In vitro antileishmanial activity and cytotoxicity of essential oil from Lippia sidoides Cham

Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. There is no vaccine available for Leishmania infections and conventional treatments are very toxic to the patients. Therefore, antileishmanial drugs are urgently needed. In this study we have analyzed the effects of essential oils from Lippia sidoides (LSEO) and its major compound thymol on the growth, viability and ultrastructure of Leishmania amazonensis. The essential oil and thymol showed significant activity against promastigote forms of L. amazonensis, with IC₅₀/48 h of 44.38 and 19.47 μg/mL respectively. However, thymol showed toxicity against peritoneal macrophages and low selectivity against the promastigotes when compared with the crude LSEO. On the other hand, no cytotoxic effect was observed in macrophages treated with the crude essential oil. Incubation of L. amazonensis-infected macrophages with LSEO showed a marked reduction in amastigote survival within the macrophages. Significant morphological alterations as accumulation of large lipid droplets in the cytoplasm, disrupted membrane and wrinkled cells were usually seen in treated parasites. The LSEO's activity against both promastigote and the amstigote forms of L. amazonensis, together with its low toxicity to mammalian cells, point to LSEO as a promising agent for the treatment of cutaneous leishmaniasis.     

Different Leishmania species determine distinct profiles of immune and histopathological responses in CBA mice

Most experimental studies on leishmaniasis compare two different inbred strains of mice that are resistant or susceptible to one species of Leishmania. In the present study we characterized some cytokines and nitric oxide production as well as histological changes related to resistance and susceptibility in isogenic CBA mice infected with Leishmania major or Leishmania amazonensis. CBA mice are capable of controlling infection with L.  major, but they succumb to infection with L. amazonensis. Cells from susceptible L. amazonensis-infected CBA mice produced interleukin (IL)-4 and IL-10 but no interferon (IFN)-γ. On the other hand, resistant L. major-infected CBA mice produced IFN-γ and IL-10, but IL-4 was detected only in the first week of infection. Histopathological studies showed patterns of tissue responses at the site of the infection and in the draining lymph nodes that correlated with resistance or susceptibility. Resistant mice showed a mixed inflammatory cell infiltration and granulomas in the lesions, whereas in susceptible mice only heavily parasitized macrophages were seen. Our results indicate an important role of the parasite species in determining the pattern of immune response. L. amazonensis induces a Th2-type immune response, whereas L.  major induces a Th1-type response. These factors must be identified and taken into account in the strategies for the development of vaccines against leishmaniasis. The model presented here will be useful for the study of such factors.     

Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance.     

Influence of long-term treatment with pravastatin on the survival, evolution of cutaneous lesion and weight of animals infected by Leishmania amazonensis

The high toxicity of current drugs for treatment of leishmaniasis is a major hindrance for controlling the disease. Pravastatin is a well-known drug with anti-inflammatory and immunomodulatory properties that may modulate host defense mechanisms against Leishmania. We evaluated the influence of prolonged pravastatin treatment on the survival of Leishmania amazonensis-infected animals (BALB/c, C57BL6 mice and Syrian hamsters), including weekly measurement of cutaneous lesions (footpad thickness) and weight. Pravastatin improved survival of Leishmania-infected BALB/c mice but not of infected C57BL6 mice or hamsters. On the 50th week of follow-up, 71% of pravastatin-treated Leishmania-infected BALB/c mice were alive against 29% of control group (p < 0.01). Low footpad thickness was found on BALB/c pravastatin treated mice from the 14th week (p < 0.05), and 20th week onward for C57BL6 treated mice. Pravastatin treatment decreased weight loss in Leishmania-infected C57BL6 mice and Syrian hamsters, but not infected BALB/c mice. Our results points to beneficial effects of pravastatin on the evolution of the disease in the murine leishmaniasis model.     

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.     

Modulation of P2X7 purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination

The purinergic P2X₇ receptor is a membrane protein of leucocytes involved in the clearance of intracellular bacteria such as Chlamydia and Mycobacterium. In this work, we investigated the role and modulation of macrophage P2X₇R in intracellular infection with the protozoan parasite Leishmania amazonensis. Upon infection, isolated murine macrophages displayed enhanced expression of P2X₇R and were significantly more responsive to extracellular ATP (ATPe)-induced pore opening, as demonstrated by the increased uptake of Lucifer Yellow. This was extended to the in vivo situation, where cells from established cutaneous lesions were more sensitive to ATPe than cells from uninfected mice. ATP treatment of infected macrophages inhibited parasite growth, and this was prevented by pre-treatment with oxidized ATP, a selective antagonist of P2X₇R. Parasite killing was unlikely due to induction of nitric oxide production or cytolysis of infected macrophage, as those functions were unaltered with parasite-effective ATPe concentrations. A direct drug effect is also unlike, as ATPe enhanced axenic parasite growth. We found that leishmanial infection rendered wild-type but not P2X₇R-deficient macrophages more prone to ATP-induced apoptosis. These results show that macrophage infection with L. amazonensis leads to enhanced expression of functional P2X₇R, that upon ligation with ATPe helps in the elimination of the parasites by an as yet unclear mechanism possibly involving host cell apoptosis.     

