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PubMed2007

A recombinant allergen chimer as novel mucosal vaccine candidate for prevention of multi-sensitivities.

BACKGROUND: As conventional immunotherapy is less efficacious in patients with allergic multi-sensitivities compared with mono-sensitized subjects, new intervention strategies are needed. Therefore, an allergen chimer was genetically engineered for treatment of multi-sensitization with birch and grass pollen on the basis of mucosal tolerance induction. METHODS: The major birch pollen allergen Bet v 1 served as a scaffold for N- and C-terminal linkage of the immunodominant peptides of the grass pollen allergens Phl p 1 and Phl p 5 and this new construct was cloned and expressed in Escherichia coli. After purification, physicochemical and immunological characterization the chimer was used for intranasal tolerance induction prior to poly-sensitization with Bet v 1, Phl p 1 and Phl p 5. RESULTS: The immunological characterization revealed that the conformation of Bet v 1 within the chimer was comparable to that of natural as well as recombinant Bet v 1. The chimer was immunogenic in mice for T and B cell responses to the three allergens. Intranasal application of the chimer prior to poly-sensitization significantly suppressed humoral and cellular allergen-specific Th2 responses and prevented development of airway inflammation upon allergen challenge. Moreover, local allergen-specific IgA antibodies were induced by the chimer. The mechanisms of poly-tolerance induction seemed to be mediated by regulatory cytokines, since TGF-beta and IL-10 mRNA in splenocytes were upregulated and tolerance was transferable with these cells. CONCLUSION: The data indicate that such allergen chimers harboring several unrelated allergens or allergen peptides could serve as mucosal polyvalent vaccines for prevention of multi-sensitivities.

Administration, Intranasal↗
PubMed1998

Demonstration of multilineage chimerism in a nonhuman primate concordant xenograft model.

Prior studies from our laboratory have demonstrated that a nonmyeloablative conditioning regimen can induce transient mixed chimerism and renal allograft tolerance between MHC disparate cynomolgus monkeys. We have also shown that this preparative regimen can be extended to a concordant baboon to cynomolgus xenograft model by adding, to the post transplant protocol, therapy designed to prevent antibody production. Here we examine the use of brequinar (BQR) for this purpose and the efficacy of two new reagents developed to demonstrate the establishment of chimerism in the xenograft recipients. The cynomolgus recipients were conditioned with WBI (300 cGy), TI (700 cGy), ATG, cyclosporine, and brequinar sodium. To detect engraftment of the donor marrow, we prepared a polyclonal cynomolgus anti-baboon antibody (CABA) and a monoclonal antibody (215.1), which distinguish baboon and cynomolgus lymphocytes and granulocytes. We employed flow cytometry analysis to detect multilineage chimerism in the xenograft recipients. Five of the six recipients monitored using our new reagents (CABA and 215.1) developed detectable chimerism and only one of these animals lost its kidney to rejection. However, other complications have not permitted assessment of long-term outcome. The features of the multilineage chimerism included the detection of donor granulocytes (1.8-77.4%) and lymphocytes (2.4-22.2%) for 9 to 37 days. Our new reagents permit the detection of multilineage mixed chimerism, which may be a predictor of xenograft tolerance. We also conclude that brequinar may be effective in preventing antibody formation, but because of its toxicity, it is probably not the drug of choice for extension of the mixed chimerism protocol to concordant xenografts.

Animals↗
PubMed1995

Tyrosine kinase activity of a chimeric insulin-like-growth-factor-1 receptor containing the insulin receptor C-terminal domain. Comparison with the tyrosine kinase activities of the insulin and insulin-like-growth-factor-1 receptors using a cell-free system.

In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the beta subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-P., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449-11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin receptor-(1142-1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor beta subunit plays a key role in regulation of the tyrosine kinase activity. The fine-tuning of the tyrosine kinase by the C-terminal tail could participate in the receptor specificity.

Adenosine Triphosphate↗
PubMed2006

Generation and characterization of a single-gene mouse-human chimeric antibody against hepatitis B surface antigen.

