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PubMed2005

Induction of stable mixed chimerism by embryonic stem cells requires functional Fas/FasL engagement.

BACKGROUND: Recent data show the efficacy of embryonic stem cells (ESC) to engraft in allogeneic recipients without host pretreatment. This property is due to their low expression of major histocompatibility complex (MHC) class I antigens and lack of MHC class II expression. Here, we tested the hypothesis that the constitutive FasL expression by ESC is a requirement for their stable engraftment in allogeneic recipients. METHODS: MRL and MRL-lpr/lpr mice (H-2k) were infused allogeneic 129SvJ RW-4 (H-2b) ESC without host preconditioning. The development of mixed chimerism was monitored over 100 days by flow cytometry. RESULTS: Mixed chimerism was detectable by day 7. The amount of donor cells detected varied between 3-5.5% and were lymphoid, but nonmyeloid. Only 50% of lpr/lpr mice engrafted and lost donor cells by day 28 post-ESC infusion. In contrast, >80% wild type mice engrafted and maintained mixed chimerism up to day 100. CONCLUSIONS: These data suggest a critical role for Fas-FasL engagement in ESC engraftment. We conclude that ESC may induce clonal deletion of alloreactive T cells by Fas-induced apoptosis in recipient T cells, protecting them from rejection. The data provide a rationale for improved protocols for the achievement of robust ESC-induced mixed chimerism.

Animals↗
PubMed2006

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence.

BACKGROUND: Use of a reduced-intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a coexistence of both host and donor hematopoietic cells may influence posttransplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced-intensity conditioning transplantation (RICT), autoantibody production, and chronic GVHD (cGVHD)-related pathology. METHODS: Chimerism state, circulating anticardiolipin, and antidouble stranded DNA autoantibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. RESULTS: We observed a novel association between mixed chimerism state, high levels of pathogenic IgG autoantibodies, and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of autoantibodies. CONCLUSION: Recipient B cell persistence may contribute to the frequency and/or severity of cGVHD after RICT.

Animals↗
PubMed2006

Long-term survival of xenogeneic heart grafts achieved by costimulatory blockade and transient mixed chimerism.

BACKGROUND: Xenotransplantation holds great promise in clinical medicine, but is limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. In this study, we investigated the role of anti-CD40L monoclonal antibody (mAb) in inducing xenogeneic mixed chimerism and donor-specific heart transplantation tolerance. METHODS: One day before heart transplantation, mice were injected intraperitoneally with anti-mouse CD8/NK1.1/Thy1.2 mAbs. On day 0, the mice received 3 Gy total body irradiation (TBI), an intravenous injection of unseparated bone marrow (BM) harvested from F344 rats, and an intraperitoneal injection of hamster antimouse CD40L mAb, MR1. Heart grafts from F344 rats were heterotopically transplanted into the abdomen of B6 mouse recipients. Using flow cytometric analysis of peripheral white blood cells, we assessed donor hematopoiesis at various times after bone marrow transplantation (BMT). RESULTS: Chimerism subsided gradually and disappeared completely 18 weeks after BMT. The cardiac graft survived permanently, even after the mixed chimerism disappeared. To determine if the mice acquired donor-specific tolerance, second rat heart grafts were transplanted 120 days after the first heart transplantation. The second transplanted hearts were also accepted over 60 days. Histological analysis revealed no remarkable vasculopathy in the coronary vessels at any stage. CONCLUSIONS: These findings clearly show that costimulatory blockade plays an important role in inducing xenochimerism, and that transient mixed chimerism can induce permanent acceptance of rat to mouse cardiac xenografts. Transplantation of xenogeneic bone marrow cells under costimulatory blockade at the time of heart transplantation may induce transplantation tolerance.

Animals↗
PubMed2001

Mixed chimerism.

