THE EFFECT OF PASSIVELY ADMINISTERED ANTIBODY ON ANTIBODY SYNTHESIS

Suppression of the primary response of rabbits to intravenously administered KLH can be achieved with very small amounts of hyperimmune anti-KLH administered a day later since the rabbit apparently rapidly eliminates most of the KLH by nonimmunologic means. The amount of passive anti-KLH needed to achieve immunosuppression was directly proportional to the dose of injected antigen. Antibody passively administered as much as 6–8 days after antigen still can be strongly immunosuppressive, which suggests that the antibody must be reacting with immunogen in or on responding cells or perhaps in the process of transfer between cells. There was no evidence that the presence of passively administered hyperimmune anti-KLH prior to the injection of antigen had any immunosuppressive action beyond the direct neutralization of the injected antigen. When KLH was injected in Freund adjuvant, anti-KLH incorporated with the KLH in the adjuvant was much more efficient in causing immunosuppression than anti-KLH given intravenously. The primary responses to 2 mg KLH given intravenously and 2 µg given in adjuvant reached approximately equal peaks and were suppressible by comparable amounts of intravenously administered anti-KLH. Two observations suggest that passive antibody neutralizes the immunogenic stimulus at the level of individual antigenic determinants and not merely by aggregating or precipitating entire antigenic molecules. First, anti-abalone hemocyanin (AH) which cross-reacts approximately 50% with KLH was only partially immunosuppressive even in extremely large amounts, i.e., amounts which could react with and precipitate much more KLH than could the smaller but more suppressive doses of anti-KLH. Second, when KLH and anti-KLH were given together in adjuvant, effective immunosuppression was achieved only with amounts of anti-KLH sufficient to saturate or cover virtually all available antigenic determinants. The immunosuppressive quality of passive antibody increases with time after immunization and with repeated immunization of the donor. In view of their relatively weak immunosuppressive properties, antibodies formed in the first weeks of a primary response may not contribute significantly to the turning off of the antibody response. In any event, results obtained by passive transfer of hyperimmune antibody to animals early in a primary response cannot be applied to the natural events in a primary response.     

REGULATION OF THE IMMUNE RESPONSE: SUPPRESSIVE AND ENHANCING EFFECTS OF PASSIVELY ADMINISTERED ANTIBODY

The ability of passively administered antibody to suppress the immune response against homologous antigenic determinants while concomitantly enhancing the response against other unrelated determinants of the same antigen molecule has been established in two distinct antigen-antibody systems: (a) guinea pig γ₂-immunoglobulin + passive anti-F(ab')₂ antibody, where suppression of anti-F(ab')₂ antibody synthesis is accompanied by enhancement of the anti-Fc response; and (b) human secretory IgA + passive anti-serum IgA antibody, where suppression of antibody production against the α and L chains accompanies augmentation of the response to the secretory component. The mechanisms of the suppressive and enhancing effects are probably unrelated for the following reasons: (a) Enhancement of the response to certain determinants may be obtained without discernible suppression of the response to the homologous determinants; and (b) the F(ab')₂ fragments of passive antibody can mediate immune suppression but were not observed to enhance the response against the unrelated determinants of the same antigen molecule. Also, the timing for achieving maximum suppression or enhancement of antibody formation is not the same; enhancement was obtained only at a later time. Both the enhancement and suppressive effects were obtained with the purified γG fraction of antisera. This finding rules out an exclusive role of γM antibody in the enhancement phenomenon.     

Phagocytosis of liposome particles by rat splenic immature monocytes makes them transiently and highly immunosuppressive in ex vivo culture conditions

Liposomes reportedly accumulate in monophagocytic systems (MPSs), such as those of the spleen. Accumulation of considerable amounts of liposome in a MPS can affect immunologic response. While developing a liposomal oxygen carrier containing human hemoglobin vesicle (HbV), we identified its suppressive effect on the proliferation of rat splenic T cells. The aim of this study was to elucidate the mechanism underlying that phenomenon and its effect on both local and systemic immune response. For this study, we infused HbV intravenously at a volume of 20% of whole blood or empty liposomes into rats, removed their spleens, and evaluated T cell responses to concanavalin A (Con A) or keyhole limpet hemocyanin (KLH) by measuring the amount of [(3)H]thymidine incorporated into DNA. Cells that phagocytized liposomal particles were sorted using flow cytometry and analyzed. Serum anti-KLH antibody was measured after immunizing rats with KLH. Results showed that T cell proliferation in response to Con A or KLH was inhibited from 6 h to 3 days after the liposome injection. Direct cell-to-cell contact was necessary for the suppression. Both inducible nitric-oxide synthase and arginase inhibitors restored T cell proliferation to some degree. The suppression abated 7 days later. Cells that trapped vesicles were responsible for the suppression. Most expressed CD11b/c but lacked class II molecules. However, the primary antibody response to KLH was unaffected. We conclude that the phagocytosis of the large load of liposomal particles by rat CD11b/c+, class II immature monocytes temporarily renders them highly immunosuppressive, but the systemic immune response was unaffected.     

