Cell-mediated and humoral immunity in mice: cross reaction between lysozyme and S-carboxymethylated lysozyme studied by a modified footpad test.

The mouse sensitized by subcutaneous (sc) injection of lysozyme in emulsion of Freund's complete adjuvant (FCA) was shown by a modified footpad test to develop three kinds of hypersensitivities. Injecting lysozyme in 2.5-mul emulsion of Freund's incomplete adjuvant (FIA) into the footpad elicited strong footpad swelling in 30 min (anaphylactic reaction), in 3 hr (Arthus-type reaction) and in 24 hr (delayed-type hypersensitivity; DTH). The mice showing anaphylactic reaction in the footpad test manifested severe active systemic anaphylaxis, and the sera of these animals showed high IgG1 antibody titers with only sparingly detectable or no IgE antibody titers. In the sensitizing system with the use of FCA, the antigenicity of S-carboxymethylated lysozyme (CM-lysozyme) devoid of the three-dimensional conformation of lysozyme was compared with that of the native molecule. CM-lysozyme and lysozyme completely cross-reacted to each other in DTH, but not at all in the anaphylactic or Arthus-type reaction or in IgG1 antibody production. CM-lysozyme was shown also to have the ability to bestow immunological memory for the induction of humoral immunity against lysozyme; intravenous (iv) injection of lysozyme in saline or sc injection of CM-lysozyme-FCA alone failed to induce immediate hypersensitivities and IgG1 antibody production against lysozyme, but pre-sensitization by sc injection of CM-lysozyme-FCA enabled the animal to induce these responses to significant levels when iv injection of lysozyme in saline was given as a booster.     

Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants

Adjuvants have been used for more than 70 yr to enhance the immune response of the host animal to an antigen. Among the mechanisms that adjuvants use to enhance the immune response are the "depot" effect, antigen presentation, antigen targeting, immune activation/modulation, and cytotoxic lymphocyte induction. The immunostimulatory properties of adjuvants result in inflammation, tissue destruction, and the potential for resulting pain and distress in the host animal. The inflammatory lesions produced by adjuvants such as Freund's complete adjuvant (FCA) have led some to conclude that pain and distress are present, even in cases where the scientific evidence fails to support this conclusion. Recommendations and regulations in the literature, based on available scientific evidence, provide guidance on total adjuvant volumes, volumes per site, routes of injection, booster injections, and adjuvants used for antibody production. Among the numerous adjuvants that are used for experimental antibody production reviewed in this article, many claim to be less inflammatory, tissue destructive, and painful than FCA while producing equal or superior antibody responses. Although no adjuvant surpasses FCA for experimental antibody production against a wide range of antigenic molecules, many produce excellent antibody responses with less inflammation and tissue destruction. Balancing the requisite degree of immuno-stimulation and the extent of inflammation, necrosis, and potential pain and distress requires consideration of the nature of the antigen, the host immune responsiveness, the adjuvant's mechanisms of action, and the desired end-product. In cases where the antigen is a weak immunogen or has a very limited availability, the type and role of adjuvant becomes a critical component in producing an acceptable immune response and humoral antibody response.     

Intracisternal administration of Mycobacterium tuberculosis potentiates the delayed type hypersensitivity in guinea-pigs

The delayed type hypersensitivity (DTH) was studied in guinea-pigs using the skin test. The mycobacterium tuberculosis (M. tbc)--was applied by various routes. The control group received ovalbumin in Freund's incomplete adjuvant (FIA) into the footpad. The first experimental group received ovalbumin in Freund's complete adjuvant (FCA) into the footpad. The other experimental groups always received, in addition to ovalbumin plus FIA into the footpad, the M. tbc. 1. intracisternally, 2. intramuscularly, 3. intraperitoneally, 4. orally. On the day of administration of the sensibilizing substance, the body temperature was monitored. The skin test was measured after 14 and 21 days. It was established that, for the study of the DTH, the 21-day interval was more significant than the 14-day interval. A 100 times smaller dose of M. tbc. given intracisternally had the same immunostimulating effect as the injection of ovalbumin with M. tbc into the footpad (p less than 0.01). The size of the skin reaction was not only significantly influenced by the intramuscular and oral administration of M. tbc. On the other hand, the intraperitoneal administration inhibited the DTH (p less than 0.01). The increase of body temperature after the administration of M. tbc. correlated with the influence on the DTH except for the intraperitoneal administration. The route of the M. tbc. administration was crucial for the development of the DTH.     

