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PubMed1992

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

Amino Acid Sequence↗
PubMed2003

Detection and analysis of spliced chimeric mRNAs in sequence databanks.

We have developed a databank screening procedure, the In Silico Trans-splicing Retrieval System (ISTReS), to identify heterologous, spliced mRNAs with potential origin from chromosomal translocations, mRNA trans-splicing and multi-locus transcription. A parsing algorithm to screen cDNA versus genome Blast outputs was implemented. Key filtering criteria were Blast scores of > or =300, match lengths of > or =95% of the query sequences, junction of the two partners at exon-exon borders and concordant 'sense/sense' reading orientation. ISTReS was validated by the successful identification of bona fide chromosomal translocation-derived fusion transcripts in the HGI and RefSeq databanks. The performance of ISTReS was verified against recently identified chimeric antisense transcripts, where it revealed essentially no independent proof of antisense transcription and absence of exon-exon borders at the chimeric join, consistent with an artefactual origin. Analysis of the UNIGENE database revealed 21 742 chimeric sequences overall that correspond to approximately 1% of the database transcripts. Novel FOP-Rho GAP and methionyl tRNA synthetase-advillin chimeric mRNAs with the canonical features of heterologous-genes spliced-transcripts were identified among 246 chimeras from the RefSeq databank. This suggests a frequency of canonically-spliced chimeras of approximately 1% of all the hybrid sequences in current databanks. These findings demonstrate the efficiency of ISTReS and the overall feasibility of sequence/structure-based strategies to search for chimeric mRNAs candidate to derive from the splicing of heterologous transcripts.

Algorithms↗
PubMed1996

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system.

In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613). We have defined, genetically constructed and expressed the chimeric protein in Escherichia coli. We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary cells. Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine. These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A. These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.

ADP Ribose Transferases↗
PubMed2002

Reconstitution of a chicken breed by inter se mating of germline chimeric birds.

Blastoderm cells from chicken embryos of a donor breed (Green-legged Partridgelike; GP) were transferred to embryos of a recipient breed (White Leghorn; WL) to form chimeric progeny that, after inter se mating, permitted successful reconstitution of the donor breed. Among 23 chimeric chicks hatched from WL embryos injected with GP cells, 20 (87%) were raised until maturity, and progeny were tested by mating with GP birds to determine the ability of blastodermal cells to form germline chimeras. Six of the tested birds (30%) produced recipient-derived and donor-derived offspring, indicating that they were germline chimeras. The mean percentages of donor-derived germ cells in these birds were 21.1 (17.6 to 50.0%) and 16.9 (5.3 to 23.1%) in males and females, respectively. Among 477 chicks, resulting from mating the germline chimeric male with four germline chimeric females, 10 chicks (2.1%) exhibited a GP phenotype, indicating that the original donor stock had been reconstituted. Only one germline chimeric hen produced GP offspring, but the expected and calculated percentages of GP offspring were similar (2.99 and 2.08, respectively). Two methods of DNA analyses (RFLP and PCR amplification of polymorphic microsatellite loci) of chimeras and their offspring indicated that through mating of a relatively small number of chimeras it is possible to reconstitute a highly diverse population.

Animals↗
PubMed1997

Chimeric strategies for the rational design of bioactive analogs of small peptide hormones.

The aim of this study was to evaluate a variety of synthetic strategies pertinent to the development of chimeric analogs of the structurally divergent nonapeptide hormones arginine vasopressin (AVP) and bradykinin (BK). Single-chain peptides combining AVP and BK directly, AVP(1-9)-BK(1-9) or via a flexible aminohexanoic acid (epsilonAhx) linker, AVP(1-9)-epsilonAhx-BK(1-9), bind with relatively high affinity to the bovine kidney medulla B2a bradykinin receptor (B2a BKR). Significantly, amino-terminal extended chimeric analogs of BK, including AVP(1-9)-BK(1-9) and galanin(1-13)-BK(1-9), are functional B2 BKR agonists. These findings illustrate that chimeric peptides can activate G-protein-coupled receptors (GPCRs) in a manner analogous to that of endogenous monomeric agonists. Further development, combining the sequences of receptor subtype-selective antagonists, produced high-affinity chimeric antagonists of the V1a vasopressin receptor (V1a VPR) and the B2a BKR. We also determined the pharmacological characteristics of high-affinity chimeric hormone analogs derivatized with the membrane targeting function of mastoparan. Homodimers of an amino-terminal extended BK analog and a V1a-selective antagonist represent the first examples of new classes of B2 BKR and V1a VPR antagonists, respectively. These findings are discussed in relation to the GPCR binding site for small peptides and the development of novel biological probes and therapeutic agents.

