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Leishmaniasis is a parasitic disease spread by the bite of an infected sandfly and caused by protozoan parasites of the genus Leishmania. Currently, there is no vaccine available for leishmaniasis in humans, and the existing chemotherapy methods face various clinical challenges. The majority of drugs are limited to a few toxic compounds, with some parasite strains developing resistance. Therefore, the discovery and development of a new anti-leishmanial compound is crucial. One promising strategy involves the use of nanoparticle delivery systems to accelerate the effectiveness of existing treatments. In this study, Amphotericin B (AmB) was incorporated into functionalized carbon nanotube (f-CNT) and evaluated for its efficacy against Leishmania major in vitro and in a BALB/c mice model. The increase in footpad thickness was measured, and real-time PCR was used to quantify the parasite load post-infection. Levels of nitric oxide and cytokines IL-4 and IFN-γ were also determined. We found that f-CNT-AmB significantly reduced the levels of promastigotes and amastigotes of the Leishmania parasite. The nanoparticle showed strong anti-leishmanial activity with an IC 50 of 0.00494 ± 0.00095 mg/mL for promastigotes and EC 50 of 0.00294 ± 0.00065 mg/mL for amastigotes at 72 h post-infection, without causing harm to mice macrophages. Treatment of infected BALB/c mice with f-CNT-AmB resulted in a significant decrease in cutaneous leishmania (CL) lesion size in the foot pad, as well as reduced Leishmania burden in both lymph nodes and spleen. The levels of nitric oxide and IFN-γ significantly increased in the f-CNT-AmB treated groups. Also, our results showed that the level of IL-4 significantly decreased after f-CNT-AmB treatment in comparison to other groups. In conclusion, our results demonstrate that AmB loaded into f-CNT is significantly more effective than AmB alone in inhibiting parasite propagation and promoting a shift towards a Th1 response., (© 2024 John Wiley & Sons Ltd.)

The golden hamster (Mesocricetus auratus) is commonly used as a promising model for Leishmania braziliensis infection developing skin-ulcerated lesions. However, different protocols using high concentration of parasites inoculated in the footpad result in severe clinical disease. Here, we further investigate the outcome of the site of infection and concentration of L. braziliensis parasites inoculated on the immunopathogenesis and clinical evolution. Initially, hamsters were infected in the ear dermis or hind footpad with a concentration of 1 × 10 5 parasites. Animals infected in the ear dermis developed a disease, with an increased parasite load that more closely resembled human cutaneous leishmaniasis lesions comparing to the group infected in the footpad. Next, we evaluated if different parasite concentrations (10 4 , 10 5 and 10 6 ) inoculated in the ear dermis would impact the course and clinical aspects of infection. Hamsters infected with 10 4 and 10 5 parasites developed mild lesions compared to the group infected with 10 6 that presented severe and persistent lesions. The parasite load varied between the different parasite concentrations. The inflammatory response was more intense when infection was initiated with 10 6 parasites accompanied by an increased initial expression of IL-4, IL-10 and arginase in the lymph node followed by expression of both pro-and anti-inflammatory cytokines comparing to groups infected with 10 4 and 10 5 parasites. In conclusion, the number of parasites inoculated, and the initial site of infection could influence the inflammatory response, and clinical presentation. Our results suggest that the ear dermis infection model induces a chronic disease that relates to immunopathological aspects of CL natural infection., (© 2022 John Wiley & Sons Ltd.)

Leishmania braziliensis is the main causative agent of American tegumentary leishmaniasis in Brazil. Current treatment includes different drugs that have important side effects and identification of cases of parasite resistance to treatment support the search for new therapeutic strategies. Recent findings have indicated that CXCL10, a chemokine that recruits and activates Th1 cells, NK cells, macrophages, dendritic cells and B lymphocytes, is a potential alternative to treat Leishmania infection. Here, we tested CXCL10 immunotherapy against experimental infection caused by an antimony-resistant isolate of Leishmania braziliensis. Following infection, mice were treated with CXCL10 for 7 days after onset of lesions. We demonstrate that mice treated with CXCL10 controlled lesion progression and parasite burden more efficiently comparing to controls. An increased IFN-γ, IL-10, TGF-β and low IL-4 production combined with a distinct inflammatory infiltrate composed by activated macrophages, lymphocytes and granulomas was observed in the CXCL10-treated group comparing to controls. However, CXCL10 and Glucantime combined therapy did not improve CXCL10-induced protective effect. Our findings reinforce the potential of CXCL10 immunotherapy as an alternative treatment against infection caused by L. braziliensis resistant to conventional chemotherapy., (© 2020 John Wiley & Sons Ltd.)

Background: Drug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non- healing Leishmania tropica isolates and on MA transfected non-healing one to PTR1., Methods: Two non-healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA-rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme-linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC 50 ) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages., Results: All three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non-healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non-healing and healing isolates (p < 0.05)., Conclusion: The results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non-healing cases. These findings have future implications in the prophylactic and therapeutic modality of non- healing Leishmania isolates to drug., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.)

Aims: The objective of this study was to determine the association of TNF-α -308 G/A, IFN-γ +874 T/A, IL-12B + 1188 A/C, IL-10 -1082 G/A and IL-4 -590 C/T polymorphisms with susceptibility to CL., Methods and Results: A total of 55 CL patients and 110 controls from Sanlıurfa province of Turkey were included to this study. Polymorphisms were genotyped by 'polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)' and 'amplification refractory mutation system-PCR (ARMS-PCR)' methods. A statistically significant difference was noted in the allele (P < .001, P = .002) and genotype (P < .001, P = .001,) frequencies of TNF-α -308 G/A and IL-4 -590 C/T, respectively. TNF-α 308 GG versus GA genotype (OR = 19.556 [95% CI 8.310-46.019] P < .001), GG versus GA + AA genotype (OR = 20.444 [95% CI 8.707-48.004] P < .001) and G versus A allele (OR = 6.968 [95% CI 3.903-12.440] P < .001) revealed significant association with CL. IL-4 -590 CC versus TT + CT genotype (OR = 2.049 [95% CI 1.025-4.096], P = .041) and C versus T allele (OR = 2.441 [95% CI 1.355-4.396], P = .002) revealed significant association with CL., Conclusion: Our study indicates that TNF-α 308 G/A and IL-4-590 C/T polymorphisms are significantly associated with susceptibility to CL. Individuals carrying A allele at TNF-α promoter -308 position and T allele at IL-4 promoter -590 position are at a higher risk for CL., (© 2020 John Wiley & Sons Ltd.)

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., (© 2020 John Wiley & Sons Ltd.)

