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The effects of clenbuterol in preserving the form and function of muscle after unilateral sciatic nerve division and epineural repair were investigated in a rat model. The drug (a β2-adrenoceptor agonist) was administered daily for six weeks by gastric gavage (10 μg/kg body weight), interrupted every 5 days by a 2 day omission of dosing to avoid drug desensitization. Clenbuterol reduced the loss of wet weight, total protein, muscle fibre cross sectional area and (in part) contractile forces in denervated hindlimb muscles, with most effects lasting until reinnervation. The effects were dependent on muscle type, with slow-twitch oxidative muscle (soleus) and mixed-fibre (gastrocnemius) showing greater sensitivity to the drug than fast-twitch muscle (extensor digitorum longus). Anabolic effects on the contralateral innervated muscles tended to be small. The results suggest a potential for the adjuvant use of selective β-adrenoceptor agonists in the management of peripheral nerve injuries in humans.

Most experimental evidence for chemopreventive activity comes from studies in which animals are administered the suspect chemopreventive agent before, after or simultaneously with a carcinogen predicted to increase tumour incidence in a particular tissue. Such experimental designs have inherent mechanistic assumptions. Evidence of chemopreventive function is also theoretically derivable from carcinogenicity assays, which in principle involve exposure to a single agent in addition to all other factors experienced by the control group. In these assays, exposure is normally for a very large proportion of the animal's lifespan, so they could indicate chemopreventive activity with much greater facility than could human trials, in which exposure for 10% of the lifespan would be considered to be a long study. Site-specific tumour incidence reductions do occur in carcinogenicity assays, often in the same experiments in which increases occur at other sites. These reductions could be due to a carcinogenic response at another site which increases mortality within the group. They could also be secondary to a noncarcinogenic toxic effect that results in either a lifespan reduction (thereby reducing the chance of tumour development) or a reduced food intake (this being the clearest example of an experimental manipulation resulting in reduction in tumour incidence). Nevertheless, if chemoprevention is possible in humans, then it should also be experimentally demonstrable at nontoxic dose levels. Studies involving experimentally reduced tumour incidence are reviewed here, with the aim of describing special requirements for their evaluation and their significance in the field of chemoprevention.

When an IgG2c monoclonal antibody specific for Trichinella spiralis muscle stage larvae was cleaved with pepsin to yield F(ab')2 fragments, the latter retained their capacity to cause mucus entrapment and rapid expulsion of larvae from the intestines of suckling rats. When fed to pups, the F(ab')2 fragments of this antibody and the F(ab')2 fragments of a similarly prepared IgG2a antibody caused mucus entrapment of muscle larvae (ML), demonstrating that trapping is not dependent upon the Fc region of the antibody molecule. Despite the fact that these two antibodies had similar specificities and that their F(ab')2 fragments caused larval entrapment in mucus, F(ab')2 fragments of the IgG2a antibody failed to protect rat pups. Fragments of the IgG2c antibody caused rapid expulsion when injected into pups, but the distribution of larvae was dramatically different from when the fragments were delivered orally. These results indicate that entrapment of T. spiralis in mucus is not in itself the cause of the expulsion. The more likely possibility is that antibody impedes a function of Trichinella spiralis that is related to the capacity of the parasite to reside in its epithelial niche.

Rat pups suckling dams parasitized by Trichinella spiralis express rapid expulsion, a protective response that is associated with the entrapment of infectious muscle larvae in intestinal mucus. Immunofluorescent studies revealed that antibodies were bound to the surfaces of the entrapped larvae. Mucus binding and rapid expulsion occurred in normal pups dosed with larvae coated with antibodies prepared from infected rat serum. Subsequent experiments revealed that entrapped larvae escaped from mucus after 2 hr in vitro incubation in saline. Escape correlated with the loss of the surface-bound antibodies, suggesting that mucus entrapment was reversible and dependent on antibody coating. Finally, when protective antibodies were injected 1, 2 or 6 hr after larvae were administered to pups, the parasites were forced to leave their epithelial niche and became enveloped in mucus. The above findings suggest that mucus trapping of T. spiralis larvae is dependent upon the coating of larvae by antibody, but that trapping is reversible, and is not in itself the pivotal event in rapid expulsion. The primary mechanism of rapid expulsion appears to be antibody-mediated inhibition of processes required for the parasite to maintain itself in the epithelium.

