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PubMed1994

A new vector for the high level expression of chimeric antibodies in myeloma cells.

We previously reported the expression of a mouse/human chimeric anti-ganglioside GD3 antibody, KM871 (IgG1,kappa) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named 'a tandem vector, pChi641HLGM4' was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50-200 nM methotrexate gave rise to several clones that stably secreted 70-100 micrograms/10(6) cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2-3 months to establish the transfectant clones which stably produced the chimeric antibody.

Animals↗

PubMed2003

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.

Anemia, Hemolytic↗

PubMed2004

Chimerism studies in HLA-identical nonmyeloablative hematopoietic stem cell transplantation point to the donor CD8(+) T-cell count on day + 14 as a predictor of acute graft-versus-host disease.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4(+) and CD8(+) T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4(+) and CD8(+) donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P =.0002 and P =.019, respectively). The rate of disappearance of recipient CD8(+) T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P =.016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8(+) T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P =.003).

Acute Disease↗

PubMed2004

Complete allogeneic hematopoietic chimerism achieved by in utero hematopoietic cell transplantation and cotransplantation of LLME-treated, MHC-sensitized donor lymphocytes.

OBJECTIVE: In utero hematopoietic cell transplantation (IUHCT) typically achieves low-level mixed hematopoietic chimerism. However, the goal of IUHCT is to achieve therapeutic levels of chimerism. We hypothesized that prenatal adoptive immunotherapy might achieve high-level donor chimerism after IUHCT. MATERIALS AND METHODS: BALB/CE15 fetal mice were transplanted with a mixture of C57BL/6 (B6) T-cell-depleted bone marrow (TCD BM) cells and splenocytes from B6 mice presensitized to BALB/C alloantigen. The splenocytes were preincubated in L-leucyl-L-leucine methyl ester (LLME), to minimize graft vs host disease (GVHD). Recipients were followed after birth for donor cell chimerism and GVHD. RESULTS: Full donor hematopoietic chimerism following a single prenatal transplant was achieved in seven transplanted animals. Fully chimeric animals were healthy, without evidence of GVHD, and maintained their engraftment for the duration of the study (48 weeks). However, the addition of presensitized LLME-treated cells decreased survival until weaning relative to TCD BM alone, suggesting that some animals were lost to acute GVHD. Surviving chimeric animals demonstrated increased frequencies of T-regulatory cell populations in their spleen and BM, suggesting that they had successfully suppressed GVHD, allowing survival. CONCLUSIONS: This study represents "proof in principle" that prenatal immunotherapeutic strategies may achieve complete hematopoietic engraftment across full MHC barriers when combined with IUHCT. However, strategies with greater hematopoietic specificity must be developed prior to consideration of clinical application.

Animals↗

PubMed2006

Prenatal transplantation of cytokine-stimulated marrow improves early chimerism in a resistant strain combination but results in poor long-term engraftment.

OBJECTIVE: In the absence of immunodeficiency, only microchimerism (<0.1%) has been achieved in human fetal recipients or nonhuman primates following in utero hematopoietic cell transplantation (IUHCT). We hypothesized that enhanced long-term engraftment might be more reliably achieved in microchimeric systems if higher levels of chimerism existed during development of adaptive immunity. To evaluate this hypothesis, we stimulated the donor cells with vascular endothelial growth factor (VEGF) and stem cell factor (SCF) prior to IUHCT in a chimerism-resistant murine strain combination. METHODS: Donor Balb/c marrow was cultured in media with or without VEGF and SCF supplementation for 12 hours prior to IUHCT into B6 fetuses at 14 days postcoitum (dpc). Donor cell phenotype, homing, and chimerism were assessed at short and long-term time points and transplanted animals received skin allografts at 8 weeks. RESULTS: In pretreated allogeneic recipients, early chimerism rates were more than double that of controls (71% vs 33%, p = 0.01). These differences were associated with higher numbers of pretransplant donor cell colony-forming cells without change in donor cell homing. Despite prolonged skin allograft survival for pretreated recipients compared with controls (mean survival = 20.8 vs 8.2 days, p < 0.001), long-term engraftment was unchanged. CONCLUSIONS: These findings demonstrate that higher levels of early chimerism in recipients of cytokine-stimulated marrow result in improved short-term chimerism and tolerance. Future studies are needed to confirm the existence of a "threshold" level of chimerism necessary to sustain long-term engraftment.