Immunoregulation in experimental interstitial nephritis: immunization with renal tubular antigen in incomplete Freund's adjuvant induces major histocompatibility complex-restricted, OX8+ suppressor T cells which are antigen-specific and inhibit the expression of disease

We utilized a model of experimental interstitial nephritis induced by renal tubular antigen in complete Freund's adjuvant to examine a mechanism of immunologic tolerance produced by priming immunization with tubular antigen in incomplete Freund's adjuvant. Brown Norway rats primed with tubular antigen in incomplete adjuvant do not develop significant nephritis after challenge with antigen in complete adjuvant, and this tolerance can be transferred to naive recipients with donor T cells. These T cells also specifically suppress a delayed-type hypersensitivity response to soluble tubular antigen in recipients immunized to produce disease. This suppression is MHC-restricted and is mediated by OX8+ T cells which bind antigen and bear idiotypes cross-reactive with those on antibodies eluted from the tubular basement membrane. Despite the suppression of histologic disease, tolerized animals were able to produce significant titers of antibodies to tubular basement membrane. Our findings demonstrate an additional strategy for altering the natural history of immune-mediated renal disease, and further refine the characterization of the suppressive effect produced by incomplete Freund's adjuvant.     

Beta-glucan derived from zymosan acts as an adjuvant for collagen-induced arthritis

Collagen-induced arthritis (CIA) is an experimental model of rheumatoid arthritis (RA) and has helped researchers to analyze the pathogenesis of inflammatory joint disease. In classical CIA, Freund's complete adjuvant (FCA), which contains heat-killed Mycobacterium tuberculosis, is used as an adjuvant. In our previous study, we reported that particles of beta-glucan, OX-CA, derived from Candida albicans, acted as a proper adjuvant in the CIA model. In this study, to establish pure beta-glucan as an adjuvant for CIA, we tested a commercially available preparation of Zymosan A (ZYM) and modified its products. beta-Glucan fractions of ZYM were prepared by oxidation with various concentrations of NaClO. The oxidized ZYM (OX-ZYM) was mainly composed of beta-glucan. In this study, we examined its effect as an adjuvant for CIA. DBA/1 mice injected with CII and OX-CA developed arthritis 7-10 days after receiving booster injections; the OX-ZYM fractions induced arthritis with the same time course. 0.01% OX-ZYM (oxidized with a 0.01% NaClO solution) caused arthritis faster than 0.1% OX-ZYM or 0.5% OX-ZYM. In conclusion, beta-glucan derived from ZYM by brief oxidation with NaClO is a suitable adjuvant for a CIA model with anti-CII antibody production.     

Humoral and peritoneal cell responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin, Vibrio anguillarum and Freund's complete adjuvant following intraperitoneal and bath immunisation

ELISA methods were used to evaluate the humoral immune responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin and Vibrio anguillarum. Antibody responses to ovalbumin administered intraperitoneally (i.p.) were inconsistent even when Freund's complete adjuvant (FCA) was used and the induction phase (4-6 weeks) of the response was longer compared with the response to V. anguillarum (<4 weeks). Significant elevation in antibody level was noted 3 weeks after bath vaccination with V. anguillarum but levels decreased thereafter. Humoral responses of greatest magnitude occurred where V. anguillarum was given in an emulsion with Freund's complete adjuvant (FCA). However, i.p. administration of FCA alone 3 weeks prior to i.p. immunisation with non-adjuvanted V. anguillarum resulted at 5 weeks in similar elevations in antibody to those in fish given V. anguillarum and FCA concurrently, suggesting that the effects of FCA were not limited to creation of an antigen depot. Proliferation of peritoneal inflammatory cell populations which included macrophages and plasma cells was detected histologically within 3 weeks of administration of FCA, but changes were not detected in the spleen or haematopoietic kidney.     