Affinity columns prepared with rabbit antibody to the F(ab′) 2 fragment of rat immunoglobulin were used to separate rat thoracic duct lymphocytes into sub-populations that differ with respect to the density of their surface membrane immunoglobulin. Using this technique, it was shown that lymphocytes in the DNA synthetic (S) phase of the mitotic cycle are added in increased number to the lymph of rats infected with Listeria monocytogenes. The great majority of these S-phase cells lacked a high density of surface immunoglobulin as indicated by their failure to bind to the immunoabsorbent. Cells which can protect recipient rats against a challenge infection with L. monocytogenes also segregated with nonadherent thoracic duct lymphocytes obtained from Listeria -immune donors. These protective cells realized their full immunological potential only in recipients that shared histocompatibility-gene-coded structures with the immune lymphocyte donors. The above findings accord with the view that immunity to L. monocytogenes is mediated in rats by activated T cells which are formed as part of the animal's cell-mediated response to infection. Although Listeria -protective lymphocytes concentrate in the nonadherent, T-cell-enriched fraction, it was consistently observed that the adherent, B-cell-enriched fractions of immune donor thoracic duct lymphocytes also could transfer a low level of antimicrobial resistance. This immunity was restricted in allogeneic recipients, a finding which implies that the protection afforded by the adherent population is related to its content of T cells. Nonadherent S-phase lymphoblasts moved in substantial numbers from the blood into peritoneal inflammatory exudates induced by L. monocytogenes. The above finding encourages the belief that recently activated T cells realize their protective function locally in centers of infection where they have secondary effects on macrophages.

Acquired resistance to the intracellular bacterial parasite, Listeria monocytogenes can be transferred to normal recipients by thoracic duct lymphocytes or peritoneal exudate cells obtained from rats infected with this organism; The appearance of protective cells in thoracic duct lymph coincides with the development in the donors of delayed-type hypersensitivity to Listeria antigens and accumulation in induced peritoneal exudates of cells which are responsive to these antigens in the migration inhibitory factor (MIF) assay. The cells in exudates that confer protection, and those that release MIF, arise at sites remote from their final destination. From their point of origin in the caudal lymph nodes of infected rats, cells with these properties are delivered to the thoracic duct and hence to the blood from where they are drawn into the peritoneal cavity in response to inflammation. The parallel observed in the appearance, increase and subsequent decline of protective lymphocytes and MIF-producing cells in exudates suggest that the two activities are mediated by a single line of T cells. However this may be, the development and deployment of the cells concerned encourages the belief that MIF has a meaningful role in the expression of cellular resistance to infection.

Soluble material enriched in tumor-associated antigen was prepared by affinity chromatography from a KCl extract of the chemically-induced D-23 rat hepatoma. Microgram quantities of the above material bound spontaneously to living BCG when the two were incubated briefly in vitro. When injected into normal syngeneic rats, the BCG-associated tumor antigen induced a measure of resistance against challenge with D-23 tumor cells. Peritoneal exudate cells (PEC) obtained from such actively immunized subjects were able to suppress the growth of D-23 tumor cells at a test site in muscle. In contrast, immunization with either BCG alone, tumor protein alone, or tumor protein admixed with BCG in circumstances designed to impede association of the protein, failed to provoke the formation of tumor suppressor PEC. The results encourage of the belief that binding of tumor antigen to BCG favors the induction of a cell-mediated tumor suppressive response.

Rats convalescing from a recent infection with Listeria monocytogenes generate T cells which can protect recipient rats against a challenge infection with that organism. Using monoclonal antibodies that react with some but not all rat peripheral T cells, the T-cell mediators of acquired resistance to infection (TCRI) were isolated by panning and characterized by using a fluorescence-activated cell sorter. Many L. monocytogenes-immune TCRI were relatively large cells as judged by their light-scattering properties. That finding accords with previously reported cytokinetic data and velocity sedimentation analyses which revealed that the majority of L. monocytogenes-immune TCRI are lymphoblasts. In the current investigation, the surface antigenic profile of these mediator T cells was revealed as W3/25+ OX8+ OX4+ RT6.1-. That phenotype is identical to that of L. monocytogenes-induced prekiller cells which are formed as part of the animal's cell-mediated response to infection. Like prekiller cells and their differentiated counterparts, L. monocytogenes-immune TCRI adhere preferentially to monolayers of syngeneic fibroblasts. The results indicate that L. monocytogenes-immune TCRI belong to a minor subset of peripheral T cells which also contains T cells that have the cytotoxic potential by which L. monocytogenes-induced prekiller cells have been defined.

Specifically sensitized lymphocytes have a focusing influence on mononuclear phagocytes that is expressed in the local accumulation and division of macrophages in bacteria-induced exudates. This was demonstrated by injecting Listeria monocytogenes into the peritoneal cavity of normal rats immediately before the animals were transfused with thoracic duct lymphocytes from either Listeria -immune donors or donors that had been infected with the unrelated parasite, Francisella tularensis . Sensitized lymphocytes originally present in the intravenous inocula were found later in the exudates. The arrival in the inflamed peritoneal cavity of specifically sensitized lymphocytes was associated with an exuberant influx of newly formed host cells and a local proliferative response that involved both immunoblasts and macrophages. These cytokinetic observations provide a plausible explanation of the delayed inflammatory response induced by the parasite, and imply that sensitized lymphocytes contribute to the host's defence by encouraging the prompt and purposeful deployment of monocyte-derived macrophages in centers of infection. In addition to their focusing influence on mononuclear phagocytes, specifically sensitized lymphocytes or their products can enhance the metabolic and microbicidal activity of macrophages. This activation process was revealed in the ability of rats infected with L. monocytogenes or BCG to control the growth of F. tularensis at a challenge site in the testis. But resistance was expressed only when the challenge organisms were injected with killed bacteria against which the recipients had been specifically immunized. The results accord with the view that macrophages are functionally activated by an immunological mechanism, and imply that the process is triggered locally by sensitized lymphocytes which are recruited from the blood.

