Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits

Background

Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS) ofLutzomyia intermedia, the natural vector ofLeishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC) of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected withL. braziliensis.

Results

Pre-treatment of non-infected human monocytes withL. intermediaSGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation andL. braziliensisinfection led to a significant increase in TNF-α, IL-6, and IL-8 production without significant alterations in co-stimulatory molecule expression. However, pre-treatment withL. intermediaSGS did not result in significant changes in the infection rate of human monocytes.

Conclusion

Our data indicate thatL. intermediasaliva is able to modulate monocyte response, and, although this modulation is dissociated from enhanced infection withL. braziliensis, it may be associated with successful parasitism.     

Antigenicity of Leishmania braziliensis Histone H1 during Cutaneous Leishmaniasis: Localization of Antigenic Determinants

The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that L. braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L. braziliensis histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant L. braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.     

Analysis of the Immune Responses of Mice to Infection with Leishmania braziliensis

Leishmania major and Leishmania braziliensis both cause cutaneous leishmaniasis, but the former kills BALB/c mice while the latter is killed by the mice. This killing of L. braziliensis occurred by a gamma interferon-dependent mechanism, potentially made possible by the observed lack of high interleukin-4 production.     

Development of a Multilocus Microsatellite Typing Approach for Discriminating Strains of Leishmania (Viannia) Species

A multilocus microsatellite typing (MLMT) approach based on the analysis of 15 independent loci has been developed for the discrimination of strains belonging to different Viannia species. Thirteen microsatellite loci were isolated de novo from microsatellite-enriched libraries for both Leishmania braziliensis and L. guyanensis. Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Markers were designed to contain simple dinucleotide repeats flanked by the minimal possible number of nucleotides in order to allow variations in repeat numbers to be scored as size variations of the PCR products. The 15 markers in total were amplified for almost all of the strains of Viannia tested; one marker did not amplify from the two L. peruviana strains included in the study. When 30 strains of L. braziliensis, 21 strains of L. guyanensis, and 2 strains of L. peruviana were tested for polymorphisms, all strains except two strains of L. guyanensis had individual MLMT types. Distance-based analysis identified three main clusters. All strains except one strain of L. guyanensis grouped together. Two clusters consisted of strains of L. braziliensis according to their geographical origins. The two strains of L. peruviana grouped together with strains of L. braziliensis from Peru and the adjacent Brazilian state of Acre. MLMT has proven capable of individualizing strains even from the same areas of endemicity and of detecting genetic structures at different levels. MLMT is thus applicable for epidemiological and population genetic studies of strains within the subgenus Viannia.Cutaneous leishmaniasis (CL) is a serious but neglected public health problem that is endemic in 22 countries of Latin America, with Brazil and Colombia being among the six countries reporting 90% of the worldwide cases. Species of the subgenus Viannia cause the majority of cases of CL and mucocutaneous leishmaniasis (MCL) in South America and often overlap in their distribution (22). The subgenus is subdivided into species complexes representing Leishmania