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At least 1,621 records · Page 90Linked to original sources
PubMed1990

Induction of donor-type chimerism in murine recipients of bone marrow allografts by different radiation regimens currently used in treatment of leukemia patients.

Three radiation protocols currently used in treatment of leukemia patients before bone marrow transplantation (BMT) were investigated in a murine model (C57BL/6----C3H/HeJ) for BM allograft rejection. These include (a) a single dose of total body irradiation (8.5 Gy TBI delivered at a dose rate of 0.2 Gy/min), (b) fractionated TBI (12 Gy administered in six fractions, 2 Gy twice a day in 3 days, delivered at a dose rate of 0.1 Gy/min, and (c) hyperfractionated TBI (14.4 Gy administered in 12 fractions, 1.2 Gy three times a day in 3 days, delivered at a dose rate of 0.1 Gy/min). Donor-type chimerism 6 to 8 weeks after BMT and hematologic reconstitution on day 12 after BMT found in these groups were compared with results obtained in mice conditioned with 8 Gy TBI delivered at a dose rate of 0.67 Gy/min, routinely used in this murine model. The results in both parameters showed a marked advantage for the single dose 8.5 Gy TBI over all the other treatments. This advantage was found to be equivalent to three- to fourfold increment in the BM inoculum when compared with hyperfractionated radiation, which afforded the least favorable conditions for development of donor-type chimerism. The fractionated radiation protocol was equivalent in its efficacy to results obtained in mice irradiated by single-dose 8 Gy TBI, both of which afforded a smaller but not significant advantage over the hyperfractionated protocol. This model was also used to test the effect of radiation dose rate on the development of donor-type chimerism. A significant enhancement was found after an increase in dose rate from 0.1 to 0.7 Gy/min. Further enhancement could be achieved when the dose rate was increased to 1.3 Gy/min, but survival at this high dose rate was reduced. These results demonstrated indirectly that dose rate affects the expression of host-type pluripotent stem cells, the progeny of which appear 3 to 6 weeks after treatment with 8 Gy TBI delivered at a dose rate of 0.1 Gy/min, but which are eradicated if radiation is delivered at a dose rate of 1.3 Gy/min.

Animals↗
PubMed1990

Effects of mixed chimeric bone marrow repopulation on platelet storage pool-associated bleeding defects in mouse mutants.

We have previously shown mouse platelet storage pool deficiency (SPD) to be associated with lesions at eight different genetic loci, each of which is sufficient to produce murine SPD. We have also shown that normal bleeding times and normal platelet functions are restored when mice with SPD are transplanted with marrow from normal mice. Conversely, when normal mice are transplanted with mutant marrow, they present symptoms of SPD. In order to determine the amount of normal platelets needed to prevent the prolonged bleeding times associated with SPD, we established stable mixed chimeric mice by transplanting various ratios of normal and mutant marrow into lethally irradiated host animals. The proportion of normal input marrow correlated well with the proportion of normal peripheral red blood cells and platelets determined in chimerae 100 days after transplantation using direct morphology and electrophoretic variants of glucose phosphate isomerase to identify normal and mutant cell populations. The proportions of normal input marrow were also reflected in the proportions of platelets with normal and mutant platelet morphology in the chimerae. This confirms that the platelet abnormality in SPD is intrinsic to the stem cell population from which the platelets are derived. When bleeding times were determined in the mixed chimeric mice, a surprisingly high percentage of normal platelets (greater than 50% and sometimes greater than 75%) were needed to stop bleeding. These results suggest that the mutant platelets in the mixed chimeric mice may interfere with normal platelet aggregation patterns. They also raise some important considerations in devising treatment for SPD. Bleeding episodes in human SPD are normally treated by platelet transfusion. The results suggest that, at least in some cases, transfusions may not be effective. Also, in future gene therapy of this disease, it is like that a functional gene will have to be present in greater than 50% of stem cells for therapy to be effective.

Animals↗
PubMed1989

Pseudomonas exotoxin: chimeric toxins.

