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PubMed1990

Bone marrow-derived cells are essential for intrathymic deletion of self-reactive T cells in both the host- and donor-derived thymocytes of fully allogeneic bone marrow chimeras.

The fate of self-reactive T cells was examined in both the host- and donor-derived thymocytes of fully allogeneic bone marrow (BM) chimeras of two strain combinations of AKR/J (H-2k, IE+, Thy-1.1, Mls-1a2b) and C57BL/6 (H-2b, IE-, Thy-1.2, Mls-1b2b). Sequential appearance of host- and donor-derived T cells occurred in the thymus of both AKR----B6 and B6----AKR chimeras in which 5 x 10(6) of T cell-depleted BM cells were used to reconstitute recipients lethally irradiated with 950 rad. Thymocytes bearing V beta 6 high, which recognize MHC class II IE-binding Ag encoded by Mls-1a allele, were detected in neither host- nor donor-derived thymocytes of AKR-B6 chimeras in which Mls-1a and IE were expressed only by the BM-derived cells. Thymocytes bearing V beta 11high capable of recognizing IE were also deleted in the host- and donor-derived thymocytes of the AKR----B6 chimeras. One million of BM cells were inadequate to deletion of the B beta 6high and V beta 11high T cells in the host-derived thymocytes of these chimeras. On the other hand, significant number of V beta 6high and V beta 11high thymocytes were detected in both the host- and donor-derived thymocytes in B6----AKR chimeras where sufficient dose of IE- stem cells were used to reconstitute irradiated Mls-1aIE+ recipients. These results suggest that clonal deletion of the host- and donor-reactive T cells in both the host- and donor-derived thymocytes is an important mechanism for the induction of transplantation tolerance in allogeneic BM chimeras and that BM-derived APC may be essential for the intrathymic elimination of both the host- and donor-reactive T cells in the BM chimeras.

Animals↗
PubMed1988

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos.

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.

Animals↗
PubMed1982

Helper cell factors restore antibody responses of allogeneic bone marrow chimeras: evidence for ineffective cellular interactions.

We investigated the nature of deficient antibody responses to SRBC in stable, fully allogeneic bone marrow chimeras. No evidence for a suppressor cell-mediated mechanism was found. Chimera spleens possessed adequate numbers of antigen-reactive B cells to produce a normal antibody response. Using separated chimera cell populations and soluble helper factors, we assessed the functional capabilities of chimera B cells, T cells, and macrophages. Our data suggest that the failure of allogeneic chimeras to produce antibody is not the result of impaired B cell, T cell, or macrophage function, but rather that it is due in ineffective cellular interactions that normally result in the generation of helper factors. In vitro stimulation of chimera macrophages with LPS, and of chimera spleen cells with Con A, resulted in the release of soluble helper factors that were capable of fully restoring chimera B cell responses.

Animals↗
PubMed2002

Schizophrenia patients' perceptual biases in response to positively and negatively valenced emotion chimeras.

BACKGROUND: In a prior study, we observed that schizophrenia patients display atypical perceptual biases in response to emotional (happy/neutral) facial chimeras. METHODS: The present study was an attempt to replicate and extend those findings, using emotional stimuli with negative affective valence (angry/neutral chimeras) as well as positive valence, and including more than one type of non-affective facial comparison task. We compared schizophrenia patients (N = 37) and controls (N = 48) on free-vision tasks that typically yield left spatial field biases indicative of right hemisphere activation. There were six chimera tasks, including two emotion (happy, angry) chimeras, two non-emotion (gender, age) chimeras and two non-face (dots, gradients) chimeras. RESULTS: We observed a Group x Task interaction, with schizophrenia patients displaying significantly less of the expected left spatial perceptual bias in response to the happy/neutral chimeras and the angry/neutral chimeras relative to the controls. In contrast, the patients and controls did not differ in terms of their response to the Gender, Age, Dots, or Gradients tasks. CONCLUSIONS: These findings are consistent with the assertion that, compared with healthy controls individuals with schizophrenia perceive emotion differently.

Adult↗
PubMed2000

Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase.

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1.

Amino Acid Sequence↗
PubMed2006

Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras.

