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PubMed2001

[Analysis of chimerism in patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation].

Serial monitoring of chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) can be performed easily and rapidly using PCR-based assays analyzing informative tandem repeat genetic markers. Sequential analysis of individual chimerism status was performed in 34 patients who underwent myeloablative allo-HSCT using a commercial multiplex short tandem repeat (STR) kit. Mixed chimerism (MC) was found in 14 of the patients for more than one month. The incidence of MC seemed to be dependent on the type of disease or pretransplantation regimen. There was no significant difference in relapse rates between MC and complete donor chimerism (CC) in all patients. However, the relapse rate was significantly higher in MC than in CC among patients with acute leukemia. The severity of acute graft-versus-host disease (aGVHD) was significantly reduced in the patients with MC. Most of the MC patients with hematologic malignancies had transient mixed T-lymphoid chimerism, and CC was achieved within 6 months after HSCT in such cases. Patients with MC beyond 6 months after HSCT and patients with reappearance of autologous signals (MC after CC) may have an enhanced risk of relapse.

Adolescent↗
PubMed2001

Contributions of tissues to expression of a major gene for growth in chimeric mice.

The objective of this research was to determine the effect of genotype of particular tissues and organs on the growth of a chimera produced by aggregating an embryo homozygous for the high-growth (hg) allele with an embryo from a control line of wild-type mice lacking the deletion responsible for the high-growth phenotype. A total of 107 chimeric mice were produced, 81 males and 26 females. The preponderance of male chimeras closely fit the expected 3:1 ratio of males to females in balanced chimeric populations, suggesting that neither of the two genetic lines appeared to have a selective advantage for contributions to chimeric tissues. No single correlation between individual tissue chimerism and mouse growth was remarkable relative to correlations of chimerism of other tissues to growth. Other statistical models, including multiple regression techniques, did nothing to identify particular organs, tissues or various combinations that improved the explanation of expression of the high-growth phenotype by percentage of high-growth cells in that organ. These observations suggest that the chromosomal deletion thought to cause the high-growth phenotype has a systemic effect, without dominant effects of or on individual organs or tissues; the precise mechanism has yet to be determined.

Animals↗
PubMed2001

Neurons lacking huntingtin differentially colonize brain and survive in chimeric mice.

To determine whether neurons lacking huntingtin can participate in development and survive in postnatal brain, we used two approaches in an effort to create mice consisting of wild-type cells and cells without huntingtin. In one approach, chimeras were created by aggregating the 4-8 cell embryos from matings of Hdh (+/-) mice with wild-type 4-8 cell embryos. No chimeric offspring that possessed homozygous Hdh (-/-) cells were obtained thereby, although statistical considerations suggest that such chimeras should have been created. By contrast, Hdh (-/-) ES cells injected into blastocysts yielded offspring that were born and in adulthood were found to have Hdh (-/-) neurons throughout brain. The Hdh (-/-) cells were, however, 5-10 times more common in hypothalamus, midbrain, and hindbrain than in telencephalon and thalamus. Chimeric animals tended to be smaller than wild-type littermates, and chimeric mice rich in Hdh (-/-) cells tended to show motor abnormalities. Nonetheless, no brain malformations or pathologies were evident. The apparent failure of aggregation chimeras possessing Hdh (-/-) cells to survive to birth is likely attributable to the previously demonstrated critical role of huntingtin in extraembryonic membranes. That Hdh (-/-) cells in chimeric mice created by blastocyst injection are under-represented in adult telencephalon and thalamus implies a role for huntingtin in the development of these regions, whereas the neurological dysfunction in brains enriched in Hdh (-/-) cells suggests a role for huntingtin in adult brain. Nonetheless, the lengthy survival of Hdh (-/-) cells in adult chimeric mice indicates that individual neurons in many brain regions do not require huntingtin to participate in normal brain development and to survive.

Animals↗
PubMed2001

Chimeric pig hearts resist hyperacute rejection in ex vivo perfusion model.

With surrogate tolerogenesis. the recipient immune system is engrafted within the donor pig before organ transplant. Chimeric pig hearts may resist hyperacute rejection by inducing accommodation. This hypothesis was tested using an ex vivo isolated piglet heart perfusion model. Processed sheep marrow was infused into fetal pigs at 45 days gestation. Heart explants from chimeric or nonchimeric pigs were suspended in a Langendorff apparatus and perfused with plasma from unsensitized sheep or sensitized sheep. Nonchimeric hearts perfused with plasma from unsensitized functioned for 240 min (N = 3). Nonchimeric hearts perfused with sensitized plasma deteriorated rapidly, functioning at 19+/-12 min (N = 6); Immunohistochemistry of heart graft revealed extensive deposition of IgG, IgM in the microvascular. In contrast, chimeric hearts perfused with sensitized plasma functioned for 183+/-46 min (N = 3)(p <.001); Deposition of IgG, IgM had substantially less. Heart grafts procured from chimeric pigs survived in the presence of antidonor IgG, IgM, and complement, demonstrating that chimeric pig hearts resist hyperacute rejection.

