Popliteal lymph node enlargement and antibody production in the mouse induced by zimeldine and related compounds with varying side chains

Structural requirements for induction of draining lymph node responses by the antidepressant drug zimeldine ((Z)-3-(4-bromophenyl)-N,N-dimethyl-3-(3-pyridyl)allylamine) in mice were determined by comparison of its activity with that of metabolites and analogues having different side chains. Mice received 1.0 mg of the compounds into the hind footpad and the popliteal lymph nodes (PLN) were removed 7 days later to determine weight, cell number and antibody production. Compounds with a methylated (Z)-(homo)allylamine side chain induced a marked PLN weight gain in C57BL/6 mice and a significant increase of the PLN cell number and of the IgM and IgG production per 10(6) PLN cells in BALB/c mice. Moderate PLN weight increase, but no significant antibody formation was induced by the doubly demethylated zimeldine metabolite, while compounds without an aliphatic amine or having a saturated side chain lacked significant activity in all assays. The (E)-diastereomer of zimeldine induced significantly less PLN weight gain than zimeldine in C57BL/6 mice, but an equal increase of PLN cell number in BALB/c mice. IgM and IgG responses to the (E)-diastereomer were moderate and absent, respectively. The antihistaminic drug, brompheniramine, having a saturated side chain and a 2-pyridyl ring, induced less PLN weight and cell gain than zimeldine and failed to increase antibody formation. The capacity of the compounds to induce PLN responses appeared not related to their pharmacological potential to inhibit the reuptake of serotonin and noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Popliteal lymph node enlargement induced by procainamide.

The ability of procainamide (PA) to induce primary local popliteal lymph node (PLN) reactions has been investigated. We employed an in vitro drug-metabolizing system, based on the hypothesis that the negative result for PA in a PLN assay (PLNA) was due to insufficient metabolizing activity at the reaction site. PA was incubated previously in vitro with the S-9 mixture derived from rat liver. The reactants were ultrafiltered in order to eliminate high molecular-weight molecules, and then the low molecular-weight fraction was subcutaneously (s.c.) injected into the hind footpad of mice. PLN reactions were assessed by weighing the popliteal lymph node of the injected side. The reactants of more than 5 mg of PA and S-9 mixture induced PLN enlargement in C3H/He mice 8 days after injection. BALB/c and C57BL/6 mice were also susceptible to PLN reaction in response to the reactants of PA (10 mg) and S-9 mixture. PLN reactions to PA were induced 2 days after the injection and sustained until 18 days. Contact of PA with the S-9 mixture for 30 min at 37 degrees C was sufficient to induce PLN enlargement. However, contact for 24 h reduced the peak reaction. On the other hand, PA which had not been incubated with the S-9 mixture and acetylprocainamide (acetylPA) gave little or no reaction. Local PNA has not been considered to be suitable for the detection of drugs with the potential to induce immune disorders in cases where a metabolite contributes to the adverse reaction. However, the employment of an in vitro drug-metabolizing system may overcome that defect.     

Induction of popliteal lymph node enlargement and antibody production in the mouse by pyridylallylamines related to zimeldine

The role of the para-brominated phenyl ring of the allylamine antidepressant drug, zimeldine, in its immunomodulating activity was studied by comparing local lymph node responses of mice to analogues with various phenyl ring substituents. The compounds (1.0 mg) were injected into the hind footpad and weight and cell number of the popliteal lymph node (PLN) and antibody production by PLN cells were determined 7 days later. Zimeldine and the para-trimethylsilylated and ortho-para-chlorinated compounds induced a marked PLN weight gain in C57BL/6 mice. The para-chlorinated and meta-brominated analogues were significantly less effective, while the para-fluorinated and the unsubstituted analogue failed to trigger PLN weight gain. The same range of potency of the compounds was seen when increases of PLN cell number in BALB/c mice and increases of IgM production by a fixed number of BALB/c PLN cells were determined. The IgG production on a per cell basis was not increased by the fluorinated and the unsubstituted compounds, while the remaining compounds stimulated the IgG production to a similar extent. Plots of the lipophilicity of the para-substituted compounds against their ability to induce PLN weight and cell gain showed a fair correlation, but induction of antibody formation tended to be counteracted at too high lipophilicity. No correlation between pharmacologic and immunologic activity of the compounds could be found. Data indicate that lipophilicity grossly favours PLN enlargement, but the divergent responses to the equilipophilic 3- and 4-brominated compounds, suggest conformational restraints as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of the age of donors and recipients on the regional graft-versus-host reaction in mice.

