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In Central America, infection by Leishmania (Leishmania) infantum chagasi causes visceral leishmaniasis and non‐ulcerated cutaneous leishmaniasis (NUCL). This work aimed to evaluate the participation of subpopulations of antigen‐presenting cells in skin lesions of patients affected by NUCL through double‐staining immunohistochemistry using cellular and intracellular markers. Twenty‐three skin biopsies from patients affected by NUCL were used. Histological sections stained by HE were used for histopathological study. Immunohistochemical studies were performed using primary antibodies against Langerhans cells, dermal dendritic cells, T lymphocytes, and the cytokines IL‐12, IFN‐γ, TNF‐α, iNOS, and IL‐10. The histopathological lesions were characterized by an inflammatory infiltrate, predominantly lymphohistiocytic, of variable intensity, with a diffuse arrangement associated with epithelioid granulomas and discreet parasitism. Double‐staining immunohistochemistry showed higher participation of dendritic cells producing the proinflammatory cytokine IL‐12 in relation to the other evaluated cytokines. Activation of the cellular immune response was marked by a higher density of CD8 Tc1‐lymphocytes followed by CD4 Th1‐lymphocytes producing mainly IFN‐γ. The data obtained in the present study suggest that antigen‐presenting cells play an important role in the in situ immune response through the production of proinflammatory cytokines, directing the cellular immune response preferentially to the Th1 and Tc1 types in NUCL caused by L. (L.) infantum chagasi. [ABSTRACT FROM AUTHOR]

In some central‐American countries, Leishmania (L.) infantum chagasi infection can cause non‐ulcerated or atypical cutaneous leishmaniasis (NUCL) in addition to the classic clinical form, visceral leishmaniasis (VL). Little is known about the host‐parasite relationship that can contribute to the determination of one or another clinical form. The present study had the objective to evaluate the humoral and cellular immunity in the sera of individuals affected by NUCL to improve the comprehension of this atypical host‐parasite interaction. Based on clinical and laboratory diagnosis, serum of 80 individuals was collected to evaluate the cytokines and immunoglobulins profile of NUCL (n = 47), VL patients (n = 5), and negative controls (n = 28). Cytokines were detected using Cytokine Bead Array (CBA) Human Th1/Th2/Th17 kit according to the manufacturer's instructions; class (IgG and IgM), and subclass of (IgG1 and IgG2) immunoglobulins was evaluated by ELISA using specific antigens. The concentration of TNF‐α, IFN‐γ, IL‐2 and IL‐4 cytokines in NUCL, VL and control was present below the detection threshold of CBA kit. IL‐6, IL‐10 and IL‐17A cytokines was lower in NUCL compared to LV patients. Regarding to immunoglobulins, NUCL patients produced 4.0 times more IgG than the control, while VL patients produced 6.6 times more; and IgM level was 1.6 times higher in NUCL and 2.6 times in VL patients compared to the control. Concerning the immunoglobulins subclass, only VL patients showed positive reaction for IgG1, and IgG2 did not show positive reaction among the groups. The results showed a weak cellular and humoral systemic immune response in NUCL patients. [ABSTRACT FROM AUTHOR]

Skin lesions in nonulcerated cutaneous leishmaniasis (NUCL) caused by Leishmania (L.) infantum chagasi are characterized by a mononuclear inflammatory infiltrate in the dermis, which is composed mainly of lymphocytes, followed by macrophages, few plasma cells and epithelioid granulomas with mild tissue parasitism. Previous studies have shown that the main population of lymphocytes present in the dermal infiltrate is CD8+ T cells, followed by CD4+ T cells, which are correlated with IFN‐γ+ cells. To improve the knowledge of cellular immune responses in NUCL, skin biopsies were submitted to immunohistochemistry using anti‐ROR‐γt, anti‐IL‐17, anti‐IL‐6, anti‐TGF‐β, and anti‐IL‐23 antibodies to characterize the involvement of Th17 cells in the skin lesions of patients affected by NUCL. ROR‐γt+, IL‐17+, IL‐6+, TGF‐β+ and IL‐23+ cells were observed in the dermal inflammatory infiltrate of NUCL skin lesions. A positive correlation between CD4+ T‐lymphocytes and ROR‐γt+ and IL‐17+ cells suggests that some of the CD4+ T‐lymphocytes in NUCL could be Th17 lymphocytes. Moreover, a positive correlation between ROR‐γt+ cells and TGF‐β+, IL‐6+, IL‐17+ and IL‐23+ cells could indicate the role of these cytokines in the differentiation and maintenance of Th17 lymphocytes. Our findings improve knowledge of the pathogenesis of this rare and atypical clinical form of leishmaniasis. [ABSTRACT FROM AUTHOR]

Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL. [ABSTRACT FROM AUTHOR]

Although serological assays have been widely used for the diagnosis of canine visceral leishmaniasis (CVL), they present different performances depending on the clinical profile of the dogs. This study evaluated the accuracy of serological tests, immunochromatographic (Dual Path Platform: DPP®) and enzyme-linked immunosorbent (ELISA EIE®), for CVL in relation to the detection of Leishmania DNA through real-time polymerase chain reaction (real-time PCR) in samples from symptomatic and asymptomatic dogs from a non-endemic area in the state of Rio Grande do Sul, Southern Brazil. Serum from 140 dogs (39 symptomatic and 101 asymptomatic) was tested by DPP and ELISA followed by real-time PCR. From a total of 140 samples evaluated, Leishmania DNA was detected by real-time PCR in 41.4% (58/140). Moreover, 67.2% of samples positive in real-time PCR were positive in both DPP and ELISA (39/58), showing moderate agreement between methods. In the symptomatic group, one sample non-reactive in both serological assays was positive in real-time PCR, whereas in the asymptomatic group, 17.8% non-reactive or undetermined samples in serological assays were positive in the molecular method. Leishmania DNA was not detected in 17.9% reactive samples by serological assays from the symptomatic group, and in 3.9% from asymptomatic dogs. Real-time PCR demonstrated greater homogeneity between symptomatic and asymptomatic groups compared with DPP and ELISA. The molecular method can help to establish the correct CVL diagnosis, particularly in asymptomatic dogs, avoiding undesirable euthanasia. [ABSTRACT FROM PUBLISHER]

Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates. [ABSTRACT FROM AUTHOR]

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1β, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3+CD4+IL-2+TNF-α-IFN-γ-, CD3+CD4+IL-2+TNF-α+IFN-γ-, CD3+CD4+IL-2+TNF-α-IFN-γ+, and CD3+CD4+IL-2+TNF-α+IFN-γ+ T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = -0.428, P = 0.05) and liver sizes (R = -0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = -0.595, P = 0.005) and F2 (R = -0.462, P = 0.04). Conversely, F1 and F3 increased the CD3+CD8+IL-2+TNF-α-IFN-γ-, CD3+CD8+IL-2+TNF-α+IFN-γ-, and CD3+CD8+IL-2+TNF-α+IFN-γ+ T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4+-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8+ T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4+ and CD8+ T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis. [ABSTRACT FROM AUTHOR]

Obesity is the main causal factor for metabolic syndrome and chronic systemic inflammation, which impacts on immune function and increases susceptibility to pathogens. Here, we investigated the effect of obesity on the outcome of visceral leishmaniasis caused by Leishmaniasis infantum chagasi. C57BL/6 mice fed with high-sugar and butter diet (HSB) showed a significant increase in body weight, adiposity index and morphological changes in adipocyte. To investigate the consequences of obesity on the specific immunity against Leishmania, both control and HSB diet groups were infected with 107L. infantum chagasi promastigotes in the eighth-week after diet started and euthanized 4 weeks later. HSB-diet fed mice exhibited a significantly higher parasite burden in both liver and spleen compared with control- diet group. Gonadal adipocyte tissue from HSB-diet mice showed increased TNF-α, IL-6 and leptin and diminished IL-10 production compared with control. Cytokines production analysis in the spleen and liver from these animals also demonstrated higher production of IFN-γ, TNF-α, IL-6 and nitric oxide and diminished production of IL-10 and TGF-β, which correlate with inflammatory foci and the cell hyperplasia observed. Taken together, obesity can interfere with responses to pathogen-derived signals and impair the development of protective anti-Leishmania immunity. [ABSTRACT FROM PUBLISHER]

