Richard M. Rothberg

Immune responses of human adults after oral and parenteral exposure to bovine serum albumin

Abstract Investigations were carried out to examine the possible causes of the low incidence and low levels of anti-BSA in human adults as compared to those of normal children. To determine if the reduced amount of BSA in the adult diet was an important factor, childhood levels of dietary BSA in the form of oral supplemental doses were administered to 17 normal human volunteers for 21 days. To learn if the manner in which dietary protein was catabolized by the gastrointestinal tract of the adult was a determining factor, selected individuals were also stimulated by intradermal injections of BSA. When anti-BSA was present before the experiment, there was a measureable antibody response following both methods of stimulation. The responsiveness of this group indicates that, among adults who retained the capacity to produce anti-BSA, the dose of antigen was an important variable in determining the amount of antibody actually produced. When no anti-BSA or very low levels existed before the experiment, the adults were unresponsive or hyporesponsive to both methods of stimulation. At least 80 per cent of these unresponsive adults had been synthesizing anti-BSA during childhood and should have resumed antibody production had their adult unresponsiveness been due to either a low dietary intake of BSA or the gastrointestinal conditions unique to adults. Circulating antigen was demonstrated in the sera of individuals without anti-BSA for as long as 21 days after intradermal injections of BSA. In subjects with trace amounts of anti-BSA prior to the intradermal injection of BSA, both BSA and anti-BSA were demonstrated to exist simultaneously in the serum for a minimum of 21 days. These data suggest that antigenantibody complexes can sometimes persist in the circulation for prolonged periods following the injection of relatively small doses of antigen into human subjects. None of the subjects demonstrated an immediate wheal-and-flare response, an Arthus reaction, or a tuberculin type of response when tested either before or after the experiment. Thus it appears that the low incidence of detectable anti-BSA among adults is due in part to an acquired immunological hyporesponsive state to BSA.

Immunoglobulin and specific antibody synthesis during the first weeks of life of premature infants

The hypothesis that antigenic exposure following birth causes the acceleration of immunoglobulin synthesis was evaluated by relating synthesis of immunoglobulin in premature infants (1,500 to 2,250 grams) to birth and not to gestational age and by demonstrating specific antibody production to a cows milk protein during the first 2 weeks of life. During the first 21 days of life the onset of IgA synthesis and the changes in IgA and IgM serum concentrations were similar in 1,500 to 1,899 grams, 1,900 to 2,250 grams, and full-term infants. A slight increase in IgG cord serum concentration was associated with increasing birth weight. The subsequent metabolism of IgG was similar in the 3 groups. In contrast to an undetectable immune response to bovine serum albumin (BSA) in infants not fed the antigen, 63 per cent of the infants fed BSA were immunized against this antigen. Anti-BSA was detected in IgG as well as in IgM immunoglobulins, demonstrating the capacity of these infants to make specific IgG antibodies. Qualitatively the anti-BSA was shown to be a poor precipitating antibody.

Morphine-3-Succinyl—Bovine Serum Albumin: An Immunogenic Hapten-Protein Conjugate

Morphine-3-hemisuccinate was synthesized by reaction of morphine with succinic anhydride. This compound was conjugated to bovine serum albumin by the mixed anhydride method, and the degree of conjugation was determined by base hydrolysis of the conjugate, extraction, and measurement of free morphine. An average of 6.5 molecules of morphine were conjugated to each molecule of protein. Eleven rabbits immunized with varying doses of the conjugate were producing antibody 8 weeks later, as determined by a modification of the ammonium sulfate method, which measures primary binding of antigen by antibody.

Effects of passive immunization against morphine on heroin self-administration.

Antimorphine antibodies produced in Rhesus monkeys immunized with morphine-6-hemisuccinate-BSA were passively administered to recipient monkeys trained to self-administer heroin and cocaine. Following antibody administration, changes in heroin self-administration behavior were observed which were similar to those achieved with low doses of naloxone. Both manipulations increased heroin self-administration without affecting cocaine responding.

Supplemental parenteral nutrition in cystic fibrosis.

The efficacy and safety of short-term supplemental peripheral hyperalimentation (PH) was evaluated in 15 hospitalized cystic fibrosis (CF) patients who exhibited varying degrees of pulmonary disease severity and nutritional impairment. An average of 1000 supplemental calories/day were administered intravenously for a 2- to 3-week period to patients being treated with parenteral antibiotics for exacerbation of their pulmonary disease. Eleven of 15 patients responded with a weight gain of greater than 2.0 kg and showed continued weight gain and stabilized pulmonary status for the 6- to 12-month follow-up period; two patients showed dramatic reversal of poor weight gain and growth following PH. Total calorie intake (oral + PH) equaled 141 +/- 40% of the recommended dietary allowances (RDA) in responders, with 45 +/- 12% RDA contributed by PH, in contrast to 68 +/- 20% of the RDA for total calories with 31 +/- 13% supplied using PH achieved in the nonresponders. Linoleic acid deficiency was documented in these patients (linoleic acid level as a percent of total fatty acid = 21.9% +/- 1.41 SEM vs 31.8% +/- 1.16 SEM in normal controls), and all seven patients achieved normalization of linoleic acid level after PH. Prior assessment of nutritional status (anthropometric measurements) or of severity of pulmonary disease (NIH clinical score) did not allow prediction of response to PH. No complications resulted from administration of PH to these hospitalized CF patients.

