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PubMed2001

Establishment of complete and mixed donor chimerism after allogeneic lymphohematopoietic transplantation: recommendations from a workshop at the 2001 Tandem Meetings of the International Bone Marrow Transplant Registry and the American Society of Blood and Marrow Transplantation.

Approaches to the measurement of lymphohematopoietic chimerism have evolved from laboratory research to important clinical tools. However, there has been no logical, consistent, and uniform set of recommendations for the measurement of chimerism in clinical transplantation. The National Marrow Donor Program and the International Bone Marrow Transplant Registry (IBMTR) sponsored a workshop to discuss the use of chimerism analysis after allogeneic transplantation. The workshop was organized in an effort to make reasonable recommendations regarding laboratory techniques, the types of specimens to be studied, and the frequency of analysis. The panel recommended the following guidelines: 1. Chimerism analysis should use sensitive, informative techniques. At present, short tandem repeats (STR) or variable number tandem repeats (VNTR) analysis is the approach most likely to give reproducible informative data. 2. Peripheral blood cells are generally more useful than bone marrow cells for chimerism analysis. 3. Lineage-specific chimerism should be considered the assay of choice in the setting of nonmyeloablative and reduced-intensity conditioning. 4. The use of T-cell depletion, nonmyeloablative or reduced-intensity conditioning, or novel graft-versus-host disease (GVHD) prophylactic regimens warrants chimerism analysis at 1, 3, 6, and 12 months, because interventions such as donor lymphocyte infusions may depend on chimerism status. 5. In nonmyeloablative transplantation, the early patterns of chimerism may predict either GVHD or graft loss. Therefore, more frequent (every 2-4 weeks) peripheral blood analysis may be warranted. 6. For nonmalignant disorders, chimerism generally should be measured 1, 2, and 3 months after transplantation. Interventions to enhance donor engraftment must be considered on a disease-specific basis in relation to concurrent GVHD and, ultimately, clinical rationale.

Blood Cells↗

PubMed2006

Immunophenotyping of chimeric cells in localized scleroderma.

OBJECTIVE: Localized scleroderma causes thickening of the skin due to excessive collagen deposition. This condition has clinical and histopathological similarities to chronic graft-vs-host disease. We wanted to identify whether chimeric cells are present in the affected tissue in localized scleroderma and to further investigate the role of chimerism by immunophenotyping the chimeric cells. We hypothesize that the presence of chimerism and immunotypic chimeric cells will lend to an understanding of the pathogenesis of localized scleroderma and possible mechanisms by which chimeric cells participate in autoimmunity. METHODS: We studied skin biopsies from 18 localized scleroderma patients and compared them with concurrent biopsies from unaffected skin in a subset of patients. Skin biopsies from morphoea and linear scleroderma patients were analysed for the presence of chimeric cells using male-female (X, Y) differences. Cell surface markers (CD4, CD8, CD19/20, CD68, S100, CD14 and CD56) were determined for cell phenotyping of chimeric cells. RESULTS: Overall, the affected tissue contained a greater number of lymphocytic inflammatory cells. In the affected tissue, 38% of the total chimeric cells were CD68+ (dendritic cell, monocyte and macrophage marker), 29% Langerin/S100+ (dendritic cell marker), 26% CD8+ (cytotoxic T-lymphocyte marker), 20% CD19/20+ (B-lymphocyte marker), 14% CD4+ (T-helper lymphocyte) and 0% CD56+ (natural killer cell marker). CONCLUSIONS: We report that not only are chimeric cells present in affected localized scleroderma lesions but they also are more likely to be dendritic cells and B lymphocytes suggesting a role in the pathogenesis of localized scleroderma.

Adolescent↗

PubMed1997

Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys.