A method is described whereby antisera raised in rabbits to rat thoracic duct lymphocytes were made specific for the plasma membrane antigens of T and B lymphocytes. These lymphocyte-specific antisera were used in immunofluorescence assays to study the distribution of B and T cells in lymphocyte containing tissues and body fluids of the rat. Rabbit antirat B-cell serum (ALSB) reacted selectively with the surfaces of lymphocytes in the lymphoid follicles of lymph node cortex and in the follicles and marginal zones of splenic white pulp, but not with the surfaces of germinal center cells or plasma cells. An identical pattern of fluorescent staining was obtained with rabbit antirat Ig serum. It was shown by blocking, absorption, and immunoprecipitation studies that ALSB was composed in large part of antibodies to rat Ig, but that it contained antibodies to other B-cell antigens as well. Rabbit antirat T-cell serum (ALST) reacted selectively with the surfaces of lymphocytes in the paracortex of lymph node and in the periarteriolar sheath of spleen, and with thymocytes. ALST did not display anti-Ig activity. ALST reacted with approximately 100% thymocytes and with 90% thoracic duct, 80% lymph node, 60% blood, 50% spleen, and 10% bone marrow lymphocytes in suspensions of cells from these sources. ALSB reacted with the remainder of the lymphocytes in the suspensions, except for bone marrow in which only 59% of lymphocytes had detectable B- or T-cell surface antigens. The population of T lymphocytes in rat bone marrow was depleted by drainage of lymphocytes from a thoracic duct fistula, thereby establishing their membership in the pool of recirculating T cells. Approximately 14% of lymphocytes issuing from the thoracic duct of TxBM donors reacted with ALST. The presence in these animals of a small number of T cells, calculated to be approximately 2% of the normal value, may account for the limited capacity of TxBM rats to respond to antigens that induce a cell-mediated immune response.

Newcastle disease virus (NDV) can interact in at least two ways with rat T cells. By adsorbing to circulating lymphocytes, the virus can transiently deflect the cells from lymph nodes and inflammatory exudates induced in the peritoneal cavity. T cells are affected regardless of age, state of activation, or position in the mitotic cycle. The effect is reversible and is mediated not only by infectious (I)-NDV, but also by UV-NDV which cannot achieve a complete replication cycle in eggs. But I-NDV has another lasting effect on activated T cells. It is revealed in the failure of virus-treated thoracic duct lymphocytes to transfer cellular resistance to Listeria monocytogenes, delayed-type hypersensitivity to soluble antigens of the parasite, and the permanent exclusion of labeled S-phase lymphocytes from inflammatory foci. Activated T cells are inhibited by virus multiplicites which have little if any effect upon the proliferative potential of antigen-sensitive T cells or localization of labeled small lymphocytes in lymph nodes. The underlying mechanism has not been determined; however, there are reasons for thinking that NDV has a lethal effect upon activated T cells, because the latter are permissive for virus replication.

Rats immunized with bacillus Calmette-Gudrin (BCG) were either treated with rifampicin plus isoniazid or received no treatment. Thoracic duct lymphocytes (TDL) obtained from these animals were tested for their ability to provide protection against Mycobacterium tuberculosis strain R1Rv in syngeneic recipients. After six months of treatment there was no difference between the two groups of donors with respect to protection conferred by their TDL. The rats receiving chemotherapy maintained their immunity despite the reduction of viable BCG to undetectably low levels in the tissues. It was concluded that effective chemotherapy for tuberculosis does not abrogate established immunity to the tubercle bacillus.

A substantial portion of the lymphocyte-like cells in induced peritoneal exudates derive from cells which enter the blood by way of the thoracic duct. The migrant cells have been identified as large and medium lymphocytes, but they may also include short-lived small lymphocytes derived from them. Small lymphocytes which have a potentially long circulating life-span are excluded from exudates, although cells of this type predominate in thoracic duct lymph. The results imply that many (perhaps all) of the small round cells in inflamed tissue are members of a line of rapidly proliferating lymphocytes. Specifically committed lymphocytes with precisely these properties are added to the blood of rats infected with Listeria monocytogenes. The localization of committed lymphocytes in inflammatory foci could be the crucial event which enables the host to focus his cellular defenses at sites of bacterial implantation.