The lack of effect of transfer factor in thymic dysplasia with immunoglobulin synthesis.

Four children with deficient thymus-dependent lymphocytes and normal parathyroid function are described. They had normal immunoglobulin levels and responded to antigenic stimulation. Delayed skin tests were negative and in vitro lymphocyte responses to PHA, C. albicans , and streptococcal M protein were deficient. Infections with C. albicans (three patients), atypical mycobacteria (two patients), and P. carinii (one patient) were documented. Treatment with transfer factor did not alter their clinical or laboratory findings. It is postulated that these patients presented a spectrum of thymic deficiency and that transfer factor was ineffective because of the absence of thymus-influenced lymphocytes.

Reconstitution of T-cell function in severe combined immunodeficiency disease following transplantation of early embryonic liver cells

In a 51/2-month-old male infant with adenosine deaminase-positive severe combined immunodeficiency disease, who had no suitable bone marrow donor, immunologic reconstitution was attempted with lymphoid cells obtained from the liver of a 4- to 5-week-old-male human embryo. A mild graft-versus-host reaction began three weeks later. T-cells, which were absent prior to infusion of hepatic lymphoid cells, rose to a maximum of 554/mm3 at 16 weeks post transplantation. A normal lymphocyte response to pokeweek mitogen was not present until 25 to 30 weeks and to allogeneic cells until 39 weeks. Postive in vitro lymphocyte responses to Candida albicans were found repeatedly after 52 weeks. Twenty months following transplantation the patient is free of clinical infection, although he requires regular injections of gamma globulin.

Antibodies in rabbits fed milk and their similarities to antibodies in some human sera

Four of six adult rabbits drinking skimmed cows milk produced antibody against BSA and ALA. Anti-BSA was first detected in some sera 14 days after the initiation of milk feedings and anti-ALA was present when first studied on the thirty-fifth day. Three of the four antisera bound more I*BSA than I*ALA during the entire study. The specificities of the antibodies which bind ALA and BSA in human serum were very similar to the specificities of the respective antibodies in the sera from rabbits drinking milk and in the sera from rabbits immunized with purified BSA or ALA. No shared antigenic groups could be demonstrated between ALA and BSA, HSA or ovalbumin. As measured by immunological methods, commercial skimmed milk contained an average of 0.29 mg. BSA per milliliter and an average of 1.3 mg. ALA per milliliter. Raw beef contained 1.36 mg, extractable BSA per gram and cooked beef contained only 0.08 mg, extractable BSA per gram. Cows milk is probably the major source of antigenie stimulation for both the anti-BSA and anti-ALA in human serum. Even when compared on a weight-for-weight basis rather than on a mole-for-mole basis, BSA appears to be a generally more effective oral antigen than ALA in both humans and rabbits. As judged by the few parameters studied, the immune response of rabbits drinking milk was similar to the human immune response to milk.

Alteration of T cell function in healthy persons with a history of thymic x-irradiation.

The possible late effects of x-irradiation to the infantile thymus were investigated by studying immune functions in 12 healthy persons with a history of thymic x-irradiation and healthy control subjects. No differences were found in serum immunoglobulin values, humoral antibody levels, lymphocyte counts, and lymphocyte reactivity to phytochemagglutinin, vaccinia virus, purified protein derivative (PPD), and allogeneic cells. The irradiation group exhibited cellular hyperresponsiveness to streptoskinase-streptodornase (SK-SD). In contrast, mean skin and in vitro lymphocyte responses to Candida albicans were depressed in the patients with thymic irradiation. A dissociation of these two Candida responses was found in only 1 of 14 healthy control subjects but in 7 of 12 irradiated individuals. While thymic irradiation did not result in impaired immunologic defenses leading to clinical disease, it caused alterations in T cell responses similar to those reported in patients with chronic mucocutaneous candidiasis.

Humoral and cellular responses to native antigen following oral and parenteral immunization with lipid-conjugated bovine serum albumin

Abstract Humoral and cellular immune responses of rabbits to bovine serum albumin (BSA) were measured following oral and parenteral immunization with either BSA or one of two dodecanoic acid conjugates of BSA. The first consisted of a mixture of lightly and heavily conjugated BSA-molecules (L-BSA-mix), while the second (L-BSA) was a homogeneous preparation of heavily conjugated BSA with more than 95% of the 60 available amino groups covalently bound to dodecanoic acid. Animals ingesting L-BSA-mix had a similar humoral immune response but enhanced cellular reactivity to BSA in comparison to animals ingesting the native antigen. No systemic immunologic responses to BSA were detected following ingestion of L-BSA in spite of the demonstration of circulating BSA antigenic groups. This lack of a detectable immune response after oral administration was not due to masking of antigenic sites by the lipid residues since both humoral and cellular immune responses to BSA were obtained in animals injected with L-BSA. Ingestion of L-BSA did not induce tolerance since a subsequent injection of BSA elicited a normal primary immune response. The differences in immunogenicity between BSA, L-BSA and L-BSA-mix following oral administration may be related to different modes of antigen recognition by the gut-associated lymphoid tissues.