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PubMed1995

Mixed allogeneic chimerism in the rat. Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts.

Graft loss secondary to chronic rejection remains a major source of morbidity and mortality in solid organ transplantation. Mixed chimerism has been suggested as one potential approach to overcome this limitation. Until now, whether long-term tolerance for primarily vascularized allografts can be achieved with mixed chimerism has not been adequately assessed due to technical limitations in the mouse and the inability to establish a reliable model of mixed chimerism in the rat. We now report that stable multilineage mixed hematopoietic chimerism can be achieved following the transplantation of a mixture of T cell-depleted syngeneic and allogeneic bone marrow cells into myeloablated rat recipients using a number of MHC plus minor antigen-disparate donor and recipient strain combinations (F344+WF-->F344, F344+ACI-->F344, WF+F344-->WF, and WF+ACI-->WF). Ninety-one percent of animals engrafted with a level of lymphoid chimerism ranging between 12% and 93% (73.3 +/- 4.8%). Peripheral blood lymphocyte chimerism remained stable for up to 13 months after reconstitution. Multilineage chimerism for lymphoid (T and B cells) and myeloid (granulocyte and macrophage) lineages was present, which suggests that engraftment of the pluripotent rat stem cell had occurred. There was no clinical or histologic evidence of graft-versus-host disease. Donor-specific skin (mean survival time [MST] > or = 177 days) and primarily vascularized cardiac (MST > or = 213 days) grafts were accepted without evidence for acute or chronic rejection. In contrast, MHC-disparate third-party skin (MST = 14 days) and cardiac grafts (MST = 13 days) were rapidly rejected. The tolerance was systemic, since donor-specific tolerance was present in vitro as assessed by the mixed lymphocyte proliferation assay. These data suggest that mixed chimerism prevents graft loss secondary to chronic rejection in skin as well as primarily vascularized grafts. Furthermore, a rat model for mixed allogeneic chimerism may provide insight into the mechanisms involved in tolerance induction for a variety of allografts (lungs, small bowel, limb, etc.) not readily transplantable in mouse recipients.

Animals↗

PubMed1997

Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon RI bearing cells.

Crosslinking of immunoglobulin E molecules that are bound to the Fc epsilon receptors expressed on mast cells or basophils triggers activation of these cells, resulting in the development of a type I hypersensitivity. Targeting this potent immune reaction towards tumors by using IgE that reacts with a tumor-associated antigen, may induce a local inflammation at the tumor site, and may therefore promote tumor regression. We have previously shown that murine IgE bound to tumor cells can activate murine mast cells to release TNF-alpha and histamine. To further investigate the therapeutic potential of IgE-mediated immunotherapy of carcinomas, we have developed human/murine chimeric versions, containing the murine variable regions and human constant regions, of both G250 and 323/A3 IgE. These chimeric IgEs are reactive respectively with the G250 renal cell carcinoma antigen and the Ep-CAM molecule, which is highly expressed by most carcinomas. Transfection of the respective chimeric heavy and light chain genes into recipient Sp2/0 myeloma cells yielded chimeric IgE-producing clones. Chimeric G250 and 323/A3 IgE reacted with tumor cells expressing the G250 antigen or Ep-CAM, respectively. To generate a cell line that expresses Fc receptors for human or chimeric IgE, the rat basophilic leukemia cell line RBL-7 was transfected with the human Fc epsilon RI alpha chain (RBL-7TZ) and subsequently tested for binding of chimeric IgE. Functional assays showed that both chimeric IgEs activated RBL-7TZ cells to release TNF-alpha when cultured with tumor cells that express the respective specific antigen. Furthermore, both chimeric IgEs were able to activate freshly isolated human basophils.

Animals↗

PubMed1998

Conventional cytogenetics and FISH evaluation of chimerism after sex-mismatched bone marrow transplantation (BMT) and donor leukocyte infusion (DLI).

