Characterization of a novel transplantable orthotopic nude mouse model with xenografted human bladder transitional cell tumor (BIU-87) / 中华肿瘤杂志

<p><b>OBJECTIVE</b>A mouse model of orthotopic bladder cancer simulating its human counterpart is of great importance in preclinical evaluation of new treatment modalities such as immunotxin therapy. The aim of the present study is to establish a novel nude mouse model with xenografted human bladder cancer.</p><p><b>METHODS</b>Single cell suspension of an established human bladder transitional cell carcinoma (TCC) cell line BIU-87 was instilled into nude mouse bladders which were pretreated with mild acid washing. The tumor growth in mouse bladder was assessed weekly by magnetic resonance imaging (MRI). At intervals following implantation and MRI tumor detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies.</p><p><b>RESULTS</b>The overall tumor establishment was 92.9% (52/56 mice) at 7 - 36 days, while in the subgroup of animals sacrificed at 12 - 13 days, 40 out of 42 animals (95.2%) developed TCC, the majority of which was superficial. The tumor stages were assessed by gross and histopathology. Histological examination confirmed the presence of grade II - III TCC. Immunocytochemistry confirmed that the tumor model maintained the biological and immunological features of BIU-87 cells. The changes seen on MRI images well correlated with the extent of tumor invasion identified by histology. Carcinoma in situ could be detected histologically at 7 - 9 days post-inoculation and progressed into papillary or invasive tumors thereafter.</p><p><b>CONCLUSION</b>The orthotopic BIU-87 TCC model in nude mice is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.</p>

Preparation of arsenic trioxide-loaded albuminutes immuno-nanospheres and its specific killing effect on bladder cancer cell in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recently, arsenic trioxide (As2O3) was considered as a novel anti-tumor agent. However, it showed severe toxicity effect on normal tissue at the same time. To improve its therapeutic efficacy and decrease its toxicity,we prepared arsenic trioxide-loaded albuminutes immuno-nanospheres [As2O3-(HAS-NS)-BDI-1] targeted with monoclonal antibody (McAb) BDI-1 and tested its specific killing effect against bladder cancer cell.</p><p><b>METHODS</b>As2O3-HAS-NS was prepared by chemical cross-linking method. Monoclonal antibody BDI-1 was purified with ammonium sulphate saltingout and chromatography. Albuminutes microspheres were conjugated with McAb by SPDP cross-linking method. Concentration of As in As2O3-(HAS-NS)-BDI-1 and As2O3-HAS-NS was measured by atomic fluometry method. As2O3-(HAS-NS)-BDI-1 and its activity were detected by SDS-PAGE reduction electrophoresis, indirect immunofluorescence test, light microscope and scanning electron microscope observation. Acridine orange staining and tritiated thymidine (3H-TdR) incorporation tests were used to indicate specific killing activity of As2O3-(HAS-NS)-BDI-1 in vitro.</p><p><b>RESULTS</b>In As2O3-(HAS-NS)-BDI-1 groups, we saw two protein bands in SDS-PAGE reduction electrophoresis. Albuminutes immuno-nanospheres were rounded with clear green fluorescence by immunofluorescence test. Under microscope, we observed that BIU-87 cells were covered with the As2O3-(HAS-NS)-BDI-1 and that As2O3-(HAS-NS)-BDI-1 moved with the BIU-87 cells. The albuminutes immuno-nanospheres were tightly junctioned with the BIU-87 cells. Specific killing activity of As2O3-(HAS-NS)-BDI-1 on bladder tumor cells was observed by acridine orange staining and 3H-TdR incorporation assays.</p><p><b>CONCLUSIONS</b>As2O3-(HAS-NS)-BDI-1 might bind specifically against BIU-87 cells, thus leading to high activity of killing bladder tumor cells.</p>

Bone marrow stromal cell line co-transfected with IL-2 and IL-3 genes can accelerate restoration of T-cell immunity in allo-BMT mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>After T-cell depleted allogeneic bone marrow transplantation, impaired immune reconstitution is a major cause of morbidity and mortality in the recipient. The purpose of this study was to observe the effects of the gene-engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 on the reconstitution of T-cell immunity in allo-BMT mice.</p><p><b>METHODS</b>The bone marrow stromal cell line QXMSC1 was co-transfected with IL-2 and IL-3 genes using a Tet-on gene expression system. T lymphocyte subset counts per spleen were analyzed by flow cytometry. Lymphocyte proliferation response to ConA was examined to evaluate T-cell function. CDR3 spectratyping techniques were performed to evaluate TCR repertoire diversity at various time points post-transplantation.</p><p><b>RESULTS</b>Gene engineered bone marrow stromal cell line QXMSC1-IL-2 + IL-3 could express IL-2 and IL-3 [1,300 ng.day(-1).10(-6) cells and 1100 ng.day(-1).10(-6) cells, respectively] under the control of doxycycline. QXMSC1-IL-2 + IL-3 in combination with allogeneic bone marrow could significantly increase the counts of CD(4)(+) and CD(8)(+) T cell, 1.72 and 1.27-fold respectively at week 3 compared with TCD-BMT group (P < 0.01); make CD(4)(+)/CD(8)(+) ratio return to normal level at week 4; enhance splenocytes mitotic response to ConA (P < 0.01), and accelerate restoration of TCR repertoire diversity in the lethally irradiated mice (P < 0.05).</p><p><b>CONCLUSION</b>The gene transduced stromal cell line QXMSC1-IL-2 + IL-3 is able to accelerate T-cell immunity in allo-BMT mice.</p>