Animals↗

PubMed2005

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers.

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.

Animals↗

PubMed2005

The immunogenicity and protective efficacy of Mtb8.4/hIL-12 chimeric gene vaccine.

DNA vaccination has emerged as a powerful approach in the search for a more efficacious vaccine against tuberculosis (TB). In this study, we evaluated the immunogenicity and protective efficacy of Mtb8.4/hIL-12 chimeric gene vaccine. The Mtb8.4/hIL-12 chimeric gene was amplified by PCR and cloned into the eukaryotic expression vector pCI-neo. C57BL/6N mice were vaccinated with Mtb8.4/hIL-12 chimeric gene vaccine for three times at 3 weeks intervals. Four weeks after the final inoculation, three mice per group were sacrificed to assess cytokine response and CTL induction and the other five mice per group were challenged intravenously in a lateral tail vein with 1 x 10(6) CFU of virulent Mycobacterium tuberculosis H37Rv. Spleen and the left lung were harvested from each mouse at 4 weeks after infection and homogenized in sterile saline. Serial dilutions of organ homogenates were plated on L-J agar and incubated 37 degrees C until colonies were visible 4 weeks later. Protective efficacies in each experiment were expressed as reduced CFU and were compared with the negative control group. The right lung was obtained from each mouse and immediately inflated with and stored in 10% formalin saline. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Mtb8.4/hIL-12 chimeric gene vaccine induced the secretion of more of Th1 cytokines, but not IL-4 and enhanced CTL activity. Mice immunized with Mtb8.4/hIL-12 chimeric gene vaccine had fewer and smaller tubercles than control groups. As expected, control mice had the highest bacterial loads in both lung and spleen. Immunization with Mtb8.4/hIL-12 chimeric gene vaccine could remarkably reduced CFU counts in organs. When it was used to construct the chimeric gene vaccine, hIL-12 could improve the immune efficacy of Mtb8.4 gene vaccine.

Animals↗

PubMed2004

Development of a new monoclonal antibody to ovine chimeric IgE and its detection of systemic and local IgE antibody responses to the intestinal nematode Trichostrongylus colubriformis.

The J558L cell line, previously transfected with the ovine Cepsilon gene, was induced to secrete a chimeric IgE protein composed of the ovine heavy chain and a mouse light chain with MW of approximately 80 and 26 kDa, respectively. After purification, the chimeric protein was used to immunise BALB-c mice and monoclonal antibodies (mAbs) were generated. The mAb 2F1, which had greatest anti-IgE activity in preliminary screens, was chosen for further characterisation and an examination of systemic and local IgE responses to the intestinal nematode, Trichostrongylus colubriformis. The chimeric IgE protein was not recognised in enzyme linked immunosorbent assay (ELISA) by mAbs raised against ovine IgG1, IgG2, IgA or IgM. However, 2F1 was highly specific to the chimeric IgE protein, and did not cross-react with ovine IgG1, IgG2 or IgA. Western blot analysis also showed that 2F1 and secretory IgA (sIgA) did not cross-react, and that 2F1 and the anti-IgA mAb identified different MW bands from colostrum (approximately 200 and 400 kDa, respectively). 2F1 bound to mucosal mast cells (MMC) isolated from the intestines of lambs infected with T. colubriformis, but cultured bone marrow-derived mast cells (BMMC) required prior incubation with the chimeric IgE protein for this binding to occur. Distinctive staining of plasma cells and putative mast cells were observed using 2F1 on immunohistological sections of mesenteric lymph node and jejunum.ELISA incorporating 2F1 was able to detect >0.4 ng chimeric protein. Total IgE in ovine colostrum and intestinal homogenates was quantified using a capture ELISA, with known amounts of chimeric protein used to produce a standard curve. Colostrum from outbred Merino ewes had 0.55-11.05 ng ml(-1) total IgE, and their lambs, at necropsy after infection with a total of 18,000 T. colubriformis infective larvae over a 9-week period, had 45-620 ng g(-1) total IgE in intestinal tissue. Compared to genetically susceptible lambs, antigen-specific levels of IgE were significantly higher in genetically resistant lambs after infection with 4500 T. colubriformis infective larvae (TcL3) per week for 9 weeks (161.4 versus 44.8 geometric mean titres; P=0.043). In western blots, distinctive bands (19-21 and 27 kDa) from T. colubriformis larval antigen were differentially recognised by IgE, as identified by 2F1, in intestinal homogenates from genetically resistant animals. These results have demonstrated the value of 2F1 for quantification of IgE responses in samples derived from ovine fluids and tissues using ELISA, western blots and immunohistology. In this respect, it recognises native ovine IgE and does not require pre-treatment of the sample with denaturing agents or ammonium sulphate.