Two species of Leishmania (L), L. tropica and L. major, are among the main causative agents of cutaneous leishmaniasis. Arginase (ARG) is an essential enzyme for cell growth, thus an attractive drug target. In this study, we tried to survey the inhibitory impact of ARG by nor-NOHA (N-ω-hydroxy-L-nor-arginine) on in vivo infection caused by L. tropica. BALB/c mice were inoculated with L. tropica EGFP-LUC (Ltrop) or L. major EGFP-LUC (Lmj) and then were treated by nor-NOHA. ARG inhibitor only indicated a delay in generation of a cutaneous lesion in inoculated footpad with nor-NOHA-Ltrop and nor-NOHA-Lmj. ARG activity has been significantly reduced in nor-NOHA-Ltrop group. In this group, ARG activity inhibition correlated with increased levels of nitric oxide (NO). In both inoculated mice with Ltrop or Lmj, parasite load showed a significant decrease at later steps during the CL course post-treatment. In vivo bioluminescence intensity did not show any ARG's inhibitory effect on treated-Ltrop. The findings verified that the ARG activity may partially control the L. tropica infection in BALB/c mice through reduction of parasite proliferation and parasite killing through NO generation. This effect is dose-dependent., (© 2019 John Wiley & Sons Ltd.)

Phenotypic and functional aspects of monocytes from Localized Cutaneous Leishmaniasis (LCL) patients were evaluated. The frequencies of monocyte subsets and TLR2/TLR4 expression were evaluated in fresh peripheral blood whereas cytokine production was evaluated in whole blood cell cultures stimulated with TLR agonists or Leishmania braziliensis antigen (Ag). CD16 + monocytes frequency was increased in patients compared with controls. A TLR4 agonist (LPS) induced expression of TNF and IL-10 in monocyte subsets of patients and controls. The CD14 + CD16 + monocytes expressed higher levels of these cytokines than CD14 + CD16 - cells. The levels of secreted TNF were higher in whole blood cell cultures from patients than controls after LPS/TLR4 or Ag stimulation. Whereas in controls there was a positive correlation between TNF and IL-10 levels, this was not observed in stimulated cell cultures from patients. The high levels of LPS-induced TNF were associated with the number of lesions and the percentages of CD14 hi CD16 + monocytes. The levels of TLR2-induced TNF were also associated with number of lesions. All monocyte subsets from patients expressed higher levels of TLR2 and TLR4 than controls. Data suggest that systemically activated monocytes contribute for an imbalance in pro- and anti-inflammatory cytokine production during LCL, participating in the immunopathogenesis of the disease., (© 2019 John Wiley & Sons Ltd.)

Aims: The polymorphism observed in Leishmania braziliensis is associated with different clinical forms of leishmaniasis. Neutrophils (PMNs) participate in the pathogenesis of leishmania infection, and here, we evaluate neutrophil function after infection with isolates of L. braziliensis from cutaneous leishmaniasis (CL) or disseminated leishmaniasis (DL) patients., Methods and Results: Neutrophils from 30 healthy subjects (HS) were infected with isolates of L. (V.) braziliensis obtained from three CL and three DL patients. They were infected at the ratio of 3:1 parasites per neutrophil, and leishmania uptake was evaluated by microscopy. The neutrophil activation markers and oxidative burst by expression of dihidrorhodamine (DHR) were evaluated by flow cytometry and cytokine production by ELISA. The frequency of infected cells and the number of amastigotes were higher in neutrophils infected with CL isolates compared to DL isolates (P < 0.05). The DHR and CD66b expression after infection with DL isolate was lower than with CL isolates. There was no difference regarding chemokine production., Conclusion: The L. (V.) braziliensis isolates of DL induced lower respiratory burst and neutrophils activation markers compared with CL isolates which may contribute to parasite survival and dissemination in DL patients., (© 2019 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

In Honduras visceral leishmaniasis and non-ulcerated or atypical cutaneous leishmaniasis (NUCL) are caused by the species Leishmania (L.) infantum chagasi. NUCL is the most common clinical form in the southern regions of the country, mainly affecting the young. In view of the lack of knowledge about the pathogenesis of the disease pattern caused by L. (L) infantum chagasi in individuals affected by NUCL, the aim of the present study was to describe in detail the histopathological features of the skin lesion caused by the parasite. Biopsies from human NUCL lesions with a positive parasitological diagnosis were collected and processed using standard histological techniques. Paraffin sections stained by haematoxylin and eosin were used to examine the histopathological alterations seen in the skin. The lesions varied between 3 and 5 mm, and the majority of the patients (60%) had a single lesion. Lesions were more frequently seen in females (65%), with an average age of 33.4 years. Microscopically, the skin lesions were characterized by mononuclear inflammatory infiltrate in the dermis composed of lymphocytes, macrophages and a few plasma cells. The intensity of the infiltration varied from discrete to intense. In both cases, the parasitic infection was discrete. Granulomas were present in 60% of cases and were associated with intense inflammation. The data revealed by the histopathological alterations in the skin of individuals affected by NUCL suggest activation of a cellular immune response that potentially controls parasite spreading., (© 2018 The Authors. International Journal of Experimental Pathology © 2018 International Journal of Experimental Pathology.)

Cutaneous leishmaniasis (CL) treatment is based on therapy with Glucantime ® , yet, there are few laboratory methods to monitor its success. In this study, ex vivo and in vitro evaluations of peripheral blood monocytes were performed in a longitudinal study to characterize the impact of Glucantime ® on overall phenotypic/functional features of these cells from CL patients to identify predictive biomarkers for post-therapeutic monitoring by flow cytometry. The ex vivo evaluation from CL patients demonstrated a modulatory profile before treatment, with a decrease in TLR-2, FcγRII, HLA-DR, CD86, IFN-γR, TNF, IL-12, NO, and an increase in FcγRIII and IL-10R. Conversely, treatment changes some of these biomarker expressions by decreasing FcγRIII and IL-10R and increasing IFN-γR, IL-12 and NO. Moreover, an in vitro analysis of these patients showed a reduced phagocytic capacity of Leishmania braziliensis and higher levels of IL-10 and TGF-β modulating functional profile. Regardless of the compromised L. braziliensis phagocytic capacity, treatment re-established the production of IL-12, IL-10, TGF-β and NO at the basal level. Notably, monocytes from patients with early cicatrization showed enhanced FcγRI and FcγRII expressions and reduced IL-10, which was further corroborated by a baseline fold change analysis. Finally, the logistic regression model emphasized the performance of FcγRI, FcγRII and IL-10 as robust predictive biomarkers for post-therapeutic cicatrization during cutaneous leishmaniasis., (© 2018 John Wiley & Sons Ltd.)

