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Our earlier observations indicate that adoptive transfer of spleen cells obtained from dengue type 2 virus (DV)-primed mice suppressed DV antigen-specific antibody secretion as detected by Jerne PFC technique. Findings of this paper indicate that the suppression was produced by non-glass-adherent cells, macrophage-depleted (by carbonyl iron) cells and by T lymphocytes of the spleen but not by the glass-adherent cells and B lymphocytes. The activity of these cells is dependent upon production of prostaglandin as shown by abrogation of their suppressor activity by pre-treatment of cells by indomethacin or aspirin which are known to block synthesis of prostaglandins. [ABSTRACT FROM AUTHOR]

There is no doubt that HLA-DR molecules are acting as the products of HLA-linked immune response genes (Ir-genes), because (i) HLA-DR molecules are the restriction elements in the interaction between CD4+ helper T cells and antigen-presenting cells (APC) to respond to many antigens such as streptococcal cell wall antigen (SCW) (Nishimura & Sasazuki, 1983; Sone et al., 1985; Hizayama et al., 1986), schistosomal antigen (Sj) (Hirayama et al., 1987), Mycobacterium leprae antigen (ML) (Kikuchi et al., 1986) and so on; and (ii) anti-HLA-DR monoclonal antibodies completely abolish the immune response to those antigens (Nishimura & Sasazuki, 1983; Sone et al., 1985). However, genetic analysis of the immune response to those antigens in families or populations revealed that responsiveness is recessive and non-responsiveness to those antigens is a dominant genetic trait that is tightly linked to HLA (Sasazuki et al., 1980a, 1983; Watanabe et al., 1988). This is completely opposite to the situation under the Ir-gene control where responsiveness is dominant and non-responsiveness is recessive. In this paper, we report evidence of how we came across the concept of HLA-linked immune suppression genes (Is-genes) besides Ir-genes, and show evidence for the epistatic interaction between HLA-DR and DQ to determine the immune response to several antigens in humans. [ABSTRACT FROM AUTHOR]

As perhaps the staunchest advocates of repertoire purging as the central mechanism of immunological tolerance, we note with satisfaction a spate of recent, elegant papers which suggest an intrathymic clonal abortion model as the explanation for at least some examples of T-cell tolerance. This view agrees with the classical formulation of the Billingham-Brent-Medawar school of tolerance as a specific, central failure of immune responsiveness. Repertoire purging within the B-lymphocyte compartment remains much more controversial. There is no doubt that experimental models exist where the B cell is the reversible target of tolerance induction. The question is, in view of the ease of inducing autoantibody formation both in vivo and in vitro, just how relevant are such clonal anergy mechanisms to authentic self-tolerance? Arguments are presented that there must be two windows of tolerance susceptibility in the ontogeny of the B cell; one while it is maturing in the bone marrow, to prevent autoreactivity of high affinity to important accessible self-antigens; and a second soon after activation of pre-memory cells by exogenous antigen, to prevent fortuitous mutations towards high-affinity anti-self-reactivity establishing a forbidden clone. [ABSTRACT FROM AUTHOR]

CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N × C3H/HeN F1 male > CBA/N female > CBA/N · C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate that T cell participation is not essential for the blockade of CBA/N PFC responses and they suggest that direct blockade of TI- and TD-responsive B cell populations is likely to occur. [ABSTRACT FROM AUTHOR]

Evidence from in vitro studies indicates that immunoglobulin A (IgA) responses are highly T dependent, yet investigations of the requirement for cognate help For IgA responses in tiro have not previously been undertaken. Experiments reported in this paper employ hapten-carrier immunization of individual Peyer's patches (PP), the site of generation of IgA antibody-containing cells (ACC) responding to lumenal antigenic challenge in the intestine, to determine the requirements for T-cell and B-cell priming under normal physiological conditions in vivo. These experiments demonstrate that both hapten-specific B-cell priming and carrier-specific T-cell priming in PP are required for an IgA-specific anti-hapten ACC response in the intestinal lamina propria to subsequent lumenal challenge with hapten-carrier conjugate. These results confirm that IgA B-cell induction in PP requires cognate T-cell help. An IgA ACC response can also be obtained when hapten and carrier priming occur in different PP, providing functional significance for our previous observations that PP-derived T-helper cells are able to migrate between PP after priming. [ABSTRACT FROM AUTHOR]

Supernatants were collected from keyhole limpet haemocyanin (KLH) primed spleen cells which had been incubated in tissue culture medium with their priming antigen KLH. These non-specific factor (NSF) containing supernatants were then tested in a microculture system for their ability to facilitate an anti-SRBC response of nu/nu or AT x BM spleen cells. It was concluded that: (a) NSF was able to engage a large number of the available pool of sheep erythrocyte (SRBC) specific B cells into an antibody response; (b) this response was characterized by the development of clones expressing plaque-forming cells (PFC), the number of PFC produced within a clone being dependent upon the amount of NSF available in that culture; (c) NSF probably acted directly on B cells, and not via other accessory cell types. [ABSTRACT FROM AUTHOR]

