Inducible nitric oxide synthase and arginase expression in heart tissue during acute Trypanosoma cruzi infection in mice: arginase I is expressed in infiltrating CD68+ macrophages

In Chagas disease, which is caused by Trypanosoma cruzi, macrophages and cardiomyocytes are the main targets of infection. Classical activation of macrophages during infection is protective, whereas alternative activation of macrophages is involved in the survival of host cells and parasites. We studied the expression of inducible nitric oxide synthase (iNOS) and arginase as markers of classical and alternative activation, respectively, in heart tissue during in vivo infection of BALB/c and C57BL/6 mice. We found that expression of arginase I and II, as well as that of ornithine decarboxylase, was much higher in BALB/c mice than in C57BL/6 mice and that it was associated with the parasite burden in heart tissue. iNOS and arginase II were expressed by cardiomyocytes. Interestingly, heart-infiltrated CD68+ macrophages were the major cell type expressing arginase I. T helper (Th) 1 and Th2 cytokines were expressed in heart tissue in both infected mouse strains; however, at the peak of parasite infection, the balance between Th1 and Th2 predominantly favored Th1 in C57BL/6 mice and Th2 in BALB/c mice. The results of the present study suggest that Th2 cytokines induce arginase expression, which may influence host and parasite cell survival but which might also down-regulate the counterproductive effects triggered by iNOS in the heart during infection.     

Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63.

Leishmania parasites cause a spectrum of diseases that afflict the populations of 86 countries in the world. The parasites can survive within the lysosomal compartments of the host's macrophages, unless those macrophages are appropriately activated. Despite the fact that protective immunity can be induced by vaccination with crude parasite preparations, little progress has been made toward a defined vaccine for humans. In this study the gene encoding the Leishmania surface proteinase gp63 was cloned and expressed as a cytoplasmic protein in a bacille Calmette-Guérin (BCG) vaccine strain. BALB/c and CBA/J mice were inoculated with a single dose of recombinant BCG and challenged with infective Leishmania major or Leishmania mexicana promastigotes. Significant protection was observed in both mouse strains against L. mexicana and in CBA/J against L. major, whereas only a delay in L. major growth was seen in BALB/c mice. Recombinant BCG also engendered a strong protective response against challenge with amastigotes of L. mexicana, demonstrating that the induced immune response recognized the intracellular form of the parasite. The results support the view that recombinant BCG expressing gp63 may prove a useful vaccine for inducing protective cell-mediated immune responses to Leishmania species causing American cutaneous leishmaniasis.     

Vaccine development against cutaneous leishmaniasis. Subcutaneous administration of radioattenuated parasites protects CBA mice against virulent Leishmania major challenge

Experiments described in this paper were aimed at determining whether subcutaneous inoculation of live, avirulent Leishmania major would protect mice against infection by the virulent parasite. To this effect, promastigotes or amastigotes of a highly virulent strain of L. major (MRHO/IR/76), used in human trials of leishmanization, and which induces non-healing skin lesions in both CBA and BALB/c mice, were rendered non-pathogenic by gamma irradiation. A dose of 150 krad was required to abrogate the virulence of the parasite as tested on BALB/c mice. Strikingly, however, not all leishmanias were completely inactivated by this procedure since live parasites were detected in the footpads and/or the inguinal lymph nodes as long as 28 days (CBA) or 18 weeks (BALB/c) after injection. Furthermore, 150 krad-irradiated promastigotes retained the capacity to transform into amastigotes intracellularly in vitro. Subcutaneous inoculation of this irradiated 'vaccine' confered onto CBA mice a high degree of protection against challenge by both the homologous and a heterologous (MRHO/SU/59/P) strains of L. major. Lymph node cells from protected animals acquired the capacity to activate infected macrophages in vitro to kill intracellular L. major. To allow for maximum development of immunoprotection, the irradiated promastigotes had to remain viable, perhaps reflecting a requirement for transformation into amastigotes in the vaccinated host.     

Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity

Leishmanial mechanisms of virulence have been proposed previously to involve two different groups of parasite molecules. One group consists of largely surface and secretory products, and the second group includes intracellular molecules, referred to as 'pathoantigens'. In the first group are invasive/evasive determinants, which protect not only parasites themselves, but also infected host cells from premature cytolysis. These determinants help intracellular amastigotes maintain continuous infection by growing at a slow rate in the parasitophorous vacuoles of host macrophages. This is illustrated in closed in vitro systems, e.g. Leishmania amazonensis in macrophage cell lines. Although individual macrophages may become heavily parasitized at times, massive destruction of macrophages has not been observed to result from uncontrolled parasite replication. This is thus unlikely to be the direct cause of virulence manifested as the clinical symptoms seen in human leishmaniasis. Of relevance is likely the second group of immunopathology-causing parasite 'pathoantigens'. These are highly conserved cytoplasmic proteins, which have been found to contain Leishmania-unique epitopes immunologically active in leishmaniasis. How these intracellular parasite antigens become exposed to the host immune system is accounted for by periodic cytolysis of the parasites during natural infection. This event is notable with a small number of parasites, even as they grow in an infected culture. The cytolysis of these parasites to release 'pathoantigens' may be inadvertent or medicated by specific mechanisms. Information on the pathoantigenic epitopes is limited. T-cell epitopes have long been recognized, albeit ill-defined, as important in eliciting CD4+ cell development along either the Th1 or Th2 pathway. Their operational mechanisms in suppressing or exacerbating cutaneous disease are still under intensive investigation. However, immune response to B-cell epitopes of such 'pathoantigens' is clearly futile and counterproductive. Their intracellular location within the parasites renders them inaccessible to the specific antibodies generated. One example is the Leishmania K39 epitope, against which antibodies are produced in exceedingly high titers, especially in Indian kala-azar. Here, we consider the hypothetical emergence of this pathoantigenicity and its potential contributions to the virulent phenotype in the form of immunopathology. Microbial virulence may be similarly explained in other emerging and re-emerging infectious diseases. Attenuation of microbial virulence may be achieved by genetic elimination of pathoantigenicity, thereby providing mutants potentially useful as avirulent live vaccines for immunoprophylasis of infectious diseases.     

Macrophage killing of Leishmania amazonensis amastigotes requires both nitric oxide and superoxide

The requirements for effective and efficient intracellular killing of Leishmania amazonensis by activated macrophages are unknown. Despite resistance to the arginase inhibitor LOHA by intracellular L. amazonensis amastigotes, enhanced replication did not account for the relative resistance of this parasite to macrophage activation. Herein we report that the presence of both superoxide and nitric oxide is necessary for efficient killing of L. amazonensis amastigotes within LPS/IFN-gamma-activated bone marrow-derived macrophages generated from C3H mice. Addition of an extracellular signal-regulated kinase (ERK) inhibitor to L. amazonensis-infected macrophages increased the ability of these activated macrophages to kill L. amazonensis amastigotes. This enhanced macrophage killing through addition of ERK inhibitor was abrogated by inhibition of superoxide or iNOS, whereas inhibiting superoxide had no effect on the killing of L. major. These results suggest that ERK activation may modulate effective macrophage killing, leading to the ability of L. amazonensis to resist elimination within activated macrophages.     

Studies on intracellular killing of Leishmania major and lysis of host macrophages by immune lymphoid cells in vitro

Exposure of Leishmania major-infected CBA/T6 mouse macrophages to lymph node cells (LNC) from infected animals led to antigen-specific killing of the micro-organisms. The effect depended on the number of added LNC, the duration of incubation with macrophages, and the presence of LPS in the incubation fluids. Incubation with immune LNC also resulted in lysis of part of the infected cells, however without release of live amastigotes, as parasites were destroyed intracellularly before macrophages were damaged. Supernates from antigen-stimulated LNC cultures activated macrophages for intracellular killing without damage to the host cells, suggesting that macrophage lysis was not a consequence of the activation process. Treatment of effector lymphocytes with anti-L3T4 antibodies abrogated both intracellular killing and macrophage lysis. Parasitized macrophages were also destroyed by alloimmune cytolytic T-lymphocytes; in this case, however, live amastigotes were released, showing that immune lysis of the infected cells would not entail parasite destruction per se. These studies support the hypothesis that recovery from L. major infection relates to the ability of lymphoid cells to generate MAF/IFN in response to parasite antigen and are compatible with the idea that lysis of parasitized macrophages may contribute to immune recovery from the infection.     

Immunological responses and the pattern of disease in mice infected with transfected Leishmania major constitutively expressing active IL-1alpha

Immunity against leishmaniasis is mediated mainly by CD4+ T lymphocytes, which function by secreting cytokines, which in turn activate various effector mechanisms. Interleukin 1 (IL-1) represents one of the most pleiotropic proinflammatory cytokines and is required for normal regulation of Th1/Th2 responses. The aim of this study was to induce the expression of the inflammatory cytokine IL-1alpha by Leishmania parasites and to determine its effect on the parasite development. Leishmania constitutively producing IL-1alpha was engineered, using the pX63Hyg-IL-1alpha vector. IL-1alpha was produced by both promastigotes and amastigotes, and remained unchanged after transformation and development in mice. The protection against the disease achieved in BALB/c mice by the transfected parasites was superior to that obtained with the wild type. One month after infection a nodule was demonstrated in 22% and 60% of the mice inoculated with transfected parasites and the wild type, respectively. This tendency continued for an additional 2.5 months, after which the rate of infection increased to 90% and 100% in these two groups, respectively. The present study suggests that, during initial infection, the pathway of IL-1alpha production and its accessibility to the immunological cells might be important in the outcome of leishmanial infection.     