HUMORAL AND CELLULAR ASPECTS OF THE IMMUNE RESPONSE TO THE SOMATIC ANTIGEN OF SALMONELLA ENTERITIDIS

A study was made of the cellular and humoral aspects of the immune response of the rabbit to the somatic polysaccharide of Salmonella enteritidis. The response to a single intravenous injection was characterized by the appearance of elevated titers of bactericidal antibody between 2 and 3 days later. The maximum titer was dose-dependent and occurred between 5 and 7 days, thereafter declining rapidly during the first month. The significant stabilized levels which then persisted for at least 1 year were also dose-dependent. Most of the antibody produced (>99 per cent) was associated with the macroglobulin fraction of serum. Plaque-forming cells (PFC) elaborating antibody specific for this somatic antigen were detected and enumerated by the technique of localized hemolysis in gel employing polysaccharide-coated sheep erythrocytes. Significant numbers of PFC were encountered in the spleen as early as 14 to 18 hours after a single intravenous injection of antigen; after 36 hours the number of PFC rose rapidly and culminated in a maximum population at 5 days, followed by a rapid decline and plateau similar to that for circulating antibody. The spleen was the principal organ involved in the systemic response, but other lymphoid tissues including bone marrow, peripheral blood leucocytes, and thymus contributed significantly. After an interval of 3 months the effect on humoral antibody titers of a second injection of antigen was dependent on the amount of polysaccharide administered; markedly greater titers were now obtained with 0.02 to 0.002 µg, whereas 0.2 to 20 µg resulted in a duplication of the initial humoral response. The cellular response to a second dose of 5 µg was accelerated; larger numbers of PFC appeared more rapidly, attained a maximum population by day 3, and exceeded the primary response by a factor of two. This acceleration in the attainment of maximum numbers of PFC and the increased bactericidal antibody titers following a second injection of limiting amounts of antigen suggest that these somatic polysaccharides may in fact evoke a "secondary" type of response in the rabbit.     

Delayed hypersensitivity. IV. Systemic reactivity of guinea pigs sensitized to protein antigens

Guinea pigs with delayed hypersensitivity to protein antigens show a specific febrile response accompanied by a lymphopenia following injection of a desensitizing dose of specific antigen. No signs of shock are observed in highly sensitive animals following this injection. The response is not prevented in sensitive guinea pigs by inducing endotoxin tolerance or by pretreating with cortisone before specific challenge. Using a suitable antigen in sufficiently sensitive animals as little as 100 µg. can elicit a pronounced febrile response. Injection of a desensitizing dose of antigen specifically abolishes systemic as well as skin reactivity for several days. Normal or hypersensitive (delayed-type) animals passively sensitized with sufficient amounts of serum antibody show hypothermia after specific challenge and may show a delayed type of fatal shock. Differences were noted between their systemic reactivities, however, and the reactivity seen in specifically challenged tuberculous animals.     

Plasmacytoid dendritic cells of melanoma patients present exogenous proteins to CD4+ T cells after Fc gamma RII-mediated uptake

Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons. Although human pDCs can induce T cell responses upon viral infection, it remains unclear if pDCs can present exogenous antigens. Here, we show that human pDCs exploit FcgammaRII (CD32) to internalize antigen-antibody complexes, resulting in the presentation of exogenous antigen to T cells. pDCs isolated from melanoma patients vaccinated with autologous monocyte-derived peptide- and keyhold limpet hemocyanin (KLH)-loaded dendritic cells, but not from nonvaccinated patients or patients that lack a humoral response against KLH, were able to stimulate KLH-specific T cell proliferation. Interestingly, we observed that internalization of KLH by pDCs depended on the presence of serum from vaccinated patients that developed an anti-KLH antibody response. Anti-CD32 antibodies inhibited antigen uptake and presentation, demonstrating that circulating anti-KLH antibodies binding to CD32 mediate KLH internalization. We conclude that CD32 is an antigen uptake receptor on pDCs and that antigen presentation by pDCs is of particular relevance when circulating antibodies are present. Antigen presentation by pDCs may thus modulate the strength and quality of the secondary phase of an immune response.     