BACKGROUND: We demonstrated previously that a nonmyeloablative preparative regimen can induce mixed chimerism and allograft tolerance in cynomolgus monkeys. METHODS: The current studies were designed to clarify the importance and toxicity of various elements of the allotolerance conditioning regimen by: fractionating or reducing the whole-body irradiation (WBI) dosage; adding deoxyspergualine; or deleting donor bone marrow, cyclosporine, irradiation, or splenectomy. RESULTS: Monkeys treated without donor bone marrow, cyclosporine, or irradiation did not develop chimerism or long-term allograft survival. One of three monkeys treated without splenectomy developed chimerism but died of a surgical complication. The other two did not develop chimerism and rejected by day 117. Six of six monkeys treated with 300 cGy of fractionated WBI developed chimerism. Five of these recipients had long-term graft survival. Only two of four monkeys treated with 250 cGy developed chimerism, so a 2-week course of deoxyspergualine was added. This led to chimerism in two monkeys, but one died of ureteral stenosis and the other died of allograft rejection. An unanticipated high incidence of ureteral complications felt to be secondary to rejection episodes and ischemic injury was observed in the long-term surviving animals. CONCLUSIONS: All parameters of the original preparative regimen seem to be essential for consistent success. The degree of lymphocyte depletion was proportional to the WBI dose. Long-term graft survival was observed only in recipients achieving lymphocyte chimerism of > 1.5%. In this model, lymphocyte depletion seems to be the best predictor of chimerism, and significant lymphocyte chimerism seems to be important in achieving tolerance.

Animals↗

PubMed2000

Chimerism in peripheral blood of sensitized patients waiting for renal transplantation: clinical implications.

BACKGROUND: Potential renal transplant recipients with preformed antibodies to HLA resulting from previous transplants, pregnancy, and/or transfusions are unlikely to receive an allograft. The factors contributing to the long-term maintenance of antibody titers in these individuals are still unknown. In the present study, we sought to determine whether chimerism in the blood correlates with maintenance of HLA sensitization in highly sensitized patients. METHODS: Qualitative analysis of chimerism in blood of sensitized patients was assessed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) to HLA-DR. PCR single-strand conformation polymorphism (PCR-SSCP) was used to confirm the extra HLA-DR antigens detected by PCR-SSOP. RESULTS: Fourteen of 36 patients (38.9%) were positive for more than two HLA-DR indicative of chimerism. The presence of extra HLA-DR was confirmed by PCR-SSCP. When patients were analyzed on the basis of their panel-reactive antibody (PRA) status, 10 of 15 (66.7%) were positive for chimerism in the sensitized group, compared with only two of eight (25%) in the unsensitized group. Of the five males in the sensitized group who had received a blood transfusion but not a transplant, three were positive for chimerism. An association was observed between chimerism and maintenance of sensitization. None of the eight normal subjects studied demonstrated chimerism. CONCLUSIONS: The results obtained with sensitized patients suggest an association between blood chimerism and maintenance of HLA sensitization. We speculate that chimerism may lead to long-term maintenance of anti-HLA antibody titers. This finding implies that abolition of chimerism could result in the eventual elimination of antigenic stimuli for antibody production against HLA antigens.

Antibodies↗

PubMed2005

High affinity mouse-human chimeric Fab against hepatitis B surface antigen.

AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: We cloned the V(H) and V(L) genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C(L) domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.

Animals↗

PubMed2000

Analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency.

BACKGROUND: A single nonfunctional chimeric gene with its 5' and 3' ends corresponding to CYP21P and CYP21, respectively, is caused by unequal gene crossover in the CYP21 genes during meiosis. The presence of the chimeric CYP21P/CYP21 molecule can not be detected by conventional PCR methods and therefore may be lost in PCR amplification. This leads to a false result and diagnostic discordance. METHODS: We developed a rapid and direct method to detect a chimeric CYP21P/CYP21 gene that uses a 3'-specific primer for the CYP21 gene and two different 5' primers for both CYP21 and CYP21P to amplify the wild-type CYP21 and the chimeric CYP21P/CYP21 genes. A secondary PCR that can differentiate the chimeric from the wild-type gene was also performed. The PCR product was directly analyzed on agarose gel. RESULTS: After careful titration, we found that earlier failure to detect the chimeric CYP21P/CYP21 gene could be caused by unequal concentrations of two independent alleles as the PCR template or by the lack of primers to amplify chimeric molecules. We successfully amplified the chimeric gene using our improved method. CONCLUSIONS: The chimeric CYP21P/CYP21 is present in a large portion of congenital adrenal hyperplasia patients. By adding a CYP21P/CYP21-specific primer, we were able to amplify and detect both homozygous and heterozygous chimeric genes. Therefore, our new PCR-based assay is a more effective way to analyze congenital adrenal hyperplasia mutations.

