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PubMed2000

Early detection of relapse and evaluation of treatment for mixed chimerism using fluorescence in situ hybridization following allogeneic hematopoietic cell transplant for hematological malignancies.

In order to detect chimerism, fluorescence in situ hybridization (FISH) and cytogenetic analyses were performed on bone marrow cells from 47 patients with hematological malignancies following allogeneic hematopoietic cell transplant (HCT). The dual-color XY, major Bcr-Abl (M-Bcr-Abl), and specific alpha-satellite probes were used for sex-mismatched HCT, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS) cases with karyotypic abnormalities before HCT, respectively. Donor cells were found using FISH analysis in all 32 cases examined within 2 months following HCT, confirming engraftment. In six cases, however, cytogenetic analysis failed to detect donor cells due to lack of metaphases. Relapse occurred in four of the six cases in which mixed chimerism was detected using FISH analysis after 6 months of HCT. In contrast, after 12 months of HCT, no relapse was found in 24 patients without host cells. For two patients with mixed chimerism, gradual reduction of immunosuppressants or donor lymphocyte infusion resulted in the disappearance of host cells as analyzed using FISH analysis. In three extramedullary relapse cases, however, cytogenetic relapse preceded morphological and FISH relapse. These findings suggest that FISH analysis is more useful for detecting residual host cells after HCT, and the combination of FISH and cytogenetic analyses provide a more detailed evaluation for HCT patients. The results also indicate that monitoring of mixed chimerism using FISH analysis after 6 months of HCT is important for allowing the early detection of hematological relapse.

Female↗
PubMed2002

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

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed "apparent gene conversion," from the neighboring CYP21Ppseudogene. A chimeric CYP21P/CYP21gene with its 5' end corresponding to CYP21P and 3' end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with -300 nucleotides of the 5' head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21Pand CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes.

Adrenal Hyperplasia, Congenital↗
PubMed1995

Perioperative donor bone marrow infusion augments chimerism in heart and lung transplant recipients.

BACKGROUND: We and others have demonstrated that a low level of donor cell chimerism was present for years after transplantation in tissues and peripheral blood of heart and lung recipients; it was associated, in the latter, with a lower incidence of chronic rejection. To augment this phenomenon, we initiated a trial combining simultaneous infusion of donor bone marrow with heart or lung allotransplantation. METHODS: Between September 1993 and January 1995, 15 nonconditioned patients received either heart (n = 10) or lung (n = 5) allografts concurrently with an infusion of unmodified donor bone marrow (3.0 x 10(8) cells/kg), and were maintained on immunosuppressive regimen consisting of tacrolimus and steroids. RESULTS: There was no complication associated with the infusion of donor bone marrow. Chimerism was detectable in 73% of bone marrow-augmented patients up to the last sample tested. Of the 5 control recipients who did not receive bone marrow infusion, only 1 had detectable chimerism by flow on postoperative day 15, which dwindled to an undetectable level by postoperative day 36. None of the patients had evidence of donor-specific immune modulation by mixed lymphocyte reaction. CONCLUSIONS: The combined infusion of donor bone marrow and heart or lung transplantation, without preconditioning of the recipient, is safe and is associated with an augmentation of donor cell chimerism.

Adult↗
PubMed1989

Convenient plasmid vectors for construction of chimeric mouse/human antibodies.

Chimeric antibodies composed of mouse-derived variable regions and human-derived constant regions have been developed for clinical use. However, construction of chimeric mouse/human genes in expression vectors is time-consuming work. In this study, we developed convenient vectors for construction of chimeric mouse/human antibodies. The protocols are as follows: In mouse hybridomas and B cells, most active VH and V kappa genes can be identified as rearranged bands by Southern hybridization of EcoRI- and HindIII-digested DNAs with JH and J kappa probes, respectively, and such fragments can be isolated in lambda-EcoRI and lambda-HindIII vectors, respectively. We constructed two plasmids: pSV2-HG 1 gpt contains human C gamma 1 and Ecogpt genes, and only one EcoRI site upstream of the C gamma 1 gene; pSV2-HC kappa neo contains human C kappa and neo genes, and only one HindIII site upstream of the C kappa gene. An isolated EcoRI fragment containing a VHDHJH gene and a HindIII fragment containing a V kappa J kappa gene are inserted into pSV2-HC kappa neo, respectively. Both resulting plasmid DNAs are co-transfected into SP2/0 cell, a non-Ig-secreting mouse myeloma. Transformants are selected by both mycophenolic acid and G418. With this procedure, it takes only 2 months to obtain chimeric antibodies.

Animals↗
PubMed1990

Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis.

To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.

Amino Acid Sequence↗
PubMed1991

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase.

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.

ADP Ribose Transferases↗
PubMed1993

Reactions of sera from laboratory, domestic and wild animals in Africa with protein A and a recombinant chimeric protein AG.