BACKGROUND AND OBJECTIVES: Sensitive and quantitative cytogenetic methods to better assess the biological significance of post-BMT chimerism have been recently developed. In this study, we compared the results of chimerism analysis and evolution employing conventional cytogenetics and fluorescence in situ hybridization (FISH) in 16 patients after sex-mismatched BMT, and in 5 patients after donor lymphocyte infusion (DLI) to treat post-BMT relapse. DESIGN AND METHODS: FISH studies were performed using separate digoxigenin labeled centromeric DNA probes for the X (pDMX1) and Y (DYZ1/DYZ3) chromosomes. To this purpose, different types of samples were used: bone marrow (BM) and peripheral blood (PB) slides processed for conventional cytogenetics, and routine BM and PB smears. RESULTS: Results of chimerism studies performed on different types of samples showed no significant differences. No significant differences in the ability to identify the sex of each cell with both pDMX1 and DYZ1/DYZ3 probes were found and the results obtained from independent experiments showed a high linear correlation. Chimerism analysis by FISH showed initial mixed chimerism after BMT in 10 patients. Seven of these patients were also studied by conventional cytogenetics and 2 of these showed mixed chimerism. Seven of the former 10 patients evolved to complete donor chimera. 6 patients showed cytogenetic or hematologic bone marrow relapse, 3 of which were preceded by mixed chimaerism as revealed by FISH studies. FISH studies permitted an easy and accurate monitorization of the response to DLI in 5 relapsed patients, showing an increase in the proportion of donor cells in 4 patients as they reached a new complete remission. INTERPRETATION AND CONCLUSIONS: Both FISH and conventional cytogenetics are quantitative methods to assess chimerism. However, FISH is more sensitive, accurate and can even be applied on routine BM and PB smears. Furthermore, its combination with immunophenotyping approaches to quantify chimerism on cell subpopulations, will help to clarify post-BMT chimerism significance.

Adolescent↗

PubMed2002

Evaluating hematopoietic chimerism following allogeneic peripheral blood stem cell transplantation with microsatellites.

OBJECTIVE: To study the feasibility of using five selected tetranucleotide repeat sequences to detect chimerism in patients who underwent allogeneic peripheral blood stem cell transplantation and the correlation between chimerism status and prognosis in patients. METHODS: Genomic DNA of umbilical cord blood was colleted from 140 normal neonates. 5 loci including CSF1PO, D3S1359, D5S818, D17S1293 and D20S161in each sample were amplified with polymerase chain reaction PCR . PCR products were electrophoresed on PAGE gels For each locus fragments of different size were collected to analyze polymorphism Genotypes of each sibling undergoing transplantation were analyzed at every locus to determine chimerism For sex-mismatched transplantation the amelogenin locus was analyzed to discriminate between kappa and Y chromosome The correlation between chimerism and prognosis was studied. RESULTS: The four polymorphic symbols including the number of alleles and genotypes the percentage of heterozygosity for each locus were as follows:for CSF1PO, 7 alleles and 16 genotypes were found; heterozygosity were separately 69.03%, for D3S1359, 12,26, 76.79%; for D5S818, 8, 21, 81.67%; for D17S1293, 12, 28, 79.09%; for D20S161, 7,17,79.67% and 0.7250 There were 12 complete chimeras of which one died from graft-versus-host disease. One mixed chimera died two weeks following a second transplantation. The patient who had no donor-derived cells died too The one whose chimerism transformed from mixed to complete after a second transplantation had no evidence of relapse. We could not determine which kind of chimerism the other two patients were. CONCLUSION: Chimerism after transplantation can be detected successfully with the five tetranucleotide repeat sequences Every kind of chimerism has its respective prognosis As the donor-derived cells decrease, the prognosis is becoming worse.

Chimerism↗

PubMed2004

[Effects of donor-specific bone marrow cell infusion on chimerism and acute rejection in kidney transplantation].