Pseudomonas exotoxin binds to and enters cells by receptor-mediated endocytosis. Within the cell it requires exposure to low pH to enable it to translocate to the cell cytoplasm where it inhibits protein synthesis by ADP-ribosylating elongation factor 2. The toxin has three main structural domains whose functions are: Ia, cell binding; II, translocation; and III, ADP-ribosylation. Key amino acids have been identified within each domain that are required for the function of the toxin. Chimeric toxins were made originally by using chemical cross-linking reagents to couple Pseudomonas exotoxin (or other toxins) to cell-binding proteins. More recently, a variety of Pseudomonas exotoxin-related chimeric toxins have been made by gene fusion technology. These chimeric toxins may be useful clinically for treating various diseases and experimentally for understanding receptor function.

ADP Ribose Transferases↗
PubMed1989

Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG.

Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications.

Animals↗
PubMed1989

Effects of T cell depletion in radiation bone marrow chimeras. III. Characterization of allogeneic bone marrow cell populations that increase allogeneic chimerism independently of graft-vs-host disease in mixed marrow recipients.

The opposing problems of graft-vs-host disease vs failure of alloengraftment severely limit the success of allogeneic bone marrow transplantation as a therapeutic modality. We have recently used a murine bone marrow transplantation model involving reconstitution of lethally irradiated mice with mixtures of allogeneic and syngeneic marrow to demonstrate that an allogeneic bone marrow subpopulation, removed by T cell depletion with rabbit anti-mouse brain serum and complement (RAMB/C), is capable of increasing levels of allogeneic chimerism. This effect was observed in an F1 into parent genetic combination lacking the potential for graft-vs-host disease, and radiation protection studies suggested that it was not due to depletion of stem cells by RAMB/C. We have now attempted to characterize the cell population responsible for increasing allogeneic chimerism in this model. The results indicate that neither mature T cells nor NK cells are responsible for this activity. However, an assay involving mixed marrow reconstitution in an Ly-5 congenic strain combination was found to be more sensitive to small degrees of stem cell depletion than radiation protection assays using three-fold titrations of bone marrow cells. Using this assay, we were able to detect some degree of stem cell depletion by treatment with RAMB/C, but not with anti-T cell mAb. Nevertheless, if the effects of alloresistance observed in this model are considered, the degree of stem cell depletion detected by such mixing studies in insufficient to account for the effects of RAMB/C depletion on levels of allogeneic chimerism, suggesting that another cell population with this property remains to be identified.

Animals↗
PubMed1988

Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G.

Chimeric Fc receptors, consisting of the IgG-binding domains of both staphylococcal protein A and streptococcal protein G, were constructed. An efficient bacterial expression system was used to produce the recombinant proteins, which vary in size and number of IgG-binding domains. The purified receptors were analyzed by immunodiffusion and a competitive enzyme-linked immunosorbent assay to establish the relative binding strength to various polyclonal and monoclonal immunoglobulins from different species. The results demonstrate that protein A and protein G have complementary binding patterns and that the chimeric receptors retain the binding capacities of both the parental constituents. This suggests that these novel chimeric receptors might be versatile reagents for immunochemical assays.

Animals↗
PubMed1986

Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons.

To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion.

Chimera↗
PubMed1988

[Chimeric drift in blood erythrocyte population in BALB/c----C57BL/10 and BALB----B10.D2 mice].

Genotypic composition of the erythrocyte population of peripheral blood in 16 aggregation chimeras: BALB/c (H-2dd)----C57BL/10(H-2bb) and BALB/c(H-2dd)----B10.D2(H-2dd) was studied during 10 months. The proportion of cells of parental components was defined visually in the coat and by electrophoresis of allozyme variants at the Gpi-1 locus in blood. The similar increase of blood cells percentage was observed in blood of both types of chimeras with age. In chimeras the skin grafts of both parental types survive. Chimeric drift is not caused by H-2 haplotypes differences between cells of two strains or disturbance of immunological tolerance in the chimeric mice. We propose that chimeric drift results from interaction of hemopoietic cells of different strains in early stages of hemopoiesis.

Animals↗
PubMed1988

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation.

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed1987

Mixed hematopoietic chimerism following bone marrow transplantation for hematologic malignancies.