Animals↗

PubMed2006

Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein.

The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

AIDS Vaccines↗

PubMed2002

Bone marrow chimerism prevents atherosclerosis in arterial walls of mice deficient in apolipoprotein E.

OBJECTIVE: apolipoprotein E (apoE) is a key regulator in cholesterol-rich lipoprotein metabolism. Inherited deficiency of this protein results in type III hyperlipoproteinemia in humans. ApoE, especially that derived from macrophages, can efficiently protect against development of atherosclerotic lesion. To use stem cell gene therapy or mini-transplant in treating abnormal lipid metabolism and preventing atherosclerosis, a minimal level of bone marrow chimerism must be determined. METHODS: lethally irradiated apoE deficient mice (12-16 weeks of age) fed on normal chow were transplanted with normal bone marrow cells (C57BL/6.Ly5.1) mixed with those of apoE deficient mice (C57BL/6.Ly5.2) at various ratios. Plasma cholesterol levels were determined every 3 weeks for up to 42 weeks. Areas of atherosclerotic lesion in the aortas were quantified 6 months post-transplant. Plasma apoE was measured by Western blot analysis. RESULTS: bone marrow transplantation (BMT) in apoE (-/-) mice resulted in a detectable level of plasma apoE as determined by Western blot analysis. The plasma cholesterol levels in mice with > or = 60% chimerism were normalized by 6 weeks post-transplant. Mice with < or = 40% chimerism showed significant reductions, but not normalization, in the plasma cholesterol levels even at 42 weeks posttransplant. However, atherosclerotic areas observed in 10%-chimeric mice were significantly smaller than those in control mice (P<0.01). Immunohistochemical studies in 10%-chimeric mice revealed foam cells derived from donor marrow (apoE (+/+)) and expressed immunoreactive apoE in the atherosclerotic lesion. The positive signals by Western blot analysis were represented in the plasma of up to 8% of the chimeric mice. CONCLUSION: chimerism of 10%, the minimum level analyzed, was sufficient to reduce the severity of atherosclerosis, although the plasma cholesterol levels were not completely normalized. The results indicate that stem cell gene therapy and mini-transplant may provide possible therapeutic approaches to treat patients with abnormal lipid metabolism and atherosclerosis.

Animals↗

PubMed2003

Monitoring of hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation (alloSCT) by dual-color FISH analysis of X and Y chromosomes.

Hematopoietic chimerism was monitored in 18 patients with various diseases after gender-mismatched allogeneic stem cell transplantation (alloSCT). To detect host and donor cells, FISH analysis of sex chromosomes was applied. X and Y chromosomes were detected simultaneously in interphase nuclei by two-color probes. Chimerism was examined sequentially in post-transplant peripheral blood and bone marrow as well as in purified T cells. Patients with complete donor or decreasing host chimerism have not rejected or relapsed but experienced a high incidence of acute graft versus host disease (aGvHD). The clinical value of stable mixed chimerism detection remains uncertain. However, it appears to be associated with a lower risk of aGvHD. Three patients with an increase in host cells rejected their grafts. The immunotherapy was introduced to four other patients with increasing host chimerism. All of them responded, however, one relapsed in CNS despite the conversion to complete donor chimerism in both bone marrow and peripheral blood. We concluded that two-color FISH analysis of sex chromosomes was a valuable tool for chimerism monitoring and provided significant clinical data.