Regulatory T cells are induced by oral administration of an antigen, but the physiological requirements and localization of the inductive sites are largely unknown. Using an adoptive transfer system of cells transgenic for ovalbumin T-cell receptor (OVA TCR tg), we found that antigen-specific CD4+ T cells were activated in the liver-draining celiac lymph node (CLN) shortly after ovalbumin feeding, and that a significantly higher proportion of the T cells in the CLN developed into the putative regulatory phenotype [co-expressing CD25 with the glucocortico-induced tumour necrosis factor (TNF) receptor family related gene (GITR), cytotoxic T-lymphocyte antigen (CTLA)-4 and CD103] than in Peyer's patches, the mesenteric and peripheral lymph nodes and the spleen. In addition, a particularly high level of expression of CD103 on the OVA-specific T cells in the CLN may favour homing to the epithelium of the intestine. While equally suppressive, OVA tg T cells isolated from the CLN of OVA-fed DO11·10 mice were less dependent on transforming growth factor (TGF)-β for suppression than cells isolated from the peripheral and mesenteric lymph nodes, which indicates the involvement of an additional suppressive mechanism. The expression of FoxP3 was not up-regulated in any of the lymph node compartments studied. Our phenotypic and functional findings suggest that the induction of regulatory T cells in the CLN may be relevant in the control of the immune response to dietary antigens. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) play critical roles in generating an immune response and in inducing tolerance. Diverse microenvironmental factors can‘polarize’ DC toward an immunogenic or non-immunogenic phenotype. Among the various microenvironmental factors, interleukin-10 (IL-10) exhibits a potent immunosuppressive effect on antigen-presenting cells (APC). Here, we show that monocyte-derived DC generated in the presence of IL-10 exhibit a profound down-regulation of many genes that are associated with immune activation and show that the IL-10-grown DC are poor stimulators of CD8+ T cells in a strictly autologous and major histocompatibility complex (MHC) class I-restricted melanoma antigen recognized by T cells (MART-1)epitope presentation system. However, these IL-10-grown DC can efficiently activate the epitope-specific CD8+ T cells when they are made to present the epitope following transduction with an adenoviral vector expressing the MART-1 antigen. In addition, we show that the MART-1 protein colocalizes with the MHC class I protein, equally well, in the iDC and in the DC cultured in presence of IL-10 when both DC types are infected with the viral vector. We also show that the vector transduced DC present the MART-127−35 epitope for a sustained period compared to the peptide pulsed DC. These data suggest that although DCs generated in the presence of IL-10 tend to be non-immunogenic, they are capable of processing and presenting an antigen when the antigen is synthesized within the DC. [ABSTRACT FROM AUTHOR]

Deficiencies in immune responses against polysaccharides can have direct consequences for patients, and therefore, a better undestanding of these immune reactions is crucial We have studied the immune response against the polysaccharide dextran B512 (Dx). Administration of immunogenic doses of thymus-independent (TI) Dx reduces the immunoglobulin G1 response to later challenges with a thymus-dependent (TD) form of Dx. We investigated if this suppression is a general phenomenon caused not only by Dx but also by other TI antigens, and examined possible mechanisms contributing to this unresponsiveness. We show that clonal exhaustion is not involved in modulating subsequent responses, nor is signalling via FcγRIIB or other antibody mediated pathways. The reduced TD response is not an exclusive Dx phenomenon; it is also induced by TI antigen oxazolone (Ox). However, responses against the hapten dinitrophenyl (DNP) are not affected, indicating that the TI priming negative effect is not a general process. This may be explained by the restricted immune response to both Dx and Ox, in contrast to the unrestricted DNP response. Our conclusion from these experiments is that the underlying mechanism for the TI-induced reduction of latter TD responses is a property of the TI activation itself. [ABSTRACT FROM AUTHOR]

CD4+ CD25+ T regulatory cells (TReg), suppress antigen-specific immune responses and are important for allograft tolerance. During pregnancy the mother tolerates an allograft expres- sing paternal antigens (the fetus) requiring substantial changes in immune regulation over a programmed period of time. We analysed whether immune-suppressive TReg cells were altered during pregnancy and therefore might play a part in this tolerant state. The presence of TReg cells was assessed in the blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. We observed an increase in circulating TReg cells during early pregnancy, peaking during the second trimester and then a decline postpartum. Isolated CD25+ CD4+ cells expressed FoxP3 messenger RNA, a marker of TReg cells, and suppressed proliferative responses of autologous CD4+ CD25­T cells to allogeneic dendritic cells. These data support the concept that normal pregnancy is associated with an elevation in the number of TReg cells which may be important in maintaining materno-fetal tolerance. [ABSTRACT FROM AUTHOR]

The idiotypic (Id) diversity of the immune response to phosphorylcholine (PC) was studied by immunization of mice with thymus-dependent (PC-keyhole limpet haemocyanin; PC-KLH) and thymus-independent (S. pneumoniae R36a; Pn) forms of the antigen. Mice with the BALB/c genetic background (BALB/c, C.B20, and BALB.B) were used because their response to PC is dominated by immunoglobulins encoded in VH-1 and Vk 22 genes, which uniformly express the T15 idiotype. The actual repertoire of the antibody was determined by idiotypic markers (Id) defined with monoclonal antibodies designated AB1–2, B36–82, MaId5–4, and B24–44. Previous studies from our laboratory have shown that these Id are present on TI5 (VS107-1/Vk22) immunoglobulins only, but that they differentiate between somatic variants of the antibody molecules. We have measured the serum concentrations of these four Id after primary (1°), secondary (2°), and tertiary (3°) immunization; all of the Id activity was associated with the PC-binding antibody, as shown by specific immunoadsorbents. However, the levels of the Id-bearing (Id+) antibody did not correlate with each other. After immunization with PC-KLH, the AB1–2+ antibody declined precipitously, whereas the levels of B24–44 and B36–82 remained steady. A similar pattern of Id heterogeneity was seen at the level of direct antibody-plaque-forming cells from the spleen, suggesting that the idiotopic (clonal) diversification occurred already during the early IgM response. A significant portion of anti-PC antibody after the 3° PC-KLH immunization was negative for all four Id, implying that the late response to the antigen involved distinct, T15-negative clones. The same idiotope repertoire changes were observed in BALB/c and in congenic strains C.B20 (Ishb) and BALB.B (H-2b). A diversification of T15+ antibody was also detectable upon immunization with a T-independent antigen, Pn, but to a much lesser extent. These observations indicate that the T15+ antibody response in BALB/c mice is heterogeneous, and that there appears to be a selection of specific clonal variants in which the T cells play a role. [ABSTRACT FROM AUTHOR]

Spleen cells, obtained 2-5 days after in vivo priming with sheep erythrocytes (SRBC), were cultured to determine the presence of plaque-forming cell (PFC) precursors capable of developing into mature PFC under the influence of various stimulants. Lipopolysaccharide (LPS), added together with SRBC at the initiation of a 48-hr in vitro culture, enhanced the PFC response of primed spleen cells. In vivo priming for a minimum of 3 days was required, and maximal numbers of PFC were obtained from spleen cells primed for 4 days. Depletion of T lymphocytes from Day 3-primed spleen cells abrogated LI'S-mediated enhancement, and addition of concanavalin A supernatants to the T-cell depleted system restored the enhancement, suggesting that LPS action required co-operation with a product(s) of activated T cells. Addition of various interleukin-2 preparations including recombinant human IL-2 to the system restored the LPS-mediated enhancement. The response of Day 3 cells from which T cells were eliminated as vigorously as possible was similarly restored by the addition of IL-2, LPS and antigen, suggesting that IL-2 reacts directly with PFC precursors that have developed IL-2 receptors. LPS-mediated enhancement, in the presence or absence of T cells, was also markedly dependent on the presence of SRBC during in vitro culture. These data suggest that, in co-operation with IL-2 and other co-factors, antigen plays a significant role in driving the later stages of differentiation and/or division of PFC precursors to mature PFC. [ABSTRACT FROM AUTHOR]