OBJECTIVE: To evaluate the effect of donor-specific bone marrow cell infusion on the production of chimerism and acute rejection in kidney transplantation. METHODS: Sixty-one patients, 48 males and 13 females, aged 38.4 (23 - 45), underwent transplantation of cadaveric kidneys, 24 of which underwent kidney transplantation combined with donor-specific bone marrow cell infusion and 37 of which underwent pure transplantation of kidney. During the kidney transplantation combined with donor-specific bone marrow cell infusion the donor bone marrow cells (DBMC) were isolated from the thoracic vertebrae and iliac bones of the donors-cadavers and infused into the recipients of the kidneys of the same donors. After operation the peripheral blood was collected from the 61 recipients every 2 - 3 months for at least 1 year to undergo PCR to detect the presence of chimerism, and the rates of chimerism and acute rejection were compared between these 2 groups. RESULTS: During the follow-up the presence rate of chimerism was 87.5% (21/24) in the marrow recipients, significantly higher than that in the control group (40.5%, 15/37, P < 0.001). The prevalence of acute rejection in the chimerism positive patients was 19.4% (7/16), significantly lower than that in the chimerism negative patients (44%, 11/25, P < 0.05). CONCLUSION: Donor-specific bone marrow cell infusion in cadaver kidney transplantation induces the production of chimerism, and increases the immune tolerance to the donor organs, thus finally decreasing the incidence of acute rejection. Chimerism is correlated with immune tolerance.

Adult↗

PubMed2004

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering.

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.

Fluorescence Resonance Energy Transfer↗

PubMed1996

Rearrangement of the AML1/CBFA2 gene in myeloid leukemia with the 3;21 translocation: expression of co-existing multiple chimeric genes with similar functions as transcriptional repressors, but with opposite tumorigenic properties.

Several recurring chromosomal translocations involve the AML1 gene at 21q22 in myeloid leukemias resulting in fusion mRNAs and chimeric proteins between AML1 and a gene on the partner chromosome. AML1 corresponds to CBFA2, one of the DNA-binding subunits of the enhancer core binding factor CBF. Other CBF DNA-binding subunits are CBFA1 and CBFA3, also known as AML3 and AML2. AML1, AML2 and AML3 are each characterized by a conserved domain at the amino end, the runt domain, that is necessary for DNA-binding and protein dimerization, and by a transactivation domain at the carboxyl end. AML1 was first identified as the gene located at the breakpoint junction of the 8;21 translocation associated with acute myeloid leukemia. The t(8;21)(q22;q22) interrupts AML1 after the runt homology domain, and fuses the 5' part of AML1 to almost all of ETO, the partner gene on chromosome 8. AML1 is an activator of several myeloid promoters; however, the chimeric AML1/ETO is a strong repressor of some AML1-dependent promoters. AML1 is also involved in the t(3;21)(q26;q22), that occurs in myeloid leukemias primarily following treatment with topoisomerase II inhibitors. We have studied five patients with a 3;21 translocation. In all cases, AML1 is interrupted after the runt domain, and is translocated to chromosome band 3q26. As a result of the t(3;21), AML1 is consistently fused to two separate genes located at 3q26. The two genes are EAP, which codes for the abundant ribosomal protein L22, and MDS1, which encodes a small polypeptide of unknown function. In one of our patients, a third gene EVI1 is also involved. EAP is the closest to the breakpoint junction with AML1, and EVI1 is the furthest away. The fusion of EAP to AML1 is not in frame, and leads to a protein that is terminated shortly after the fusion junction by introduction of a stop codon. The fusion of AML1 to MDS1 is in frame, and adds 127 codons to the interrupted AML1. Thus, in the five cases that we studied, the 3;21 translocation results in expression of two coexisting chimeric mRNAs which contain the identical runt domain at the 5' region, but differ in the 3' region. In addition, the chimeric transcript AML1/MDS1/EVI1 has also been detected in cells from one patient with the 3;21 translocation as well as in one of our patients. Several genes necessary for myeloid lineage differentiation contain the target sequence for AML1 in their regulatory regions. One of them is the CSF1R gene. We have compared the normal AML1 to AML1/MDS1, AML1/EAP and AML1/MDS1/EVI1 as transcriptional regulators of the CSF1R promoter. Our results indicate that AML1 can activate the promoter, and that the chimeric proteins compete with the normal AML1 and repress expression from the CSF1R promoter. AML1/MDS1 and AML1/EAP affect cell growth and phenotype when expressed in rat fibroblasts. However, the pattern of tumor growth of cells expressing the different chimeric genes in nude mice is different. We show that when either fusion gene is expressed, the cells lose contact inhibition and form foci over the monolayer. In addition, cells expressing AML1/MDS1 grow larger tumors in nude mice, whereas cells expressing only AML1/EAP do not form tumors, and cells expressing both chimeric genes induce tumors of intermediate size. Thus, although both chimeric genes have similar effects in transactivation assays of the CSF1R promoter, they affect cell growth differently in culture and have opposite effects as tumor promoters in vivo. Because of the results obtained with cells expressing one or both genes, we conclude that MDS1 seems to have tumorigenic properties, but that AML1/EAP seems to repress the oncogenic property of AML1/MDS1.

