and Smad4 was detected by immunohistochemistry in paraffin-embedded cervical samples, and by RT-PCR and Western blotting in cervical cancer cell lines. The effect of TGF-beta1 on the growth of cervical cancer cells were tested by methyl thiazolyl tetrazolium (MTT), and the effects of HPV-16 E7 protein on normal and malignant cervical cells were investigated by flow cytometry. RESULTS:During the progression from benign to malignant lesions, the expression levels of TGF-beta1 and Smad4 increased significantly in cervical carcinoma tissues. The expression of TGF-beta1 was positively correlated with E7 expression. In vitro experiments showed that TGF-beta1 could not inhibit the proliferation of several cervical carcinoma cell lines in long-term regulation, but could inhibit immunologic reactions of peripheral blood mononuclear cells (PBMCs). Blocking E7 expression could lower the expression level of TGF-beta1 and induce cells to enter apoptosis. CONCLUSIONS:Our data indicate that HPV-16 E7 protein plays an important role during the development of cervical cancer by immuno-inhibition and stimulation of tumor cell proliferation through the TGF-beta1/Smads signaling pathway.

BACKGROUND:DC are potent APC that can activate both CD4 and CD8 T cells in vitro and in vivo. Although the efficacy of DC-based cancer vaccines is currently being evaluated in clinical trials, the systemic immune suppression in cancer patients negatively impacts the clinical benefit of this therapeutic approach. Therefore, in this study we tested the feasibility and anti-tumor effect of adoptive immunotherapy using in vitro-activated CD62L(low) lymph node cells that were isolated from DC-vaccinated draining lymph nodes (VDLN).METHODS:DC were prepared from BM cells and loaded with tumor lysate for inoculating into naive mice. Subsequently, the VDLN were removed and CD62L(low) cells in the VDLN population isolated, expanded in vitro by 5-day culture with IL-2 and immobilized anti-CD3 stimulation, then injected into mice with established pulmonary tumors. Eighteen days after treatment, mice were killed in order to enumerate pulmonary tumor nodes.RESULTS:DC phagocytosed the tumor lysate efficiently and induced detectable T-cell responses and significant cell expansion in the draining lymph nodes. After induction of maturation by LPS treatment, DC expressed higher levels of CD40, CD86 and MHC class II molecules. When CD62L(low) VDLN cells that had been isolated and expanded in vitro were transferred into tumor-bearing mice, as few as 3 x 10(6) cells were able to cure metastatic pulmonary tumors in vivo.DISCUSSION:DC-based VDLN T cells are an important source of anti-tumor effector for adoptive immunotherapy. This study provides a novel and an effective protocol using T-cell adoptive immunotherapy for application in cancer patients; therefore, clinical trials based on this protocol may be warranted.

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, have a direct immunosuppressive effect on T-cell proliferation in vitro. However, it is unclear whether they also modulate the immune system by acting on the very first step. In this investigation, we addressed the effects of human MSCs on the differentiation, maturation, and function of dendritic cells (DCs) derived from CD14+ monocytes in vitro. Upon induction with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4), MSC coculture could strongly inhibit the initial differentiation of monocytes to DCs, but this effect is reversible. In particular, such suppression could be recapitulated with no intercellular contact at a higher MSC/monocyte ratio (1:10). Furthermore, mature DCs treated with MSCs were significantly reduced in the expression of CD83, suggesting their skew to immature status. Meanwhile, decreased expression of presentation molecules (HLA-DR and CD1a) and costimulatory molecules (CD80 and CD86) and down-regulated IL-12 secretion were also observed. In consistence, the allostimulatory ability of MSC-treated mature DCs on allogeneic T cells was impaired. In conclusion, our data suggested for the first time that human MSCs could suppress monocyte differentiation into DCs, the most potent antigen-presenting cells (APCs), thus indicating the versatile regulation of MSCs on the ultimate specific immune response.

Objective. Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34(+) cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34(+) cells from UCB in vitro.Materials and Methods. Human placenta MPC was isolated and characterized by morphologic, immunophenotypical, and functional analysis. UCB CD34(+) cells were expanded by coculture with placental MPC. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay.Results. In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin, and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and alpha-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes. In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of cytokines, was significantly enhanced: CD34(+) cells by 14.89 +/- 2.32 fold; colonyforming cell (CFC) by 36.73 +/- 5.79 fold; and LTC-IC by 7.43 +/- 2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli.Conclusion. These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc.

To explore the feasibility of nonmyeloablative conditioning regimens, hematopoietic reconstitution, chimera level and the occurrence of GVHD after nonmyeloablative allogeneic stem cell transplantation in H-2 haploidentical mice, CB6F1 mice were used as the recipient and were divided into 3 groups, mice were pretreated five days before transplantation. Group A was pretreated with myeloablative conditioning regimens (TBI with 10.5 Gy), group B was pretreated by TBI (2 Gy) + Ara-C + Cy and group C-TBI (2 Gy) + Ara-C + CY + Flu, respectively. For all recipient mice, the prevention of GVHD was not given, and 2 x 10(7) bone marrow cells mixed 1 x 10(7) spleen cells from C57BL/6 mice were injected through tail vein on day 0, and then hematopoietic recovery, engraftment and GVHD of recipients were observed. The results of chimera detection after transplantation showed that the engraftment of group A remained full donor chimerism, and engraftments of group B and group C were associated with mixed chimerism or full donor chimerism, but the chimerism of group B remained below 80% and tended to decrease after 50 days whereas chimerism of group C was above 80% (chimerism close to or being full donor type) and preserved even after 50 days. GVHD occurred in all the recipient mice due to that prevention was not given, wherein the occurrence and death rate of GVHD in group A was obviously higher than that of group B and group C (P <0.01), but there was no statistical difference between group B and group C. In conclusion, the nonmyeloablative conditioning regimens mainly based on fludarabine can form stable and lasting engraftment in the body of recipients. The mixed chimerism established in recipients induce tolerance of transplantation and decrease or avoid the occurrence of GVHD.