Manipulation of costimulatory and surface molecules that shape the extent of immune responses by Leishmania is suggested as one of the mechanisms of evading the host's defences. The experiments reported here were designed to evaluate the expressions of CD11b, CD11c, CD14, CD18, CD54, CD80, CD86, CD206, MHC class II and TLR-2 (Toll-like receptor 2) in human macrophages infected with L. amazonensis. Phenotypic evaluation revealed a negative modulation in CD11b, CD11c, CD14, CD18, CD54 and MHC class II molecules, depending on the level of infection. The results showed that as early as 1 hour after infection no reduction in marker expression occurs, whereas after 24 hours, downregulation of these molecules was observed in macrophages. No significant changes were observed in the expressions of CD80, CD86, CD206 and TLR2. Evidence of the differential modulation of markers expression and that after parasite uptake no reduction in surface marker expression occurs indicates that parasite internalization is not involved in the phenomena of down-modulation., (© 2018 John Wiley & Sons Ltd.)

This study aimed to identify the effects of olive leaf extract (OLE) on IFNγ, TNFα, TGFβ and nitric oxide (NO) resulted from macrophages infected with Leishmania major (L. major) amastigotes in the culture medium. High-performance liquid chromatography (HPLC) was used to analyse the level of Oleuropein in plant extract. To evaluate the immunomodulatory effects of OLE, the isolated BALB/c mice peritoneal macrophages were infected with L. major promastigotes and treated with 6.25, 12.5 and 25 μg/mL concentrations of OLE. To assess the cytokines, supernatants of cell cultures were harvested after 12, 24 and 48 hours. Cytokine production was evaluated by ELISA. Nitrite accumulation in the culture medium was assessed using the Griess reaction. The level of Oleuropein in the extract was 18.45% by HPLC. According to results, the production of IFNγ and TNFα was significantly increased when the infected and/or not infected macrophages with L. major promastigotes were affected by different concentrations of OLE. Conversely, the production of TGFβ was significantly decreased under the same conditions. Furthermore, the colorimetric determination of NO accumulation in the culture medium indicated that OLE has no effect on NO production. The study corroborates the immunomodulatory effects of OLE on L. major-infected macrophages., (© 2018 John Wiley & Sons Ltd.)

The immunopathogenesis of lupoid leishmaniasis is challenging. Although an appropriate immune response is critical for controlling these parasites, inappropriate inflammatory reactions can also promote increased pathology. The role of immune modulatory effect of the main transcription factors and cytokines of T regulatory and Th17 cells in pathogenesis of leishmaniasis chronicity was investigated in this study. The gene expression of interleukin-10 (IL-10), transforming growth factor-β (TGF-β1), forkhead box P3 (Foxp3), interleukin-17(IL-17A) and retinoic acid-related orphan receptor gamma t (ROrC) was assessed in peripheral blood mononuclear cells of eighty blood samples from cutaneous leishmaniasis (CL) patients with usual lesions (n = 31), lupoid lesions (n = 29) and healthy volunteers (n = 20). Quantitative relative real-time PCR (qRT-PCR) was performed using the Taqman and Sybergreen methods for expression of target genes. Expression of Foxp3 (P = .013), IL-10 (P < .001) and IL-17A (P < .001) was significantly higher in lupoid patient compare to the nonlupoid group. Expression of Foxp3 (P < .001), IL-10 (P < .001) and IL-17A (P = .033) was significantly more in nonlupoid subjects than in healthy volunteers, except for RORγt. These findings suggest that Foxp3 + cells, IL-10 and IL-17 play important roles in the immunopathogenesis of CL and that these roles differ depending on the causal leishmania species and different body compartments., (© 2017 John Wiley & Sons Ltd.)

Cutaneous lesions of leishmaniasis are easy to diagnose when clinically obvious or when amastigotes are numerous in the biopsy. However, this is not always the case. In difficult cases, the diagnosis of leishmaniasis requires a reliable tool to identify the microorganisms. The identification of the parasite via microscope has a superior sensitivity to that of culture, and molecular techniques, such as polymerase chain reaction (PCR), highly improve the sensitivity of the diagnosis. Alternatively, immunohistochemistry has emerged as an affordable alternative to PCR. Several laboratories have produced their own antibodies against Leishmania and seem satisfied with the results. Nevertheless, most of these antibodies are not commercialized or standardized. Pathology also welcomed the unexpected positivity of amastigotes with certain clones of anti-CD1a. The latter does not universally stain all species of Leishmania, with a low sensitivity for New World species. In conclusion, although anti-CD1a is a reliable complementary tool in the diagnosis of leishmaniasis, pathologists should familiarize themselves with one of the specific antibodies against Leishmania and globalize its use, standardizing and adapting the technique., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Positive CD1a staining of Leishmania has been reported in Old World leishmaniasis, but the sensitivity of such staining for other Leishmania species is unknown., Methods: A retrospective review was done on skin biopsies of proven cutaneous leishmaniasis based on histology, immunohistochemistry, culture and/or polymerase chain reaction (PCR). We assessed the pattern of inflammation present and assessed for CD1a (MTB1 clone) positivity in amastigotes. Patients without a clearly documented travel history to delineate Old vs New World leishmaniasis and cases without tissue for CD1a staining were excluded., Results: Various patterns of granulomatous inflammation were observed including sarcoidal (31%), diffuse (25%), suppurative and granulomatous (25%), palisaded (13%) and lichenoid (6%). CD1a staining was positive in amastigotes in 9 of 16 cases (56%). Five of 7 (71%) cases of Old World disease were CD1a positive, while 4 of 9 cases (44%) of New World cases were positive., Conclusions: Multiple patterns of granulomatous inflammation occur in cutaneous leishmaniasis. Our results confirm CD1a (MTB1 clone) can be a diagnostic adjunct to highlight amastigotes in biopsies of cutaneous leishmaniasis, with variable positivity in both Old World and New World forms of the disease. As 44% of cases were CD1a negative in our cohort, there are significant limitations to this screening approach., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries., (© 2017 John Wiley & Sons Ltd.)

