细胞因子体外扩增脐血CD+34细胞移植SCID鼠人类免疫功能的建立

目的：探讨扩增的脐血ＣＤ３４细胞体内造血功能。方法：ＦＬＴ３ 配体联合血小板生成素体外培养脐血ＣＤ＋３４细胞２～３ 周后移植ＳＣＩＤ鼠,采用ＦＣＭ 检测ＳＣＩＤ鼠骨髓、外周血、脾脏中的人类ＣＤ４５、ＣＤ３、ＣＤ１９抗原。结果：脐血ＣＤ＋３４细胞扩增１０～３７ 倍,移植６．６×１０４～６．９×１０５ 脐血ＣＤ＋３４细胞于ＳＣＩＤ鼠,６ 周后１０ 只ＳＣＩＤ鼠骨髓中均检测到人类ＣＤ４５抗原（０．２％ ～４．０％ ）,６ 只ＳＣＩＤ鼠外周血、脾脏中检测到人类ＣＤ３ 抗原（０．１％ ～２．３％ ）,３ 只ＳＣＩＤ鼠中外周血、脾脏中检测到人类ＣＤ１９抗原（０．３％ ～１．３％ ）。结论：ＦＬＴ３ 配体联合血小板生成素体外扩增的脐血ＣＤ＋３４细胞能重建ＳＣＩＤ鼠人类免疫功能。     

Replication of human cytomegalovirus in severe combined immunodeficient mice implanted with human retinal tissue.

Because human cytomegalovirus (HCMV) infection and replication are limited to human cells, few animal models can be used to specifically examine the biology of HCMV in vivo. In these studies, fetal human retinal tissue was implanted into the anterior chamber of the severe combined immunodeficient (SCID) mouse eye and subsequently was inoculated with HCMV. Viral replication, localized to glial cells in the xenografts, was first detected 7 days after infection. Thereafter, HCMV replication increased to peak levels through days 21-28 and then gradually decreased to undetectable levels by 8 weeks after infection. The clinical isolate Toledo replicated to higher titers than did strain AD169 or Towne. A comparison of implant age indicated that older tissue could support higher levels of HCMV replication than could younger implants. SCID mice implanted with human retinal tissue provide an excellent model for evaluation of HCMV infection of an ocular structure in vivo.     

Effect of FK-506 on xenografted human Graves' thyroid tissue in severe combined immunodeficient mice

OBJECTIVE We studied the macrolide antibiotic FK-506, an immunosuppressive agent, in an attempt to ameliorate the lesion of autoimmune thyroid disease in human thyroid tissue xenografted into severe combined immunodeficient (SCID) mice. It was not felt appropriate to employ this agent directly in patients with autoimmune thyroid disease because adequate therapeutic modalities are available and the introduction of new, experimental agents could not be justified. Moreover, the study of the tissue before and after treatment could not have been undertaken directly in patients. DESIGN Human thyroid xenografts from four patients with Graves' disease and two normal persons were xenografted into SCID mice. Two weeks after xenograft-ing, human immunoglobulin G (IgG) was detectable in all SCID mice xenografted with Graves' thyroid tissue. Mice were divided into two groups with human IgG levels similar to each other. Mice in the first group were treated with FK-506 daily for 6 weeks; mice in the second (similar) group were given phosphate-buffered saline (PBS) only (control group). MEASUREMENTS Blood samples were taken every 2 weeks from the tail veins for human IgG, thyroid stimulating antibody, thyroperoxidase antibodies, thyroglobulin antibodies, and interferon-gamma (IFN-7). After 8 weeks treatment, animals were sacrificed; thyroid tissue was examined histologically and for thyrocyte HLA-DR expression. FK-506 was also added to thyrocytes in in-vitro tissue culture conditions. RESULTS After 4–6 weeks of FK-506 therapy, human IgG, all thyroid antibodies and IFN-7 were suppressed, while the levels remained elevated in the control group. Lymphocytic infiltration virtually disappeared in the human thyroid tissue of the FK-506-treated mice and thyrocyte HLA-DR expression markedly declined; in the control mice, lymphocytic infiltration remained heavy and HLA-DR expression remained high. On the other hand, FK-506 added directly to thyrocytes in vitro (without lymphocytes) did not reduce thyrocyte HLA-DR expression. CONCLUSIONS FK-506 appears to suppress the activation of intrathyroidal lymphocytes, but not thyrocytes. From these observations, it is concluded that this agent, by its action on intrathyroidal lymphocytes, is able to ameliorate the immunologically mediated histological and serological disturbance in human autoimmune thyroid disease, at least under these circumstances.     

