Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Chimerism”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed2007

In vivo drug metabolism model for human cytochrome P450 enzyme using chimeric mice with humanized liver.

We previously clarified that major human drug metabolizing enzymes were expressed in a chimeric urokinase-type plasminogen activator (uPA)+/+/severe combined immunodeficient (SCID) mouse line established recently, in which the liver could be replaced by more than 80% with human hepatocytes. In the present study, we investigated the in vivo drug metabolism of a CYP2D6 substrate, debrisoquin (DB), in chimeric mice with high (High) or low (Low) human albumin (hAlb) concentrations and in control uPA-/-/SCID mice. The hAlb in the mouse blood is one of the indices of humanized liver because the chimeric mice produce hAlb. After oral administration of DB at 2.0 mg/kg, the AUC0-8 value of a major CYP2D6 metabolite of DB, 4'-hydroxydebrisoquin (4-OH DB), in High was 3.6-fold higher than those of Low and uPA-/-/SCID mice. By pre-treatment with a typical CYP2D6 inhibitor, quinidine, the AUC0-8 value of 4-OH DB in High was decreased although such values in Low and uPA-/-/SCID mice did not change. The in vitro kinetic analyses and the Ki values of quinidine on the DB 4'-hydroxylase activity in liver microsomes also supported the humanization of the chimeric mice. In conclusion, the chimeric mice exhibited a humanized profile of drug metabolism and the inhibition of P450.

Adrenergic Agents↗
PubMed1991

Cryopreservation of mouse half-morulae and chimeric embryos by vitrification.

Mouse half-morulae were cryopreserved less than or equal to 1, 3, 6, and 12 hr after bisection by the vitrification method using 25% glycerol and 25% 1,2-propanediol as cryoprotectant. The developmental rates of the frozen-thawed half-embryos to blastocysts in vitro were 77.8% (63/81), 82.0% (41/50), 92.1% (117/127), and 0% (0/37), respectively. Sixty-one of the half-embryos that had been vitrified 6 hr after the bisection followed by transfer to five recipients resulted in a total of ten (16.4%) normal fetuses. Chimeric mouse embryos constructed by two half-morulae were also vitrified 6 and 16 hr after aggregation. Survivors were obtained from the former case: 40 (80.0%) of 50 frozen-thawed embryos developed in vitro to blastocysts, and, after transfer, six chimeric offspring were obtained from the 34 vitrified chimeric embryos. These results showed that mouse half-morulae and chimeric embryos could be cryopreserved by the vitrification method. It seems possible to manufacture a chimeric mouse embryo of defined genotypic composition that can be analyzed during its frozen state using the identical half-embryos of the components.

Animals↗
PubMed1993

Quantitative estimation of chimerism in mice using microsatellite markers.

An embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ-line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring. The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample. Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice.

Animals↗
PubMed1988

Chimerization of highly asynchronous murine blastomeres: developmental alteration?

Previous studies have shown that early embryos contain information that can alter the developmental fate of adjacent cells and transferred nuclei. In this report we show that a specific combination of cells from early murine embryos, a single blastomere from an eight-cell embryo placed under the zona pellucida with a two-cell embryo, results in a difference in incorporation of 3H-uridine and expression of two protein bands between the chimeric treatment group and the nonchimeric controls, a single blastomere from an eight-cell embryo in a separate zona pellucida and a two-cell embryo. The incorporation of 3H-uridine in the chimeric group and nonchimeric control group was significantly different at 45 hours after chimerization (P less than .02). A stage-specific protein band (52k) on a polyacrylamide gel detected with fluorography was found to be qualitatively different (present more often; P less than .01) and another stage-specific protein band (48k) was found to be quantitatively different (more protein; P = .07) in the chimeric treatment vs. the nonchimeric controls at 45 hours after chimerization. These results suggest communication between the cells resulting in a change in their incorporation of uridine and protein synthetic profiles.

Animals↗
PubMed1988

Production of chimeric rabbits from morulae by a simple procedure.