Induction of mixed chimerism has the potential to overcome the current limitations of transplantation, namely chronic rejection, complications of immunosuppressive therapy and the need for xenografts to overcome the current shortage of allogeneic organs. Successful achievement of mixed chimerism had been shown to tolerize T cells, B cells and possibly natural killer cells, the lymphocyte subsets that pose major barriers to allogeneic and xenogeneic transplants. Current understanding of the mechanisms involved in tolerization of each cell type is reviewed. Considerable advances have been made in reducing the potential toxicity of conditioning regimens required for the induction of mixed chimerism in rodent models, and translation of these strategies to large animal models and in a patient are important advances toward more widespread clinical application of the mixed chimerism approach for tolerance induction.

Animals↗
PubMed1995

Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication.

We constructed a series of human immunodeficiency virus 1 (HIV-1)/simian immunodeficiency virus strain mac (SIVmac) chimeric viruses having vpr and/or nef genes of either HIV-1 or SIVmac based on a chimeric virus with LTRs, gag, pol, vif and vpx derived from SIVmac and tar, rev, vpu and env from HIV-1. All of the chimeric viruses replicated in human and macaque peripheral blood mononuclear cells (PBMCs) and in several CD4+ human cell lines, though their growth potentials were slightly different depending on whether vpr and nef were from HIV-1 or SIVmac, or were defective. The presence of nef accelerated replication in all the cells used and the replication of each chimera appeared to reflect that of the parental virus from which nef was derived. The presence of vpr had no clear effect in human and monkey PBMCs, but the replication of each chimera was influenced by the origin of vpr in H9 and A3.01 cells. NM-3rN, which carries HIV-1 vpr and SIVmac nef, was inoculated intravenously into three rhesus monkeys, three cynomolgus monkeys and two pig-tailed monkeys. From 2 to 14 weeks after inoculation, viruses were consistently re-isolated from all the monkeys and virus loads were as high as that of SIVmac reported previously. The results indicate that infection with NM-3rN is more efficient than any of our previous chimeric viruses and suggest that NM-3rN, having HIV-1 Env, will be a useful challenge virus for evaluating AIDS vaccines based on HIV-1 Env in macaque monkeys instead of chimpanzees.

Animals↗
PubMed1997

Chimeric coxsackie B3 virus genomes that express hybrid coxsackievirus-poliovirus 2B proteins: functional dissection of structural domains involved in RNA replication.

The 2B proteins of coxsackievirus and poliovirus (PV) share significant structural similarity and exhibit similar biochemical activities, namely inhibition of protein secretion and modification of membrane permeability. Both proteins contain two hydrophobic domains in the carboxy-terminal two-thirds of their sequence, of which one has the potential to form a cationic amphipathic alpha-helix. To gain more insight into the structural requirements of enterovirus protein 2B for its functioning in viral RNA replication, a chimeric cDNA approach was used. Chimeric coxsackie B3 virus (CBV3) genomes were constructed that expressed either the entire PV 2B protein or hybrid proteins in which specific segments of CBV3 2B were substituted by their corresponding PV counterparts. In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities. CBV3 genomes carrying the entire PV 2B gene failed to replicate. A chimeric genome that expressed a hybrid 2B protein consisting of the amino-terminal one-third of PV and the remainder of CBV3 yielded viable viruses. In contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed to drive replication. These data imply that a sequence-specific interaction with another viral protein is required to drive RNA replication and suggest that the proposed sites of contact reside in the carboxy-terminal two-thirds of 2B. Hybrid genomes in which either the amphipathic alpha-helix or the other hydrophobic domain was replaced failed to replicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.

Amino Acid Sequence↗
PubMed2003

Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5'NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5'NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5'NTRs. A two-step analysis of these 5'NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5'NTRs, while replication of subgenomic replicons with these chimeric 5'NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.

5' Untranslated Regions↗
PubMed2005

Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury.

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE66(4Glu), an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE66(4Glu). However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.

Animals↗
PubMed1992

Effector cell analysis of human multidrug-resistant cell killing by mouse-human chimeric antibody against P-glycoprotein.

