Failure of limiting antigen doses to selectively stimulate high-avidity memory cells.

Mouse spleen cells secondarily stimulated with a small dose of antigen, which elicited few antibody-forming cells, produced antibodies of avidity no greater than that of antibodies synthesized by cells stimulated with an optimal antigen dose. This result is in conflict with the hypothesis that maturation of the immune response is based on competition for limiting amounts of antigen among cells with receptors of varying avidity.

Cloning and sequence analysis of cDNAs encoding the heavy and light chain variable regions of an Ab2beta anti-idiotypic monoclonal antibody possessing an internal image of cocaine.

We report here the cloning and sequence analysis of cDNAs encoding the variable regions of an Ab2beta anti-idiotypic monoclonal antibody (K1-4c, gamma1kappa) that mimics the configuration of cocaine. The Ab2beta specifically binds to the human dopamine transporter as shown by confocal immunofluorescence microscopy. The sequence of the heavy chain complementarity-determining region 3 of K1-4c is strikingly similar to that of a monoclonal antibody (F11.2.32) specific for HIV-1 protease. Three or four amino acids in the epitope recognized by the anti-HIV-1 protease antibody are also present in the third extracellular loop of the dopamine transporter. This epitope is within the conserved region of the known transporters for dopamine, norepinephrine and serotonin in Homo sapiens, Rattus norvegicus, Caenorhabditis elegans and Drosophila melanogaster.

Age-related changes in B and T lymphocytes and decline of humoral immune responsiveness in aged mice.

The activity of the immune system declines in an age-related fashion, after reaching a peak in young-adult animals. Although this pattern has been well documented, the mechanisms responsible for the decline of immune potential in senescence have not been fully elucidated. At the same time, it has become apparent that the immune response depends on complex interactions among different cell types. We review the results of the analyses of B- and T-lymphocyte function and activity in the humoral responsiveness of aged mice that have been performed in a number of laboratories, with a variety of experimental techniques. We conclude that an increase in T-cell suppressor function is the first immunologic lesion of aging in mice. This is followed by a decrease in T-cell helper function and finally by a loss of B-cell function.

Effects of long-term treatment of mice with anti-I-J monoclonal antibody and dialyzable leukocyte extract on immune function and lifespan.

In 1969 Walford hypothesized that age-related dysfunctions of the immune system may be involved in the pathogenesis of the lesions and disease of aging. Studies were initiated to test whether immunologic interventions intended to maintain the integrity of the immune system would delay the onset of diseases of aging and prolong lifespan. Adult BC3F1 mice were treated with anti-I-J monoclonal antibody, with human dialyzable leukocyte extract, or with saline once a week for one year. Spleen cells from the mice were then assayed for suppressor, T-helper and B-cell activity. Treatment with dialyzable leukocyte extract decreased the elevated nonspecific suppressor activity. Mice treated with anti-I-J antibody had elevated T-helper cell activity. In another experiment, mice were treated weekly with anti-I-J antibody, dialyzable leukocyte extract, or saline from 18 months of age until natural death. The mice were immunized with avian gammaglobulin at 27 and again at 29 months of age. Both types of immunologic intervention resulted in a greater secondary antibody response than that of the saline-treated control mice. Mice treated with anti-I-J antibody survived longer than did mice of the other two groups. There was a correlation between the magnitude of the secondary response of individual mice and their lifespan. The results provide support for the immunologic theory of aging.

Detection and enumeration of immunoglobulin secreting cells.

A method is described in which sheep red blood cells (SRBC) are coated with anti-immunoglobulin (Ig) antibodies (Ab) for use in reverse hemolytic plaque assays (RHPA) as follows. The non-complement fixing F(ab')2 fragments of rabbit anti-mouse Ig Ab are derivatized with the N-hydroxysuccinimide ester of palmitate. The hydrophobic palmitate tails spontaneously insert into the SRBC membranes, thus coating the cells with anti-Ig F(ab')2 molecules. The SRBC are lysed by successive additions of mouse Ig, rabbit anti-mouse Ig and complement. When this procedure is carried out in agar gel, Ig-secreting mouse cells produce localized hemolytic areas (plaques). The procedure is more reproducible and more sensitive than RHPA performed with protein A-coated SRBC. In principle, this procedure should be adaptable to the detection of cells secreting any molecule for which specific antibodies are available.

Dopamine uptake by mouse neuroblastoma N1E-115 cells stably expressing human dopamine transporter is differentially inhibited by anti-idiotypic ab2beta antibodies mimicking the configuration of cocaine.

