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PubMed2006

An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials.

The generation of germline competent chimeric mice via embryonic stem (ES) cells is a crucial step in developing gene-manipulated mouse models. To date, techniques for generating chimeric mice include direct microinjection of ES cells into the cavity of 3.5-d post-coitum (dpc) blastocysts and aggregating or coculturing 2.5 dpc zona pellucida-free (denuded) embryos with ES cells. We present here a procedure that is simple and reproducible for mass producing (10-150 embryos/vial/time) chimeric embryos by coculturing denuded 8-cell embryos and morula in 0.8 mL KSOM-AA medium containing 5 x 10(5)mL-1 purified green fluorescence protein-expressing ES cells (either fresh or thawed) in an 1.7 mL Eppendorf vial for 3h. The resulting chimeras had substantial levels of chimerism and high germline transmission rates. Therefore, the method developed in this study can provide a simple and mass reproducible alternative method (to germline transmitter chimeric mice), without technological and instrumental difficulties, for generating chimeric embryos.

Animals↗
PubMed2004

Ability of donor splenocytes with costimulation blockade to induce mixed hematopoietic chimerism and transplantation tolerance.

We reported stable mixed chimerism and specific tolerance to a fully allogeneic graft after a minimally myelosuppressive regimen including costimulation blockade (CB), donor bone marrow cells (BMC), and busulfan (Bu), a chemotherapeutic conditioning agent that makes niches for engraftment of BMC. For clinical application, the strategy may have the limitation of the number of donor BMC when a deceased donor offers transplants to multiple recipients. Herein, we examined whether donor splenocytes can serve as an alternative source to induce mixed chimerism and tolerance. When a C57BL/6 (H-2b) recipient was treated with CB (CTLA4-Ig and anti-CD154 mAb, on days 0, 2, 4, 6) and donor BALB/c (H-2d) BMC (2 x 10(7) cells on day 0) in the absence of Bu, survival of BALB/c skin graft was remarkably prolonged but not indefinite (median survival time [MST]: 138 days). The recipients never showed durable chimerism. When the recipient was treated with CB and donor splenocytes ([DST] 2 x 10(7) cells on day 0), survival was not indefinite either (MST: 114 days). When the dose of DST was increased to 2 x 10(8) cells, survival was further prolonged; two of six recipients had indefinite survival (MST: 132 days). Moreover, one recipient showed a low level of chimerism. When treated with CB, donor DST (2 x 10(7) cells on day 0) and Bu (20 mg/kg, day -1), six of seven recipients showed a stable, high level of chimerism and enjoyed tolerance of skin allografts. DST combined with CB and Bu may be an alternative source of hematopoietic stem cells to induce mixed chimerism and transplantation tolerance in our model.

Animals↗
PubMedDate not supplied

Different routes of donor-derived hematopoietic stem cell transplantation for donor-specific chimerism induction across MHC barrier.

Transplantation of donor-derived stem cells can improve organ allograft survival in animal models. This study was designed to investigate the effect of different routes of bone marrow cell (BMC) transplantation on donor-specific tolerance induction across MHC barrier under short-term CsA monotherapy and alphabetaTCR/CsA treatment protocols. Forty-eight BMC transplantations were performed between BN(RT1(n)) donors and LEW(RT1(1)) recipients. Intraosseous and intravenous BMC transplantation was studied in six groups of eight animals each receiving 35 x 10(6) (n = 4) and 70 x 10(6) (n = 4) bone marrow cells. Groups I and II (controls) received BMC transplantation but no treatment, groups III and IV CsA monotherapy, and groups V, VI alphabetaTCR/CsA protocol for 7 days. Flow cytometry monitored immunodepletion and donor-specific chimerism for MHC class I RT1(n)/CD4, RT1(n)/CD8 and RT1(n)/CD45RA antigens. All animals survived without graft-versus-host disease. At day 63 under CsA monotherapy a low level of chimerism for RT1(n)/CD4 was induced after intraosseous (1.9%) and intravenous (0.8%) transplantation of (70 x 10(6)) BMC. Under alphabetaTCR/CsA protocol chimerism for RT1(n)/CD4 revealed 6.5% and 0.9% in intraosseous and intravenous (70 x 10(6)) BMC transplantation, respectively. The total number of chimerism in intraosseous and intravenous (70 x 10(6)) BMC transplantation groups was 9.9% and 3.4%, respectively. Following intraosseous BMC transplantation under alphabetaTCR/CsA protocol chimerism was 50% higher in a group receiving 70 x 10(6) (9.9%) vs 35 x 10(6) (4.9%) BMC. Intraosseous transplantation of donor BMC under alphabetaTCR/CsA protocol was 75% more efficient in induction of donor-specific chimerism compared to intravenous transplantation.

