Human Tegumentary Leishmaniasis Research Articles

Dogs Harbor Leishmania braziliensis and Participate in the Transmission Cycle of Human Tegumentary Leishmaniasis.

Dogs play an important role in transmission of Leishmania infantum, but epidemiologic and clinical studies of canine tegumentary leishmaniasis (CTL) are scarce. In an endemic area of human American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis, we determine the prevalence and incidence of both CTL and subclinical (SC) L. braziliensis infection in dogs and evaluated if the presence of dogs with CTL or SC L. braziliensis infection is associated with the occurrence of human ATL. SC infection in healthy animals and CTL in animals with ulcers were determined by PCR on biopsied healthy skin or on ulcers or by detecting antibodies against soluble leishmania antigen. We compared the occurrence of human ATL in homes with dogs with CTL or SC infection with control homes without dogs or with dogs without CTL or SC infection. The prevalence of SC infection was 35% and of CTL 31%. The incidence of SC infection in dogs was 4.6% and of CTL 9.3%. The frequency of ATL in humans was 50% in homes with infected dogs and 13% in homes without L. braziliensis infection in dogs. CTL and SC infection is highly prevalent, and dogs may participate in the transmission chain of L. braziliensis.

Anti-Leishmania amazonensis Activity, Cytotoxic Features, and Chemical Profile of Allium sativum (Garlic) Essential Oil.

Human tegumentary leishmaniasis (HTL) is a serious tropical disease caused by Leishmania amazonensis. Developing new leishmanicidal agents can help overcome current treatment challenges, such as drug resistance and toxicity. Essential oils are a source of lipophilic substances with diverse therapeutic properties. This study aimed to determine the anti-L. amazonensis activity, cytotoxicity, and chemical profile of Allium sativum essential oil (ASEO). The effect of ASEO on parasite and mammalian cells viability was evaluated using resazurin and MTT assays, respectively. The oil's effect against intracellular amastigotes was also determined. Transmission electron microscopy was used to assess the ultrastructural changes induced by ASEO. In addition, the chemical constituents of ASEO were identified by gas chromatography-mass spectrometry (GC-MS). The cytotoxic potential was evaluated in vitro and in silico. The oil displayed IC50 of 1.76, 3.46, and 3.77 µg/mL against promastigotes, axenic, and intracellular amastigotes, respectively. Photomicrographs of treated parasites showed plasma membrane disruption, increased lipid bodies, and autophagic-like structures. ASEO chemical profiling revealed 1,2,4,6-tetrathiepane (24.84%) and diallyl disulfide (16.75%) as major components. Computational pharmacokinetics and toxicological analysis of ASEO's major components demonstrated good oral bioavailability and better toxicological endpoints than the reference drugs. Altogether, the results suggest that ASEO could be an alternative drug candidate against HTL.

Leishmania (Viannia) braziliensis in Migonemyia migonei and Cortelezzii complex (Diptera: Phlebotominae) from Chaco, Argentina

Chaco province is included in Argentinean ecoregions with human tegumentary leishmaniasis case records. During 2012-2014 in Pampa del Indio town an ecoepidemiological study was carried out including phlebotomine dynamics and its natural infection with Leishmania demonstrated by sand flies dissection, polymerase chain reaction and sequencing. The species recorded were: Migonemyia migonei (72.79%), Nyssomyia neivai (23.6%), Evandromyia cortelezzii (0.94%), Evandromyia sallesi (0.39%), Cortelezzii complex (1.61%), Evandromyia aldafalcaoae (0.05%), Psathyromyia bigeniculata (0.02%), Brumptomyia brumpti (0.6%) and Corumbaensis complex (0.01%). A total of 380 females sand flies (Cortelezzii complex, Mg. migonei and Ny. neivai) from peridomicile and extradomicile were individually dissected and no flagellates were observed in the intestinal tract. Later, these females were arranged in 38 pools for molecular analyses and Leishmania braziliensis DNA was amplified in 3 pools with a minimum infection rate of the total females of 0.8%, while specific rates were 0.5% for the Cortelezzii complex and 1.5% for Mg. migonei. In conclusion, our results would strengthen the hypothesis that, in the study area, these species are candidates to be incriminated as vectors, while further studies will be required to fulfill the criteria to characterize both species as proven vectors of Le. braziliensis.

Development of a chimeric protein based on a proteomic approach for the serological diagnosis of human tegumentary leishmaniasis.