Experiments were conducted to develop a simple and reliable technique to produce chimeric rabbits from morula stage embryos. In Experiments 1 and 2, an in-vitro test of viability was initially performed by culturing embryos to the blastocyst stage. Ninety-three percent of the "chimeric" embryos developed to the blastocyst stage compared to 94% for controls when embryos were manipulated soon after collection (Exp. 1). Eight-one percent chimeric embryos and 78% control embryos developed to blastocyst stage when embryos were held at room temperature for 4 hr (Exp. 2). In Experiment 3, enough morula-stage embryos were available from true breeding Dutch-belted and albino rabbits to form potentially 67 diverse "color" pairs. These micromanipulated pairs of morulae were successfully combined to produce 64 chimeric embryos (96%, 64/67). They were transferred to the uteri of seven recipient does and three became pregnant producing 13 young. Four of the young exhibited substantial overt chimerism (31%) and one more was a possible chimera.

Animals↗
PubMed1996

A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface.

3-Isopropylmalate Dehydrogenase↗
PubMed1993

The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.

A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees C), ranges from 0.29 x 10(7) M-1s-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) to 1.08 x 10(7) M-1s-1 (tcr-[KuPA-->K4HPgK5HPg]uPA), for the tcr-chimeric variants. Neither wtr-uPA nor any of its chimeric r-variants interacted macroscopically with a fibrin clot under conditions that allowed binding of 74% of single-chain r-tissue-type plasminogen activator. However, the tcr-chimeric uPA variants provided HPg-enriched clot lysis times between 0.2 (r-[KuPA-->K1HPgK4HPg]uPA) and 2.4 (r-[KuPA-->K2HPgK3HPg]uPA) relative to that of wtr-uPA.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminocaproic Acid↗
PubMed2000

Inhibition of HIV-1 replication by a new type of circular dumbbell RNA/DNA chimeric oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters," containing two alkyl loop structures with RNA/DNA base pairs [sense (RNA) and antisense (DNA)] in the double helical stem. The reaction of nicked (NDRDON-gag-AUG) and circular (CDRDON-gag-AUG) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA. The circular dumbbell RNA/DNA chimeric oligoncleotide showed more nuclease resistance and cellular uptake than the linear antisense phosphorothioate oligodeoxynucleotide (S-ODN-gag-AUG) and nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON-gag-AUG with an AUG initiation codon sequence, as the target of the HIV-1 gag-gene (779-801), was synthesized and tested for inhibitory effects using peripheral blood mononuclear cells. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON-gag-AUG) showed highly inhibitory effects compared to the antisense phosphorothioate oligonucleotide (S-ODN-gag-AUG), indicating sequence-specific inhibition of HIV-1 replication without the inhibition of reverse transcriptase and/or the viral entry process such as antisense phosphorothioate oligonucleotides.

Base Sequence↗
PubMed2001

Chimeric synthetic peptides containing two immunodominant epitopes from the envelope gp46 and the transmembrane gp21 glycoproteins of HTLV-I virus.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-I virus were synthesized. Monomeric peptides P7 and P8 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P7 is a gp21 (374-400) sequence and the peptide P8 is a gp46 (190-207) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P7-GG-P8 and P8-GG-P7), separated by two glycine residues as spacer arms. The antigenic activity of these peptides were evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-I-positive sera (n = 22), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-II-positive sera (n = 11), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and monomeric peptides together. The chimeric peptide P7-GG-P8 proved to be the most reactive with anti-HTLV-I-positive sera. These results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-I diagnostics.

Amino Acid Sequence↗
PubMed2001

Chimeric synthetic peptides from the envelope (gp46) and the transmembrane (gp21) glycoproteins for the detection of antibodies to human T-cell leukemia virus type II.