Acute Disease↗

PubMed1998

Properties of nicked and circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligodeoxynucleotides.

We have designed a new class of oligonucleotides, 'dumbbell RNA/DNA chimeric phosphodiester oligonucleotides', consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the Nicked (NDRDON) and Circular (CDRNON) dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 45 mer RNA, which gave 45 mer RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide (anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligodeoxynucleotide. The circularization, achieved by joining the 3' and the 5' ends of RNA/DNA chimeric oligonucleotides containing two hairpin loop structures, increases the oligonucleotide uptake into cells, as compared with the nicked dumbbell RNA/DNA chimeric oligonucleotide and the linear antisense phosphodiester oligodeoxynucleotides. When the circular dumbbell RNA/DNA chimeric oligonucleotide is directly delivered into retrovirus infected cells, its antisense phosphodiester oligodeoxynucleotide function appears.

DNA, Antisense↗

PubMed2002

A novel heparin-binding, human chimeric, superoxide dismutase improves myocardial preservation and protects from ischemia-reperfusion injury.

BACKGROUND: The plasma membranes of endothelial cells are sites of physiologic injury caused by superoxide attack, whether the radicals are generated within the cell (i.e., from enzymatic sources such as xanthine oxidase or from ischemically injured mitochondria) or are generated within the interstitial spaces by activated neutrophils or macrophages. An extracellular superoxide dismutase (ECSOD) electrostatically bound to endothelial surfaces partially protects against this oxidative attack. To provide a therapeutic equivalent of this ECSOD activity, we evaluated the product of a fusion gene encoding a chimeric manganese SOD (chimeric-SOD) and the carboxyl-terminal 26-amino acid basic "tail" from ECSOD with high affinity for heparin-like proteoglycans on cell surfaces. METHODS: We tested the chimeric-SOD in isolated rabbit hearts during warm and cold ischemia. RESULTS: When perfused through an isolated rabbit heart, chimeric-SOD bound to endothelial surfaces and was displaced by a bolus dose of heparin. In an established model of no-flow ischemia followed by reperfusion of the isolated rabbit heart, the chimeric-SOD was as protective as native Mn-SOD or Cu,Zn-SOD, but at doses nearly 2 orders of magnitude lower. In a rabbit-heart preservation model, the chimeric-SOD provided better recovery of function after 4 hours of cold ischemia than did University of Wisconsin cardioplegia solution. CONCLUSION: This chimeric-SOD can bind to cell surfaces and may aid in preventing superoxide-mediated endothelial damage and may function as a rational therapeutic agent for treating free-radical-mediated diseases.

Analysis of Variance↗

PubMed2000

Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies.

