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At least 973 records · Page 54Linked to original sources
PubMed2000

Molecular analysis of the rearranged genome and chimeric mRNAs caused by the t(6;11)(q27;q23) chromosome translocation involving MLL in an infant acute monocytic leukemia.

Chromosomal analysis of acute monocytic leukemia cells in a female infant revealed a t(6;11)(q27;q23) translocation. Southern blot analysis with a cDNA probe of the MLL gene at chromosome band 11q23 indicated that the breakpoint was in an 8.3-kb BamHI fragment that contained exons 5-11 of the MLL gene. Northern blot analysis showed a faint band corresponding to the MLL chimeric transcript. Structural analysis of a genomic clone with the rearranged MLL gene from der(11) chromosome demonstrated the breakpoint to be localized between exons 6 and 7 of the the MLL gene and to lie in an Alu sequence of this region. The partner gene fused to the 3' part of MLL was shown to be the AF6 gene on chromosome 6q27 by in situ chromosome hybridization and nucleotide sequencing of chimeric MLL cDNA clones. However, it was shown that MLL exon 5 was fused to AF6 in one clone, whereas most clones were MLL exon 6/AF6 chimeric cDNA clones. These findings indicate that exon 6 of MLL is spliced out in the process of transcription in a variant MLL/AF6. In addition, we were able to detect the splicing of exon 6 in either this chimeric MLL/AF6 or MLL transcripts from untranslocated chromosomes by reverse transcriptase-polymerase chain reaction. The detailed genetic map of AF6 was determined by in situ chromosome hybridization and radiation hybrid mapping.

Amino Acid Sequence↗
PubMedDate not supplied

[Molecular study of bone marrow chimerism and expression of EVI-1 gene as manifestations of minimal residual disease after bone marrow transplantation (BMT)].

Leukaemias are the most frequent tumour diseases in children. Bone marrow transplantation (BMT) is the most effective therapy for many patients with leukaemia. Highly polymorphic microsatellite markers provide useful genetic markers for detection of complete or mixed chimerism in patients after (BMT). Chimerism can be monitored successfully using several polymerase chain reaction (PCR) techniques and cytogenetic analysis, especially fluorescent in situ hybridization (FISH). It is still unclear whether individuals with mixed chimerism after bone marrow transplantation have an increased risk of developing leukaemic relapse or graft rejection. EVI-1 gene was mapped in human chromosome 3q26. It encodes zinc finger, DNA binding protein detected only in the nucleus. The EVI-1 function is unknown. It is not expressed in normal human haematopoietic cells, but is expressed in leukaemias especially with 3q26 abnormalities. Expression of the EVI-1 may play a significant role in pathogenesis of human leukaemias. Molecular study of chimerism and expression of EVI-1 gene may be useful for monitoring residual disease after bone marrow transplantation.

Bone Marrow↗
PubMed2000

Follow-up of chimerism status after allogeneic HLA-mismatched stem cell transplantation by detection of non-shared HLA alleles.

BACKGROUND AND OBJECTIVES: Chimerism studies after allogeneic transplantation are usually performed using cytogenetic analysis, PCR-VNTR or PCR-STR. Here, we report an alternative method for following the chimerism status after an HLA-mismatched stem cell transplantation (SCT), detecting the presence of non-shared HLA alleles by reference-strand mediated conformation analysis (RSCA). DESIGN AND METHODS: We tested this new approach on allogeneic related haploidentical SCT, unrelated cord blood transplantation, and HLA-mismatched unrelated donor SCT. The quantification of the chimerism was performed by laser detection of fluorescent-labeled primers on an automated DNA sequencer. RESULTS: In all cases this technique was able to detect mixed chimeras. The technique detected above 5% of residual cells when the analysis was based on HLA-class I and above 3% for HLA-class II. This sensitivity is similar to that of the PCR-VNTR analysis. INTERPRETATION AND CONCLUSIONS: This method avoids the need to search for an informative locus (which is essential for PCR-VNTR or -STR). Moreover, we did not find the phenomenon of preferential amplification that is observed with most VNTR, thus avoiding the need for construction of standard curves to quantify mixed chimeras. We conclude that the detection of the non-shared HLA alleles by RSCA is a useful approach for chimerism follow-up after HLA-mismatched SCT.

