David M. Neville

Insulin interactions with its receptors: experimental evidence for negative cooperativity.

Summary A simple method is reported to detect cooperative interactions in the binding of polypeptide hormones to their membrane receptors. The dissociation of radioiodinated hormone from the receptor is studied under two conditions: first, by diluting the hormone-receptor complex sufficiently to prevent rebinding of the dissociated tracer; second, by dilution to the same extent in a medium containing an excess of unlabeled hormone. If the sites are independent, the dissociation rates must be the same in both cases. If the presence of unlabeled hormone increases the dissociation rate of the tracer, negatively cooperative interactions must occur. Insulin receptors on cultured lymphocytes and liver plasma membranes show negative cooperative interactions. Growth hormone receptor sites lack these interactions.

ACTINOMYCIN BINDING TO DNA: MECHANISM AND SPECIFICITY.

The interaction, of actinomycin with DNA is found to involve two classes of sites. Measurements are reported here of the properties of the strongly binding sites, which appear to be the ones involved in the biological activity of actinomycin as an inhibitor of DNA-dependent RNA synthesis. It is found that the binding of actinomycin to dGMP involves a favorable enthalpy change and a decrease in entropy, whereas the binding to the DNA sites which are studied here involves a large increase in entropy and no enthalpy change. The probable source of the entropy increase is the disruption of actinomycin-solvent interaction; the entropy of solution of actinomycin is shown to be large and negative at room temperature. Measurements of the effect of disruption of helical structure upon actinomycin binding demonstrate that this structure is essential to the binding. Titration experiments with a series of DNAs reveal the dependence of the number of strongly binding sites in the helical structure upon base composition. The dependence can be accounted for either by a site which consists of a sequence of two base pairs, involving one G-C pair and one A-T pair, or by a site consisting entirely of G-C pairs, in which there is a large amount of steric interference between sites spaced more closely than five to six base pairs apart.

FN18-CRM9 immunotoxin promotes tolerance in primate renal allografts

BACKGROUND Transplant tolerance, rather than immunity, may be favored in the setting of a lower mature lymphoid mass in the recipient induced by anti-T cell agents. A novel immunosuppressive agent, FN18-CRM9, known to specifically kill T cells with great potency, was evaluated in a transplant model. METHODS In order to ablate recipient T cells, the immunotoxin FN18-CRM9 was administered to rhesus monkey recipients of MHC-mismatched renal allografts. Donor lymphocytes were injected intrathymically into some animals. RESULTS All monkeys with T-cell depletion by immunotoxin had prolonged allograft survival, and tolerance confirmed by skin grafting has been confirmed in five of six long-surviving recipients. CONCLUSIONS In this clinically relevant model, profound but transient T-cell depletion by a single agent substantially promotes tolerance.

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3-immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts

A major challenge in clinical transplantation today is to design a practical and effective protocol for tolerance induction compatible with cadaver organ transplantation. A preclinical rhesus monkey kidney allograft model using immediate peritransplant anti-CD3 immunotoxin (anti-CD3-IT) and donor bone marrow (DBM) is shown here to induce operational tolerance with prolonged graft survival in the absence of chronic immunosuppressive drugs. Bone marrow harvested from the kidney donor was depleted of mature alloantigen-presenting cells and T cells by removing DR(bright) cells and CD3(bright) cells, respectively. In outbred, major histocompatibility complex-incompatible donor-recipient pairs with high pretransplant mixed lymphocyte response and cytotoxic T lymphocyte precursor activity, four of six allografts survived for periods of 120 days to >1.5 years. Graft acceptance after peritransplant treatment followed robust elimination of both peripheral blood T cells and lymph node T cells. In most recipients given anti-CD3-IT and DBM infusion, anti-donor immunoglobulin G responses were completely inhibited. Microchimerism was observed in all recipients studied, including those not given DBM, but levels of microchimerism did not correlate with graft survival. Anti-CD3-IT induction in combination with modified DBM protocols such as the depletion of mature T cells and DR(bright) antigen-presenting cells may offer new opportunities to improve clinical tolerance protocols beyond those attempted in the clinic to date. Overall, these results with anti-CD3-IT show promise for development of cadaver transplant tolerance induction.

The interaction of acridine dyes with DNA: An X-ray diffraction and optical investigation

The interaction of DNA with acridine orange and with proflavine has been investigated by X-ray diffraction and optical techniques. Fibers of the complexes containing one dye molecule per three base pairs can be observed under conditions where their diffraction patterns closely resemble the B -form pattern of sodium-DNA. At higher relative humidities these fiber diffraction patterns become more disordered and show an increase in the layer-line spacing of the first three layer lines. It has been demonstrated by the use of optical transforms that these results are qualitatively consistent with the intercalation hypothesis of Lerman (1961), although one other model is considered which can also account for the changes in the diffraction pattern, but not for the results of other investigators (Luzzati, Masson & Lerman, 1961; Cairns, 1962). Measurements of the absorption spectra of solutions and of films of these dye-DNA complexes have been interpreted in conjunction with the X-ray diffraction measurements, and demonstrate that not all of the bound dye is intercalated, an appreciable fraction being bound on the outside. The changes in the absorption spectra and in the layer-line spacings which occur upon wetting the fiber show that the distribution of dye between the external and intercalated site is a function of the hydration of the fiber. A comparative study with the non-planar triphenylmethane dye, p -rosaniline, failed to reveal layer-line changes characteristic of intercalation.

Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris.

The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensitive to the toxin. However, Pichia pastoris has a tolerance to levels of DT (diphtheria toxin) that were previously observed to intoxicate wild type eukaryotic cells, including Saccharomyces cerevisiae. This tolerance has permitted the optimization of the secreted expression of A-dmDT390-bisFv(G(4)S) in P. pastoris under the control of AOX1 (alcohol oxidase 1) promoter. The original DNA sequence of A-dmDT390-bisFv(G(4)S) was not expressed in P. pastoris because of several AT-rich regions, which induce an early termination of transcription. After DNA rebuilding for abolishing AT-rich regions and codon optimization, the immunotoxin could be expressed up to 10mg/L in the shake flask culture. No differences in the expression levels of immunotoxin were observed by using different secretional signal sequences, Mut(s) (methanol utilization slow phenotype) or Mut(+) (methanol utilization plus phenotype) phenotypes. Buffered complex medium (pH 7.0) having 1% casamino acids provided the highest expression in shake flask culture and PMSF (phenylmethylsulfonyl fluoride) in the range of 1 to 3mM further improved the expression level presumably by inhibiting protein degradation. The immunotoxin was purified by DEAE (diethylaminoethyl) Sepharose ion exchange chromatography and Protein L affinity chromatography. The immunotoxin purified from P. pastoris culture was as fully functional as that expressed in a toxin resistant mutant CHO (Chinese hamster ovary) cell line. Our results demonstrate that P. pastoris is an ideal system for expression of toxin-based fusion proteins.

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine

Background. Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. Methods. Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. Results. All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. Conclusions. This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.

Reversal of naturally occurring diabetes in primates by unmodified islet xenografts without chronic immunosuppression

BACKGROUND Isolated pancreatic islet transplantation (IPITx) is an attractive alternative for treatment of insulin-dependent diabetes mellitus (IDDM). However, IPITx has been difficult to implement clinically because islets frequently fail to function, have a high incidence of rejection, and are susceptible to autoimmune recurrence and damage by chronic immunosuppressive therapy. Tolerance induction may be a rational approach to resolve several of these limitations. Because anti-CD3 immunotoxin (IT) has been successful in promoting stable primate kidney transplant tolerance in our experience, we considered that tolerance induction with IT might be duplicated in IPITx. MATERIALS AND METHODS Three monkeys with spontaneous IDDM (two Macaca fascicularis and one Ceropithecus aethiops) were treated with xenogeneic pancreatic islets (Macaca mulatta). Intrahepatic islet transplantation was performed at a mean of 13136+/-3860 islet equivalents/kg. Islet xenograft acceptance was accomplished by tolerance induction with two injections of IT given on day 0 at 2 hr before transplantation and on day +1, respectively. IT treatment was supplemented with cyclosporine and steroids administered on days 0 through 4. No additional immunosuppression was given thereafter. Two additional control macaques with spontaneous IDDM received the immunosuppressive protocol without islet infusion. RESULTS All recipients were restored to stable euglycemia, off exogenous insulin, within 1-2 weeks after transplantation. Glucose tolerance, C-peptide, and glycosylated hemoglobin tests confirmed the restoration of normal glucose homeostasis after islet transplantation. All three islet recipients have remained euglycemic at 410, 255, and 100 days of follow-up despite recovery of peripheral T cells to normal levels. In contrast, none of the controls presented changes in the diabetic status 4 and 8 months after treatment. CONCLUSIONS These results represent the first demonstration in nonhuman primates of stable, long-term acceptance of nonencapsulated xenogeneic islets off all immunosuppression, suggesting operational tolerance. The findings have potential implications for islet transplantation as well as improved and more cost-effective therapy for IDDM.

Insulin receptor defect in insulin resistance: Studies in the obese-hyperglycemic mouse

Abstract To evaluate the possible role of the insulin receptor in the pathophysiology of disease, the interaction of insulin with plasma membranes of liver has been studied in the obese hyperglycemic mouse and their thin litter mates. The obese hyperglycemic syndrome is characterized by marked resistance to both endogenous and exogenous insulin. Under identical conditions of purification and incubation, plasma membranes of the obese mouse bind only 16–35% as much 125I-insulin as membranes of the thin mouse. This is in contrast to other characteristics of the membrane which are similar to these two animals.