Use of adjuvants in modulating the behaviour of Plasmodium berghei

Different adjuvants were assessed for their role in conferring protection against the rodent malarial parasite P. berghei and compared with the classical Freund's complete adjuvant (FCA). Pretreatment of mice with trehalose dimycolate (TDM) mixed with antigen (Ag), sulpholipids (SL) mixed with Ag, muramyl dipeptide (MDP) alone, liposomes containing Ag and phosphomannoinositides (PIM) mixed with Ag were ineffective in conferring protection. However, MDP given with squalane (Sq) and Ag, MDP with incomplete Freund's adjuvant (IFA) and Ag, palmitoyl-MDP with Sq and Ag, aluminium hydroxide adsorbed Ag, and FCA with Ag were effective in conferring varying degrees of protection to mice. Complete protection in rats was obtained with MDP mixed with Sq and Ag, and FCA mixed with Ag, and a partial protection with liposomes containing Ag.     

Immunization of cattle against Hyalomma anatolicum anatolicum using larval antigens

Development of immunity in cross-bred (Bos taurus x Bos indicus) calves against Hyalomma anatolicum anatolicum, vector of bovine tropical theileriosis, was studied using larval antigen (LS) in Freund's complete adjuvant (FCA). Calves immunized with LS + FCA showed significant rejection of larvae (57.25 +/- 6.8) and nymphs (45.75 +/- 5.16). Abnormally fed larvae (11.4 +/- 0.8) and nymphs (8.25 +/- 1.2) were also recovered from immunized calves. This abnormal feeding may possibly be attributed to their inability to gain access to the blood vessels owing to the host immunological reactions. Consequently, feeding of extravascular fluid leads to white colour of fed ticks. Sera from all immunized calves after a week of immunization were positive for anti-LS antibodies in ELISA. The investigation indicates that LS in FCA enhanced anti-tick immunity.     

Circadian disorganization in experimental arthritis

This review discusses the experimental evidence indicating that arthritis disrupts circadian organization, which was mainly derived from animal studies employing Freund's complete mycobacterial adjuvant (FCA). The defense response to antigenic challenge, mediated in part by cytokines, includes changes in chronobiological central nervous system function, like depressed daily activity, superficial sleep or anorexia. Interferon (IFN)-gamma receptors are detectable in the central circadian pacemaker, the hypothalamic suprachiasmatic nuclei, at a time when the capacity for photic entrainment of the pacemaker became established. The disruptive effects of the systemic injection of IFN on the circadian rhythms of locomotor activity, body temperature and clock-gene mRNA expression have been documented. In the last few years we have examined a number of immune and neuroendocrine circadian rhythms in FCA-injected rats, both in the preclinical phase of arthritis (2-3 days after FCA injection) as well as in the acute phase of the disease (18 days after FCA injection). In arthritic rats, the 24-hour organization of immune and neuroendocrine responses becomes altered. A hormonal pathway involving the circadian secretion of melatonin and a purely neural pathway including, as a motor leg, the autonomic nervous system innervating the lymph nodes were identified. The significant effects of the immune-mediated inflammatory response on the diurnal rhythmicity of adenohypophysial and hypophysiotropic hormones occurred in arthritic rats. Melatonin treatment prevented the alteration in 24-hour rhythms of serum ACTH, prolactin and luteinizing hormone in rats injected with FCA. In addition, melatonin pretreatment prevented the alteration in the 24-hour variation in hypothalamic serotonin and dopamine turnover during the preclinical phase of Freund's adjuvant arthritis in rats. Some pinealectomy-induced immune changes in arthritic rats were also prevented by physiological concentrations of melatonin. Melatonin may play the role of an 'internal synchronizer' for the immune system.     