Leishmania braziliensis is responsible for most cases of human tegumentary leishmaniasis (HTL) and has caused a wide range of clinical manifestations, including cutaneous (CL) and mucosal leishmaniasis (ML). The diagnosis is based on criteria that consider epidemiological data, clinical findings, and laboratory tests and is hard to establish. For laboratory tests, none of the assays available can be considered gold standards for disease detection. In addition, the Montenegro skin test, essential to supporting infectologists in the clinical management of the disease, is no longer available in Brazil. Thus, the aim of this study was to develop new targets to be used in diagnostic tests for HTL. In the first step, we carried out two-dimensional gel electrophoresis, followed by mass spectrometry, combined with heat map analysis and immunoproteomics approach, and disclosed eight proteins expressed in the amastigote stage specifically recognized by serum from CL and ML patients. A chimeric protein was designed based on the combination of thirteen linear B-cell epitopes, identified by immunoinformatics analysis, from L. braziliensis proteins. Our results showed that the strategy used in this work was successful in developing an antigen to be used in immunological assays (100.0% sensitivity and specificity) in the detection of HTL cases and in comparison with results obtained from an ELISA using soluble L. braziliensis antigen (SLb-Antigen) and immunofluorescence assay (Bio-Manguinhos/FIOCRUZ). The present technology opens the door for its use in field exams by means of an immunochromatographic test, which will be even more helpful in regions without laboratory structures.Key points• Rational strategy to develop antigens.• Integration between immunoproteomic and immunoinformatics analysis.• Chimeric protein shows high performance in HTL diagnosis.

Potential antigenic targets used in immunological tests for diagnosis of tegumentary leishmaniasis: A systematic review.

Immunological tests may represent valuable tools for the diagnosis of human tegumentary leishmaniasis (TL) due to their simple execution, less invasive nature and potential use as a point-of-care test. Indeed, several antigenic targets have been used with the aim of improving the restricted scenario for TL-diagnosis. We performed a worldwide systematic review to identify antigenic targets that have been evaluated for the main clinical forms of TL, such as cutaneous (CL) and mucosal (ML) leishmaniasis. Included were original studies evaluating the sensitivity and specificity of immunological tests for human-TL, CL and/or ML diagnosis using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies to detect Leishmania-specific antibodies or antigens. The review methodology followed PRISMA guidelines and all selected studies were evaluated in accordance with QUADAS-2. Thirty-eight original studies from four databases fulfilled the selection criteria. A total of 79 antigens were evaluated for the detection of antibodies as a diagnostic for TL, CL and/or ML by ELISA. Furthermore, three antibodies were evaluated for the detection of antigen by immunochromatographic test (ICT) and immunohistochemistry (IHC) for CL-diagnosis. Several antigenic targets showed 100% of sensitivity and specificity, suggesting potential use for TL-diagnosis in its different clinical manifestations. However, a high number of proof-of-concept studies reinforce the need for further analysis aimed at verifying true diagnostic accuracy in clinical practice.

Autochthonous and imported tegumentary leishmaniasis in Catalonia (Spain): Aetiological evolution in the last four decades and usefulness of different typing approaches based on biochemical, molecular and proteomic markers.

Leishmaniasis is a transmissible disease caused by Leishmania protozoa. Spain is endemic for both visceral and cutaneous leishmaniasis, the autochthonous aetiological agent being Leishmania infantum. Around the world, the L.donovani complex is associated with visceral symptoms, while any species of the Leishmania or Viannia subgenera affecting human can produce tegumentary forms. In a context of growing numbers of imported cases, associated with globalisation, the aim of this study was to analyse the aetiological evolution of human tegumentary leishmaniasis in a region of Spain (Catalonia). Fifty-six Leishmania strains, isolated from 1981 to 2018, were analysed using MLEE, gene sequencing (hsp70, rpoIILS, fh and ITS2) and MALDI-TOF. The utility of these different analytical methods was compared. The results showed an increase in leishmaniasis over the two last decades, particularly imported cases, which represented 39% of all cases studied. Leishmania infantum, L.major, L.tropica, L.braziliensis, L.guyanensis and L.panamensis were identified. The combination of molecular and enzymatic methods allowed the identification of 29 different strain types (A to AC). Strain diversity was higher in L. (Viannia), whilst the different L.major types were relatable with geo-temporal data. Among the autochthonous cases, type C prevailed throughout the studied period (39%). Minor types generally appeared within a short time interval. While all the techniques provided identical identification at the species complex level, MALDI-TOF and rpoIILS or fh sequencing would be the most suitable identification tools for clinical practice, and the tandem hsp70-ITS2 could substitute MLEE in the epidemiological field.