Effect of T suppressor cells on the maintenance phase of tolerance to cardiac allografts in the rats / 中华外科杂志

<p><b>OBJECTIVE</b>To study the role of T suppressor cells in immune tolerance to cardiac allografts in the rats.</p><p><b>METHODS</b>Male DA rat hearts were transplanted to male Lewis rats using Ono's model and randomly divided into five groups: group 1: untreated, group 2: portal venous injection of 3 x 10(8) DA splenocytes to Lewis rat, group 3: intraperitoneal injection of cyclophosphamide (80 mg/kg) to Lewis rat, group 4: portal venous injection of 3 x 10(8) DA splenocytes combined with intraperitoneal injection of cyclophosphamide (80 mg/kg) to Lewis rat, 15 days later heart transplantation was performed. Group 5: intravenous injection 3 (108 splenocytes of group 4 to normal recipient, and then heart transplantation was performed. Mean survival time (MST), histological changes, mixed lymphocyte reaction (MLR) were measured after operation.</p><p><b>RESULTS</b>The survival time of heart allografts in the group 4 [MST: (71.5 +/- 29.1) d, t = -14.063, -13.915, -13.777; P < 0.01] was significantly longer than in the groups of 1 [MST: (7.3 +/- 1.0) d], 2 [MST: (7.8 +/- 0.8) d], 3 [MST: (8.2 +/- 1.1) d ]. Only a few inflammatory cells infiltrated in cardiac allografts in the groups of 4 and 5. MLR in the groups of 4 and 5 were significantly decreased compared with those of normal control (t = 29.902, 23.047; P < 0.01).</p><p><b>CONCLUSIONS</b>Portal venous injection of donor splenocytes combined with intraperitoneal injection of cyclophosphamide could induce immune tolerance to cardiac allografts. The immune tolerance could be transferred through splenocytes. T suppressor cells play an important role in the immune tolerance.</p>

Induced tolerance to cardiac allografts with multiple intravenous injection of donor spleen cells / 中华外科杂志

<p><b>OBJECTIVE</b>To study the methods and mechanisms of immune tolerance in cardiac transplantation.</p><p><b>METHODS</b>Male DA rat hearts were transplanted to male Lewis rats using Ono's model and randomly divided into five groups: untreated, intravenous injection of 1 x 10(8) DA splenocytes to Lewis rat, intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat, intravenous injection of 1 x 10(8) DA splenocytes combined with intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat, multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide, 11 days later heart transplantation was performed. Mean survival time (MST), histological changes, mixed lymphocyte reaction (MLR), the role of interleukin-2 (IL-2) to MLR and the role of tolerant rat splenocytes to MLR were measured after operation.</p><p><b>RESULTS</b>The survival time of heart allografts in the group of multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide [MST: (85.3 +/- 7.5) d, t = 0, P < 0.01] was significantly longer than in the groups of untreated [MST: (7.3 +/- 1.0) d], intravenous injection of 1 x 10(8) DA splenocytes to Lewis rat [MST: (7.9 +/- 0.9) d], intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat [MST: (8.1 +/- 1.2) d], intravenous injection of 1 x 10(8) DA splenocytes combined with intraperitoneal injection of cyclophosphamide (100 mg/kg) to Lewis rat [MST: (25.8 +/- 3.5) d]. Only a few inflammatory cells infiltrated in cardiac allografts in the group of multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide. MLR in the group of multiple injection of DA rat splenocytes with intraperitoneal injection of cyclophosphamide were significantly decreased compared with those of normal control (t = 0, P < 0.01). IL-2 could partly reversed the hyporesponsiveness of MLR in tolerant rats, the tolerance could be transferred in vitro.</p><p><b>CONCLUSIONS</b>Multiple injection of donor splenocytes combined with intraperitoneal injection of cyclophosphamide to recipients could induce immune tolerance to cardiac allografts.</p>

The improving effect of bone marrow stromal cell transfected with IL-3 gene on hematopoietic reconstitution in bone marrow transplantation of mice / 中国实验血液学杂志