Regional graft versus host (GVH) reaction in mice induced by injection of parental strain spleen cells into the footpad of F1, recipients, was measured 7 days later by a popliteal lymph node (PLN) enlargement index. Young, 6-week-old F1 hosts can be used for studying the age- and sex-dependent GVH reactivity of parental strain donors. On the other hand, senescent, 21-24-month-old F1 recipients are able to mount a host versus graft response against parental antigens which may obscure the typical GVH reaction. The PLN enlargement in syngeneic old F1 mice points to an autoimmune mechanism of this phenomenon.     

T-cell-dependent popliteal lymph node reactions to platinum compounds in mice.

The requirements for sensitization to complex salts of platinum were investigated in a mouse model by means of the popliteal lymph node (PLN) assay. A single subcutaneous injection of dissolved hexachloroplatinates without adjuvant induced a vigorous primary immune reaction in the draining PLN. Dose-dependent lymph node activation was determined by an increase in both PLN weight and cellularity. In C57BL/6 mice, peak reactions were obtained around day 6 after administration of 90-180 nmol Na2[PtCl6] or (NH4)2[PtCl6] per animal. Mice primed to [PtCl6]2- mounted an enhanced response upon local restimulation with suboptimal doses of the same but not unrelated compounds, indicating a specific secondary response. T cells were required to elicit PLN reactions to [PtCl6]2-, because athymic nude mice completely failed to respond, in contrast to their +/nu littermates. Differences between various inbred strains of mice revealed that Pt-induced PLN responses are genetically controlled. Moreover, the immunogenicity of Pt salts in mice is not confined to hexachloroplatinates, but other compounds, such as the antineoplastic agent cis-dichlorodiamine platinum, are able to induce comparable PLN reactions.     

Immunopathological signs in mice treated with mercury compounds--I. Identification by the popliteal lymph node assay of responder and nonresponder strains

In the present study we screened mice from 22 different inbred strains for potential differences in their immunological reaction to HgCl2. As a rapid screening test, we used the popliteal lymph node assay (PLNA). Mice were injected s.c. into one hind footpad with 3-60 micrograms of Hg2+, given as HgCl2 in phosphate-buffered saline (PBS); contralateral hindfeet remained uninjected. Control mice received PBS alone. On day 6 the weights of the draining and contralateral PLN were determined and the PLN index calculated. While we found linear dose-response curves in some strains, these curves had a different shape in others. Out of the total of 21 euthymic strains tested only strain DBA/2 (H-2d) proved to be a nonresponder to HgCl2; it remained a nonresponder over the whole dose range (3-180 micrograms Hg2+) and period of time (days 2-12) studied. The other H-2d strains tested, i.e. NZB, BALB/c and B10.D2/n, showed absent or low PLN responses only in the lower dose range (3-30 micrograms Hg2+). F1 hybrids of strain DBA/2 and the responder strain C57BL/6 gave an intermediate response. While C3H nu/nu mice failed to respond to HgCl2, C3H +/nu mice did. The weight increase of the draining PLN after HgCl2 injection was preceded in time by an increased 3H thymidine uptake by the PLN. Histologically, enlarged PLNs revealed increased cellularity in both the T-cell and the B-cell areas. When CH3HgCl, instead of HgCl2, was injected all three strains tested, including DBA/2, responded by PLN enlargement. We conclude that (1) HgCl2 is an immunostimulatory agent in mice in that it induces T-cell-dependent enlargement of the draining PLN upon local injection, (2) there are striking genetic differences between inbred strains of mice in the PLN response to HgCl2, but these differences are not paralleled by similar differences in the response to CH3HgCl, (3) responsiveness to HgCl2 appears to be inherited in a codominant fashion, and (4) there is suggestive evidence that the observed genetic differences are determined by both H-2 and non-H-2 genes.     