Growth of primary human acute leukemia in severe combined immunodeficient mice

Seven populations of human leukaemic cells were implanted i.v. into sublethally irradiated severe combined immunodeficient (scid) mice. Growth of leukaemia was monitored by labelling murine peripheral blood (PB) cells with an anti-HLA monoclonal antibody and flow cytometric analysis. Two of the populations transplanted were fresh acute lymphoblastic leukaemia (ALL) bone marrow (BM) cells which both caused sustained proliferative growth in scid mice. Human cells accounted for up to a mean of 87% of the total nucleated cells (TNC) in the PB of these mice between weeks 12-15. One of these populations was passaged into fresh mice and frank leukaemia was again established. Three populations of cryopreserved acute myeloblastic leukaemia (AML) cells (2 obtained from PB and 1 from BM) and one population of cryopreserved biphenotypic acute leukaemia BM cells, only grew to a maximum of 4% within the 15 week period of the experiment. A cell population from an AML cell line (HL60), however, did engraft and proliferate resulting in a rapid deterioration of these mice between weeks 3-6 when the proportion of human cells accounted for 9% of the TNC in the PB.     

Epstein-Barr virus (EBV)-associated lymphoproliferations in severe combined immunodeficient mice transplanted with Hodgkin's disease lymph nodes: implications of EBV-positive bystander B lymphocytes rather than EBV-infected Reed-Sternberg cells

To establish an in vivo model for the study of Hodgkin's disease and Reed-Sternberg (RS) cells, 25 lymph node tissue samples involved by Hodgkin's disease were grafted into severe combined immunodeficiency (SCID) mice. Ten Epstein-Barr virus (EBV)-associated tumors were obtained in SCID mice. EBV-positive tumors growing in SCID mice were correlated with the presence of EBV-positive nonneoplastic B cells in patient tumors (90% v 26.6%; P<.01) and was independent of the EBV status of RS cells. Our results suggested that EBV-positive tumors growing in SCID mice originated from normal EBV-positive small lymphocytes (bystander B lymphocytes). We also compared the characteristics of these tumors with those obtained after transplantation of 15 non-Hodgkin's lymphoma and four reactive lymph nodes. The latent period to observe a growing tumor in SCID mice was similar between the two groups (12.86 +/- 5.59 weeks for Hodgkin's disease v 13.6 +/- 5.36 weeks for non-Hodgkin's lymphoma and reactive lymph nodes). The relatively high number of EBV-positive small lymphocytes detected in Hodgkin's disease and T-cell lymphoma compared with B-cell lymphoma may account for the greater percentage of EBV- positive tumors obtained in SCID mice. Our results show that SCID mice do not provide the growth conditions that are required for in vivo growth of RS cells. We noted in some SCID tumors, the presence of binucleated and/or multinucleated giant cells resembling RS cells. However, the presence of such cells was not restricted to mice grafted with lymph nodes involved by Hodgkin's disease. We postulate that in previous reports, cells resembling RS cells were just binucleated EBV- positive lymphoma blastoid cells rather than actual RS cells.     