Human CD8+ T Cells Release Extracellular Traps Co-Localized With Cytotoxic Vesicles That Are Associated With Lesion Progression and Severity in Human Leishmaniasis.

Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation and exert microbicidal activity. While etosis is often associated with innate cells, recent studies showed that B cells and CD4+ T cells can release ETs. Here we investigate whether CD8+ T cells can also release ETs, which might be related to cytotoxicity and tissue pathology. To these ends, we first employed an in vitro system stimulating human CD8+ T cells isolated from healthy volunteers with anti-CD3/anti-CD28. Using time-frame video, confocal and electron microscopy, we demonstrate that human CD8+ T cells release ETs upon stimulation (herein LETs – lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form long strands that co-localize with CD107a, a marker of vesicles containing cytotoxic granules. In addition, these structures connect the LET-releasing cell to other neighboring cells, often resulting in cell death. After demonstrating the release of LETs by human CD8+ T cells in vitro, we went on to study the occurrence of CD8-derived LETs in a human disease setting. Thus, we evaluated the occurrence of CD8-derived LETs in lesions from patients with human tegumentary leishmaniasis, where CD8+ T cells play a key role in mediating pathology. In addition, we evaluated the association of these structures with the intensity of the inflammatory infiltrate in early and late cutaneous, as well as in mucosal leishmaniasis lesions. We demonstrated that progression and severity of debilitating and mutilating forms of human tegumentary leishmaniasis are associated with the frequency of CD8+ T cells in etosis, as well as the occurrence of CD8-derived LETs carrying CD107a+ vesicles in the lesions. We propose that CD8+ T cell derived LETs may serve as a tool for delivering cytotoxic vesicles to distant target cells, providing insights into mechanisms of CD8+ T cell mediated pathology.

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23.

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.

Potential application of small myristoylated protein-3 evaluated as recombinant antigen and a synthetic peptide containing its linear B-cell epitope for the serodiagnosis of canine visceral and human tegumentary leishmaniasis

Serological tests are important tools for the diagnosis of Leishmania infection. However, they are not effective markers to diagnose asymptomatic cases of visceral leishmaniasis (VL) and patients developing tegumentary leishmaniasis (TL), since antileishmanial antibodies can be encountered in low levels resulting in false-negative results in the serological trials. In this context, antigens able to be recognized by antibodies in sera from both VL and TL patients will be desirable to be employed in a more sensitivity and specific diagnosis of disease. In the present study, a conserved Leishmania protein, small myristoylated protein-3 (SMP-3), which was showed to be conserved in different Leishmania species and an effective vaccine candidate against Leishmania infantum infection in a murine model, was cloned and the recombinant protein was evaluated as a serological marker for the diagnosis of human TL and canine VL. In addition, a linear B cell-specific epitope (MQKDEESGEFKCEL) was identified, synthetized and also investigated as a serological marker. As antigen controls, rA2 protein and antigenic Leishmania extracts (SLA) were used. Results showed that ELISA-rSMP-3 and ELISA-Peptide presented sensitivity and specificity values higher than 90% in both diseases in humans and canids, having identified all asymptomatic cases and did not present cross-reaction with cross-reactivity diseases in both mammalian hosts. On the other hand, sensitivity and specificity values were worst when rA2 or SLA were used as antigens in humans and dogs. In conclusion, results showed the efficacy and Leishmania SMP-3 protein, employed as a recombinant antigen or a B cell epitope, for the improvement of the serodiagnosis of human TL and canine VL. This candidate can be tested in other diagnostic platforms, such as rapid immunochromatographic dipstick tests, aiming its use in epidemiological studies in remote areas where laboratories are not readily accessible for conventional assays.

The Binomial Parasite-Host Immunity in the Healing Process and in Reactivation of Human Tegumentary Leishmaniasis.

