Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. The Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.

Current treatment of cutaneous leishmaniasis includes pentavalent antimonials as first-line drugs, but this therapy has shown severe adverse effects. An alternative to minimize this issue is based on combination therapy scheme with other drugs. In this study we analyzed the potential of the association of meglumine antimoniate (MA) with the oxiranes epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW). Results demonstrated that association between these drugs enhanced leishmanicidal activity on Leishmania (Leishmania) amazonensis infection. The compounds were tested in monotherapy or in combinations (3:1; 1:1 and 1:3) and reduced intracellular parasite numbers, measured by the endocytic index, in all tested conditions. The most effective combination regimens were MA/LAP or MA/LAW in 3:1 ratio, which achieved a reduction of 98.3% and 93.6% in the endocytic index, respectively. BALB/c mice challenged with L. (L.) amazonensis showed significant reduction in lesion size and parasite load in both footpad and lymph nodes, after four weeks of treatment. Although, MA, LAP or LAW monotherapy were able to control the evolution of lesions when compared to untreated animals (30%, 40% and 40% of reduction, respectively), the combination of MA/LAP and LAW in 3:1 ratio showed better results reducing 61.7 and 54.4%, respectively. The results indicate that the association of meglumine antimoniate to oxiranes lead to an increment in the antileishmanial activity and represent a promising approach for the cutaneous leishmaniasis treatment.

Background: Leishmania (Vianna) braziliensis,Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasiare important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction betweenLeishmaniaspp. promastigotes and Lulo cellsin vitro, specifically focusing on adhesion events occurring between threeLeishmaniaspecies and this cell line.Methods: Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy.Findings: The results indicate that species from the subgeneraLeishmaniaandVianniahave great potential to adhere to Lulo cells. The highest adherence rate was observed forL. (L.) chagasiafter 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed byL. (L.) amazonensis(16.0 ± 0.7%) andL. (V.) braziliensis(3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells withLeishmania (L.) chagasishowed the participation of cytoplasmic projections from the former closely associating the parasites with the cells.Conclusions: We present evidence that Lulo cells can be useful in studies of insect-parasite interactions forLeishmaniaspecies.

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min⁻¹ mg of protein⁻¹ and Suc-AFK-AMC = 252 ± 20 µmol.min⁻¹ mg of protein⁻¹) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min⁻¹ mg of protein⁻¹ and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min⁻¹ mg of protein⁻¹). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.

The protein profiles of the New Guinea "C" dengue virus type 2 (DENV-2)prototype and those of a Brazilian DENV-2 isolated in the State of Rio de Janeiro in 1995 were compared. SDS-PAGE analysis showed that the virus from Rio de Janeiro expresses NS5 (93.0 kDa), NS3 (66.8 kDa) E (62.4 kDa) and NS1 (41.2 kDa) proteins differently from the New Guinea "C" virus. The immunoblot revealed specificity and antigenicity for the NS3 protein from DENV-2 Rio de Janeiro mainly in primary infections, convalescent cases, and in secondary infections in both cases and only antigenicity for E and NS1 proteins for both viruses in primary and secondary infections.

Background/Objectives: Cutaneous leishmaniasis (CL) remains a global health challenge, with treatment options often limited by drug resistance and systemic toxicity. Amphotericin B (AmB) represents a promising alternative. but intravenous administration causes severe systemic adverse effects. Despite growing interest in topical therapies, knowledge gaps remain regarding the comparative efficacy of delivery systems, including the influence of treatment timing and potential intrinsic effects. This study aimed to develop and characterize different topical AmB formulations (polymeric nanoparticles (PCL-AmB), a lipid-based (Oil_AmB) formulation, and a gel emulsion) to evaluate their in vivo efficacy against CL in a murine model, considering treatment initiation timing and potential intrinsic effects of the delivery systems. Methods: Formulations were prepared and characterized in terms of hydrodynamic size, polydispersity index, and AmB content. Antileishmanial activity was assessed in two independent in vivo experiments, with topical monotherapy administered five days per week for four weeks, starting either 10 or 30 days post-infection, representing early and established chronic stages of infection, respectively. Results: All formulations exhibited nanoscale dimensions and high homogeneity, with the lipid system demonstrating superior AmB solubilization. Both PCL-AmB and Oil_AmB reduced parasite load in the footpad, with Oil_AmB also reducing parasite load in draining lymph nodes. Conclusions: PCL-AmB and Oil_AmB reduced lesions and parasite burden in L. amazonensis-infected mice. Treatment timing was critical, with early Oil_AmB also reducing parasite loads in draining lymph nodes. These findings suggest that topical AmB formulations may provide a promising alternative for CL treatment, though further studies are required to optimize efficacy and administration schedules.