braziliensis, L. guyanensis, and L. naiffi (8). The L. guyanensis complex includes some named species, such as L. panamensis and L. shawi, which were found to be very similar to each other (7). On the other hand, L. peruviana was always considered to be closely related to L. braziliensis (14, 30). L. lainsoni was shown to be very distinct from the other species (8). In addition, numerous interspecies hybrids have been reported, such as L. braziliensis/L. peruviana (26), L. braziliensis/L. guyanensis (10), L. braziliensis/L. panamensis (4), and L. lainsoni/L. naiffi (35).Many of the Leishmania (Viannia) species are capable of producing a wide spectrum of diseases, with the severity varying from self-limiting CL to severe MCL (9, 15). MCL is principally caused by L. braziliensis, but less than 5% of patients with a primary cutaneous lesion will develop metastatic mucosal involvement during their life course (17, 23, 24). Other species of the subgenus Leishmania (Viannia), such as L. guyanensis, may be associated with MCL (34), but the exact risk and frequency are unknown. Interestingly, L. peruviana, the species that is the closest to L. braziliensis, has not yet been reported to cause MCL.The transmission of leishmaniasis in South America occurs through sand flies of the genus Lutzomyia, with each Leishmania species usually being transmitted by more than one sand fly species, and the preferred mammalian hosts also vary according to the Leishmania species (15). For some of the Leishmania (Viannia) species, such as L. guyanensis and L. panamensis, the patterns of transmission are well understood, but for other species they are not. Knowledge of the patterns of transmission are necessary, however, to develop disease control measures and surveillance activities.Markers that are able to discriminate Leishmania (Viannia) organisms to the species and strain levels are needed to resolve the diversity and structure of Leishmania (Viannia) populations. Multilocus microsatellite typing (MLMT) has proven highly discriminatory in the typing of strains (5) and has been successfully used in population genetic studies of different Old World species of Leishmania (2, 19, 20, 32). This approach could be performed directly with clinical materials without prior cultivation of the parasites. Its results are reproducible, can be stored in databases, and can be exchanged between different laboratories. The first three microsatellite markers for the subgenus Leishmania (Viannia) were published in 1999 (30). Two of them, AC01 and AC52, were polymorphic in L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis; however, marker AC16 was monomorphic for the seven strains of L. guyanensis analyzed. Marker AC52 had a composite repeat and needed to be sequenced because electrophoretic alleles of the same size could consist of different repeat compositions. This marker was also not very reliable when it was used to type strains of L. braziliensis and L. peruviana from Peru (26). Recently, an additional eight microsatellites designed by using genomic libraries of L. braziliensis genome project loci were found to be polymorphic in L. braziliensis (28). However, the variation of these markers in other Leishmania (Viannia) species was not tested.The study described here aimed to develop an MLMT approach that could be used for epidemiological and population studies of different Leishmania (Viannia) species. For this purpose, 13 microsatellite loci were identified by searching microsatellite-enriched libraries for L. braziliensis and L. guyanensis. Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Variations in repeat numbers should be scorable as size variations of the PCR products. The 15 markers in total were amplified for all Leishmania (Viannia) species tested and tested for polymorphisms in 31 strains of L. braziliensis, 20 strains of L. guyanensis, and 2 strains of L. peruviana.     