STUDIES ON ANTIBODY PRODUCTION : VIII. THE INHIBITORY EFFECT OF CHLORAMPHENICOL ON THE SYNTHESIS OF ANTIBODY IN TISSUE CULTURE

When lymph node fragments from previously immunized rabbits were stimulated in vitro to produce a secondary response, the continuous presence of 50 µg/ml (0.15 mM) of chloramphenicol in the medium during the entire incubation period of 15 to 21 days produced nearly complete suppression of the response. Concentrations as low as 5 µg/ml (0.015 mM) produced approximately 80 per cent suppression of the response. When 50 µg/ml of chloramphenicol was present during only the first 6 days of culture, the secondary response was reduced 90 per cent. When it was absent for the first 6 days but present for the next 9 to 15 days, the response was reduced only 40 per cent. Since over 95 per cent of the antibody of the secondary response in most experiments appeared in the medium after the 6th day, chloramphenicol apparently inhibits antibody production by interfering with some early phase of the response. It is suggested that this interference involves messenger RNA and that animal cells have appeared resistant to this drug only because their complement of messenger RNA present when the drug has been added is stable over the short periods during which protein synthesis has usually been studied.     

THE RELATIVE AFFINITY OF ANTIBODIES SYNTHESIZED IN THE SECONDARY RESPONSE

In response to a second injection of rabbits with a dinitrophenylated antigen, given 2 months to 2 yr after the first injection, there was rapid synthesis of large amounts of antibody high in relative affinity for the dinitrophenyl (DNP) determinant. The antibodies formed 3 days after restimulation were already high in affinity. Amounts of antigen too small to elicit detectable antibody production may prime the animal for a partial secondary response characterized by the formation of antibody of intermediate affinity after a second antigenic stimulus. Investigations into the specificity requirements for the secondary response indicated that variation in the carrier protein and in the haptenic determinant could be tolerated. Thus, after immunization with DNP-bovine γ-globulin, DNP-hemocyanin elicited the vigorous production of high affinity anti-DNP antibodies. However, DNP serum albumin was much less effective: it elicited a secondary response in some animals primed with DNP-bovine γ-globulin only when the interval between injections was increased from 10 to 28 wk. A secondary response was also evoked when the haptenic determinent of the second immunogen differed slightly from that of the one injected initially (i.e., 2,4,6-trinitrophenyl versus 2,4-dinitrophenyl).     

ROLE OF THE THYMUS IN TOLERANCE : III. TOLERANCE TO BOVINE GAMMA GLOBULIN AFTER DIRECT INJECTION OF ANTIGEN INTO THE SHIELDED THYMUS OF IRRADIATED RATS

Rats subjected to high doses of whole-body irradiation, with simultaneous shielding of the thymus or spleen, recovered at 3 wk the ability to develop delayed sensitization and to form hemagglutinating and precipitating antibody following foot-pad injection of BγG in complete adjuvant. Injection of BγG into the shielded thymus immediately after irradiation, in amounts between 20 γg and 40 mg, inhibited these response to later challenge partially or completely. A comparable effect on immune responses to BγG was not seen after injection of heterologous antigen (Ea) intrathymically, BγG intraperitoneally, or BγG into the shielded spleen. However high doses (20 or 40 mg) of antigen given by the latter routes resulted in some diminution of later response. Arthus reactivity recovered partially in the spleen-shielded group and was readily suppressed by intrasplenic administration of antigen.     