The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465-1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.

Arabidopsis↗
PubMedDate not supplied

Wortmannin, a phosphoinositide 3-kinase inhibitor, selectively enhances cytotoxicity of receptor-directed-toxin chimeras in vitro and in vivo.

BACKGROUND: Generalized resistance of some neoplastic cell lines to treatment with ligand-toxin chimeras has been attributed to an increased rate of lysosomal uptake and degradation following endocytosis of the chimera-receptor complex. Because phosphoinositide 3-kinase (Pl 3-kinase) activity is known to play a role in intracellular trafficking, particularly from endosomes to lysosomes, we hypothesized that co-exposing cells to the Pl 3-kinase inhibitor, wortmannin, might enhance cytotoxicity of ligand-toxin chimeras. METHODS: In vitro, cytotoxicity of five receptor directed-toxin chimeras (bFGF-SAP, bFGF-PE, aFGF-PE, HBEGF-SAP, bFGF-gelonin) and an immunotoxin (11A8-SAP) was examined in the presence or absence of this Pl 3-kinase inhibitor against a panel of human neoplastic cell lines: SK-MEL-5 (melanoma), PA-1 (ovarian teratocarcinoma), DU145 (prostatic carcinoma) and MCF-7 (breast carcinoma). In vivo, antitumor activity of a treatment regimen combining wortmannin (1 or 2 mg/kg i.p.) and bFGF-SAP (10 micrograms/kg i.v.) once a week for 4 weeks was evaluated compared to administration of each agent alone in C3H/HeN mice implanted with the FSallC murine fibrosarcoma. RESULTS: At concentrations greater than the reported Ki for Pl 3-kinase inhibition (1-10 microM), wortmannin enhanced cytotoxicity when combined with saporin or gelonin chimeras, but produced subadditive cytotoxicity when combined with Pseudomonas exotoxin chimeras. When low nanomolar concentrations selective for Pl 3-kinase inhibition (5-100 nM) were examined for effects on one receptor directed-toxin chimera, wortmannin dramatically enhanced bFGF-SAP cytotoxicity in three of the four cell lines. A different Pl 3-kinase inhibitor, LY294002 (Ki approximately 1 microM), however, failed to potentiate bFGF-SAP. When administered to mice, wortmannin combined with bFGF-SAP resulted in a significant decrease in tumor volumes compared to vehicle-treated controls that was not observed in mice treated with either agent alone. CONCLUSIONS: Taken together, these results suggest that although wortmannin increases the cytotoxic efficacy of some receptor-directed chimeras, potentiation may occur through an alternative pathway not involving Pl 3-kinase inhibition.

Adenocarcinoma↗
PubMed1985

Immune responses of thymus/lymphocyte embryonic chimeras: studies on tolerance and major histocompatibility complex restriction in Xenopus.

A new model has been developed to address the question of whether T cells that traverse an allogeneic thymus during early and late life become restricted to interact, in vivo, with other leukocytes and target cells that display the major histocompatibility complex (MHC) antigens of the thymus haplotype. Chimeras were made microsurgically with pairs of 24-h-old Xenopus embryos such that the anterior region of an embryonic chimera contained the thymus anlagen and was of one MHC genotype, whereas the posterior region contained the anlagen of all hemopoietic cells and was of another genotype. Assays to determine the MHC haplotype restriction specificity of T cells in chimeras that had been reared through metamorphosis involved: specific antibody responses (IgM and IgG) to dinitrophenylated keyhole limpet hemocyanin; rejection of minor H locus disparate skin grafts that expressed the MHC antigens of either the thymus donor or the lymphocyte donor; and mixed leukocyte culture. MHC-mismatched chimeras displayed split tolerance since they accepted skin grafts of the thymus haplotype but had lymphocytes that proliferated in response to MHC antigens of the thymus donor strain as well as to MHC antigens of third-party donors. IgM responses of MHC-matched and MHC-mismatched chimeras and of nonchimeric controls did not differ. However, the IgG responses of MHC-mismatched thymus/lymphocyte chimeras peaked later than those of MHC-matched chimeras and normal controls. Data from skin grafting protocols were consistent with the proposition that there may be in vivo selection of T cells reactive to minor H antigens presented in association with the MHC antigens of the thymus rather than the MHC antigens of the lymphocytes themselves. These data suggest that although it is not absolute, there is thymic selection of the T cell repertoire in Xenopus.