Environment influences the composition, distribution, and behavior of the vectors and mammalian hosts involved in the transmission of visceral leishmaniasis (VL), affecting the epidemiology of the disease. In Brazil, the urbanization process and canine cases of VL are indicators for local health authorities. This study aimed to investigate the occurrence of the canine visceral leishmaniasis (CVL) in Maranhão State, Brazil. Blood samples collected from 960 dogs from six municipalities and six different ecosystems (Baixada Maranhense, Mangue, Mata dos Cocais, Amazônia, Cerrado, and Restinga) to serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescence antibody test [IFAT], and chromatographic immunoassay methods [Dual Path Platform technology, DPP®]) and parasitological diagnosis. From serological tests, 11.14% (107) of the dogs were positive for CVL, with 59.16% (568), 14.5% (148), and 131% (126) positives to ELISA, DPP, and IFAT tests, respectively. Only seven animals (0.73%) were positive in a parasitological test. We also performed parasite isolation and phylogenetic characterization. All isolates of dogs obtained from Maranhão were grouped in a single branch with Leishmania infantum chagasi from Brazil. The ecosystem Amazonia presented the highest positivity rates to CVL in serological and parasitological tests. Brazilian biomes/ecosystems suffer large degradation and may favor, depending on climatic conditions, the installation of new diseases. In the case of VL, dogs are reservoirs of parasites and sentinels for human infection. [ABSTRACT FROM AUTHOR]

Trypanosoma and Leishmania are obligate parasites that cause important diseases in human and domestic animals. Wild mammals are the natural reservoirs of these parasites, which are transmitted by hematophagous arthropods. The present study aimed to detect the natural occurrence of trypanosomatids through serological diagnosis, PCR of whole blood and blood culture (hemoculture), and phylogenetic relationships using small subunit ribosomal DNA (SSU rDNA), cytochrome b, and glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. Samples from 131 wild animals, including rodents, marsupials, and bats, were sampled in six areas in the state of Maranhão, in a transition zone of semiarid climates northeast of the equatorial humid Amazon. Serological analysis for Leishmania (Leishmania) infantum chagasi was performed in opossums by indirect fluorescent antibody test (IFAT), and all animals were serologically negative. Nine positive hemocultures (6.77%) were isolated and cryopreserved and from mammals of the Didelphimorphia and Chiroptera orders and positioned in phylogenies on the basis of sequences from different genes with reference strains of Trypanosoma cruzi marinkellei and T. cruzi. From primary samples (blood and tissues) only one bat, Pteronotus parnellii, was positive to SSU rDNA and gGAPDH genes and grouped with the L. infantum chagasi branch. The studies conducted in Maranhão State provide knowledge of parasite diversity. It is important to determine the presence of trypanosomatids in wild mammals with synanthropic habits. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a potentially fatal disease caused by Leishmania infantum chagasi in the New World. Anti-leishmanial treatment is problematic because anti-leishmanial drugs are toxic and costly and drug resistance is emerging. Moreover, there is no effective and safe vaccine approved for human use against any form of leishmaniasis. It seems likely that both a subunit vaccine and an optimized diagnostic test would benefit from the production of recombinant antigens. However, for recombinant protein production, the composition of the cell growth medium must be carefully formulated and monitored, because it may have significant metabolic effects on both the cells and protein expression. Thus, the aim of this study was to optimize the culture medium for cell growth and the expression of the 648 antigen from Leishmania i. chagasi in recombinant Escherichia coli M15. The induction allowed the expression of the 648 antigen; this was confirmed by electrophoresis, which showed a protein with a molecular mass of 24 kDa. The results showed that the higher cell concentration obtained in TB medium can be explained by the larger number of components such as amino acids and vitamin precursors contained in this complex medium, as well as glycerol as a carbon and energy source. Acetic acid excretion, detected at the end of fermentation, resulted in an inhibitory effect for both cell growth and protein production, because this was above the concentration limit of 0.9 g L. [ABSTRACT FROM AUTHOR]

