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1.
Roger Magno Macedo-Silva Carina de Lima Pereira dos Santos Vanessa Alvaro Diniz Jorge José de Carvalho Camila Guerra Suzana C?rte-Real 《Memórias do Instituto Oswaldo Cruz》2014,109(1):61-69
Fibrocytes are important for understanding the progression of many diseases because
they are present in areas where pathogenic lesions are generated. However, the
morphology of fibrocytes and their interactions with parasites are poorly understood.
In this study, we examined the morphology of peripheral blood fibrocytes and their
interactions with Leishmania (L.) amazonensis . Through
ultrastructural analysis, we describe the details of fibrocyte morphology and how
fibrocytes rapidly internalise Leishmania promastigotes. The
parasites differentiated into amastigotes after 2 h in phagolysosomes and the
infection was completely resolved after 72 h. Early in the infection, we found
increased nitric oxide production and large lysosomes with electron-dense material.
These factors may regulate the proliferation and death of the parasites. Because
fibrocytes are present at the infection site and are directly involved in developing
cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based
therapies that control and treat leishmaniasis. 相似文献
2.
Lilian Motta Cantanhêde Cipriano Ferreira da Silva Júnior Marcos Massayuki Ito Kátia Paula Felipin Roberto Nicolete Juan Miguel Villalobos Salcedo Renato Porrozzi Elisa Cupolillo Ricardo de Godoi Mattos Ferreira 《PLoS neglected tropical diseases》2015,9(9)
Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia''s Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71.1% (n = 27) mucosal lesions were positive for LRV1, and 28.9% (n = 11) were negative. In cutaneous lesions, 36.7% (n = 40) were positive and 63.3% (n = 69) were negative for LRV1. The ratio P(ML|LRV1+)/P(ML|LRV1-) was 2.93 (CI95% 1.57…5.46; p<0.001), thus corroborating the hypothesis of the association between LRV1 and the occurrence of mucosal leishmaniasis, as previously described in animal models; it also indicates that LRV1 is not the only factor contributing to the disease outcome. 相似文献
3.
Carla Maia Lídia Dionísio Maria Odete Afonso Luís Neto José Manuel Cristóv?o Lenea Campino 《Memórias do Instituto Oswaldo Cruz》2013,108(4):481-487
The Algarve Region (AR) in southern Portugal, which is an
international tourist destination, has been considered an endemic region of
zoonotic leishmaniasis caused by Leishmania infantum since the
1980s. In the present study, phlebotomine and canine surveys were conducted to
identify sandfly blood meal sources and to update the occurrence of
Leishmania infection in vectors and dogs. Four sandfly
species were captured: Phlebotomus perniciosus,
Phlebotomus ariasi, Phlebotomus sergenti
and Sergentomyia minuta. In one P. perniciosus
female, L. infantum DNA was detected. Blood meal tests showed
that this species had no host preferences and was an opportunistic feeder. An
overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the
seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that
attended veterinary clinics. The simultaneous occurrence of dogs and P.
perniciosus infected with L. infantum in the AR
indicates that the region continues to be an endemic area for CanL. Our results
reinforce the need for the systematic spatial distribution of phlebotomine
populations and their Leishmania infection rates and the need
to simultaneously perform pathogen monitoring in both invertebrate and
vertebrate hosts to investigate the transmission, distribution and spreading of
Leishmania infection. 相似文献
4.
5.
Fernando Real Ramon Oliveira Vidal Marcelo Falsarella Carazzolle Jorge Maurício Costa Mondego Gustavo Gilson Lacerda Costa Roberto Hirochi Herai Martin Würtele Lucas Miguel de Carvalho Renata Carmona e Ferreira Renato Arruda Mortara Clara Lucia Barbiéri Piotr Mieczkowski José Franco da Silveira Marcelo Ribeiro da Silva Briones Gon?alo Amarante Guimar?es Pereira Diana Bahia 《DNA research》2013,20(6):567-581
We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment. 相似文献
6.