Animals↗
PubMed1996

Induction of diabetes in NOD<-->C57BL/6 embryo aggregation chimeras by cyclophosphamide through preferential depletion of C57BL/6 lymphocytes.

The majority of embryo aggregation (EA) mouse chimeras between non-obese diabetic (NOD) mice and C57BL/6 (B6) mice show clear signs of insulitis frequently accompanied by beta-cell destruction. Less than 5% of these chimeras, however, spontaneously progress to autoimmune diabetes, an incidence far lower than observed in NOD mice. The resistance in chimeras can be accounted for by the target organ chimerism and/or the immune system chimerism. To investigate the mechanism(s) controlling diabetes resistance in these mice, we studied a total of 92 NOD<-->B6 EA chimeras that showed overt lymphoid chimerism and treated 34 chimeras with cyclophosphamide (CY), a compound known to precipitate an acute form of insulin-dependent diabetes mellitus (IDDM) in pre-diabetic NOD mice, by interfering with regulatory mechanisms. We found that CY-treated EA chimeras displayed an increase in the NOD:B6 lymphocyte ratio and 32% of them developed diabetes that could be adoptively transferred to irradiated NOD or NOD-rag-2-/- mice. These findings suggest that lymphocyte chimerism rather than beta-cell chimerism accounts for diabetes resistance in NOD<-->B6 EA chimeras and that the susceptibility to CY-induced diabetes may be related to the proportion of NOD versus B6 lymphoid cells.

Animals↗
PubMed2006

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis.

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.

Brassica↗
PubMed1989

Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2----P1) irradiation chimeras.

Mixed irradiation bone marrow chimeras were prepared by reconstituting lethally irradiated C57BL/10 (B10) or B10.D2 mice with T cell-depleted bone marrow cells of B10 plus B10.D2 origin. These chimeras were healthy and survived well under conventional housing conditions and after experimental laboratory infections. Of a total of 17 chimeras tested, 2 died spontaneously or from the injected virus. Twelve of fifteen chimeras mounted a measurable cytotoxic T cell response to virus. Despite approximately equal percentages of B10 and B10.D2 lymphocytes in chimeras, cytotoxic T cell responses to vaccinia virus and lymphocytic choriomeningitis virus were mediated variably by either syngeneic or allogeneic donor lymphocytes; thus the H-2 type of effector T cells frequently did not correspond to the 50:50 distribution of spleen or peripheral blood lymphocytes. Cytotoxic responses were restricted exclusively to recipient H-2 type. All mixed chimeras examined were able to mount a good IgG response to vesicular stomatitis virus. These results confirm previous data suggesting that such mixed chimeras are healthy and immunocompetent and demonstrate strict recipient-determined restriction specificity of effector T cells; they also suggest that if T help is necessary for induction of virus-specific cytotoxic T cells, it does not require host-restricted interactions between helper T cells and precursor cytotoxic T cells.

Animals↗
PubMed1984

Mouse trisomy 16 as an animal model of human trisomy 21 (Down syndrome): production of viable trisomy 16 diploid mouse chimeras.