Antibody-targeted lymphokine-activated killer cells inhibit liver micrometastases in severe combined immunodeficient mice

Animal models for hepatic metastases can facilitate the investigation of lymphokine-activated killer (LAK) cell-based immunotherapy. The aim of this study was to investigate the efficacy of ccM4 antibody-targeted LAK cells in inhibiting hepatic micrometastases. Hepatic micrometastases were generated after the intrasplenic injection of HM7 colon carcinoma cells. TAG72 expression was detected in these hepatic micrometastases using ccM4 antibody. The ccM4 antibody was conjugated onto LAK cells by treatment with 17.5% polyethylene glycol 8000. After the intrasplenic injection of HM7 cells, severe combined immunodeficient mice were randomized into five groups (i–v) and received either 10⁷ ccM4-LAK cells plus 1000 U interleukin 2 (IL-2; group i), LAK cells plus 50 μg ccM4 and IL-2 (group ii), LAK cells plus IL-2 (group iii), IL-2 alone (group iv), or only phosphate-buffered saline (group v). The ccM4-LAK cells retained cytolytic activity and acquired TAG72-binding reactivity. The results showed that group i had significantly fewer hepatic metastases compared with group ii or group iii (P < 0.05) and even fewer hepatic metastases compared with group iv or group v (P < 0.001). These results show that ccM4 antibody-targeted LAK cells significantly inhibited tumor growth in vivo; thus, they can be potentially useful in treatment of hepatic micrometastases.     

Epstein-Barr virus induced lymphoproliferative tumors in severe combined immunodeficient mice are oligoclonal.

Severe combined immunodeficient (SCID) mice reconstituted with lymphocytes from Epstein-Barr virus (EBV) negative human donors develop aggressive tumors after the chimeric mice are infected with EBV. The tumors were composed of human B cells that expressed EBV encoded antigens (latent membrane protein and EBV nuclear antigen2). Southern blot analysis of DNA from 16 SCID/hu tumors with human Ig gene probes showed that each tumor contained multiple heavy and light chain gene rearrangements. Ig kappa gene rearrangements were frequent, while clonal lambda gene rearrangements were infrequent. Analysis of EBV terminal repeat sequences indicated two or more fused termini in each tumor, consistent with a multiclonal origin. Linear terminal repeat segments and viral antigens (EA-D and EA-R) associated with EBV replication were not detected in the tumors. High levels of human Igs in the SCID/hu serum were oligoclonal and primarily contained kappa light chains. Before the appearance of overt tumors, circulating cells with human and EBV DNA could be detected in the SCID/hu mice by the polymerase chain reaction. We conclude that EBV infection in SCID/hu chimeric mice produces a limited number of transformation events, which give rise to oligoclonal tumors resembling EBV-associated lymphoproliferative disorders in some immune-deficient patients.     

Shiga-like toxin purges human lymphoma from bone marrow of severe combined immunodeficient mice

Shiga-like toxin-1 (SLT-1) is a bacterial toxin that kills cells by inhibiting protein synthesis. SLT-1 is composed of one cytotoxic A- subunit and five B-subunits that bind to CD77, a cell-surface glycolipid. In the human hematopoietic system, CD77 expression is restricted to a subset of activated B cells and derived cancers. Here we report that SLT-1 treatment of murine bone marrow ex vivo effectively cures severe combined immunodeficient mice of a human B- cell lymphoma xenograft while sparing normal hematopoietic precursor cells. Flow cytometry results using fluorescein isothiocyanate-labeled SLT-1 B-subunit show the high prevalence of expression of SLT-1 receptors (CD77) in human non-Hodgkin's lymphomas, especially follicular lymphomas. These results suggest the use of SLT-1 for the purging of human bone marrow before autologous bone marrow transplant in the case of CD77+ B-cell lymphomas as just one of many possible uses.     