Adrenal Hyperplasia, Congenital↗

PubMed2001

Early full donor myeloid chimerism after reduced-intensity stem cell transplantation using a combination of fludarabine and busulfan.

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate lineage-specific chimerism reconstitution after reduced-intensity allogeneic stem cell transplantation (RIST) using a combination of fludarabine (30 mg/m2 for 6 days) and busulfan (4 mg/kg for 2 days). DESIGN AND METHODS: We prospectively enrolled 8 consecutive patients with hematologic malignancies who were not candidates for conventional transplantation because of either high age or organ dysfunction. Host-donor chimerism was evaluated using polymerase chain reaction-based amplification of a polymorphic short tandem repeat region. RESULTS: All of our patients achieved engraftment within a median of 11 days after transplantation. On day 30, full donor myeloid cell chimerism (>90%) was achieved in 7 patients whereas full donor T-cell chimerism was achieved in only one patient. Thus, in contrast to other reported results, full donor chimerism was achieved earlier in the myeloid lineage than the T-cell lineage. On day 60, however, T-cell chimerism caught up with myeloid chimerism. Two patients developed grade II-IV acute graft-versus-host disease (GVHD) before the detection of full donor T-cell chimerism. INTERPRETATION AND CONCLUSIONS: Our findings suggest that the kinetics of lineage-specific chimerism depend on the agents used in the conditioning regimen, and may provide insight into the chimerism kinetics and pathogenesis of GVHD. Thus, the strategy for controlling immunosuppression after RIST should be modified according to the type of conditioning regimen applied.

Adult↗

PubMed1992

Myeloid and lymphoid chimerism after T-cell-depleted bone marrow transplantation: evaluation of conditioning regimens using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA.

Determining both myeloid and lymphoid chimerism after T-cell-depleted allogeneic bone marrow transplantation (BMT) could be helpful in the understanding of the biology of engraftment and could provide a rational method of assessing the ability of different conditioning regimens to promote engraftment. We prospectively investigated the role of different pretransplant conditioning regimens in 29 leukemic patients post-BMT by assessing myeloid and T-cell chimerism using a rapid and sensitive polymerase chain reaction (PCR) method. Minisatellites are hypervariable regions of DNA consisting of tandem repeats of a core nucleotide sequence, and allelic polymorphism results from differences in the number of the repeats. We used this variation to distinguish between donor and recipient cells post-BMT. Seventeen patients (9 sibling and 8 unrelated donors) received conditioning with hyperfractionated total body irradiation (TBI), thiotepa, and cyclophosphamide (Cy). Of the other 12 patients (all sibling donors), 11 received TBI plus Cy plus another agent: VP16, carboplatinum, or AZQ. One patient received TBI plus thiotepa plus VP16. All but one of the patients studied received marrow from HLA-identical donors. PCR analysis confirmed donor lymphoid engraftment within 8 days of transplant in six of six patients studied. All granulocyte DNA was of donor origin within the first 4 weeks of transplant, regardless of the conditioning regimen. The day +28 T cells were exclusively of donor origin in 14 of 17 patients who received TBI plus thiotepa plus Cy, but were mixed chimeric in 10 of 12 patients who received other conditioning regimens (P < .001). Early graft rejection was seen in one unrelated transplant recipient conditioned with TBI plus thiotepa plus Cy. Late graft failure was observed in 3 of 12 patients with mixed T-cell chimerism and in none of 16 patients with full donor chimerism at day +28. However, 5 of 16 patients who had complete T-cell chimerism at day +28 developed acute graft-versus-host disease (GVHD), whereas no patient with mixed chimerism had acute GVHD. Our results indicate that minisatellite PCR is a rapid and sensitive method for assessing chimerism post-BMT, that the donor T cells are important for consistent durable engraftment, and that TBI plus thiotepa plus Cy may be superior to the other regimens studied in inducing full donor chimerism. Larger numbers and longer follow-up are necessary to confirm these data and also to assess the relationship between complete donor T-cell chimerism and leukemia-free survival.