Animals↗

PubMed2005

Requirement of a higher degree of chimerism for skin allograft tolerance in cyclophosphamide-induced tolerance.

By using a cyclophosphamide (CP)-induced tolerance system, we previously raised the possibility that the degree of chimerism might determine the induction of heart and skin allograft tolerance. When C3H (H-2k; Thy1.2, Mls-1b) mice were intravenously primed with 1 x 10(8) spleen cells (SCs) from H-2 matched AKR (H-2k; Thy1.1, Mls-1a) mice and then treated intraperitoneally with 200 mg/kg CP, the survival of AKR skin grafts was permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism and the clonal destruction of Mls-1a-reactive CD4+Vbeta6+ T cells in the periphery were observed. When AKR SCs and 100 mg/kg CP were used for conditioning, the survival of the AKR skin grafts was mildly prolonged. The clonal destruction of CD4+Vbeta6+ T cells in the periphery was induced and a minimal degree of mixed chimerism was detectable. The degree of mixed chimerism induced with AKR SCs and 200 mg/kg CP was significantly higher than that with AKR SCs and 100 mg/kg CP during the observation. On the other hand, neither skin allograft prolongation nor permanent mixed chimerism could be induced when C3H mice were treated with AKR SCs and 50 mg/kg CP. In order to increase the degree of mixed chimerism, we injected 1 x 10(8) tolerant AKR SCs on day 3 into the recipient C3H mice that had been treated with AKR SCs on day 0 and with 100 mg/kg CP on day 2. The reason that we used tolerant SCs was that untreated AKR SCs caused graft-versus-host disease in most of the recipients. Tolerant AKR SCs were harvested from AKR mice that had been treated with C3H SCs and 200 mg/kg CP 2 weeks earlier, and did not contain regulatory cells. By adoptive transfer, the degree of chimerism was stably and significantly increased in all recipients, and AKR skin graft tolerance was induced in half of the recipients. T-cell-depleted bone marrow cells (BMCs) from untreated AKR mice induced skin allograft tolerance in 83% of recipients. Thus, the present study strongly confirmed the hypothesis that a higher degree of chimerism is required for the induction of skin allograft tolerance in CP-induced tolerance.

Adoptive Transfer↗

PubMed1995

Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.

Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed. To examine the properties of chimeric Fab derived from these monoclonal antibodies, we have developed an expression system which allows the rapid production of milligram quantities of chimeric Fab. Both the chimeric light chain and the chimeric Fd were co-expressed from the same vector, pAPEX-3P. This vector contains the SV40 origin of replication, which allows the rapid production of chimeric Fab in COS cells for preliminary characterization. Additionally, pAPEX-3P contains the Epstein-Barr virus origin of replication and a puromycin selectable marker for maintenance as a stable episome in human cell lines. A production system consisting of transfected 293-EBNA cells cultured in serum free medium followed by protein G-Sepharose chromatography of the conditioned medium was found to be sufficient for the rapid production of purified chimeric Fab. Here we have utilized this expression system to demonstrate that an anti-human C5 chimeric Fab was a potent inhibitor of complement activation in both in vitro activation assays and an ex vivo model of complement-mediated tissue damage.

Animals↗

PubMed1994

Expression and characterization of a chimeric bispecific antibody against fibrin and against urokinase-type plasminogen activator.