Recent findings have demonstrated the suitability of interferon-gamma-induced protein 10 (IP-10) or CXCL-10 as an immunotherapy tool in treatment of leishmaniasis. This chemokine can overcome Leishmania (L.) infection through inducing nitric oxide (NO) production for parasite elimination. This study was undertaken to investigate the therapeutic effects of recombinant Leishmania tarentolae expressing CXCL-10 and an expression vector encoding CXCL-10 (pcDNA-CXCL-10-EGFP) in a model of BALB/c mice susceptible to infection by Leishmania major. The outcome of intervention was examined at 3 weeks post-treatment by evaluating the parameters of parasite burden (PB), arginase activity, NO and various cytokines such as IFN-γ, IL-4, IL-6 and IL-10. The results have shown that despite the efficacy of CXCL-10 expression vector as gene therapy, the live therapy strategy using L. tarentolae expressing CXCL-10 was more effective in terms of decreasing PB. Nitric oxide production increased, especially in the live therapy approaches. Arginase activity also decreased in all regimens, which demonstrates the potency of the treatment. The overall cytokine production shifted in favour of Th1 responses in the treated mice. Altogether, recombinant L. tarentolae expressing CXCL-10 represents a promising therapeutic strategy to improve treatment of cutaneous leishmaniasis., (© 2017 John Wiley & Sons Ltd.)

Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL., (© 2017 John Wiley & Sons Ltd.)

The inflammasome is a multiprotein signalling platform involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated gene and protein expression of the inflammasome molecules AIM2 and NLRP3 in active lesions from patients with L. (V.) braziliensis-associated tegumentary leishmaniasis (TL) and correlated these findings with the clinical presentations and responses to therapy. Real-time PCR assays showed a significantly higher AIM2 gene expression in mucosal leishmaniasis (ML) compared with that in cutaneous leishmaniasis (CL). Additionally, AIM2 mRNA expression was significantly higher in lesions from poor responders than in lesions from good responders. In situ protein quantification analyses revealed greater AIM2 expression in ML lesions than in CL lesions. The percentage of AIM2-producing cells was higher in poor responders than in good responders. Although not quite significant, IL-1β+ cells were slightly more prominent in poor responders than in good responders. Similar results were observed when patients were evaluated according to clinical form. GP63 immunostaining was identified in all samples, but no significant variation between mucosal and cutaneous lesions was observed. GP63 could be associated with reduced NLRP3 inflammasome expression in CL and ML patients. Taken together, these data demonstrate that AIM2 is an important component of the inflammasome in TL patients and is directly associated with the severity of lesions., (© 2017 John Wiley & Sons Ltd.)

Cutaneous leishmaniasis caused by Leishmania mexicana is associated with an important inflammatory response. We here analysed the kinetics of Th17 cells and neutrophils in ear lobe lesions caused by Leishmania mexicana throughout 90 days of disease progression in susceptible BALB/c and semi-resistant C57BL/6 mice infected with 1 × 10 5 Leishmania mexicana promastigotes. Cells in the lesions were extracted and quantified by flow cytometry, whereas their distribution in the tissues in relation to the parasites was analysed by immunohistochemistry. Our results show that in BALB/c mice, both Th17 cells and neutrophils increase concomitantly and to significantly higher levels on day 90 post-infection, as compared to C57BL/6 mice. Our results provide novel evidence on the cells causing chronic inflammation throughout Leishmania mexicana infections, resulting as a consequence of neutrophil recruitment together with Th17 cell differentiation and recruitment, both of which remain in the infection site throughout the late phase of the infection. We conclude that the more enhanced levels of Th17 cells and neutrophils during chronic inflammatory lesions in BALB/c mice participate in their enhanced susceptibility towards a progressive disease evolution, whereas the more controlled response of these cells in C57BL/6 mice possibly relates to the more resistant profile of this mouse strain., (© 2017 John Wiley & Sons Ltd.)

TNF-α, IFN-γ, IL-10, IL-17, CD68 and CD57 were evaluated in biopsies of patients with American cutaneous leishmaniasis living in Sorocaba, Brazil. The analyses were performed considering the time of lesions from 23 patients with recent lesions (Group I) and 19 patients with late lesions (Group II). All patients were infected with Leishmania (Viannia) braziliensis. Immunostaining cells for CD68, CD57, TNF- α, IFN-γ, IL-10 and IL-17 were performed by immunohistochemistry. Except for CD68 and IL-17, the distribution of in situ for CD57, IL-10, TNF-α and IFN-γ showed that patients with recent lesions expressed higher levels than those with late lesions. The comparison of cytokine expression/group showed that IL-10 was significantly higher than IL-17 and IFN-γ (similar data were shown in IL-17 compared with TNF-α), suggesting an immunological balance between inflammatory-anti-inflammatory agents. This balance was similar for two groups of patients. In conclusion, these data suggested that (i) patients from Group I had recent lesions (in the beginning of chronic phase) compared to those from Group II and (ii) the modulation of inflammatory response in patients with recent American cutaneous leishmaniasis was correlated with IL-10 expression in skin lesions preventing the development of mucosal forms. The parasite treatment also prevented the evolution of severe forms., (© 2017 John Wiley & Sons Ltd.)

Photodynamic therapy (PDT) has proven to be an effective alternative for the treatment of cutaneous leishmaniasis. Skin lesions consist of ulcers with well-defined raised edges, and granular floor. Th1 immune response is the protective profile in patients infected with Leishmania. In this study, the photodynamic therapy with 5-aminolevulinic acid, the parasitic load, and the modulation of the immune response was evaluated in mice infected with Leishmania braziliensis. Balb/c mice were infected with L. braziliensis and subsequently treated with three sections of PDT. The parasite load and mRNA expression of cytokines (IFN-γ, IL-4, IL-17, IL-22, IL-27, IL-10) and transcription factors (GATA-3, Foxp3 and T-bet) were analysed by quantitative PCR. The parasite load in the treated group was significantly lower than in the untreated group (P<.0001); in PDT treated animals, we observed an increase in IFN-γ and T-bet mRNA (P=.012 and P=.0071). There was a significant reduction in mRNA expression of IL-22 associated with an increased expression of IL-27 mRNA in the animals treated with light only (P=.0001). 5-ALA associated with photodynamic therapy promotes a reduction in parasite load and an increased expression of IFN-γ and T-bet mRNA., (© 2016 John Wiley & Sons Ltd.)

Cutaneous leishmaniasis in Sri Lanka is a newly established parasitic disease caused by the usually visceralizing Leishmania donovani. Skin lesions manifest as non-itchy, non-tender papules, nodules or ulcers. In situ cytokine expression provides clues for immunopathogenesis of this localized form of disease. Skin biopsies from 58 patients were analyzed for histological appearance and in situ cytokine expression of T-helper 1 (Th1) and T-helper 2 (Th2) cytokines, namely interferon (IFN)-γ, interleukin (IL)-12A, tumor necrosis factor (TNF)-α, IL-4 and IL-10 by real-time RT-PCR. Significant up-regulation of the Th1 cytokine IFN-γ and down-regulation of the Th2 cytokine IL-4 were seen in patients compared to healthy controls. Significantly elevated tissue expression of IFN-γ and TNF-α was seen in lesions that presented later than 6 months from the time of onset, while IL-4 expression was more prominent in lesions that responded poorly to antimony therapy. A prominent Th1 response appears to support resolving of lesions, whereas a Th2-biased milieu tends to favor poor responsiveness to antimony and delayed lesion healing in L. donovani infections in Sri Lanka., (© 2017 John Wiley & Sons Ltd.)