Thymectomized adult rats which have been heavily irradiated and reconstituted with syngeneic bone marrow cells rapidly regain the ability to defend themselves against a primary infection with the intracellular bacterial parasite, Listeria monocytogenes. They do so by a cell-mediated immunological mechanism as evidenced by the protective immunity transferred adoptively by thoracic duct lymphocytes or peritoneal exudate cells from donors infected with this organism. But peritoneal exudate cells from thymus-derived donors convey only a fraction of the immunity transmitted by exudate cells from similarly infected intact rats. Since thymectomized irradiated animals can mobilize their cellular defenses more effectively when they are injected with a modest number of thoracic duct lymphocytes, an effect that cannot be duplicated with a massive infusion of bone marrow, it is argued that thymusdependent lymphocytes or T cells have an influential role in the development of cellular resistance to infection.

The cellular deficit in rats thymectomized at birth is primarily one of circulating small lymphocytes. The lymphocyte deficiency is similar to that induced in adult rats by chronic drainage from a thoracic duct fistula. In both cases, the animals show a reduction of small lymphocytes in peripheral blood, thoracic duct lymph, and in circumscribed areas of lymphoid tissue. The lympocyte deficiency in lymphoid tissue can be corrected by an intravenous injection of thoracic duct lymphocytes. The evidence suggests that the deficiency is corrected by small lymphocytes. Small lymphocytes pass from blood to lymphoid tissue along a route which includes the marginal sinus in splenic white pulp and postcapillary venules in the cortex of lymph nodes and Peyer's patches. Neither the ability of small lymphocytes to colonize lymphoid tissue nor their ability to traverse postcapillary venules are thymus-dependent phenomena. However, movement of small lymphocytes across postcapillary venules appears to modify the structure of endothelium. Intravenously injected small thymocytes migrate to lymphoid tissue in smaller numbers than small lymphocytes inoculated by the same route. The few thymocytes which localize in lymphoid tissue follow the same pathway as circulating small lymphocytes.

Rat bone marrow is deficient in immunologically competent cells when compared with the peripheral pool of circulating small lymphocytes. However, circulating lymphocytes capable of initiating a lethal graft vs. host reaction can develop from myeloid precursors. In adult rats inoculated at birth with parental strain bone marrow cells, the thoracic duct lymph contains cells having the immunological capability of the marrow donor. The evidence suggests that immunologically competent lymphocytes derive from cells normally resident in bone marrow and probably not from precursors in the peripheral lymphocyte pool.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.

Intraperitoneal stimulation of adoptively sensitized rats with bacterial antigen promotes the localization of lymphoblasts at the site of antigen deposition. Lymphoblast extravasation activity (LEA) is generated only when specifically immune donor lymphocytes and the recipients of these cells share at least on Ag-B haplotype. However, if the specificity criteria for its formation are satisfied, LEA promotes the local development of lymphoblasts of all available specificities and irrespective of their Ag-B genotype. Allogeneic lymphoblasts do not participate actively in the delayed inflammatory reaction even when they are passively recruited into exudates. The results suggest that LEA is a T cell-derived mediator that amplifies the delayed type hypersensitivity reaction by directing recently activated lymphocytes into lesions.

The genetic basis of the restriction imposed on T cell mediating acquired antimicrobial resistance and delayed-type hypersensitivity (DTH) to Listeria in the rat was investigated. Sharing of MHC-coded genes between donors of sensitized T cells and antigen-stimulated recipients was both necessary and sufficient for efficient transfer of both resistance and DTH. Evidence to support this assumption was derived from experiments involving allogeneic transfers within major histocompatibility complex (MHC)-compatible strains and across MHC-barriers. Further support came from linkage studies with backcrossed rats and with the progeny of F1 rats mated with an unrelated strain. An unexpected difference in the compatibility requirements for effective transfer of DTH and resistance was noted in experiments involving the BI strain (formerly called B3). Thus, while B-region compatibility was obligatory for expression of DTH in recipients of sensitized T cells, considerable levels of protection could be transferred to either A-region or B-region compatible hosts.

A soluble extract of Listeria monocytogenes bound firmly and in similar amounts to a variety of rat cells. Cells that bound this material differed in their capacity to stimulate the in vitro proliferation of lymphocytes obtained from the thoracic duct of Listeria-immune donors. The capacity of cells to serve as antigen-presenting cells in this system coincided or closely overlapped the expression on these cells of an Ia antigen-like structure. Three lines of evidence indicate that T cells respond to L. monocytogenes antigen: the responder cells are members of a nylon-wool nonadherent population that lacks readily detectable surface immunoglobulin; they express determinants recognized by the W3/25 monoclonal antibody (a surface marker of rat peripheral T cells); and they are stimulated optimally by L. monocytogenes antigen when the latter is displayed on cells that share a haplotype with the responder lymphocytes.