The effect of adjuvant and microbial challenge on the expression of antimicrobial polypeptides in channel catfish (Ictalurus punctatus)

While antimicrobial polypeptides (AMPPs) are increasingly recognized as one of the most important components of innate immunity, there is very little information in vertebrates that documents their upregulation to levels that are microbicidal in vivo. Here we demonstrate that intraperitoneal injection of either Freund's complete adjuvant (FCA) or live Tetrahymena pyriformis (a parasitic ciliate) upregulated AMPP expression in channel catfish skin. FCA induced significant upregulation of total antibacterial activity, anti-Edwardsiella ictaluri activity (the fraction of antibacterial activity active against E. ictaluri), and HLP-1 (the major AMPP in channel catfish skin). Tetrahymena induced a similar upregulation, except that HLP-1 was not significantly induced and the response appeared to be more transient than FCA immunostimulation. AMPP levels were increased up to five-fold higher than resting levels and levels expressed were well within concentrations known to be inhibitory to many important pathogens in vitro. These results provide encouragement that AMPP upregulation may be a promising tool in aquaculture for enhancing the resistance of fish to disease.     

Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin

Delayed-type hypersensitivity (DTH) and cell migration inhibition (MI) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemagglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund's complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI of their induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 micrograms) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of 10(-3) or 10(-2) micrograms/ml OVA and another peak at 1 or 10 micrograms/ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.     

The influence of various adjuvants on antibody synthesis following immunization with an hapten.

For the production of specific antibodies to the hapten MATP (4-Amino-1,2,2-trimethyl-phenylphosphonate) in Balb/c mice various non-toxic adjuvants were compared to Freund's complete adjuvant (FCA). For immunization the hapten MATP was coupled to the carrier human serum albumin (HSA). The immunostimulating effect of the synthetic lipopeptides Pam3Cys-OH, Pam3Cys-Ser-Ser-Asn-Ala and different concentrations of the lipohexapeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys = S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine as well as of aluminium hydroxide were tested. IgG antibody titers in serum were determined in ELISA. In dose-response studies 50 micrograms Pam3Cys-Ser-(Lys)4 per mouse was the most effective dose with a long period of high antibody levels after the second booster. Pam3Cys-Ser-Ser-Asn-Ala provoked only low antibody titers. Immunostimulation with Pam3Cys--OH did not result in an increased production of specific antibodies. Compared to the control group an enhanced antibody synthesis could be provoked with aluminium hydroxide. However, the increase was much smaller than by using FCA. The lipopeptide Pam3Cys-Ser-(Lys)4 turned out to be a very potent adjuvant. One week after booster injection into mice 50 micrograms of this substance helped to elicit a higher antibody titer than FCA. Hence, as far as the degree of antibody production is concerned, Pam3Cys-Ser--(Lys)4 represents an alternative adjuvant to FCA.     

Sequence analysis of the Bs-Ag1 gene of Baylisascaris schroederi from the giant panda and an evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag1 antigen in mice

The Baylisascaris schroederi infection rate among wild giant pandas may reach over 50% or even 100%, making it one of the leading causes of death from primary or secondary infection in wild populations. Until now, little was known about how protective immunity to B. schroederi infection could be achieved. The present study was conducted to evaluate the immunogenicity and protective efficacy of recombinant Bs-Ag1 from B. schroederi, by cloning the full-length Bs-Ag1 gene of B. schroederi and expressing it in a heterologous host, Escherichia coli BL21. In mice vaccinated with rBs-Ag1 coupled with Freund's complete adjuvant (FCA), there was a significant reduction (69.2%) in the recovery of challenged B. schroederi L3 compared with either nonvaccinated controls or mice vaccinated with FCA alone. Our study supports the use of Bs-Ag1 as a potential candidate for vaccination against B. schroederi infection and provides basic data for further vaccination trials with mixtures of antigens (with Bs-Ag2 and Bs-Ag3) to B. schroederi.     