Adoptive transfer of immunity with intraepithelial lymphocytes in Cryptosporidium parvum-infected severe combined immunodeficient mice

BACKGROUND: Intestinal infections with the protozoan parasite Cryptosporidium parvum are prevalent in both immunocompetent and immunocompromised hosts. Although C parvum is an important cause of outbreaks and opportunistic infections worldwide, little is known about protective mucosal immune responses. This is in part because animal models of infection are limited to those with genetic or induced immunodeficiencies. METHOD: In this report, we isolated immune (primed) or nonimmune (unprimed) intraepithelial lymphocytes (IEL) from BALB/cJ mouse intestines, adoptively transferred them into C parvum-infected severe combined immunodeficient (SCID) mice, and evaluated infection and cell phenotype responses. RESULTS: Control SCID mice that received no IEL shed large numbers of oocysts throughout the experimental period (day 18 to day 72). Transfer of primed IEL significantly reduced fecal oocyst shedding in recipient SCID mice compared with SCID mice that received unprimed IEL or no IEL. SCID mice transferred with unprimed IEL shed variable numbers of fecal oocysts that increased and decreased in bursts until day 57 after infection. SCID mice transferred with primed IEL exhibited significantly higher proportions of T-cell receptor (TCR) alphabeta+, CD8+, and CD8alphabeta+ EL compared with inoculated SCID mice that received unprimed or no IEL. CONCLUSION: We conclude that primed IEL from immunocompetent mice may influence protective mucosal response against cryptosporidiosis when transferred into SCID mice. In addition, the increased percentage of TCR alphabeta+, CD8+, CD8alphabeta+ IEL in recipient SCID mice may reflect mucosal cell populations involved in these responses during chronic C parvum infection.     

Establishment of human T-cell leukemia virus type I T-cell lymphomas in severe combined immunodeficient mice

Human T-cell leukemia virus type I (HTLV-I) is recognized as the etiologic agent of adult T-cell leukemia (ATL), a disease endemic in certain regions of southeastern Japan, Africa, and the Caribbean basin. Although HTLV-I can immortalize T lymphocytes in culture, factors leading to tumor progression after HTLV-I infection remain elusive. Previous attempts to propagate the ATL tumor cells in animals have been unsuccessful. Severe combined immunodeficient (SCID) mice have previously been used to support the survival of human lymphoid cell populations when inoculated with human peripheral blood lymphocytes (PBL). SCID mice were injected intraperitoneally with PBL from patients diagnosed with ATL, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or from asymptomatic HTLV-I-seropositive patients. Many of these mice become persistently infected with HTLV-I. Furthermore, after human reconstitution was established in these mice, HTLV-I-infected cells displayed a proliferative advantage over uninfected human cells. Lymphoblastic lymphomas of human origin developed in animals injected with PBL from two ATL patients. The tumor cells represented outgrowth of the original ATL leukemic clone in that they had monoclonal or oligoclonal integrations of the HTLV-I provirus identical to the leukemic clone and predominantly expressed the cell surface markers, CD4 and CD25. In contrast, cell lines derived by HTLV immortalization of T cells in vitro did not persist or form tumors when inoculated into SCID mice, indicating differences between in vitro immortalized cells and ATL leukemic cells. This system represents the first small animal model to study HTLV-I tumorigenesis in vivo.     

Variables to predict engraftment of umbilical cord blood into immunodeficient mice: usefulness of the non-obese diabetic--severe combined immunodeficient assay

Umbilical cord blood is an alternative stem cell source for patients without matched family donors. In this study, we examined several parameters that have not been studied in detail -- radiation dose, cell dose, age of mice, and maternal and neonatal characteristics of the cord blood donor -- that affect engraftment of cord blood in non-obese diabetic-severe combined immunodeficient (NOD--scid) mice. Engraftment, measured using flow cytometry analyses of human CD45(+) cells, was highest in 400 cGy-treated mice. Successful engraftment was demonstrated up to 6 months, with a mean engraftment of 31% (range 0--67%) of human cells in recipient bone marrow. Engraftment was skewed to B lymphocytes. The radiation dose of 350 cGy resulted in superior survival of the murine recipients compared with 400 cGy (P = 0.03). The sex of the NOD--scid recipients had a significant effect on survival (female superior to male, P = 0.01), but not on engraftment. There were high levels of variability among different cord units and among animals injected with the same cord unit. This variability may limit the clinical usefulness of the NOD--scid mice as hosts for the quantification of human stem cells.     