We have successfully constructed a mouse neuronal N1E-115 cell line stably expressing a fully functional human dopamine transporter (hDAT). Previous studies in our laboratory have produced several anti-idiotypic Ab2beta antibodies that mimic the configuration of the cocaine molecule at their antigen-combining site. In the present study, we observed that some anti-idiotypic Ab2beta antibodies inhibited dopamine uptake by the hDAT-transfected line. Each antibody showed a different level of inhibition (between 40% and 90%). These findings suggest that the internal images of these antibodies can be used as analog peptides that may compete with cocaine for its binding site on the transporter but not impair dopamine uptake as much as cocaine does.

Individual and combined effects of ethanol and cocaine on the human dopamine transporter in neuronal cell lines.

Concurrent use of cocaine and alcohol results in reinforced behavioral consequences, but the molecular mechanisms associated with the co-use of both drugs are not clear. We report here that a 24-h exposure of the human dopamine transporter (hDAT)-transfected mouse neuroblastoma N1E-115 cell line (6C6) to cocaine (1 microM), or ethanol (1%) or both, increased dopamine re-uptake by approximately 25, 29 and 44%, respectively. The same treatment also increased dopamine re-uptake by the hDAT-transfected mouse neuroblastoma Neuro2A cell line (3B7) by approximately 36, 41 and 77%, respectively. However, no increase of dopamine re-uptake was observed in the hDAT-transfected non-neuronal CHO cell line (10E9). These data support the hypothesis that the DAT may be a common neural substrate for cocaine and ethanol in dopaminergic neurons and it may be involved in the psychological effects of both addictions.

The influence of age on the canine immune system.

Immune function was assessed in a group of 47 Labrador Retrievers, ranging in age from 0.8 to 11.5 years, in order to establish baseline data on canine immunosenescence. Natural killer cell activity, lymphocyte subset distributions, antibody production, and mitogen-induced lymphoproliferative responses, all of which have been demonstrated to undergo age-related changes in humans and mice, were chosen as indicators of immune function. Dogs were categorized by age as young (mean 2.4 years), middle-aged (mean 5.8 years), and old (mean 9.1 years). Natural killer cell activity was not affected significantly by age. Lymphocyte subset analysis revealed a significant age-related increase in the percentage of cells staining with a pan T-cell reagent, accompanied by a corresponding increase in the percentage of CD8 cells from youth to middle age. An age-related decrease in the percentage of B-cells was observed concomitant with the increases in T-cell percentages. A gender-related difference in pan T-cell distribution was also observed, with females having a higher percentage than males. Lymphoproliferative responses of both young and middle-aged dogs to the mitogens concanavalin A, phytohemagglutinin, pokeweed mitogen, and staphylococcal enterotoxin B were significantly higher than those of old dogs. In general, the mitogen responses of male dogs were affected more dramatically by age than those of females. A significant age-related decline in in vivo antibody responses to the protein antigen, keyhole limpet hemocyanin, was not observed, although the mean titers of the young dogs were higher than those of the old.

The influence of age and gender on the immune system: a longitudinal study in Labrador Retriever dogs.

While aging studies employing a cross-sectional design have been informative in documenting many age-related alterations in immune function between different age cohorts within a population, longitudinal studies are invaluable for verifying changes at the level of the individual and for defining the precise periods of life during which particular changes occur. In the present study, a battery of immunological parameters were evaluated in a group of Labrador Retrievers as part of a comprehensive longitudinal aging study. Twenty-three dogs (14 females, 9 males; from 4 to 11 years of age) were evaluated annually for total WBC counts, lymphocyte subset distributions, natural killer cell activity and neutrophil phagocytic activity, and biannually for lymphoproliferative activity. An age-related decline in absolute numbers of lymphocytes, T-cells, CD4-cells and CD8-cells was observed in both genders. The distribution of lymphocyte subsets shifted with age, most dramatically in the females; percentages of B-cells declined while those of T-cells increased. Changes in percentages of CD4- and CD8-cells over the 8-year period were not dramatic; in females, percentages of CD8-cells increased significantly in early- to mid-life and then stabilized. Lymphoproliferative responses to mitogens declined over time in both genders. Males demonstrated higher levels of NK cytolytic activity than females; a marginal decline in activity with age was observed. No significant age-related changes in the phagocytic capacity of PMN were observed. These longitudinal findings help to discriminate between those immune parameters which change most dramatically in early-life versus those which either change more dramatically later in life or change gradually over the entire span of life. In addition they identify significant gender differences in several parameters and corroborate our previously published cross-sectional aging data in the same species.