Animals↗
PubMed2006

Frequency of recipient-derived chimerism and relationship with acute rejection and HLA tissue typing in transplanted livers.

BACKGROUND: We sought to determine the extent and time course of recipient-derived chimerism after transplantation and the relationship with acute rejection episodes (ARE) and HLA typing in hepatic allograft patients. PATIENTS AND METHODS: We studied 18 needle liver biopsy specimens from patients who had undergone orthotopic liver transplantation. Fluorescent in situ hybridization (FISH) analysis for X and Y chromosomes was performed in all cases with a sex mismatch. To evaluate the HLA matching, we used serological and polymerase chain reaction (PCR) methodology. RESULTS: There was a sex mismatch between the recipients and donors in all cases. X and Y chromosome chimerism was detected in 14 of 18 (83%; 31.14 +/- 27.4) patients. Also, no statistical association was found between the presence and the extent of chimerism and clinicopathological parameters (P < .05). CONCLUSIONS: Our results suggest that chimerism was frequently seen in liver allografts, but it did not influence the occurrence of ARE, tissue compatibility, or histopathological changes in the posttransplantation period. The clinical, immunological, and histopathological relevance of chimerism remain unclear. These results may relate to the small number of patients and disproportion of chimerism-positive versus-negative cases. Further prospective studies will be required to clarify these findings in a larger population of liver transplant patients.

Adolescent↗
PubMed2006

T-cell depletion eliminates the development of cardiac allograft vasculopathy in mice rendered tolerant by the induction of mixed chimerism.

We previously demonstrated that cardiac allografts to fully tolerant chimeric mice developed cardiac allograft vasculopathy (CAV). Here we begin to examine which components of the immune system are responsible for the pathogenesis of CAV in such tolerant recipients. B10.A/B6 mixed chimeric mice were created by receiving injections of bone marrow cells from B10.A (H-2k) mice given to C57BL/6 (B6; H-2b) mice with some preparations. B10.A skin grafts were first placed onto B10.A/B6 mixed chimeric recipients. When the donor strain skin grafts had survived perfectly for at least 56 days, B10.A hearts were transplanted heterotopically into B10.A/B6 mixed chimeric recipients. Hearts were examined for the presence of CAV 56 days later. To determine the effector cells that contribute to the development of CAV, they were treated weekly with a combination of anti-CD4/CD8 monoclonal antibodies (mAbs) or anti-NK1.1 mAb continuing until 56 days. 14 B10.A cardiac transplants of 18 otherwise untreated B10.A/B6 chimeric recipients developed CAV; concurrent B6 isografts were unaffected (0/7). In chimeric recipients treated with anti-CD4/8 mAbs, the prevalence of CAV was greatly reduced (0/6, P < .01 compared to the untreated group). Anti-NK1.1 mAb was not effective in the prevention of CAV (4/5). These data suggest that T cells may contribute in some way to the development of CAV that occurs in those fully tolerant recipients. Host T cells that may still be responsive to non-major histocompatability complex antigens, including tissue-specific antigens presented not on skin but on heart, may also be responsible for the development of CAV in tolerant animals.

Animals↗
PubMed2006

Enhanced efficacy of an AAV vector encoding chimeric, highly secreted acid alpha-glucosidase in glycogen storage disease type II.

Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid alpha-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 x 10(10) vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3 x 10(10) vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice.

Animals↗
PubMed2000

Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis.

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.

Animals↗
PubMed2003

Prevention of autoimmune insulitis by delivery of a chimeric plasmid encoding interleukin-4 and interleukin-10.

The combined administration of interleukin-4 (IL-4) and interleukin-10 (IL-10) expression plasmids has demonstrated synergistic effects on the prevention of autoimmune diabetes. To this end, we constructed a co-expression 'chimeric' plasmid, pCMV-IL4-IL10, in which the expression of IL-4 and IL-10 was driven by two separate CMV immediate early promoters by using the biodegradable polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) as a gene carrier to optimize gene delivery. In vitro transfection assays of the chimeric plasmid in 293T cells showed higher expression levels as well as dose dependence than the single gene expression plasmids. To evaluate the in vivo efficacy of the chimeric plasmid, the pCMV-IL4-IL10/PAGA complex was intravenously injected into 4-week-old non-obese diabetic (NOD) mice and compared to the co-administration group. While both groups had persistent gene expression longer than 5 weeks, the IL-4 and IL-10 serum levels of the chimeric group were higher than those in the co-administration group. Furthermore, the degree of insulitis in the chimeric group was improved over both the co-administration and non-injected control groups. These results suggest that the chimeric IL-4 and IL-10 expression plasmid can effectively reduce the incidence of autoimmune insulitis.