To address the question whether there exists mesenchymal stem cells in adult mouse skeletal muscle, mononuclear cells from muscle were obtained by digestion and density gradient centrifugation and plated in alpha-MEM/F12 medium containing 10% fetal bovine serum. Cell biological properties including morphology, cytochemistry, growth pattern and phenotypes as well were evaluated. Likewise, the osteogenesis of cultured cells was also observed. The results showed that adherent cells homogenous in shape proliferated quickly in the culture system. The phenotypes of the cells were unique, which were positive for CD29 and Sca-1, and negative for CD34 and CD45. Cytochemistry evaluation showed that they were homogeneously positive for acid alpha-naphthl acetate esterase (ANAE) and acid phosphatase (ACP), and negative for alkaline phosphatase (ALP) and that, around 5% of them were positive for glycogen (periodic acid-Schiff reaction, PAS). Cells became ALP-positive after the induction by ascorbic acid, beta-phosphoglycerol and dexamethasone. It is concluded that mesenchymal stem cells exist in murine skeletal muscle and compose the complex heterogenous population of stem cells in muscle.

In order to research the prophylactic effect of cyclosporine A (CSA) and mycophenolate mofetile (MMF) on GVHD in mice with H-2 haploidentical nonmyeloablative bone marrow transplantation, a murine model was established by using of C57BL/6J mouse as donor and (C57BL/6J x BALB/C) F(1) mouse as the recipient. The recipient mice were given CSA + MTX or CSA + MMF for prophylaxis of acute GVHD (aGVHD). The survival rate, hematopoietic recovery, and morbidity and mortality of aGVHD were observed for 50 days after transplantation. The results showed that typical aGVHD developed in the transplanted mice without prophylactic treatment during 22 to 25 days after transplantation. The morbidity of aGVHD was 75% (15/20), 40% (8/20) and 30% (6/20) and mortality was 100% (15/15), 62.5% (5/8) and 50% (3/6) respectively in unprophylactic group (control), CSA + MTX and CSA + MMF groups. In conclusion, CSA and MTX reduce the morbidity and mortality of aGVHD in mice with haploidentical nonmyeloablative bone marrow transplantation, and the effect of CSA + MMF is better than that of CSA + MTX.

The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors associated closely with stem cell proliferation including SCL, GATA-2 and AML1 as well as various receptors of hematopoietic growth factors such as c-kit, GM-CSF receptor and interleukin 3 receptor et al. Finally, in order to understand the in vivo hematopoietic capacity of the ES cells-derived HPP-CFC, spleen colony-forming unit (CFU-S) assay was performed. Nevertheless, typical CFU-S was not observed after transplantation of the day 12 EB cells or HPP-CFC colonies into lethally irradiated adult murine. In conclusion the HPP-CFC differentiated from murine ES cells displayed robust hematopoietic activity in vitro, however their in vivo reconstitution ability was not detected. The difference between in vitro and in vivo hematopoietic activities of ES cells-derived primitive hematopoietic precursors deserves further investigation.

The purpose of this study was to evaluate whether the DNA vaccine containing idiotypic gene fragment of human B-cell lymphoma cell line Namalwa could elicit the specific anti-idiotypic immune response in vivo. The candidate gene fragment of the lymphoma cell, variable region of heavy chain (VH) of the membranous immunoglobulin, was amplified using Ig superfamily primers by means of RT-PCR. Also, the intact cDNA of murine monocyte chemoattractant protein (MCP-3) was cloned and used as the adjuvant molecular. The two gene fragments of VH and MCP-3 were fused together by 8aa linker peptide with recombinant PCR. Subsequently, the fusion gene fragment was cloned into eukaryonic expression vector pcDNA3.1 to construct DNA vaccine plasmid. Prior to the immunization, the transient transfection coupled with RT-PCR was performed to prove that the recombinant plasmid could express in eukaryonic cells in right way. Then two groups of mice were immunized by intramuscular injection with DNA vaccine and mock plasmid pcDNA3.1 respectively. Three times of injection were performed with 100 micro g plasmid respectively at the beginning of the experiment and 2, 4 weeks after the first injection for all the groups. FACS analysis was chosen to detect the antibodies recognizing lymphoma cells at different time following vaccination. The results demonstrated that specific anti-idiotypic antibody could be detected in the group of DNA vaccine immunized mice as early as eight weeks after the first immunization. Further test demonstrated that the anti-idiotypic antibody could maintain for at least twenty weeks with high titer. Anti-idiotypic antibodies were elicited in three of five mice of the DNA vaccine immunized group. The Abs of DNA vaccine immunized mice could only recognize Namalwa cell line instead of another unrelated human cell line A549. There is no cellular response detected in the DNA vaccine immunized mice. It is concluded that the DNA vaccine containing fused MCP3-VH sequence could elicit specific anti-idiotypic antibody against B-cell lymphoma in vivo and could be used in further study of DNA vaccine against B-cell lymphoma. The results would provide the basis for further studies and optimization of this therapeutic strategy on patients with B-lymphoproliferative disease.