Soon after rats are infected with Listeria monocytogenes (LM), Listeria antigen- (LMA) responsive lymphocytes are delivered to the animal's thoracic duct. These LM-responsive lymphocytes can be restimulated in vitro by LMA to generate cells that have a potent cytolytic capability. The activation of LMA responsive lymphocytes is immunologically specific and dependent upon the activity of histocompatible accessory cells. Neither cell-free LMA nor LMA-pulsed allogeneic accessory cells can promote a significant cytotoxic response by negatively selected responder lymphocytes. LM-dependent cytolytic lymphocytes differ from natural killer (NK) cells inasmuch as their activation is not facilitated by interferon (IF). Likewise, supernatants from cultures containing specifically sensitized thymus-derived (T) lymphocytes and histocompatible LMA-pulsed accessory cells fail to augment (day 2 and day 3 supernatants actually inhibit) the activation process. The results imply that the successful activation of LM dependent cytolytic lymphocytes requires the cooperative interplay of responder T cells and specific-antigen-pulsed accessory cells.

In order to identify and characterize the mediator(s) of rapid expulsion, infant rats were passively immunized against oral challenge with 200 infectious Trichinella spiralis muscle larvae by oral or intraperitoneal (i.p.) administration of secretions or sera from infected rats. Milk whey from infected dams provided a modest level of protection when fed to pups. Immunoglobulins from T. spiralis-infected rat serum protected suckling pups when injected intravenously (i.v.) into lactating dams 2 days prior to pup challenge. Intraperitoneal injection of pups with serum immunoglobulins also enabled them to express rapid expulsion. The protective component of serum was precipitated with 40% (NH4)2SO4 and was not affected by heating to 56 degrees, although antibodies mediating passive cutaneous anaphylaxis were inactivated by both treatments. Oral transfer of biliary immunoglobulins collected from infected rats at various times during a primary infection provided no protection to pups. However, serum immunoglobulins from rats infected for 42 days or longer transferred rapid expulsion to pups. Absorption of protective serum immunoglobulins with subclass-specific reagents revealed that IgG1 played a significant role in protection.

Thymus-dependent lymphocytes, or T cells, of rats infected with Listeria monocytogenes acquire a potent cytolytic capability when the cells are restimulated in vitro by Listeria antigens. Activation of such Listeria monocytogenes-dependent cytotoxic T lymphocytes requires both antigen and histocompatible accessory cells, but only when the responder T cells have been specifically depleted of lymphocytes that are responsive to accessory cell alloantigens. When unselected T cells are used, significant activation occurs in specific-antigen-free cultures containing allogeneic accessory cells. This seeming paradox is explained by the fact that a lymphokine generated in mixed leukocyte cultures can promote the terminal differentiation of cytotoxic T lymphocytes precursors. This lymphokine has tentatively been identified as interleukin-2. The results suggest that a three stage process is involved in the activation of Listeria monocytogenes-dependent cytotoxic T lymphocytes. The first stage occurs in response to an immunizing Listeria monocytogenes infection and itself involves two significant events, the polyclonal priming of prekiller cells and the generation of Listeria antigens-specific helper T cells. During the second stage, helper T cells are stimulated by Listeria antigens to release interleukin-2. This process is efficiently executed only in the presence of histocompatible accessory cells. The third stage in the activation process requires neither accessory cells nor antigen and involves the interleukin-2-driven differentiation of prekiller cells.

Cytotoxic thymus-derived lymphocytes (CTL) generated in vitro by restimulating rat cells with Listeria antigen- (LMA) pulsed syngeneic accessory cells were characterized in respect to their surface membrane markers. LM-dependent CTL were devoid of detectable surface immunoglobulin (Ig) and receptors for the Fc region of rabbit IgG. Experiments with monoclonal antibodies to rat T cell markers revealed that these cytotoxic cells have the phenotypic profile W3/13+, W3/25-, MRC OX 8+. LM-dependent CTL also bind the monoclonal antibody, MRC OX 3, which recognizes an Ia-antigen-like determinant on rat cells. Although LM-dependent CTL lack the W3/25 marker, their generation depends on the cooperative interplay of W3/25+ and W3/25- T cells.

Rat thoracic duct lymphocytes (TDL) were separated into two fractions by passing the cells through a column of rabbit anti-rat F (ab′) 2 antibody coupled to Sephadex G-200. Cells with readily detectable surface immunoglobulin (Ig) were retained on the gel, whereas those without surface Ig were recovered in the effluent. Adherent cells were retrieved by eluting the column with rat Ig. Both dividing and nondividing lymphocytes were separated by this procedure. The adherent and non-adherent fractions contained functionally active lymphocytes as judged by a thymidine incorporation technique and the immunological performance of the cells after transfer to normal recipients. Antibody forming cells and B memory cells were concentrated in the adherent fraction. The non-adherent fraction contained antigen-sensitive T cells which initiate graft versus host reaction and specifically sensitized lymphocytes of the kind which transfer resistance to L. monocytogenes .