IMMUNE RESPONSES IN VITRO : IV. SUPPRESSION OF PRIMARYγM, γG,ANDγA PLAQUE-FORMING CELL RESPONSES IN MOUSE SPLEEN CELL CULTURES BY CLASS-SPECIFIC ANTIBODY TO MOUSE IMMUNOGLOBULINS

The suppressive effects of monospecific goat anti-mouse globulins on primary immunoglobulin class-specific plaque-forming cell responses in mouse spleen cell cultures were investigated. Anti-µ suppressed responses in all immunoglobulin classes, whereas anti-γ₁ and anti-γ₂ suppressed the γ₁ and γ₂ responses but not γM or γA responses, and anti-γA suppressed only γA responses. The mechanism of action of the anti-µ was studied in detail because of its suppression of responses in all immunoglobulin classes. The anti-µ was specific for µ-chain determinants; its activity was dose dependent, but was not mediated by killing cells with surface µ-chain determinants. Free γM but not γG myeloma proteins in solution effectively competed with µ-bearing cells for the anti-µ. An excess of anti-µ was necessary in the cultures for 48 hr to insure complete suppression of 5-day responses. However, after removal of excess anti-µ at 48 hr, responses could be stimulated by newly added antigen in cultures where incubation was prolonged to 7 days. Anti-µ was most effective when added at the initiation of cultures and had no suppressive effect when added at 48 hr. Excess antigen did not effectively compete with anti-µ for antigen receptors. Precursors of antibody-forming cells were shown to be the cell population where the suppressive activity of anti-µ was mediated. The experiments suggest that anti-µ combines with µ-chain determinants in antigen-specific receptors on the surfaces of antibody-forming cell precursors, prevents effective stimulation by antigen and subsequent antibody production. To explain suppression of responses in all Ig classes by anti-µ, several models were proposed. It is not possible to determine from the data whether stimulation of precursor cells with γG or γA receptors requires concommitant stimulation of separate cells with only γM receptors, or whether cells bearing γM receptors are precommitted to or differentiate into cells capable of synthesis of other Ig classes, or whether receptors of γM and another Ig class are present on some virgin precursors or the second Ig receptor appears after antigenic stimulation.     

Immunochemical studies of antitoxin produced in normal and allergic individuals hyperimmunized with diphtheria toxoid. III. Studies of the passive Arthus reaction in guinea pigs using human precipitating and nonprecipitating diphtheria antitoxin

The Arthus reaction was studied in guinea pigs passively immunized with human diphtheria antitoxin. Diphtheria toxoid was given intradermally 24 hours following the intravenous administration of antitoxin and the subsequent reactions were graded and measured. The intensity of Arthus reactions was dependent upon the relative amounts of precipitating antitoxin and toxoid used. Severe lesions were caused by intravenous sensitization with 0.48 mg. precipitating antitoxin N and intradermal challenge with 0.05 or 0.17 toxoid N. Reactions of lesser intensity were caused by smaller amounts of antitoxin and toxoid. The nature of the antibody used for sensitization was of importance in the severity of Arthus reactions. In contrast to the behavior of precipitating antitoxin, amounts of non-precipitating antitoxin equivalent to 0.48 mg. N did not cause severe Arthus reactions when 0.05 or 0.17 mg. toxoid N was given intradermally. Precipitating antitoxic whole serum is altered by heating at 56°C. for 5 hours so that its precipitability is lost without any appreciable loss of antitoxic strength. This modified antitoxin produced Arthus reactions of only intermediate severity in guinea pigs sensitized with 0.48 mg. antitoxin N. When fractions of precipitating antitoxic serum were obtained using a cold ethanol technique described by Deutsch (16), a mixture of the purified γ₂-globulin and the crude albumin fraction heated together at 56°C. for 5 hours behaved similarly to whole serum. However, γ₂-globulin alone was not affected by the heating procedure, remained precipitable by toxoid, and was able to cause a severe Arthus reaction following sensitization with 0.48 mg. antitoxin N.     

EXPERIMENTAL GLOMERULONEPHRITIS : VI. THE AUTOLOGOUS PHASE OF NEPHROTOXIC SERUM NEPHRITIS

Nephrotoxic serum nephritis was studied in rats receiving variable amounts of kidney-fixing antibody and active or passive immunization to the heterologous gamma globulin of the species supplying the nephrotoxic antibody. Rats injected with a moderate to a large amount of kidney-fixing antibody (180 to 380 µg) and immunized to the heterologous gamma globulin developed a more severe nephritis than rats receiving similar amounts of antibody without further immunization. Rats injected with minimal amounts of kidney-fixing antibody (2 to 45 µg) and immunized to the heterologous gamma globulin developed a moderate nephritis in contrast to rats receiving similar amounts of antibody without further immunizations which showed no evidence of renal injury. In the rats receiving small doses of kidney-fixing antibody and immunization to the heterologous gamma globulin a lag period existed between the appearance of circulating antibody and kidney-fixed host antibodies and the appearance of renal injury. This delay apparently reflects the need for a continuing antigen-antibody reaction for some time in order to produce detectable injury with the small amounts of reactants involved. From these data it appears that, in the presence of excess circulating antibody, antigens occupying at most a few per cent of the glomerular capillary surface can provide an antigen-antibody interaction which will over a period of time cause detectable morphological and functional alterations of the glomerulus.     