Twenty-nine of 172 patients (17%) who received an allogeneic bone marrow transplant (BMT) from histocompatible sibling donors for hematologic malignancies were mixed hematopoietic chimeras; ie, they had a mixture of donor and host hematopoietic or lymphohematopoietic cells at greater than or equal to 14 days after transplantation. Twenty-four of the 29 mixed chimeras (83%) have remained in continuous complete remission for up to 116 months (greater than 9 years) following BMT. Four of the 29 patients (14%) have had recurrent leukemia, and 7 of the 29 (24%) have had moderate or severe graft-v-host disease (GVHD). Twelve of these 29 patients have persisted as stable mixed chimeras for greater than or equal to 2 years after BMT, whereas other patients converted to all donor-type hematopoiesis. The incidence of mixed chimerism was independent of the pretransplant regimen, the donor or recipient age (less than 20 v greater than 20 years), remission status (first complete remission of acute leukemia and first chronic phase of chronic myelocytic leukemia v later stages of disease), and type of leukemia. Our data indicate that mixed hematopoietic chimerism is not rare after BMT for hematologic malignancies and that its presence is compatible with long-term disease-free survival. Prospective studies of mixed chimerism after BMT are warranted to achieve better understanding of its biologic importance.

Acute Disease↗
PubMed1988

Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.

A bovine recombinant phage library was constructed and screened with rabbit S mu and human C gamma probes. IgM, IgA, and IgE H chain constant region (CH) genes were isolated with the rabbit S mu probe and three IgG CH genes were isolated with the C gamma probe. The CH genes were individually cloned into an expression vector which contained a murine VDJ gene cloned from a hybridoma producing anti-dansyl hapten antibody. The resulting constructs were transfected into murine hybridoma cells producing L chain of the anti-dansyl antibody and stable transfectomas secreting chimeric bovine-murine IgA, IgE, or IgG subclass anti-dansyl antibodies were obtained. The chimeric antibodies, immunoprecipitated with Ag or with anti-bovine H chain antibodies, were analyzed by SDS-PAGE and were shown to contain H and L chains of expected size. Of the three chimeric antibodies derived from the C gamma genes, one reacted with anti-IgG1 antibody, another reacted with anti-IgG2 antibody and the third did not react with either anti-IgG1 or anti-IgG2. This third IgG appears to represent a "new" subclass of bovine IgG, IgG3. Southern blot analysis indicated that the bovine genome contains a fourth C gamma gene. These experiments demonstrate the usefulness of molecular genetic techniques for the isolation and characterization of Ig which are not readily purified from biologic fluids. These techniques will be useful for isolation and characterization of Ig genes from other outbred mammals.

Animals↗
PubMed1986

Enzymatic synthesis of chimeric tRNAs with unusual numbers of base pairs in the anticodon stem; their structure and properties.

Chimeric tRNATyr molecules have been constructed by enzymatic procedures in vitro from the 5'-half fragment of T. utilis tRNATyr and the 3'-half fragment of yeast tRNATyr, and vice versa. These chimeric tRNAs contain base-mismatching(s) in the anticodon stem and, therefore, have only 3 or 4 base pairs in the stem. Although the Tm of these chimeras are largely decreased, there seems to be no gross difference between the structure of native and chimeric tRNATyrs at physiological temperatures in the presence of 10 mM MgCl2. Aminoacylation assays also revealed that the tyrosine-acceptance of the chimeras are fully comparable to that of native tRNATyrs. However, the possibility remains that the properties of the chimeras are considerably different from those of native ones at lower Mg++ concentrations.

Anticodon↗
PubMed1985

A human-mouse chimeric immunoglobulin gene with a human variable region is expressed in mouse myeloma cells.

We have constructed and obtained expression of a chimeric human-mouse immunoglobulin gene after transfection into mouse myeloma cells. A human VDJH gene segment was joined to a mouse C kappa gene in the plasmid vector pSV2-gpt, and the construct was transfected into J558L cells by protoplast fusion. Analyses of six transformants by RIA and SDS-PAGE indicated that the chimeric protein was synthesized in large amounts in five. A kappa-specific transcript was observed by Northern blot analysis. Four out of five clones were stable producers of this chimeric chain over a period of 10 mo.