We have produced a chimeric bispecific antibody that has dual specificity of human fibrin and urokinase-type plasminogen activator (u-PA). Complementary DNAs for variable regions of both anti-fibrin and anti-u-PA antibodies were cloned from two murine hybridomas secreting respective antibodies using polymerase chain reaction (PCR) techniques, and joined to cDNAs for human constant regions to form chimeric antibody genes. Both of two expression vectors for chimeric anti-fibrin and chimeric anti-u-PA antibodies were sequentially introduced into Chinese hamster ovary cells, and stable transfectants secreting the chimeric bispecific antibody were obtained. The highest producer transfectant (SULF/C2-30) secreted high level (about 40 micrograms ml-1) of total chimeric IgG and about 2% of the IgG had the bispecific activity of binding with both antigens. The chimeric bispecific antibody was purified by a combination of affinity chromatographies employing antigen-coupled columns and hydroxyapatite high-performance liquid chromatography. The purified chimeric bispecific antibody significantly enhanced the thrombolytic potency of single chain u-PA in an in vitro clot lysis assay as well as the original murine bispecific antibody.

Amino Acid Sequence↗

PubMed1992

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Amino Acid Sequence↗

PubMed2006

Busulfan-conditioned bone marrow transplantation results in high-level allogeneic chimerism in mice made tolerant by in utero hematopoietic cell transplantation.

OBJECTIVE: In utero hematopoietic cell transplantation (IUHCT) is a non-ablative approach that achieves mixed allogeneic chimerism and donor-specific tolerance. However, clinical application of IUHCT has been limited by minimal engraftment. We have previously demonstrated in the murine model that low-level allogeneic chimerism achieved by IUHCT can be enhanced to near-complete donor chimerism by postnatal minimally myeloablative total body irradiation (TBI) followed by same-donor bone marrow transplantation. Because of concerns of toxicity related to even low-dose TBI in early life, we wondered if a potentially less toxic strategy utilizing a single myelosuppressive agent, Busulfan (BU), would provide similar enhancement of engraftment. METHODS: In this study, mixed chimerism was created by IUHCT in a fully allogeneic strain combination. After birth, chimeric mice were conditioned with BU followed by transplantation of bone marrow cells congenic to the prenatal donor. RESULTS: We demonstrate that: 1) low-level chimerism after IUHCT can be converted to high-level chimerism by this protocol; 2) enhancement of chimerism is BU dose-dependent; and 3) BU reduces the proliferative potential of hematopoietic progenitor cells thus conferring a competitive advantage to the non-BU-treated postnatal donor cells. CONCLUSION: This study confirms the potential of IUHCT for facilitation of minimally toxic postnatal regimens to achieve therapeutic levels of allogeneic engraftment.

Animals↗

PubMed2006

Kinetics of plasma-cell chimerism after allogeneic stem cell transplantation by highly sensitive real-time PCR based on sequence polymorphism and its value to quantify minimal residual disease in patients with multiple myeloma.

OBJECTIVE: To investigate lineage-specific chimerism of plasma cells after allogeneic transplantation by real-time PCR based on bi-allelic sequence polymorphism or, in case of female-to-male transplantation, on the detection of the DFFRY gene and to determine its value to quantify minimal residual disease in myeloma patients. METHODS: Forty-eight samples from bone marrow samples and peripheral blood from 34 nonmyeloma patients were analyzed at different times after transplantation. Sixty-two samples from 22 myeloma patients were analyzed at different times after allogeneic stem cell transplantation, and results were compared with immunofixation and, in some cases, with PCR data using patient-specific primers. RESULTS: The median chimerism for T cells at day +100 was greater than 99.9% and remained stable on day +180 and 1 year after transplantation. In contrast, the median donor plasma cell chimerism at day +100 was 95.5%, at day +180 98.6%, at day +360 99.8%, and 2 or more years after transplantation greater than 99.9%. Sensitivity of real-time PCR using human short insertion/deletion polymorphisms (SIDP) was 10(-4) and in case of Y-PCR 10(-5). Sequential monitoring of donor plasma cell chimerism showed that increasing and stable chimerism were associated with ongoing remission in 15 out of 16 samples (93%), and decreases in chimerism predicted relapse in 5 out of 6 patients. CONCLUSION: We conclude that plasma cell chimerism after allogeneic stem cell transplantation is delayed in comparison to T-cell chimerism. Sequential quantitative measurement of plasma cells after allogeneic stem cell transplantation with highly sensitive real-time PCR allows monitoring of residual host-tumor cells in patients with multiple myeloma and allows guiding adoptive immunotherapy strategies to enhance remission status and to prevent clinical relapse.