We have previously proposed that mice trisomic for chromosome 16 will provide an animal model of human trisomy 21 (Down syndrome). However, the value of this model is limited to some extent because trisomy 16 mouse fetuses do not survive as live-born animals. Therefore, in an effort to produce viable mice with cells trisomic for chromosome 16, we have used an aggregation technique to generate trisomy 16 diploid (Ts 16 2n) chimeras. A total of 79 chimeric mice were produced, 11 of which were Ts 16 2n chimeras. Seven of these Ts 16 2n mice were analyzed as fetuses, just prior to birth, and 4 were analyzed as live-born animals. Unlike nonchimeric Ts 16 mouse fetuses which die shortly before birth with edema, congenital heart disease, and thymic and splenic hypoplasia, all but 1 of the Ts 16 2n animals were viable and phenotypically normal. The oldest of the live-born Ts 16 2n chimeras was 12 months old at the time of necropsy. Ts 16 cells, identified by coat color, enzyme marker, and/or karyotype analyses, comprised 50-60% of the brain, heart, lung, liver, and kidney in the 7 Ts 16 2n chimeric fetuses and 30-40% of these organs in the 4 live-born Ts 16 2n animals. Ts 16 cells comprised an average of 40% of the thymus and 80% of the spleen in the Ts 16 2n chimeras analyzed as fetuses, with no evidence of thymic or splenic hypoplasia. However, we observed a marked deficiency to Ts 16 cells in the blood, spleen, thymus, and bone marrow of live-born Ts 16 2n chimeras as compared to 2n 2n controls. These results demonstrate that although the Ts 16 2n chimeras were, with one exception, viable and phenotypically normal, each animal contained a significant proportion of trisomic cells in a variety of tissues, including the brain. Furthermore, our results suggest that although the abnormal development of Ts 16 thymus and spleen cells observed in Ts 16 fetuses is largely corrected in Ts 16 2n fetuses, Ts 16 erythroid and lymphoid cells have a severe proliferative disadvantage as compared to diploid cells in older live-born Ts 16 2n chimeras. Ts 16 2n chimeric mice will provide a valuable tool for studying the functional consequences of aneuploidy and may provide insight into the mechanisms by which trisomy 21 leads to developmental abnormalities in man.

Animals↗
PubMed1990

In vivo administration of cytokine in allogeneic bone marrow chimeras.

When we analyzed the in vivo efficacy of cytokine administration in murine allogeneic bone marrow chimeras, mitogen-induced responses to ConA, PHA, LPS, or PWM were increased by the in vivo administration of human recombinant granulocyte colony-stimulating factor (rG-CSF), human recombinant interleukin 2 (rIL-2), or WEHI-3B conditioned medium (CM). Furthermore, we found increased alloreactive mixed lymphocyte reactions (MLRs) against donor and/or host type alloantigens in spleen cells from (BALB/c----C3H/He) chimeras, although cytotoxic activity against BALB/c or C3H/He target cells was not detected in spleen cells from these chimeras. Since no significant increase of T cells or Ia positive cells was observed, some functional activation, rather than changes in the cell count, appeared to relate to increase immunoreactivity. An increased IL-2 production in spleen cells from chimeras injected with cytokine was observed shortly after the cessation of cytokine administration. Thereafter, an IL-2 production in these chimeras decreased around 45 days after bone marrow transplantation and then recovered nearly to the control level. An increased IL-2 responsiveness was also observed in spleen cells from these chimeras. These findings suggest that the in vivo administration of rG-CSF as well as rIL-2 or WEHI-3B CM (IL-3) can modulate the immunoreactivity in chimeras via the network of immune systems.

Animals↗
PubMed1989

Donor and recipient specific tolerance in cells from semi-allogeneic, H-2 subregion compatible or fully allogeneic bone marrow chimeras attributable to clonal deletion.

Specificities of tolerance induced in allogeneic bone marrow (BM) chimeras which had been established by injecting allogeneic BM cells pretreated with anti-Thy-1 mAb alone (without complement (C)) were analyzed using Simonsen's splenomegaly assay. Lymphocytes from fully allogeneic, semi-allogeneic and H-2 subregion compatible BM chimeras were specifically unresponsive to donor and recipient antigens (Ag). However, cells from H-2 subregion compatible chimeras initiated as vigorously a GVHR in F1 recipient mice, which were disparate at H-2K and I-A regions, as did spleen cells of donor mice, which were incompatible at the entire H-2 and minor histocompatibility regions of the recipients. The donor cells from such chimeras that initiated these considerable GVHR were either CD4+ or CD8+ T cells. Furthermore, synergistic effects by the CD4+ and CD8+ T lymphocytes were also observed. We found no evidence for a suppressive mechanism(s) in maintenance of the specific tolerance in allogeneic chimeras. Further, when lymphoid cells from these chimeras were adoptively transferred to irradiated mice of the donor strain and maintained for 5 days in the absence of recipient Ag (tolerogen), the adoptively transferred cells were shown to retain their unresponsiveness to the recipient Ag. These results reveal that T lymphocytes from allogeneic BM chimeras prepared by our method had been specifically induced to a tolerant state to both donor and recipient Ag and that the major mechanism of induction and maintenance of long-lasting tolerance is attributable to clonal deletion of both CD4+ and CD8+ T cell subsets rather than to the development of a population of suppressor cells of any sort.

