Non-healer Mice Research Articles

Ear wound healing in MRL/MpJ mice is associated with gut microbiome composition and is transferable to non-healer mice via microbiome transplantation.

Adult elastic cartilage has limited repair capacity. MRL/MpJ (MRL) mice, by contrast, are capable of spontaneously healing ear punctures. This study was undertaken to characterize microbiome differences between healer and non-healer mice and to evaluate whether this healing phenotype can be transferred via gut microbiome transplantation. We orally transplanted C57BL/6J (B6) mice with MRL/MpJ cecal contents at weaning and as adults (n = 57) and measured ear hole closure 4 weeks after a 2.0mm punch and compared to vehicle-transplanted MRL and B6 (n = 25) and B6-transplanted MRL (n = 20) mice. Sex effects, timing of transplant relative to earpunch, and transgenerational heritability were evaluated. In a subset (n = 58), cecal microbiomes were profiled by 16S sequencing and compared to ear hole closure. Microbial metagenomes were imputed using PICRUSt. Transplantation of B6 mice with MRL microbiota, either in weanlings or adults, improved ear hole closure. B6-vehicle mice healed ear hole punches poorly (0.25±0.03mm, mm ear hole healing 4 weeks after a 2mm ear hole punch [2.0mm-final ear hole size], mean±SEM), whereas MRL-vehicle mice healed well (1.4±0.1mm). MRL-transplanted B6 mice healed roughly three times as well as B6-vehicle mice, and half as well as MRL-vehicle mice (0.74±0.05mm, P = 6.9E-10 vs. B6-vehicle, P = 5.2E-12 vs. MRL-vehicle). Transplantation of MRL mice with B6 cecal material did not reduce MRL healing (B6-transplanted MRL 1.3±0.1 vs. MRL-vehicle 1.4±0.1, p = 0.36). Transplantation prior to ear punch was associated with the greatest ear hole closure. Offspring of transplanted mice healed significantly better than non-transplanted control mice (offspring:0.63±0.03mm, mean±SEM vs. B6-vehicle control:0.25±0.03mm, n = 39 offspring, P = 4.6E-11). Several microbiome clades were correlated with healing, including Firmicutes (R = 0.84, P = 8.0E-7), Lactobacillales (R = 0.65, P = 1.1E-3), and Verrucomicrobia (R = -0.80, P = 9.2E-6). Females of all groups tended to heal better than males (B6-vehicle P = 0.059, MRL-transplanted B6 P = 0.096, offspring of MRL-transplanted B6 P = 0.0038, B6-transplanted MRL P = 1.6E-6, MRL-vehicle P = 0.0031). Many clades characteristic of female mouse cecal microbiota vs. males were the same as clades characteristic of MRL and MRL-transplanted B6 mice vs. B6 controls, including including increases in Clostridia and reductions in Verrucomicrobia in female mice. In this study, we found an association between the microbiome and tissue regeneration in MRL mice and demonstrate that this trait can be transferred to non-healer mice via microbiome transplantation. We identified several microbiome clades associated with healing.

The murine ear wound cartilage superhealer trait, mediated by the gut microbiome, is transgenerationally heritable following cecal transplantation

Purpose: MRL/MpJ mice are substantially protected from developing post-traumatic osteoarthritis (OA), a trait with strong correlation to the ability to heal ear wounds. We have previously shown that this ear wound healing trait is partially determined by the gut microbiome, and that gut microbial transplantation into non-healer mice can confer the healing phenotype. In this study, we sought to examine whether the earhole healing phenotype associated with microbiome transplantation can be transgenerationally inherited.

Cecal microbiome transplantation from MRL/MPJ mice prevents post-traumatic osteoarthritis in C57BL6/J mice

Purpose: MRL/MpJ mice are substantially protected from developing post-traumatic osteoarthritis (OA), a trait with strong correlation to the ability to heal ear wounds. We have previously shown that this ear wound healing trait is partially determined by the gut microbiome, and that gut microbial transplantation into non-healer mice can confer the ear wound healing phenotype. In this study, we sought to examine whether microbiome-mediated cartilage regeneration extended to protection against post-traumatic osteoarthritis.