Animals↗
PubMed2001

[Development of donor cells in chicken-duck chimeric embryos].

Chicken W chromosome-specific DNA fragments were obtained after transformation, extraction, purification and digestion with EcoR I of pUGD1201. The W chromosome-specific DNA probe was a 1.2 kb insert, which was labelled by the random-technique using a Digoxigenin Labeling Kit. Chicken PGCs from a line of White Leghorns were microinjected into Maya duck blastoderms at stage X. The embryos at 6,8,9,12,16,18 days of incubation were used to study the development of chicken PGCs in the chimeric embryos. Used chicken W chromosome-specific DNA probes by in situ hybridization, development of donor cells in chicken-duck chimeric embryos were studied. In 54 chimeric embryos, there were different degrees of chimerism in the organs. The chimeric rate of the livers was the highest, which of the gonads was the lowest. It is demonstrated that somatic and germline chimeras can be yielded successfully by transferring stage X chicken blastodermal cells.

Animals↗
PubMed1999

[Effect of anti-CD4+ human/murine chimeric antibody on the proliferation and apoptosis of lymphocyte in patients with asthma].

OBJECTIVE: To investigate the effects of anti-CD4+ human/murine chimeric antibody on the proliferation and the apoptosis of peripheral blood lymphocytes (PBL) in patients with asthma. METHODS: PBL isolated from patients with asthma were cultured with 50 mg/L, 100 mg/L anti-CD4+ human/murine chimeric antibody and anti-CD3+ McAb (10 mg/L) for 0, 24, 48 h in vitro, the apoptosis cell percentage were detected by flow cytometry and in situ end-labeling technique of fragmental DNA as well as the proliferation of PBL were detected by MTT method. RESULTS: Comparing with normal controls, the apoptosis cell percentage was significantly decreased by stimulation with anti-CD3+ McAb for 24 h and 48 h, the proliferation of lymphocyte was increased in patients with asthma; the apoptosis cell percentage of patients with asthma was significantly increased (P < 0.01, respectively) with the cell cultured with 50 mg/L or 100 mg/L anti-CD4+ human/murine chimeric antibody for 24 h or 48 h At the same time the lymphocyte proliferation was inhabited (P < 0.01, respectively). CONCLUSIONS: It is demonstrated that the proliferation of PBL in patients with asthma were increased and the apoptosis cell percentage were decreased, anti-CD4+ human/murine chimeric antibody had inhibited the proliferation of T lymphocyte and had enhanced the T lymphocyte apoptosis. The anti-CD4+ human/murine chimeric antibody may be potentially useful for the biotherapy of asthma.

Adult↗
PubMed2002

[Immunocontraceptive effect of a synthetic chimeric peptide of sperm].

OBJECTIVE: To investigate the immunocontraceptive ability of a new synthetic chimeric peptide with 35 amino acid residues. METHODS: A chimeric peptide with 35 amino acid residues was synthesized based on the amino acid sequence of SP17, a protein specific to murine testis/sperm, and cyritestin. The purified product, with keyhole limpet hemocyanin (KLH), was injected subcutaneously into the pad of female and male Balh/C mice. Blood was extracted from the ophthalmic arteries of the mice to identify the corresponding proteins from organs of mice and/or rat and human being by Western blotting. The chimeric peptide was injected into other female mice that were then raised together with male mice in the same cages to observe their status of pregnancy. Freund' adjuvant or normal saline were injected into the controls. RESULTS: The purity of the new chemiric peptide was 95%. The highest specific IgG titre in mouse serum was 1:6000 and the highest titre of specific IgA in the washing of vaginal mucous membrane of female mice was 1:300. The specific antiserum from the mice immunized by the chimeric peptide and KLH could identify two proteins with the molecular weights of 22 000 and 55 000 from testes of mice, rats, and human being. Western blotting showed no band for the extracts of mouse liver, spleen, and kidney. The pregnancy rate of female immunized mice was 61.11%, significantly lower that those in the controls (P < 0.05). The average number of cubs in a litter was less in the experimental group than in the controls (P < 0.05). CONCLUSION: The new chimeric peptide with 35 amino acid residues stimulates powerfully humoral and mucous immunity in mice and obviously decreases the pregnancy rate and the number of cubs in each litter in mice.