A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells. The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells. The ADCC reaction was assessed by a 6-h 51Cr release assay. Highly purified lymphocytes (greater than 99%), monocytes (greater than 99%) and neutrophils (greater than 96%) were obtained from peripheral blood of the same healthy donors. A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils. But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells. The lymphocytes responsible for this ADCC had CD16+ Fc receptors. Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells. Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells. These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Establishment and characterization of mouse-human chimeric monoclonal antibody to erbB-2 product.

A mouse-human chimeric antibody for erbB-2 product was established by a new procedure using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The E401 hybridoma secreted anti-erbB-2 product monoclonal antibody (MoAb) (IgG1, kappa). The gene of the mouse variable regions of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the E401 hybridoma RNA. The variable region of heavy chain was joined with the expression vector, which contains human gamma 1 constant gene. The expression vector was transfected into heavy chain loss mutant cells E401-12, which produced only murine immunoglobulin light chains. A chimeric monoclonal antibody CH401 retained full binding reactivity to erbB-2 product, compared with murine E401 parental antibody. Furthermore, the chimeric MoAb CH401 was much more efficient in supporting antibody dependent cell-mediated cytotoxicity activity against erbB-2-bearing human adenocarcinoma cells than murine MoAb E401. These suggest that a chimeric monoclonal antibody CH401 may be a potent reagent for therapy of human adenocarcinomas.

Adenocarcinoma↗
PubMed1997

Mouse/human chimeric IgG1 and IgG4 antibodies directed to the house dust mite allergen Der p 2: use in quantification of allergen specific IgG.

BACKGROUND AND OBJECTIVE: Chimeric mouse/human monoclonal IgG1 and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains of the original murine monoclonal antibody 2B12, whereas the heavy chain constant domains have been replaced by the human IgG1 or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 microg/mL, respectively. METHODS AND RESULTS: Since all IgG in these culture supernatants is allergen-specific, they are useful reference reagents and enable the calculation of the amount of allergen specific IgG1 and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgG1 and IgG4 RAST and with reversed IgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found. CONCLUSION: With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.

Animals↗
PubMed1991

A chimeric nucleotide-binding protein, encoded by a hisP-malK hybrid gene, is functional in maltose transport in Salmonella typhimurium.

We have isolated a hybrid gene, composed of the first 455 nucleotides of hisP and nucleotides 275-1107 of malK, the genes coding for the nucleotide-binding components of the high-affinity transport systems for histidine and maltose in Salmonella typhimurium, respectively. The fusion had occurred by recombination within 11 homologous base pairs located between the two DNA fragments. In the chimeric protein peptidic motifs A and B, proposed to be part of the nucleotide-binding fold, originate from HisP and MalK, respectively. Plasmid pES42-39, harbouring the hybrid gene, was shown to complement only a malK mutation but failed to complement a hisP deletion mutation. The chimeric protein was identified by immunoblotting as a protein with an apparent molecular mass of 49kDa. Removal of the C-terminal 77 amino acid residues from the chimeric protein resulted in the loss of function in transport. In contrast, 51 amino acid residues could be removed from the C-terminus of wild-type MalK without any effect. Upon overproduction the chimeric protein, as wild-type MalK, inhibited expression of the malB regulon. However, both truncated proteins, when overproduced, did not exhibit this activity. Based on these results, a tentative model of the functional domains of MalK is presented.

ATP-Binding Cassette Transporters↗
PubMed1993

Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme.

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminidases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-binding protein (CLC) that has been purified using affinity chromatography on DEAE-cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was choline-independent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin-defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.

Amino Acid Sequence↗
PubMed1992

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗
PubMed1995

Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential.

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226,000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively). The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively). In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir. Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Structural properties of chimeric peptides containing a T-cell epitope linked to a fusion peptide and their importance for in vivo induction of cytotoxic T-cell responses.