The murine ear wound cartilage superhealer trait is associated with gut microbiota changes and is transferrable to non-healer mice by gut microbiome transplant

The murine ear wound cartilage superhealer trait is associated with gut microbiota changes and is transferrable to non-healer mice by gut microbiome transplant

Using impedance to track fracture healing rates in mice in vivo: A pilot study.

Fracture injuries are highly prevalent worldwide, with treatment of problematic fractures causing a significant burden on the U.S. healthcare system. Physicians typically monitor fracture healing by conducting physical examinations and taking radiographic images. However, nonunions currently take over 6 months to be diagnosed because these techniques are not sensitive enough to adequately assess fracture union. In this study, we display the utility of impedance spectroscopy to track different healing rates in a pilot study of an in vivo mouse tibia fracture model. We have developed small (56 μm) sensors and implanted them in an externally-stabilized fracture for twice-weekly measurement. We found that impedance magnitude increases steadily over time in healing mice but stalls in non-healing mice, and phase angle displays frequency-dependent behavior that also reflects the extent of healing at the fracture site. Our results demonstrate that impedance can track differences in healing rates early on, highlighting the potential of this technique as a method for early detection of fracture nonunion.

Metabolic, immune, and gut microbial signals mount a systems response to Leishmania major infection.

Parasitic infections such as leishmaniasis induce a cascade of host physiological responses, including metabolic and immunological changes. Infection with Leishmania major parasites causes cutaneous leishmaniasis in humans, a neglected tropical disease that is difficult to manage. To understand the determinants of pathology, we studied L. major infection in two mouse models: the self-healing C57BL/6 strain and the nonhealing BALB/c strain. Metabolic profiling of urine, plasma, and feces via proton NMR spectroscopy was performed to discover parasite-specific imprints on global host metabolism. Plasma cytokine status and fecal microbiome were also characterized as additional metrics of the host response to infection. Results demonstrated differences in glucose and lipid metabolism, distinctive immunological phenotypes, and shifts in microbial composition between the two models. We present a novel approach to integrate such metrics using correlation network analyses, whereby self-healing mice demonstrated an orchestrated interaction between the biological measures shortly after infection. In contrast, the response observed in nonhealing mice was delayed and fragmented. Our study suggests that trans-system communication across host metabolism, the innate immune system, and gut microbiome is key for a successful host response to L. major and provides a new concept, potentially translatable to other diseases.

Undifferentiated human adipose‐derived stromal/stem cells loaded onto wet‐spun starch–polycaprolactone scaffolds enhance bone regeneration: Nude mice calvarial defect in vivo study

The repair of large bony defects remains challenging in the clinical setting. Human adipose-derived stromal/stem cells (hASCs) have been reported to differentiate along different cell lineages, including the osteogenic. The objective of the present study was to assess the bone regeneration potential of undifferentiated hASCs loaded in starch-polycaprolactone (SPCL) scaffolds, in a critical-sized nude mice calvarial defect. Human ASCs were isolated from lipoaspirate of five female donors, cryopreserved, and pooled together. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an SPCL scaffold alone, or SPCL scaffold with human ASCs. Histological analysis and Micro-CT imaging of the retrieved implants were performed. Improved new bone deposition and osseointegration was observed in SPCL loaded with hASC engrafted calvarial defects as compared to control groups that showed little healing. Nondifferentiated human ASCs enhance ossification of nonhealing nude mice calvarial defects, and wet-spun SPCL confirmed its suitability for bone tissue engineering. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

Kinetics of Cryptosporidium parvum-specific cytokine responses in healing and nonhealing murine models of C. parvum infection

Susceptibility or resistance to infection with Cryptosporidium parvum correlates with the ability of mice to produce characteristic panels of cytokines in response to infection. Both adult healing and nonhealing mouse models of cryptosporidiosis were used to study the cell-mediated immune response during the course of C. parvum infection. Mesenteric lymph node (MLN) lymphocytes from both mouse models were proliferated after ex vivo re-stimulation with C. parvum sporozoite antigen. Study of the cytokine profile from the supernatant of proliferated MLN cells revealed that healing mice produced greater levels of Th1 (IFN-gamma and IL-2) and moderate amounts of Th2 (IL-4, IL-5, IL-6, and IL-10) cytokines throughout the course of infection. Whereas, MLN cells from nonhealing mice produced no IFN-gamma, low levels of IL-2 and IL-4, and higher levels of IL-5, IL-6, and IL-10 cytokines. These results suggest that the capacity to produce both Th1 and Th2 cytokines, rather than the presence of Th2 cytokines alone, determines the effective immune response against C. parvum infection.