BACKGROUND: Antibody against hepatitis B surface antigen (HBsAg) is used for passive immunotherapy in certain cases of hepatitis B infection. The authors have earlier reported a high-affinity mouse monoclonal (5S) against HBsAg. However, this mouse antibody cannot be used for therapeutic purposes because it may elicit antimouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: A single-chain variable fragment (scFv), derived from the mouse monoclonal 5S, was fused with the fragment crystallisable (Fc) fragment of human IgG1. The scFv region is expected to bind to the antigen, whereas the Fc fragment can provide the effector functions required for virus neutralization. This chimeric molecule was expressed in Chinese hamster ovary (CHO) cells in serum-free medium. It was purified by affinity chromatography and characterized by in vitro binding studies. RESULTS: Purification and characterization indicated that this chimeric scFv-Fc fusion protein is secreted as a disulfide-linked, glycosylated, homodimeric molecule. The yield of the purified chimeric antibody was approximately 4.6 mg/L. In vitro analyses confirmed that this chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. CONCLUSION: Because it is a single-gene product, this chimeric scFv-Fc has the advantage of stable expression. Being chimeric and bivalent, it is expected to be less immunogenic and therefore suitable for further in vivo studies on virus neutralization.

Animals↗
PubMed2006

A SYBR green-based real-time PCR method for detection of haemopoietic chimerism in allogeneic haemopoietic stem cell transplant recipients.

OBJECTIVE: To develop a reliable and rapid real-time quantitative polymerase chain reaction (Q-PCR) method using SYBR green to quantify chimerism in allogeneic haemopoietic stem cell transplant recipients. METHODS: Twelve specific nucleotide polymorphisms (NPs) were selected to identify informative markers for detecting chimerism in transplant donor/recipient pairs. One informative marker was then used in SYBR green Q-PCR to detect chimerism post-transplantation in each patient. The percentage of donor cells was calculated using a standard curve, constructed using artificially mixed donor/recipient chimeric DNA in 12 serial dilutions (0.01-100%). RESULTS: DNA from 37 donor/recipient pairs was screened for informative markers and 18 post-transplant samples were monitored for chimerism with SYBR green Q-PCR method. The Q-PCR was able to discriminate between recipient and donor genetic profiles in all 18 samples, and quantify the chimerism. These results were confirmed by at least one independent method, such as TaqMan Q-PCR, microsatellite and fluorescent in situ hybridisation (FISH) methods. The detection limit of this method was 0.1%, which was more sensitive than the two currently used microsatellite and FISH methods. CONCLUSION: The new single platform SYBR green Q-PCR method is capable of detecting all haemopoietic chimerism with high accuracy; hence, it may be used to replace the current detection methods.

Adult↗
PubMed2002

Chimeric human immunodeficiency virus type 1 and feline immunodeficiency virus reverse transcriptases: role of the subunits in resistance/sensitivity to non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and do not inhibit HIV-2. Given that the amino acids lining the NNRTI-specific pocket of HIV-1 RT display higher similarity to the corresponding feline immunodeficiency virus (FIV) RT amino acids than to HIV-2 RT, the susceptibility of FIV RT and chimeric HIV-1/FIV RTs to NNRTIs and the role of the p51 subunit in the inhibitory action of NNRTIs were investigated. We found that the wild-type FIV RT and the FIVp66/HIVp51 chimeric enzyme showed no susceptibility for NNRTIs. On the other hand, the chimeric HIVp66/FIVp51 RT retained a sensitivity spectrum for NNRTIs similar to that of the wild-type HIV-1 RT. The noncompetitive nature of inhibition of HIV-1 RT by nevirapine was also observed with the HIVp66/FIVp51 chimeric enzyme. Inhibition of the chimeric RTs by nucleoside reverse transcriptase inhibitors and foscarnet was in the same range as observed for the corresponding HIVp66/HIVp51 and FIVp66/FIVp51 wild-type enzymes. The chimeric RTs had an affinity (K(m)) for their dNTP substrate and template/primer comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy (k(cat)) was markedly decreased. This decreased catalytic efficacy of the RT chimeras may suggest suboptimal interactions between p66 and p51 in the chimeric enzymes. Our results point to a minor role of the p51 subunit in the sensitivity to RT inhibitors.

Amino Acid Sequence↗
PubMed1991

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons.

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange of the fiber head domain is a viable approach to the production of adenovirus vectors with cell-type-selective transduction properties. It may be possible to extend this approach to the use of ligands for a range of different cellular receptors in order to target gene transfer to specific cell types at the level of transduction.

Adenoviridae↗
PubMed2004

A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs.

