Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1ß, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Myeloid-Restricted AMPKα1 Promotes Host Immunity and Protects against IL-12/23p40-Dependent Lung Injury during Hookworm Infection.

How the metabolic demand of parasitism affects immune-mediated resistance is poorly understood. Immunity against parasitic helminths requires M2 cells and IL-13, secreted by CD4(+) Th2 and group 2 innate lymphoid cells (ILC2), but whether certain metabolic enzymes control disease outcome has not been addressed. This study demonstrates that AMP-activated protein kinase (AMPK), a key driver of cellular energy, regulates type 2 immunity and restricts lung injury following hookworm infection. Mice with a selective deficiency in the AMPK catalytic α1 subunit in alveolar macrophages and conventional dendritic cells produced less IL-13 and CCL17 and had impaired expansion of ILC2 in damaged lung tissue compared with wild-type controls. Defective type 2 responses were marked by increased intestinal worm burdens, exacerbated lung injury, and increased production of IL-12/23p40, which, when neutralized, restored IL-13 production and improved lung recovery. Taken together, these data indicate that defective AMPK activity in myeloid cells negatively impacts type 2 responses through increased IL-12/23p40 production. These data support an emerging concept that myeloid cells and ILC2 can coordinately regulate tissue damage at mucosal sites through mechanisms dependent on metabolic enzyme function.

In Ivorian school-age children, infection with hookworm does not reduce dietary iron absorption or systemic iron utilization, whereas afebrile Plasmodium falciparum infection reduces iron absorption by half.

BACKGROUND: In sub-Saharan Africa, parasitic diseases and low bioavailable iron intake are major causes of anemia. Anemia results from inflammation, preventing iron recycling and decreasing dietary iron absorption. Hookworm, Plasmodium, and Schistosoma infections contribute to anemia, but their influence on dietary iron absorption and recycling is unknown. OBJECTIVE: The objective was to measure inflammation biomarkers, hepcidin, iron absorption, and utilization pre- and posttreatment in children with afebrile malaria, hookworm, and Schistosoma haematobium infection. DESIGN: Ivorian children aged 11-17 y with afebrile Plasmodium falciparum (n = 17), hookworm (n = 16), or S. haematobium infection (n = 8) consumed a syrup containing 3 mg 57Fe as ferrous sulfate and received an intravenous infusion of 50 µg 58Fe as ferrous citrate. Children were treated for their respective infection, and the iron studies were repeated 4 wk later. Iron and inflammation biomarkers and hepcidin were measured. RESULTS: Geometric mean iron absorptions in the afebrile malaria and hookworm groups were 12.9% and 32.2% (P < 0.001) before treatment and 23.6% and 30.0% (P = 0.113) after treatment, respectively. Treatment of afebrile malaria reduced inflammation (P < 0.001) and serum hepcidin (P = 0.004) and improved iron absorption (P = 0.003). Treatment of hookworm infection neither affected inflammation biomarkers nor altered iron absorption. Similarly, there was a lack of treatment effects in the S. haematobium-infected group; however, the small sample size limits conclusions. Geometric mean iron utilization ranged between 79.1% and 88.0% in the afebrile malaria and hookworm groups with no significant differences pre- and posttreatment. CONCLUSIONS: In school-age children, hookworm infection does not produce inflammation or increase serum hepcidin, and it does not influence iron absorption or utilization. In contrast, afebrile malaria causes inflammation, increases hepcidin, and reduces iron absorption but not utilization. These findings provide insights into the iron metabolism and the etiology of anemia in parasitic infections.

Age-Stratified Profiles of Serum IL-6, IL-10, and TNF-α Cytokines Among Kenyan Children with Schistosoma haematobium, Plasmodium falciparum, and Other Chronic Parasitic Co-Infections.