Granulocyte-macrophage and macrophage colony-stimulating factors activate intramacrophage killing of Leishmania mexicana amazonensis

Leishmania organisms are important pathogens, causing diseases worldwide. Standard therapies are often toxic and are not always effective. The effect of recombinant human granulocyte-macrophage and macrophage colony-stimulating factors (GM-CSF and M-CSF) on intramacrophage survival of Leishmania mexicana amazonensis (Lma) were compared with those of interferon-gamma (IFN-gamma). Macrophages previously infected with Lma were treated with or without GM-CSF and M-CSF. Compared with no cytokine treatment, treatment with GM-CSF (0.1-100 ng/ml) or M-CSF (1:3.5 X 10(6) - 1:3.5 X 10(3) dilutions) caused a significant dose-dependent reduction in intracellular parasites, 427 +/- 20 (mean +/- SE) Lma/100 macrophages. GM-CSF or M-CSF in combination with IFN-gamma resulted in more effective inhibition of intracellular parasites. Thus, the cytostatic activity appears to require interaction between cytokines, macrophages, and amastigotes. These cytokines are potential therapeutic agents for visceral leishmaniasis.     

Sambucus ebulus extract stimulates cellular responses in cutaneous leishmaniasis

This is the first study aiming to determine the therapeutic effects of the Sambucus ebulus aquatic extract as an antileishmanial herbal drug and evaluate the immune responses in Leishmania major major infected BALB/c mice. The antileishmanial activity of S ebulus aquatic extract was evaluated using MTT test as well as parasite rescue and transformation assay. Footpad swelling and parasite load of infected mice were measured by several techniques. The immune responses were evaluated by measuring the levels of IFN‐γ, IL‐4, nitric oxide and arginase. The results indicated that S. ebulus can significantly decrease L. major promastigotes and amastigotes viability, but it was not toxic to macrophages. The lesion size, parasite burden and the level of ARG decreased in the treated infected mice, while the IFN‐γ‐to‐IL‐4 ratio and the level of NO increased significantly. Altogether, the S. ebulus extract is an effective compound for killing Leishmania parasite without excessive toxicity to the host cells and can cure the CL by switching the host immune responses towards Th1 response. Thus, it may be a perfect therapeutic option for CL treatment.     

Leishmania braziliensis amastigotes stimulate production of IL‐1β, IL‐6, IL‐10 and TGF‐β by peripheral blood mononuclear cells from nonendemic area healthy residents

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL‐1β, IL‐6, TGF‐β and IL‐23 production, whereas IL‐10 and TGF‐β are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon‐γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL‐10 production in PBMCs; however, only amastigotes induced IL‐1β, IL‐6 and TGF‐β. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.     

Factors influencing Leishmania major infection in IL-4-deficient BALB/c mice

The outcome of Leishmania major infection in IL-4-deficient BALB/c mice has been a controversial subject. We have shown that IL-4-deficient BALB/c mice infected with Leishmania major developed progressive lesions and could not contain the replication of the parasites, whereas other studies have reported that IL-4-deficient mice were able to resist infection. Therefore, we examined different factors that can influence the course of Leishmania major infection. We tested different lines of IL-4-deficient BALB/c mice and show that the reported differences in the outcome of infection were not due to the different genetic origin of the embryonic stem cells used to disrupt the IL-4 gene. In addition, we infected IL-4-deficient mice with different isolates of L. major parasites and show that none of the parasite strains tested were cleared, although some of them caused milder pathology. Interestingly, this milder pathology was paralleled by a reduced arginase activity of the parasites. We also tested the influence of age on the course of Leishmania major infection in IL-4-deficient BALB/c mice and show that older mice express a transient resistance. Thus, we conclude that differences in the age of the mice and in the arginase activity of the different isolates of parasites are factors that can influence the non-healing phenotype of IL-4-/- BALB/c mice.     

Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.     

Variants with reduced virulence derived from Leishmania major after mutagen treatment

After several in vitro treatments of a virulent population of Leishmania major with the mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), five clones (vir-) were obtained that did not produce cutaneous lesions after subcutaneous injection of 10(6) promastigotes. All the control clones (vir+) obtained from the non-mutagenized parasite population produced progressive cutaneous lesions with as few as 10(3) parasites. Late lesions were observed occasionally after injection of 10(7) vir- parasites. These late lesions appeared to result from the selection of virulent revertants, since parasites isolated from these lesions produced progressive lesions in BALB/c mice almost as readily as the control parasites. Two vir- clones, one vir+ clone and one revertant clone were examined for survival in BALB/c macrophages in vitro. All clones were taken up by the macrophages and transformed into amastigotes. However, vir- clones failed to multiply inside the macrophages. A vir- clone was found to protect mice against a subsequent challenge with vir+ parasites.     