Animals↗
PubMed1984

Altered differentiation, indefinite growth potential, diminished tumorigenicity, and suppressed chimerization potential of hybrids between mouse teratocarcinoma cells and thymocytes.

Hybrids between normal mouse thymocytes and a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) -deficient, pseudodiploid, multipotent mouse teratocarcinoma cell line displayed predominately hypotetraploid modes with differential patterns of karyotypic variation and no apparent consistent pattern of chromosome segregation. The hybrids failed to express thymocyte antigens Thy-1.2, Lyt-1, and Lyt-2, indicating suppression of thymocyte differentiation. Like the teratocarcinoma parent, the hybrids had an embryonal carcinoma (EC) stem cell component which exhibited indefinite growth potential in vitro and produced tumors with multiple pathways of tissue differentiation when injected subcutaneously. Hybrids had decreased proportions of EC cells and individually unique patterns of differentiation. The tumorigenicity of hybrids was diminished, indicating partial suppression of the neoplastic phenotype by the genome of the thymocyte parent. The EC parent gave chimerization frequencies of 18.5-19.2%, confirming the findings of Dewey et al. that mutant teratocarcinoma cells can be used to synthesize chimeric mice. "Selfed" near-tetraploid EC cells gave a frequency of 2.3%, showing that an increase in ploidy can reduce, although not eliminate, chimerization potential. No chimeras could be detected with hybrids (frequency less than 0.65%). Thus, there was no evidence that a thymocyte could be "reprogrammed" to participate in new developmental pathways.

Animals↗
PubMed1982

Effect of splenectomy on establishment of hematopoietic chimerism in nonirradiated mice.

When spleen cells (1.5 x 10(8)) from beige C57BL/6 (bgJ/bgJ, Chediak-Higashi syndrome) mice were injected into nonirradiated (C57BL/6 x CBA)F1 hybrid mice, the hematopoietic stem cells of the hybrid host were eradicated by graft-versus-host (GVH) reaction, and peripheral neutrophils of the host changed from normal type to beige type with giant sudanophilic granules in 20 days after the cell injection. Effect of splenectomy on the establishment of such hematopoietic chimerism was investigated. The splenectomy carried out either before or after the cell injection reduced the proportion of mice in which more than 90% of peripheral neutrophils became of beige type. Since the mortality of mice which received splenectomy at various days prior to or after the cell injection paralleled the proportion of establishment of chimerism, the splenectomy does not seem to affect directly the establishment of chimerism but indirectly through the modification of the intensity of GVH reaction.

Animals↗
PubMed1995

Biological properties of chimeric domain-deleted anticarcinoma immunoglobulins.

CC49 is a second-generation monoclonal antibody (MAb) that has high affinity for the tumor-associated pancarcinoma antigen tumor-associated glycoprotein-72. In clinical trials using gamma scanning, radiolabeled CC49 has facilitated the detection of more than 90% of carcinomas. We report here the development of a constant heavy-chain 2 (CH2) domain-deleted chimeric (c) CC49 MAb by transfecting an expression construct consisting of the CC49 murine variable region and a CH2 domain-deleted human IgG1 constant region into cCC49 kappa producing SP2/0 murine myeloma cells. As determined by SDS-PAGE, the intact cCC49 delta CH2 has a molecular weight of 153,000 and, under reducing conditions, molecular weights of 43,000 and 27,000. The plasma clearance and tumor-targeting properties of cCC49 delta CH2 were evaluated and compared with those of mouse/human chimeric forms cCC49 delta CH1 and intact cCC49. Previous studies have shown that the in vitro antigen-binding properties of cCC49 delta CH1 are similar to those of cCC49. Biodistribution studies reported here, using 131I-labeled cCC49 delta CH1 and 125I-labeled cCC49 in athymic mice bearing human colon carcinoma xenografts, demonstrated that both cMAbs localized to the tumor and cleared from the normal tissues similarly. However, in comparison with 125I-labeled cCC49, 131I-labeled cCC49 delta CH2 localized to tumors earlier and had a significantly lower percentage of the injected dose of cMAb/g (%ID/g) in normal tissues than cCC49. Immunoscintigraphy of 131I-labeled cCC49 delta CH2 and 125I-labeled cCC49 in athymic mice bearing human tumor xenografts demonstrated a clear image of the tumor by 24 h after i.v. administration of the delta CH2 cMAb versus the 72 h required for cCC49. Biodistribution studies using 177Lu-conjugated cCC49 delta CH1 and cCC49 showed no significant difference between the radiolocalization indices (% ID/g in tumor divided by % ID/g in normal tissue). 177Lu-conjugated cCC49 delta CH2, however, had lower % ID/g values in tumor xenografts and lower radiolocalization indices than either 177Lu-conjugated cCC49 delta CH1 or 177Lu-conjugated cCC49. Pharmacokinetic studies in non-tumor-bearing athymic mice using cCC49 delta CH1 and cCC49 revealed no significant difference between these cMAbs. However, the plasma clearance of cCC49 delta CH2 in non-tumor-bearing mice was significantly faster than that of cCC49. These results were similar when the cMAbs were labeled with either iodine or lutetium. In nonhuman primates, 131I-labeled cCC49 delta CH2 cleared significantly faster than 125I-labeled cCC49. The similar plasma clearance and tumor localization of cCC49 and cCC49 delta CH1 suggest that these two cMAbs may be used in similar clinical settings. However, because of the unique pharmacokinetics and tumor targeting of cCC49 delta CH2 versus cCC49 or cCC49 delta CH1, this chimeric immunoglobulin form may be useful in clinical settings that require efficient tumor targeting and rapid serum and whole-body clearance.