In a study of children having polyparasitic infections in a Schistosoma haematobium-endemic area, we examined the hypothesis that S. haematobium-positive children, compared with S. haematobium-negative children (anti-soluble worm antigen preparation [SWAP] negative and egg negative) have increased systemic production of pro-inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α) and decreased down-regulatory IL-10. A total of 804 children, 2-19 years of age, were surveyed between July and December 2009 and tested for S. haematobium, Plasmodium falciparum, filariasis, and soil-transmitted helminth infections. Plasma levels of IL-6, TNF-α, and IL-10 were compared for S. haematobium-positive and S. haematobium-negative children, adjusting for malaria, filaria, and hookworm co-infections, and for nutritional status, age group, sex, and geographic location. IL-10 was significantly elevated among children infected with S. haematobium, showing bimodal peaks in 7-8 and 13-14 years age groups. IL-10 was also higher among children who were acutely malnourished, whereas IL-10 levels were lower in the presence of S. haematobium-filaria co-infection. After adjustment for co-factors, IL-6 was significantly elevated among children of 5-6 years and among those with P. falciparum infection. Lower levels of IL-6 were found in malaria-hookworm co-infection. High levels of TNF-α were found in children aged 11-12 years regardless of infection status. In addition, village of residence was a strong predictor of IL-6 and IL-10 plasma levels. In adolescent children infected with S. haematobium, there is an associated elevation in circulating IL-10 that may reduce the risk of later morbidity. Although we did not find a direct link between S. haematobium infection and circulating pro-inflammatory IL-6 and TNF-α levels, future T-cell stimulation studies may provide more conclusive linkages between infection and cytokine responses in settings that are endemic for multiple parasites and multiple co-infections.

Suppression of basophil histamine release and other IgE-dependent responses in childhood Schistosoma mansoni/hookworm coinfection.

BACKGROUND: The poor correlation between allergen-specific immunoglobulin E (asIgE) and clinical signs of allergy in helminth infected populations suggests that helminth infections could protect against allergy by uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan schoolchildren coinfected with Schistosoma mansoni and hookworm. METHODS: Skin prick test (SPT) sensitivity to house dust mite allergen (HDM) and current wheeze were assessed pre-anthelmintic treatment. Nonspecific (anti-IgE), helminth-specific, and HDM-allergen-specific basophil histamine release (HR), plus helminth- and HDM-specific IgE and IgG4 responses were measured pre- and post-treatment. RESULTS: Nonspecific- and helminth-specific-HR, and associations between helminth-specific IgE and helminth-specific HR increased post-treatment. Hookworm infection appeared to modify the relationship between circulating levels of HDM-IgE and HR: a significant positive association was observed among children without detectable hookworm infection, but no association was observed among infected children. In addition, hookworm infection was associated with a significantly reduced risk of wheeze, and IgG4 to somatic adult hookworm antigen with a reduced risk of HDM-SPT sensitivity. There was no evidence for S. mansoni infection having a similar suppressive effect on HDM-HR or symptoms of allergy. CONCLUSIONS: Basophil responsiveness appears suppressed during chronic helminth infection; at least in hookworm infection, this suppression may protect against allergy.

Effects of treatment on IgE responses against parasite allergen-like proteins and immunity to reinfection in childhood schistosome and hookworm coinfections.

Naturally occurring human immunity to both schistosomiasis and hookworm infection has been associated with IgE responses against parasite allergen-like proteins. Since the two helminths frequently coinfect the same individuals, there is growing advocacy for their concurrent treatment. However, both helminths are known to exert strong immunomodulatory effects; therefore, coinfected individuals could have immune responses different from those characteristically seen in monoinfected individuals. In this study, we measured changes in IgE, IgG1, and IgG4 responses to schistosome and hookworm antigens, including the allergen-like proteins Schistosoma mansoni tegumental-allergen-like 1 protein (SmTAL1), SmTAL2, and Necator americanus Ancylostoma-secreted protein-2 (Na-ASP-2), following concurrent treatment of schoolchildren coinfected with Schistosoma mansoni and hookworm. Antibody responses to schistosome egg (soluble egg antigen and SmTAL2) or somatic adult hookworm (AHW) antigens either decreased after treatment or were unchanged, whereas those to schistosome worm antigens (soluble worm antigen and SmTAL1) increased. The observed different effects of treatment likely reflect the different modes of drug action and sites of infection for these two helminths. Importantly, there was no evidence that the simultaneous treatment of coinfected children with praziquantel and albendazole affected schistosome- and hookworm-specific humoral responses differently from those characteristic of populations in which only one organism is endemic; schistosome- and hookworm-specific responses were not associated, and there was no evidence for cross-regulation. Posttreatment increases in the levels of IgE to schistosome worm antigens were associated with lower Schistosoma mansoni reinfection intensity, while no associations between humoral responses to AHW antigen and protection from hookworm reinfection were observed in this sample of school-aged children.