Animals↗
PubMed1999

Dynamics of gap junctions observed in living cells with connexin43-GFP chimeric protein.

To study the aggregation of cell-to-cell channels into gap junctions at individual cell-cell contacts, we transfected cells with an expression vector for a chimeric protein composed of the cell-to-cell channel protein connexin43 and a green fluorescent protein. The chimeric channel protein was visualized in the fluorescence microscope and was found to form gap junctions at the cell-cell contacts just like wild-type connexin43. Cells expressing the chimeric protein had functional cell-to-cell channels. Using timelapse videomicroscopy on live cells we observed individual gap junctions over long periods and recorded the time course of aggregation of the chimeric channel protein into gap junctions at newly formed cell-cell contacts. We found that individual small gap junctions were very dynamic, moving about or becoming assembled and disassembled in the course of minutes. Larger gap junctions were more stable than small punctate ones. In control condition, stable new gap junctions were not formed during observation times of 30 min or longer. But at elevated levels of cyclic adenosine monophosphate, the chimeric channel protein began aggregating at new junctions 5-10 minutes after cell-cell contact and continued to concentrate there for at least one hour. Also already established junctions grew in size. The fluorescent chimeric channel protein will be an excellent tool to investigate the regulation of trafficking of connexin from and to the membrane and the mechanism of connexin channel aggregation into gap junctions.

Animals↗
PubMedDate not supplied

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase.

A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.

Binding Sites↗
PubMed2001

Combined host-conditioning with CTLA4-Ig, tacrolimus, anti-lymphocyte serum, and low-dose radiation leads to stable mixed hematopoietic chimerism.

The toxic dose of irradiation required to achieve stable mixed hematopoietic chimerism is the major limitation to its clinical application in transplantation and other nonmalignant conditions such as hemoglobinopathies. This study examines the additive effect of costimulatory blockage, to our previously described tacrolimus-based conditioning regimen, in further reducing the dose of total-body irradiation to achieve stable mixed chimerism in rats. Fully mismatched, 4- to 6-week-old ACI and Wistar Furth rats were used as donors and recipients, respectively. Recipients were administered CTLA4-Ig 2mg/kg/day (alternate days) in combination with tacrolimus 1 mg/kg/day (daily) from day 0 through day +10, anti-lymphocyte serum 10 mg at day +10 (single dose), and total-body irradiation ranging from 100-600 cGy, prior to bone marrow transplantation (day 0) with 100 x 10(6) of T-cell-depleted bone marrow cells. Levels of donor chimerism were determined over a period of 12 months. The short course of CTLA4-Ig, tacrolimus, and ALS led to dramatic engraftments at reduced doses of irradiation: 100% (5/5) and 93% (13/14) of the animals developed mixed chimerism at 400 cGy and 300 cGy, respectively. At 300 cGy, recipients exhibited durable, multilineage mixed chimerism at 365 days with donor cells ranging from 19-42% (mean 23.4%) with no evidence of graft-vs-host disease. These mixed chimeras exhibited in vitro (mixed lymphocyte reaction) and in vivo (skin grafts) donor-specific tolerance. This study suggests that addition of costimulatory blockade to a tacrolimus-based conditioning regimen reduces the dose of irradiation required to achieve stable multilineage chimerism in rats.

Abatacept↗
PubMed2003

Mixed chimerism following in utero hematopoietic stem cell transplantation in murine models of hemoglobinopathy.

OBJECTIVE: Mixed hematopoietic chimerism after bone marrow transplantation can provide effective treatment for beta-thalassemia because of the selective advantage that exists for donor erythropoiesis. In utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism, particularly when a selective advantage exists for donor cells. To investigate the biology of IUHSCTx in hemoglobinopathies, we performed fully allogeneic IUHSCTx in murine models of beta-thalassemia (Thal) and sickle cell disease (SCD). MATERIALS AND METHODS: We serially assessed and compared levels of mononuclear cell (MNC) and erythroid chimerism after IUHSCTx of either adult bone marrow (BM)- or fetal liver (FL)-derived allogeneic donor cells in the two hemoglobinopathy models, which differ significantly in their degree of anemia (Thal>>SCD) and red cell half-life (Thal<<SCD). RESULTS: The mean level of donor MNC chimerism was higher for SCD and Thal chimeras receiving FL- compared to adult BM-derived donor cells and tended to increase over time in the FL recipients. Donor hemoglobin (Hb) levels also were higher in all groups receiving FL compared to adult BM. Donor Hb levels in chimeric Thal mice were significantly higher than those in SCD or wild-type mice. Hematologic parameters such as Hb, hematocrit (Hct), mean cell volume (MCV), membrane-associated denatured Hb, and the oxygen equilibration curve were improved in chimeric hemoglobinopathy mice. However, the improvement in Hb, Hct, and MCV was not sustained despite stable levels of donor leukocyte engraftment. CONCLUSION: The severity of the hemoglobinopathy being treated and the source of donor cells may be important determinants of success in the treatment of hemoglobinopathy by IUHSCTx.