Klebsiella 03 lipopolysaccharide (LPS) and Escherichia coli 09 LPS were previously shown to have potent adjuvant activities in augmenting antibody response and delayed-type hypersensitivity to protein antigens. The R form LPS extracted from the O-specific polysaccharide-less mutants derived from Klebsiella 03 and E. coli 09 strains were characterized by chemical and electrophoretic analyses. The adjuvant activities of the R-LPS in augmenting antibody response and DTH to protein antigens were much weaker than those of the parental LPS. The strength of the adjuvant activities of the R-LPS was similar to that of the activities of Salmonella minnesota LPS and Ra-LPS. By contrast, polyclonal B-cell activation effects of the R-LPS were stronger than those of the parental LPS. The common feature of the parental LPS is that their O-specific polysaccharides are mannose homopolymers (mannans). From these results, it is suggested that mannose homopolymers as the O side-chains of LPS contribute to the action of LPS in enhancing strongly the T-cell dependent immune responses to protein antigens. [ABSTRACT FROM AUTHOR]

The role of suppressor cells in control of persistent infections may be of profound importance. Whether a positive immune response or suppression of immunity is generated at the time of initial exposure to pathogens causing such infections may in part be due to the nature of the initial antigen presentation to the specific cells of the immune system. We have shown that in vivo modulation of epidermal APCs by an environmentally encountered stimulus (UV-B light exposure) and subsequent transfer of these APCs together with live HSV-1 to naive syngeneic recipients is sufficient to generate suppression of DH rather than DH to HSV-1. This suppression is T-cell mediated and specific for HSV-l. The phenotype of the Ts cell induced by epidermal cell transfer is Thyl+ L3T4+ Ly2-. [ABSTRACT FROM AUTHOR]

Current approaches to the analysis of antigens involved in the cellular immune response to mycobacterial infection rely on the initial identification and isolation of molecular components using monoclonal antibodies. In order to overcome the constraints of this approach, we have utilized a procedure involving T-cell recognition of antigens fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) and added to proliferation assays after blotting onto nitrocellulose membranes. Analysis of human T-cell responses to Mycobacterium tuberculosis and Mycobacterium bovis BCG by this procedure revealed distinctive patterns of reactivity to different molecular weight components indicative of the selective recognition of immunodomin ant and species-specific determinants. Human T-cell clones were subsequently derived, and SDS-PAGE immunoblotting was used to identify the antigen recognized by each clone. Three epitopes defined by individual T-cell clones were identified on separate polypeptides with molecular weights 16,000-18,000 (clone P5 3), 18,000-20000 (clone P57) and 52,000-55,000 (clone P35). This study demonstrates the potential application of T-cell cloning in conjunction with SDS-PAGE immunoblotting for the dissection and analysis of the cellular immune response to pathogenic agents during human infection. [ABSTRACT FROM AUTHOR]

Using cloned human T lymphocytes reactive with a 24 amino acid peptide (p20) of the carboxyl terminus of the HA-1 molecule of influenza haemagglutinin (HA), we have investigated the ability of solid-phase antigen to induce antigen-specific T-cell proliferation. The activation by nitrocellulose- bound virus and p20 was accessory-cell dependent and was not caused by immobilized antigen directly cross-linking the specific receptors. Furthermore, we report that separation of complex antigen mixtures such as influenza virus and HA by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) followed by transfer to a nitrocellulose membrane can be used to allow direct screening of individual polypeptides in T-cell proliferation assays. With this immunoblotting procedure the antigenic site recognized by HA-reactive T cells was confirmed to reside in the HA-i molecule of influenza virus of only the appropriate subtype. The general application of this approach is discussed in the case of infections and autoimmune diseases in which the immune response is predominantly T-cell mediated and where antibody studies may fail to identify key antigenic determinants involved in the activation of T cells. [ABSTRACT FROM AUTHOR]

Multiple minor antigen incompatibilities produce unexpectedly strong DTH reactions in mice. These experiments showed that this is probably not due to the effect of a single minor incompatibility, and that the response to the MUC is relatively weak compared with that against multiple minor antigens. Assessment of the in vivo response to multiple minor antigens in delayed-type hypersensitivity responses and in priming for subsequent mixed lymphocyte culture generation of lymphocyte- mediated cytotoxicity revealed a major degree of cross-reactivity for antigens not present on the initial in vivo stimulus. Recruitment of cross-reactive cells in vivo might play a role in these strong responses to multiple minor antigens. [ABSTRACT FROM AUTHOR]

Affinity maturation was studied by the analysis of the kinetics of the appearance of antibody subpopulations with different affinities during the immune response, using an hapten-inhibition ELISA. The immune response in KLH-Ar-immunized A/J mice was used as a model system. Five antibody subpopulations of different affinity (103-107 M-1) could be detected, the relative concentrations of which changed during affinity maturation. The high-affinity antibody subpopulations did not represent the major fraction at any stage during affinity maturation. The appearance of the highest affinity subpopulation (107 M-1), despite exhibiting relative concentrations no higher than 12%, produced an important increase in average affinity. On the other hand, its disappearance at the end of the maturation process could explain the average affinity decrease observed at this stage. Our results indicate that affinity maturation cannot be explained by the dominance of high-affinity clones, as proposed by Siskind & Benacerraf (1969). The increase in affinity could rather be due to the progressive appearance of low percentages of high-affinity clones, which are not present in the primary response and never become dominant. [ABSTRACT FROM AUTHOR]