Copyright of Revista de Ciências Farmacêuticas Básica e Aplicada is the property of Revista de Ciencias Farmaceuticas Basica e Aplicada and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Lipid bodies (LB; lipid droplets) are cytoplasmic organelles involved in lipid metabolism. Mammalian LBs display an important role in host-pathogen interactions, but the role of parasite LBs in biosynthesis of prostaglandin F2α (PGF2α) has not been investigated. We report herein that LBs increased in abundance during development of Leishmania infantum chagasi to a virulent metacyclic stage, as did the expression of PGF2α synthase (PGFS). The amount of parasite LBs and PGF2α were modulated by exogenous arachidonic acid. During macrophage infection, LBs were restricted to parasites inside the parasitophorous vacuoles (PV). We detected PGF2α receptor (FP) on the Leishmania PV surface. The blockage of FP with AL8810, a selective antagonist, hampered Leishmania infection, whereas the irreversible inhibition of cyclooxygenase with aspirin increased the parasite burden. These data demonstrate novel functions for parasite-derived LBs and PGF2α in the cellular metabolism of Leishmania and its evasion of the host immune response. [ABSTRACT FROM AUTHOR]

Copyright of Revista Brasileira de Higiene e Sanidade Animal is the property of Associacao Cientifica dos Medicos Veterinarios do Ceara and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (), Li-txn1 () and Li-ntf2 (). Initial vaccine validation studies were performed with the r Li-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases. [ABSTRACT FROM AUTHOR]

Summary: This prospective study was carried out from October 2003 to December 2005 and involved a cohort of 946 individuals of both genders, aged 1–89 years, from an endemic area for American visceral leishmaniasis (AVL), in Pará State, Brazil. The aim of the study was to analyze the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection represented by the following clinical-immunological profiles: asymptomatic infection (AI); symptomatic infection (SI=AVL); subclinical oligosymptomatic infection (SOI); subclinical resistant infection (SRI); and indeterminate initial infection (III). Infection diagnosis was determined by the indirect fluorescent antibody test and leishmanin skin test. In total, 231 cases of infection were diagnosed: the AI profile was the most frequent (73.2%), followed by SRI (12.1%), III (9.9%), SI (2.6%) and SOI (2.2%). The major conclusion regarding evolution dynamics was that the III profile plays a pivotal role from which the cases evolve to either the resistant, SRI and AI, or susceptible, SOI and SI, profiles; only one of the 23 III cases evolved to SI, while most evolved to either SRI (nine cases) or SOI (five cases) and eight cases remained as III. [Copyright &y& Elsevier]

Summary: The objectives of this study were to identify individuals with symptomatic and/or asymptomatic infection due to Leishmania (L.) infantum chagasi; to study the two types of infection, both clinically and immunologically, and to determine the prevalence rate of infection at the beginning of the study. This was a cross-sectional study with a cohort of 946 individuals, of both genders, from the age of 1 year, living in the municipality of Barcarena, PA, Brazil, an area endemic for American visceral leishmaniasis (AVL). The leishmanin skin test (LST) and the indirect fluorescent test (IFAT), were used for the diagnosis of infection. One hundred and twenty cases of infection were diagnosed, with a prevalence rate of 12.6%; eight cases showed high seroreactivity (1280–10240, IgG) in IFAT and no LST reaction; four of these cases were typical AVL and four had subclinical oligosymptomatic infection. Using two immunological methods with a clinical examination of the infected individuals enabled the identification of five clinical-immunological profiles which may promote a better understanding of the interaction between L. (L.) i. chagasi and the human immune response: asymptomatic infection (AI) 73.4%; subclinical resistant infection (SRI) 15%; subclinical oligosymptomatic infection (SOI) 3%; symptomatic infection (AVL) 3% and indeterminate initial infection (III) 5%. [Copyright &y& Elsevier]

Copyright of Acta Veterinaria Brasilica is the property of Acta Veterinaria Brasilica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)