FETAL RESPONSE TO ANTIGENIC STIMULUS : II. ANTIBODY PRODUCTION BY THE FETAL LAMB

The fetal lamb in utero is able to form large amounts of specific antibody in response to antigenic stimulus as early as the 66th to 70th day of the 150 day gestation period. Among the several antigens employed, the fetal lamb responded earliest, and with the highest titers, to bacteriophage ϕX. Slightly less effective as an antigen was horse ferritin, while ovalbumin proved to be a weak antigen, especially in younger fetuses. Ineffective in stimulating an antibody response at any time during fetal or early neonatal life were diphtheria toxoid, Salmonella typhosa, and BCG. Thus, it may not be feasible to fix precisely the time of onset of immunologic responsiveness in a species, inasmuch as it appears to differ so greatly from one antigen to another. The quantity of antibody found 10 days after ϕX immunization was not significantly different in fetuses injected at 60 to 120 days of gestation. The earliest anti-phage antibody produced by the lamb fetus is a macroglobulin sensitive to the action of 2-mercaptoethanol. Only in older fetuses with longer lasting stimuli were appreciable amounts of 7S γ-globulin antibodies formed. The conformity of these observations to theories on the ontogenesis of the immune response is discussed.     

Schistosoma mansoni shares a protective carbohydrate epitope with keyhole limpet hemocyanin

The glycanic epitope of the 38,000 Mr Schistosoma mansoni schistosomula major immunogen defined by the IPLSm1 protective mAb was identified in the hemocyanin of the marine mollusc Megathura crenulata, better known as KLH. This antigenic community was exploited to investigate further the biological properties of this epitope. KLH was shown to strongly inhibit the binding of IPLSm1 mAb to its 38,000 Mr target antigen. Immunization of naive LOU rats with KLH elicited the production of anti-S. mansoni antibodies capable of immunoprecipitating the 38,000 Mr schistosomulum antigen. Antibodies to KLH mediated a marked eosinophil-dependent cytotoxicity and passively transferred immunity towards S. mansoni infection. Finally, rats immunized with KLH were significantly protected against a challenge with S. mansoni cercariae. The deglycosylation of KLH completely abolishes its immunological and functional KLH properties, indicating the participation of an oligosaccharidic epitope of the native KLH that is also recognized by the sera of S. mansoni-infected patients. These observations provide new opportunities of access to the well-defined structure of a glycanic epitope potentially available for the immunoprophylaxis and seroepidemiology of schistosomiasis, and a new approach to the isotypic response towards a well-chemically defined epitope.     

THE ROLE OF COMPLEMENT IN THE PASSIVE CUTANEOUS REACTION OF MICE

Intradermal injection of mice with ribonuclease antibody, followed by intravenous injection with ribonuclease, resulted in permeability increase, demonstrable by "blueing." The size of the blued area depends on the quantity of antibody injected and on the interval between the two injections. If antigen was injected first and antibody was injected subsequently, a similar increase in permeability was observed in animals having a complete complement system (MuB1-positive) and in animals which have a deficient complement system (MuB1-negative). Marked differences in response were observed between these two types of mice if antigen was injected some hours after the antibody. In MuB1-negative mice, a blueing reaction was not observed at intervals between injections (2½ hours if 3 µg N antibody and 15 hours if 25 µg N antibody were injected intradermally) at which MuB1-positive animals showed a marked permeability increase. At these intervals, blueing did occur in MuB1-negative animals if they were injected with the serum of MuB1-positive mice or with fresh guinea pig serum. Blueing was not induced if the serum of MuB1-negative mice or heated guinea pig serum was injected. The occurrence of two distinct phases of the cutaneous reaction, of which only one involves the complete hemolytic complement system, was deduced from these observations.     