Leishmaniasis is a vector-borne infectious disease caused by different species of protozoa from the Leishmania genus. Classically, the disease can be classified into two main clinical forms: Visceral (VL) and Tegumentary (TL) leishmaniasis. TL is a skin/mucosal granulomatous disease that manifests mainly as cutaneous localized or disseminated ulcers, papules diffusely distributed, mucosal lesions or atypical lesions. Once the etiology of the infection is confirmed, treatment can take place, and different drugs can be administered. It has already been shown that, even when the scar is clinically evident, inflammation is still present in the native tissue, and the decrease of the inflammatory process occurs slowly during the 1st years after clinical healing. The maintenance of residual parasites in the scar tissue is also well documented. Therefore, it is no longer a surprise that, under some circumstances, therapeutic failure and/or lesion reactivation occurs. All over the years, an impressive amount of data on relapses, treatment resistance and lesion reactivation after healing has been collected, and several factors have been pointed out as having a role in the process. Different factors such as Leishmania species, parasite variability, Leishmania RNA virus 1, parasite load, parasite persistence, age, nutritional status, gender, co-morbidities, co-infection, pregnancy, immunosuppression, lesion duration, number and localization of lesions, drug metabolism, irregular treatment and individual host cellular immune response were described and discussed in the present review. Unfortunately, despite this amount of information, a conclusive understanding remains under construction. In addition, multifactorial influence cannot be discarded. In this context, knowing why leishmaniasis has been difficult to treat and control can help the development of new approaches, such as drugs and immunotherapy in order to improve healing maintenance. In this sense, we would like to highlight some of the findings that may influence the course of Leishmania infection and the therapeutic response, with an emphasis on TL.

A proteomic road to acquire an accurate serological diagnosis for human tegumentary leishmaniasis

Diagnostic tools are important for clinical management and epidemiological evaluation of Tegumentary (TL) and Visceral (VL) Leishmaniasis. Serology is not frequently used for the diagnosis of the TL form because low antibody titers and cross-reaction with VL. Therefore, it is crucial to identify specific and immunogenic antigens from species associated with the TL form. Here we employed a proteomic approach coupled to an in silico analysis and identified the most abundant and immunogenic proteins from Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum. Of 16 species specific proteins, nine were from the species causative of the TL form (L. amazonensis and L. braziliensis). In silico analysis revealed 18 B-cell epitopes with 0% similarity to Trypanosoma cruzi orthologs and, therefore, less likely to crossreact with sera of patients with Chagas disease. Two proteins reacted exclusively with serum from TL patients and presented several B-cell epitopes without similarity to T. cruzi orthologs: the hypothetical protein GI 134063939 and the metallo-peptidase Clan MA(E)-Family M3. The immunoassay revealed nine peptides with strong reactivity to sera from TL patients. These proteins and peptides may be good candidates to improve the specificity and sensibility of serological tests aiming to diagnose the TL of this neglected human disease. Biological significanceAs no gold-standard test for tegumentary leishmaniasis (TL) exists, a combination of different diagnostic techniques is often necessary to obtain precise results. Thus, the identification of species-specific, highly immunogenic and abundant proteins that stimulate the humoral immune response in the host should help in the development of serological tests for human TL. Herein we searched for these potential antigens in Leishmania species related to American Leishmaniasis (L. amazonensis, L. braziliensis and L. infantum). To this end, we employed an immunoproteomic approach using proteins from these Leishmania species and sera from TL and Visceral Leishmaniasis (VL) patients. Our study unveils specific proteins and peptides that may represent antigens that will help the efforts to improve the accuracy of serological tests to diagnose the TL form.

New serological tools for improved diagnosis of human tegumentary leishmaniasis

Human tegumentary leishmaniasis (HTL), characterized by skin ulcers that may spread and cause dreadful and massive tissue destruction of the nose and mouth, is considered a neglected tropical disease, and it is a serious threat to global health due to its continuous expansion, favored by the lifecycle of its causative organism that is maintained in domestic animal reservoirs and anthropophilic sand fly species. Serodiagnosis of HTL is a great challenge due to many biological factors, including hampered specificity and/or sensitivity. This investigation addresses the unmet need for new diagnostic markers of HTL, and describes a simple platform to improve the serodiagnosis. A constrained conformational phage display random peptide library combined with a magnetic microsphere-based subtraction strategy was used to identify ligands with potential diagnostic applications. Six clones were selected against IgG antibodies from HTL patients, characterized by sequencing and confirmed by a phage-ELISA using sera from patients developing visceral leishmaniasis (n=20), Chagas disease (n=10), mucosal (n=30) and cutaneous (n=20) leishmaniasis; as well as from healthy subjects living in endemic (n=20) and non-endemic (n=30) areas of leishmaniasis. A wild-type M13-phage clone and a soluble Leishmania antigenic extract were used as negative and positive controls, respectively. Three clones reached 100% sensitivity and specificity, without any cross-reactivity with sera from patients with leishmaniasis-related diseases. Briefly, we describe for the first time a set of serological markers based on three immunodominant mimotopes that showed 100% accuracy, and that could be used in a phage-ELISA assay for the HTL serodiagnosis.