Adolfo Vásquez Trujillo Angélica E González Reina Agustín Góngora Orjuela Edgar Prieto Suárez Jairo Enrique Palomares Luz Stella Buitrago Alvarez 《Memórias do Instituto Oswaldo Cruz》2013,108(4):463-469
Lutzomyia antunesi has been commonly reported in outbreaks of
cutaneous leishmaniasis (CL) in the Orinoquia region of Colombia. The bionomics
of this species were studied in the municipality of Villavicencio (Meta,
Colombia). Sandflies were captured over the course of one week per month for one
year in intradomiciliary, peridomiciliary and extradomiciliary housing areas.
The captures were performed from 06:00 pm-06:00 am using CDC light traps and the
females were processed for polymerase chain reaction (PCR) to detect
Leishmania spp. A total of 22,097 specimens and 19 species
were captured of which Lu. antunesi (89%) and Lutzomyia
walkeri (5%) were the most abundant. Other species recognised as
anthropophilic (Lutzomyia panamensis, Lutzomyia
gomezi, Lutzomyia flaviscutellata and
Lutzomyia fairtigi) were present in very low abundance
(< 2%). Natural infection with Leishmania spp was detected using PCR in
Lu. antunesi, Lu. panamensis and
Lu. flavicutellata, showing infection rates of 1%, 4.8% and
7.5%, respectively. The present paper provides information on various ecological
aspects of Lu. antunesi. An analysis of seasonality shows that
this species increases in abundance in the hottest months (December, January and
February), directly correlating with the maximum temperature and inversely
correlating with precipitation. The natural infection rate is associated with
the peaks of highest abundance. 相似文献
7.
8.
Braulio Mark Valencia Rachel Lau Ruwandi Kariyawasam Marlene Jara Ana Pilar Ramos Mathilde Chantry Justin T. Lana Andrea K. Boggild Alejandro Llanos-Cuentas 《PLoS neglected tropical diseases》2022,16(1)
American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL. 相似文献
9.
Aliehsan Heidari Mehdi Mohebali Kourosh Kabir Hojatallah Barati Yousef Soultani Hossein Keshavarz Behnaz Akhoundi Homa Hajjaran Hosein Reisi 《The Korean journal of parasitology》2015,53(4):379-383
Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of ≥1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P<0.002). Two children with anti-Leishmania infantum antibodies titers of ≥1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran. 相似文献
10.
Haroun Zangger Catherine Ronet Chantal Desponds F. Matthew Kuhlmann John Robinson Mary-Anne Hartley Florence Prevel Patrik Castiglioni Francine Pratlong Patrick Bastien Norbert Müller Laurent Parmentier Nancy Gore Saravia Stephen M. Beverley Nicolas Fasel 《PLoS neglected tropical diseases》2013,7(1)
Background
Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.Methodology/Principal Findings
This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.Conclusions/Significance
We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis. 相似文献11.
Mary-Anne Hartley Eliane Bourreau Matteo Rossi Patrik Castiglioni Remzi Onur Eren Florence Prevel Pierre Couppié Suzanne M. Hickerson Pascal Launois Stephen M. Beverley Catherine Ronet Nicolas Fasel 《PLoS pathogens》2016,12(9)
Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5–10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients. 相似文献
12.
Giselle Aparecida Fagundes-Silva Gustavo Adolfo Sierra Romero Elisa Cupolillo Ellen Priscila Gadelha Yamashita Adriano Gomes-Silva Jorge Augusto de Oliveira Guerra Alda Maria Da-Cruz 《Memórias do Instituto Oswaldo Cruz》2015,110(6):797-800
In the Brazilian Amazon, American tegumentary leishmaniasis (ATL) is endemic and
presents a wide spectrum of clinical manifestations due, in part, to the circulation
of at least seven Leishmania species. Few reports of
Leishmania (Viannia) naiffi infection suggest that its occurrence
is uncommon and the reported cases present a benign clinical course and a good
response to treatment. This study aimed to strengthen the clinical and
epidemiological importance of L. (V.) naiffi in the Amazon Region
(Manaus, state of Amazonas) and to report therapeutic failure in patients infected
with this species. Thirty Leishmania spp samples isolated from
cutaneous lesions were characterised by multilocus enzyme electrophoresis. As
expected, the most common species was Leishmania (V.) guyanensis (20
cases). However, a relevant number ofL. (V.) naiffi patients (8
cases) was observed, thus demonstrating that this species is not uncommon in the
region. No patient infected withL. (V.) naiffi evolved to
spontaneous cure until the start of treatment, which indicated that this species may
not have a self-limiting nature. In addition, two of the patients experienced a poor
response to antimonial or pentamidine therapy. Thus, either ATL cases due to
L. (V.) naiffi cannot be as uncommon as previously thought or
this species is currently expanding in this region. 相似文献
13.