The idiotypic profile of anti-phosphorylcholine plaque-forming cell responses and their evolution with ageing were studied in (NZB × NZW) F1 mice. Our results showed that the anti-phosphorylcholine plaque-forming cell response induced by phosphorylcholine coupled to keyhole limpet haemocyanin and, paralleling, the T15 idiotype clonal dominance declined with ageing. This loss of immune competence was also observed with another thymus-dependant (phosphorylcholine coupled to egg globulin) as well as thymus-independent (capsular polysaccharide of Streptococcus pneumoniae strain R36a) antigens. In contrast, old mice challenged with an antigenic preparation of Neisseria meningitidis showed an immune response not significantly different from that elicited by the same antigen in young mice. The hapten-augmentable plaque-forming cells were assayed to determine whether a putative auto-antiidiotypic regulation underlies this loss of immune competence. Only minimal numbers and non-significant differences between young and old mice immunized with any antigen could be detected. Further studies using an adoptive transfer system demonstrated that cells from aged mice were able to support a normal anti-phosphorylcholine response when transferred into lethally irradiated young recipients. Our results suggest that no permanent cellular defects, but rather internal environment or/and radioresistant suppressor cells, are involved in this loss of immune competence. The role played by these factors and their effect on distinct subpopulations of B cells are discussed. [ABSTRACT FROM AUTHOR]

The contribution of gut-associated lymphoid tissue (GALT) to the local immune response in the respiratory mucosa of sheep has been investigated. Sheep were primed intraperitoneally (i.p.) with antigen in Freund's complete adjuvant, a procedure known to produce a large IgA-specific antibody-containing cell (ACC) response in intestinal lymph. ACC and their class specificity were then enumerated by double fluorochrome immunofluorescence in respiratory tissues after intratracheal (i.t.) antigen administration. This immunization procedure produced an enhanced IgA-specific ACC response in the upper respiratory tract mucosa compared with either i.t. or i.p. immunization alone and this was not reflected in the regional lymph nodes. Furthermore, chronic drainage of the intestinal efferent lymphatic duct for the duration of the immunization period abrogated the enhanced response in the respiratory mucosa. These data are consistent with the concept of an intermucosal cell circuit with respect to IgA cell precursors, and provide indirect evidence that IgA responses in the respiratory tract can be enhanced by harnessing the immune potential of GALT as a source of IgA precursors by appropriate immunization strategies. [ABSTRACT FROM AUTHOR]

Examines the formation of antibodies to the respective idiotypes of myeloma proteins produced by MOPC plasmocytoma tumours of BALB/c origin. Description of the biological characteristics and morphology of the IR202 transplantable immunocytoma tumour in rats; Presentation of the 202M protein molecules on the macrophage surface; Development of antibodies specific to individual antigenic determinant of the monoclonal IgM.

Passive administration of monoclonal IgM anti-sheep red blood cell antibody 2 hr prior to a low dose of sheep red blood cells (SR BC) markedly enhances the specific antibody response. This IgM enhancement of the response to SRBC is highly reproducible and can furthermore be demonstrated in vitro. The time course and dose dependency of the response indicate that a critical antibody: antigen ratio is necessary for enhancement to occur. The IgM enhancement phenomenon has been demonstrated in several strains of mice, providing strong evidence against an idiotype-related mechanism. [ABSTRACT FROM AUTHOR]

Inducibility of immunological tolerance to a T-dependent antigen, human serum albumin, was studied in autoimmune mice (NZB, MRL/Mp-1pr/1pr and male BXSB mice), comparing with non-autoimmune mice (BALB/c, DBA/2, MRL/Mp- +/+ and female BXSB mice). Weekly injections of increasing doses of the tolerogen into DBA/2 mice induced T-cell tolerance without the participation of suppressor cells and without affecting B cells. All of the autoimmune mice tested were refractory to the tolerogen; partial tolerance (40%-60% responsiveness) was attained in mice aged 1 month, but not in older mice. By comparison, tolerance was induced in non-autoimmune mice to a similar degree irrespective of the age of the mice. The tolerance inducibility was also examined in reciprocal bone marrow cell (BMC) transfer experiments between NZB and DBA/2 mice and also BMC transfer into B10.D2 mice. It was shown that recipients of NZB BMC were more resistant to tolerance induction than those of DBA/2 BMC, while irradiated NZB mice receiving DBA/2 BMC were susceptible. Hence, it is suggested that the tolerance-resistant nature of the NZB mice was inherent in T-cell precursors in the bone marrow and not in the host environment. Similar findings in support of this notion were also observed in BSXB mice. The significance of tolerance resistance in the development of autoimmunity is discussed. [ABSTRACT FROM AUTHOR]

Some suppressor cells are reported to lose their activity when precultured without stimulus in vitro. We have investigated the role of such suppressors in responsiveness to mycobacterial antigens of peripheral blood mononuclear cells (PBMNC) from patients with leprosy or tuberculosis, or from normal donors. Delayed addition of mycobacterial antigens (Mycobacterium leprae, Mycobacterium vaccae and Mycobacterium tuberculosis), but not of a fungal antigen (Candida albicans) caused enhanced responses using PBMNC from most normal donors, or tuberculoid leprosy (TT/BT) patients. However, the effect was less common using PBMNC from the lepromatous leprosy (BL/LL) group. (P < 0·01, using M. leprae, relative to the TT/BT group), suggesting that this type of suppression reflects a normal mechanism, which is diminished rather than increased in anergic patients. Delayed addition of antigens to ‘E’-rosetting cells did not result in enhanced responses. However, the different effects of ‘E’-rosetting on the responses to the mycobacterial antigens of cells from normals, TT/BT and BL/LL patients, suggested that there may be two types of proliferative response to these antigens. [ABSTRACT FROM AUTHOR]

Anti-red blood cells (RBC) and anti-hapten antibody synthesis were studied in the goldfish, Carassius auratus. Spontaneous haemagglutination titres were found against all the antigens tested. A weak secondary response was observed in RBC-primed fish boosted during the end-phase of the primary antibody production. However, when the second antigenic challenge was performed during the early exponential phase of a primary stimulation, an important amplified response was obtained. The antibody production and immunological memory can be dissociated: no antibody synthesis occurred in glutaraldehyde-fixed RBC (F-RBC) primed animals but a characteristic amplified response was obtained when untreated or F-RBC were given to F-RBC primed animals. The amplified response to sheep red blood cells (SRBC) was significantly inhibited when fish were primed with a mixture of SRBC and Xenopus red blood cells (SRBC). demonstrating an antigenic competition phenomenon. Studies on anti-trinitrobenzene responses confirm the efficiency of E. coli lipopolysaccharide as a carrier for fish anti-hapten immunization. The kinetics and regulation of antibody synthesis in fish are discussed in relation to the described results. [ABSTRACT FROM AUTHOR]