Popliteal lymph node enlargement and antibody production in the mouse induced by drugs affecting monoamine levels in the brain

Drugs that affect monoamine levels in the brain were screened for their potential to cause immunological changes in the popliteal lymph node (PLN) of mice after injection into the hind paw. The tricyclic antidepressant (TCA) drugs, imipramine and amitriptyline, and the serotonin reuptake blocker, zimeldine, induced a prominent PLN weight gain in C57BL/6 mice. Ketanserin and ritanserin appeared less effective while nomifensine, serotonin and the antigen, sheep erythrocytes, lacked significant activity. In BALB/c mice all agents induced an increase of PLN cell number, the TCA drugs and zimeldine appeared superior in this respect. Increased IgM as well as IgG production on a per cell basis was only induced by the TCA drugs, zimeldine and, especially, sheep erythrocytes. Data indicate that induction of PLN responses is not a general property of agents affecting monoamine levels. Structural, i.e. antigenic, characteristics of the drugs rather than their pharmacological properties are probably at play.     

The effect of localized injection of adjuvant material on the draining lymph node

The histological accompaniments of adjuvant activity were studied in popliteal nodes of CBA mice at various intervals after footpad injection. Substances tested possessed either extrinsic adjuvanticity but little or no antigenicity (alum, vitamin A alcohol), antigenicity but no extrinsic adjuvanticity (bovine γA-like globulin), both (Freund's complete adjuvant, killed B. pertussis organisms, alum-precipitated BGG), or neither (paraffin oil BP).

Substances possessing extrinsic adjuvanticity produced marked persistent enlargement of the draining lymph node, with prominent expansion, hypercellularity and blast transformation in the paracortical areas by the 4th day after injection. This was followed by germinal centre formation and medullary plasmacytosis occurring between the 4th and 10th day. Substances possessing little or no antigenicity produced a feeble germinal centre response and minimal medullary expansion. Bovine γA-like globulin, which lacks extrinsic adjuvanticity, produced a prominent germinal centre and medullary response but a minimal paracortical response. Only mild, transient lymph node enlargement, lasting 1–2 days, was seen after paraffin oil BP.

Passive immunization of CBA mice with CBA anti-pertussis hyperimmune serum inhibited the adjuvant action of pertussis, and diminished and delayed the histological changes in the draining lymph node.

These data suggested that paracortical expansion and hyperplasia were characteristic histological accompaniments to the injection of substances with extrinsic adjuvanticity. Because paracortical hypercellularity was observed in some instances to precede blast cell transformation, it is suggested that paracortical expansion induced by adjuvants is partly due to an augmented cellular traffic, with a net flux of recirculating lymphocytes into these areas.

     

Transplantation of T cell-mediated, lymphoreticular disease from the scurfy (sf) mouse.