Macrophage chemotactic activity (MCA) is generated in situ in peritoneal inflammatory exudates induced by antigens of the intracellular parasite, Listeria monocytogenes. Chemotactic and chemokinetic activity is formed locally in response to an immunologically specific signal. In rats that have been immunized adoptively with thoracic duct lymphocytes (TDL) from specifically immunized donors, the production of MCA depends upon stimulation by LMA of exudate-seeking S-phase lymphocytes of their progeny. The sequential appearance, increase, and subsequent decline of MCA in the peritoneal cavity parallels the influx of lymphocytes and precedes maximal recruitment of labeled monocytes from the blood. The MCA response in peritoneal exudates induced in adoptively immunized rats correlates with the level of macrophage accumulation in the peritoneal cavity and at sites of LMA injection in the pinna of the ear. The results suggest that MCA is released locally by antigen-activated lymphocytes and imply that the factor(s) concerned has a purposeful role in the host's defense by promoting the rapid deployment and/or retention of monocyte-derived macrophages in centers of infection.

Microgram quantities of bovine serum albumin (BSA) and hen egg albumin (OA) associate spontaneously with living BCG in aqueous suspension. The association is stable insofar as bound protein cannot be readily dissociated from the organism by repeated washing. Association of BSA or OA is dependent upon the concentration of specific protein or competing protein in the incubation mixture, pH and the amount of protein already bound to the organisms. Washed BCG "complexes" containing either BSA or OA are potent inducers of delayed-type hypersensitivity (DTH). The same soluble proteins are far less effective inducers of DTH, even when injected with BCG. The capacity of OA-BCG complexes to provoke a cell-mediated response seems to be related, at least in part, to "stabilization" of the antigen on an appropriate carrier and its concentration at the site of BCG injection.

Rats can be solidly immunized against Trichinella spiralis by a series of methyridine-terminated oral infections with T. spiralis larvae. Injections of thoracic duct lymphocytes (TDL) obtained from such animals can protect normal rats against a Trichinella challenge. The protective cells belong to two populations which differ with respect to their adherence to affinity columns prepared with rabbit antibody to rat F(ab')2. Immune lymphocytes in the column-adherent B cell fraction are inhibited by vinblastine, whereas those in the non-adherent, T cell fraction are resistant to this drug. The above observations suggest that acquired resistance to T. spiralis is mediated by two classes of lymphocytes: B cells which are delivered to the thoracic duct and hence to the blood while still in active cycle, and T cells which have a potentially long life-span and presumably belong to a pool of recirculating small lymphocytes.

Rat dams infected with 1000 Trichinella spiralis muscle larvae, 4 weeks prior to breeding, provided their suckling offspring with immunity to challenge with 200 muscle larvae at 2 weeks of age. The immunity was expressed in the elimination of 75-99% of the challenge dose within 24 hr. The intestinal worm burden did not decline significantly after the initial expulsion. Infected dams continued to protect their offspring during three breeding cycles, for as long as 26 weeks after infection. Immunity was conferred upon pups by dams that had been selectively immunized with the parenteral phase of the parasite's life cycle. Immunization with a drug-terminated enteral infection was ineffective as was enteral immunization followed by the parenteral phase. Further analysis revealed that rapid expulsion by pups was dependent on the number of mature muscle larvae recovered from dams immunized with NBL. By comparison, the expulsive capacity of the same dams was not improved by increasing the numbers of NBL within the range tested.

Rats infected with the live vaccine strain (LVS) of Francisella tularensis develop in vivo and in vitro evidence of cellular hypersensitivity and a concomitant state of cellular resistance to infection. They key role of sensitized lymphocytes in cellular resistance was domonstrated in transfer experiments. Using this technique, it was shown that thoracic duct lymphocytes from Francisella immune donors conferred specific antimicrobial resistance on normal recipients, whereas antiserum afforded no protection whatsoever. Further evidence for the participation of sensitized lymphocytes in the host's defence emerged from experiments in which a comparative analysis was made of the immunogenic properties of living and heat-killed LVS organisms. Rats stimulated with the living parasite developed cellular hypersensitivity and specific antibodies. Throacic duct lymphocytes obtained from such animals could immunize adoptively. By comparison, rats stimulated with a substantially larger number of dead organisms failed to develop cellular hypersensitivity and their lymphocytes were devoid of protective activity. Dead organisms, however, provoked an antibody response similar to that observed in infected rats. The development of cellular hypersensitivity in Francisella-infected rats is associated with enhanced resistance to Listeria monocytogenes. This finding accords with the results of similar studies of infection immunity to other intracellular parasites, and implies that the expression of cellular resistance to F. tularensis is a cooperative venture involving specifically sensitized lymphocytes and non-specific inflammatory cells, presumably macrophages.

A dissociation between the in situ generation of lymphocyte-dependent macrophage chemotactic activity (MCA) and the accumulation of macrophages in peritoneal inflammatory exudates was demonstrated in rats stimulated intraperitoneally with a saline suspension of killed Listeria monocytogenes (LM). Treatment of specifically immunized animals with cobra venom factor (CoVF) erased the hemolytic activity of serum complement (C) and the generation of peritoneal MCA. Such C-deficient rats nonetheless marshaled a substantial number of monocyte-derived macrophages into LM-induced exudates. The results suggest that MCA does not have an obligatory role in the attraction of macrophages into lesions in which there is a delayed inflammatory component. CoVF not only abrogated lymphocyte-dependent MCA in antigen-induced exudates but also decreased MCA of fresh and of heated normal rat serum. The serum of venom-treated animals could not be rendered chemotactic by C activation. It remains to be determined whether lymphocyte-dependent MCA is a product of antigen-stimulated T cells or is generated extracellularly by the interaction of T-cell factors with a humoral precursor. In any event, lymphocyte-dependent MCA differs from C-dependent MCA insofar as it is inactivated by heating (30 min at 56 °C).