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells. When the fusion peptide was linked to the C-terminus of the CTL epitope, the chimeric peptide (M2-F) adopted a marked beta-sheet conformation. In contrast, when the fusion peptide was linked to the N-terminus of the T-cell epitope (F-M2), the chimeric peptide adopted an alpha-helical conformation in the presence of trifluoroethanol. The immunogenicity of the two chimeric peptides for class-I restricted CTL was also significantly different, the one adopting the alpha-helical conformation being more immunogenic. Probably due to its obvious conversion to an alpha-helical conformation, the F-M2 peptide could have a higher propensity to insert into membranes, as shown by microspectrofluorometry, with a resultant better immunogenicity than the M2-F peptide.

Amino Acid Sequence↗
PubMed1994

Mechanisms of unresponsiveness associated with pretransplant blood transfusion-cyclosporine-induced mixed lymphocyte chimerism.

Multiple pretransplant blood transfusions while under limited daily cyclosporine cover (PTBT-CsA) induce extensive rat renal allograft survival and antigen-specific non-responsiveness. The underlying mechanisms of this extensive allograft survival are not yet fully understood. We hypothesized that one of the potential contributing mechanisms to tolerance induction in PTBT-CsA-treated kidney recipients is the development of stable mixed chimerism, putatively due to the proliferation of stem cells capable of haematopoiesis in the transfused blood. BN rats served as whole blood and kidney donors. LEW rats served as recipients of the PTBT-CsA protocol and BN kidney transplants. Three weekly transfusions were given under concomitant limited CsA cover. Following these multiple primary sensitizations, antigen-specific splenic cellular responsiveness in vivo was normal in comparison with naive animals. However, these experimental splenocytes were non-specifically suppressed against third-party allodeterminants. At 100 days post-transplantation (T100) following tolerance induction to kidney allografts (secondary challenge), in vivo adoptive transfer experiments demonstrated the existence of potent splenic suppressor cells. In vitro suppressor cell assays confirmed that these cells were non-specific suppressor cells. However, following chimerism stabilization at T130, splenic antigen-specific suppressor cells became exclusively expressed in the tolerant animals, replacing the non-specific suppressor cells. At this time, splenic microchimerism was at peak levels and remained stable from T100 to T130. In conclusion, these findings demonstrate that sequential mechanisms of suppressor cell network expression are induced within a chimeric environment by blood-CsA immune modulation. Stable mixed lymphocyte chimerism and related immunomodulatory mechanisms may, therefore, play an important tolerogenic role in blood-CsA-induced non-responsiveness and in the beneficial effect of blood transfusion.

Animals↗
PubMed1994

Split tolerance in chimeric GVH mice.

The i.v. inoculation of parental spleen cells into unirradiated adult F1 hybrid mice results in a graft-versus-host reaction (GVHR). In the strain combination B10D2 --> (B10.BRx B10.D2) F1, this reaction is associated with thymic injury and transient but profound cellular immune deficiency. We further analysed the immune status of these mice 60 days after GVHR induction. Phenotypic studies of spleen cells showed that these mice were repopulated with parental lymphocytes resulting in a high degree of chimerism (85%). At this time, the mice looked healthy and recovered a normal cytotoxic T cell response (CTL) against allogeneic cells. GVH chimeric splenocytes were unresponsive against F1 hybrid cells in mixed lymphocyte culture (MLC), but exhibited anti-F1 CTL reactivity. We also analysed the anti-F1 reactivity of these mice in vivo. GVH chimeric splenocytes were unable to induce GVHR after injection into a new F1 hybrid and F1 GVH mice specifically rejected F1 bone marrow (BM) cells after lethal irradiation. Grafting a neonatal parental thymus prevented the rejection of F1 BM cells and restored CTL alloreactivity. It is concluded that the chimeric state induced by GVHR is associated with a split tolerance and that a radiosensitive thymic-dependent mechanism is involved in maintaining self-tolerance in these mice.

Animals↗