The X-linked mutation, scurfy (sf), causes a fatal lymphoreticular disease characterized by runting, lymphadenopathy, splenomegaly, hypergammaglobulinemia, exfoliative dermatitis, Coombs'-positive anemia, and death by 24 days of age. T lymphocytes are required to mediate this syndrome as shown by a total absence of disease in mice bred to be scurfy and nude (sf/Y; nu/nu). The scurfy phenotype is not transmitted by sf/Y bone marrow transplants, though cells of scurfy origin do reconstitute all lymphoid organs in the recipient mouse. These data suggest that scurfy disease results from an abnormal T cell development process and not from an intrinsic stem cell defect. We therefore tested the ability of transplanted scurfy thymuses to transmit scurfy disease to congenic euthymic mice, to athymic (nude) mice, and to severe combined immunodeficiency (SCID) mice. Euthymic recipients of sf/Y thymic grafts remained clinically normal as did all SCID and nude recipients of normal thymus transplants. Morphological lesions similar to those found in scurfy mice occurred in all H-2-compatible nude and SCID recipients of sf/Y thymic grafts. Intraperitoneal injections of scurfy thymocytes, splenocytes, and lymph node cells also transmitted the scurfy phenotype to H-2-compatible nude mice and SCID mice. Our findings indicate that scurfy disease can be transmitted to T cell-deficient mice by engraftment of scurfy T cells, but that pathogenic scurfy T cell activities can be inhibited (or prevented) in immunocompetent recipient mice.     

Induction of lymph follicle formation with endotoxin lipopolysaccharide in the draining lymph node of athymic nude mice

Formation of lymph follicles in draining popliteal lymph nodes in athymic nude mice and their phenotypically normal littermates (PN mice) was investigated after footpad injection with either endotoxin lipopolysaccharide (LPS) or phytohemagglutinin in soluble and precipitated forms (PHA). The dose of LPS injected ranged from 10 to 100 micrograms, and that of PHA was 50 micrograms. After any dose of LPS, draining nodes of nude mice produced new lymph follicles in the peripheral cortex outside pre-existing follicles, though the number of follicles induced was somewhat less than in the case of PN mice treated with LPS. After injection of PHA in soluble or precipitated form, PN mice developed a significant number of new follicles in the draining nodes, but nude mice failed to do so. The present results are consistent with the view that nude mice have the ability to develop lymph follicles by way of a thymus-independent mechanism in response to exogenous stimuli.     

Kinetics of germinal center development in lymph nodes of young and aging immune mice

Recent findings imply that germinal center paucity in old mice, at least in part, results from a defect in the mechanisms responsible for the transport of antigens to lymphoid nodules (follicles) and the consequent impairment of the antigen retaining reticulum (ARR) of follicular dendritic cells (FDCs). The present objective was to observe the kinetics of lymph node germinal center development in old mice having antigen transport and ARR deficits. Germinal center development was monitored in popliteal (PLN) and axillary (AXLN) lymph nodes of 6-8 wk and 23-mo-old horseradish peroxidase (HRP) immune C57BL/6 mice. Using the selective binding of germinal center B cells for peanut agglutinin (PNA), germinal centers were identified in serial vibratome sections following histochemical labeling with PNA-peroxidase conjugates at times 0, 15 min, 1, 3, 5, and 10 days after footpad challenge with 8 micrograms HRP. To follow the fate of preexisting (environmental antigen-induced) germinal centers and the development of de novo (HRP-induced) germinal centers, it was essential to distinguish between these germinal centers. Accordingly, PNA positive germinal centers associated with HRP-retaining (peroxidase positive) ARR were identified as de novo germinal centers and germinal centers not associated with a peroxidase positive ARR were classified as preexisting germinal centers. Kinetic analysis of PNA positive germinal centers showed the following: 1) Preexisting, environmentally-induced germinal centers dissociated and disappeared by day 3 as indicated by a decline in their numbers after antigen injection: the process of germinal center dissociation remained unaffected by aging. 2) The latency of de novo germinal center appearance was approximately equal in duration (approximately 3 days) to the disappearance of pre-existing germinal centers. 3) The number and size of de novo HRP-induced germinal centers increased through the experimental period in young lymph nodes, but in old mice these parameters were depressed, resulting in a significant germinal center deficit. 4) The ratio of HRP-retaining ARR to de novo induced germinal centers was 1:1 in young and responder old mice. This ratio was not affected by aging. This finding favored the concept that antigen retention in ARR is a requirement of germinal center development. The observations supported our hypothesis that germinal center development, at least in part, depends on a normal antigen transport by showing that in aged mice with defective antigen transport-related ARR and iccosome deficits there is an impaired development of germinal centers.     