The introduction of an antigenic determinant strongly recognized by T cells on to a cell enhances the immune response to weak cellular antigens. Tuberculin (PPD) is a particularly suitable antigenic determinant for this purpose since it behaves in many ways as a ‘T-cell hapten’. It has been found that direct chemical coupling of PPD to cell surfaces damages their antigenicity, but that this can be circumvented by coupling PPD to the lectin Concanavalin A and using this as the ligand for binding PPD to the cell. Techniques for preparing Con-A/PPD using little glutaraldehyde or SPDP as cross-linking agents are described. [ABSTRACT FROM AUTHOR]

T- and B-cell participation in the immune response induced by immune ribonucleic acid (iRNA) preparations against 1-dependent antigens was studied using athymic nude, neonatally thymectomized (NT) and cyclophosphamide-treated (CY) mice. The IRNA(T + B) preparations were made from the spleen of BALD/c mice immunized with these antigens. Injection of the IRNA into nude or NT mice caused an increase in the number of specific rosette-forming cells (RFC) and of memory cells capable of responding to secondary stimulus with a small dose of the corresponding antigen. Injection with T-dependent antigens or with iRNA(T + B) did not cause any immune response in CY mice, suggesting depletion of the if-cell function. The (RNA(T) and iRNA(B) were prepared, respectively, from the thymuses of BALD/c mice and from the spleens of nude mice which had been immunized with 1-dependent antigens. Injection of nude mice with both IRNA(T) and iRNA(S) caused an increase in the number of specific RFC and the secondary antibody formation response after boosting with a small dose of the corresponding antigen. Injection of IRNA(T) preparation into nude mice could induce the anamnestic response after boosting. However, neither of the iRNA(T) or iRNA(B) preparations could induce in nude mice the proliferation of the number of specific RFC. These results indicate the presence of at least two kinds of IRNA preparations against 1-dependent antigens and that the cooperation of IRNA(T) and IRNA(B) was required for the induction of immune response against T-dependent antigens. [ABSTRACT FROM AUTHOR]

The primary immune response to SRBC in BALB/c mice was depressed when they were injected with a fraction (FAd) obtained from Trypanosoma cruzi epimastigotes grown in LIT medium. Plaque- forming cell (PFC) number was 50% less than controls when FAd was injected i.v. 15 mm before antigen in doses ranging from 70 μg up to 400 μg of protein. Similar depression was observed when 100 μg FAd was injected up to 6 h before antigen. There was no shift in the peak response to SRBC, neither was depression detected, when a total of 100 μg FAd protein was given in 20 μg amounts twice a day before immunization. Mice injected with FAd fraction only showed no increase in background PFC. Both secondary IgM and secondary IgG PFC were depressed when FAd was given before the boosting injection. However, only IgG PFC were depressed when FAd was injected before the priming dose. The delayed- type hypersensitivity reaction to DNFB was depressed when animals were injected either during the 3 days after sensitization or with a single dose of 100 μg of protein of FAd on day of challenge. Bone marrow colony-forming units in spleens of mice injected with FAd were depressed and nodules in the treated animals were smaller than in controls. We conclude that FAd affects humoral and cell-mediated immune responses by interfering with cell division at some stage of the cell cycle. [ABSTRACT FROM AUTHOR]

Endotoxin (1–10 μg intravenously) increased the number of strongly peroxidase-positive cells in the white pulp of the mouse spleen, demonstrable by the Graham Karnovsky technique. The increase was greatest after 10 h, the cells being found most frequently around the central vessels of the white pulp. The appearance of such cells at this critical immunological site may relate to the known adjuvant activity of endotoxin in promoting immune responses against protein antigens. [ABSTRACT FROM AUTHOR]

TN BSA injected at tolerogenic doses with a trace label of 14C showed an immediate fixation on serum proteins, red cells, lymphoid cells and. as far as could be determined, all organs in the body. The fixation on lymphoid cells was non-specific, at least initially. These results are discussed in relation to the mechanism of tolerance induction by TNBSA. [ABSTRACT FROM AUTHOR]

The delayed footpad reaction to sheep erythrocytes (SRBC) was studied in mice whose mononuclear phagocyte system (MPS) was blocked or stimulated. Colloidal carbon or carrageenan was used for the blockade of MPS and Corynebacterium parvurn or diethylstilbestrol used for the stimulation. The optimal dose of SRBC for the induction of the delayed footpad reaction was lower in MPS-blocked mice than in non-treated mice, whereas a high dose of SRBC was required for the induction of the strongest delayed footpad reaction in MPS-stimulated mice. These results suggest that non-specific phagocytic function of MPS modulates subsequent immune responses after administration of antigens. [ABSTRACT FROM AUTHOR]

The immune response of pigs to oral immunization with dinitrophenylated bovine gamma globulin (DNP-BGG) was assessed using the indirect macrophage migration assay. Two days' exposure to the antigen resulted in the inhibition of macrophage migration by supernatants of cultures from the intestine but not the mesenteric lymph node or spleen. Stimulation for 7 days resulted in macrophage inhibition by supernatants of lymphocyte cultures prepared from the intestine, mesenteric lymph node and spleen. Supernatants of splenic lymphocytes from animals stimulated for 2 or 3 days produced enhancement of macrophage migration. [ABSTRACT FROM AUTHOR]

The intensity of the cellular immune response to strong transplantation antigens of the H-2 complex is related to the histological type of cells taken for immunization. Splenic cells from C3H mice immunized BALB/c mice better than cells of non-lymphoid origin from the same donor. The reverse was observed when, instead of BALB/c mice, C57B1/6 and A/He were used in immunization experiments. Analysis of the inheritance of this immunization pattern studied in mice of the first generation from various crosses between strains (BALB/c, C57B1/6 and A/He) and in mice from backcrosses has demonstrated that this immunization pattern is not inherited as a simple Mendelian character. Therefore, it could be supposed that efficiency of immunological control of cells having different histological types, may be genetically determined. [ABSTRACT FROM AUTHOR]