Adolescent↗

PubMed2004

[Application of sequential and quantitative monitoring of chimerism in allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To establish multiple short tandem repeat (STR) amplification by fluorescence labeling polymerase chain reaction (PCR) combined with capillary electrophoresis for quantitative determination of chimerism, and to evaluate the status of engraftment and predict the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Thirty-one patients received bone marrow transplantation (BMT) or nonmyeloablative allogeneic stem cell transplantation (NST) were evaluated. Peripheral blood and bone marrow were co-llected before and after transplantation in different period. Nine different STR markers were co-amplified in a single reaction by using a commercial AmpF/STR Profiler Plus PCR amplification kit. Separation of the PCR products and fluorescence detection were performed by ABI prism 310 Genetic Analyzer with capillary electrophoresis. The Genescan and Genotype software were used for size calling and quantification of peak areas. The formula to calculate donor chimerism values was based on the different allelic distribution type between donor and recipient. RESULTS: 48.4% of the patients received sex-matched transplantation and the quantification of donor chimerism could only be performed by STR-PCR method. Comparison of values obtained by FISH analysis with that by STR-PCR in patients transplanted from sex-mismatched donors showed an excellent correlation. The median number of informative alleles was 6.7 (range 2 - 10). The donor's alleles appeared in all the patients on day 7 post-transplant. The median values of donor chimerism in BMT group were inferior to that in NST group on day 7, day 14 and 1 month post-transplant. However the difference disappeared in the midterm or later period of transplant. On day 21, all of the 31 patients had stable engraftment and the percentage of donor chimerism was more than 92%. Median follow-up was 17 (3.5 - 29.0) months after transplantation. Twenty-six of 31 patients had durable engraftment and donor chimerism ratio was more than 90%. So for all of them survived leukemia-freely. Four of the 31 patients had unstable mixed chimerism and relapsed within 6 months post allo-HSCT. Another patient with unstable mixed chimerism appeared graft rejection. Decreasing values of donor chimerism were detected prior to the occurrence of graft rejection and disease relapse. The incidence of GVHD was much higher in the group of full donor chimerism. CONCLUSION: Sequential and quantitative monitoring of STR is a valuable tool for studying engraftment dynamics, graft rejection, and relapse and for predicting GVHD. Furthermore it can provide a basis for early intervention of clinical treatment.

Adolescent↗

PubMed1990

Comparative binding and preclinical localization and therapy studies with radiolabeled human chimeric and murine 17-1A monoclonal antibodies.