Interleukin-4 enhances the survival of severe combined immunodeficient mice engrafted with human B-cell precursor leukemia

Human interleukin-4 (huIL-4) has been shown to inhibit the growth in vitro of cells from patients with acute lymphoblastic leukemia (ALL). With the aim of determining whether this cytokine might be useful in the treatment of patients with ALL, the effects of huIL-4 on human B- cell precursor ALL engrafted in severe combined immunodeficient (SCID) mice were examined. The inhibition of [3H] thymidine uptake of primary ALL cells by huIL-4 was maintained following engraftment and passage of leukemia in SCID mice. Five of seven xenograft leukemias showed significant inhibition in vitro by huIL-4 at concentrations as low as 0.5 ng/mL; furthermore, huIL-4 counteracted the proliferative effects of IL-7. When used to treat two human leukemias engrafted in SCID mice, huIL-4 200 microgram/kg/d, as a continuous 14-day subcutaneous infusion, suppressed the appearance of circulating lymphoblasts and extended survival of mice by 39% and 108%, respectively, the first demonstration of IL-4 activity against human leukemia in vivo. The mean steady-state huIL-4 level in mouse plasma during the infusion was 1.46 ng/mL (SEM +/- 0.14 ng/mL), which was similar to concentrations found to be effective in vitro. ALL cells obtained from mice relapsing after huIL-4 treatment continued to show inhibition by the cytokine in vitro. These data suggest that IL-4 may be useful in the treatment of patients with ALL.     

Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.     

Circulation of human hematopoietic cells in severe combined immunodeficient mice after Cl2MDP-liposome-mediated macrophage depletion

Intravenous injection of dichloromethylene diphosphonate (Cl2MDP) encapsulated in liposomes results in specific elimination of macrophages in the spleen and liver of normal mice. Severe combined immunodeficient (SCID) mice were treated with Cl2MDP-liposomes followed by injection of human peripheral blood leukocytes. Control SCID mice had no detectable human cells within 72 hours as determined by fluorescence-activated cell sorting (FACS) analysis. However, Cl2MDP- liposome-treated animals maintained a large proportion (%) of human cells in peripheral blood and spleen for at least 12 days. Cl2MDP- liposome-injected SCID mice that had previously been implanted with human fetal thymus and liver showed a transient increase in human cell content in peripheral blood, and an accumulation of human cells specific to the white pulp of the spleen. These results indicate that murine mononuclear phagocytic cells may play an important role in the clearance of human cells injected intravenously or generated endogenously in SCID mice and that Cl2MDP-liposome-mediated macrophage depletion allows human hematopoietic cells to circulate and survive in SCID mice, thereby expanding the potential for studying human cellular processes in vivo.     

Growth and immortalization of human myeloma cells in immunodeficient severe combined immunodeficiency mice: a preclinical model

Human multiple myeloma (MM) purified tumour cells readily undergo apoptosis in vitro. Interleukin 6 (IL-6), a main growth factor of tumour cells, has enabled the development of IL-6-dependent MM cell lines. Recently, we developed anti-gp130 monoclonal antibodies (mAbs), two of which (B1 + I2) were able to dimerize gp130 and replace IL-6 in vitro. We show here that the injection of B1 + I2 IL-6 agonistic mAbs via the inguinal subcutaneous (SC) route efficiently produced tumours in severe combined immunodeficiency (SCID) mice grafted with IL-6-dependent myeloma cell lines compared with either the intraperitoneal (IP) or abdominal surgical bursa (SB) routes. The SC tumour graft, together with Matrigel and vascular endothelial growth factor (VEGF), leads to a strong vascularization and early detection of serum human immunoglobulins (huIgs). SCID mice treated with B1 + I2 mAbs were injected with fresh MM cells from five patients, four of whom had consistent levels of huIgs, and tumour growth was present in two. For one patient, tumour plasma cells that were passed several times subcutaneously in new SCID mice, still expressed their initial markers after several months. They remained unable to grow in vitro in the presence of B1 + I2 or IL-6. The nature of the SCID factors involved and the triggered genes are under investigation.     

Human growth hormone promotes engraftment of murine or human T cells in severe combined immunodeficient mice.