Evaluation of two recombinant Leishmania proteins identified by an immunoproteomic approach as tools for the serodiagnosis of canine visceral and human tegumentary leishmaniasis

Serological diagnostic tests for canine and human leishmaniasis present problems related with their sensitivity and/or specificity. Recently, an immunoproteomic approach performed with Leishmania infantum proteins identified new parasite antigens. In the present study, the diagnostic properties of two of these proteins, cytochrome c oxidase and IgE-dependent histamine-releasing factor, were evaluated for the serodiagnosis of canine visceral (CVL) and human tegumentary (HTL) leishmaniasis. For the CVL diagnosis, sera samples from non-infected dogs living in an endemic or non-endemic area of leishmaniasis, sera from asymptomatic or symptomatic visceral leishmaniasis (VL) dogs, from Leish-Tec®-vaccinated dogs, and sera from animals experimentally infected by Trypanosoma cruzi or Ehrlichia canis were used. For the HTL diagnosis, sera from non-infected subjects living in an endemic area of leishmaniasis, sera from active cutaneous or mucosal leishmaniasis patients, as well as those from T. cruzi-infected patients were employed. ELISA assays using the recombinant proteins showed both sensitivity and specificity values of 100% for the serodiagnosis of both forms of disease, with high positive and negative predictive values, showing better diagnostic properties than the parasite recombinant A2 protein or a soluble Leishmania antigen extract. In this context, the two new recombinant proteins could be considered to be used in the serodiagnosis of CVL and HTL.

Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis.

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL.

Avaliation the therapeutic efficacy of Fluconazol on Human Tegumentary Leishmaniasis

Leishmaniasis is a group of diseases with a large spectrum, cosmopolitan, but with broad and signifiant impact in the tropical zone. Leishmaniasis presents itself in two forms, one visceral involving hemolinfopoietic system structures, other with skin and/or mucosa involvement, often without visceralizing. The latter, known as American Cutaneous Leishmaniasis (ACL) is the aim of this study. One of the major problems in the ACL is treatment procedure using injectable formulations, with risk for complications from the injection and the risk of damage to the liver and kidney function and cardiac complications. Several tests have demonstrated satisfactory results using Fluconazole®. In order to consolidate the results described in the literature, this study sought to demonstrate the therapeutic effiacy of Fluconazole® with high-dose treatment of ACL in patients from an endemic area in the Southern State of Ceara, Brazil, City of Barbalha. It’s conducted a prospective randomized study with two groups of patients. Sixty of the Group I: they were treated with 300mg or 450mg of Fluconazole® for six weeks. Sixty Group II: they were treated with Glucantime 20mg/kg/ day for 20 consecutive days. The diagnosis of ACL was performed with Imprint, culture, biopsy and histopathological stain with Giemsa and immunohistochemistry. Montenegro’s Intradermoreaction was also performed. All patients were evaluated clinically and followed up for ninety days. Continuous variables were evaluated by Student's t test, and for the correlation of variables the Pearson correlation coeffiient (r) was used. The time healing of each group had its evaluation by Kaplan-Meier method. In all tests the hypothesis α was considered signifiant when less than 5% (p <0.05). The therapeutic effiacy (TE) was calculated based on the reduction of relative risk. The study population was predominantly made up of individuals between 30 and 40 years, brown skin color, peasants, rural people, with a slight prevalence of females. The results showed therapeutic effiacy of 38.7%. The cure was related to the size of the lesion, relevant factor in the statistical p <0.0001. The lesions smaller than 30 mm (alone or sum of multiple lesions) responded promptly to Fluconazole®

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.

Towards a More Precise Serological Diagnosis of Human Tegumentary Leishmaniasis Using Leishmania Recombinant Proteins

BackgroundExposure to Leishmania induces a humoral immune response that can be used as a marker of parasite exposure.Methodology/Principal FindingsHerein, ELISA was used to screen sera from patients with Tegumentary Leishmaniasis (TL) against different L. infantum-chagasi-derived recombinant proteins (rHSP70, rH2A, rH2B, rH3, rH4 and rKMP11). Among the recombinant proteins, rHSP70 and rH2A showed the best reactivity against human sera obtained from endemic areas of TL. Receiver-Operator Characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for serodiagnosis of TL. ROC curves confirmed the superior performance of rHSP70 and rH2A, in comparison to the other tested recombinant proteins. Additionally, we evaluated the specificity of the response to rHSP70 and rH2A by testing sera obtained from patients with Chagas' disease, Tuberculosis, Leprosy or Systemic Lupus Erythematosus. In this case, rHSP70 displayed an increased ability to discriminate diseases, in comparison to SLA.ConclusionOur results raise possibility of using rHSP70 for the serodiagnosis of TL