Diagnosis of histoplasmosis and blastomycosis by an antiglobulin hemagglutination test.

An antiglobulin hemagglutination test was developed for detection of antibody directed to Histoplasma capsulatum and Blastomyces dermatitidis. The substances responsible for spontaneous agglutination of erythrocytes were removed from histoplasmin and blastomycin by vacuum dialysis, and partial purification of the antigens by gel filtration chromatography on Sephadex G-150 allowed removal of additional nonantigenic material which competed with the antigens for binding on the erythrocyte surface. The test was sensitive enough to detect antibodies in sera which were negative by complement fixation, immunodiffusion, or both, but it failed to discriminate reliably between antibody directed to H capsulatum and antibody directed to B dermatitidis. Erythrocytes sensitized with partially purified blastomycin produced some false-positive reactions with normal canine sera; this was corrected by diluting the antigen before sensitization of the erythrocytes.

Reflections on bioethics: consolidation of the principle of autonomy and legal aspects.

The author highlights the importance of emotions in all ethical reflections. He describes the most common positions of ethicists employing duties and rights as the basis for ethical thought. The author, goes to Freudian theory as viewed by the utilitarians, stating that the 'quest for pleasure' is not necessarily egocentric, especially for adults. For example, the feeling of solidarity emerges 'from the inside out', making irrelevant all the emphasis laid on obedience to duty (from the outside in). The article questions the essence of Kantian theory, based exclusively on 'reason' with disregard for feelings, by establishing what he considers a 'positivist' view of rational thought. It emphasizes the principle of autonomy, which it seen as basically opposing the principles of beneficence and fairness. It is proposed that the latter should be seen as what he calls heteronomy (a concept different from that of the rational ethicists). In theory, autonomy is not assigned to anyone on the basis of an external assessment. Any intervention in individual autonomy must be made (by the intervenor) when it becomes imperative in the defense of social or cultural values. The article distinguishes between ethics and morals) and states that the sole acceptable ethical principle is that ethics (theoretically) has no principle.

[The concept of health]. / O conceito de saúde.

Objections to the present WHO (World Health Organization) definition of HEALTH, as "the state of perfect physical, mental and social well-being", are expressed. It is considered to be anachronistic, first because it aims at perfection which is unaltainelle because of district personality characteristics. As the main support for this idea, the necessary renunciation of part of man's drive to liberty in exchange for the lesser insecurity provided by social life (Freud, Castoriadis and McDougall), is groted. The validity of distinguishing between "soma", "psyche" and "society" is questioned and the concept of the "integrated man", alluding to Pierre Marty and to Freud himself is adapted, and situations are recalled in which the interaction of the three aspects mentioned above is actually evident. Finally, the notion of the quality of life, in accordance with an antipositivistic taken from Bion, point of view, is discussed, and the concept that reality is that of each human being, is adapted. This priority and the proposal to rescue subjectivism which was also observed by Foucault when he studied mental disease, leads to a last criticism of the present definition of health, based exclusively on external, objective evaluations.

Hematology values from clinically healthy Peromyscus leucopus.

Reported herein are hematocrit and total and differential WBCs obtained from 132 clinically healthy male and female white-footed mice (Peromyscus leucopus) of four different age groups: 4, 5-8, 9-20, and >32 wk old. Minimal differences were identified between the age groups. The number and percentage of neutrophils in the 4-wk-old mice were significantly higher than those in the other groups; the relative percentage of lymphocytes in the 4-wk-old mice was significantly lower than in the 9-20-wk-old mice. The only significant gender effects identified were higher numbers of WBCs and lymphocytes in females of the 4-wk-old group and higher hematocrits in males of the 5-8- and >32-wk-old groups.

[Ethics in occupational health]. / Etica em saúde ocupacional.

The author considers the doctor-patient relationship according to modern ethic concepts. He underscores the total respect owed to his individuality, providing him with all the data available on his health, so he may use it at his discretion. Next, he presents the concept of occupational health, pointing out its multiprofessional and multidisciplinary character, and specifying its level of activity (factory, labor union, government departments, etc.). He then details the extraordinary divergence of interests between the employer and the factory employee which often places the factory doctor, at least as viewed by workers, as an employer's accomplice. He stresses the importance of existing legal instruments, such as the Federal Constitution, the Labor Act, and the Medical Ethics Code as being ideologically progressive. However, he also points out the difficulties faced for an effective performance.