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas PCV1 is nonpathogenic. We previously demonstrated that a chimeric PCV1-2 virus (with the immunogenic capsid gene of PCV2 cloned into the backbone of PCV1) induces an antibody response to the PCV2 capsid protein and is attenuated in pigs. Here, we report that the attenuated chimeric PCV1-2 induces protective immunity to wild-type PCV2 challenge in pigs. A total of 48 specific-pathogen-free piglets were randomly and equally assigned to four groups of 12 pigs each. Pigs in group 1 were vaccinated by intramuscular injection with 200 microg of the chimeric PCV1-2 infectious DNA clone. Pigs in group 2 were vaccinated by intralymphoid injection with 200 microg of a chimeric PCV1-2 infectious DNA clone. Pigs in group 3 were vaccinated by intramuscular injection with 10(3.5) 50% tissue culture infective doses (TCID(50)) of the chimeric PCV1-2 live virus. Pigs in group 4 were not vaccinated and served as controls. By 42 days postvaccination (DPV), the majority of pigs had seroconverted to PCV2 capsid antibody. At 42 DPV, all pigs were challenged intranasally and intramuscularly with 2 x 10(4.5) TCID(50) of a wild-type pathogenic PCV2 virus. By 21 days postchallenge (DPC), 9 out of the 12 group 4 pigs were viremic for PCV2. Vaccinated animals in groups 1 to 3 had no detectable PCV2 viremia after challenge. At 21 DPC the lymph nodes in the nonvaccinated pigs were larger (P < 0.05) than those of vaccinated pigs. The PCV2 genomic copy loads in lymph nodes were reduced (P < 0.0001) in vaccinated pigs. Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs but in only 1 of 36 vaccinated pigs. Mild-to-severe lymphoid depletion and histiocytic replacement were detected in lymphoid tissues in the majority of nonvaccinated group 4 pigs but in only a few vaccinated group 1 to 3 pigs. The data from this study indicated that when given intramuscularly in pigs, the attenuated chimeric PCV1-2 live virus, as well as the chimeric PCV1-2 infectious DNA clone, induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.

Animals↗
PubMed1986

Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice.

Actins↗
PubMed2002

High-level allogeneic chimerism achieved by prenatal tolerance induction and postnatal nonmyeloablative bone marrow transplantation.

Clinical application of allogeneic bone marrow transplantation (BMT) has been limited by toxicity related to cytoreductive conditioning and immune response. In utero hematopoietic stem cell transplantation (IUHSCT) is a nonablative approach that achieves mixed chimerism and donor-specific tolerance but has been limited by minimal engraftment. We hypothesized that mixed chimerism achieved by IUHSCT could be enhanced after birth by nonmyeloablative total body irradiation (TBI) followed by same-donor BMT. To test this hypothesis, mixed chimerism was created by IUHSCT in a major histocompatibility complex-mismatched strain combination. After birth, chimeric animals received nonmyeloablative TBI followed by transplantation of donor congenic bone marrow cells. Our results show that: (1) low-level chimerism after IUHSCT can be enhanced to high-level chimerism by this strategy; (2) enhancement of chimerism is dependent on dose of TBI; (3) the mechanism of TBI enhancement is via a transient competitive advantage for nonirradiated hematopoietic stem cells; (4) engraftment observed in the tolerant, fully allogeneic IUHSC transplant recipient is equivalent to a congenic recipient; and (5) host-reactive donor lymphocytes are deleted with no evidence of graft-versus-host disease. This study supports the concept of prenatal tolerance induction to facilitate nonmyeloablative postnatal strategies for cellular therapy. If clinically applicable, such an approach could dramatically expand the application of IUHSCT.

Animals↗
PubMed2002

A novel rapid single nucleotide polymorphism (SNP)-based method for assessment of hematopoietic chimerism after allogeneic stem cell transplantation.

A major end point of nonmyeloablative hematopoietic stem cell transplantation is the attainment of either mixed chimerism or full donor hematopoiesis. Because the majority of human genetic disparity is generated by single nucleotide polymorphisms (SNPs), direct measurement of SNPs should provide a robust tool for the detection and quantitation of chimerism. Using pyrosequencing, a rapid quantitative sequencing technology, we developed a SNP-based assay for hematopoietic chimerism. Based on 14 SNPs with high allele frequencies, we were able to identify at least 1 informative SNP locus in 55 patients with HLA-identical donors. The median number of informative SNPs in related pairs was 5 and in unrelated pairs was 8 (P <.0001). Assessment of hematopoietic chimerism in posttransplantation DNA was shown to be quantitative, accurate, and highly reproducible. The presence of 5% donor cells was reliably detected in replicate assays. Compared with current measures of engraftment based on identification of short tandem repeats (STRs), variable number of tandem repeats (VNTRs), or microsatellite polymorphisms, this SNP-based method provides a more rapid and quantitative assessment of chimerism. A large panel of SNPs enhances the ability to identify an informative marker in almost all patient/donor pairs and also facilitates the simultaneous use of multiple markers to improve the statistical validity of chimerism measurements. The inclusion of SNPs that encode minor histocompatibility antigens or other genetic polymorphisms that may influence graft-versus-host disease or other transplantation outcomes can provide additional clinically relevant data. SNP-based assessment of chimerism is a promising technique that will assist in the analysis of outcomes following transplantation.