Cutaneous leishmaniasis (CL) is endemic in the northwest of Isfahan province, Iran. Increase in the incidence of the disease in Kashan has made it necessary to find out the best method for diagnosis and molecular characterization of Leishmania species. In the present study, 130 patients suspected to cutaneous leishmaniosis referred to health care centers of Kashan were examined. Serosity of lesion was collected for smear preparation and cultured in Novy-Nicolle-McNeal medium. DNA was extracted from serosity, and Leishmania species was determined by polymerase chain reaction (PCR) and nested PCR using kinetoplast DNA (kDNA) specific primers. The diagnostic criteria of CL were based on the observation of amastigotes in the smear, promastigotes in culture, presence of expected bands in PCR, or nested PCR. Of 130 specimens, 87 (66.9%), 72 (56.2%), 98 (75.4 %), 96 (73.8%), and 99 (76.2%) were positive for microscopic culture, PCR, nested PCR, and combined PCR and microscopy (proposed method), respectively. Sensitivity, specificity, positive and negative predictive values of PCR were 99%, 100%, 100%, 96.9%, respectively, for microscopy 87.9%, 100%, 100%, 72.1%, for culture 72.7%, 100%, 100%, 53.4 %, and for nested PCR 97%, 100%, 100%, 91.2%, respectively. Based on the results of the study, kDNA-PCR was the most sensitive method for diagnosis of CL., (© 2016 Wiley Periodicals, Inc.)

Previous studies have shown that Leishmania elongation initiation factor (LeIF) antigen causes a partial immunity against leishmaniasis. The antigen develops type I immunity by overexpression of inflammatory cytokines such as interleukin-12 (IL-12), IFN-γ and TNF-α. Therefore, We evaluated immune responses induced by the LeIF gene against Leishmania major infection. Immunization with LeIF gene alone or with IL-12 induced Th1 response and produced higher IFN-γ and lower IL-4 levels by splenocytes than control groups (P < 0·05) and also ratios of IFN-γ/IL-4 were 11·68 to 18·53 times more in immunized groups than control groups after challenge. In addition, analysis of humoral immune response revealed that immunized mice had more IgG2a levels than both control groups (P < 0·05). On the other hand, lesion size was less for immunized animals than control groups from 4th week after challenge (P < 0·05). The percentage reduction in lesion size was 29·30% for LeIF and 51·98% for LeIF + IL-12 than PBS at 12th week post-infection. Spleen parasite burden decreased in all immunized groups in comparison with control groups (P < 0·05). The results indicated that LeIF gene induced partial immunity against L. major infection in BALB/c mice. However, LeIF plus IL-12 group showed more potent immunity with smaller lesions than other groups., (© 2016 John Wiley & Sons Ltd.)

During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less prominent in the groups of mice infected in the presence of both SGHs when compared to the control group. The results showed that the presence of SGHs of Lu. flaviscutellata and Lu. (P.) complexus led to induction of processes that were disadvantageous to parasite establishment during infection by L. (L.) amazonensis and L. (V.) braziliensis. An inhibitory effect on Leishmania infection could be observed in both groups inoculated with SGHs, especially when the SGH from Lu. (P.) complexus was used., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Cutaneous leishmaniasis (CL) manifests as localized skin lesions, which lead to significant tissue destruction and disfigurement. In the Yucatan Peninsula, Mayan traditional healers use Pentalinon andrieuxii Muell.-Arg. (Apocynaceae) roots for the topical treatment of CL. Here, we studied the effect of P. andrieuxii root hexane extract (PARE) on the parasites and host cells in vitro and examined its efficacy in the topical treatment of CL caused by Leishmania mexicana. PARE exhibited potent antiparasitic activity in vitro against promastigotes as well as amastigotes residing in macrophages. Electron microscopy of PARE-treated parasites revealed direct membrane damage. PARE also activated nuclear factor kappaB and enhanced interferon-γ receptor and MHC class II expression and TNF-α production in macrophages. In addition, PARE induced production of the Th1 promoting cytokine IL-12 in dendritic cells as well as enhanced expression of the co-stimulatory molecules CD40, CD80, and CD86. In vivo studies showed that L. mexicana-infected mice treated by topical application of PARE resulted in the significant reduction in lesion size and parasite burden compared to controls. These findings indicate that PARE could be used as an alternative therapy for the topical treatment of CL., (Copyright © 2013 John Wiley & Sons, Ltd.)

Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post-infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6-8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti-L. amazonensis IgG and increased production of IL-10 and IL-17. Because IL-10 is a mediator of susceptibility to Leishmania infection, we blocked IL-10 signalling in neutrophil-depleted mice using anti-IL-10R. Interestingly, inhibition of IL-10 signalling abrogated the increase in parasite loads observed in neutrophil-depleted mice, suggesting that parasite proliferation is at least partially mediated by IL-10. Additionally, we tested the effect of IL-17 in inflammatory macrophages and observed that IL-17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice., (© 2013 John Wiley & Sons Ltd.)

Leishmania major is an aetiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives antileishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared with WT cells upon lipopolysaccharide (LPS) stimulation. We therefore investigated multiple signalling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1 or IRF-8 activation in WT or CD11b-deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major-mediated inhibition of IL-12 occurs through CR3 engagement; however, the mechanism of inhibition is independent of NFκB, MAPK, IRF and ETS., (© 2013 John Wiley & Sons Ltd.)

The effect of alterations in virulence and transformation by long-term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down-regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL-22, Tgad51, Cox2, IL-1, IL-10, TGF-β, TNF-α, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up-regulated Th2-associated cytokines, but down-regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host-parasite interaction., (© 2013 John Wiley & Sons Ltd.)