TGF-ß-responsive myeloid cells suppress type 2 immunity and emphysematous pathology after hookworm infection.

Transforming growth factor ß (TGF-ß) regulates inflammation, immunosuppression, and wound-healing cascades, but it remains unclear whether any of these functions involve regulation of myeloid cell function. The present study demonstrates that selective deletion of TGF-ßRII expression in myeloid phagocytes i) impairs macrophage-mediated suppressor activity, ii) increases baseline mRNA expression of proinflammatory chemokines/cytokines in the lung, and iii) enhances type 2 immunity against the hookworm parasite Nippostrongylus brasiliensis. Strikingly, TGF-ß-responsive myeloid cells promote repair of hookworm-damaged lung tissue, because LysM(Cre)TGF-ßRII(flox/flox) mice develop emphysema more rapidly than wild-type littermate controls. Emphysematous pathology in LysM(Cre)TGF-ßRII(flox/flox) mice is characterized by excessive matrix metalloprotease (MMP) activity, reduced lung elasticity, increased total lung capacity, and dysregulated respiration. Thus, TGF-ß effects on myeloid cells suppress helminth immunity as a consequence of restoring lung function after infection.

Trefoil factor 2 rapidly induces interleukin 33 to promote type 2 immunity during allergic asthma and hookworm infection.

The molecular mechanisms that drive mucosal T helper type 2 (T(H)2) responses against parasitic helminths and allergens remain unclear. In this study, we demonstrate in mice that TFF2 (trefoil factor 2), an epithelial cell-derived repair molecule, is needed for the control of lung injury caused by the hookworm parasite Nippostrongylus brasiliensis and for type 2 immunity after infection. TFF2 is also necessary for the rapid production of IL-33, a T(H)2-promoting cytokine, by lung epithelia, alveolar macrophages, and inflammatory dendritic cells in infected mice. TFF2 also increases the severity of allergic lung disease caused by house dust mite antigens or IL-13. Moreover, TFF2 messenger RNA expression is significantly increased in nasal mucosal brushings during asthma exacerbations in children. These experiments extend the biological functions of TFF2 from tissue repair to the initiation and maintenance of mucosal T(H)2 responses.

Exposure to hookworms in patients with Crohn's disease: a case-control study.

BACKGROUND: Helminths have been used to inhibit intestinal inflammation in patients with Crohn's disease. AIM: This study was undertaken to determine if there is a protective association of prior hookworm infection with Crohn's disease, in a region where there is epidemiological transition from parasitic and infectious diseases to increased auto-inflammatory diseases. METHODS: Hookworm exposure was assessed by peripheral blood mononuclear cell (PBMC) activation by hookworm antigens in 78 patients with Crohn's disease and 75 healthy control participants. The change in proportion of T cells exhibiting CD69 after exposure to crude hookworm antigens was measured. Interferon-γ ELISPOT response to a panel of six recombinant hookworm antigens was analysed. RESULTS: Patients with Crohn's disease were more often from an urban background (P=0.005) compared to controls, while their socioeconomic status was not significantly different. T cell activation (increase in CD3(+) CD69(+) population) by hookworm antigen was significantly higher in controls compared to Crohn's disease patients (P=0.017), while activation by the nonspecific mitogen phytohemagglutinin was similar in both groups. Circulating T memory cells (CD3(+) CD45RO(+)) after exposure to hookworm antigens were not significantly different between the two groups. Mirroring these changes, interferon-γ ELISPOT responses to hookworm antigens were seen in 36 of 75 controls compared to 20 of 78 Crohn's disease patients (Fisher's exact P=0.005). Multivariate analysis indicated that CD3CD69 shifts (P=0.019), ELISPOT reactivity (P=0.039) and place of residence (P=0.024) were all independently associated with Crohn's disease. CONCLUSION: The inverse association between Crohn's disease and hookworm antigen reactivity is consistent with the hygiene hypothesis, but requires further exploration.