Case-Control Studies↗
PubMed1990

Beta-endorphin chimeric peptides: transport through the blood-brain barrier in vivo and cleavage of disulfide linkage by brain.

Water soluble peptides are normally not transported through the brain capillary wall, i.e. the blood-brain barrier (BBB). Chimeric peptides may be transportable through the BBB and are formed by the covalent coupling of a nontransportable peptide, e.g. beta-endorphin, to a transportable peptide vector, e.g. cationized albumin, using disulfide-based coupling reagents such as N-succinimidyl 3-[2-pyridyldithio(propionate)] (SPDP). The transcytosis of peptide into brain parenchyma, as opposed to vascular sequestration of blood-borne peptide, was quantified using an internal carotid artery perfusion/capillary depletion method. It is shown that [125I]beta-endorphin is not transported through the BBB, but is rapidly cleaved to free [125I] tyrosine via capillary peptidase. Therefore, chimeric peptide was prepared using [125I] [D-Ala2]beta-endorphin (DABE), owing to the resistance of this analogue to peptidase degradation. The [125I] DABE-cationized albumin chimeric peptide is shown to enter brain parenchyma at a rate comparable to that reported previously for unconjugated cationized albumin. When the [125I] DABE-cationized albumin chimeric peptide was incubated with rat brain homogenate at 37 C, the free [125I] DABE was liberated from the cationized albumin conjugate prior to its subsequent degradation into free [125I] tyrosine. Approximately 50% of the chimeric peptide was cleaved within 60 sec of incubation at 37 C. These studies demonstrate that 1) [125I]beta-endorphin is not transported through the BBB in its unconjugated form, 2) a [125I] DABE-cationized albumin chimeric peptide is transported through the BBB into brain parenchyma at a rate comparable to the unconjugated cationized albumin, and 3) brain contains the necessary disulfide reductases for rapid cleavage of the chimeric peptide into free beta-endorphin and this cleavage occurs before degradation of the [125I] DABE into [125I] tyrosine.

Albumins↗
PubMed2003

Diagnostic significance and clinical applications of chimeric genes in Ewing's sarcoma.

Ewing's sarcoma (ES) is one of the most malignant bone and soft tissue tumors in childhood. Morphologically, ES belongs to the small round cell tumors (SRCT). ES, peripheral primitive neuroectodermal tumor (PNET), and Askin's tumor are classified as ES family tumors (ESFT) because they share a common chromosomal translocation. The EWS-FLI1 chimeric gene is generated by t (11; 22). Other reciprocal translocations resulting in formation of chimeric genes between EWS and ETS family genes (ERG, ETV1, E1AF, and FEV) are t (21; 22), t (7; 22), t (17; 22), and t (2; 22), respectively. Although it is generally difficult to distinguish ES from SRCT, we could easily and quickly distinguish ES from other SRCT by using reverse transcription polymerase chain reaction (RT-PCR). We looked for specific chimeric genes in 23 tumor samples, including three ES clinical samples. We detected five chimeric genes in the three ES samples. Three chimeric genes, all EWS-FLI1, were detected in one ES sample. Different chimeric genes, EWS-ERG and EWS-ETV1, were detected in the other two ES samples. Moreover, because we could not detect specific chimeric genes in samples from non-ESFT, it may be possible to use this technique to diagnose ESFT and to detect tumor cell contamination before hematopoietic stem cell transplantation.

Adolescent↗
PubMed2006

Evaluation of human CYP1A2 and CYP3A4 mRNA expression in hepatocytes from chimeric mice with humanized liver.