Leishmaniasis is an important tropical disease composed of several clinical forms that adversely affect millions of people globally. Critical cells involved in the host-Leishmania interaction are monocytes and macrophages, which act to protect against infections due to their ability to both control intracellular infections and regulate the subsequent adaptive immune response. Both soluble factors and cell surface receptors are keys in directing the immune response following interaction with pathogens such as Leishmania. Toll-like receptors (TLRs) have an essential role in immune responses against infections, but little is known about their role in human infection with Leishmania braziliensis. In this work, we evaluated peripheral blood CD14+ monocytes for the expression of immunoregulatory cytokines, co-stimulatory molecules and TLR9 from cutaneous leishmaniasis patients infected with L. braziliensis and noninfected individuals. Our results showed that patients present decreased expression of co-stimulatory molecules such as CD80 and CD86 following culture with media alone or after stimulus with soluble Leishmania antigen. Interestingly, TLR9 expression was higher after culture with soluble Leishmania antigen (SLA), suggesting a role of this molecule in immunoregulation of active disease. Lastly, higher frequencies of TLR9+ monocytes were correlated with greater lesion size. These findings demonstrate a peripheral monocytes profile compatible with important immunoregulatory potential., (© 2012 Blackwell Publishing Ltd.)

Through their receptors, prostaglandins play crucial roles in various infections. Although prostaglandin E₂ (PGE₂) is implicated as a susceptibility factor in Leishmania infection, the relative contributions of its four receptors--EP1, EP2, EP3 and EP4--to this infection remain unknown. We report that Leishmania major infection of BALB/c-derived peritoneal macrophages up-regulated EP1 and EP3 expressions but down-regulated EP2 and EP4 expressions. EP2 and EP4 agonists reduced parasite load, but EP1 and EP3 agonists increased parasite load in macrophages in vitro. Agonists of EP2 and EP4, antagonists of EP1 and EP3, or lentivirally expressed EP1-shRNA and EP3-shRNA significantly reduced parasite burden in susceptible BALB/c mice. These novel data suggest differential regulation and counteractive functions of EP receptor subsets., (© 2012 Blackwell Publishing Ltd.)

Four genotypically distinct strains of L. major collected from persons residing in different endemic areas of cutaneous leishmaniasis in Iran were evaluated in BALB/c mice. Parasite virulence was evaluated by measuring the parasite burden in the lymph nodes. Immunogenicity of the strains was assessed by analysis of cytokines mRNA expression levels in popliteal lymph nodes of the mice in early (3, 16, 40 h) and late (week 1, W3, W5 and W8) time periods after infection. The expression of cytokines mRNA, namely Ifng, Il2,Il4,Il10 and Il12, was quantitated by real-time PCR. The lowest and the highest parasite loads were induced by Damghan (2·15 × 10⁷) and Shiraz (9·59 × 10⁹) strains, respectively. Moreover, Damghan strain elicited higher expression levels of Ifng and Il2 mRNA and the highest ratio of Ifng/Il4 mRNA expression compared with the other strains at 40 h and 8 weeks post-infection. The results indicate that the inoculation of BALB/c mice with different strains induced high diversity in parasite burden and cytokines gene expression. Amongst the four strains, Damghan strain showed the lowest parasite load and the highest tendency to induce expression of Th1 cytokines gene and might be considered as a safe and immunogenic strain., (© 2012 Blackwell Publishing Ltd.)

Cutaneous leishmaniasis (CL) is caused by parasitic infection of dermal macrophages resulting in intense immune-mediated tissue inflammation and skin ulceration. The severity of the disease is dependent on parasite species as well as the immune responses evoked by the host. Most cases of CL heal spontaneously. In rare cases, the ulcer/s become chronic, and some Leishmania species may induce mucosal leishmaniasis (MCL) leading to severe tissue damage. Due to difficulties in obtaining skin tissue, most human studies of CL have been limited to the analysis of peripheral blood. While systemic responses may be good correlates of immunity, tissue damage and local immune responses at the site of infection is seldom reflected in alterations in the peripheral blood. In this review, we discuss the different forms of CL focusing on the in situ responses in established disease and the mechanisms involved in pathology and healing of Leishmania infection. Great effort has been put into animal models dissecting the immune events behind the evolution of disease, tissue pathology and parasite control. These models of genetically engineered, immune deficient mice or mice given therapy prior to onset of overt disease poorly reflect the clinical situation, where patients seek treatment once infection is well established. Models of established disease are needed to address the clinical challenge of identifying new therapeutic targets in treatment CL. Through understanding immune deviations during CL potential benefits and risks of emerging biological drugs in leishmaniasis can be addressed., (© 2012 Blackwell Publishing Ltd.)

American cutaneous leishmaniasis (ACL) has different clinical manifestations and these manifestations are dependent on the immunological status of the host. As CD4(+) and CD8(+) T cells and their mediators play a fundamental role in the host response to Leishmania and there is also a search for antigenic molecules to be used as future vaccines and tools for prognostic tests, this study characterized ACL patients' immune response after stimulation with soluble and insoluble fractions of L. (V.) braziliensis. We demonstrated a prevailing production of the Th2 cytokines, IL-4 and IL-10 and a specific production of IFN-γ and TNF-α in patients before treatment. There was also a predominance of CD4(+) T cells and a small percentage CD8(+) T cells. The insoluble antigenic fraction primarily stimulated CD4(+) T cells, while the soluble antigenic fraction showed a mixed profile, with CD4(+) T cells being the main responsible for Th2 cytokines and CD8(+) T cells for Th1 cytokines. Therefore, our results showed that a down-modulation of the Th1 type of response occurs in the initial phase of L. braziliensis disease, being the antigenic fractions capable of stimulating a specific immune response., (© 2011 Blackwell Publishing Ltd.)

Background: Transepidermal elimination has been documented in a myriad of infectious diseases; however, its occurrence in cutaneous leishmaniasis has not been evaluated., Methods: Skin biopsies (n = 212) with cutaneous leishmaniasis in Lebanon (n = 46), Syria (n = 53), Saudi Arabia (n = 45) and Pakistan (n = 68) were evaluated. Clinical data collected included age, gender, eruption type (papule, nodule, verrucous or scar), duration and anatomic location. Histopathologically, multiple parameters were recorded including Ridley's parasitic index and pattern, transepidermal elimination, interface changes, ulceration and necrosis. Transepidermal elimination was defined as the presence of amastigotes in the epidermis in all layers, limited to the basal layer or present in a perforating plug. All cases were confirmed by polymerase chain reaction (PCR) analysis followed by restriction fragment length polymorphism analysis for molecular subspeciation., Results: Leishmania tropica was identified in 88.2% and Leishmania major in 11.8% of all cases. Transepidermal elimination was observed in 28.3% of cases (29 perforating plug, 19 all layers and 12 basal layer) with a significant prevalence of L. major in this group (35 vs. 2%, p < 0.001). Cases with transepidermal elimination were associated with interface changes and higher parasitic index (p < 0.001) but not with an increased ulceration rate (p > 0.05). Multivariate analysis showed that transepidermal elimination was independently predicted by L. major [OR (95% confidence interval) = 80 (9-712); p < 0.001], parasitic index [OR = 3.4 (2.1-5.3); p < 0.001], interface changes [OR = 6.24 (2.2-17.8); p < 0.001] and necrosis [OR = 0.2 (0.1-0.8);p = 0.026]., Conclusions: We report the largest multiregional cutaneous leishmaniasis series with a 28.3% documented transepidermal elimination incidence of which 48% were perforating plug; a significant prevalence of L. major was also identified in the transepidermal elimination group. The association of transepidermal elimination with interface changes and a higher parasitic index, without an increased ulceration rate, may reflect a unique biologic alteration in the epidermis, serving to facilitate the extrusion of the parasites through the skin., (Copyright © 2012 John Wiley & Sons A/S.)