Alleles↗
PubMed2001

The usefulness of X-linked polymorphic loci as gene markers to track X allele and chimerism in a post-allogeneic peripheral blood stem cell transplant patient with Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS), an X-linked recessive disorder, is characterized by progressive T-cell immunodeficiency. Laboratory findings generally demonstrate reduced response to T-cell mitogens, markedly decreased serum concentration of IgM, and thrombocytopenia with small platelet volume. Allogeneic HLA-matched sibling bone marrow transplantation (BMT) can correct this disorder. We report the usefulness of X-linked polymorphic loci to detect X-allele gene tracking among WAS siblings and chimerism between a pre- and post-allogeneic matched sibling peripheral blood stem cell transplantation (PBSCT). A 3 1/2 year old boy with clinical and laboratory findings consistent with WAS underwent allogeneic matched sibling PBSCT. We used BclI restriction fragment length polymorphism (RFLP) of intron 18 of factor VII gene and MseI RFLP of the 5' flanking region of factor IX gene to detect X-allele gene tracking among siblings and family members and chimerism in patients between pre-and post-allogeneic matched sibling PBSCT. We were able to demonstrate that determination of BclI and MseI RFLP can be employed to recognize the difference in X-allele genes between the recipient and donor for allogeneic matched sibling PBSCT. The authors also were able to demonstrate that these polymorphic loci can detect full chimerism of donor hematopoietic cells in recipient blood after allogeneic PBSCT. This finding was correlated with improvement of post-PBSCT clinical and laboratory findings. BclI and MseI RFLP associated with X-chromosome can effectively track X-allele, detect carrier state, and demonstrate the different X-allele among male siblings, and chimerism of hematopoietic cells between donors and recipients in a setting of allogeneic matched sibling BMT or PBSCT for X-linked hereditary diseases such as Wiskott-Aldrich syndrome.

Child, Preschool↗
PubMed2001

[Study of the time course of mixed chimerism by fluorescent in situ hybridization in patients with chronic myeloid leukemia after allogenic transplantation of bone marrow].

AIM: To determine the type of chimerism in patients with chronic myeloid leukemia (CML) in various periods after allogenic transplantation of bone marrow (TBM) and its association with subsequent relapse. MATERIALS AND METHODS: Ten patients were examined after allogenic TBM, which was performed during the chronic phase of CML in 9 patients and during acceleration phase in 1. Two patients received therapy with donor lymphocytes during relapse after transplantation. Time course of chimerism and minimum residual illness was studied by standard cytogenetic methods, fluorescent in situ hybridization (FISH) with DNA probes to centromer sites of X and Y chromosomes and BCR and ABL genes. The studies were carried out 30, 60, 90, 180 days, 9 months, 1 year, and then every 6 months after transplantation. RESULTS: Mixed chimerism was observed in all patients during 9 months after TBM. The count of host cells was 0.1-5.8% in 8 patients; later the count of autologous cells was less than 1% in 5 patients, and in 3 patients complete donor chimerism was observed. Clinical hematological remission was stable in these patients. Relapses of leukemia with 40 and 83.1% host cells occurred in 2 patients 13 and 23 months after transplantation, respectively. Donor lymphocytes were transfused in order to induce the graft versus host effect, and in patient No. 2 restoration of donor hemopoiesis was attained. CONCLUSION: Highly sensitive FISH method with DNA probe to centromer sites of X and Y chromosomes detects early relapse of the disease and demonstrates the time course of donor hemopoiesis recovery after transfusion of donor lymphocytes. The data indicate that 9 months after transplantation molecular cytogenetic studies should be carried out more often (once a month), particularly in patients with poor prognosis, for earlier detection of the relapse and beginning of immunotherapy.

Adult↗
PubMed1999

[Construction of anti-human VEGF165 chimeric antibodies and expression in eukaryotic cells].

OBJECTIVE: To express chimeric anti-VEGF165 antibody genes in eukaryotic cells. METHODS: The variable region genes of light and heavy chains from mouse anti-human VEGF165 monoclonal antibody VmD11 were cloned into eukaryotic expression vectors and transfected into dihydrofolate reductase-deficient Chinese hamster ovary(CHO-dhfr-) cells to express chimeric antibody. The antibody expressed was examined for the presence of human constant regions and specificity against human VEGF165. RESULTS: The chimeric antibody with human constant regions and specificity against human VEGF was detected in the culture supernatant of transfected CHO cells by ELISA and Western blot. The chimeric antibody gene was also detected at mRNA level by RT-PCR. CONCLUSION: The mouse anti-human VEGF165 monoclonal antibody is successfully humanized and expressed in eukaryotic cells.