Distinct Roles for MyD88 and Toll-Like Receptor 2 during Leishmania braziliensis Infection in Mice

We have previously reported that Leishmania braziliensis infection can activate murine dendritic cells (DCs) and upregulate signaling pathways that are essential for the initiation of innate immunity. However, it remains unclear whether Toll-like receptors (TLRs) are involved in L. braziliensis-mediated DC activation. To address this issue, we generated bone marrow-derived DCs from MyD88^−/− and TLR2^−/− mice and examined their responsiveness to parasite infection. While wild-type DCs were efficiently activated to produce cytokines and prime naïve CD4⁺ T cells, L. braziliensis-infected MyD88^−/− DCs exhibited less activation and decreased production of interleukin-12 (IL-12) p40. Furthermore, MyD88^−/− mice were more susceptible to infection in that they developed larger and prolonged lesions compared to those in control mice. In sharp contrast, the lack of TLR2 resulted in an enhanced DC activation and increased IL-12 p40 production after infection. As such, L. braziliensis-infected TLR2^−/− DCs were more competent in priming naïve CD4⁺ T cells in vitro than were their controls, findings which correlated with an increased gamma interferon production in vivo and enhanced resistance to infection. Our results suggest that while MyD88 is indispensable for the generation of protective immunity to L. braziliensis, TLR2 seems to have a regulatory role during infection.Leishmaniasis is a vector-borne disease that has a great socioeconomic impact in many tropical and neotropical countries (40). Leishmania parasites multiply as flagellated promastigotes in the midguts of sand flies and are transmitted to the vertebrate host via the bites of parasite-carrying female flies (3, 22). The insult at the bite site initiates a strong neutrophil influx and parasite capture by these cells (38). Interestingly, some of the captured parasites remain viable, and these infected neutrophils actually facilitate the silent entry of parasites into macrophages (Mφs) (29), where parasites survive and replicate as intracellular amastigotes (3). The magnitude and nature of inflammatory responses at the infection site and the profile of subsequent T-cell responses determine the outcome of the infection. In South America, Leishmania braziliensis infection causes cutaneous leishmaniasis in most cases and mucocutanous leishmaniasis in some individuals. The latter is a severe and disfiguring form of the disease. At present, it remains unclear why the infection is controlled in some individuals but progressive in others (40).Dendritic cell (DC)-pathogen interactions are initiated by interaction between receptors on DCs and pathogen-associated molecular patterns, including lipopolysaccharide (LPS), glycolipids, and nucleic acids. Signals through Toll-like receptors (TLRs) can induce DC maturation and the production of proinflammatory cytokines (20, 39), thereby bridging the innate and adaptive immune responses (9). Upon ligand binding, downstream signaling of all TLRs (with the exception of TLR3) uses the adaptor protein MyD88 (32). Gene knockout studies in mice have suggested that TLR signaling is essential for the immune responses against Leishmania parasites (52). For example, MyD88 and TLR4 contribute to the control of Leishmania major infection in C57BL/6 mice (27, 33). TLR9 is involved in NK cell activation in animal models of visceral (Leishmania donovani) and cutaneous (L. major and L. braziliensis) leishmaniasis (30, 45), while TLR2 and TLR3 are required for the intracellular killing of L. donovani in gamma interferon (IFN-γ)-primed Mφs (15). Leishmania lypophosphoglycan (LPG), an abundant molecule in the surfaces of promastigotes, not only is a virulence factor for some Leishmania species (e.g., L. major and L. donovani) (49) but also acts as a ligand for TLR2-mediated signaling (5). However, different species of Leishmania display relatively high variations (biochemical modifications) in LPG molecules (7). In the case of L. braziliensis, the procyclic form of the parasite lacks side chain sugar substitutions on its LPG, whereas the metacyclic form appears to contain decreased amounts of LPG compared to other Leishmania species (47). On the DC surface, TLR2 is present as preexisting heterodimers of TLR2/1 and/or TLR2/6, recognizing triacylated and diacylated lipoproteins, respectively (51). TLR2 has been shown to be important for NK cell activation in vitro by purified L. major LPG (5); however, the functional roles of TLR2 remain largely unclear during both parasite-DC interactions and the course of Leishmania infection in vivo.Most inbred strains of mice are genetically resistant to L. braziliensis infection, due to the capacity of mice to establish a strong Th1 response (43). This self control of infection is accompanied by the selective expansion of IFN-γ-producing CD4⁺ T cells, which induce nitric oxide production in infected Mφs to promote parasite killing (3, 12). We have previously revealed that several key molecules in the innate immunity pathways (e.g., STAT1, STAT3, and ISG15) were upregulated in L. braziliensis-infected DCs and that such DCs were highly efficient in priming CD4⁺ T cells in vitro and in vivo (53). However, it remains unclear whether DC-Leishmania cell interactions in the absence of MyD88 and TLR2 impact T-cell functions and in vivo containment of infection. In the present study, we generated bone marrow-derived DCs (BMDCs) from MyD88^−/− and TLR2^−/− mice and examined their responsiveness to L. braziliensis infection. We found that infected MyD88^−/− DCs showed low levels of cell activation and interleukin-12 (IL-12) p40 production, which correlated with increased susceptibilities of these mice to L. braziliensis infection and decreased expansion of IFN-γ-producing and IL-17-producing CD4⁺ T cells during the course of infection. Given that most TLR pathways share MyD88 and that TLR2 is involved in LPG recognition, we then examined the role of TLR2 in L. braziliensis recognition. Contrary to MyD88^−/− DCs, the lack of TLR2 enhanced DC activation, IL-12 p40 production, and T-cell priming in vitro. Consequently, TLR2^−/− mice were more resistant to infection than were the control mice, a finding that was associated with enhanced IFN-γ production in the draining lymph nodes (dLN). Collectively, our results show that while MyD88 is critical for L. braziliensis recognition in vitro and in vivo, TLR2 appears to have a regulatory role in modulating immune responses to the parasite.     