Amino Acid Sequence↗
PubMed1999

Characterization of a chimeric T-cell receptor with specificity for the Hodgkin's lymphoma-associated CD30 antigen.

Recombinant receptors with antibody-like specificity for tumor-associated antigens were shown to direct specifically T cells to target tumor cells. Hodgkin and Reed-Sternberg cells, the malignant cell population in Hodgkin's lymphoma, express high amounts of the cell surface antigen CD30. An anti-CD30 T-cell receptor with cellular activation properties is expected to graft T cells with specificity to Hodgkin cells. Here, the authors characterize a chimeric T-cell receptor with an extracellular domain consisting of the single-chain antibody fragment HRS3-scFv with specificity for the CD30 antigen and intracellular domain of the signal transducing part of the Fc-epsilon-I-gamma receptor. The HRS3-scFv was derived from the monoclonal anti-CD30 antibody HRS3 and retained specificity for the CD30 antigen. The recombinant HRS3-scFv-gamma receptor was expressed under control of the RSV-LTR after transfection into MD45 T-cells. The chimeric receptor protein is detected and analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Expression of the chimeric receptor converts MD45 T cells to specificity for CD30+ lymphoma cells. Specific cross-linking of the chimeric receptor with antigen resulted in cytolytic reactivity against CD30+ tumor cells in vitro. The results demonstrate that the chimeric receptor HRS3-scFv-gamma converts T cells to a specific MHC-unrestricted cytolytic response against CD30+ tumor cells offering an alternative strategy in cellular immunotherapy of Hodgkin's disease.

Animals↗
PubMed1993

Free combined composite flaps using the lateral circumflex femoral system for repair of massive defects of the head and neck regions: an introduction to the chimeric flap principle.

Chimeric composite flaps combined using microanastomoses consist of two or more flaps or tissues, each with an isolated pedicle and a single vascular source. Free combined chimeric flaps using the lateral circumflex femoral system were used to treat massive composite defects of the head and neck in 10 cases. A combined anterolateral thigh flap and vascularized iliac bone graft based on the lateral circumflex femoral system and the deep circumflex iliac system was the most commonly used combination. An anteromedial thigh flap and a paraumbilical perforator-based flap were also combined with this principal combination. The advantages of this chimeric flap over other osteocutaneous flaps are: (1) The flap is relatively thin and the pedicle vessels are up to 10 cm longer and are wider than those of other flaps for easier harvesting of the flap. (2) It is unnecessary to reposition the patient. (3) The vascular pedicle to the skin can be separated from that of the bone. (4) The donor site is not close to the recipient site. (5) The donor scar is in an unexposed area. (6) The skin territory of this flap is extremely wide. (7) A combined anterolateral and anteromedial thigh flap and vascularized iliac bone graft can be easily obtained as an extended combined osteocutaneous flap. (8) Other neighboring skin flaps, such as a groin flap, a paraumbilical perforator-based flap, or a medial thigh flap, can be combined with this chimeric flap because several major muscle branches to be anastomosed derive from the lateral circumflex femoral system. Chimeric composite flaps using the lateral circumflex femoral system are considered suitable for the repair of massive composite defects of the head and neck.

Adult↗
PubMed1990

Allogeneic versus semiallogeneic F1 bone marrow transplantation into sublethally irradiated MHC-disparate hosts. Effects on mixed lymphoid chimerism, skin graft tolerance, host survival, and alloreactivity.