Chimera↗

PubMed2004

Bone marrow chimerism and tolerance induced by single-dose cyclophosphamide.

BACKGROUND: Establishment of hematopoietic chimerism is the most stable strategy for donor-specific tolerance. Safer pretreatment regimens are needed for clinical application. We evaluated the efficacy of a simple protocol using cyclophosphamide (CYP) on induction of chimerism and organ transplant tolerance across major histocompatibility complex (MHC) barriers in the rat. MATERIALS AND METHODS: Bone marrow cells from BN (RT1(n)) donors were infused to LEW (RT1(l)) recipients on day 0 after a single injection of CYP at various doses on day -1. Donor-derived hematopoietic chimerism was evaluated by flowcytometry. The recipients received BN or third party (BUF) heart allografts on day 100. RESULTS: While pretreatment with 200 mg/kg of CYP induced high levels of hematopoietic chimerism, six of eight recipients died of severe graft-versus-host-disease (GVHD). CYP at dose of 150 mg/kg induced 36.5 +/- 24.1% of donor-derived chimerism on day 10, and sustained macrochimerism was seen until day 100 without GVHD. Pretreatment with 100 mg/kg of CYP resulted in only transient chimerism (4.8 +/- 5.2%) which disappeared by day 20. In the recipients with 50 mg/kg of CYP, donor bone marrow cells were rapidly rejected and no chimerism was observed. The recipients with 150 mg/kg of CYP accepted BN heart allografts (>100 days x 5), while rejecting BUF allografts by day 12 (n = 4). BN heart allografts were rejected in the recipients with 100 (MST: 57 days, n = 5) and 50 mg/kg (MST: 7 days, n = 5) of CYP. CONCLUSIONS: A single dose of CYP can induce hematopoietic chimerism across MHC-barriers. The dose of 150 mg/kg seems to be optimal to induce organ transplant tolerance without developing GVHD.

Animals↗

PubMed2004

The CD28 peptidemimic can induce mixed chimerism and prolong the survival of cardiac allografts.

Costimulatory blockade with CD28 peptidemimic (CD28PM, CD28 PM was synthesized by solid phase synthetic methods) prolongs cardiac allograft survival in mice, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding B7 blockade to a chimerism inducing nonmyeloablative regimen in mice and observed a significant improvement of donor bone marrow cells (BMC) engraftment, which had been associated with mixed chimerism and long-term survival of cardiac allografts. The mixed lymphocyte reaction (MLR) and the ear pinna cardiac transplantation model were performed to evaluate the effects of CD28PM in induction of specific immune hypo-response and extension of allograft survival. The expressed rates of B7.1 and B7.2 on the C57BL/6 splenocytes were 56.25% and 20.52%, respectively. The specific hypo-response status was established after immunization with CD28PM pre-treated donor splenocytes and the average inhibition rate was only 43% compared with normal control. Subsequently, a total number of 2 x 10(7) bone marrow cells per mouse were implanted to the recipients. The allogenic chimerism was obviously observed with the rate as high as 8.84% (mean) at the time point of day 14. During the first 50 days post bone marrow transfusion (BMT) the chimerism rate declined stepwise. But from 50 to 100 days, the chimerism rate sustained in a range of 3.35% to 4.6%. The results of transplantation experiments showed the survival of allgenic cardiac grafts were maintained over 100 days in recipients. Thus, donor BMC engraftment with mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Mixed chimerism approach, by the addition of CD28-B7 costimulatory blockade with CD28PM, has been shown to establish mixed chimerism and induce cardiac allograft tolerance in mice.

Animals↗

PubMed2000

DNA-RNA chimeric hammerhead ribozyme to transforming growth factor-beta(1) mRNA inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats.