Basophils protect against reinfection with hookworms independently of mast cells and memory Th2 cells.

Hookworms infect several hundred million people worldwide, causing malnutrition, anemia, and growth retardation. Infections generally result in a strong type 2 immune response, but the effector mechanisms that mediate worm expulsion remain poorly characterized. In this study, we determined the role of mast cells and basophils in protective immunity against the murine hookworm, Nippostrongylus Brasiliensis, during primary and secondary infection. Mast cell-deficient c-Kit(W-sh) mice had lower serum IgE levels compared with wild-type mice under steady-state conditions and after N. brasiliensis infection. Worm expulsion was delayed during primary but not during secondary infection of c-Kit(W-sh) mice, even in the absence of CD4 T cells. However, protective immunity was lost when basophils were depleted before reinfection of c-Kit(W-sh) mice. We conclude that basophils play a crucial role for worm expulsion during a memory type 2 immune response independently of mast cells and memory Th2 cells.

Malaria, hookworms and recent fever are related to anemia and iron status indicators in 0- to 5-y old Zanzibari children and these relationships change with age.

In Zanzibar and other tropical regions, iron deficiency, malaria and multiple helminth infections coexist. We addressed the following questions: 1) What are the predictors of low hemoglobin in Zanzibari preschool children? 2) Are indicators of iron status informative in this population? 3) Does malaria modify the relation of iron indicators to hemoglobin? We used multivariate regression to analyze cross-sectional data from a community-based sample of rural Zanzibari children who were not ill (n = 490; 4-71 mo of age) in whom we assessed hemoglobin, serum ferritin (SF), erythrocyte protoporphyrin (EP), serum transferrin receptor (TfR), recent fever, malaria parasitemia and helminth fecal egg counts. Of hemoglobin values, 80% were <100 g/L and 15.5% were <70 g/L. In children <18 mo of age, 40.2% of hemoglobin values were <70 g/L. Our primary findings were as follows: 1) In children <30 mo old, hemoglobin was associated with malaria but not hookworms, whereas in children >/=30 mo, hemoglobin was related to hookworms but not malaria. In the younger age group, male sex and recent fever also predicted lower hemoglobin. 2) The three iron indicators were informative in this population but did not reflect only iron status. Malaria elevated SF in younger children and TfR and EP in both age groups. Fever elevated SF in older children and EP in both age groups, but not TfR. 3) Malaria modified the relation of all three indicators to hemoglobin. The relation of SF to hemoglobin was weak overall, and absent in malaria-infected children. EP and TfR were strongly related to hemoglobin, but this relation was attenuated by malaria.

Measurement of hookworm infection intensity and circulating levels of IgE and autoantibodies to IgE in atopics and nonatopics living in a parasitized community in Papua New Guinea.

We have compared hookworm infection intensity, as determined by fecal egg count, and circulating levels of IgE and autoantibodies to IgE in atopic and nonatopic parasitized (predominantly hookworm-infected) patients from Kebasob village on Karkar Island, Papua New Guinea. Our study has clearly established that parasitized atopic individuals have significantly higher levels of IgE and autoanti-IgE than their nonatopic counterparts, and that atopy does not appear to influence accumulated levels of hookworm infection. These data, therefore, do not support an earlier report that suggested that the atopic state may confer increased resistance to hookworm infestation in a parasitized community in Papua New Guinea.

Role of specific immunoglobulin E to excretory-secretory antigen in diagnosis and prognosis of hookworm infection.