In models of tolerance associated with mixed lymphoid chimerism, depletion of Thy 1+ cells from the allogeneic donor inoculum may decrease the level of chimerism achieved and the capacity of donor cells to induce tolerance. To determine whether the apparent role of Thy 1+ cells in the facilitation of bone marrow engraftment and induction of skin graft tolerance is related to alloaggression, the capacity of fully allogeneic C57BL/6J, H-2b BM cells to establish mixed lymphoid chimerism and skin graft tolerance in sublethally irradiated (2.5 Gy x 3) BALB/c, H-2d hosts was compared with that of semi-allogeneic BALB/c x C57BL/6J F1 H-2d/b BM cells which genetically lack the potential for graft-versus-host reactivity against parental recipients. The levels of mixed chimerism observed with allogeneic and semi-allogeneic F1 BM cells were nearly identical: 21.0 +/- 9.7% of spleen cells in H-2b BM-injected and 18.6 +/- 8.8% of spleen cells in H-2d/b BM-injected H-2d hosts were of donor allotype. There was no difference in the fraction of hosts rendered tolerant to C57BL/6J, H-2b skin grafts by H-2b vs. H-2d/b BM at either excess (94% vs. 92% tolerant) or threshold (37% vs. 40% tolerant) numbers of donor cells. Spleen cells from both types of mixed chimeras failed to respond to donor antigens in MLR. Both H-2b and H-2d/b BM-injected H-2d hosts rejected third party C3H/HeJ, H-2k skin grafts and responded to third party stimulators in MLR. Although these nonspecific allo-immune responses were not as strong as the responses of normal animals, they were suppressed to an equivalent degree in both types of chimeras. Graft-versus-host disease, if present in irradiated H-2b BM-injected hosts, did not significantly affect survival compared with survival of irradiated H-2d/b BM-injected animals. These results suggest that the tolerizing capacity of allogeneic BM does not depend upon GVHD and that allogeneic and semi-allogeneic BM establish mixed lymphoid chimerism and induce skin graft tolerance by similar mechanisms across a complete MHC disparity in sublethally irradiated adult hosts.

Animals↗
PubMed1992

The requirement for allogeneic chimerism for second transfer of tolerance from mixed allogeneic chimeras (A+B-->A) to secondary recipients.

We have applied the model of mixed allogeneic chimerism (A+B-->A), in which stem cells from both allogeneic and syngeneic donor engraft, to determine the in vivo cellular requirements for transfer of tolerance from mixed chimeras to secondary recipients. Using two approaches, we have demonstrated that the persistence of donor-specific transplantation tolerance is dependent on the presence of bone-marrow-derived cells. When untreated bone marrow from mixed chimeras was transferred to irradiated secondary recipient mice, most of the secondary recipients were rescued, but only 48% were demonstrably chimeric. This pattern of repopulation, therefore, allowed us to examine whether chimerism was required to maintain transplantation tolerance. In all of our studies, the presence of allogeneic chimerism was required for successful transfer of tolerance from mixed allogeneic chimeras to irradiated secondary recipients. Only those secondary recipients which repopulated with demonstrable allogeneic chimerism exhibited in vivo and in vitro evidence for transfer of donor-specific transplantation tolerance. These results were confirmed by using transfer of bone marrow from mixed chimeras depleted of allogeneic class I elements. In an attempt to identify a putative population of suppressor cells, second transfer of splenic lymphoid cells from mixed allogeneic chimeras, containing approximately 6 times more T-lymphocytes that were functionally tolerant to donor alloantigens, was also performed with similar results. These data suggest that the in vivo maintenance of tolerance to MHC transplantation alloantigens requires persistence of donor bone marrow-derived alloantigens.

Animals↗
PubMed1996

Additional monoclonal antibody (mAB) injections can replace thymic irradiation to allow induction of mixed chimerism and tolerance in mice receiving bone marrow transplantation after conditioning with anti-T cell mABs and 3-Gy whole body irradiation.