Animals↗
PubMed1997

Separate Expression and Characterization of the Heavy- and the Light-chain of A HBsAg Specific Murine-human Chimeric Antibody in Insect Cells.

Both the heavy- and light-chain of a murine-human chimeric antibody with specificity for hepatitis B virus surface antigen have been expressed separately in a baculovirus expression system. The heavy- and light-chain variable region (VH and VL) genes encoding the marine-monoclonal antibody OH3 were combined with human gamma3 and kappa constant region genes to construct the murine-human chimeric genes, respectively. The transfer vectors containing the chimeric genes were cotransfected into Sf9 cells with linearized virus DNA and recombinant viruses were isolated by dot hybridization, PCR and Southern blot analysis. The chimeric heavy- and light-chain was expressed respectively in the recombinant viruses-infected insect cells and the characterization by Western blot and competitive ELISA demonstrated that both of the expressed chimeric heavy- and light-chain have the specificity of HBsAg binding.

Journal Article↗
PubMed2002

[Expression of TPO mimetic peptide chimeric proteins with human IgG1 Fc fragments and their biological characters].

Many antineoplastic agents can cause myelosuppression and thrombocytopenia. Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels. We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo. For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments. The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5' terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5' 648 bp; Fc2: Fc 5' 270 bp; Fc3: Fc 5' 267 bp; Fc4: Fc 5' 90 bp), and cloned into expression plasmid pET28a (+) for constructing four recombinant plasmids. By transforming the four recombinant plasmids into E. coli. BL21 (DE3) respectively, we got 3 kinds of engineered E. coli which express TPO + Fc chimeric proteins(28 kD TPO + Fc1, 12 kD TPO + Fc2 and 12 kD TPO + Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF column. The bioactivities of the expressed chimeric proteins(TPO + Fc1, TPO + Fc2 and TPO + Fc3), TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo, the EC50 on Ba/F3-mp1 cells were 13, 10, 10, 50, and 25 nmol/L respectively, all of them can increase circulating platelet counts. Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO + Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc. These studies have laid basis for production of TPO mimetic peptide by genetic engineering.

Animals↗
PubMed2002

Detection of relapse by sequential monitoring of chimerism in circulating CD34+ cells.

Relapse of the underlying malignant disease represents one of the major causes of treatment failure in patients treated with stem cell transplantation, especially in patients with acute leukemia. Detection of minimal residual disease (MRD) after allogenic stem cell transplantation (SCT) is important to schedule therapeutic intervention, e.g. donor lymphocyte infusion. We asked, whether chimerism analysis in circulating CD34+ cells might be a feasible method to monitor MRD in patients after allogeneic SCT. Eighty-seven patients undergoing allogeneic SCT were prospectively analyzed. CD34+ cells were prepared from peripheral blood samples and sorted after immunomagnetic preenrichment. Results were correlated to overall chimerism and results of nested RT-PCR for the bcr-abl rearrangement in Ph1-cases. In the 87 patients a median of 8 analyses (range 2-22) covering a median period of 295 days (range 28-1152) were performed. A hematological relapse was observed in 22/84 engrafting patients (26%). In twenty patients, the relapse was detected in the CD34+ fraction, in 14 of these patients, donor chimerism in CD34+ cells decreased 12-97 days (median 52 days) before the clinical diagnosis. In two cases, CD34+ chimerism failed to demonstrate the relapse. In patients with relapsing CML, the decrease of donor CD34+ cells was associated with reappearance of bcr-abl transcripts. Treatment (reduction of immunosuppression, DLI or STI571) was associated with an increase of donor derived CD34+ cells and clearance of bcr-abl positive cells. Sequential chimerism analysis in CD34+ cells is feasible and sensitive, allowing early detection of relapse and monitoring of therapeutic intervention. This method appears especially useful in patients with high risk leukemia lacking other markers for detection of MRD.

Antigens, CD34↗
PubMedDate not supplied

[Inhibition by IFN-(Asp)4-Lys-HIV chimeric protein of hydrolysis of the low molecular substrate by the enteropeptidase light chain].