Mixed lymphohematopoietic chimerism can be induced in mice with bone marrow transplantation (BMT) after a nonmyeloablative preparative regimen that includes cyclophosphamide, anti-T-cell antibody therapy, and thymic irradiation. These mixed chimeras are resistant to the induction of graft-versus-host disease (GVHD) after delayed donor leukocyte infusions (DLIs), despite a potent lymphohematopoietic graft-versus-host reaction that converts the mixed chimeric state to a full donor one. Based on this animal model, we initiated a trial of nonmyeloablative therapy with HLA-matched or -mismatched donor BMT and DLI for refractory hematologic malignancies. Twenty-one of 36 patients enrolled in this trial received a genotypically (n = 20) or phenotypically (n = 1) HLA-matched donor transplant; results reported here are for those patients only. Preparative therapy consisted of cyclophosphamide in doses of 150 to 200 mg/kg; peritransplant antithymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal radiation therapy); and cyclosporine. Eighteen of 20 evaluable patients developed persistent mixed lymphohematopoietic chimerism as defined by >1% donor peripheral white blood cells until at least day 35 posttransplantation. Ten patients received prophylactic DLI beginning 5 to 6 weeks after BMT for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-leukemia effect. Fourteen of 20 evaluable patients (70%) achieved an antitumor response; 8 of these responses were complete, and 6 were partial. Of the 8 evaluable patients who received prophylactic DLI, 6 showed conversion to full donor chimerism. Five of the 9 evaluable patients (56%) who received prophylactic DLI achieved a complete response, compared with 3 of 11 patients (27%) who did not receive prophylactic DLI. Currently 11 patients are alive, and 7 of these are free of disease progression at a median follow-up time of 445 days (range, 105-548 days) posttransplantation. Transplantation-related complications included cyclophosphamide-induced cardiac toxicity in 3 of 21 patients (14%) and grade II or greater GVHD in 6 patients (29%). One patient (5%) died from a complication of BMT, and 1 patient (5%) died from GVHD after 2 prophylactic DLIs were given for conversion of chimerism. In summary, mixed lymphohematopoietic chimerism was reproducibly induced after a novel nonmyeloablative preparative regimen incorporating chemotherapy, peritransplant antithymocyte globulin, and thymic irradiation, allowing for early administration of DLI in 10 of 21 patients. After treatment, striking antitumor responses were observed in the majority of patients with chemotherapy-refractory hematologic malignancies.

Adult↗

PubMed2003

Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation.

In an attempt to capture graft-versus-tumor effects without graft-versus-host disease (GVHD), the authors initiated a trial of nonmyeloablative allogeneic bone marrow transplantation (BMT) in patients with advanced hematologic malignancies, with the majority of patients having chemotherapy-refractory disease. Forty-two patients received an HLA-matched related donor BMT after a cyclophosphamide and antithymocyte globulin-based conditioning that also included thymic irradiation for patients who had not received prior mediastinal radiotherapy. Prophylactic donor leukocyte infusion (pDLI) at a dose of 1 x 10(7) CD3(+) cells per kilogram were given beginning 5 weeks post-BMT to 16 patients with mixed chimerism (MC) but without GVHD, whereas 26 patients did not receive pDLI, either because of GVHD or early relapse. Twelve of 16 patients (75%) receiving pDLI had T cell chimerism at the time of pDLI >/=40%. These patients, by day 100 post-BMT, either converted to full donor chimerism (FDC) (n = 10) or had an increase in or stable donor chimerism (n = 2) after pDLI. Four of 4 patients whose T cell chimerism was </=20% at the time of pDLI, lost the graft. In contrast, only 5 of 18 evaluable patients (28%) not receiving a pDLI converted to FDC by day 100 post-BMT, 7 maintained MC, and 10 of an evaluable 22 lost the graft. Patients who had undergone a previous autologous stem cell transplant had a higher rate of conversion to FDC (69% v 31%) and higher incidence of GVHD (69% v 34%) compared with those who did not have a previous autologous SCT. Eleven of 16 patients (69%) who received a pDLI achieved a remission with 50% 1-year progression-free survival rate and 44% 3-year overall survival rate. Nineteen of 42 patients (45%) had >/=grade II acute GVHD, including 12 after BMT and 7 after pDLI. Approximately one third of patients, after having initial MC, eventually lost their donor graft. The authors conclude that (1) pDLI has the potential to convert MC to FDC; (2) sustained remissions can be achieved in patients with chemorefractory hematologic malignancies who receive a pDLI, albeit with a significant risk of acute GVHD; and (3) the degree of donor T cell chimerism at the time of pDLI is predictive of the fate of MC, ie, donor T cell chimerism >/=40% or </=20% at the time of pDLI correlates with conversion of MC or loss of the graft, respectively.

Adult↗

PubMed2003

Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine.

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

Animals↗

PubMed2005

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres.

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p < 0.05), but significantly lower than in TR<>N chimeric blastocysts (p < 0.001). Similarly, a higher proportion (p < 0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

Animals↗

PubMedDate not supplied

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification.

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.

Bacterial Proteins↗