Total immunoglobulin E (IgE) and specific IgE were assayed by a radioimmunosorbent test and a reverse enzyme immunoassay in patients with hookworm infections before and after treatment. A total of 77 subjects (30 patients with hookworm infections and 47 subjects as controls) were studied. Both specific and total IgE levels in serum and jejunal juice were raised in hookworm patients. There was a significant decrease in IgE levels after therapy. Total IgE levels were raised in other nematode infections, but specific IgE levels were low. The reverse enzyme immunoassay for specific IgE was highly specific (96%) and sensitive (100%) and may be used in the serodiagnosis of hookworm infections.

In vivo kinetics and nature of rat IgE-bearing lymphocytes after IgE stimulation.

Surface IgE-bearing (sIgE+) cells were studied in BN and rnu/rnu athymic rats after Nippostrongylus brasiliensis infection or i.p. injection of unpurified or purified IgE from plasmacytoma ascitic fluid. The number of sIgE+ cells increased markedly after a serum IgE increase without change in the proportion of sIgM+ and sIgD+ cells. A high percentage of the sIgE+ cells bore cytophilic IgE. Receptors for IgE were induced with a 2.56-micrograms IgE injection/100 g body weight and reached a maximum with 1.6 mg IgE/100 g body weight. They appeared less than 4 hr after a single injection of purified IgE. The number of IgE receptor-bearing cells reached a maximum plateau at 24 hr to day 3 after injection and declined thereafter, to reach the control level on day 9 or 11 after injection. Nearly all the sIgE+ cells of BN rats also bore sIgD, but the number of triple sIgE-sIgM-sIgD+ cells varied in a wide range. Maximum 4.5% of the sIgE+ cells of euthymic rats were T cells. More than 98% of the sIgE+ cells of nude rats were triple sIgM-sIgD-sIgE+ cells, and the majority were cytophilic IgE+. For the most part, the sIgM-sIgD-sIgE+ cells are probably not cells that can differentiate, as generally accepted, in IgE-producing cells. New interpretations of the role of these triple sIgM-sIgD-sIgE+ cells in IgE immune responses are necessary.

Increased expression of the IgE Fc receptors on rat macrophages induced by elevated serum IgE levels.

Macrophages (M phi) from rats with elevated serum IgE levels induced by (i) Nippostrongylus brasiliensis (Nb) infection, (ii) IgE-secreting plasmacytoma IR 162, or (iii) i.p. injection of purified rat IgE, and M phi from normal animals cultured in the presence of 10 micrograms/ml IgE were analysed for Fc IgE receptors (Fc epsilon R) expression. To detect Fc epsilon R-bearing cells, a rosette assay employing fixed ox erythrocytes coated with rat IgE was used. With undersensitized indicator cells a significantly (P less than 0.002) greater number of M phi from animals having elevated serum IgE levels or of M phi cultured in the presence of IgE formed IgE rosettes than M phi from normal donors. The IgE rosettes were IgE class-specific, since they were inhibited by rat IgE in a dose-dependent manner, but not by any other rat Ig class, heat-denatured rat IgE or human IgE. The modulating effect of Fc epsilon R expression on M phi was IgE specific, because neither rat IgG nor heated rat IgE induced increased IgE rosette formation. Furthermore, elevated serum IgE levels did not increase the expression of Fc receptors for IgG subclasses. Studies of 125I-IgE binding showed that alveolar macrophages (AM phi) from Nb-infected rats bind IgE with similar affinity (Ka 1.1 X 10(7) M-1) as AM phi from normal animals, but they have increased numbers of IgE binding sites. Collectively, the results demonstrate that in vivo and in vitro elevated serum IgE concentrations induce increased IgE rosette formation as a result of a marked increase in the number of Fc epsilon R per macrophage.

Gut mucosal mast cells. Origin, traffic, and differentiation.