Applicability of the Popliteal Lymph Node Assay in the Brown-Norway Rat

A number of drugs and chemicals can induce autoimmune disorders. Previous studies suggested the popliteal lymph node (PLN) assay might be a suitable and reliable model to detect such compounds in the mouse. the applicability of this assay has been examined in the Brown-Norway rat using a pannel of positive as well as negative reference compounds.

Popliteal lymph nodes were excised and weighed seven days after subcutaneous injection of 5 mg of each compound into one footpad. Interestingly, all negative reference compounds produced no significant increase in PLN weight index whereas four positive reference compounds were associated with PLN-weight indices greater than 2.3.

However, no increase in PLN weight was noted in procainamide-and isoniazide-treated rats as previously reported in the mouse. This first set of experiments indicates that the PLN assay is applicable to Brown-Norway rats and warrants further investigation.     

ADDITION OF SERUM TO THE MEDIUM USED FOR PREPARATION OF CELL SUSPENSIONS AS A POSSIBLE SOURCE OF ARTIFACTS IN CELL-MEDIATED REACTIONS STUDIED BY MEANS OF THE POPLITEAL LYMPH NODE TEST

Conditions for preparations of cells used in immunogenetic studies may substantially affect their properties and the results obtained. It was found that short-term incubation or a mere preparation of parental spleen cells in medium containing either xenogeneic serum or concavalin A (Con A) increase their ability (after footpad injection) to induce enlargement of the popliteal lymph node in (B10.LP x A/Ph)F₁ hybrid recipients. Under similar conditions, enlargement of PLN could be induced also by syngeneic F₁ hybrid cells. The greater lymphadenomegaly following the injection of parental or syngenneic cells prepared in serum-containing medium may be explained by the host reaction against additional foreign antigens rather than increased GVH reactivity or autoimmunity.     

Structural and cell population changes in the lymph nodes of the athymic nude mouse

A recent tridimensional analysis of the lymph node demonstrated that its deep cortex is composed of grossly hemispherical "units," adjoining a portion of its peripheral cortex. Each deep cortex unit can be distinguished into a center and a periphery. The periphery was concluded to be a site for migration of circulating lymphocytes, the center, a site where T cells would participate in cellular immune responses. The aim of the present work was to determine the influence of the congenital athymic state on the development of the units and of other components in the lymph nodes of the nude mouse. For this, the lymph nodes at various anatomical locations in adult athymic nude mice were analyzed. The present study revealed that the athymic state did not inhibit the development of the units but severely depleted the lymphocyte population of their center only. However, it did inhibit the development of an area of peripheral cortex located over the middle part of a unit. Such an area of peripheral cortex is, thus, concluded to be thymus dependent, as is the center of a deep cortex unit. The athymic state also prevented the development of the cells of the nodules (germinal centers) and of much of the plasmocytes. On the other hand, it yielded to the enlargement of the follicles, the formation of new structures: medullary "lymphocyte clusters" and the transformation of the medullary venules into high endothelial venules. The various modifications of the nodal structures resulting from the congenital athymic state are discussed in relation to some functions of the organ.     

The production of contact sensitivity by the injection into the footpads of recipients of the lymph node cells from mice 1 day after painting the skin with contact sensitizing agent: requirement for matching at the major histocompatibility complex between donor and recipient mice.

Donor mice were painted on the skin of the abdomen with the contact sensitizing agent, oxazolone. One day later 2-5 x 10(6) cells from the regional lymph nodes were injected into the footpads of recipient mice. Contact sensitivity was detected 6 days later by challenging the ears of the recipients and measuring the increase of thickness at 24 h. Good contact sensitivity was obtained when CBA cells were injected into CBA mice and BALB/c cells injected into BALB/c mice; the injection of BALB/c (H-2d) cells into CBA (H-2k) mice and vice versa failed to give rise to contact sensitivity. Hybrid F1 cells gave intermediate responses. The contact sensitivity caused by the injection of small numbers of lymph node cells into the footpad is interpreted as a mode of active immunization and the present results show that this only occurs when there is genetic matching at the major histocompatibility complex between the donor and the recipient mouse.     