Animals↗
PubMed2001

[Construction and characterization of the chimeric protein consisting RGD-containing peptide and proUK].

On the basis of structure analysis and computer modeling of proUK, two-chain DNA fragment encoding RGD peptide was inserted into the corresponding proUK cDNA site between Gly118-Leu119, using site-directed mutagenesis and DNA recombinant techniques. The chimeric gene was expressed in methylotrophic yeast Pichia pastoris expression system. The chimeric protein was purified after two step purification of Zn2+ chelating column and SP cation exchange column. The specific activity was 65,000 IU/mg protein. The chimeric protein had somewhat lower catalytic efficiency (kcat/Km) on the substrate S2444 as compared to Urokinase. But it had high anti-platelet aggregation activity, and the half inhibit constant was 2.1 mumol/L. The results showed that the chimeric protein not only had higher thrombolytic activity but also obtained anti-thrombus function. Further evaluation of the thrombolytic and antithrombolytic potential in appropriate animal models seemed to be investigated.

Amino Acid Sequence↗
PubMedDate not supplied

[Perception of chimeric faces in schizophrenia and right hemispheric dysfunction].

The aim of this study was the assessment of cerebral specialisation in perception of emotional chimeric drawings in 50 non-chronic schizophrenics (S), 50 chronic schizophrenics (CS), 30 right brain-damaged inpatients (P), and 50 normal controls. All were marked right handers. The assessment was performed after a four-week treatment. Structure and intensity of schizophrenia symptomatology were scored on the PANSS scale. Happy-sad chimeric face drawings (David 1989) were viewed twice in free vision. A perceiver bias towards left hemiface of chimeric drawings (LHF bias) and sad bias were scored in all subjects. Subjects rated their mood at the time of testing on a visual analogue scale. The schizophrenics and right brain-damaged inpatients showed significantly weaker LHF bias compared with healthy subjects, which may suggest right cerebral hemisphere dysfunction in perception of emotional chimeric drawings. Moreover, chronic schizophrenic patients revealed significantly weaker LHF bias and sad bias compared to non-chronic subjects. There was no correlation of left perceptual bias with clinical ratings: PANSS scale, MMSE, number of hospitalisations, neuroleptic dose, and current mood, which suggests stable properties of the perceptual deficit.

Adult↗
PubMed1996

Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Human Activated Platelets.

SZ-51, a murine monoclonal antibody (McAb) specific for alpha-granule membrane protein (GMP-140) on the surface of the activated human platelets, has shown promise for thrombus imaging and thrombolysis. In order to reduce the immunogenicity of the murine McAb SZ-51 in man and to obtain a high level of antibody production, we constructed two chimeras (alpha-Lys17-51BVK/Hu, alpha-Lys30-51VH/Hu) by joining the variable regions gene of mouse antibody to the constant regions gene of human immunoglobulin (Ig)(gamma1,k). Both chimeric genes were cloned into two selectable expression vectors separately, which were co-transfected into a non-Ig secreting murine myeloma cell line SP2/0 with the Lipofectin reagent. One transfectoma, which showed stable antigen (GMP-140) binding ability and a high level expression of 5 mg/L, was obtained. Immunoblotting analysis demonstrated that the chimeric antibody in the supernatant, like the native mouse SZ-51, had the characteristic of binding to GMP-140. In addition, the chimeric antibody can bind competitively to activated platelets with (125)I-labeled mouse SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for the diagnosis and therapy of thrombotic diseases in future.

Journal Article↗
PubMed2002

[Persistent T-cell mixed chimerism in a case of malignant lymphoma after nonmyeloablative allogeneic peripheral blood stem cell transplantation].

We describe a 51-year-old woman with recurrent follicular lymphoma from the age of 47 despite chemo-radio therapy, who subsequently underwent nonmyeloablative stem cell transplantation with conditioning consisting of fludarabine and low-dose total body irradiation (2 Gy). Myelosuppression was very mild, so the patient required no transfusions. Chimerism analysis from peripheral blood showed that T-cell mixed chimerism continued over 12 months after stem cell transplantation (the percentage of recipient T-cells was approximately 20%). Despite this, the lymphadenopathy disappeared, and the patient developed grade II acute GVHD (graft versus host disease). It has been considered that the establishment of full donor chimerism is required to induce GVHD and GVM (graft versus malignancy) effects. In this case, however, an allo-response was observed despite the persistence of T-cell mixed chimerism.