The full length enteropeptidase or it's light chain have often used for the limited proteolysis of recombinant chimeric proteins incorporating the linker-(Asp)4Lys- to obtain the target protein. Any chimeric proteins were not cleaved by the full length enteropeptidase efficiently. The resistant to the hydrolysis chimeric protein IFN-(Asp)4Lys-HIV earlier was shown to be the competitive inhibitor (Ki = 3,4 x 10(-6) M) in relation to the low molecular substrate. In present study we were determined this chimeric protein competitive inhibited the same substrate hydrolysis by enteropeptidase light chain (Ki = 2,7 x 10(-5) M). Comparison the Ki values for the substrate hydrolysis by full length enzyme and its light chain suggests that the enteropeptidase heavy chain may participate in chimeric protein binding.

Aspartic Acid↗
PubMed1992

Mixed allogeneic chimeras prepared by a non-myeloablative regimen: requirement for chimerism to maintain tolerance.

We have recently described a non-myeloablative conditioning regimen permitting engraftment of allogeneic bone marrow in mice which involves administration of anti-CD4 (GK1.5) plus anti-CD8 (2.43) monoclonal antibodies in vivo, 3 Gy whole body irradiation, plus 7 Gy thymic irradiation. B10 (H-2b) mice prepared by this regimen and infused with unmanipulated B10.D2 (H-2d) bone marrow develop permanent mixed lymphohematopoietic chimerism and specific tolerance to donor skin grafts. We now demonstrate that mixed chimerism persists longer than 170 days in the lymphoid tissues including spleen, thymus and bone marrow of such animals, and that equivalent levels of donor chimerism are observed in both T and B cell compartments. In addition stable mixed chimeras were found to be unresponsive to host (B10) and donor (B10.D2) stimulator cells in mixed lymphocyte reaction and in cell mediated lympholysis assays, while responses to a third party (B10.BR, H-2k) were intact. Persistent chimerism was found to be necessary for the maintenance of skin graft tolerance in these animals, since in vivo depletion of donor cells by treatment with an anti-H-2d (34-2-12) monoclonal antibody resulted in the subsequent rejection of donor skin grafts. These studies demonstrate that mixed allogeneic chimeras produced using this regimen are specifically tolerant to donor in vitro and in vivo, and that persistence of donor chimerism is critical for the maintenance of tolerance.

Animals↗
PubMed2003

Long-term follow-up of chimerical state of the patients transplanted for different haematologic diseases.

Long-term follow-up of peripheral cellular chimerism in patients treated with BMT or PBSCT revealed the usefulness of their continuous monitoring at molecular level. Our results are based on monitoring of 120 patients, who were followed for at least 24 months. Comparison of the patients treated for chronic myelogenous leukemia (CML), acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS) and aplastic anaemia (AA) revealed that mixed chimerism was practically absent in MDS and relatively long-lasting in ALL and AA (regardless to substantially different post-transplantation treatment). The first disease relapses signalized by molecular checking of mixed peripheral chimerism were observed also after a period of remission lasting for several years. Molecular watching enables us to detect relapses at their very beginning that would remain hidden to less sensitive methods. We believe that all of the transplanted patients ought to be monitored for residual disease i.e. cellular chimerism using molecular methods without time limits. On the other hand low level of mixed cellular chimerism is not necessarily a sign of disease progression and can remain unchanged as "status quo" for a very long period.

Adult↗
PubMed2003

[Analysis of the effects of mutant hairless genes in chimeric mice].

The autosomal recessive gene hairless (hr) is responsible for the complete hairlessness in mice homozygous for this gene. Hair shedding that begins at the age of 10 days is caused by an abnormal cycle of hair follicle development disturbed at the catagen stage. This results in enhanced programmed cell death (apoptosis) and ultimately leads to the complete hair follicle destruction and shedding of all hairs by the age of three weeks. To study the phenotypic expression of the hr gene in a chimeric organism, we have obtained 12 chimeric mice hr/hr <--> +/+ by means of aggregation of early embryos hr/hr and +/+. In chimeric mice, the hair shedding has begun two days later than in the hr/hr mice. By day 23 of postnatal development, hairless areas were present on the coat of chimeric mice or the latter were completely hairless depending on the percentage of the hr/hr mutant component. In four chimeras with high content of the mutant component (68-76%), the hair shedding process was similar to that in the hr/hr mice, though it was accomplished two days later. In three chimeras with 48-51% of the mutant component, alternating hairless and hair-covered bands were observed. These data suggest that the hr gene acts in epidermal cells of a hair follicle, because epidermal cell clones in embryonic skin migrate in the lateral-ventral direction coherently and without mixing. However, some chimeras displayed a pattern which was not so clear-cut: the band borders were illegible and hairs partly covered the hairless areas. In some chimeras, the uniform thinning of the coat was observed. Analysis of the effects of the hr mutant gene in chimeric mice differing in the ratio between mutant (hr/hr) and normal (+/+) components in tissues suggests that the hr gene acts in the epidermal cells of the hair follicle. The interactions between cells have an essential effect on the mode and degree of the hr gene expression, which leads to distortion of the "ectodermal" coat pattern in chimeras.