Rohoušová I Hostomská J Vlková M Kobets T Lipoldová M Volf P 《International journal for parasitology》2011,41(5):481-485
Under laboratory conditions, hosts exposed twice to sand fly saliva are protected against severe leishmaniasis. However, people in endemic areas are exposed to the vector over a long term and may experience sand fly-free periods. Therefore, we exposed mice long- or short-term to Phlebotomus duboscqi bites, followed by Leishmania major infection either immediately or after a sand fly-free period. We showed that protection against leishmaniasis is limited to short-term exposure to sand flies immediately before infection. Our results may explain the persistence of leishmaniasis in endemic areas and should be taken into account when designing anti-Leishmania vaccines based on sand fly saliva. 相似文献
14.
Everton Falc?o de Oliveira Aline Etelvina Casaril Nathália Lopes Fontoura Mateus Paula Guerra Murat Wagner Souza Fernandes Elisa Teruya Oshiro Alessandra Gutierrez de Oliveira Eunice Aparecida Bianchi Galati 《Memórias do Instituto Oswaldo Cruz》2015,110(8):1051-1057
Studies on natural infection by Leishmania spp of sandflies
collected in endemic and nonendemic areas can provide important information on the
distribution and intensity of the transmission of these parasites. This study sought
to investigate the natural infection by Leishmaniain wild female
sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do
Sul (Brazil) between October 2012-March 2014, and dissected to investigate
flagellates and/or submitted to molecular analysis to detect
Leishmania DNA. A total of 1,164 females (77.56% of which were
Lutzomyia cruzi) representing 11 species were investigated using
molecular analysis; 126 specimens of Lu. cruziwere dissected and
also submitted to molecular analysis. The infection rate based on the presence of
Leishmania DNA considering all the sandfly species analysed was
0.69%; only Leishmania (Leishmania) amazonensis was identified in
Lu. cruzi by the molecular analysis. The dissections were
negative for flagellates. This is the first record of the presence of L. (L.)
amazonensis DNA in Lu. cruzi, and the first record of
this parasite in this area. These findings point to the need for further
investigation into the possible role of this sandfly as vector of this parasite. 相似文献
15.
Veruska Nogueira de Brito Arleana do Bom Parto Ferreira de Almeida Luciano Nakazato Rosemere Duarte Cladson de Oliveira Souza Valéria Régia Franco Sousa 《Memórias do Instituto Oswaldo Cruz》2014,109(7):899-904
Visceral leishmaniasis (VL) in Brazil is transmitted by the phlebotomine
Lutzomyia longipalpis and in some midwestern regions by
Lutzomyia cruzi. Studies of the phlebotomine fauna, feeding
habits and natural infection rate by Leishmania contribute to
increased understanding of the epidemiological chain of leishmaniases and their
vectorial capacity. Collections were performed in Jaciara, state of Mato Grosso from
2010-2013, during which time 2,011 phlebotomines (23 species) were captured (68.70%
Lu. cruzi and 20.52% Lutzomyia whitmani).
Lu. cruzi females were identified by observing the shapes of the
cibarium (a portion of the mouthpart) and spermatheca, from which samples were
obtained for polymerase chain reaction to determine the rates of natural infection.
Engorged phlebotomines were assessed to identify the blood-meal host by ELISA. A
moderate correlation was discovered between the number of Lu. cruzi
and the temperature and the minimum rate of infection was 6.10%. Twenty-two females
were reactive to the antisera of bird (28%), dog (3.30%) and skunk (1.60%). We
conclude that Lu. cruzi and Lu. whitmani have
adapted to the urban environment in this region and that Lu. cruzi
is the most likely vector of VL in Jaciara. Moreover, maintenance of
Leishmania in the environment is likely aided by the presence of
birds and domestic and synanthropic animals. 相似文献
16.
