Anti-idiotypes against anti-H-2 monoclonal antibodies: structural analysis of the molecules induced by in vivo anti-idiotype treatment.

Previous studies have shown that treatment of mice in vivo with xenogeneic anti-idiotype produced against a monoclonal anti-H-2Kk antibody, 11-4.1, leads to the induction of molecules (Id') that inhibit the binding of anti-idiotype to idiotype. To investigate the nature of these Id' molecules, spleens from such anti-idiotype-treated mice were fused with the SP2/0 myeloma to produce monoclonal Id' antibodies. All four monoclonal Id' antibodies were found to react with goat and rabbit anti-11-4.1 in addition to the pig anti-idiotype used for their induction and one of the four, J1-8-1, reacted with syngeneic BALB/c anti-11-4.1. Partial amino acid sequences were determined for the heavy and light chains of these monoclonal antibodies. J1-8-1 heavy chain had an NH2-terminal amino acid sequence identical to that of 11-4.1 for the 39 NH2-terminal residues assigned, whereas its light chain and the heavy and light chains of the other Id' molecules differed markedly from those of the 11-4.1 antibody. Isolated heavy chains and light chains of J1-8-1 and 11-4.1 were reassociated in homologous and heterologous pairs. When J1-8-1 heavy chains and 11-4.1 light chains were mixed in equimolar concentrations, anti-H-2Kk reactivity was found at a level approximately 10% of that observed for reassociated 11-4.1 homologous heavy and light chains. The finding that in vivo anti-idiotype treatment can trigger Id' molecules structurally similar to the original idiotype has implications regarding the mechanism of induction of Id' molecules and the regulation of repertoire expression by idiotypic networks.     

Molecular heterogeneity in acute leukemia lineage switch

Six cases of acute leukemia that underwent lineage switch from acute lymphocytic leukemia to acute myelogenous leukemia are reported. The mean age of the patients was 24 years, time to conversion was 36 months, and survival after conversion was only 3 months. Of the three cases which showed abnormal metaphases at both diagnosis and conversion, two (cases 2, 5) showed related cytogenetic abnormalities, and the third showed (case 3) independent chromosomal changes. Molecular analysis for immunoglobulin heavy chain and T-cell receptor beta chain genes showed that five of the six cases had rearrangement of at least one of these lymphoid associated genes at conversion to acute myelogenous leukemia. The single case (case 3) in which there were no lymphoid gene rearrangements at conversion was also the only case in which independent karyotypic abnormalities at diagnosis and conversion were demonstrated. Our findings suggest that lineage switch can represent either relapse of the original clone with heterogeneity at the molecular level or the emergence of a second new leukemic clone without molecular heterogeneity.     

Experimental evolution of conflict mediation between genomes

Transitions to new levels of biological complexity often require cooperation among component individuals, but individual selection among those components may favor a selfishness that thwarts the evolution of cooperation. Biological systems with elements of cooperation and conflict are especially challenging to understand because the very direction of evolution is indeterminate and cannot be predicted without knowing which types of selfish mutations and interactions can arise. Here, we investigated the evolution of two bacteriophages (f1 and IKe) experimentally forced to obey a life cycle with elements of cooperation and conflict, whose outcome could have ranged from extinction of the population (due to selection of selfish elements) to extreme cooperation. Our results show the de novo evolution of a conflict mediation system that facilitates cooperation. Specifically, the two phages evolved to copackage their genomes into one protein coat, ensuring cotransmission with each other and virtually eliminating conflict. Thereafter, IKe evolved such extreme genome reduction that it lost the ability to make its own virions independent of f1. Our results parallel a variety of conflict mediation mechanisms existing in nature: evolution of reduced genomes in symbionts, cotransmission of partners, and obligate coexistence between cooperating species.     

Teleantagonism: A pharmacodynamic property of the primary nociceptive neuron

Previous work from our group showed that intrathecal (i.t.) administration of substances such as glutamate, NMDA, or PGE₂ induced sensitization of the primary nociceptive neuron (PNN hypernociception) that was inhibited by a distal intraplantar (i.pl.) injection of either morphine or dipyrone. This pharmacodynamic phenomenon is referred to in the present work as “teleantagonism”. We previously observed that the antinociceptive effect of i.t. morphine could be blocked by injecting inhibitors of the NO signaling pathway in the paw (i.pl.), and this effect was used to explain the mechanism of opioid-induced peripheral analgesia by i.t. administration. The objective of the present investigation was to determine whether this teleantagonism phenomenon was specific to this biochemical pathway (NO) or was a general property of the PNNs. Teleantagonism was investigated by administering test substances to the two ends of the PNN (i.e., to distal and proximal terminals; i.pl. plus i.t. or i.t. plus i.pl. injections). We found teleantagonism when: (i) inhibitors of the NO signaling pathway were injected distally during the antinociception induced by opioid agonists; (ii) a nonselective COX inhibitor was tested against PNN sensitization by IL-1β; (iii) selective opioid-receptor antagonists tested against antinociception induced by corresponding selective agonists. Although the dorsal root ganglion seems to be an important site for drug interactions, the teleantagonism phenomenon suggests that, in PNNs, a local sensitization spreads to the entire cell and constitutes an intriguing and not yet completely understood pharmacodynamic property of this group of neurons.     

Kidney‐Induced Cardiac Allograft Tolerance in Miniature Swine is Dependent on MHC‐Matching of Donor Cardiac and Renal Parenchyma

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full‐MHC barrier in miniature swine. However, the renal element(s) responsible for kidney‐induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic‐derived “passenger” cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co‐transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC‐mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC‐matched to each other but MHC‐mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC‐mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC‐mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.     

Pilot Study Evaluating Regulatory T Cell–Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model

The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1‐ethyl‐3‐(3′‐dimethylaminopropyl)‐carbodiimide (ECDI)‐treated donor lymphoid cell infusion (ECDI‐DLI) with thymoglobulin, anti‐interleukin‐6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor‐specific T cell hyporesponsiveness and a transient absence of donor‐specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.