Gut mucosal mast cells (MMC), which are nearly absent in normal mice are abundant during nematode infection. In normal mice, study of MMC precursors (MMC-P: cells giving rise to MMC colonies in the presence of IL-3) show that: (a) their frequency, judged by limiting dilution is very high in bone marrow (BM) and gut, and very low in most lymphoid organs and thoracic duct lymph (TDL); (b) gut MMC-P are Thy-1- Lyt-1-2- and are not rapidly replicating; (c) they are the progeny of less differentiated BM MMC-P which are attracted from the blood to the gut mucosa by local factor(s), other than antigen and T cell factors (since normal amounts of gut MMC-P are found in germ-free, nude, and newborn mice). In mice bearing the Wehi 3 tumor (which releases enough IL-3 to produce detectable blood levels) spleen and mesenteric lymph nodes (LN) show increased MMC-P frequency, the greatest increase being in the gut and BM, where numerous differentiated MMC are found. In Nippostrongylus brasiliensis (Nb)-infested mice (known to develop a large, T cell-dependent, gut MMC infiltration), gut MMC-P proliferation is induced by IL-3 released from gut mucosal Thy-1+ Lyt-2- cells, whose in vitro IL-3 release capability is much higher than that of similar cells from normal mice. Both Nb-stimulated T blasts and proliferating MMC-P undergo cyclic traffic, migrating into the TDL and then seeding the whole length of the gut (a process which allows a widespread immune defense after a local antigenic stimulus). Experiments using 2-d interruption of this traffic and fetal gut grafts, suggest that the continuous homing of T blasts back to the gut which leads to permanent Nb-stimulated IL-3 release, is essential for the full maturation of MMC. Transfer experiments in the rat show that TDL circulating MMC-P rapidly mature into MMC when they home back to the Nb-infested gut. It is proposed that gut MMC arise after several stages of progressive differentiation of MMC-P, influenced both by IL-3 and unidentified gut factor(s).

Immunomodulation by nematodes: a review.

There is current evidence that infections with Trichinella spiralis, Ascaris suum, Nippostrongylus brasiliensis, Nematospiroides dubius (syn. Heligmosomoides polygyrus) and diverse filariae affect the immune responsiveness of their hosts. T. spiralis, or its extracts, can depress or enhance the heterologous humoral or cell-mediated immunities, and affect macrophage activity or the response to other invaders. These effects are induced by products of the migratory and early muscle larvae and appear to obey more than one single-mechanism. A suum acute infections or extracts depress responses involving T cell activity, but stimulate polygonal expansion of B-cells. Nippostrongylus brasiliensis causes polyclonal stimulation of IgE-producing cells, enhances immune responses during the first week of infection and inhibits them later on. Nematospiroides dubius depresses homologous and heterologous immunity and facilitates the permanence of other intestinal nematodes. Filarial worms appear to depress the homologous cell-mediated immunity and the heterologous humoral response by induction of suppressor cells and humoral factors. These phenomena are probably the result of evolutionary pressures on the parasites that facilitate their survival. In the host, they are likely to aggravate the homologous infection, facilitate intercurrent conditions and interfere with immunoprophylaxis procedures.

Bone marrow origin of mucosal mast cells.

Infection with the intestinal parasite Nippostrongylus brasiliensis stimulates an accumulation of mucosal mast cells (MMC) in the villi of the small intestine of normal but not athymic or W/Wv anemic mice. W/Wv mice are congenitally deficient in both MMC and skin and connective tissue mast cells (CTMC). Athymic mice have normal or elevated numbers of CTMC but are severely deficient in MMC. CTMC derive from the bone marrow. To determine the origin of MMC, athymic and W/Wv mice were given various hematopoietic or lymphoid tissues from normal littermate or beige mice and the MMC response to N. brasiliensis infection was evaluated. The MMC defect in athymic mice was repaired by grafts of thymus cells, thymus gland, or spleen cells, but not by bone marrow cells or anti-Thy 1-treated bone marrow or spleen cells. The MMC and CTMC defects of W/Wv mice were repaired by grafts of bone marrow, spleen cells, or anti-Thy 1-treated bone marrow or spleen cells. Neither the MMC nor the CTMC defect in W/Wv mice was repaired by grafts of thymus cells or thymus glands. These results indicate the following, MMC, like CTMC, derive from the bone marrow and not from the thymus. MMC require a thymic influence for development. Athymic mice possess bone marrow precursors for both MMC and CTMC but lack a thymus-dependent component necessary for MMC development. W/Wv mice lack both MMC and CTMC mast cell precursors but possess the thymus-dependent component required for MMC development.