The MRL Mouse Heart Healing Response Shows Donor Dominance in Allogeneic Fetal Liver Chimeric Mice

We previously demonstrated that after a severe cryoinjury to the right ventricle of the heart, adult MRL mice display structural and functional recovery with myocardial tissue replacement resembling that seen in amphibians. The control non-regenerating adult C57BL/6 (B6) mouse shows a predominant scar response. In the present study, radiation chimeras reconstituted with fetal liver cells from either healer MRL or nonhealer B6 mice were generated to test for a transfer of phenotype. Allogeneic MRL fetal liver cells were injected into x-irradiated (9 Gy) B6 mice and B6 fetal liver cells were injected into x-irradiated MRL mice. In these allogeneic chimeras, the healing response to cardiac cryoinjury was predominantly of the donor phenotype. Thus, MRL fetal liver cells transferred the healing phenotype to the B6 nonhealer with the appearance of Y-chromosome positive, donor-derived cardiomyocytes in the injury site and MRL-like healing with little scar. Similarly, B6 fetal liver cells transferred the nonhealing phenotype to the MRL with little cardiomyocyte growth and an acellular B6-like scar. These results are in contrast to the ear hole closure response which was of the recipient phenotype. We conclude that, in the case of the heart, fetal liver-derived stem cells regulate regenerative healing.

Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major

Experimental cutaneous leishmaniasis is a useful model in studying the mechanism regulating immune responses between T helper type 1 (Th1) and Th2. Mice susceptible to Leishmania major infection such as BALB/c (H-2 d) are associated with the induction of the disease-promoting Th2 response, while the resistant mice such as DBA/2 (H-2 d) develop the protective Th1 response. To understand the induction mechanism of Th1 and Th2 responses, it is necessary to establish an immunization scheme by which the induction of each Th response can be easily and experimentally controlled. Adjuvants are known to enhance the immune responses through the combined effect of several factors: prolonged release of antigen, migration of cells, mitogenic effect and so forth. When the genetically resistant DBA/2 mice were immunized twice with soluble leishmanial antigen (SLA), emulsified in incomplete Freund's adjuvant (IFA) before L. major inoculation, these mice mounted a Th2 cell response and suffered from progressive infection. While IL-4 and IL-13 were upregulated early after the infection in both healer and non-healer groups of mice, IL-5 and IL-10 were upregulated only in non-healer mice. From these results, IL-5 and IL-10 appear to have an important role, at least in the early phases of the infection, rather than IL-4 and IL-13 in establishing the disease-promoting Th2 response in leishmaniasis. Further, IL-9 was found to be expressed in both BALB/c and DBA/2 mice immunized with IFA/SLA. This cytokine may support the establishment of a Th2 response in these mice. Therefore it is suggested that Th2 cytokines play different roles between priming and maintaining the Th2 immune response after the infection.

Profiles of healing and nonhealing Cryptosporidium parvum infection in C57BL/6 mice with functional B and T lymphocytes: the extent of gamma interferon modulation determines the outcome of infection.

This study describes healing and nonhealing models of Cryptosporidium parvum infection with adult mice that have functional T and B lymphocytes. In our nonhealing model, mice on a C57BL/6 background which have a targeted disruption in the gamma interferon (IFN-gamma) gene (GKO mice) are utilized. C. parvum-infected GKO mice shed extremely high levels of oocysts and displayed overwhelming infection of the entire small intestine. The majority of these mice succumbed within 2 to 3 weeks due to severe acute infection and profound mucosal destruction. In our healing murine model, C57BL/6J mice treated with a single injection of the neutralizing anti-IFN-gamma monoclonal antibody XMG 1.2 prior to infection were used. These mice developed two peaks of oocyst shedding but were ultimately free of parasites on day 30 of infection. Again, the small intestine was the primary site of infection. Mesenteric lymph node (MLN) cells isolated from C. parvum-infected nonhealing GKO mice proliferated and secreted interleukin 2 (IL-2) but not IFN-gamma or IL-4 in response to ex vivo restimulation with intact C. parvum sporozoites or a C. parvum sporozoite antigen preparation. In contrast, parasite-specific MLN cells isolated from healing C57BL/6J mice secreted IL-2 and IFN-gamma but not IL-4. These results suggest that IFN-gamma, either directly or indirectly, is important for resistance to and resolution of cryptosporidiosis. Moreover, these models now allow the analysis of parasite-specific cell-mediated and humoral mucosal immune responses to determine what constitutes protective immunity to C. parvum.