Effect of the Synadenium carinatum latex lectin (ScLL) on Leishmania (Leishmania) amazonensis infection in murine macrophages

Antiparasitic effect of a lectin isolated from Synadenium carinatum latex (ScLL) was evaluated against Leishmania (Leishmania) amazonensis promastigotes/amastigotes. Pretreatment of murine inflammatory peritoneal macrophages with ScLL reduced by 65.5% the association index of macrophages and L. (L) amazonensis promastigotes. Expression of cytokines (IL-12, IL-1 and TNF-α) was detected in infected macrophages pretreated with ScLL (10 μg/mL). ScLL also reduced the growth of L. (L) amazonensis amastigote intracellular forms, showing no in vitro cytotoxic effects in mammalian host cells. ScLL treatment in infected murine inflammatory peritoneal macrophages did not induce nitric oxide production, suggesting that a nitric oxide independent pathway is activated to decrease the number of intracellular Leishmania.     

A suitable model for the utilization of Duddingtonia flagrans fungus in small-flock-size sheep farms

Effective alternatives to anthelmintic treatment of nematode parasite infections of sheep are required because of the high prevalence of drug resistance. Within this context, the nematode-trapping fungus Duddingtonia flagrans has become a valuable component of various integrated control strategies. Toward this objective, a small quantity of lyophilized D. flagrans chlamydospores (10⁶ spores per animal) was administered to sheep in a one-year plot study. Animals grazing on native pasture were divided into two homogeneous groups and were kept in 1-ha paddocks in the southern region of Brazil. The oral administration of chlamydospores led to a significant reduction (p < 0.05) in the number of nematode eggs per gram of feces and in the larval availability on herbage (difference of 37.6%) in comparison to the control group. Control animals needed to be dewormed three times during the experiment, whereas the fungus-treated animals maintained a low parasite load, independent of seasonal variation. Although D. flagrans cannot serve as a panacea for nematode parasite control of livestock, it represents a significant advance toward rationalizing the use of endoparasitic drugs in small animals.     

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 μM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 μM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.     

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.     

Reactive oxygen species and imbalance of calcium homeostasis contributes to curcumin induced programmed cell death in Leishmania donovani

Curcumin, a polyphenol compound, has been recognized as a promising anti-cancer drug. The purpose of the present study was to investigate the cytotoxicity of curcumin to Leishmania donovani, the causative agent for visceral leishmaniasis. Flow cytometric analysis revealed that curcumin induced cell cycle arrest at G2/M phase. Incubation of Leishmania promastigotes with curcumin caused exposure of phosphatidylserine to the outer leaflet of plasma membrane. This event is preceded by curcumin-induced formation of reactive oxygen species (ROS) and elevation of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (ΔΨm), release of Cytochrome c into the cytosol and concomitant nuclear alterations that included deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) and DNA fragmentation. Taken together, these data indicate that curcumin has promising antileishmanial activity that is mediated by programmed cell death and, accordingly, merits further investigation as a therapeutic option for the treatment of leishmaniasis.     