Glutaraldehyde (GA)-treated sheep red blood cells (SRBC) or H-2 allogeneic spleen cells (SC), induced immunological memory with absent or markedly reduced primary antibody production. In contrast, a normal secondary response was obtained when GA-SR BC or GA-SC were given to mice primed with the corresponding untreated antigens. The secondary response of mice primed and boosted with GA-treated cells was relatively high with GA-SR BC, and negative or very low with GA-SC. Morphological studies of the fate of intraperitoneally injected cells showed that endocytosed GA-SRBC persisted much longer in pen- toneal macrophages than untreated SRBC. Simultaneous challenge of mice with untreated and GA- treated SRBC revealed that phagocytosis and digestion of both types of cells in the same macrophage proceeded independently of each other. The primary response of mice receiving both SRBC and GA-SRBC was entirely similar to the response when SRBC alone was given. [ABSTRACT FROM AUTHOR]

Experiments have been carried out to find out if the administration of an anti-IgD serum to mice interferes in anyway with their immune response to sheep red blood cells. This was done to test a hypothesis that a biological role for IgD might be as a critical cellular receptor for antigen. Our results show that the injection of anti-IgD two days before antigen results in suppression of primary responses and priming (the antigen dependent generation of memory cells) but has no suppressive effect on a secondary response. On this indirect evidence we conclude that it is likely that IgD is present on antigen-sensitive precursor cells but not on memory cells. [ABSTRACT FROM AUTHOR]

A total of fifty-two foetal sheep between 49 and 126 days gestation were injected with polymeric and monomeric flagellin, dinitrophenylated monomeric flagellin, chicken red blood cells, ovalburain, ferritin, chicken γ-globulin and the somatic antigens of Salmonella typhimurium in a variety of combinations. Immune responses were followed in these animals by taking serial blood samples from them through indwelling vascular cannulae and measuring the circulating titres of antibody. Of the antigens tested, ferritin induced immune responses in the youngest foetuses. A short time later in gestation, the majority of foetuses responded to chicken red blood cells, polymeric flagellin, monomeric flagellin and dinitrophenylated monomeric flagellin. Only older foetuses responded regularly to chicken γ-globulin and ovalbumin. However, antibodies to all these antigens were first detected over the relatively short period of development between 64 and 82 days gestation and this made it difficult to define any precise order in the development of immune responsiveness. Of the antigens tested only the somatic antigens of S. typhimurium failed to induce a primary antibody response during foetal life. The character and magnitude of the antibody responses in foetuses changed throughout in utero development. Both the total amount of antibody produced and the duration of the response increased with foetal age. Foetuses younger than 87 days gestation did not synthesize 2-mercaptoethanol resistant antibodies or IgG1 immunoglobulin to any of the antigens tested, whereas most foetuses older than this regularly did so. [ABSTRACT FROM AUTHOR]

This study was designed to examine aberrations of immune responses in autoimmune NZB strain mice during ageing, at the level of individual B cell clones. The response to phosphorylcholine (PC) was chosen because murine responses to PC are restricted to a few B cell clones and can be characterized with idiotypic markers, Responses to both thymus-dependent (TD) and thymus-independently (TI) PC-containing antigens were measured in mice ranging from 1 to 62 weeks of age. We found that: (1) TD responses to phosphorylcholine keyhole limpet haemocyanin (PC-KLH) decreased markedly (about 17-fold) between 28 and 62 weeks of age. TI responses to the R36a strain pneumococcus decreased only slightly during the same period. (2) The PFC responses to both antigens became markedly prolonged in mice older than 24 weeks. (3) The NZB response to either antigen is essentially monoclonal, as measured by inhibition of PFC with specific anti-idiotype serum and PC hapten. No age-related alteration in avidity or idiotype expression was observed. Our results demonstrate that no aberrant PC reactive B cell clones appear in old NZB, and lend support to the notion that the abnormalities observed are due to defective regulatory mechanisms. [ABSTRACT FROM AUTHOR]

The influence of route of administration and dose of antigen on the generation of humoral and cell-mediated immune (CMI) responses has been studied in the lizard, Calotes versicolor. Intra- muscular (i.m.) immunization was found to be more suitable for the induction of both plaque-forming cell (PFC) and migration inhibition (MI) responses to sheep erythrocytes (SRBC) than the intraperitoneal (i.p.) route. The i.m. route favoured the development of quicker PFC as well as MI responses. The latter was observed to be sustained up to a month after immunization. In contrast, with the i.p. route of immunization, the peak PFC and MI responses appeared slowly and were followed by a sudden decline. An inverse relationship between PFC and MI functions was noted. The maximum level of MI was obtained with 104 SRBC while the PFC response was greater with the higher doses of antigen. Low dose of antigen induced a strong and quick MI response with concomitant postponement of the PFC response. Incorporation of SRBC into Freund's complete adjuvant did not influence the MI level. After an injection of a supra-optimal dose of SRBC, the MI level was very low at a time when the PFC response was at its maximum and the degree of MI rose again after the decline in PFC number. Thus, the induction of antibody synthesis needs a higher threshold level of antigen for its full expression, in contrast to the minimal antigen dose required for MI response to SRBC in lizards. [ABSTRACT FROM AUTHOR]

A new mathematical model for antigen antibody interactions has been developed. The new model is based on the assumption that the formation of complexes between a bivalent antibody and a multivalent antigen is determined thermodynamically by the concentrations and valences of antigen as well as antibody, together with one association constant which is common to all mutual interactions. Formulae have been derived for calculation of the distributions of compositionally different antigen-antibody complexes either from knowledge of equilibrium concentrations of free antigen and antibody, or from knowledge of total amounts of antigen and antibody in the system. A computer program for these calculations is described. The model is found to yield precise predictions of the formation of soluble immune complexes, as studied by zonal centrifugation. It is found through use of the model that ‘complex formation’ as such differs in binding characteristics from adsorption, especially for high concentrations of antigens and antibodies. ‘Complex formation’ implies that association constants estimated through a Sips plot method will vary with antibody concentration, and that certain curvatures of the lines in a Sips plot reflect inherent properties of complex-forming systems. [ABSTRACT FROM AUTHOR]