An ELISA was developed to determine the reactivity of peroxidase labelled Protein A and a recombinant Protein A + Protein G construct, to sera from a variety of laboratory, domestic and wild animals from Africa. There was variability in the binding capacity of sera from individuals of the same species, but four groups could be recognized. Sera from birds and crocodiles were at most weakly reactive with either Protein A or the chimeric construct. Sera from some domestic animals such as horse, goat and cat, and sera from some wild ungulates including buffalo, wildebeest, waterbuck and impala were reactive with Protein A, but reacted to a much greater degree with the chimeric construct. Sera from larger wild animals such as elephant, rhinoceros and giraffe were strongly reactive with the chimeric protein and moderately reactive with Protein A. Sera from primates and dog, pig, guinea pig and rabbit reacted strongly with both proteins. Chimeric proteins that combine the IgG binding capacities of Protein A and Protein G can be used to detect immunoglobulin from a wide variety of African wild animal species. They may thus be of great value in seroepidemiological investigations of these animal populations.

Africa↗
PubMed1990

Production of murine V-human Cr1 chimeric anti-TAG72 antibody using V region cDNA amplified by PCR.

A mouse/human chimeric B72.3-1-3 antibody was produced by construction of a novel expression vector mpSV2neo-EP1-V-Cr1. This vector contains the neo gene as a selection marker, the murine immunoglobulin heavy chain promoter and enhancer, the murine V region cDNA containing mRNA splicing joint sequences, amplified and cloned by the PCR technique directly from the B72.3 hybridoma RNA, and the human genomic Cr1 region. The expression vector containing the murine/human chimeric immunoglobulin heavy chain gene was transfected into heavy chain loss mutant cell line, B72.3Ml. Chimeric B72.3-1-3 antibody was produced at 2 micrograms/ml and retained full binding reactivity to TAG72 compared to the murine B72.3 parental antibody. Using this method, chimeric immunoglobulin molecules can be produced rapidly in comparison with the cDNA and genomic cloning techniques.

Amino Acid Sequence↗
PubMed1994

Chimeric immunoglobulin light chains are secreted at different levels: influence of framework-1 amino acids.

Immunoglobulin light chain proteins are generally thought to be readily secreted without their corresponding heavy chains; non-secreted light chains have been viewed as aberrant forms. We have re-examined this assumption by expressing chimeric mouse-human light chains constructed for 12 mouse antibodies (mouse variable regions fused to a human kappa light chain constant region) in Sp2/0 and CHO cells. Five of the 12 light chains were either poorly secreted or not secreted at all. There was approximately a five-fold difference in the levels of secreted light chain between the highest poor secretor and the lowest good secretor. All of these light chains formed functional chimeric IgGs, which were secreted at similar levels, when co-expressed with their respective chimeric mouse-human heavy chains (mouse variable regions fused to a human gamma-1 heavy chain constant region). The influence of variable region amino acids on light chain secretion was examined by replacing the Framework-1 region of three poorly-secreted chimeric light chains with that of a readily-secreted light chain. For two of the light chains, secretion levels increased approximately 30- and 100-fold relative to that of the unmodified light chains. Comparison of the Framework-I amino acid sequence of the poorly- and readily-secreted light chains revealed an asparagine (N) and proline (P) at positions 11 and 12, respectively of these poorly-secreted light chains and a leucine (L) and serine (S) in the same region for some of the readily secreted light chains. Alteration of the NP to LS for one of the poorly-secreted light chains resulted in an approximately seven-fold increase in light chain secretion over that of the native form of the poorly-secreted light chain. We conclude from these studies that poor secretion can be a naturally occurring state for normal light chains and that amino acids within Framework-1 contribute to poor secretion for some of the light chains.

Amino Acid Sequence↗
PubMed1994

Restoration of CD2-mediated signaling by a chimeric membrane protein including the cytoplasmic sequence of CD45.

We showed previously that the TCR and CD2 fail to couple efficiently with their signal transduction machinery in J45.01, a CD45-deficient variant of the Jurkat T-cell line. Transfection into J45.01 of a cDNA encoding a chimeric membrane protein containing the cytoplasmic sequence of CD45 and extracellular and transmembrane sequences derived from the A2 allele of MHC class I rescues proximal signaling events after TCR stimulation. In this report, we describe rescue of CD2-mediated signaling and evaluate further the characteristics of TCR signaling in J45.01 after expression of the chimeric protein. Cells expressing the chimeric molecule demonstrate TCR- and CD2-mediated increases in PTK activity and PI turnover. Stimulation of the TCR and CD2 on the transfected cells also results in the expression of CD69 on the cell surface, a more distal signaling event. Although these measures of signal transduction via the TCR and CD2 are restored in the transfected cells, the magnitude of the responses are less than those seen in the wild-type Jurkat cells. These findings demonstrate that the cytoplasmic domain of CD45, expressed as a chimeric membrane protein, is sufficient for mediating signal transduction through CD2 as well as through the TCR complex. In addition, these results suggest that the extracellular and/or transmembrane domains of CD45 may contribute to the efficiency of signal transduction.

Antigens, CD↗
PubMed1993

Transgenic mice and transhybridomas producing chimeric mouse/human anti-human interleukin-2 receptor recombinant antibodies.