TGF-beta is important in determining the in vivo patterns of susceptibility or resistance in mice infected with Candida albicans.

Resistance and susceptibility of mice to systemic infection with the fungus Candida albicans are associated with the preferential expansion of Th1 and Th2 cells, respectively. In this study, endogenous production of TGF-beta was found to be increased soon after infection of healer mice with a live vaccine strain of the fungus, but down-regulated in nonhealer mice with virulent yeast challenge. Although not affecting the outcome of primary challenge, serologic ablation of TGF-beta in the former animals abrogated development of acquired resistance and resulted in impaired production of IL-12/IFN-gamma and higher expression of IL-4/IL-10 at the time of reinfection with virulent yeast. A CD4+ population expressing the memory phenotype, CD44highMEL-14low, which appeared to be expanded by yeast infection of nonhealer mice, was similarly increased in the healer mice by anti-TGF-beta treatment. In vitro rTGF-beta impaired the candidacidal function of IFN-gamma-activated macrophages. Yet in nonhealer mice infected with virulent C. albicans, administration of rTGF-beta delayed progression of the disease, which was concomitant with the detection of lower levels of IL-4. In addition to previous evidence for an obligatory role of IFN-gamma and IL-12 in Candida-driven Th1 cell differentiation in vivo, the present data establish TGF-beta as a third cytokine, the presence of which may be required for optimal Th1 development leading to long-lived anticandidal resistance.

Interleukin‐4 and ‐10 exacerbate candidiasis in mice

Neutralization of endogenous interleukin (IL)-4 or IL-10 in mice with Candida albicans infection initiates or accelerates development of a T helper (Th)1-associated protective response. Here, we report the effect of IL-4 and IL-10 administration on the course of systemic or gastrointestinal (GI) candidiasis and on the development of Th immunity using yeast/host combinations that result either in Th1-associated self-limiting infection (healer mice) or in Th2-associated progressive disease (nonhealer mice). Treatment with IL-4 or IL-10 greatly exacerbated the course of systemic infection in nonhealer mice and rendered healer mice, inoculated with attenuated yeast cells, susceptible to infection. Under the latter conditions of yeast challenge and IL-4/IL-10 administration, the development of a fatal disease was associated with inhibition of IL-12 production and detection of progressive Th2 cell dominance. In contrast, in healer mice allowed to resolve their infections and to develop long-lived anti-candidal resistance, the expression of this acquired resistance was not impaired by IL-4 and/or IL-10, as shown by the outcome of reinfection with virulent yeast cells. In the GI model of infection, both IL-4 and IL-10 were found to exacerbate the course of infection and to induce the appearance of CD4+ T cells producing high levels of IL-4 and IL-10 in Peyer's patches. These findings demonstrate that exogenous IL-4 and IL-10 may greatly affect the development of Th responses to C. albicans in vivo, but do not modify the expression of established and predominant Th1 cell reactivity.

Site-specific immunity to Leishmania major in SWR mice: the site of infection influences susceptibility and expression of the antileishmanial immune response