Generation of suppressor T cells during the primary response of murine spleen cells in diffusion chambers.

A vigorous secondary response to DNP is elicited by dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) in spleen cells from primed mice cultured in Millipore diffusion chambers. However, the same antigen under the same conditions does not elicit a primary response in normal spleen cells. In contract, foreign erythrocytes elicit a primary response and function as an effective carrier for the primary response to a hapten. We found that the failure of haptenated protein to induce a primary response in diffusion chambers can be attributed to the generation of suppressor T cells by the protein. Cultures of T depleted normal spleen cells, supplemented with carrier-specific helper T cells, developed a DNP-specific primary response upon stimulation with DNP-KLH.

Behavior of the idiotypic network in conventional immune responses. III. Detection and enumeration of cells producing idiotypic and anti-idiotypic antibodies by a spot ELISA technique.

The idiotypic (Ab1) and anti-idiotypic (Ab2) responses of spleen cells of mice immunized with DNP-Ficoll were assayed with the spot ELISA technique in wells coated with DNP-BSA or with affinity-purified rabbit anti-DNP antibodies, respectively. In agreement with results previously obtained with the hemolytic plaque technique, large numbers of Ab1 and Ab2 ELISA spots were found. The cytokinetics of the Ab1 and Ab2 responses were very similar and the peak responses occurred simultaneously. The isotypes of Abs1 and Abs2 were also similar. Both responses were specifically inhibited by soluble DNP-lysine. Similar results were obtained with spleen cells from mice immunized with the T-dependent antigen DNP-KLH and with the T-independent antigen fluorescein-Ficoll. In contrast, no Ab2 response was detected when spleen cells from mice immunized with ovalbumin were assayed, probably because the affinity-purified rabbit anti-ovalbumin antibodies used to coat the wells for the Ab2 assay contained molecules of several specificities, corresponding to the several epitopes of the ovalbumin molecule. The many similarities between the Ab1 and Ab2 responses to the haptens suggest that they reflected two different reactivities of the same antibody molecules.

A time study of the requirement for carrier-specific helper cells in the response of mice to dinitrophenylated keyhole limpet hemocyanin.

The anti-hapten response of spleen cells from mice primed with hapten-carrier (dinitrophenylated keyhole limpet hemocyanin) was decreased by depletion of T cells during the first four days after secondary antigenic stimulation, but was unaffected when the T cells were depleted on day 5. The response of cultures that were depleted of T cells prior to secondary antigenic stimulation was restored by addition of carrier-primed cells during the first two days of culture, partially restored on day 3, but not at later times. Thus, T cells were required only at about day 4 of a secondary response to a T-dependent antigen that peaked on day 8. Autoradiography experiments showed that the frequency of antigen-binding cells increased with time regardless of the presence of T cells in the cultures. We conclude that helper T cells are not required for B cell proliferation, but are required for the differentiation of B cells to antibody-secreting cells.

Humoral immunity in aged mice. I. Age-related decline in the secondary response to DNP of spleen cells propagated in diffusion chambers.

The secondary response of young-adult and old BC3F1 mice to DNP was assessed by boosting spleen cells from primed mice with DNP-KLH, propagating them in diffusion chambers implanted into irradiated recipient mice, and by counting the DNP-specific PFC. There was no difference between young-adult and old cells in the antigen dose response and in the kinetics of the immune response. There were marked differences in the magnitude of the peak response. The response of 2-year-old mice was significantly smaller than the response of young-adult mice. Two and one half-year-old mice had the smallest response. In the old mice, there was an inverse correlation between the number of nucleated cells in the spleen and the log of the number of PFC.

Humoral immunity in aged mice. II. Increased suppressor T cell activity in immunologically deficient old mice.

Mixed cultures of DNP-primed spleen cells from old and young BC3F1 mice in diffusion chambers yielded significantly fewer secondary PFC than expected from the numbers of PFC found when young and old spleen cells were cultured alone. There was a negative correlation between the log of the number of PFC found in cultures of old spleen cells alone and the degree of immunosuppression exerted by the cells. The immunosuppressive effect was abolished by pretreatment of the old spleen cells with anti-theta serum and complement. Thus, the effect was attributed to the presence of large numbers of suppressor T cells in the spleen of primed old mice. No suppression was found in mixed cultures of spleen cells from old unimmunized mice and young primed mice.