Female↗
PubMed2002

Chimeric Newcastle disease virus nucleocapsid with parts of viral hemagglutinin-neuraminidase and fusion proteins.

The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles. Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer. These chimeric proteins were expressed efficiently in soluble form in E. coli as detected by Western blot analysis. Electron microscopy of the purified products revealed that they self-assembled into ring-like particles. These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles. Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.

Animals↗
PubMed2001

[Purification and immunological characterization of hepatitis E virus recombinant chimeric antigen encoded by ORF2 fragments and ORF3].

OBJECTIVE: To study immunological characteristics of recombinant chimeric HEV antigen. METHODS: Constructed recombinant plasmids pThioHisORF(2.1 + 2.2 + 3), which contains three HEV antigen gene fragments (ORF2.1:6287-6403nt, ORF2.2:6743-7126nt, ORF3), was transformed into E. coli and induced with IPTG. Expressed product P(2.1 + 2.2 + 3) existed in inclusion bodies, was purified by denature SP Sepharose FF cation exchange chromatography. Rabbits and rats were immunized with renatured P(2.1 + 2.2 + 3). The level of IgG in sera from experimental animals and clinical patients were examined with P(2.1 + 2.2 + 3) by ELISA. The characteristics of IgG of immunized animals interacted with recombinant antigen expressed by baculovirus system as well as recombinant chimeric antigen interacted with clinical patients sera were evaluated by Western-blotting. RESULTS: High titer of IgG antibodies, 1:25,600 in rabbits and 1:12,800 in rats, were detected after immunized with P(2.1 + 2.2 + 3). Furthermore, recombinant antigen expressed by baculovirus system was specifically recognized by IgG of experimental animal immunized with P(2.1 + 2.2 + 3), and the purified recombinant chimeric antigen P(2.1 + 2.2 + 3) was specifically reacted with the IgG of clinical patients. CONCLUSIONS: Recombinant chimeric antigen appears a promising strategy for detection of and prevention from HEV infection.

Animals↗
PubMed1992

Unidirectional distribution of mosaicism in chimeric rats.

Experimental rat chimeras were produced by aggregation of eight-cell embryos from two inbred strains, ACI/Hkm and WKAH/Hkm, which differ from each other in their major histocompatibility complexes and coat colors, and their mosaicism was analyzed. The existence of the isozyme Es-1, a serum cholinesterase specifically produced by WKAH-derived cells, and the agouti coat color due to ACI cells, indicated that all of the rats analyzed were unequivocal chimeras. The proportion of ACI cells in the red blood cell populations of the chimeras varied from 45% to 98%, as determined with a fluorescence-activated cell sorter and a monoclonal antibody against class I (RT1) antigen. Digital analysis of the coat color revealed that the proportion of the ACI type of coat color ranged from 72% to 98% in these chimeric rats. Each phenotype expressed in the coat color was complex and varied in size. The ratios of red blood cells and the coat color inclined toward the ACI type of cell population. Conversely, the rate of the WKAH-cell-type population was less than 50%. A breeding test disclosed chimerism of germ cells in two chimeric rats, and there were more pups with agouti coats than with albino coats. Taken together, it was shown in most of the phenotypes analyzed that the ACI type of cells was predominant in all of the chimeric rats. We discuss the possible causes for this unbalanced distribution in the rats.

Animals↗
PubMed1992

Effect of erythropoietic stress on donor hematopoietic cell expression in chimeric rhesus monkeys transplanted in utero.

We have previously reported the successful development of hematopoietic chimerism after the in utero transplantation of fetal hematopoietic stem cells (HSC) in rhesus monkeys (Macaca mulatta). These animals exhibit sustained engraftment without immunosuppression or graft-versus-host disease (GVHD). To assess the functional response of the donor-derived erythropoietic population, we assayed the relative expression of donor and recipient hematopoietic progenitors in chimeric monkeys before and after anemic stress. Anemia in our chimeric animals resulted in increased erythropoietin (EPO) production comparable to controls. This was accompanied by changes in erythroid progenitor profiles, again similar to controls. Chimeric animals demonstrated normal reticulocytosis and reconstituted their hematocrit after hemorrhage at the same rate as controls. The donor-derived erythropoietic population exhibited normal responses to recipient regulatory signals and did not seem to expand at the expense of other hematopoietic lineages. Thus the proportions of engraftment for the myeloid and erythroid precursors in bone marrow and for blood lymphocytes remained stable. Our results demonstrate that the in utero transplantation of fetal HSC results in stable engraftment of donor erythropoietic progenitors, which appear to be fully integrated within the recipient's regulatory system. The abnormalities reported in the postnatal transplantation setting can then be attributed to immunologic reactions requiring conditioning myeloablative regimens. Fetal transplantation bypasses all these factors.