Long-term antigen retention by dendritic cells in the popliteal lymph node of immunized mice.

Antigen retention by follicular dendritic cells (FDC) was studied in the popliteal lymph nodes (PLN) of mice actively or passively immunized against human serum albumin (HSA) or horse spleen ferritin. Electron microscopic autoradiography was used to locate a challenge dose (1 microgram) of 125I-labelled HSA in the draining PLN following injection into the hind footpad of specifically immune mice. In both actively and passively immunized mice, the radiolabelled antigen was localized to the follicles in the cortex of the draining node. In actively immunized mice, the antigen formed a crescent of label on the superficial aspect of germinal centres, while in passively immunized mice label was seen in primary follicles. In electron microscope autoradiographs, the silver grains were concentrated in areas of dendritic cell processes which emanated from a cell body containing a characteristic irregular nucleus. The size and complexity of the dendritic cell processes increased in actively immunized mice suggesting that the FDC could hypertrophy. High resolution studies using the electron-dense antigen, ferritin, showed that it was localized to the extracellular space of the FDC processes and was associated with amorphous electron-dense material; presumably immune complexes. Antigen was not present uniformly distributed in the extracellular material but rather it was in discrete patches occupying small segments of the FDC processes. Large amounts of label-free electron-dense material were present suggesting that immune complexes of various specificities were present on each DC. Ferritin was seen more than 3 months after challenge but only on the FDC. The data suggest that the antigen retaining mechanism in the lymph node of immune mice is antigen non-specific, is capable of hypertrophy in response to active immunization and provides a mechanism for stable long-term retention of antigen.     

Use of Peyer''s patch and lymph node fragment cultures to compare local immune responses to Morganella morganii.

Lymphoid tissue fragment cultures were established to analyze the differentiative processes among B cells in Peyer's patches (PP) and peripheral lymph nodes (PLN), especially those in germinal centers. PP cultures from both conventionally reared mice and formerly germ-free mice colonized with Morganella morganii could be maintained for greater than 12 days with continued B-cell division, especially among cells binding high levels of peanut agglutinin, a characteristic of germinal center cells. PLN cultures from conventionally reared mice injected with a heat-killed vaccine of M. morganii could be maintained for the same amount of time. Over this period, PP cultures continued to secrete immunoglobulin A (IgA) as well as smaller amounts of IgM. PP cultures from formerly germ-free mice colonized with M. morganii showed net increases of IgA antiphosphocholine (anti-PC) antibodies with avidities as high as those of the prototypic T15 monoclonal antibody. Similar PLN fragment cultures from conventionally reared mice given footpad injections of M. morganii showed net increases of IgM and IgG anti-PC antibodies in the culture fluid. Thus, although M. morganii stimulated lymphoid tissues in vivo to produce an anti-PC response in vitro when given by either the oral or the parenteral route, the antibody isotypes differed between PP and PLN fragment cultures. Fragment culturing may offer a complementary and simpler way to detect a local secretory IgA response than does either measuring IgA antibody in secretions or detecting IgA antibody in the cytoplasm of plasma cells in the lamina propria of gastrointestinal or respiratory tissue.     

Local immune response to Mycobacterium lepraemurium in C3H and C57Bl/6 mice.