Characterization of regulatory T cell (Treg) function in patients infected with Leishmania braziliensis

Th1 immune responses are crucial for eliminating Leishmania parasites. However, despite strong Th1 responses, cutaneous leishmaniasis (CL) patients infected with Leishmania braziliensis develop the disease, while milder Th1 responses are found in sub-clinical (SC) infections. Therefore, CL patients may experience impaired regulatory T cell (Treg) function, causing excessive Th1 responses and tissue damage. To address this hypothesis, we characterized the function of circulating Tregs in L. braziliensis infected CL patients and compared them to Tregs from uninfected controls (UC) and SC subjects. The frequency of circulating Tregs was similar in CL patients, UC and SC subjects. Moreover, CL patients Tregs suppressed lymphocyte proliferation and PBMC pro-inflammatory cytokine production more efficiently than UC Tregs, and also produced higher levels of IL-10 than UC and SC Tregs. Furthermore, PBMC and mononuclear cells from lesions of CL patients responded normally to Treg-induced suppression. Therefore, the lesion development in CL patients infected with L. braziliensis is not associated with impairment in Treg function or failure of cells to respond to immunomodulation. Rather, the increased Treg activation in CL patients may impair parasite elimination, resulting in establishment of chronic infection. Thus, immunological strategies that interfere with this response may improve leishmaniasis treatment.     

Occurrence of a conserved domain in ATP diphosphohydrolases from pathogenic organisms associated to antigenicity in human parasitic diseases

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.     

Molecular and biologic characterization of Leishmania parasites implicated in an epidemic outbreak in northwestern Argentina

Leishmania (Viannia)braziliensis and its variants were implicated in the epidemic outbreak of mucocutaneous leishmaniasis that occurred in Salta, northwestern Argentina, in 1985. A total of 24 suspected, untreated cases were evaluated clinically and parasitologically. Four of five stable isolates were consistent with the reference strain of L. (V.)braziliensis as determined by monoclonal antibodies and indirect immunofluorescence or radioimmunobinding assays. Zymodeme analysis in agarose gels showed a close relationship with L. (V.)guyanensis and L. (V.)panamensis. All zymograms obtained with polyacrylamide gels belonged to the subgenus Viannia; the patterns were different from, but very closely related to, the reference strains of L. (V.)braziliensis as determined by dendrogram analysis. Hamsters infected with two isolates showed a pattern consistent with L. (V.)braziliensis. The pattern of development in the gut of Lutzomyia longipalpis was consistent with members of Viannia. Received: 6 October 1999 / Accepted: 22 October 1999     

A vaccine composed of a hypothetical protein and the eukaryotic initiation factor 5a from Leishmania braziliensis cross-protection against Leishmania amazonensis infection

In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4⁺ and CD8⁺ T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.     

Immunocytochemical and immunohistochemical methods as auxiliary techniques for histopathological diagnosis of cutaneous leishmaniasis

A significant increase in cutaneous leishmaniasis (CL) and its geographic expansion has motivated the development of techniques to help with diagnosis of the disease. Here we describe immunocytochemical (ICC) and immunohistochemical (IHC) techniques for the diagnosis of CL in the laboratory. Polyclonal antibodies and a modified avidin-biotin complex (Ultra Streptavidin^®) for Leishmania (V.) braziliensis or Leishmania (L.) amazonensis were developed for the present study. In vitro culture and histological sections from experimentally infected tissues were submitted to ICC/IHC techniques. The polyclonal antibody specificity, stability and immunostaining were evaluated. The polyclonal antibodies purified by chromatography (Sephadex^®) and obtained from L. (V.) braziliensis and L. (L.) amazonensis insoluble antigens presented 83.3% sensitivity, when the presence of antigens was evaluated, i.e., higher than histopathology or any equivalent method (in vitro culture). The polyclonal antibody presented 100% specificity when used against species frequently found in CL lesions. The ICC/IHC techniques developed in the current study were able to recognize amastigotes and antigens from in vivo and in vitro cultures and from biopsies, offering additional help in the diagnosis of CL. This methodology could be beneficially adopted in public health laboratories.     