Murine MoAb 17-1A is an IgG2a antibody reactive with a gastrointestinal cancer-associated cell surface antigen. Human-mouse chimeric 17-1A MoAbs were constructed in which the murine variable region of 17-1A was joined with human IgG1, IgG2, IgG3, and IgG4 constant regions. Human-mouse IgG1, IgG2, and IgG4 chimeric antibodies were compared with the parental murine antibody and its F(ab')2 fragments for their ability to bind to colon carcinoma cells in vitro, for their blood clearance in normal nude mice, and for their localization and tumor growth inhibition of colon carcinoma xenografts in nude mice. Indirect immunofluorescence experiments with fluorescein-conjugated goat anti-mouse or goat anti-human antibody verified that the substitution of human constant regions in the chimeric MoAbs did not significantly alter the ability of the murine variable region to bind to colon adenocarcinoma cell lines (LS174T, SW948, and C0112). The immunoreactivities of 125I-labeled murine and chimeric 17-1A MoAbs measured in a live cell-binding assay with LS174T, SW948, and C0112 cells revealed that chimeric IgG1, IgG4, and 17-1A F(ab')2 were comparable to murine 17-1A while chimeric IgG2 showed lower binding. The blood half-lives of 125I-labeled murine 17-1A, its F(ab')2 fragments, and chimeric IgG1, IgG2, and IgG4 in normal nude mice determined by serial eye bleeding were 7.5, 0.5, 5.2, 6.9, and 1.9 days, respectively. In biodistribution studies at 4 days after injection of 125I-labeled MoAbs in nude mice bearing LS174T tumors, chimeric IgG1 had the highest tumor concentration of 20.5% injected dose/g with a tumor/blood ratio of 3.2. 131I-labeled murine 17-1A administered in a single injection of 300 microCi or 3 injections of about 300 microCi each to nude mice bearing established LS174T tumors inhibited tumor growth, whereas a comparable amount of unlabeled murine 17-1A did not inhibit tumor growth. 131I-labeled chimeric IgG1 MoAb showed a similar level of tumor growth inhibition. The results of the present study indicate that 17-1A chimeric IgG1 antibody may be the best choice for clinical radioimmunodetection and radioimmunotherapy studies.

Animals↗

PubMed1994

Chimeric murine-human antibodies directed against folate binding receptor are efficient mediators of ovarian carcinoma cell killing.

The MOv18 (gamma 1, kappa) and MOv19 (gamma 2a, kappa) murine monoclonal antibodies (MAbs) recognize different epitopes on the human folate binding receptor which is overexpressed on 90% of nonmucinous epithelial ovarian tumors. A chimeric murine-human (human gamma 1, kappa) version of both antibodies was constructed and expressed. The genes encoding the murine heavy and light chain variable regions of the MOv18 and MOv19 MAbs were cloned from the parental hybridomas, fused with genes encoding the human heavy (gamma 1) and light (kappa) chain constant regions, respectively, and expressed in the SP2/0 murine myeloma cell line. Using human peripheral blood mononuclear cells as effector cells and conditions that provide for maximum lysis (effector target = 50:1, saturating antibody concentration), the murine MOv18 MAb (IgG1) mediated variable levels of specific cytolysis of the target ovarian cancer cell line IGROV1. In contrast, the chimeric MOv18 MAb mediated higher and more consistent lysis even at a 10-100-fold lower antibody concentration. The murine MOv19 MAb (IgG2a) mediated specific lysis of IGROV1 cells, and the chimeric version of this antibody mediated an amount of lysis at least equal to that mediated by its murine counterpart. A comparison of the ED50 values obtained for the murine MOv19 and chimeric MOv19 antibodies indicates that the chimeric MOv19 MAb was 3 to 10 times more potent than the murine MOv19 antibody. In addition, the ED50 values obtained for the chimeric MOv18 and chimeric MOv19 MAbs were similar, indicating that these MAbs are equally potent. The level of maximal lysis obtained was dependent on the number of target molecules/cell; the same high level of lysis mediated by cMOv18, MOv19, and cMOv19 was observed with both IGROV1 and OvCA432 target cells. However, only low levels of lysis were obtained when the SW626 cell line, which expresses 1 x 10(4) folate binding protein sites/cell, was used as a target. An equimolar mixture of the chimeric MOv18 and MOv19 MAbs was no more effective in the mediation of lysis than an equivalent amount of either chimeric MAb alone. These data suggest that the folate binding receptor is expressed on IGROV1 cells at a density sufficient to provide for optimal levels of antibody-mediated lysis using a single chimeric antibody directed at the folate binding receptor.

Animals↗

PubMed1997

Chimeric influenza viruses incorporating epitopes of outer membrane protein F as a vaccine against pulmonary infection with Pseudomonas aeruginosa.