OBJECTIVE: The purpose of this study was to develop DNA-RNA chimeric hammerhead ribozyme against transforming growth factor-beta(1) (TGF-beta(1)) mRNA as a gene therapy agent for arterial proliferative diseases. METHODS: A 38-base hammerhead ribozyme against rat TGF-beta(1) mRNA, to produce cleavage at the GUC sequence at nucleotide 825 according to the secondary structure of rat TGF-beta(1) mRNA was designed. To enhance its stability, we synthesized a DNA-RNA chimeric ribozyme with two phosphorothioate linkages at the 3'-terminal. We also synthesized a mismatch ribozyme with single base change in the catalytic loop region as a control. These ribozymes were delivered into rat vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats by lipofectin-mediated transfection, and their biological effects were investigated. RESULTS: According to in vitro cleavage studies, the synthetic ribozyme can cleave the synthetic substrate RNA into two RNA fragments. Chimeric ribozyme significantly inhibited DNA synthesis in VSMC from SHR but not in cells from WKY rats. Mismatch ribozyme showed only a little effect on growth of VSMC from SHR. Chimeric ribozyme significantly inhibited proliferation of VSMC from SHR; in contrast, the proliferation of VSMC from WKY rats was significantly increased by this chimeric ribozyme. Mismatch ribozyme did not affect proliferation of VSMC from either rat strain. Chimeric hammerhead ribozyme to rat TGF-beta(1) dose-dependently inhibited TGF-beta(1) mRNA expression detected by reverse transcription and polymerase chain reaction analysis in VSMC from both rat strains. Chimeric hammerhead ribozyme to rat TGF-beta(1) also dose-dependently inhibited TGF-beta(1) protein production detected by Western blot analysis. CONCLUSIONS: The present results demonstrated that our designed DNA-RNA chimeric hammerhead ribozyme to TGF-beta(1) mRNA might be a useful gene therapy agent for hypertensive vascular diseases.

Analysis of Variance↗

PubMed2002

The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant.

Intranasal (i.n.) immunization of BALB/c mice with chimeric murine rotavirus EDIM (epizootic diarrhea of infant mice) VP6 and attenuated E. coli heat-labile toxin (LT), LT(R192G), stimulated >99% protection against rotavirus shedding after EDIM challenge. Here, we evaluated other potential adjuvants with chimeric VP6 administered by two mucosal routes: i.n. and oral. Besides LT(R192G), the adjuvants examined included Adjumer, CpG oligodeoxynucleotides (CpG ODN), chimeric A1 subunit of cholera toxin (CTA1)-DD, and QS-21. All except QS-21 significantly (P<0.05) increased VP6-specific serum IgG responses after i.n. immunization, but none significantly increased these responses when administered orally. The i.n. delivery of chimeric VP6 alone induced both rotavirus IgG1 and IgG2a whose relative titers suggested a skewed Th2-like response. Inclusion of Adjumer greatly increased Th2-like responses, while CpG ODN shifted the response to a less Th2-like response. The adjuvants CTA1-DD, LT(R192G), QS-21 had no significant effect on ratios of IgG1/IgG2a titers. Following EDIM challenge of mice immunized i.n. with chimeric VP6 and either LT(R192G), CTA1-DD, Adjumer or CpG ODN, shedding was reduced >99, 95, 80, 74, respectively, relative to that found in unimmunized mice (P<0.05). QS-21 induced less protection (43%, not significant (N.S.)) while immunization with chimeric VP6 alone reduced shedding by only 16% (N.S.). Oral immunization with chimeric VP6 and all selected adjuvants except QS-21 was less effective than after i.n. immunization, with protection levels of 94 (P<0.05), 71 (P<0.05), 55, 35 and 28% for LT(R192G), QS-21, CpG ODN, CTA1-DD, and Adjumer, respectively, while immunization with chimeric VP6 alone gave no protection. Thus, different adjuvants induced different degrees of protection and oral immunization was generally less effective then the i.n. route.

Adjuvants, Immunologic↗

PubMed2003

Substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys.

Antigenic chimeric viruses in which the structural genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3' untranslated region. The two DEN2/4 chimeric viruses lacking the delta 30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta 30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta 30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta 30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta 30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta 30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials.

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