Two chimeric synthetic peptides incorporating immunodominant sequences from HTLV-II virus were synthesized. Monomeric peptides P2 and P3 represent sequences from transmembrane protein (gp21) and envelope protein (gp46) of the virus. The peptide P2 is a gp21 (370-396) sequence and the peptide P3 is a gp46 (178-205) sequence. Those peptides were arranged in a way that permits one to obtain different combinations of chimeric peptides (P2-GG-P3 and P3-GG-P2), separated by two glycine residues as spacer arms. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels anti-HTLV-II-positive sera (n = 11), anti-HTLV-I/II-positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 2), and anti-HTLV-I-positive sera (n = 22), while specificity was evaluated with anti-HIV-positive samples (n = 19) and samples from healthy blood donors (n = 30). The efficacy of the chimeric peptides in solid-phase immunoassays was compared with the monomeric peptides and a mixture of the monomeric peptides. Higher sensitivity was observed for chimeric peptide Q5 assay. Those results may be related to a higher peptide adsorption capacity to the solid surface and for epitope accessibility to the antibodies. This chimeric peptide would be very useful for HTLV-II diagnostic.

Amino Acid Sequence↗
PubMed1994

Chimeric parvovirus B19 capsids for the presentation of foreign epitopes.

Chimeric proteins consisting of the VP2 capsid protein of human parvovirus B19 and defined linear epitopes from human herpes simplex virus type 1 and mouse hepatitis virus A59 inserted at the N-terminus and at a predicted surface region were expressed by recombinant baculoviruses. The chimeric proteins expressed the inserted epitopes and assembled into empty capsids. Immunoelectron microscopy indicated that the epitopes inserted in the loop were exposed on the surface of the chimeric particles. The chimeric capsids were immunogenic in mice and antibodies specific for the inserted sequences were induced. In the case of MHV, antibodies were produced that recognized the epitope in the context of native virus. Mice immunized with the chimeric capsids were partially protected against a lethal challenge infection with either MHV or HSV.

Amino Acid Sequence↗
PubMed1997

Increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into Pr55gag virus-like particles by an Epstein-Barr virus gp220/350-derived transmembrane domain.

Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.

Amino Acid Sequence↗
PubMed1995

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.

Animals↗
PubMed1995

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays.

The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')2 (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3 mg). Serum samples were taken before injection and 2-3 and 6-14 weeks after administration. A double-antigen or bridging assay was developed to detect responses against both murine as well as chimeric antibodies. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) as well as three commercially available assays were used to study antibody response against the murine antibody OV-TL 3. With both the double-antigen (bridging) assay and the indirect ELISA 1 of the 28 patients (4%) injected with murine OV-TL 3 F(ab')2 showed a HAMA reaction 6 weeks after injection, which was demonstrated to be a mixed anti-isotypic and anti-idiotypic response. None of the 24 patients injected with the chimeric MOv18 showed an anti-chimeric antibody response. The various commercially available assays demonstrated conflicting results. The double-antigen- or bridging assay is a reliable method to detect anti-murine and anti-chimeric antibodies. The assay can be easily adapted for use with human antibodies. The immunogenicity of OV-TL 3 F(ab')2 and cMOv18 in patients is low, making both antibodies candidates for immunotherapy.

Adult↗
PubMed1994

Differences in biodistribution of the anti-(carcinoembryonic antigen) murine monoclonal antibody CE-25, its F(ab')2 fragment and its intact mainly human chimeric form CE 4-8-13. Dependence on tumour size and amount of antibody injected.

The effect of the size of the tumour and the amount of antibody injected on the biodistribution of a family of radioiodinated antibodies was studied. The intact mouse anti-(carcinoembryonic antigen) (anti-CEA) monoclonal antibody CE-25, its F(ab')2 fragment and the intact human-mouse chimeric from CE 4-8-13 were evaluated in a model system using the human CEA-producing colon xenograft T 380 grown in nude mice. The relative retention (the percentage of the injected dose per gram of tissue), of mouse mAb and F(ab')2 in tumour and most normal tissues 1 day after injection was independent of the antibody dose; after 4 days the mAb values increased with increasing antibody dose. The relative retention of chimeric mAb increased with increasing antibody dose 1 day after injection and also slightly after 4 days. The relative retention in tumour tissue was lower in bigger xenografts for all antibodies. The relative retention of mouse mAb in small tumours increased from day 1 to day 4; for chimeric mAb this value decreased. In normal tissues the relative retention of mouse mAb decreased from day 1 to day 4, but the relative retention of chimeric mAb in normal tissue dropped rapidly and changed little afterwards. Thus the biokinetics of antibodies is "species"-dependent: foreign, mainly human, chimeric antibody clears faster from normal mouse tissue than mouse antibody and reaches lower concentrations.