We investigated and compared the expression of human CYPs mRNA in primary cultures of cryopreserved human hepatocytes and in chimeric mice constructed by transplanting hepatocytes from the same human donors. Analysis was performed by real-time reverse-transcription polymerase chain reaction. Initial expression levels for the 12 human CYPs mRNA in chimeric mouse hepatocytes were higher than those in human hepatocytes, but a low correlation coefficient was observed (r=0.690). After 24 h of culture, the correlation remained low (r=0.699). The medium was replaced with fresh medium without human epidermal growth factor, and after 48 h of culture, expression of the 12 human CYPs mRNA were very similar in human hepatocytes and chimeric mouse hepatocytes, and a higher correlation coefficient was observed (r=0.809). After 72 h of culture, the correlation remained high (r=0.873). The ratio of human CYP1A2 mRNA to beta-actin mRNA in chimeric mouse hepatocytes decreased quickly during the first 24 h of culture, and then remained constant. Expression profiles of human CYP1A2 mRNA in chimeric mouse hepatocytes were similar to those in human hepatocytes after exposure of beta-naphthoflavone. CYP3A4 mRNA expression was increased significantly by rifampicin (Rif) exposure in human hepatocytes, whereas Rif-induced increases in CYP3A4 mRNA expression in chimeric mouse hepatocytes was seen for two of the three donors. In conclusion, we demonstrated that expression and induction of human CYPs in human hepatocytes can be reproduced in chimeric mouse hepatocytes.

Animals↗
PubMed1998

Chimeric anti-TAG72 receptors with immunoglobulin constant Fc domains and gamma or zeta signalling chains.

We recently described the generation and expression of a chimeric T cell receptor with specificity for the tumor antigen TAG72 consisting of the single chain antibody (scFv) B72.3-scFv and the gamma chain of the FcepsilonRI receptor. The corresponding chimeric receptor containing the zeta chain of the TCR as signalling unit is not functionally expressed reflecting that the requirements for functional expression of chimeric receptors containing the gamma signalling chain are apparently different compared to those containing the CD3zeta signalling chain of the TCR. We describe a novel set of chimeric anti-TAG72 receptors including in their extracellular moiety the constant immunoglobulin CH2/3 domains that allow stable expression of chimeric gamma as well as zeta receptors. We designed anti-TAG72 receptors that consist of a scFv fragment derived from an anti-TAG72 second generation antibody (CC49) and of the CH2/3 domains of the human IgG and intracellularily either of the zeta or gamma signalling chain. The recombinant CC49-CH2/3-zeta and CC49-CH2/3-gamma DNA, respectively, was transfected into MD45 T cells and expressed under control of the RSV LTR. Both receptors were found on the cell membrane of transfected cells as demonstrated by flow cytometry analysis using an anti-human IgG Fc antibody directed to the CH2/3 immunoglobulin domains of the chimeric receptor. Specific cross-linking of the chimeric zeta as well as the gamma receptor by antigen or anti-human Ig antibodies resulted in specific activation of transfected cells. Our results demonstrate that both the gamma chain and the zeta chain++ containing receptor are stably expressed and convert T cells to specificity for the TAG72 antigen. This receptor design will facilitate efficient generation of genetically modified peripheral T cells and may provide valuable tools for the cellular immunotherapy of TAG72+ tumors.

Animals↗
PubMed1998

Engraftment and chimerism, particularly of T- and B-cells, in children undergoing allogeneic bone marrow transplantation.

The spectrum of pediatric bone marrow transplantation has changed in recent years. Mismatched and unrelated donor transplants are common, demanding an increased vigilance to detect incipient graft failure, secondary lymphoma as well as relapse and other problems, which now are potentially treatable. To diagnose these complications it may be important to know whether blood and marrow cells are of recipient or donor origin. To evaluate the role of mixed donor-recipient chimerism in relation to clinical problems we adapted a polymerase chain-reaction technique, using fluorescent primers analyzing DNA fragment length polymorphisms, to follow prospectively 17 bone marrow grafted children. To increase the precision of chimerism analysis, immunomagnetically isolated leukocyte populations were assayed in selected cases. Five patients encountered clinical problems related to chimerism. One infant with adenosine deaminase deficiency failed to engraft stem cells, yet succumbed to graft-versus-host disease, mediated by mature donor T-cells. Three children developed significant mixed chimerism. One of these three patients died in relapse of leukemia, while the two other patients who had received T-cell depleted grafts had persistent recipient T-cells, in spite of engraftment. After 5 months, these were displaced by donor T-cells in one of the patients. In the fifth patient, also after T-cell depleted BMT, a fatal donor cell lymphoma occurred. Twelve children had stable full chimerism or in one case a low grade mixed chimerism and remain disease-free throughout follow up (median 9 months). In conclusion, the analysis of chimerism, particularly of separated leukocyte populations, offers an almost indispensable insight and a basis for therapeutic decisions in complicated situations such as grafting involving unrelated or mismatched donors, graft manipulation, adoptive immunotherapy and in immunodeficiency patients.

B-Lymphocytes↗
PubMed1999

Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses.

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.

Adult↗