Background: Cutaneous leishmaniasis is endemic in the Middle East and North Africa. Confirming the diagnosis histologically depends on amastigote identification, which varies significantly depending on the inoculum, strain type, host response and disease stage. Accurate histological diagnosis is mandatory for appropriate therapy., Methods: Skin biopsies from 122 patients from Lebanon, Syria and Saudi Arabia with clinical diagnosis of untreated leishmaniasis were reviewed and clinical data extracted. Cases were classified according to the modified Ridley's parasitic index. DNA was extracted from formalin-fixed paraffin-embedded blocks. Polymerase chain reaction (PCR) was performed using Leishmania-specific ribosomal internal transcribed spacer 1 (ITS1-PCR). Nested ITS1-PCR was performed on cases negative for conventional ITS1-PCR. ITS1-PCR amplicons were digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation., Results: Of 122 cases, 54 (44.3%) showed a parasitic index of 0-1+ (no unequivocal amastigotes). ITS1-PCR (conventional and nested) was positive for all cases as compared with negative control tissue. RFLP identified Leishmania tropica in all cases. Patients with clinically suspected leishmaniasis, whose skin biopsies failed to detect amastigotes represented 44.3% of our cases., Conclusions: In this study, we describe a rapid and optimized protocol from DNA extraction to leishmaniasis subspeciation. ITS1-PCR showed high sensitivity and specificity in confirming clinically suspected cases., (Copyright © 2012 John Wiley & Sons A/S.)

Background: Cutaneous leishmaniasis displays considerable variation in its histopathological and clinical presentation. Clinically, it progresses from a papule into a painless ulcerated and crusted nodule/papule. Microscopically, it progresses from sheets of amastigote-filled histiocytes to granulomatous inflammation., Methods: The study was conducted on 145 skin biopsies from untreated patients with histopathological and/or clinical suspicion of cutaneous leishmaniasis in Lebanon, Syria and Saudi Arabia (1992-2010). The pre-biopsy clinical diagnosis and demographic data were collected. Biopsies were evaluated for the major microscopic pattern, and the parasitic index (PI) was also determined. Diagnosis was confirmed by polymerase chain reaction (PCR) followed by molecular sub-speciation., Results: Of the 145 patients, 125 were confirmed as cutaneous leishmaniasis by PCR. Eighteen cases presented with a pre-biopsy clinical diagnosis other than cutaneous leishmaniasis that ranged from dermatitis to neoplasm. Of the 125 cases, 57 showed a major histopathological pattern other than cutaneous leishmaniasis. Identification of amastigotes was equivocal (PI ≤1) in 38 of the 57 cases. Of interest, all the 18 cases with a pre-biopsy clinical diagnosis other than cutaneous leishmaniasis also showed atypical histopathology for cutaneous leishmaniasis., Conclusions: The manifestations of cutaneous leishmaniasis are broad and may mimic other inflammatory and neoplastic diseases. Pathologists and dermatologists should be aware of such pitfalls and can utilize PCR to confirm the diagnosis of leishmaniasis., (Copyright © 2011 John Wiley & Sons A/S.)

This study was aimed at identifying the Leishmania species using serosity materials punctured from skin lesions of cutaneous leishmaniasis (CL) patients by using internal transcribed spacer1 (ITS1) polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). We used the PCR-RFLP on 60 parasitological confirmed CL patients who referred to leishmaniasis laboratory from the School of Public Health, Tehran University of Medical Sciences. The PCR-RFLP could correctly detect 51 Leishmania species of the 60 confirmed positive specimens, where all the other 10 parasitological (microscopy and culture) negative samples that were prepared from other bacterial- and fungal-infected lesions had negative results. The results also revealed that Leishmania major was the dominant species (53.3%). This study suggests that the PCR-RFLP assay with serosity materials punctured from CL patients using Hae III enzyme is useful for the rapid identification of Leishmania species., (© 2011 Wiley-Liss, Inc.)

Skin inflammation plays an important role during the healing of American tegumentary leishmaniasis (ATL), the distribution of cells in active lesions may vary according to disease outcome and parasite antigens in ATL scars have already been shown. We evaluated by immunohistochemistry, 18 patients with 1- or 3-year-old scars and the corresponding active lesions and compared them with healthy skin. Small cell clusters in scars organized as in the active lesions spreaded over the fibrotic tissue were detected, as well as close to vessels and cutaneous glands, despite a reduction in the inflammatory process. Analysis of 1-year-old scar tissue showed reduction of NOS2, E-selectin, Ki67, Bcl-2 and Fas expression. However, similar percentages of lymphocytes and macrophages were detected when compared to active lesions. Only 3-year-old scars showed reduction of CD3(+), CD4(+) and CD8(+)T cells, in addition to reduced expression of NOS2, E-selectin, Ki67 and BCl-2. These results suggest that the pattern of cellularity of the inflammatory reaction observed in active lesions changes slowly even after clinical healing. Analysis of 3-year-old scars showed reduction of the inflammatory reaction as demonstrated by decrease in inflammatory cells and in the expression of cell-activity markers, suggesting that the host-parasite balance was only established after that period.

The diagnosis of American tegumentary leishmaniasis (ATL) is based on the visualization or isolation of the parasite, which is a time-consuming and poorly sensitive method. In this study, we evaluated the accuracy and reliability of ELISA for the diagnosis of ATL using soluble (SF) and membrane-enriched (MF) antigen fractions obtained from an infectious strain of Leishmania (Viannia) braziliensis. A total of 152 serum samples investigated at a referral center in Rio de Janeiro, Brazil, between 2005 and 2007 were studied. Each sample was tested twice with each fraction for the calculation of reliability (intraclass coefficient (ICC)). Cut-off values of 0.22 (SF) and 0.33 (MF) were defined. The use of the fractions resulted in good discrimination between patients, with a large area under the curve (P<0.0001), but no difference was observed between the two fractions (P=0.45). Sensitivity was 89.5% for each fraction, specificity was 89.5% for SF and 93.4% for MF, and the positive likelihood ratio was 8.5 for SF and 13.6 for MF. The ICCs were excellent (SF: 0.96 and MF: 0.90). The antigens tested provided precision and accuracy for the diagnosis of ATL, with SF being recommended due to its lower cost and greater practicality., (J. Clin. Lab. Anal. 24:289-294, 2010. © 2010 Wiley-Liss, Inc.)

The immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis were reviewed in the light of more recent features found in the clinical and immunopathological spectrum of American cutaneous leishmaniasis. It was shown a dichotomy in the interaction between these Leishmania species and human T-cell immune response; while L. (V.) braziliensis shows a clear tendency to lead infection from the localized cutaneous leishmaniasis (LCL), a moderate T-cell hypersensitivity form at the centre of the spectrum, toward to the mucocutaneous leishmaniasis (MCL) at the T-cell hypersensitivity pole and with a prominent Th1-type immune response, L. (L.) amazonensis shows an opposite tendency, leading infection to the anergic diffuse cutaneous leishmaniasis (ADCL) at the T-cell hyposensitivity pole and with a marked Th2-type immune response. Between the central LCL and the two polar MCL and ADCL, the infection can present an intermediary form known as borderline disseminated cutaneous leishmaniasis, characterized by an incomplete inhibition of T-cell hypersensitivity but with a evident supremacy of Th1 over Th2 immune response (Th1 > or = Th2). These are probably the main immunopathogenic competences of L. (V.) braziliensis and L. (L.) amazonensis regarding the immune response dichotomy that modulates human infection outcome by these Leishmania parasites.

In this study, we have the objective of evaluating the lymphoproliferative response and determining interferon (IFN)-gamma and interleukin (IL)-10 cytokine production in the peripheral blood mononuclear cells (PBMC) of patients with American tegumentary leishmaniasis prior and post 12 months of chemotherapy treatment with meglumine antimoniate compared with the PBMC of noninfected donors. Lymphoproliferation, such as cytokine production, was evaluated through in vitro stimulus with the soluble antigenic fraction from Leishmania (Viannia) braziliensis promastigotes (1.25 microg/ml) and Concanavalin A (2.5 microg/ml). Patients showed a significant lymphoproliferative response prior and post treatment compared with the control group. Similar result, prior to chemotherapy treatment, was observed in IFN-gamma and IL-10 production when patients were compared with the control group. After chemotherapy treatment, PBMC lymphoproliferative response of the patients revealed an increase, whereas patients have shown a decrease in IFN-gamma levels and an increase in IL-10, although without statistical difference. These results may indicate that the patients produced a specific cellular response to the soluble antigenic fraction suggesting that besides Th1 and Th2 dichotomy, immunological regulation mechanisms with the participation of memory T cells and regulatory T cells could be present in the clinical evolution of these patients. This understanding will allow the study and identification of new L. (V.) braziliensis molecules potentially candidates to vaccines., (Copyright 2009 Wiley-Liss, Inc.)

In the conventional mouse model for cutaneous leishmaniasis involving infection with stationary phase Leishmania major promastigotes at the base of the tail, mice congenic for leishmaniasis resistance loci designated lmr1,2,3 cured their lesions more rapidly and laid down more ordered collagen fibres than the susceptible parental BALB/c mice, while the opposite was the case for the congenic mice carrying the susceptibility loci on the resistant C57BL/6 background. In that model, we showed that wound healing and not T cell responses played a major role in determining the resolution of skin infection. Here, we show a similar disease phenotype in the mouse model that mimics more closely the situation in humans, that is, strictly intradermal infection in the ear pinna with small numbers of metacyclic promastigotes. The data show that at the site of infection the innate and adaptive immune responses act in concert to clear parasites, and induce tissue repair and wound healing. Importantly, the data show that the host responses controlled by the lmr loci, which act locally to control infection in the skin, are distinct from the host responses operating systemically in the draining lymph node.

In humans, Leishmania chagasi parasites can produce subclinical infections, atypical cutaneous leishmaniasis (ACL) and visceral leshmaniasis that is potentially fatal if not treated in a timely fashion. L. chagasi parasites that cause both ACL and visceral disease appear to be genetically similar, which suggests that host factors such as the immune response play an important role in controlling infection. We evaluated the immunologic response in ACL using peripheral blood mononuclear cells (PBMCs) of 37 subjects divided into three groups: (i) active ACL cases, (ii) asymptomatic cases and (iii) persons with no history of Leishmania infection. The supernatants of stimulated PBMCs were analysed for production of IL-10, IFN-gamma and IL-2. Robust production of IL-10 in response to Leishmania stimulation was observed in active ACL cases, compared to low levels in asymptomatic cases and negative controls. Serum IgE levels, measured by ELISA, were not significantly different among the three groups. In addition, ACL cases displayed depressed levels of all cytokines in response to mitogen. Thus, this first characterization of the immune response in ACL suggests a role for IL-10 as well as partial immunosuppression.

The chemokine CCL2 (MCP-1) and its receptor CCR2 modulate leucocyte migration and T helper differentiation. CCL2 or CCR2 knockout (KO) mice have divergent phenotypes following infection with the intracellular parasite Leishmania major (L. major). Compared to wild-type (WT) mice, intradermally infected CCR2 KO mice in the L. major-resistant C57BL/6j background become susceptible and fail to generate protective Th1 responses. In contrast, subcutaneously infected CCL2 KO mice in the L. major-susceptible BALB/c background are resistant and exhibit reduced pathogenic Th2 responses. Here we explore two variables that may account for this contrasting outcome, namely background strain and route of infection. We found that the CCR2-null state, both in the BALB/c and the C57BL/6j background, was associated with increased susceptibility to intradermal or subcutaneous L. major infection. Notably, the CCL2-null state did not change the ability of C57BL/6j mice to mount protective responses following intradermal infection. Dual genetic inactivation of CCR2 and CCL2 in the L. major-resistant C57BL/6j background resulted in a shift to a susceptible phenotype analogous to that of CCR2 KO in the C57BL/6j background. We concluded that CCL2-independent effects of CCR2 are indispensable for the control of L. major infection and the generation of protective immune responses.

We report the rare instance of four family members with numerous cutaneous lesions of Leishmania major contracted while on holiday in Algeria. Treatment was successful with oral itraconazole for the children and intralesional sodium stibogluconate for the mother. Cutaneous leishmaniasis should be considered in those with apparently sterile plaques returning from endemic areas. These results suggest that itraconazole, which is ideally suited for use in children, is an effective monotherapy for L. major.