Anemia↗
PubMed1990

Comparison of CD composition produced by chimeric CGTases.

Using 12 chimeric cyclomaltodextrin glucanotransferases (CGTases) constructed from two genes, the CGTase gene from the alkalophilic Bacillus sp. strain No. 38-2 (CGTase 38-2 gene) and the CGTase gene from the alkalophilic Bacillus sp. strain No. 17-1 (CGTase 17-1 gene), we compared the effect of those chimeric enzymes on cyclodextrin (CD) production, especially on the composition of the CDs produced. It was found that the N-terminal and the C-terminal segments were important for CD production. Chimeric enzymes that contained the N-terminal and the C-terminal segments derived from CGTase 38-2 produced large amounts of CD, and especially a higher proportion of alpha-CD than those of other chimeric enzymes.

Bacillus↗
PubMed2003

Maxizymes and small hairpin-type RNAs that are driven by a tRNA promoter specifically cleave a chimeric gene associated with leukemia in vitro and in vivo.

Many leukemias result from chromosomal translocations that lead to the generation of chimeric oncoproteins. Chimeric gene for the promyelocytic leukemia-retinoic acid receptor alpha (PML-RAR alpha) is the hallmark of acute promyelocytic leukemia. Specific gene silencing of an oncogene is desirable for the treatment of these diseases. We have previously constructed an allosterically controllable ribozyme (designated maxizyme) targeted for bcr-abl chimeric mRNA, whose cleavage activity is induced only in the presence of a specific RNA sequence of interest. It has been demonstrated recently that RNA interference induced by short hairpin-type RNAs provides a powerful method for sequence-specific gene silencing. Here we report that DNA vector-based maxizymes and short hairpin-type RNAs driven by the promoter of a human gene for tRNA(Val) specifically inhibit the expression of the chimeric gene for PML-RAR alpha both in vitro and in vivo. Our findings confirm the potential utility of maxizymes and shRNAs as therapeutic agents.

Base Sequence↗
PubMed1992

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Animals↗
PubMed2004

[The mechanism of tetragametic chimerism in a true hermaphroditism with 46, XX/46 ,XY].

OBJECTIVE: To report a true hermaphroditism due to a teragametic chimerism and to discuss the pathogenesis of tetragametic chimerism. METHODS: Chromosomal analysis and fluorescence in situ hybridization(FISH) were carried out on the lymphocytes from the blood and on the fibroblasts from the cultured skin and on fibroblasts from two different kinds of gonadal tissues of the patient with ambiguous genitalia respectively. Blood groups, human leukocyte antigen (HLA) haplotyping and 77 short tandem repeat (STR) microsatellite markers were tested. The two kinds of tissues in the gonad were detected by histopathological examination. Blood groups, HLA haplotying and 77 STR microsatellite markers parents of the patient's were also analyzed. RESULTS: Either 46,XX or 46,XY karyotype was found in the lymphocytes of the blood and in the fibroblasts of the cultured skin and of the two different kinds of gonadal tissues. Two X chromosome-specific signals or one X and one Y signal were detected in each interphase nucleus by FISH from the lymphocytes of the blood and the fibroblasts of three different tissue cultures. The karyotype of the 46,XY cell line predominated in all cultures except the cultured-fibroblasts from yellow gonadal tissues. STR marker analysis, ABO grouping and HLA study from the patient were identified a single haplotype in the patient from the mother and two different haplotypes from the father. Two kinds of tissues in the gonad were observed by histopathological examination. The yellow tissue was ovary and the white one was testis. CONCLUSIONS: Histopathological examination and chromosomal analysis combined with FISH are very useful methods for the diagnosis of true hermaphroditism. Blood typing, HLA and short tandem repeat microsatellite markers afford strong evidence for confirming tetragametic chimerism. The mechanism of tetragametic chimerism in true hermaphroditism can be explained by a parthenogenetic division of a haploid nucleu into two identical gametes, followed by fertilization with both X and Y spermatozoa and then developed into an organism.

ABO Blood-Group System↗