Animals↗
PubMed2004

Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles.

OBJECTIVE: To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). METHODS: The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization. RESULTS: The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg. CONCLUSION: The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes↗
PubMed2004

[A study of quadriplex PCR with chimeric primers for short tandem repeats loci].

OBJECTIVE: To establish a simplified method of multiplex PCR based on chimeric primers for STR loci. METHODS: A set of chimeric primers and universal primers were designed to carry out a multiplex PCR for amplifying four short tandem repeats(STR) loci. Then the amplified STR loci were detected by polyacrylamide gel electrophoresis and the gels were sliverstained. RESULTS: A quadriplex STR system was developed on the basis of both chimeric and universal primers. CONCLUSION: The STR multiplex PCR based on both chimeric and universal primers hss been achieved readily and reproducibly by simple adjustment of the individual primer concentrations. The use of chimeric primers provides a method for primer design that eliminates the multiple optimization steps involved in developing the multiplex PCR.

DNA Primers↗
PubMed2004

[The kinetics of hematopoietic cell chimerism in the early period after non-myeloablative transplantation and its clinical implications].

OBJECTIVE: To analyze the kinetics of hematopoietic cell chimerism in the early period after non-myeloablative stem cell transplantation (NAST) and to investigate the correlation between molecular and hematologic assessment of engraftment or rejection. METHOD: Short tandem repeat-polymerase chain reaction (STR-PCR) analysis of chimerism status was carried out in 6 patients who received NAST from HLA-matched sibling donors. RESULTS: In 5/6 patients, the peripheral blood samples collected on the first day after allograft infusion displayed the presence of mixed chimerism. STR-PCR analysis revealed a gradual increase of the donor-specific allelic signal which became dominant over the recipient-specific allele by day +7. On day +14, hematologic chimerisms were completely donor origin. Their molecular engraftments (ME) were detected at a median time of 6 days, preceding hematologic engraftment by a median of 5 days (P > 0.05). But the sixth patient showed more than 50% host residual cells on day +7 and had no signs of ME on day +14. CONCLUSION: It suggested that molecular monitoring of the early dynamics of chimerism after NAST could be useful in predicting engraftment, or rejection. If the engraftment was less than 50% on day +7 and failed to get ME on day +14, the graft rejection would occur.

Adult↗
PubMed2004

[Study on the cleavage activity of U1 small nuclear RNA chimeric ribozyme against HCV RNA in vitro].

OBJECTIVE: To study the cleavage activity on the HCV RNA of a chimeric recombinant of HCV specific ribozyme and U1 small nuclear RNA, which compartmentalizes within the nucleolus. METHODS: The third stem-loop sequence of human U1 snRNA (position 95-116) within pBSIISK+ U1 was substituted by hammerhead ribozyme against HCV RNA by PCR and cloning methods, and the constructed plasmid was named pBSIISK+ (U1-Rz). Then the whole gene fragment of the chimeric ribozyme was cloned into a pGEM-T vector under the control of T7 promoter, and the constructed plasmid was named pGEM- (U1-Rz). The pGEM- (U1-Rz) and pGEM-Rz (containing the same ribozyme sequence as that in U1-Rz) transcripts as enzyme were transcribed in vitro. Also the (32)P-labeled pCMV/T7-NCRC luc (containing the gene sequence of the whole 5'-NCR and part core of HCV RNA) transcripts as target-RNAs were transcribed in vitro. The enzymes were incubated with the target RNAs under different conditions and autoradiographed after denaturing gel-electrophoresis. RESULTS: The sequencing result showed that the construction of U1 snRNA chimeric ribozyme was correct. Compared with the ribozyme alone, both of them were active at 37 degree C and with Mg2+ (10 mmol/L) and TrisCl (10 mmol/L, pH7.9), and there was no remarkable difference between them. The cleavage activity of the chimeric ribozyme increased with the prolongation of reaction time and increment of enzyme concentration. CONCLUSION: Both ribozyme and U1 snRNA chimeric ribozyme exhibited specifically catalytic activity against HCV RNA in vitro. There was no remarkable difference between their cleavage efficiencies.

Chimera↗