Growth hormone (GH) has previously been implicated in T-cell development, and here we test its efficacy in promoting T-lymphocyte engraftment in mice with severe combined immune deficiency (SCID). SCID mice receiving syngeneic thymocytes and treated with recombinant human GH (rhGH) had significantly better T-cell engraftment in their lymph nodes. Human T-cell engraftment was also strongly promoted by rhGH in SCID recipients receiving human peripheral blood lymphocytes. Additionally, although mature human cells have not been thought to enter the murine thymus, human T cells were detected in the SCID thymus after rhGH treatment. Thus, rhGH can be used to optimize long-term peripheral T-cell engraftment in these human-mouse chimeras and may also be useful clinically in treating T-cell deficiencies.     

P选择素L-EGF单抗对SCID小鼠胃癌转移抑制作用

目的:研究黏附分子P选择素L-EGF单抗在SCID小鼠胃癌转移中的治疗作用及其机制．方法:用SGC-7901人胃癌组织原位移植SCID小鼠建立转移模型．术后第3天开始,动物分别静脉注射生理盐水(生理盐水组,n=11)或P选择素L-EGF单克隆抗体(L-EGF单抗组,n =9),6 wk末取部分胃癌组织、肿大淋巴结和可疑转移脏器作病理检查;同时取部分胃癌组织作荧光定量PCR检测．结果:生理盐水组肿瘤转移率为81．8％(9／11),L-EGF单抗组转移率为11．1％(1／9),两组相比有显著性差异(P<0．05)．荧光定量PCR测定显示,SCID小鼠伴有癌转移者其胃癌中P选择素mRNA表达较不伴癌转移者明显增强;L-EGF单抗组P选择素mRNA表达较生理盐水组低(Ct值:20．54±2．20 vs 17．09± 1．40,P<0．05)．结论:P选择素与癌转移密切相关,其L-EGF单抗具有抗转移作用;其机制可能与阻断癌细胞与血管内皮的黏附以及抑制 P选择素基因表达有关．     

Nonlethal transfusion associated graft-versus-host disease in a severe combined immunodeficient patient

An X-linked severe combined immunodeficient (SCID) patient received a nonirradiated erythrocyte transfusion and developed transfusion-associated graft-versus-host disease (TAGVHD), which was controllable with high-dose corticosteroids. Haplo-identical SCT was performed, after a myeloablative conditioning regimen. At day +26, he developed GVHD. Chimerism studies revealed DNA of the erythrocyte transfusion donor (ETD) and recipient only. Because of early nonengraftment and the presence of alloreactive T cells of ETD origin, the patient was treated with an immunosuppressive conditioning regimen followed by a second SCT from the same donor. While tapering immunosuppression, he again developed mild GVHD, and DNA of ETD and bone marrow donor origin were both present. On cyclosporin, the ETD-DNA signal finally disappeared. High-resolution HLA typing revealed haplo-identity between BMD, ETD and the patient, which might have contributed to the relative mild course of the TAGVHD.     

Eradication of minimal disease in severe combined immunodeficient mice with disseminated Daudi lymphoma using chemotherapy and an immunotoxin cocktail

Severe combined immunodeficient (SCID) mice injected intravenously with a human Burkitt's lymphoma cell line (Daudi) develop disseminated lymphoma (SCID/Daudi), which is fatal in 100% of the mice. Early treatment of these mice with either an immunotoxin (IT) cocktail (consisting of anti-CD19-ricin A chain plus anti-CD22-ricin A chain) or chemotherapy significantly prolonged survival but was not curative. Combination therapy with the IT cocktail and any one of three chemotherapeutic drugs (doxorubicin, cytoxan, or camptothecin) cured the mice. Cure was demonstrated by both histopathologic examination of treated mice and, more importantly, by adoptive transfer of cells from organs of the cured mice to naive SCID mice where 100 tumor cells would have caused disease in the recipients. These results provide a strong rationale for combining IT therapy with conventional chemotherapy in the treatment of B-cell neoplasia.