Anemia, Sickle Cell↗
PubMed2003

NK cell recovery, chimerism, function, and recognition in recipients of haploidentical hematopoietic cell transplantation following nonmyeloablative conditioning using a humanized anti-CD2 mAb, Medi-507.

OBJECTIVE: Natural killer (NK) cells kill allogeneic cells that lack a class I MHC ligand for clonally distributed killer inhibitory receptors (KIR). Following HLA-mismatched hematopoietic cell transplantation (HCT), donor NK cells might mediate graft-vs-host (GVH) reactions that promote donor chimerism and mediate anti-tumor effects. Additionally, recipient NK cells might mediate donor marrow rejection. We have developed a nonmyeloablative approach to haploidentical HCT involving recipient treatment with a T cell-depleting mAb, Medi-507, that can achieve donor engraftment and mixed hematopoietic chimerism without graft-vs-host disease (GVHD). Donor lymphocyte infusions (DLI) are later administered in an effort to achieve graft-vs-leukemia/lymphoma (GVL) effects without GVHD. It is unknown whether NK cell "tolerance" develops in human mixed chimeras. METHODS: We have addressed these issues in 12 patients receiving Medi-507-based nonmyeloablative haploidentical HCT. RESULTS: NK cells recovered relatively early, despite the presence of circulating anti-CD2 mAb, but the majority of initially recovering cells lacked CD2 expression. These NK cells showed a reduced capacity, compared to those from normal donors, to kill class I-deficient targets. No association was detected between KIR mismatches in the host-vs-graft (HVG) or GVH direction and graft or tumor outcomes in this small series. NK cell chimerism did not correlate with chimerism in other lineages in mixed chimeras. NK cell tolerance to the host was not observed in a patient with full donor chimerism. One patient developed NK cell reactivity against donor-derived lymphoblast targets after loss of chimerism, despite the absence of an HVG KIR mismatch. CONCLUSION: Our results do not show an impact of NK cells on the outcome of nonmyeloablative, even T cell-depleted, HCT across haplotype barriers using an anti-CD2 mAb. Our data also raise questions about the applicability of observations made with NK cell clones to the bulk NK cell repertoire in humans.

Antibodies, Monoclonal↗
PubMed1999

Radiotoxicity of systemically administered 211At-labeled human/mouse chimeric monoclonal antibody: a long-term survival study with histologic analysis.

PURPOSE: The antitenascin human/mouse chimeric monoclonal antibody labeled with the alpha-particle-emitting radionuclide 211At is of interest as an endoradiotherapeutic agent for the treatment of brain tumors. To facilitate the investigation of 211At-labeled chimeric 81C6 in patients, the long-term radiotoxicity of this radiopharmaceutical has been evaluated. METHODS AND MATERIALS: Antibody labeling was performed using N-succinimidyl 3-[211At]astato-benzoate. After an initial dose-finding experiment, a second toxicity study was carried out at 4 dose levels in groups of 30 nonthyroid blocked B6C3F1 mice per group (15 males, 15 females). Male mice received either saline or 15-81 kBq/g and females received either saline or 16-83 kBq/g of 211At-labeled antibody. Ten animals (5 males, 5 females) were followed for 6 months and the remainder for 1 year. RESULTS: The lethal dose in 10% of animals (LD10) for 211At-labeled chimeric 81C6 was 46 kBq/g in females and 102 kBq/g in males. Toxic effects--perivascular fibrosis of the intraventricular septum of the heart, bone marrow suppression, splenic white pulp atrophy, and spermatic maturational delay--generally were confined to a few animals receiving the highest doses of labeled antibody. CONCLUSIONS: The LD10 of 211At-labeled chimeric 81C6 in this mouse strain was about half that of [211At]astatide. These results establish the preclinical maximum tolerated dose of 211At-labeled chimeric 81C6 and define in the mouse the target organs for toxicity. These studies will be useful for determining starting doses for clinical studies with 211At-labeled chimeric 81C6.

