Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.

Ascorbate peroxidase overexpression protects Leishmania braziliensis against trivalent antimony effects

Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the trivalent antimony (SbIII)-resistance phenotype. Western blot results demonstrated that APX-overexpressing parasites had higher APX protein levels in comparison with the wild-type line (LbWTS). APX-overexpressing clones showed an 8-fold increase in the antimony-resistance index over the parental line. In addition, our results indicated that these clones were approximately 1.8-fold more tolerant to H2O2 than the LbWTS line, suggesting that the APX enzyme plays an important role in the defence against oxidative stress. Susceptibility tests revealed that APX-overexpressing L. braziliensis lines were more resistant to isoniazid, an antibacterial agent that interacts with APX. Interestingly, this compound enhanced the anti-leishmanial SbIII effect, indicating that this combination represents a good strategy for leishmaniasis chemotherapy. Our data demonstrate that APX enzyme is involved in the development of L. braziliensis antimony-resistance phenotype and may be an attractive therapeutic target in the design of new strategies for leishmaniasis treatment.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

effect of antimony on the vision, intraocular pressure and hair growth in a sample of Yemenis with myopia

Clinical trials were performed on a sample of twenty-six Yemenis with myopia to study the effect of antimony eye definer [painter] on vision and intraocular pressure [IOP]. In the first trial each patient applied traditional eye definer containing lead daily for three months, in the second trial each patient applied antimony eye definer daily for three months. Vision parameters including the IOP were measured before and after each trial, and a washout period of one month was kept between the two trials. The results showed that antimony eye definer did not change the power of spherical lenses in both eyes, while it significantly improved the power of cylindrical lenses by 20% in the right eye and 23% in the left eye. Moreover it lowered the IOP by 9% in both eyes, these results were statistically significant. The traditional eye definer containing lead did not change these parameters in the patients. Another trials were formed to the study the effect of antimony ointment 5% on human scalp hair growth, in another sample of healthy volunteers, the results revealed that this ointment has stimulated hair growth by 56%, while the standard trichogenic agent minoxidil 5% solution improved hair growth by 62%. In rabbits the effects were more dramatic, where antimony cream stimulates hair growth by 97%, while the standard trichogenic agent minoxidil 5% solution improved hair growth by 78%. Finally antimony did not show antibacterial effects when tested on streptococci [gram+ve] and E coli [gram-ve]

Studies of the leishmanin skin test positivity in cases with treatment anti-Leishmania

Leishmanin skin test (LST) was applied in 115 (40.5, percent) females and 169 (59.5 percent) males who suffered from cutaneous leishmaniosis (CL), with 1 to 7 cutaneous lesions, with > 211 days of evolution (mean= 47; DE= 36.7). Their ages ranged from 0 to 75 years (mean= 21). The clinical, parasitological, serological and immunological diagnosis of CL were studied. The LST reactivity was compared with respect to years age, sex, number and evolution of the lesion, before and after the treatment with glucantime (G) injected intramuscularly (IM). Second and third injections the G were necessary in 61.97 percent and 12.76 percent of the patients respectively. The time required for healing ranged from 30 to 120 days. The diameter of the induration was of 5 to 27 mm before the treatment, and up to 27 mm of induration when no clinicas manifestations were observed in all cases of CL. A higher proportion of positive skin test was obtained in the > 8 years age group (79.22 percent) than in the younger group. The relatively higher prevalence of LST in children from 1 to 10 years correlates with the percentage distribution of positive parasitaemias, and is associated with the mafe patients. These findings suggest that it is the intensity rather than the simple occurrence of parasitaemia which determine, the association into the LST and number and evolution of lesion in these cases of CL. The frequency of LST positivity was higher in individual males (55.63 percent). When these infections were grouped according to years age, sex and time of infection, and relationship with LST, the differences were no longer significant

In vitro activity of meglumine antimoniate, a pentavalent antimonial drug, on Leishmania promastigotes

The activity of a pentavalent antimonial drug glucantime on the growth of promastigote forms of Leishmania strains involved in South American cutaneous and mucocutaneous leishmaniasis was investigated. A marked difference in susceptibility to glucantime among four different strains and cloned lines obtained from a single strain was observed. For the sensitive strains (L.braziliensis M2903 and L. guyanensis M1176), the cell growth was reduced in a dose-dependent manner for drug concentrations at a range of 0.23 to 23 mM. However, despite the relative sensitivity of the assay, no significant increase of effect was observed in the presence of higher drug concentrations. For the resistant strains (L. amazonensis M10996 and L. braziliensis LYB259) a dose-response line is obtained at a higher concentration range (20 mM to mM). The influence of the drug on surface properties, respiratory activity and incorporation of radiolabelled leucine by a sensitive strain - L-guyanensis M1176-was studied as an approach to its site of action. Despite the increased intensity of self-aglutination for cells growing in the presence of glucantime, no significant change was observed in electrophoretic mobility or Concanavian A reactivity. Since the oxygen uptake of glucose-stimulated promastigotes was only slightly reduced in the presence of 23 mM glucantime at 28-C, the reduction was not sufficient to explain the total growth inhibition observed. A significant decrease of 14C-leucine incorporation into the cold TCA-insoluble fraction of drug-treated cells was observed within the same concentration range that reduces promastigote growth

Ensaio terapêutico com glucantime em sagüis (Callithrix jacchus) infectados com uma cepa de Leishmania donovani aparentemente resistente ao tratamento / Therapeutic assay with glucantime in marmosets (Callithrix jacchus) infected with a strain of Leishmania donovani apparently resistent to treatment

Os autores relatam o resultado de um ensaio terapêutico em sangüis com uma cepa de Leishmania donovani isolada de um caso humano fatal de calazar, clinicamente resistente aos antimoniais (Glucantime e Pentostam), Afotericina B e Pentamidina. Os testes cutâneos realizados no paciente para avaliaçäo da imunidade celular foram negativos à exceçäo do DNCB a 2%. Quatro sangüis adultos (Callithrix jacchus) foram inoculados por via intraperitoneal com uma suspensäo de formas amastigotas de L. donovani. Duzentos e dez dias após, todos os animais mostravam formas amastigotas do protozoário, evidenciadas através de punçäo hepática por aspiraçäo. Iniciou-se entäo um esquema terapêutico com glucantime (28 mgSbV/kg/dia) em três séries de 10 dias com cinco dias de intervalo entre elas em três dos primatas, ficando o quarto animal como controle. Nos três animais tratados houve cura parasitológica da doença, o mesmo näo ocorrendo com o controle. O fato de a amostra de L. donovanir ter sido, no paciente, resistente aos vários tratamentos e ter sido sensível em modelo experimental à terapêutica com glucantime, sugere a possibilidade de que fatores imunológicos do paciente possam ter contribuído para a evoluçäo fatal da doença