Candida amazonensis FLP/FRT基因敲除系统的构建及初步验证

【目的】构建一个适用于Candida amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并通过敲除C.amazonensis的丙酮酸脱羧酶基因(Pyruvate decarboxylase,PDC)对该系统进行初步验证。【方法】以gfpm(绿色荧光蛋白基因)为报告基因,通过添加相应诱导剂评估Spathaspora passalidarum来源启动子(SpXYLp、SpMAL6p、SpMAL1p、SpGAL1p)和Saccharomyces cerevisiae来源Sc GAL1p启动子在C.amazonensis中的诱导调控性能。选择严格诱导型启动子调控FLP重组酶的表达,并在FLP表达盒和潮霉素(Hygromycin B)抗性标记基因(hphm)两端添加同向重复的FRT位点,以PDC基因作为靶基因构建敲除盒PRFg HRP,转化宿主菌C.amazonensis CBS 12363,筛选得到阳性转化子后,通过添加诱导剂,表达FLP重组酶,实现FRT位点间片段切除。【结果】诱导调控实验表明启动子SpGAL1p(受半乳糖诱导)和SpMAL1p(受麦芽糖诱导)是适用于C.amazonensis的严格诱导型启动子。以SpGAL1p调控FLP基因表达,构建的敲除盒PRFg HRP成功转化宿主菌,获得阳性转化子C.amazonensis PDC01,通过添加半乳糖诱导,成功切除基因组中FLP表达盒和抗性标记盒,获得突变株C.amazonensis PDC02。【结论】首次建立了一个适用于C.amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并利用该系统成功敲除了C.amazonensis内的PDC基因,为进一步利用代谢工程改造C.amazonensis酵母奠定了良好基础。     

Leishmania amazonensis: Partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage

Telomeres are protein–DNA complexes that protect chromosome ends from degradation and fusion. In Leishmania spp., telomeric DNA comprises a conserved TTAGGG repeat and is maintained by telomerase. Telomerase is a multisubunit enzymatic complex that ensures the complete DNA replication by adding new telomeric repeats to the G-rich strand. In this report we aimed to purify and study the biochemical properties of Leishmani amazonensis telomerase. In a first trial we used affinity chromatography with antisense 2′-O-methyl oligonucleotide without success since the Leishmania telomerase, similarly to Trypanosoma cruzi enzyme, was not eluted by competition, but instead, it remained bound to the column. Partially purified L. amazonensis telomerase activity was achieved by fractionation of extracts on complementary ion exchange and Heparin columns. Further purification of these fractions on a G-rich telomeric DNA affinity chromatography enriched for telomerase activity. The knowledge of telomerase characteristics in Leishmania could help to develop new strategies to overcome leishmaniasis.     

UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

Pre-clinical validation of a vaginal cream containing copaiba oil (reproductive toxicology study)

The aims of this study was to evaluate the effects of oil-resin of Copaiba (Copaifera duckei Dwyer), aired in vaginal cream on the reproductive performance of female rats (Rattus norvegicus). To determine the components of the C. duckei oleoresin, gas chromatography coupled with mass spectrometry (CG-MS) was used, and considering the trans-caryophyllene sesquiterpene as a phytochemical marker in the oleoresin. Due to the extensive use of copaiba oleoresin in the suppository form for gynecological infections, an evaluation was carried out on the effects of copaiba oleoresin (Copaifera duckei Dwyer), delivered in a vaginal cream, on the reproductive performance of female Wistar rats. For this purpose, three groups (n = 5-6/group) of female rats were treated as follows: 1 - vaginal cream of copaiba oleoresin (28.6 mg/kg), 2 - base vaginal cream and 3 - control (physiological saline 0.9%), administered intravaginally, for 30 days before pregnancy, and from day zero to day 20 during pregnancy. Laparotomy was performed on the 21st day of pregnancy, followed by the determination of reproductive variables: number of live and dead fetuses, mass of the fetuses and placentas, number of implantations and resorptions, number of corpora lutea, pre- and post-implantation loss, and analyses of the fetuses with regard to external and internal anomalies and/or malformations (skeletal and visceral). The trans-caryophyllene present in the sample is suggested as a phytochemical marker and the results of this study demonstrate an absence of maternal toxicity and foetotoxicity embryofoetotoxicity at the dose administered, corresponding to ten times the recommended dose for use in humans. Accordingly, no significant statistical difference was observed between the treated and control groups, for the variables analyzed.Thus, it is concluded that the vaginal cream containing 2.5% copaiba oleoresin is safe during gestation, in female rats (Rattus norvegicus) of the Wistar strain.     

In vivo induction of the autophagic machinery in human bone marrow cells during Leishmania donovani complex infection

Autophagy is a homeostatic process promoting cell survival in periods of stress. The induction of the autophagic machinery has also been implicated in both innate and adaptive immunity. Leishmania donovani, which is the causative pathogen of visceral leishmaniasis, is an intracellular parasite that invades and multiplies in bone marrow macrophages. We describe the induction of host cell autophagic machinery during acute natural bone marrow infection by L. donovani complex, detected by LC3B immunoblot. The successful treatment with liposomal amphotericin B resulted in the resolution of this phenomenon. Even though the role of autophagy in parasite biology has been previously studied, our findings show for the first time the in vivο host cell LC3B conversion as a marker of the induction of the autophagic machinery during infection with Leishmania parasite in real time conditions.     