Peptide 10 (NATAEGRAINRRVE, residues 305-318 of mature protein F) is one of two linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa both of which have been shown to elicit whole cell-reactive antibodies and to afford protection in animal models against P. aeruginosa infection. Influenza A virus was chosen as a vector to present this epitope in a human-compatible vaccine. Various lengths of the peptide 10 epitope ranging from a 5-mer (GRAIN), 7-mer (AINRRVE), 8-mer (TAEGRAIN), 9-mer (GRAINRRVE), 11-mer (AEGRAINRRVE) to a 12-mer (TAEGRAINRRVE) were attempted to be presented into the antigenic B-site of the hemagglutinin (HA) of live recombinant influenza virus. Using PCR, DNA sequences encoding these various peptide 10 lengths were inserted into the HA gene of influenza A/WSN/33 virus. By using a reverse-genetics transfection system, RNA transcribed in vitro from these chimeric HA genes was reassorted into infectious virus. To date chimeric viruses have been rescued and purified containing the peptide 10 5-mer, 7-mer, 8-mer, and 11-mer. RT-PCR and sequencing have confirmed the presence of P. aeruginosa sequences in the HA RNA segment of each chimeric virus. Each of the four chimeric viruses produced to date was used to immunize mice to determine the ability of each chimeric virus to elicit antibodies reactive with whole cells of P. aeruginosa. The immunization protocol consisted of a series of three intranasal inoculations, followed by two intramuscular injections of the chimeric virus. The chimeric virus incorporating the 11-mer elicited IgG antibodies that reacted with various immunotype strains of P. aeruginosa in a whole cell ELISA at titers of 80 to 2,560, whereas the chimeric virus incorporating the 8-mer elicited whole cell-reactive IgG antibodies at titers of 320 to 2,560. These data suggest that these two chimeric viruses may have vaccine efficacy against P. aeruginosa infection. These studies may result in the development of a chimeric influenza virus-protein F vaccine which would prove to be suitable for use in children with cystic fibrosis for the prevention of pulmonary colonization of these children with P. aeruginosa.

Amino Acid Sequence↗

PubMed2006

Endothelial chimerism in transplantation: Looking for needles in a haystack.

Endothelial chimerism in transplanted organs is a fascinating phenomenon, indicative of a mechanism by which progenitor recipient cells replace the donor endothelium. It has been hypothesized that this replacement could lead to a decrease in alloreactivity and thus would positively influence graft outcome. However, recent studies have shown that the amount of recipient-derived endothelial cells found in donor organs is relatively small. What effect on graft survival can we expect from this low number of chimeric cells? There are several hypotheses that address this question, but distinguishing the true effect of donor endothelial replacement on outcome from other factors affecting graft survival is difficult. Furthermore, "contamination" of chimeric cells from sources other than the recipient would have to be excluded before the effect of donor endothelial replacement by recipient cells can be accurately assessed. Pregnancies and blood transfusions are the other sources that may induce chimerism. Most of the techniques currently used to detect chimeric cells in donor organs are not specific enough to distinguish chimeric cells that may have been present in the graft before transplantation and recipient-derived chimeric cells that replace the endothelium after transplantation. Also, the sensitivity of these techniques may be questioned: do we really detect all chimeric cells that are present? This review will elaborate on these questions and discuss future perspectives of research into chimerism.

Chimerism↗

PubMed2006

Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFbeta1 in cell-free system and in hepatic stellate cells.

Transforming growth factorbeta1 (TGFbeta1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFbeta1 in these processes, the non-chimeric hammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFbeta1 were designed to down-regulate TGFbeta1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFbeta1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFbeta1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFbeta1 in many cellular processes and a potential therapeutic candidate for TGFbeta1-related diseases.

Base Sequence↗

PubMed1994

Generation and characterization of a mouse/human chimeric antibody directed against extracellular matrix protein tenascin.