Acceptor-suppressor T cell hybridoma with a receptor recognizing antigen-specific suppressor factor

An acceptor hybridoma with a receptor that recognizes the keyhole limpet hemocyanin (KLH)-specific suppressor T cell factor (KLH-TsF) was established after the fusion of C57BL/6 splenic T cells enriched with KLH-coated petri dishes. The cloned hybridoma (34S-281) could be specifically activated by stimulation with the conventional KLH-TsF or monoclonal KLH-TsF from three different hybridomas in the absence of the relevant antigen (KLH) and it started to produce another factor that suppresses the antibody response against DNP-KLH in a KLH-specific fashion. The KLH specificity of the TsF was required for activation. The new factor was found not to bind the KLH but to be absorbed with the KLH-TsF-producing hybridoma. It is thus strongly suggested that the acceptor site has a complementary structure (antiidiotype) for the KLH-TsF. Moreover, the idiotypic determinant on KLH-TsF was found to have a structure similar to that on some of the anti-KLH antibodies, since the acceptor hybridoma was specifically killed by the conventional anti-KLH antibodies and complement. Drawing on the above results, the idiotype-antiidiotype network in the conventional antigen system is discussed.     

ANTIGEN RECOGNITION AND ANTIBODY SPECIFICITY : CARRIER SPECIFICITY AND GENETIC CONTROL OF ANTI-DINITROPHENYL-OLIGOLYSINE ANTIBODY

The exact specifiicity of anti-DNP antibody produced by Hartley guinea pigs immunized with a series of defined α,DNP and ε,DNP-oligolysines was studied by fluorescence quenching. All responder animals made anti-DNP antibody which recognized the precise chain length, ± 1 lysyl residue, of the DNP-oligolysines used to induce the immune response as measured by an increase in binding energy (–ΔF°) for that antigen. The ability of the immune system to detect the smallest possible change in oligolysine chain length suggests that the anti-hapten antibody-forming cell possesses a highly specific recognition system for carrier conformation. When DNP-oligolysines are incorporated in an adjuvant containing M. tuberculosis H37Rv, both responder and nonresponder produce anti-DNP antibody, but only the responder develops delayed skin sensitivity. In addition to their failure to develop delayed hypersensitivity, nonresponders produced anti-DNP oligolysine antibody which did not show the increase in –ΔF° for the immunizing antigen characteristic of responder antibody. These observations support a local environment hypothesis for antigen recognition at the level of the anti-hapten antibody-forming cell and suggest that the polylysine gene exerts its control at the same cell.     

IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS : II. PROPERTIES OF THE BLOOD GROUP A SUBSTANCE FROM POOLS OF HOO STOMACHS AND OF SPECIFIC PRECIPITATES COMPOSED OF "A" SUBSTANCE AND HOMOLOGOUS HUMAN ANTIBODY

1. The microquantitative precipitin method can be used to compare the relative activity of different preparations of the blood group A substance from hog stomachs and to study the effect of chemical treatment upon its stability. 2. With samples of about 25 µg. antibody nitrogen, an error of ±1.7 µg. antibody nitrogen will result in an error of ±12 per cent in the estimation of the amount of A substance. 3. The blood group A substance showed no significant loss of activity at 37°C. after 48 hours at pH 1.07 to 10.7 or after 2 hours at 100°C. over a pH range from 2.97 to 7.58. Exposure at 100°C. at pH 1.03 or at 9.03 or higher resulted in loss of activity. Parallel results were obtained by the hemagglutination inhibition and quantitative precipitin methods. 4. The solubility of specific precipitates of the blood group A substance from hog stomach and its homologous antibody formed in man was found to be about 1.6 µg. antibody N/ml. 5. A comparison is given of the chemical properties and activity of blood group A substances obtained by several procedures from pools of hog stomachs.     