While allogeneic bone marrow transplantation (BMT) has long been known to be capable of inducing donor-specific tolerance and hence permitting allograft acceptance without immunosuppressive pharmacotherapy, the toxicity of conditioning regimens required to achieve marrow engraftment has precluded the application of this approach to clinical organ transplantation. A relatively nontoxic method of conditioning mice that allows allogeneic bone marrow engraftment and induction of donor-specific skin allograft tolerance has recently been described. This regimen included anti-CD4 and anti-CD8 mAbs administered on day -5, followed by 3-Gy whole body irradiation (WBI) and 7-Gy thymic irradiation (TI) on day 0. To further reduce the potential toxicity of this regimen, we have now attempted to overcome the requirement for TI by administering additional mAb injections before and after BMT. Mixed chimerism and prolonged donor-specific skin graft acceptance were induced in 90% of B10 mice conditioned with anti-CD4 and -CD8 mAbs on days -6 and -1 and 3-Gy WBI on day 0 without TI. Despite long-term acceptance of donor-specific skin grafts, however, some of these animals showed a gradual decline in donor-type hematopoietic repopulation, and 2 of 10 mice regrafted with a second donor-type skin graft 5-9 months after BMT rejected the second and/or the original graft. This rejection after repeat donor-specific skin grafting correlated with a decline in the percentage of donor-type T cells between 6 and 12 weeks after BMT. In contrast, all animals receiving additional mAb injections 7 and 14 days following BMT after conditioning with mAbs on days -6 and -1 and 3-Gy WBI showed stable chimerism and accepted both primary and secondary donor-specific skin grafts. Animals receiving TI in addition to mAb and 3-Gy WBI also showed stable chimerism and long-term acceptance of initial (at 7 weeks) and later repeat donor-specific grafts. In contrast, the majority of mice receiving mAbs only on day -5 or on day -1 only, followed by 3-Gy WBI on day 0 without TI, did not accept initial donor-specific skin grafts, and showed only transient chimerism. Thus, the requirement for thymic irradiation to allow permanent mixed chimerism and donor-specific tolerance induction can be overcome by the administration of additional T cell-depleting mAb injections. These results establish a less toxic method of inducing donor-specific tolerance, thus increasing the potential clinical applicability of this approach to inducing organ allograft acceptance without chronic immunosuppressive therapy.

Animals↗
PubMed1996

Neonatal transplantation tolerance is associated with a systemic reduction in memory cells, altered chimeric cell phenotype, and modified eicosanoid and cytokine production.

Certain B10 background mice are resistant to tolerance induction following a neonatal inoculation of semiallogeneic class I/II MHC-disparate cells despite early thymic clonal deletion of alloreactive cells. The emergence of memory T cells and persistence of particular chimeric cells in the thymus has an association with this resistance. In these studies, we utilized a hemisplenectomy technique to examine systemic cell populations of adult Bl0.S (H2s, H2E-) mice that received (Bl0.S x B10.A)F1 cells at birth and before and following application (and rejection or acceptance) of Bl0.A (H2k/d, H2E+) skin grafts. Prior to skin graft challenge, tolerant mice had reduced splenic levels of memory (CD45hi, PgP-1hi, Mel-14neg) T cells as compared with the rejecting recipients and following B10.A graft challenge, the nontolerant mice showed a further increase in these cells. Elevated pretransplant levels of donor H2Kk+ cells coexpressing B220, CD11b, or CD3 were seen in the tolerant mice. Following skin grafting, splenic chimerism was reduced with differing chimeric cell phenotypes between the tolerant and nontolerant mice. In vitro production of PGE2 in a MLC was delayed in the tolerant mice with minimal production of IL-2 and IL-4. Nontolerant mice made high levels of TxB2 and heightened, early production of IL-2 and IL-4 during the MLC. Thus, tolerance induction is associated with increased numbers of particular chimeric cells, fewer peripheral lymphoid immunocompetant memory T cells, impaired eicosanoid secretion, and reduced alloreactivity and alloantigen-driven IL-2/IL-4 production. It appears that alloreactive cells necessary to break tolerance are generated when fewer class II+ (e.g., B220+, CD11b+) chimeric cells are present and that there is a coexistence of effector and regulatory T cell subpopulations in the nontolerant mice. By comparison, tolerance acquisition does not appear associated with the presence or generation of a predominant subtype of T cell but rather is likely more dependent upon clonal deletion processes.