The murine anti-tenascin monoclonal antibody 81C6, following iodination, has been shown to be an efficient localizing and therapeutic agent in both subcutaneous and intracranial human glioma xenograft models in athymic mice and rats. Similarly, effective monoclonal antibody 81C6 localization has been demonstrated in glioma patients, and Phase I trials with the intact murine IgG2b kappa molecule are currently in progress. In order to maximize the potential for repeated administration by minimizing murine Fc-mediated immunogenicity and reducing Fc-mediated immune effects, we created murine 81C6 variable region/human IgG2 chimeric monoclonal antibodies by the molecular cloning of the variable region genes of mouse 81C6 and their genetic linkage to human constant region exons. The resulting chimeric constructs were introduced into SP2/0 cells, and stable transfectomas were selected by G418 and mycophenolic acid resistance. The resistant clones were screened for anti-tenascin activity on tenascin-coated plates by enzyme-linked immunosorbent assay. The N-terminal amino acid sequence of both heavy and light chains of the purified chimeric 81C6 antibody matched exactly with that of the native mouse 81C6 as well as with that deduced from the nucleotide sequence. The production level of chimeric 81C6 (13.9 mg/ml) from ascites in the highest expressing transfectoma was much higher than that of native mouse 81C6 (2.5 mg/ml). The chimeric antibody showed the same specificity and equivalent affinity for human intact tenascin or tenascin-expressing cells as the native mouse 81C6 antibody. Direct comparison of radioiodinated chimeric and radioiodinated mouse 81C6 biodistribution in subcutaneous and intracranial xenograft-bearing mice showed higher tumor-to-normal tissue ratios for chimeric 81C6 as compared with native mouse 81C6. The improved localizing and clearance characteristics of chimeric 81C6 in xenograft model systems suggests that chimeric 81C6 would be an improved reagent for intracompartmental therapy of tenascin-expressing tumors in the human central nervous system.

Animals↗

PubMed1996

An examination of tissue chimerism in the ACI to Lewis rat cardiac transplant model.

While the existence of chimeric cells in host tissue following organ transplantation is well documented, its distribution, temporal evolution and relationship to allograft survival is less clear. To explore this phenomenon, Lewis recipients of ACI cardiac allografts representing a wide range of immunosuppressive protocols and graft survival times were examined for the presence of chimerism using a sensitive polymerase chain reaction assay. Four groups of animals were examined: untransplanted animals receiving donor specific transfusion (DST)/cyclosporine A (CsA); allograft recipients with no treatment; recipients treated with DST/CsA/supplementary immunosuppression with rejection at 21-183 days; and recipients sacrificed with functioning allografts, treated with DST/CsA/supplementary immunosuppression and surviving > 200 days. To elucidate variations in the tissue distribution of chimeric cells, bone marrow, skin, liver, spleen, and thymus were examined in each animal. Untransplanted animals receiving DST/CsA displayed no evidence of chimerism. In animals receiving a cardiac allograft but no treatment, there was extensive evidence of chimerism in four of five animals. Chimerism was also detected in seven of nine animals with intermediate graft survival at the time of rejection. In animals with long-term graft survival, only four of eight displayed chimerism. These results suggest that, without immunosuppression, early chimerism does not lead to prolonged graft survival and that, even when graft survival is moderately prolonged, these cells are not sufficient to prevent rejection. In conclusion, chimerism appears to be a common phenomenon following transplantation, is not a result of DST, and may not be necessary for maintenance of long-term graft survival.

Animals↗

PubMed1975

Construction and characterization of a chimeric plasmid composed of DNA Pfrom Escherichia coli and Drosophila melanogaster.