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.     

Leishmania parasites and their ploys to disrupt macrophage activation

Leishmania are intracellular protozoan parasites of macrophages. At the cellular level, the disease leishmaniasis involves the invasion of tissue macrophages by the parasite, the avoidance of cellular killing mechanisms, and the subsequent intracellular replication of parasites, with the eventual spread of the organisms to adjacent macrophages. This paper describes the process by which Leishmania organisms invade macrophages, with an overview of some of the molecules involved in this process; the mechanisms available to macrophages that have the potential to restrict the growth of Leishmania within them; and the ways that Leishmania and Leishmania-derived molecules can modulate macrophage functions and circumvent leukocyte antimicrobial responses.     

Phosphatidylserine exposure on the surface of Leishmania amazonensis amastigotes modulates in vivo infection and dendritic cell function

Leishmania amazonensis parasites can cause diverse forms of leishmaniasis in humans and persistent lesions in most inbred strains of mice. In both cases, the infection is characterized by a marked immunosuppression of the host. We previously showed that amastigote forms of the parasite make use of surface‐exposed phosphatidylserine (PS) molecules to infect host cells and promote alternative macrophage activation, leading to uncontrolled intracellular proliferation of the parasites. In this study, we demonstrated that treatment of infected mice with a PS‐targeting monoclonal antibody ameliorated parasite loads and lesion development, which correlated with increased proliferative responses by lymphocytes. In addition, we observed an enhanced dendritic cell (DC) activation and antigen presentation in vitro. Our data imply that the recognition of PS exposed on the surface of amastigotes plays a role in down‐modulating DC functions, in a matter similar to that of apoptotic cell clearance. This study provides new information regarding the mechanism of immune suppression in Leishmania infection.     

Characterization of a Leishmania isolate from the rodent host Neotoma micropus collected in Texas and comparison with human isolates.

We report the biological and biochemical parameters of Leishmania parasites (MNEO/US/90/WR972) isolated from a rodent host, Neotoma micropus, collected in Texas. Footpad inoculations of WR972 promastigotes into BALB/c mice and Syrian hamsters resulted in ulcerating lesions six and eight weeks post-inoculation, respectively. Using monoclonal antibody-stained touch preparations, amastigotes were found in the liver of both laboratory hosts. Infection of J774 macrophages with WR972 promastigotes supported the growth of amastigotes for 12 days at 35 degrees C. The WR972 parasite was identified by enzyme electrophoresis as L. mexicana. Isozyme comparison of WR972 with 42 L. mexicana isolates (from humans and rodents) from four different endemic areas, including Texas, suggest that these parasite populations are identical for approximately 97% of their genetic loci. Pulse field gel electrophoresis (PFGE) of WR972 resolved 18 chromosomes with a size range of 300- greater than 2,000 kb. The karyotype strongly resembles that of two other Texas L. mexicana isolates from humans. Taken together, the PFGE, hybridization, and isoenzyme data suggest that the wood rat isolate (WR972) is identical to parasites from human cutaneous lesions isolated in Texas and Central America. In addition, the biological characteristics of WR972, its infectivity of BALB/c mice and the Syrian hamster, and the potential of the isolate to infect, transform, and divide in J774 macrophages indicate that WR972 will be pathogenic in humans if transmission occurs. Health care providers should consider this possibility when studying the epidemiology and control of cutaneous leishmaniasis in Texas.     

Activity of azithromycin against Leishmania major in vitro and in vivo

Azithromycin, an azalide antibiotic of the macrolide family, concentrates in the tissues and especially in macrophages. Because Leishmania parasites reside in these cells, we tested this antibiotic for a possible antileishmanial activity in vitro and in vivo. Azithromycin decreased the Leishmania major promastigote count in cell-free cultures at log phase approximately 50-fold. In macrophage cultures infected with L. major amastigotes, azithromycin caused a significant decrease in parasite levels with an ED50 of 12 microg/ml. The activity in vivo was evaluated after infection of the footpads of susceptible BALB/cByJ mice and resistant C57BL/6J mice with L. major. Treatment of BALB/cByJ mice with azithromycin, 100 to 200 mg/kg/d, resulted in a significant decrease in lesion size and in the number of parasites per lesion, whereas no effect was seen in the treated C57BL/6J mice. Azithromycin has activity against L. major in vitro and in vivo. Given the severity of the disease and the limitations of the available therapeutic agents, azithromycin may have a significant role in the treatment of this group of diseases.