Subcutaneous footpad inoculation of living M. lepraemurium (L.MLM) induced, in high responder C57Bl/6 mice, a local granulomatous reaction associated with the production of effector cells which stopped the multiplication of bacilli in the draining popliteal node with the concurrent development of 24--48 hr delayed type hypersensitivity (DTH). The thymus-dependent local reaction did not occur after the injection of heat-killed M. lepraemurium (HK.MLM) or after the inoculation of L.MLM in nude mice. However, HK.MLM injection interfered with the onset of the local reaction and enhanced acid-fast bacteria (AFB) counts in the draining node. In low responder C3H mice, L.MLM produced a local and delayed footpad swelling but no restriction of bacilli multiplication in the draining lymph node was observed. This unresponsiveness was not due to an overloading of the inoculum dose since doses ranging from 3 x 10(4) to 3 x 10(7) MLM did not produce any granulomatous local reaction as in C57Bl/6 mice. The injection of dead bacilli in the contralateral footpad of subcutaneously (s.c.) infected C3H mice revealed Arthus-like and 18--24 hr delayed reactions. When 10(6) L.MLM per mouse were injected intravenously (i.v.), systemic infection, measured in the spleen, was found to be less restricted in C57Bl/6 than in C3H mice. Moreover, in C57Bl/6 mice low doses of L.MLM injected i.v. delayed the local reaction at first, then enhanced footpad swelling and AFB counts in the draining nodes, indicating some acquired defect of peripheral immunity. When a high dose of L.MLM (2 x 10(8)/mouse) was injected i.v., C57Bl/6 mice died sooner than C3H mice, indicating certain discrepancies between local resistance and systemic susceptibility.     

Adoptively transferred reactivity to M. leprae in nude mice infected with M. leprae.

Reversal reactions are manifestations of delayed hypersensitivity to M. leprae and are thought to be usually accompanied by manifestations of effective cell-mediated immunity (CMI) as measured by bacterial clearing. These experiments were designed to study the induction of reversal reactions in M. leprae-infected, congenitally athymic nude mice using adoptive transfer of CMI. Splenic cell suspensions derived from unimmunized heterozygous nu/+ mice, and those vaccinated with heat-killed M. leprae, viable BCG and a mixture of the two antigens were diluted to contain 10(4), 10(5), 10(6), 10(7) lymphocytes/0.1 ml and infused intravenously into multibacillary nude mice. The production of reversal reactions in leprous nude mice in response to adoptively transferred CMI was studied in a quantitative fashion. Dose responsive induction of reversal reactions, apparent by footpad inflammation and swelling, decreased morphological indices (MI) of the bacteria and mononuclear cell infiltrations, histopathologically, were observed. For nude mice receiving cells primed with 3.9 X 10(5) living BCG alone, the effective dose 50% (ED50) was 1.0 x 10(6) lymphocytes to induce reversal reactions. For those receiving cells primed with 10(7) M. leprae the ED50 was 3.7 x 10(5) lymphocytes. For nude mice receiving cells primed with a mixture consisting of 1/2 the above dose of BCG + 1/2 the above dose of M. leprae, the ED50 was 6.8 x 10(4) lymphocytes.     

Cellular immune responses in mice immunized with Anisakis simplex larval antigens

Cellular immune responses to Anisakis simplex L3 antigens were investigated in BALB/c mice injected subcutaneously with a homologous crude extract (CE). Popliteal lymph nodes (PLN) were found to be increased in size and weight after A. simplex CE footpad injection. The effects of A. simplex CE in vitro proliferation were assayed with non-fractionated PLN cells or nylon-wool purified T cells derived from pooled lymph node cells of mice subcutaneously injected with CE. Spleen cells from immunized animals (antigen alone, or larva alone, or antigen plus larva) were studied by flow cytometry. The immunization induced a high proportion of CD4 + and TCRalphabeta + T cells. The number of B cells (CD45 + and TCRalphabeta-) in pre-immunized and infected mice was lower than that observed in animals subjected to infection only. The number of CD4+ T cells increased in the infected and in the pre-immunized and infected mice. In the latter, a decrease of CD8a + T cells was noted. The greatest increase in CD8a+ and TCRalphabeta- T cells was found in mice that had been subjected to infection only. Histological analysis showed that the most prominent lesions were gastric and intestinal in animals infected orally with one larva.