Animals↗
PubMed1997

Lung rejection occurs in lung transplant recipients with blood chimerism.

BACKGROUND: It has been postulated that chimerism after transplantation might promote graft acceptance. In the present study, we prospectively assessed blood chimerism in 10 lung transplant recipients during the first posttransplant year and investigated whether chimerism was associated with an immunologically stable situation of the graft. METHODS: The recipients' peripheral blood mononuclear cells were obtained before transplantation and at various time points during the first postoperative year. Donor cells were detected using nested polymerase chain reaction amplification of a donor-specific HLA-DRB1 allele. Clinical graft acceptance was determined by the number of rejection episodes. RESULTS: The incidence of blood chimerism was high during the first 3 postoperative months and then decreased over time. All patients experienced at least one acute rejection episode, and three patients developed chronic rejection. CONCLUSION: We, thus, conclude that rejection of the lung allograft may occur in the presence of blood chimerism.

Adolescent↗
PubMed1999

Origin, kinetics, and function of chimeric B lymphocytes in liver allografts.

BACKGROUND: Despite the well-recognized concordance of chimerism with spontaneous acceptance of rat liver allografts, the active role and the identity of chimeric cells mediating liver allograft tolerance are unknown. Because resting B cells are endowed with a tolerogenic antigen-presenting capacity, we assessed whether donor B cells propagated from the grafted liver may be responsible for liver allograft tolerance. METHODS: Dark Agouti or Lewis rats were grafted with Lewis or Dark Agouti livers as a tolerogenic or a rejection combination, respectively. We followed the kinetics of donor B cells in recipients by flow cytometry, and we examined the fate of liver allografts depleted of passenger B cells in either B cell-sufficient or -deficient recipients. B-cell depletion was achieved by treatment of animals with polyclonal goat anti-rat IgM antibody from birth. RESULTS: During the first 3 days after liver allografting, donor B cells rapidly migrated from graft-infiltrating cells and appeared in systemic circulation in both the tolerogenic and rejection combinations. However, systemic chimerism was detectable in the tolerogenic combination by day 14, whereas it was undetectable in the rejection combination by day 7. In graft-infiltrating cells, a significant expansion of chimeric IgM+ (newly formed) B cells was observed on day 5 in the tolerogenic, but not in the rejection, combination. However, depletion of B cells from liver grafts and the absence of antibodies failed to alter the outcome of liver allograft survival in the tolerogenic or immunogenic combination. CONCLUSION: Although intragraft chimeric B cells proliferated in tolerogenic liver allografts, their clonal expansion does not seem to be essential for the promotion of liver allograft tolerance.

Animals↗
PubMed2000

Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation.

BACKGROUND: Induction of tolerance to organ transplants will increase graft survival and decrease patient mortality and morbidity. Radiation-induced cytoreduction/ablation followed by donor hematopoietic cell reconstitution has been the most consistently successful approach to experimental tolerance induction. However, reluctance of clinicians to expose recipients to radiation has hampered its clinical application. METHODS: In the studies described, administration of polyclonal antilymphocyte serum (ALS), donor-specific bone marrow (DSBM) (150x10(6) cells), and sirolimus (24 mg/kg) in a completely mismatched murine model (B10.A donor, C57B/10 recipient) produced 100% indefinite (>250 days) skin graft survival. The level and character of donor-specific chimerism was evaluated with flow cytometry. RESULTS: Specific tolerance was confirmed by continued acceptance of primary and secondary donor-specific skin allografts and rejection of third-party grafts. The level and duration of chimerism induced was directly related to the dose of DSBM administered. Mice given 150x10(6) DSBM cells showed levels of 8-10% donor peripheral blood mononuclear cell chimerism by 30 days, and these levels persisted indefinitely (>250 days) in association with permanent tolerance of donor grafts. Eighty percent of donor chimeric cells were B lymphocytes (MHC class I and II positive, Fc receptor positive, CD45/B220 positive but negative for CD4, CD8 and Thy 1.2) and 20% were sorted in the macrophage monocyte population. CONCLUSIONS: These studies demonstrate for the first time that cytoreduction/ablation with ALS combined with sirolimus and reconstitution with donor bone marrow induces tolerance and chimerism in a completely mismatched murine combination. The use of ALS and sirolimus, currently employed therapies in clinical transplantation, and the lack of requirement for radiation make this tolerance protocol attractive for clinical application.