Protective and pathologic immune responses in human tegumentary leishmaniasis

Studies in the recent years have advanced the knowledge of how host and parasite factors contribute to the pathogenesis of human tegumentary leishmaniasis. Polymorphism within populations of Leishmania from the same species has been documented; indicating that infection with different strains may lead to distinct clinical pictures and can also interfere in the response to treatment. Moreover, detection of parasite genetic tags for the precise identification of strains will improve diagnostics and therapy against leishmaniasis. On the host side, while a predominant Th1 type immune response is important to control parasite growth, it does not eradicate Leishmania and, in some cases, does not prevent parasite dissemination. Evidence has accumulated showing the participation of CD4+ and CD8+ T cells, as well as macrophages, in the pathology associated with L. braziliensis, L. guayanensis, and L. major infection. The discovery that a large percentage of individuals that are infected with Leishmania do not develop disease will help to understand how the host controls Leishmania infection. As these individuals have a weaker type 1 immune response than patients with cutaneous leishmaniasis, it is possible that control of parasite replication in these individuals is dependent, predominantly, on innate immunity, and studies addressing the ability of neutrophils, macrophages, and NK cells to kill Leishmania should be emphasized.

Leishmania (Viannia) panamensis-specific IgE and IgA antibodies in relation to expression of human tegumentary leishmaniasis.

Leishmania (Viannia) panamensis-specific IgE and IgA antibodies were quantified in patients with parasitologically confirmed American tegumentary leishmaniasis using a radioallergosorbent test (RAST) and an enzyme-linked immunosorbent assay (ELISA), respectively. The RAST values, presented as the mean +/- SEM percentage of total isotope added, were significantly elevated in patients having disease evolution greater than 12 months (3.14 +/- 0.91), as compared with those with an evolution time of 12 or fewer months (1.66 +/- 0.15) (P = 0.017). A separate group of patients, those with eosinophils in the biopsy specimen of their lesion, also had elevated mean RAST values (2.55 +/- 0.58) when compared with patients who did not demonstrate these cells in their biopsy specimens (1.32 +/- 0.24) (P = 0.038). Leishmania-specific IgA levels, presented as the mean +/- SEM optical density, were significantly higher for patients with mucocutaneous disease (0.40 +/- 0.03) than for patients with cutaneous disease (0.28 +/- 0.023) (P = 0.0063). Inhibition testing with homologous and heterologous antigens confirmed the specificity of these assays, and were used to assess cross-reactivity among L. (Viannia) subspecies and other kineto-plastic hemoparasites. Results demonstrate that patients with more severe forms of American tegumentary leishmaniasis, defined either as increased duration of disease or invasion of the mucosa, have elevated levels of Leishmania-specific IgE and IgA antibodies, respectively.

The relationship of Leishmania braziliensis subspecies and immune response to disease expression in New World leishmaniasis.

Multivariate analyses of clinical presentation, subspecies identity of the causal organism, and the Leishmania-specific immune response parameters (indirect fluorescent antibody test [IFAT], cutaneous delayed type hypersensitivity [DTH], and in vitro lymphocyte transformation [LT]) of 441 patients with tegumentary leishmaniasis were used to examine the human host-parasite interaction in L. braziliensis infection. Mucocutaneous disease (P less than .002) and L. braziliensis braziliensis infection (P less than .001) were independently associated with significantly higher IFAT titers and cutaneous DTH than were cutaneous disease or L. braziliensis panamensis infection. Lesion size was also correlated with IFAT titer (P. less than .001). Although time of lesion evolution was highly correlated with all parameters, differences associated with subspecies and disease form were independent of lesion duration (three-way analysis of variance). In contrast with the cutaneous DTH response, the in vitro lymphocyte proliferative response to Leishmania antigen did not correlate with disease form and only weakly with infecting subspecies when time of evolution and subspecies were controlled. The association of mucosal disease presentation with a particular subspecies and the independent correlation of both variables with heightened IFAT titers and cutaneous DTH to Leishmania antigen supports the possibility of immune mechanisms of pathogenesis in human tegumentary leishmaniasis.