Animals↗
PubMed1982

Production of viable adult trisomy 17 reversible diploid mouse chimeras.

Aneuploid mouse embryos and fetuses are an important system for investigating the pathogenesis of the developmental and functional consequences of chromosome imbalance in mammals. However, the fact that almost all mouse aneuploids die in embryonic or fetal life restricts their usefulness for studies of loci and functions that are expressed only after birth. As an approach to rescuing aneuploid cells, we have prepared chimeras by aggregating aneuploid embryos with diploid embryos at the 8-16 cell stage. This technique has allowed us to produce trisomy 17 reversible diploid (Ts17 reversible 2n) chimeras containing cells of a trisomic state, which is ordinarily lethal at 10-12 days of gestation. All of the analyzed organs from the chimeras, including brain, liver, kidney, lung, muscle, heart, thymus, spleen, bone marrow, blood, and skin, contained both Ts17 and 2n cells. The proportion of Ts17 cells in each organ as estimated from coat color, enzyme markers (glucosephosphate isomerase), and karyotypes ranged from 5% to 85%, most commonly 20-40%. This is in contrast to the control 2n reversible 2n chimeras in which the 2n component on the same genetic background as the Ts17 generally comprised 60-90% of the cell population. The growth rates of the living Ts17 reversible 2n chimeras were in the lower half of the normal range, and the oldest animals are now age 14 mo. No progeny were obtained from Ts17 germ cells in the two fertile T217 reversible 2n chimeras. In comparison with analogous human situations, it is striking that, except for a kink in the tail of one living animal, none of the Ts17 reversible 2n chimeras had any discernible structural abnormalities. Our findings indicate that it is feasible to rescue cells from autosomal aneuploidies that otherwise result in early fetal death.

Aneuploidy↗
PubMed1992

Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos.

To determine whether embryonic cells transplanted from one zebrafish embryo to another can contribute to the germ line of the recipient, and to determine whether pigmentation can be used as a dominant visible marker to monitor cell transplants, we introduced cells from genetically pigmented (donor) embryos to albino recipients at midblastula stage. By 48 hr many of the resulting chimeras expressed dark pigment in their eyes and bodies, characteristics of donor but not albino embryos. By 4-6 weeks of age pigmentation was observed on the body of 23 of 70 chimeras. In contrast to fully pigmented wild-type fish, pigmentation in chimeras appeared within transverse bands running from dorsal to ventral. Pigmentation patterns differed from one fish to another and in almost every case were different on each side of a single fish. At 2-3 months of age chimeras were mated to albino fish to determine whether pigmented donor cells had contributed to the germ line. Of 28 chimeric fish that have yielded at least 50 offspring each, 5 have given rise to pigmented progeny at frequencies of 1-40%. The donor cells for some chimeras were derived from embryos that, in addition to being pigmented, were transgenic for a lacZ plasmid. Pigmented offspring of some germ-line chimeras inherited the transgene, confirming that they descended from transplanted donor cells. Our ability to make germ-line chimeras suggests that it is possible to introduce genetically engineered cells into zebrafish embryos and to identify the offspring of these cells by pigmentation at 2 days of age.

Albinism↗
PubMed1981

Self recognition in allogeneic radiation bone marrow chimeras. A radiation-resistant host element dictates the self specificity and immune response gene phenotype of T-helper cells.