Animals↗
PubMed2000

Association of natural killer cell depletion with induction of mixed chimerism and allograft tolerance in non-human primates.

BACKGROUND: Nonmyeloablative T cell depletion followed by donor bone marrow infusion has proved to be an effective approach to induction of mixed chimerism and tolerance of organ allografts in non-human primates. To help define the mechanisms involved we have compared T cell depletion with ATG versus anti-CD2 monoclonal antibody with respect to establishment of mixed chimerism and induction of tolerance. METHOD: Both nonmyeloablative regimens included low dose total body irradiation (1.5 Gy x 2), thymic irradiation (7 Gy), splenectomy and kidney plus donor bone marrow transplantation, followed by a 4-week posttransplant course of cyclosporine. In addition, the ATG group (13 recipients) received antithymocyte globulin, although the LOCD2b group (10 recipients) were treated with an anti-CD2 monoclonal antibody (LOCD2b). RESULTS: In the ATG group, 11 of 13 monkeys developed multilineage chimerism and 9 survived for more than 100 days without kidney allograft rejection. In contrast, 0/10 monkeys in the LOCD2b group developed chimerism, 5 died of infection and 5 suffered progressive rejection; only 1 recipient survived beyond 100 days. Sequential monitoring of peripheral blood mononuclear cells revealed greater T cell (CD3+) depletion in the LOCD2b-treated animals compared to those receiving ATG. However, NK cells (CD16+CD8+) were significantly more depleted in the ATG group and NK function remained abrogated longer after ATG than LOCD2b treatment (3 weeks vs. <5 days). CONCLUSION: Despite excellent T cell depletion by LoCD2b, ATG was more effective in inducing chimerism and tolerance. This difference correlated with anti-NK activity of the two reagents. These data suggest that NK cells may also resist engraftment of allogeneic bone marrow cells in this model.

Animals↗
PubMed2001

Will mixed chimerism cure autoimmune diseases after a nonmyeloablative stem cell transplant?

BACKGROUND: Mixed chimerism after allogeneic bone marrow transplantation has been shown to cure a number of genetic disorders in both the clinical and experimental settings. Although encouraging results have been reported from animal experiments, the role of mixed chimerism in eliminating autoimmune disorders is not clear. METHODS: A 50-year-old man with extensive psoriasis received an allogeneic transplant from his brother after nonmyeloablative conditioning with fludarabine, melphalan, and Campath-1H for relapsed non-Hodgkin's lymphoma. The chimerism status and the immunological recovery after the transplant were serially monitored. RESULTS: Twenty-one months after the transplant, the patient continues to be in complete remission from psoriasis and lymphoma with stable mixed chimerism (30% to 40% donor cells), despite significant recovery of T-cell subsets and antigen-specific response. CONCLUSIONS: If mixed chimerism can be achieved safely with novel low-intensity conditioning regimens and results in sustained remission of autoimmune diseases, allogeneic transplantation may become a realistic therapy in the management of some patients with autoimmune disease.

Alemtuzumab↗
PubMed2002

Effect of living-related donor bone marrow infusion on chimerism and in vitro immunoregulatory activity in kidney transplant recipients.