Naturally occurring double-stranded RNA (ds-RNA) was immunogenic when injected into mice, rats, guinea-pigs, rabbits, dogs and baboons. The response to native material administered intravenously (i.v.) was strongest in rabbits and mice, and weakest in baboons. Mice, guinea-pigs and baboons injected with ds-RNA complexed with methylated BSA emulsified in Freund's complete adjuvant all gave high antibody responses. When ds-RNA was given in aerosol form to mice and guinea-pigs the response was weaker than that following i.v. injection, and baboons did not respond to antigen given as an aerosol. In most species the immune response obtained was predominantly IgM in nature, and there was no evidence for cell-mediated immunity in any species. The only evidence of an adverse reaction associated with repeated administration of ds-RNA was a systemic anaphylactic-type response in a small group of mice given ds-RNA repeatedly in aerosol form and challenged with ds-RNA i.v. [ABSTRACT FROM AUTHOR]

The affinity of anti-DNP antibodies produced by mice of various ages has been studied at the cellular level by the plaque inhibition technique. The affinity of PFC produced shortly after a single injection of antigen was found to increase during the first 6 weeks of life. The responses of 1- and 2-week-old animals also showed apparently restricted heterogeneity. The difference in the affinity of anti-DNP-PFC between young and adult mice could not be attributed to different (serum) levels of antigen or to differences in the rate of maturation of affinity during the immune response. Cell transfer experiments suggested that the age-dependent increase was due to a change in the population of antibody-forming (B) cell precursors and not to a progressive improvement of T-cell function. This finding is interpreted as favouring somatic mutation theories of antibody diversity. [ABSTRACT FROM AUTHOR]

Limiting dilution analysis has been applied to the study of T-cell 'helper' function in vitro. Using the microculture system one can estimate the numbers of (a) 'helper' T cells involved in specific collaboration with B cells and (b) those T cells which are able, on being activated by their specific antigen, to facilitate the response of B cells to another antigen. Such studies have enabled us to demonstrate that: (1) a single helper' T cell was able to activate a single B-cell precursor to detectable antibody production; (2) the `helper' function of primed T cells was radio-resistant; (3) a minimal estimate of `helper' frequencies could be obtained in defined cell populations; (4) non- specific facilitation was directed towards virtually all available B cells of a given specificity if these were challenged with their appropriate particulate antigen; (5) the microculture system offers the opportunity to determine whether specific and non-specific T-cell helper' effects are a consequence of the activity of one T-cell type or of different subpopulations of T cells. [ABSTRACT FROM AUTHOR]

The bird's antibody response to a thymus-dependent antigen (sheep erythrocytes) (SRBC) and a thymus-independent antigen (Salmonella adelaide O antigen) were characterized: whereas the former proceeded through a brief 19S response to a declining 7S response, the latter failed to switch from 19S to 7S for several weeks and consisted in repeated excursions of 19S antibodies. When injected intravenously and simultaneously an injection of S. adelaide-killed organisms and SRBC interact, so that the response to the latter fails to switch from 19S to 7S and consists of repeated excursions of 19S antibodies. The changed character of the SRBC response is interpreted to be due to the relative lack of 7S antibody: passive 7S antibody to S. adelaide O antigen or 7S anti-SRBC produces a negative feedback inhibition of their respective responses, so that only one excursion of 19S antibody is observed. The effect is not, however, symmetrical; the thymus-independent antigen is dominant. Thus, whereas 7S antibody to S. adelaide produces the same negative feedback inhibition on the response to S. adelaide and the response to SRBC (when injected with adelaide), 7S antibody to SRBC inhibits only the response to SRBC and not the response to S. adelaide. These results are discussed in relation to current hypotheses of antibody biosynthesis and mechanisms of adjuvant action. They are also discussed in relation to the function of the germinal centres of the spleen which may function to mediate the negative feedback of 7S antibody on the antibody response. [ABSTRACT FROM AUTHOR]

To better understand the contribution of T cells to the immunity of pigs to pseudorabies virus (PRV), we examined the lymphoproliferation response to this virus. Depletion studies demonstrated that both CD2+ CD8+ and CD2+ CD4+ cells contributed to lymphoproliferation, but to varying degrees upon stimulation with live and ultraviolet (UV) light-inactivated PRV. Flow cytometric analysis revealed the emergence of both CD2+CD8+ and CD2+CD4+ lymphoblastoid cells. To examine the contribution of specific viral proteins, we prepared immortalized porcine B cells of haplotype d/d that stably expressed a single PRV protein, and used these cells for in vitro stimulation of lymphocytes from PRV-immune miniature pigs of the same haplotype. Cells expressing PRV gB or gC induced proliferation. An immunization/challenge experiment showed that the lymphoproliferation response was stronger upon immunization with the virulent NIA-3 strain than with the attenuated Bartha strain. Upon challenge inoculation, the NIA-3-immunized pigs were almost completely immune, in contrast to the Bartha-immunized pigs. Such poorly protected pigs showed secondary B- and T-cell immune responses upon challenge. In contrast, the better protected NIA-3-immunized pigs did not show a secondary B-cell response. However, they developed a secondary lymphoproliferation response, which was quicker and stronger than in the Bartha-immunized pigs. This dichotomy between secondary B- and T-cell responses indicates that an effective T-cell memory response is able to quickly eliminate challenge virus in immune pigs, so preventing a secondary B-cell response. [ABSTRACT FROM AUTHOR]