Mixed adjuvant formulations reveal a new combination that elicit antibody response comparable to Freund's adjuvants

Adjuvant formulations capable of inducing high titer and high affinity antibody responses would provide a major advance in the development of vaccines to viral infections such as HIV-1. Although oil-in-water emulsions, such as Freund's adjuvant (FCA/FIA), are known to be potent, their toxicity and reactogenicity make them unacceptable for human use. Here, we explored different adjuvants and compared their ability to elicit antibody responses to FCA/FIA. Recombinant soluble trimeric HIV-1 gp140 antigen was formulated in different adjuvants, including FCA/FIA, Carbopol-971P, Carbopol-974P and the licensed adjuvant MF59, or combinations of MF59 and Carbopol. The antigen-adjuvant formulation was administered in a prime-boost regimen into rabbits, and elicitation of antigen binding and neutralizing antibodies (nAbs) was evaluated. When used individually, only FCA/FIA elicited significantly higher titer of nAbs than the control group (gp140 in PBS (p<0.05)). Sequential prime-boost immunizations with different adjuvants did not offer improvements over the use of FCA/FIA or MF59. Remarkably however, the concurrent use of the combination of Carbopol-971P and MF59 induced potent adjuvant activity with significantly higher titer nAbs than FCA/FIA (p<0.05). This combination was not associated with any obvious local or systemic adverse effects. Antibody competition indicated that the majority of the neutralizing activities were directed to the CD4 binding site (CD4bs). Increased antibody titers to the gp41 membrane proximal external region (MPER) and gp120 V3 were detected when the more potent adjuvants were used. These data reveal that the combination of Carbopol-971P and MF59 is unusually potent for eliciting nAbs to a variety of HIV-1 nAb epitopes.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Delayed hypersensitivity against hamster erythrocyte antigen was examined after sensitization with hamster erythrocytes (HRBC) in Freund's complete adjuvant (FCA). Extent of the delayed hypersensitivity was determined by the migration inhibition test, the peritoneal macrophage disappearance test and the skin test in the ear using solubilized HRBC as the test antigen. 1) Delayed hypersensitivity against HRBC developed earlier in high-responder SL mice than in low-responder C57BL/6 mice after sensitization. The period required for development of the delayed hypersensitivity in AKR mice was intermediate between periods in high-responder SL mice and low-responder C57BL/6 mice. 2) After sensitization with HRBC in FCA, a delayed hypersensitive state without detectable antibody production persisted until day 12 in high-responder SL mice and until day 16 or later in low-responder C57BL/6 mice. 3) Delayed hypersensitivity against HRBC antigen persisted even after the appearance of circulating antibody which occurred late after sensitization with HRBC in FCA or after intravenous injection of HRBC into sensitized mice.     

Use of a styrene-maleic anhydride copolymer as an adjuvant with testosterone-serum albumin immunogens and its application to increasing ovulation rate in the ewe

A synthetic poly(styrene-maleic anhydride) copolymer of average molecular weight 470,000 potentiated a testosterone-binding antibody response during immunization of sheep with immunogenic testosterone-3-carboxymethyloxime-serum albumin conjugates. The copolymer had weaker immunostimulatory activity than Freund's complete adjuvant (FCA). The minimum effective dose of the copolymer was about 30 mg at which secondary but not primary immune responses could be detected by radioimmunoassay. There was a generally weak anamnestic response in immune sheep as secondary and teritary responses tended to decline promptly from peak values; tertiary antibody titres usually did not exceed secondaries. The use of the copolymer as a solution or emulsion had no apparent effect on its immunoadjuvant activity when administered intramuscularly but the soluble form was inactive when given intraperitoneally. The testosterone-binding antibody that was produced using either the copolymer or FCA had considerable sensitivity to deactivation by mercaptoethanol. The ovulation rate of a group of 20 Merino ewes following immunization with testosterone-serum albumin using the copolymer adjuvant was significantly higher than an equal group of untreated control ewes.     