A chimeric plasmid has been constructed in vitro from colicin E1 factor (Col E1), nontransmissible R-factor RSF-1010, and Drosophila melanogaster DNAs by the sequential action of Escherichia coli endonuclease RI(Eco RI) and T4 phage DNA ligase. The chimeric plasmid was assembled in two stages--first, a composite plasmid consisting of Col E1 and RSF 1010 was constructed, followed by partial digestion of the composite with Eco RI (in order to open one of the susceptible cleavage sites) and ligation with an Eco RI-digested D. melanogaster DNA preparation. The chimeric plasmid was selected and amplified in vivo by sequential transformation of E. COLI C with the ligated mixture, selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-CsCl solution. Treatment of the chimeric plasmid with Eco RI yields three fragments with mobilities corresponding to the linear forms of the constituents--COL E1, mol wt 4.2 times 106, RSF 1010, mol wt 5.5 times 106 and D. melanogaster DNA, mol wt 4.0 times 106. The buoyant densities of the three constituents are respectively 1.706, 1.719, and 1.697 g/cm3, while the buoyant density of the composite factor is 1.712 and that of the chimeric plasmid is 1.705. Serratia marscesens endonuclease R (Sma) which introduces a single cut in Col E1, but not in RSF 1010, converts the chimeric plasmid to a single linear molecule (mol wt 13.7 times 106) and sequential digestion with both Sma and Hin III yields two distinct fragments, mol wt 3.7 and 10.0 times 10.6, respectively; this implies that the two sites are unique and occur at distinctly different positions. Sequential digestion with both Hin III and Eco RI reveals that the Hin III cut is in the D. melanogaster segment; neither Col E1 nor RSF 1010 contain sites susceptible to digestion with Hin III. In the presence of chloramphenicol, the chimeric plasmid continues toreplicate for 9 hr while bacterial chromosomal DNA replicates at a much slower rate. As in the case of the composite plasmid, continued synthesis is the presence of chloramphenicol suggests that the replicator of Col E1 is functional in the chimeric plasmid as well. Examination of the chimeric plasmid by partial denaturation mapping permits identification of its constituents, each of which presents a characteristic profile. The D. melanogaster segment reveals a wealth of detail at the molecular level pertaining to the distribution of AT-rich regions.

Animals↗

PubMed1997

Temporal and Quantitative Analysis of Chimerism in Liver and Kidney Transplant Patients: An Enhanced Fluorescence Detection System Allows for More Sensitive Quantitative Detection.

Background: Because spontaneous microchimerism has been reported in stable renal and hepatic allografts, the presence of donor-derived cells in recipient tissues was investigated in kidney and liver tranplant recipients. Methods and Results: Human lymphocyte antigen class II markers and Y-chromosome sequences in male donor-to-female recipient transplants were used for chimeric analysis. Human lymphocyte antigen typing was performed by group-specific polymerase chain reaction amplification and restriction fragment length polymorphism analysis, X-chromosome- and Y-chromosome-specific primers were used in a multiplex polymerase chain reaction analysis. Quantitative Y-chromosome analysis was performed using energy-transfer fluorescence from a nested primer system. Patients who had rejected their grafts were also analyzed, as were a group who were analyzed for chimerism at the time of transplant (day 1) and sequentially at various intervals for up to 3 months. Of 23 long-term kidney patients analyzed, 16 were chimeric by human lymphocyte antigen or sex-determination analysis. In 2 patients whose graft had failed no chimerism was observed. Chimerism in liver patients was detectable on the day of transplant and was maintained for 30 to 120 days as measured at 5-day intervals (these patients continue to be monitored). Quantititative analysis suggested that the ratio of donor to recipient cells was variable in a patient and ranged from greater than 1 in 10(4) to less than 1 in 10(5). An enhanced fluorescence energy-transfer detection system was adopted to increase sensitivity of the polymerase chain reaction detection of chimerism and to quantitate the results. Conclusions: The results indicate that cells from the donor organ migrate into recipient tissues early after transplantation. These cells persist in a majority of patients for at least 3 to 4 years. It has been proposed that tolerance is related to the presence of these "passenger" leukocytes and that dendritic cells play the most important role. The data suggest that the establishment of chimerism plays an important role in graft acceptance in a majoritiy of the kidney and liver patients in this study. These findings also suggest that the levels of chimeric cells, "a quantitative chimerism," may be important in establishing tolerance but further studies are required to support this contention.

Journal Article↗