Animals↗
PubMed1991

Detection of engraftment and mixed chimerism following bone marrow transplantation using PCR amplification of a highly variable region-variable number of tandem repeats (VNTR) in the von Willebrand factor gene.

Detection of host cells in peripheral blood and/or bone marrow (mixed chimerism) of patients who have undergone bone marrow transplantation (BMT) is possible using either immunological methods or cytogenetic or molecular genetic analysis. We shall report a new method for the detection of mixed chimerism, which makes use of the fact, that the von Willebrand factor (vWF) gene has a highly variable region-variable number of tandem repeats (VNTR)--within intron 40. vWF-VNTR amplification by the polymerase chain reaction (PCR) was performed as described by Peake et al. We have studied 185 peripheral blood and/or bone marrow samples of 26 patients. Median time after BMT was 14 months (range 1-83 months). Of the 11 patients who were studied sequentially during the first 100 days following BMT, mixed chimerism was detected in four, but only transiently. None of these patients has relapsed so far. Of 18 patients who were studied more than 100 days after BMT mixed chimerism was found in three; two of these patients have subsequently relapsed. The advantages of this new method are: (a) it is informative in a high percentage of patients; (b) it requires only small amounts (200 microliters) of peripheral blood; (c) reliable results can be obtained at leukocyte counts of even less than 50 per microliters. The clinical relevance and sensitivity of the method compared with established methods for detection of mixed chimerism remain to be determined.

Bone Marrow↗
PubMed2002

Real-time PCR for monitoring minimal residual disease and chimerism in patients after allogeneic transplantation.

Real-time PCR is a new fluorometric method for cycle-to-cycle quantification of PCR product growth rates. The real-time PCR method is fast and associated with a high reproducibility rate. It is used more often for monitoring MRD and chimerism in patients after allogeneic stem cell transplantation (SCT). There are real-time PCR methods for patients with CML, AML and ALL patients with inv(16), t(8;21), t(15;17); t(1;19) and other chromosomal aberrations. For patients with AML monitoring MRD is useful to identify patients who were at high risk for relapse after receiving chemotherapy. In patients with CML monitoring MRD might be helpful to assess success of after allogeneic SCT, or response to therapies with interferon alfa or STI 571. We found, that it is possible to estimate the relapse stage in CML after SCT by the amount of bcr-abl fusion transcript detected using a real-time PCR method. The median measured bcr-abl amount differ significantly (P<0.001) between the various stages, which has relevant clinical implications because it enables early therapeutic decisions in relapsing patients after transplant as e.g. the application of DLI to induce graft-versus-leukemia effects. Using real-time PCR it is possible to detect differences at alleles between recipient and donor at a single nucleotide basis (SNP) for chimerism analysis. The real-time PCR method enables to achieve a high a sensitivity of up to 1x10(-4), which is much more sensitive than all other chimerism methods including VNTR-PCR, STR-PCR. Furthermore, chimerism in male recipients with a female donor can be monitored also by detecting y-chromosome specific sequences by real-time PCR after transplant, which might be the most sensitive method to detect host type gene sequences. All in all, new real-time PCR methods offer a fast, reliable and very sensitive method to evaluate MRD and chimerism in patients after allogeneic SCT and therefore, to help to identify patients who are at high risk for leukemic relapse.

Hematopoietic Stem Cell Transplantation↗
PubMed2004

Lipid-membrane affinity of chimeric metal-binding green fluorescent protein.

The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.

Amino Acid Sequence↗