Delayed-type hypersensitivity (DTH) to Listeria monocytogenes was measured in rats that were recipients of syngeneic, semisyngeneic, and allogeneic immune thoracic duct lymphocytes (TDL). DTH could be transferred only to recipients that shared at least one haplotype with the TDL donors. The restriction was expressed in an inability of sensitized lymphoblasts to localize efficiently at antigen injection sites in the pinna of the ear and peritoneal cavity. Failure of allogeneic lymphoblasts to extravasate in more than trace numbers into Listeria-antigen-induced exudates was reflected in an absence of other lymphocyte-mediated expressions of DTH. Thus, lymphocyte-dependent MCA was not detected in Listeria-antigen-induced peritoneal exudates borne by recipients of allogeneic immune TDL and blood monocytes were not recruited in increased numbers into such exudates as they were in exudates borne by syngeneic rats. But allogeneic restriction of the delayed inflammatory response to Listeria antigen was overcome, at least in part, when antigen-presenting macrophages of the same MHC type as the immune TDL donors were implanted in the peritoneal cavity. The results encourage the belief that the observed failure of immune TDL to transfer DTH to allogeneic recipients is related to the inability of sensitized donor T cells to recognize antigen displayed by allogeneic macrophages.

Cells that carry immunologic memory of phiX 174 have been studied in the rat. Memory cells could not be detected in thoracic duct lymph before the 10th day after priming. Thereafter, they increased rapidly in number to reach a plateau by the 4th week. This long latency was not due to a protracted process of maturation of cells formed and delivered to the lymph during the early post-induction period because memory never appeared in rats immunized adoptively with cells obtained before the 11th day. The fact that phiX memory cells are resistant to inhibition by vinblastine also indicates that memory is carried by cells which are released from regionally stimulated lymphoid tissue only after they have ceased dividing and become functionally mature. Adoptively acquired memory decays rapidly at first, but slowly from the 2nd week after cell transfer. This suggests that two cell populations of differing half-life are involved. If two distinct populations are necessary to the full expression of phiX memory, both populations recirculate because the memory cells in lymph draining the sites of the primary response and those reaching the central lymph of adoptive hosts give parallel dose-response curves.

An immunizing infection with Listeria monocytogenes provides a potent stimulus for the formation of prekiller lymphocytes. Their cytolytic potential is revealed when the cells are restimulated in vitro by Listeria antigens. Listeria monocytogenes-induced cytotoxic lymphocytes and the prekiller cells from which they are derived were characterized in respect to their surface antigenic markers. Using monoclonal antibodies, B-cell depleted lymphocytes from the thoracic duct of Listeria immune rats were fractionated into subsets by a combination of panning and sorting techniques. Listeria monocytogenes-induced cytotoxic lymphocytes and their prekiller cell precursors were demonstrated to have the phenotype W3/25-, OX8+, OX4+, W3/13+ (high density), OX19+ (low density), RT6.1-. The OX8+, RT6.1- subset, which contained prekiller cells, constituted approximately 6% of lymph-borne T cells. The data indicate that these microbial antigen-induced cytotoxic lymphocytes belong to a minor subset of peripheral T cells whose surface antigenic properties distinguish them from natural killer cells.

Pups born to rats immunized with the excretory/secretory antigens (ESA) of Trichinella spiralis L1 larvae expressed rapid expulsion when challenged orally. Rat monoclonal antibodies specific for ESA were produced and tested for their specificity in Western blots, binding to intact larvae and protective capacity in suckling rats. Eight antibodies had apparently identical specificity in Western blots, each recognizing a polypeptide family that included three molecular weight species sized at 41,000-50,000 MW. These polypeptides formed a series of higher molecular weight aggregates that were also bound by the monoclonal antibodies. Four of eight antibodies were protective when serially transferred to suckling pups. Each protective antibody was able to bind to intact larvae. Antibodies of two subclasses, IgG1 and IgG2c, were strongly protective, delivering to pups the capacity to expel as much as 94% of the challenge dose.

A variety of inhibitors was examined for their ability to interfere with the expression of rapid expulsion (RE) of challenge Trichinella spiralis infections in rats. Inhibitors of immediate hypersensitivity, prostaglandin release, peristalsis, or complement function, did not impair RE when administered to immune rats. Induction of intestinal anaphylaxis against T. spiralis or ovalbumin by passive serum transfer to intestinally primed rats (prior infection with Heligmosomoides polygyrus) or administration of the histamine liberator 48/80 also failed to stimulate RE. In contrast, irradiation or cortisone treatment 1, 3 or 5 days before challenge inhibited RE. We conclude that immediate hypersensitivity is not the terminal mediator of RE and plays a minor role or none at all. The effects of cortisone and irradiation suggest a major involvement by lymphoid cells in the RE reaction.