Protective and Pathological Functions of CD8+ T Cells in Leishmania braziliensis Infection

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8⁺ T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ T cells. Monocytes from PBMC were infected with L. braziliensis and cocultured with CD8⁺ T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8⁺ IFN-γ⁺ cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8⁺ T cells induced more apoptosis of infected monocytes than did SC CD8⁺ T cells. Granzyme B production in CD8⁺ T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8⁺ T cells are more cytotoxic and may be involved in pathology.     

Evaluation of a hypothetical protein for serodiagnosis and as a potential marker for post-treatment serological evaluation of tegumentary leishmaniasis patients

The serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.     

Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.     

Antigen‐triggered interferon‐γ and interleukin‐10 pattern in cured mucosal leishmaniasis patients is shaped during the active phase of disease

An exacerbated type 1 response to leishmanial antigens is the basis of tissue destruction observed in mucosal leishmaniasis (ML). After therapy, a persistent production of high levels of inflammatory cytokines can confer a poor prognosis. Herein we investigated whether the clinical conditions defined during the active phase of ML affect the magnitude of long-term anti-Leishmania immune response. Twenty clinically cured ML cases were studied. Peripheral blood mononuclear cells (PBMC) were cultured with L. braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), concanavalin-A (Con-A) or medium alone, and the lymphocyte proliferative response and cytokine secretion were quantified. Medical records were reviewed for Montenegro skin test (MST) during diagnosis, duration of ML disease or time elapsed after clinical cure. The duration of disease was correlated positively with MST (r = 0·61). Lb-Ag induced interferon (IFN)-γ was correlated positively with duration of illness (r = 0·69) as well as the frequency of secreting cells [enzyme-linked immunospot (ELISPOT)] assay. No association was observed for Tg-Ag or Con-A. Disease duration was correlated negatively with interleukin (IL)-10 production (r = −0·76). Moreover, a negative correlation between length of time after clinical cure and TNF levels (r = −0·94) or the IFN-γ : IL-10 ratio (r = −0·89) were also seen. We suggest that the magnitude of the IFN-γ inflammatory response triggered by ML can be driven by the time of leishmanial antigens exposition during the active phase of the disease. This pattern could persist even long-term after cure. However, despite IFN-γ levels, the decrease of the TNF and IFN-γ : IL-10 ratio reflects the control of proinflammatory responses achieved by cure of ML, possibly preventing disease relapses.     

Single-Step Multiplex PCR Assay for Characterization of New World Leishmania Complexes

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.     

Presence of parasite DNA in clinically unaffected nasal mucosa during cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis

Objectives

We aimed to detect Leishmania DNA carriage in nasal mucosa of individuals with cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis.

The Effect of Peritoneal Macrophages from Mice Injected with Leishmania Braziliensis on the in Vitro Growth of Tumor Cells

Leishmania braziliensis reportedly is capable of producing a reduction in the growth of solid and ascites murine leukemia and lymphomas. The possibility that the inhibitory effect on the ascites tumor was produced by the activation of the macrophage peritoneal cell population was explored. It was observed that adherent cells from the peritoneal cavity of L. braziliensis injected mice caused a marked inhibitory effect on the in vitro growth of the 6C3HED lymphoma and EL-4 leukemia cells. This effect was dependent on the degree of the macrophage activation, was not produced by supernatants from cultures of activated macrophages, and it seems that contact cell between target and effector cells is necessary. In addition to being cytostatic, this effect was also cytotoxic. The L. braziliensis activated macrophages were also capable of suppressing the multiplication of normal cells induced by mitogen, but this was not observed if the cells were already undergoing multiplication. A similar cytostatic effect on the tumor cells was observed to be produced by the peritoneal non-adherent cell population of the L. braziliensis injected mice.