Epinephrine-induced Insulin Resistance in Man

Endogenous release of epinephrine after stress as well as exogenous epinephrine infusion are known to result in impaired glucose tolerance. Previous studies of man and animals have demonstrated that this effect of epinephrine results from inhibition of insulin secretion and augmentation of hepatic glucose production. However, the effect of epinephrine on tissue sensitivity to insulin, and the relative contributions of peripheral vs. hepatic resistance to impaired insulin action, have not been defined. Nine young normal-weight subjects were studied with the insulin clamp technique. Plasma insulin was raised by ~100 μU/ml while plasma glucose concentration was maintained at basal levels by a variable glucose infusion. Under these conditions of euglycemia, the amount of glucose metabolized equals the glucose infusion rate and is a measure of tissue sensitivity to insulin. Subjects received four studies: (a) insulin (42.6 mU/m²·min), (b) insulin plus epinephrine (0.05 μg/kg·min), (c) insulin plus epinephrine plus propranolol (1.43 μg/kg·min), and (d) insulin plus propranolol. During insulin administration alone, glucose metabolism averaged 5.49±0.58 mg/kg·min. When epinephrine was infused with insulin, glucose metabolism fell by 41% to 3.26 mg/kg·min (P < 0.001). After insulin alone, hepatic glucose production declined by 92% to 0.16±0.08 mg/kg·min. Addition of epinephrine was associated with a delayed and incomplete suppression of glucose production (P < 0.01) despite plasma insulin levels >100 μU/ml. When propranolol was administered with epinephrine, total glucose metabolism was restored to control values and hepatic glucose production suppressed normally. Propranolol alone had no effect on insulin-mediated glucose metabolism. These results indicate that epinephrine, acting primarily through a β-adrenergic receptor, markedly impairs tissue sensitivity to an increase in plasma insulin levels, and that this effect results from both peripheral and hepatic resistance to the action of insulin.     

Reversal of Advanced Digitoxin Toxicity and Modification of Pharmacokinetics by Specific Antibodies and Fab Fragments

The effects of Fab fragments of high-affinity specific antibodies have been studied in a canine experimental model of lethal digitoxin toxicity. Selected antiserum from sheep immunized and boosted with a digoxin-serum albumin conjugate contained antibodies that cross-reacted with digitoxin with an average intrinsic association constant of 1.4 × 10¹⁰ M⁻¹ as determined by equilibrium dialysis. Rapid second-order association kinetics (k_f = 3.7 × 10⁶ M⁻¹ per s) and slow dissociation kinetics (k_r = 1.9 × 10⁻⁴ per s) were documented for the antibody-digitoxin complex. Eight dogs given 0.5 mg/kg digitoxin intravenously developed ventricular tachycardia after 23±4 (SEM) min. Control nonspecific Fab fragments were then given. All animals died an average of 101±36 min after digitoxin administration. Another eight dogs given the same digitoxin dose similarly developed ventricular tachycardia after 28±3 min. This group then received a molar equivalent dose of specific Fab fragments intravenously over 3 min, followed by a 30-min infusion of one-third of the initial dose. All dogs survived. Conducted sinus beats reappeared 18±4 min after initial Fab infusion, and stable normal sinus rhythm was present at 54±16 min. Plasma total digitoxin concentrations increased threefold during the hour after initial Fab infusion, while plasma free digitoxin concentration decreased to less than 0.1 ng/ml. Effects on digitoxin pharmacokinetics of these Fab fragments and the antibody population from which they were derived were further investigated in a primate species. Unlike common laboratory animals previously studied, the rhesus monkey was found to have a prolonged elimination half-life, estimated at 135 and 118 h by radioimmunoassay and [³H]digitoxin measurements, respectively, similar to man and thus providing a clinically relevant experimental model. Intravenous administration of 2 mol of specific Fab fragments per mole of digitoxin 6 h after 0.2 mg of digitoxin produced a rapid 4.3-fold increase in plasma total digitoxin concentration followed by a rapid fall (t_½ 4 h) accompanied by a 14-fold enhancement of urinary digitoxin excretion over control values during the 6-h period after Fab was given. Analytical studies were consistent with increased excretion of native digitoxin rather than metabolites, and the glycoside was found in equilibrium dialysis studies to be excreted in the urine in Fab-bound form. Administration of 2 mol of specific antibody binding sites per mole of digitoxin as intact IgG caused a greater and more prolonged increase in plasma total digitoxin concentration, peaking 13-fold above control levels. In contrast to the effects of Fab, however, specific IgG reduced the rate of urinary digitoxin excretion substantially below control values. We conclude that Fab fragments of antibodies with high affinity for digitoxin are capable of rapid reversal of advanced, otherwise lethal digitoxin toxicity, and are capable of reducing the plasma half-life and accelerating urinary excretion of digitoxin.