Animals↗
PubMed1994

Chimerization of antitumor antibodies via homologous recombination conversion vectors.

Homologous recombination vectors were designed to convert murine hybridoma cell lines expressing IgG3, IgG1, or IgG2a heavy chains into chimeric human IgG1 producers. These conversion vectors included homology both upstream and downstream of the target sequences and consistently resulted in a higher frequency of successful gene targeting than an insertion vector bearing a single region of homology. A human kappa light chain conversion vector was also constructed and used to complete chimerization of the anticarcinoma hybridoma cell line BR96. The resulting cell line expressed antigen-specific chimeric antibody at comparable levels to those found in the murine parental cell line. Southern blots confirm that recombination occurred within the upstream and downstream regions of homology for both vectors, resulting in the loss of murine constant region sequences.

Animals↗
PubMed1994

Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma.

The B-cell antigen CD20 is expressed on normal B cells and by nearly all B-cell lymphomas. This nonmodulating antigen provides an excellent target for antibody-directed therapies. A chimeric anti-CD20 antibody (IDEC-C2B8), consisting of human IgG1-kappa constant regions and variable regions from the murine monoclonal anti-CD20 antibody IDEC-2B8, has been produced for clinical trials. It lyses CD20+ cells in vitro via complement and antibody-dependent cell-mediated lysis. Preclinical studies have shown that the chimeric antibody selectively depletes B cells in blood and lymph nodes in macaque monkeys. In this phase I clinical trial, 15 patients (3 per dose level) with relapsed low-grade B-cell lymphoma were treated with a single dose (10, 50, 100, 250, or 500 mg/m2) of antibody administered intravenously. Treatment-related symptoms correlated with the number of circulating CD20 cells and grade II events consisted of fever (5 patients); nausea (2), rigor (2), orthostatic hypotension (2), bronchospasm (1), and thrombocytopenia (1). No significant toxicities were observed during the 3 months of follow-up. Serum C3, IgG, and IgM levels, neutrophils, and T cells were largely unchanged. At the three higher dose levels, pharmacokinetics of the free antibody showed a serum half-life of 4.4 days (range, 1.6 to 10.5). Levels greater than 10 micrograms/mL persisted in 6 of 9 patients for more than 14 days. No quantifiable immune responses to the infused antibody have been detected. CD20+ B cells were rapidly and specifically depleted in the peripheral blood at 24 to 72 hours and remained depleted for at least 2 to 3 months in most patients. Two-week postinfusion tumor biopsies showed the chimeric antibody bound to tumor cells and a decrease in the percentage of B cells. Tumor regressions occurred in 6 of 15 patients (2 partial and 4 minor responses). The results of this single-dose trial have been used to design a multiple-dose phase I/II study.

Adult↗