The injection of cell-free extracts of larval Trichinella spiralis with Freund complete adjuvant immunized rats against a challenge infection with T. spiralis. Protection was expressed by a reduction of adult worms in the intestines 8 days after a challenge infection. The quantitatively more important rapid expulsion reaction could also be produced by immunization with antigen, provided immunized rats were also given an intestinal priming infection with adult Trichinella spiralis or an unrelated nematode, Heligmosomoides polygyrus. These parasites stimulated a local change in the intestine that synergistically interacted with immunity to give up to 90% protection through the rapid expulsion response. Intestinal priming had to follow antigen administration by not less than 5 days and was optimal when 10 days elapsed. These experiments provide evidence for an immunological component in the rapid expulsion process. In addition, the technique suggests that the utilization of intestinal priming may provide a new approach for the development of intestinal antihelminth vaccines.

Allogeneic restriction of acquired antimicrobial resistance was demonstrated by infusing Listeria-immune thoracic duct lymphocytes (TDL) into syngeneic, semisyngeneic, or allogeneic recipients that had been infected i.v. with Listeria monocytogenes (LM). Syngeneic and semisyngeneic recipients enjoyed a high level of protection (3 to 5 log10 reduction in viable organisms within 64 hr, whereas allogeneic recipients were poorly protected (0.5 to 2.5 log10 reduction). Sharing one haplotype between TDL-donors and challenged recipients resulted in a 4- to 8-fold increase in the level of resistance transferred. Histoincompatibility reactions could be excluded as a cause of the observed allogeneic restriction. Therefore, it is likely that LM-immune TDL are unable to respond efficiently to the antigens of this organism in an allogeneic environment. But allogeneic immune TDL do confer a low level of protection that cannot be ascribed to B cell activity nor to GvH and/or HvG reactions. Evidence is provided that the modest protection afforded by allogeneic lymphocytes is conveyed by the same T cell subset that operates with much higher efficiency in genetically related rats.

Monoclonal antibodies were used to select T-cell subsets that mediate delayed-type hypersensitivity (DTH) and acquired cellular resistance (CRI) in rats infected with Listeria monocytogenes. The mediators of DTH were identified as W3/25+ OX8- T cells. The latter comprised a subset distinct from that which could protect recipient rats against a Listeria challenge. The protective T cells had a W3/25- OX8+ phenotype. The T-cell mediators of cellular resistance to infection (TCRI) failed to augment the expression of DTH; however, the mediators of DTH (TDTH) significantly enhanced the protective capacity of TCRI. This property of TDTH correlated with the ability of the cells to promote the focal deployment of TCRI and macrophages at sites of soluble Listeria antigen injection in skin, and in peritoneal exudates induced by killed L. monocytogenes. These findings illustrate the co-operative interaction of activated T cells in acquired resistance to L. monocytogenes, and imply that DTH has a purposeful role in the host defence against infection.

Immunospecific T cells cooperate with macrophages in the expression of delayed type hypersensitivity and cellular resistance to Listeria monocytogenes. The mediator cells involved are generated in response to an immunizing Listeria infection. Immunospecific T cells are formed in lymphoid tissue and appear briefly in the blood. Many localize at sites of microbial implantation, but others leave the blood spontaneously and infiltrate tissues throughout the body. Both circulating T cells and those in tissues participate in the expression of delayed type hypersensitivity and cellular resistance. Several lines of evidence suggest that these two phenomena are mediated by heterogeneous but possibly cooperating T cell populations.

Rats subjected to a 7-day abbreviated enteral infection with Trichinella spiralis subsequently reject more than 90% of a challenge infection within 24 h. This process is known as rapid expulsion. In these experiments parabiotic rats were used to examine the factors that establish rapid expulsion in the intestine. Induction with low to moderate doses of worms required exposure to two separate stimuli. These initiated different responses; one was readily transferred between parabiotic rats, whereas the second response was sessile and restricted to the intestine. These two responses interacted synergistically to produce strong rapid expulsion. Stage-specific exposure of parabiotic rats to preadult or adult trichinae (or the unrelated parasite Heligmosomoides polygyrus) showed that only preadult worms induced the transferable factor. Exposure to adult worms or to H. polygyrus induced a strictly local intestinal effect that was nonspecific. It is suggested that preadult worms initiated an immune response specific for preadults. This was transferable between parabionts but was unable to produce rapid expulsion unless the intestine had been non-specifically stimulated. Intestinal stimulation is accomplished by exposure to adult worms in natural infections or artificial regimes. These results suggest novel techniques for the development of enteral antihelminth vaccines.