Tamoxifen is effective in the treatment of Leishmania amazonensis infections in mice

Background

Chemotherapy is still a critical issue in the management of leishmaniasis. Until recently, pentavalent antimonials, amphotericin B or pentamidine compounded the classical arsenal of treatment. All these drugs are toxic and have to be administered by the parenteral route. Tamoxifen has been used as an antiestrogen in the treatment and prevention of breast cancer for many years. Its safety and pharmacological profiles are well established in humans. We have shown that tamoxifen is active as an antileishmanial compound in vitro, and in this paper we analyzed the efficacy of tamoxifen for the treatment of mice infected with Leishmania amazonensis, an etiological agent of localized cutaneous leishmaniasis and the main cause of diffuse cutaneous leishmaniasis in South America.

Methodology/Principal Findings

BALB/c mice were infected with L. amazonensis promastigotes. Five weeks post-infection, treatment with 15 daily intraperitoneal injections of 20 mg/kg tamoxifen was administered. Lesion and ulcer sizes were recorded and parasite burden quantified by limiting dilution. A significant decrease in lesion size and ulcer development was noted in mice treated with tamoxifen as compared to control untreated animals. Parasite burden in the inoculation site at the end of treatment was reduced from 10^8.5±0.7 in control untreated animals to 10^5.0±0.0 in tamoxifen-treated mice. Parasite load was also reduced in the draining lymph nodes. The reduction in parasite number was sustained: 6 weeks after the end of treatment, 10^15.5±0.5 parasites were quantified from untreated animals, as opposed to 10^5.1±0.1 parasites detected in treated mice.

Conclusions/Significance

Treatment of BALB/c mice infected with L. amazonensis for 15 days with tamoxifen resulted in significant decrease in lesion size and parasite burden. BALB/c mice infected with L. amazonensis represents a model of extreme susceptibility, and the striking and sustained reduction in the number of parasites in treated animals supports the proposal of further testing of this drug in other models of leishmaniasis.     

Saliva of laboratory-reared Lutzomyia longipalpis exacerbates Leishmania (Leishmania) amazonensis infection more potently than saliva of wild-caught Lutzomyia longipalpis

In order to compare the saliva effect from wild-caught and lab-reared L. longipalpis on the development of experimental cutaneous leishmaniasis, C57BL/6 mice were inoculated subcutaneously into the hind footpads with promastigotes of L. (L.) amazonensis plus salivary gland lysate from wild-caught (SGL-W) and lab-colonized (SGL-C) vectors. Lesion sizes were significantly larger in the mice infected with both saliva compared to mice infected with parasites alone; moreover, the lesions caused by parasite + SGL-C were significantly larger than the lesions caused by parasite + SGL-W. Histopathological morphometric studies regarding the acute phase of infections showed lower numbers of polymorphonuclear cells, greater numbers of mononuclear cells and parasites in SGL-C infected mice compared to SGL-W infected mice. In the chronic phase of infection, the number of mononuclear cells was lower and the number of parasites was greater in SGL-C infected mice than SGL-W infected mice. In vitro studies showed increased infection index of macrophages infected with parasites plus saliva compared to infection with parasites alone, with no difference between the saliva infection indices. SDS-PAGE gel for SGL-C and SGL-W showed differences in the composition and quantity of protein bands, determined by densitometry. These results call attention to the experimental saliva model, which shows exacerbation of infection caused by sandfly saliva.     

IL-2 limits IL-12 enhanced lymphocyte proliferation during Leishmania amazonensis infection

C3H mice infected with Leishmania amazonensis develop persistent, localized lesions with high parasite loads. During infection, memory/effector CD44^hiCD4⁺ T cells proliferate and produce IL-2, but do not polarize to a known effector phenotype. Previous studies have demonstrated IL-12 is insufficient to skew these antigen-responsive T cells to a functional Th1 response. To determine the mechanism of this IL-12 unresponsiveness, we used an in vitro assay of repeated antigen activation. Memory/effector CD44^hiCD4⁺ T cells did not increase proliferation in response to either IL-2 or IL-12, although these cytokines upregulated CD25 expression. Neutralization of IL-2 enhanced CD4⁺ T cell proliferation in response to IL-12. This cross-regulation of IL-12 responsiveness by IL-2 was confirmed in vivo by treatment with anti-IL-2 antibodies and IL-12 during antigen challenge of previously infected mice. These results suggest that during chronic infection with L. amazonensis, IL-2 plays a dominant, immunosuppressive role independent of identifiable conventional T_reg cells.