Inbred strains of mice usually develop either of two divergent patterns of infection in response to Leishmania major. Resistant mice, which develop self-limiting infections, respond immunologically with the activation of gamma interferon-secreting Th1 helper T cells, while nonhealing infections in susceptible mice are characterized by the proliferation of interleukin-4-secreting Th2 cells. Development of these divergent responses is dependent primarily on the strain of mouse infected, although factors such as the infective dose, species, and strain of parasite can also influence the degree of resistance. In this study, we show that a single mouse strain, SWR, can develop totally divergent patterns of infection depending on the site of parasite inoculation. Both SWR mice and highly susceptible BALB/c mice developed progressive, ultimately fatal disease when inoculated in the dorsal skin over the base of the tail. However, SWR mice infected in the hind footpad developed far less severe infections, which were for the most part controlled, whereas BALB/c mice infected in this site developed severe, nonhealing lesions. Production of gamma interferon and interleukin-4 and measurement of immunoglobulin E levels in serum were used to assess the degree of Th1 and Th2 cell activation in infected mice. Cytokine profiles early in infection had characteristics of a mixed Th1-Th2 response and were similar in SWR mice infected at either site. These early cytokine responses were not predictive of the ultimate disease outcome, since lymph node cells from healing mice eventually produced higher levels of gamma interferon than did those from nonhealing mice, and healing mice had lower levels of immunoglobulin E in serum, suggesting a functional bias toward Th1 cell activity in these animals. The differential ability of SWR mice to heal infections at different cutaneous sites provides a new model for the study of resistance to cutaneous leishmaniasis. Unlike traditional models of infection in which resistant and susceptible strains of mice are compared, this model allows for the study of factors that contribute to healing and nonhealing infections in a genetically identical strain of mouse.

Interleukin-12 but not interferon-gamma production correlates with induction of T helper type-1 phenotype in murine candidiasis.

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-gamma (IFN-gamma), IL-4, and IL-10 in CD4+ and CD8+ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection ("healer mice") or in a Th2-associated progressive disease ("nonhealer mice"). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-gamma or IL-10 mRNA in CD4+ and CD8+ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4+ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4+ and CD8+ cells was assessed in vitro, while IFN-gamma-producing cells were enumerated in CD4- CD8- cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-gamma protein in vitro by CD4+ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4+ cells would expand in nonhealer mice in the face of high levels of circulating IFN-gamma, likely released by CD4- CD8- lymphocytes; (iii) a finely regulated IFN-gamma production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-gamma-producing CD4+ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-gamma by early CD4+ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-gamma production may be an indicator of Th1 differentiation.

Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets.

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.

Macrophage activation in vitro by lymphocytes from Leishmania major infected healer and non-healer mice.

Peritoneal macrophages from CBA/T6 (healer) and BALB/c (non-healer) mice were infected with Leishmania major (LV39) in vitro. The microorganism replicated at the same rate in macrophages from either strain. Exposure of infected cells to lymph node cells (LNC) from infected syngeneic animals led to intracellular killing of the parasite by macrophages from both strains, provided LPS was present in the incubation medium. In vitro-propagated L.major-specific T-cell blasts activated macrophages from either strain in the absence of LPS. On a per cell basis, lymphoid cells from BALB/c mice were less efficient, however, than cells from CBA/T6 mice. Lysis of parasitized macrophages was also more marked in CBA/T6 than in BALB/c cell mixtures. LNC exposed to parasite antigen or to infected macrophages secreted macrophage-activating factor (MAF); incubation with antigen also induced lymphocyte proliferation. MAF production and LNC proliferation decreased with progression of the infection of BALB/c mice, but always remained significant. The reduction in relative T-cell numbers in the lymph nodes of infected animals was moderate; the absolute number of T-cells increased markedly in the lymphoid organs of both strains, however. These results suggest that failure to heal may coexist together with active cell-mediated immune response in non-healer mice.

Murine cutaneous leishmaniasis : Susceptibility correlates with differential expansion of helper T-cell subsets

BALB/c mice develop fatal illness following infection with Leishmania major despite expansion of helper L3T4+ T cells in the draining lymph nodes and spleen. Healer mice, either genetically resistant C57BL/6 or BALB/c that have been pretreated with monoclonal antibody GK 1.5, also develop expanded numbers of L3T4+ T cells at the time of healing. Lymph node cells from healer mice produce gamma-interferon in vitro and message for gamma-interferon can be recovered from the lymph nodes during healing in vivo. Conversely, cells harvested from non-healer mice during the course of infection produce minimal gamma-interferon in vitro and have little message for gamma-interferon detectable in vivo. When the same Northern blots are hybridized for IL-4, large amounts of IL-4 message are detected only in cells from non-healer mice. The data are consistent with the expansion of type 1 helper cells (Th1) during healing and type 2 helper cells (Th2) during progressive leishmania infection.

Leishmania tropica: Pathogenicity and in vitro macrophage function in strains of inbred mice

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10 6 active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C 3H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu +). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.