To study the improving effect of regulatable gene of IL-3 engineered bone marrow stromal cell on the hematopoietic reconstitution in allogeneic bone marrow transplantation, an inducible gene expression system was established in a bone marrow stromal cell line which expressed IL-3 gene induced by doxycycline (Dox). The lethally irradiated mice C57BL/6 (H-2(d)) were co-transplanted with allogeneic bone marrow (BALB/c, H-2(d), 1 x 10(7)/mice) in which T cell were depleted by monoclonal antibody anti-Thy1.2 added with complement and the gene engineered stromal cell QXMSC1tet-on + IL-3 (5 x 10(5)/mice) at the same time. Dox was administrated continuously for 15 days to induce the expression of IL-3. The hematopoiesis in the bone marrow transplanted mice were observed at 30, 60 days post-transplantation, respectively. The numbers of RBC and WBC in peripheral blood were counted, and nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM were measured in recipient bone marrow. The results showed that the engineered stromal cell line achieved high-level and controllable IL-3 expression. Co-graft with QXMSC1tet-on + IL-3 significantly increased the number of RBC, WBC in recipient peripheral blood, and the nucleated cells, CFU-S, CFU-GM, CFU-E, CFU-GEMM in bone marrow, compared with those coinfused with QXMSC1 or QXMSC1tet-on-TRE as control. In conclusion, regulatable gene IL-3 engineered bone marrow stromal cells accelerates hematopoietic reconstitution after allogeneic bone marrow transplantation.

The enhancing effects of IL-3 gene transfected murine bone marrow stromal cells on hematopoiesis under control of doxycycline / 中国实验血液学杂志

To study enhancing effect of IL-3 gene transfected bone marrow stromal cell which can be induced by doxycycline (Dox) to express IL-3 cytokine on the proliferation and differentiation of hematopoietic stem cell, retrovirus vector system contained mIL-3 cDNA was established and bone marrow stromal cell line was transfected, and obtained QXMSC1Tet-on-IL-3, in which expression level of IL-3 can be modulated by Dox. The activities of IL-3 were measured under different Dox concentrations. The numbers of hematopoietic progenitors (CFU-GM, CFU-E, CFU-GEMM and LTC-IC) were measured and the capacity of QXMSC1Tet-on-IL-3 sustaining hematopoietic progenitor cell growth was evaluated. The results showed that IL-3 gene transfected stromal cell line QXMSC1-Tet-on + IL-3 expressed high concentration of IL-3 in vitro under control of Dox. The supernatant of QXMSC1-Tet-on + IL-3 was able to increase the number of CFU-GM, CFU-E and CFU-GEMM. The total numbers of nucleated cells and long-term cultured colonies increased in LTC-IC assay. It is concluded that in the culture of QXMSC1-Tet-on + IL-3 cells, Dox actually enhanced IL-3 expression, and thus augmented the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro.

Accelerating hematopoietic recovery in allogeneic bone marrow transplantation mice by co-infusion of marrow stromal cells transduced with dual genes of IL-3/IL-6 / 中国实验血液学杂志

To observe whether bone marrow stromal cell line QXMSC1 (H-2(d)) engineered to secrete IL-3 and IL-6 can improved the hematopoiesis in allogeneic bone marrow transplantation (allo-BMT) mice, the stromal cell line QXMSC1IL-3/IL-6 was established by QXMSC1 cells transduced with the recombined retrovirus vector pL3SN containing mouse IL-3 cDNA and pL6SN containing human IL-6 cDNA. The lethally irradiated C57BL/6 (H-2(b)) mice were engrafted with bone marrow cells (1 x 10(7) cells/mouse BALB/c mice, H-2(d)) in which T cells were depleted by anti-Thy1.2 monoclonal antibody adding complement, and QXMSC1IL-3/IL-6 cells (5 x 10(5)/mouse) were co-infused at same time. The hematopoiesis of recipient mice was observed in 20 and 40 days post-transplantation. Blood RBC and WBC were counted, and nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM were assayed in recipient bone marrow. Results showed that IL-3 and IL-6 were stably expressed in QXMSCQIL-3/IL-6 cells. Compared with BMT and co-infusion with QXMSC1 or QXMSC1 pLXSN cell groups, co-graft with QXMSC1IL-3/IL-6 cells increased the number of blood RBC and WBC in the recipients, and also significantly increased nucleated cells, CFU-S, CFU-GM, CFU-E and CFU-GEMM in recipient bone marrow. It is concluded that the marrow stromal cells transduced with IL-3/IL-6 cDNA improve the hematopoiesis in allo-BMT mice. Co-graft with QXMSC1IL-3/IL-6 cells has synergistic effect in accelerating hematopoietic reconstitution.