Animals↗
PubMed2003

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer.

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.

Animals↗
PubMed2002

Allogeneic chimerism established with a mixture of low dose bone marrow cells and splenocytes in sublethally irradiated mice.

BACKGROUND: Allogeneic chimerism has been established in graft-accepting recipients and the donor cells in the host may act in a major way to facilitate the induction of tolerance. In this study, we examined the effects of allogeneic chimerism after injecting donor bone marrow cells (BMCs) mixed with splenocytes (SPLCs) to the sublethally conditioned recipients. METHODS: In BALB/c(H-2(d)) to B6(H-2(b)) combination, B6 recipients were irradiated at 7.5 Gy and were injected a mixture of donor BMCs and SPLCs intravenously. On day 90 after injection, the degree of chimerism in peripheral blood lymphocytes (PBL) and in the splenocytes was checked by flow cytometry. RESULTS: In groups which were injected varying BMCs, when > 45 x 10(6) BMCs were injected into B6, a large percentage of donor cells were detected in PBL and in the spleen. In contrast, when < 30 x 10(6) BMCs were injected into B6, only a small percentage of donor cells were detected. In the groups which were injected 3 x 10(6) BMCs with varying SPLCs, when > 10 x 10(6) SPLCs were added, a large percentage of donor cells were detected in PBL and SPLCs, but a small percentage of donor cells were detected with the addition of < 3 x 10(6) SPLCs. A high percentage of chimeric mice showed donor specific tolerance in vitro, mixed lymphocyte responses, and in vivo, skin grafting. In contrast, only a small percentage of chimeric mice showed no donor specific tolerance by skin grafting. CONCLUSION: These findings suggested that even a low dose of BMCs can establish a state of allogeneic chimerism and donor specific tolerance if combined with SPLCs.

Animals↗
PubMed1994

Increased cytotoxicity of interleukin 2-pseudomonas exotoxin (IL2-PE) chimeric proteins containing a targeting signal for lysosomal membranes.

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a truncated form of pseudomonas exotoxin lacking its cell recognition domain (PE40). IL2-PE40 is extremely cytotoxic to IL2 receptor positive cells. This chimeric protein was found to be an effective and selective immunosuppressive agent for IL2 receptor targeted therapy in many models of disorders of the immune response where activated T-cells play a crucial role. In an attempt to produce an improved IL2-PE40 chimeric protein, we constructed new IL2-PE derivatives. This was done by inserting defined DNA sequences within the chimeric gene encoding IL2-PE40. Inserted sequences represent motifs of other proteins known to be targeted and/or sorted to specific compartments inside or outside the cell. One of the proteins, IL2-PE40(LAP+DUP), containing a targeted signal for lysosomal membrane, was 2-3-fold more active than IL2-PE40. The insertion of the LAP sequence also increased the cytotoxicity of another IL2-PE derivative, IL2-PE664Glu. Our results suggest that a selective targeting of IL2-PE chimeric proteins to lysosomes may enable the proteins to reach the cytosol more efficiently, thus improving its specific cytotoxicity. The LAP (lysosomal alkaline phosphatase) sequence may be used as a common motif for increasing the cytotoxicity of other chimeric proteins to be used for targeted immunotherapy.

ADP Ribose Transferases↗
PubMed2004

Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands.

To understand the influences of the ribonucleotide on a duplex conformation and its stability, we systematically studied the CD spectra and the thermodynamics of nucleic acid duplexes formed by the chimeric RNA-DNA strand in which ribonucleotides and deoxyribonucleotides were covalently attached. It was found that the duplex stability was context-dependent and independent of the number of ribonucleotides in the chimeric strand, whereas the CD spectra showed less overall structural perturbation by the chimeric junctions. Combining the results of the CD and the thermodynamic data revealed a stability-structure relationship for the duplexes. Importantly, DeltaG(o)37 values estimated for the chimeric junction formation in the RNA-DNA/DNA and the RNA-DNA/RNA duplexes were close to those of RNA/DNA and RNA/RNA interactions, respectively. Furthermore, DeltaG(o)37s of the DNA-RNA/DNA and DNA-RNA/DNA-RNA junctions were similar to those of the DNA duplex, and the values of DNA-RNA/RNA-DNA were similar to those of the DNA/RNA. The thermodynamic analyses suggest that the 5'-nucleotide may be the crucial factor that determines the stability at the chimeric junction. Our results not only suggest influences of the ribonucleotide on a duplex conformation and its stability but also are useful for the design of RNA-DNA chimeric strands applicable to biotechnology.

Base Pairing↗