BACKGROUND: In a previously reported series of donor-specific bone marrow cell (DBMC) infusions in cadaver kidney transplant recipients, there appeared to be an improvement in long-term graft survival (6 years) and fewer chronic rejections, which correlated with increasing DBMC chimerism (approximately 1.4% in the iliac crest bone marrow compartment now at 6 years). Prompted by this, we embarked on a study of DBMC infusion in living-related donor (LRD) kidney transplant recipients. METHODS: Between November 1996 and May 2000, 47 LRD kidney transplant recipients received donor iliac crest marrow (1.8 x 10(8)+/-1.9 x 10(8) cells/kg body weight+/-SD) in a single infusion 4 days postoperatively. Either OKT3 (n=26) or daclizumab (n=21) were used for induction therapy, with maintenance tacrolimus, mycophenolate mofetil, and methylprednisolone immunosuppression. These recipients were prospectively compared with 39 noninfused LRD kidney transplants (control group), which received equivalent immunosuppression in the same time period. Clinical follow-up ranged from 19.0 months to 61.6 months (mean 33.2 months). Polymerase chain reaction-flow chimerism analysis and in vitro assays of immunoregulatory activity of chimeric cells were performed. RESULTS: The incidence of acute rejection over this period of time was 10.6% and 10.3%, respectively (i.e., did not differ between groups). Immunosuppressive dosages were somewhat (but not statistically) lower over time in the DBMC group. Four-year actuarial patient and graft survival for the DBMC-infused group was 98% and 98%, and 98% and 95% for the control group, respectively ( =NS). DBMC infusion was well tolerated, with no increase in infectious episodes. DBMC chimerism in recipient iliac crest marrow has increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused. DBMCs and (donor) peripheral blood mononuclear cells purified by immunobeads from recipient blood or bone marrow (recipient-derived donor cells) inhibited mixed leukocyte responses of the recipient to the donor more strongly than freshly obtained peripheral blood cells drawn from the donors or even compared with bone marrow cells aspirated from the donors in a previously reported group of experiments. Additionally, similarly purified recipient-derived recipient cells from the same chimeric recipient more strongly inhibited the same mixed leukocyte response reactions autologously than a large group of nonchimeric (autologous) bone marrow modulating cells in similar reactions. CONCLUSIONS: These observations confirm that an immunoregulatory process appears to have been generated by DBMC infusion, encouraging a further decrease in immunosuppressive dosing using such assays in the future.

Adult↗
PubMed2003

Donor lymphocyte infusion-mediated graft-versus-leukemia effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of graft-versus-leukemia effects after conversion to full donor chimerism.

BACKGROUND: We investigated an approach to separating graft-versus-lymphoma (GVL) effects from graft-versus-host disease (GVHD) in mice receiving a nonmyeloablative conditioning regimen allowing establishment of mixed hematopoietic chimerism. METHODS: We evaluated the ability of donor lymphocyte infusions (DLI) to mediate GVL effects without GVHD in mixed chimeras prepared with cyclophosphamide, anti-T-cell antibodies, and thymic irradiation. To examine the fate of GVH-reactive donor CD8+ T cells, we used the 2C T-cell receptor (TCR) transgenic mouse strain, which carries an Ld-specific transgenic TCR on the B6 background. RESULTS: Administration of DLI on day 35 post-BMT led to conversion from mixed to full donor chimerism and mediated a powerful GVL effect with complete protection (100% survival) against mortality induced by a host-type lymphoma (EL4) administered 7 days later (100% mortality in non-DLI controls; P<0.001). No GVHD occurred in DLI recipients. Rechallenging the surviving DLI recipients, which had converted to full chimerism, with the same tumor dose 17 weeks later led to rapid tumor mortality. Long-term DLI recipients had anti-host proliferative responses, but not CTL responses in vitro. When given as DLI together with wild-type spleen cells, marked expansion of GVH-reactive 2C CD8+ T cells was observed on day 10, followed by a marked decline in their numbers by week 10 post-DLI. CONCLUSIONS: Nonmyeloablative induction of mixed chimerism followed by administration of DLI can mediate powerful GVL effects. The late loss of DLI-mediated GVL effects may reflect the eventual loss of donor-derived GVH-reactive CTL, which occurs in association with conversion to full donor chimerism.

Animals↗