The specificity of the self-recognition repertoire in fully allogeneic (A {arrow} B), semiallogeneic (A {arrow} A x B and A x B {arrow} A), and double donor (A + B {arrow} A) radiation bone marrow chimeras was assessed by the ability of their spleen cells to generate in vitro primary plaque-forming cell (PFC) responses to trinitrophenyl- keyhole limpet hemocyanin. In contrast to spleen cells from semiallogeneic and double donor chimeras, intact spleen cells from fully allogeneic BI0 {arrow} B10.A and B10.A {arrow} B10 chimeras were not capable of generating responses to trinitrophenyl (TNP)-keyhole limpet hemocyanin. However, cultures containing a mixture of both B10 {arrow} B10.A and B10.A {arrow} B10 spleen cells did respond, demonstrating that all the cell populations required for the in vitro generation of T-dependent PFC responses were able to differentiate into functional competence in a fully allogeneic major histocompatibility complex (MHC) environment. The self recognition repertoire of T-helper cells from fully allogeneic A {arrow} B chimeras was determined to be specific for the recognition of host, not donor, MHC determinants in that they were able to collaborate with cells expressing only host MHC determinants but not with cells expressing only donor MHC determinants, even though the functional lymphocytes in these chimeras were shown to be of donor origin. Experiments utilizing double donor A + B {arrow} A chimeras further demonstrated that the ability of chimeric T cells to recognize allogeneic MHC determinants as self structures was a function of a radiation-resistant host element and not simply a consequence of the tolerization of T cell precursors to allogeneic MHC determinants, because strain A lymphocytes isolated from A + B {arrow} A chimeras were tolerant to both A and B MHC determinants but were restricted to the self recognition of syngeneic host type A MHC determinants. Finally, the Ir gene phenotype expressed by B10 {arrow} B10.A and B10.A {arrow} B10 chimeric lymphocytes was determined by their ability to function in the Ir gene controlled response to TNP-poly-L-(Tyr,Glu)-poly-D,L-Ala-poly- L-Lys [(T,G)-A--L]. The ability of lymphocytes to function in TNP-(T,G)-A--L responses was not determined by their genotype but rather paralleled the specificity of their self recognition repertoire for high responder (H-2 (b)) determinants. The possible degeneracy of the MHC-specific self recognition repertoire is discussed, and a model is proposed for Ir gene regulation in which expression of Ir gene function by lymphocytes is an antigen-nonspecific consequence of the specificity and cross-reactivity of their self recognition repertoire.

Animals↗
PubMed1990

Strong T cell tolerance in parent----F1 bone marrow chimeras prepared with supralethal irradiation. Evidence for clonal deletion and anergy.

T cell tolerance induction was examined in long-term H-2-heterozygous parent----F1 chimeras prepared with supralethal irradiation (1,300 rad). Although these chimeras appeared to be devoid of host-type APC, the donor T cells developing in the chimeras showed marked tolerance to host-type H-2 determinants. Tolerance to the host appeared to be virtually complete in four assay systems: (a) primary mixed lymphocyte reactions (MLR) of purified lymph node (LN) CD8+ cells (+/- IL-2); (b) primary MLR of CD4+ (CD8-) thymocytes; (c) skin graft rejection; and (d) induction of lethal graft-vs.-host disease by CD4+ cells. Similar tolerance was observed in chimeras given double irradiation. The only assay in which the chimera T cells failed to show near-total tolerance to the host was the primary MLR of post-thymic CD4+ cells. In this assay, LN CD4+ cells regularly gave a significant antihost MLR. The magnitude of this response was two- to fourfold less than the response of normal parental strain CD4+ cells and, in I-E(-)----I-E+ chimeras, was paralleled by approximately 70% deletion of V beta 11+ cells. Since marked tolerance was evident at the level of mature thymocytes, tolerance induction in the chimeras presumably occurred in the thymus itself. The failure to detect host APC in the thymus implies that tolerance reflected contact with thymic epithelial cells (and/or other non-BM-derived cells in the thymus). To account for the residual host reactivity of LN CD4+ cells and the incomplete deletion of V beta 11+ cells, it is suggested that T cell contact with thymic epithelial cells induced clonal deletion of most of the host-reactive T cells but spared a proportion of these cells (possibly low affinity cells). Since these latter cells appeared to be functionally inert in the thymus (in contrast to LN), we suggest that the thymic epithelial cells induced a temporary form of anergy in the remaining host-reactive thymocytes. This anergic state disappeared when the T cells left the thymus and reached LN.

Animals↗