In areas endemic for schistosomiasis, there is great heterogeneity in antibody isotype responses to parasite antigens amongst infected individuals. At the population level, the isotype composition of antibody responses undergoes dynamic changes which are associated with the age of infected individuals. Here we examine the IgG subclass responses to Schistosoma mansoni eggs (soluble egg antigens; SEA) of infected individuals by immunoblot and ELISA. By controlled treatment of SEA-coated ELISA plates and immunoblot nitrocellular strips with sodium periodate, in order to oxidize terminal carbohydrate residues selectively, we were able to relate individuals subjects' isotype responses to the different antigens that they responded to, and to the presence of putative carbohydrate and peptide epitopes on those antigens. IgG2 responses were restricted strictly to sodium periodate-sensitive carbohydrate epitopes and antigens of relatively high molecular weight. These antigens were not usually recognized by other isotypes and, therefore, they were only recognized by individuals who had high levels of IgG2. IgG1 and IgG3 responses were directed against both carbohydrate and peptide epitopes, whereas IgG4 responses were restricted to periodate-resistant epitopes. This suggests that the fall in IgG2 responses, and reciprocal rise in IgG4 antibodies, seen in young children as their intensities of schistosome infection increase, is not the result of isotype switching, and that, if these two subclasses are involved in blocking immunity to schistosomiasis, they are operating independently. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) isolated from the lymph nodes or spleens of mice and pulsed with contact sensitizers or protein antigens stimulate primary proliferative responses by syngeneic T cells and responses to alloantigens in the mixed leucocyte reaction (MLR). Using enriched human peripheral blood DC, we attempted to stimulate primary immune responses to contact sensitizers by autologous lymphocytes/n vitro. No significant proliferation above background levels or CD69 expression (an early activation antigen on lymphocytes) was detected despite using a wide range of donors, chemicals, antigens and cell concentrations. Culture of DC for up to 5 days in vitro in the presence of phytohaemagglutinin (PHA)-conditioned culture supernatants, or recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) also failed to induce primary proliferative responses to contact sensitizers. Comparisons were made between blood and lymph node DC from mice to explore whether the lack of stimulation was the result of differences between mouse and human DC or between DC isolated from different tissues. DC from lymph nodes stimulated primary responses to contact sensitizers in both blood and lymph node lymphocytes whereas blood DC did not stimulate responses. Both lymph node and blood DC stimulated an allogeneic MLR, although blood DC were less efficient than those from lymph node. The data show that DC from different tissues exhibit variable functional activity. DC from blood and lymph nodes were examined to determine whether surface antigen expression is related to functional activity. Murine blood DC expressed similar levels of LFA-1, LECAM-1 and CD44 compared with lymph node DC but lower levels of MHC class if, B7 and ICAM-1. These results may therefore have important implications for antigen processing and presentation in cells from different tissue compartments. [ABSTRACT FROM AUTHOR]

Information about interleukin-3 (IL-3) effects in vivo is limited compared with the in vitro effects. We found that a repetitive injection of a Iow dose of recombinant IL-3 induced protection against intestinal worms of Strongyloides ratti in C57BL/6 mice. When mice were injected i.p. with different doses of recombinant IL-3 twice a day from day -5 to day -1 and infected orally with larvae recovered from the head of infected rats on day 0, worm recovery from the small intestine was markedly reduced by a total of 104 U IL-3 or more on day 2 post-infection. The number of intestinal mucosal mast cells (MMC) was increased by the protective dose of IL-3. The IL-3 treatment, however, was ineffective in protecting mice against tissue migrating larvae, as assessed by recovery from the head. The protective effect of IL-3 on intestinal worms was observed within 6 hr post oral infection, suggesting little concern with antigen-specific immune responses. The effective dose of IL-3 treatment increased the number of MMC progenitors five times in the spleen and the mesenteric lymph nodes. An MMC-specific protease, MMCP-1, was secreted 200 times more than in controls in the intestinal lumen by the IL-3 treatment. The IL-3 treatment induced no protection or mastocytosis in mast cell-deficient W/Wv mice. These results suggest that the IL-3-induced intestinal protection against S. ratti is mediated by MMC. [ABSTRACT FROM AUTHOR]

The present study focused on the influence of maternal immunity during pregnancy on the responsiveness of the immune system(s) in offspring. Maternal immunization of pregnant mice with T-dependent foreign antigen sheep red blood cells (SRBC) induced suppression of anti-SRBC plaque-forming cell (PFC) responses in their offspring. We attempted to identify the cell species among the maternal lymphoid cells of the immunized pregnant mice that induced this suppression in their offspring, by separating the maternal cells into T cells, B cells and macrophages, or T-cell subsets, and then adoptively transferring them into other normal pregnant mice. The results demonstrated the following: first, maternal CD4+ T cells of immunized pregnant mice induced immune suppression in their offspring. Second, maternal T cells could be activated during pregnancy in the same fashion as in non-pregnant mice. The T-cell factor(s) for the immune suppression in offspring is produced not only by maternal T cells of immunized pregnant mice but also by T cells activated in non-pregnant mice. Third, cellular organization was required for maternal T cells to induce this immune suppression in their offspring. [ABSTRACT FROM AUTHOR]

The article summarizes the work from many laboratories in the identification of antigenic regions from HIV, a variety of methods of identification and interpretation of results means that not all groups agree on the importance of certain epitopes. However, consensus agreement on many regions exist where independent laboratories have confirmed results from others. The knowledge of these regions can be applied in several ways to improve understanding of the natural immune response to HIV, to contemplate manipulation of immune responses in infected individuals, either stimulation as active immunotherapy or suppression of deleterious responses, and in the design of a subunit prophylactic vaccine.

A putative synthetic vaccine for foot-and-mouth disease (FMDVI5) has proved less successful in a host species, cattle, than predicted by results in a small-animal model. Possible reasons for this include non-recognition by T cells influenced by major histocompatibility complex (MHC)-linked immune response gene control. It is now possible to type for human leucocyte antigen (HLA) DR-like bovine MHC (BoLA) class II polymorphisms with a one-dimensional isoelectric focusing (IEF) technique. Using this method 14 unrelated cattle were selected with eight different BoLA class II IEF types. After immunization with FMDV15, 13 cattle generated a T-cell response to FMDV15. However, the fine specificity and magnitude of the response was related to BoLA class II type. The non-response by one animal and low response by two other animals were associated with two of the BoLA class II types. Response to the region 149-158 was immunodominant and animals which did not respond to this region had low responses to the whole peptide. Using FMDV-specific T-cell lines five BoLA class II types associated with responder animals were able to present FMDV15 in an MHC class II-restricted fashion, indicating that this peptide is capable of binding to different MHC class II molecules and may account for the broad response observed. The restriction patterns of the lines indicated that the IEF method does not distinguish all functional polymorphisms. At least two of the IEF-defined types could each be split into two distinct specificities and revealed that the three sets of animals with identical IEF types in fact expressed distinct restriction elements. [ABSTRACT FROM AUTHOR]