Induction of latent immunological memory in genetically nonresponsive mice

C57BL/10 mice exhibit major histocompatibility complex linked nonresponsiveness to hen egg white lysozyme (HEL). When these animals are primed with HEL in Freund's complete adjuvant (FCA), their secondary splenic plaque forming cell responses to aqueous HEL challenge are minimal to nonexistent. This notwithstanding, we show here that concomitant priming with both HEL and keyhole limpet hemocyanin (KLH) leads to an enhanced response to the HEL component following secondary challenge with an HEL-KLH conjugate. This enhancing effect can be transferred by nylon wool nonadherent spleen cells from HEL/FCA primed animals. Adoptive transfer studies with fractionated spleen cell populations suggest also that B cells are primed in these animals. Thus, animals which are incapable of mounting a secondary response to this antigen nevertheless appear to be primed at both the T-cell and B-cell levels following exposure to the antigen in FCA. The implications of this finding are discussed.     

Comparison of immunospecific antibody response in young and old chickens immunized with human IgG

Three groups of 18-month-old chickens and three groups of 5-month-old chickens were immunized with human immunoglobulin G (IgG) using one of three adjuvants in the first injection (Freund's Complete Adjuvant (FCA), Freunds Incomplete Adjuvant (FIA) and Hunter's TiterMax (HTM)) following the same immunization scheme. The specific antibody response in serum was measured by ELISA. In both older and younger chickens the serum antibody response in the FCA group reached a significantly higher level (P < 0.01) than in the FIA group and in the HTM group on week 5. The FCA group also had a significantly higher (P < 0.01) response on week 10 compared to the HTM group. Other than that, there was no significant difference between the three adjuvant groups in specific serum antibody response in older chickens. In the younger chickens the specific serum antibody response in the FCA group was significantly higher (P < 0.05) than the response in the HTM group. There was no significant difference in the chicken serum antibody response between the FCA and the FIA groups, nor was there a significant difference between the FIA and the HTM groups. Comparing the younger chickens and the older chickens immunized using the same adjuvant, the older chickens had consistently higher titres than the younger chickens, although the difference was not always significant.     

Prevention of experimental allergic encephalomyelitis by bacterial lipopolysaccharides: inhibition of cell-mediated immunity

The immunization of Lewis rats with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant inhibits the development of experimental allergic encephalomyelitis (EAE) in these animals. These protected animals fail to manifest significant in vivo delayed-type hypersensitivity skin tests and in vitro lymphocyte proliferative responses to BP. Our results indicated that LPS induces a nonspecific reduction in immune reactivity of BP in Lewis rats.     

Assessment of the lymphocyte response to silicone

The biocompatibility of silicone is once again the focus of increased interest. Long considered inert, silicone has now been reported to be responsible for macrophage inhibition in rats and to possibly cause adjuvant disease in humans, and the related compound silica has elicited an antibody response in mice. The present study evaluates lymphocytic response to silicone as expressed by the demonstration of immunologic memory, or changes in specific lymphocyte subpopulations. Thirty-six female Lewis rats (250 gm body weight) were used as test animals. Group 1 (n = 12) was injected subcutaneously with 2.5 ml Freund's Complete Adjuvant (FCA) alone. Group 2 (n = 12) was injected with 2.5 ml FCA sonicated with silicone gel. Group 3 (n = 6) was injected with 2.5 ml FCA, and at 4 weeks, gel-filled silicone implants were placed subcutaneously. Group 4 (n = 6) was injected with 2.5 ml FCA sonicated with silicone gel, and gel-filled silicone implants were placed at 4 weeks. An additional group of six rats (group 5) served as control for the experimental animals, and a group of four rats (group 6) served as naive control. Groups 1 and 2 were sacrificed at 4 weeks, and splenic lymphocytes were obtained for lymphocyte transformation assays performed against silicone. Assays also were run with the addition of the known mitogens Con A, PHA, LPS, and pokeweed. Cytofluorographic analysis of pan-T, T-helper, T-suppressor, and B-cell populations was performed. Groups 3, 4, 5, and 6 were harvested at 8 months, and splenic lymphocytes were subjected to lymphocyte transformation assay.(ABSTRACT TRUNCATED AT 250 WORDS)