Luana Leandro Gois Sanjay Mehta Maria Zilma Andrade Rodrigues Robert T Schooley Roberto Badaró Maria Fernanda Rios Grassi 《Memórias do Instituto Oswaldo Cruz》2014,109(1):9-14
The effects of human immunodeficiency virus (HIV) on the immune response in patients
with cutaneous leishmaniasis have not yet been fully delineated. This study
quantified and evaluated the function of memory T-cell subsets in response to soluble
Leishmania antigens (SLA) from patients coinfected with HIV and
Leishmania with tegumentary leishmaniasis (TL). Eight TL/HIV
coinfected subjects and 10 HIV seronegative subjects with TL were evaluated. The
proliferative response of CD4+and CD8+T-cells and naïve, central memory (CM) and
effector memory (EM) CD4+T-cells in response to SLA were quantified using flow
cytometry. The median cell division indices for CD4+and CD8+T-cells of coinfected
patients in response to SLA were significantly lower than those in patients with
Leishmania monoinfection (p < 0.05). The proportions of CM and
EM CD4+T-cells in response to SLA were similar between the coinfected patients and
patients with Leishmania monoinfection. However, the median CM and
EM CD4+T-cell counts from coinfected patients were significantly lower (p < 0.05).
The reduction in the lymphoproliferative response to Leishmania
antigens coincides with the decrease in the absolute numbers of both EM and CM
CD4+T-cells in response to Leishmania antigens in patients
coinfected with HIV/Leishmania. 相似文献
17.
Cutaneous leishmaniases present similar clinical appearances, but differing prognosis in the course of infection. Ulcers caused by parasites of the subgenus Viannia are more aggressive than ulcers caused by parasites of the subgenus Leishmania. Another problem is distinguishing between true Leishmania infection and other skin diseases in endemic areas, where cutaneous lesions and a single positive Montenegro intradermal test are enough to submit patients to specific treatment for cutaneous leishmaniasis. This study evaluated the efficacy of PCR in detecting in Leishmania in patients with cutaneous lesions. Leishmania (V.) braziliensis complex was determined by a primer pair from the multicopy spliced leader RNA. The results were compared to those of traditional methods. We analyzed biopsies of 109 patients with cutaneous lesions in the second most endemic region of Sao Paulo State, Brazil. Definitive diagnosis was established by clinical and “consensus laboratory criteria” (positive culture, stained tissue smears or PCR). Of 52 patients with cutaneous leishmaniasis, 96% had positive PCR, 69%, positive parasitological tests and 100%, positive Montenegro intradermal tests. Histopathological examination (only in 32 samples) were positive in 14 samples, suggestive in 14 and negative in 4 samples. All 57 patients with other etiologies had negative results in parasitological methods, PCR and histopathological examination (in 39 samples), but Montenegro intradermal tests were positive in 35%. PCR was highly sensitive and specific for L. (V.) braziliensis complex detection compared with other laboratory methods. Despite the specificity of the parasitological tests, the sensitivity was less than 70%. Montenegro intradermal reaction was highly sensitive, but with low specificity, only 65%. As suggestive results in histopathological examinations were shown in 14 samples, it was difficult to determine the true result. PCR applied to biopsies proved to be useful for differential diagnosis of cutaneous lesions of other etiologies in patients living in endemic areas. The advantages are most striking in clinical specimens with scarce amastigotes for which conventional methods have low sensitivity and should be considered for clinical and epidemiological patterns. On the other hand, both Montenegro intradermal test and parasitological methods are only modestly effective in cutaneous leishmaniasis diagnosis. 相似文献
18.
Elsy Nalleli Loría-Cervera Erika Ivett Sosa-Bibiano Liliana Estefanía Villanueva-Lizama Nicole Raymonde Van Wynsberghe Silvia Beatriz Canto-Lara José Luis Batún-Cutz Fernando José Andrade-Narváez 《Memórias do Instituto Oswaldo Cruz》2013,108(2):172-177
Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 102
and 2.5 x 106
promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection. 相似文献
19.
20.
Surendra K. Jain Rajnish Sahu Larry A. Walker Babu L. Tekwani 《Journal of visualized experiments : JoVE》2012,(70)
Leishmaniasis is one of the world''s most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished). 相似文献