Transgenic mice were developed that secreted chimeric mouse/human anti-human interleukin-2 receptor (IL-2R) antibodies (Ab) into their serum. In addition, hybridomas producing the chimeric Ab in tissue culture were generated from the transgenic mice. The presence of the mouse/human immunoglobulin (Ig) transgene did not appear to affect rearrangement of endogenous murine Ig in the hybridomas. Serum levels of the chimeric Ab correlated with transgene copy number. Although many of the transgenic lineages had serum titers of the chimeric Ab comparable to endogenous mouse IgG, there was no apparent correlation with endogenous mouse IgG levels.

Animals↗
PubMed1995

Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase beta.

A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Pol beta and TdT to investigate the region of Pol beta required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of POL beta. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of POL beta represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Pol beta protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Pol beta are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Pol beta possesses ssDNA-binding activity [Kumar et al., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar et al., Biochemistry 29 (1990b) 7156-7159; Prasad et al., J. Biol. Chem. 268 (1993) 22746-22755].

Amino Acid Sequence↗
PubMedDate not supplied

Structural and functional characterization of the OmpF and OmpC porins of the Escherichia coli outer membrane: studies involving chimeric proteins.

The roles of submolecular regions of OmpF and OmpC, major outer membrane proteins of Escherichia coli, as concerns their biogenesis, structure and function were studied using a large number of chimeric genes constructed from the ompF and ompC genes through single or double homologous in vivo recombination. When recombination between the two genes took place at certain regions of their central regions, no chimeric protein was detected, irrespective of whether the amino-terminal and carboxy-terminal regions were derived from OmpF or OmpC. Biochemical studies revealed that these proteins were synthesized and exported across the cytoplasmic membrane normally, but that they were not properly assembled into the outer membrane and hence were degraded rapidly. Characterization of these chimeric proteins, in which recombination between OmpF and OmpC took place once or twice, suggested that the central region of each of these proteins plays an important role in the respective assembly, whereas the roles of the amino-terminal and carboxy-terminal regions may be marginal. Functional characterization of these chimeric proteins revealed the regions important for the receptor functions of OmpF and OmpC for phages TuIa and TuIb, respectively.

Amino Acid Sequence↗
PubMed1995

A minimal conditioning approach to achieve stable multilineage mouse plus rat chimerism.

Transplantation of untreated rat bone marrow into lethally irradiated (950 cGy) mouse recipients results in durable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent xenografts. The purpose of the present study was to characterize the minimal dose of total body irradiation (TBI) which would allow engraftment of rat bone marrow in mouse recipients. We report here that durable and stable lymphohaematopoietic cross-species chimerism can be achieved using a less than totally ablative radiation-based conditioning approach. The percentage of B10 mouse recipients which engrafted with rat bone marrow cells correlated with the dose of TBI. Engraftment of rat bone marrow stem cells occurred in all animals receiving 750 cGy prior to bone marrow transplantation, while no engraftment was detected at doses less than 650 cGy. Although most of the recipients were repopulated with mixed mouse and rat multilineage chimerism, some exhibited a predominance of rat cells. Although mixed xenogeneic rat/mouse chimeras prepared by lethal TBI produced only mouse derived RBC (red blood cells), chimeras prepared by sublethal conditioning produced both rat and mouse RBC. Only animals with detectable chimerism exhibited specific functional transplantation tolerance to donor xenoantigens, as assessed in vitro by mixed lymphocyte reaction assay. This model may offer an in vivo approach to study the role of species-specific growth factors in stem cell biology as well as the mechanisms for the induction of tolerance across species barriers.

Animals↗
PubMed1989

Modification of strain-specific femoral bone density by bone marrow chimerism in mice: a study on the spontaneously osteoporotic mouse (SAM-P/6).

The role of marrow-derived cells in determining strain differences in femoral bone density in mice was investigated by inducing marrow chimerism in a newly developed, osteoporotic strain of mouse: SAM-P/6. As a normative study prior to the chimeric experiment, bone density of F1 hybrids between SAM-P/6 and SAM-P/2 (strain with a high bone density) was assessed. Microdensitometrically, the F1(P2P6F1) exhibited a significantly higher bone density than did the SAM-P/6. P2P6F1 marrow cells, injected into SAM-P/6 neonates, increased the bone density at age 60 days while the syngeneic cells were without effect. The successful induction and maintenance of chimerism was confirmed by flow cytometry in which a donor-specific H-2K haplotype was used as the marker. This study shows that, at least in certain combinations of strains of mice, strain-specific bone density can be modified by bone marrow chimerism. Also, it suggests that the low bone density observed in the SAM-P/6 might reflect functional characteristics of the marrow-derived cells.

Animals↗
PubMed2004

Addition of treosulfan to a nonmyeloablative conditioning regimen results in enhanced chimerism and immunologic tolerance in an experimental allogeneic bone marrow transplant model.

Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.

Animals↗
PubMed2005

Lack of correlation between an assay used to determine early marrow allograft rejection and long-term chimerism after murine allogeneic bone marrow transplantation: effects of marrow dose.

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unit-granulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2b) mice were lethally irradiated and transplanted with various doses of BALB/c (H2d) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.

Animals↗
PubMed2005

Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy.

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development.

1,2-Dipalmitoylphosphatidylcholine↗