Heterologous Booster with BNT162b2 Induced High Specific Antibody Levels in CoronaVac Vaccinees.

Immune responses after COVID-19 vaccination should be evaluated in different populations around the world. This study compared antibody responses induced by ChAdOx1 nCoV-19, CoronaVac, and BNT162b2 vaccines. Blood samples from vaccinees were collected pre- and post-vaccinations with the second and third doses. The study enrolled 78 vaccinees, of whom 62.8% were women, with the following median ages: 26 years-ChAdOx1 nCoV-19; 40 years-CoronaVac; 30 years-BNT162b2. Serum samples were quantified for anti-RBD IgG and anti-RBD IgA and anti-spike IgG by ELISA. After two vaccine doses, BNT162b2 vaccinees produced higher levels of anti-RBD IgA and IgG, and anti-spike IgG compared to ChAdOx1 nCoV-19 and CoronaVac vaccinees. The third dose booster with BNT162b2 induced higher levels of anti-RBD IgA and IgG, and anti-spike IgG in CoronaVac vaccinees. Individuals who reported a SARS-CoV-2 infection before or during the study had higher anti-RBD IgA and IgG production. In conclusion, two doses of the studied vaccines induced detectable levels of anti-RBD IgA and IgG and anti-spike IgG in vaccinees. The heterologous booster with BNT162b2 increased anti-RBD IgA and IgG and anti-spike IgG levels in CoronaVac vaccinees and anti-RBD IgA levels in ChAdOx1 nCoV-19 vaccinees. Furthermore, SARS-CoV-2 infection induced higher anti-RBD IgA and IgG levels in CoronaVac vaccinees.

Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress.

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC50 and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (KM/W), and the concentration of the compound in the membrane (cm50) and in the aqueous phase (cw50), which inhibits cell growth by 50%. The KM/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 108 parasites/mL the compounds did not show significantly different results. The cm50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.

Plasma membrane rigidity effects of 4-hydroxy-2-nonenal in Leishmania, erythrocyte and macrophage.

4-hydroxy-2-nonenal (HNE) is a reactive aldehyde produced by cells under conditions of oxidative stress, which has been shown to react with proteins and phosphatidylethanolamine in biological membranes. Using electron paramagnetic resonance (EPR) spectroscopy of a spin label it was demonstrated that 2 h of treatment with HNE causes membrane rigidity in promastigotes of Leishmania (L.) amazonensis, J774.A1 macrophages and erythrocytes. Remarkable fluidity-reducing effects on the parasite membrane were observed at HNE concentrations approximately 4-fold lower than in the case of erythrocyte and macrophage membranes. Autofluorescence of the parasites in PBS suspension (1 × 107 cell/mL) with excitation at 354 nm showed a linear increase of intensity in the range of 400 to 600 nm over 3 h after treatment with 30 µM HNE. Parasite ghosts prepared after this period of HNE treatment showed a high degree of membrane rigidity. Bovine serum albumin (BSA) in PBS treated with HNE for 2 h showed an increase in molecular dynamics and suffered a decrease in its ability to bind a lipid probe. In addition, the antiproliferative activity of L. amazonensis promastigotes, macrophage cytotoxicity and hemolytic potential were assessed for HNE. An IC50 of 24 µM was found, which was a concentration > 10 times lower than the cytotoxic and hemolytic concentrations of HNE. These results indicate that the action of HNE has high selectivity indices for the parasite as opposed to the macrophage and erythrocyte.

New world Leishmania spp. infection in people living with HIV: Concerns about relapses and secondary prophylaxis.

Coinfection with the human immunodeficiency virus (HIV) and Leishmania impairs immune responses, increases treatment failure and relapse rates in patients with American tegumentary leishmaniasis (ATL), as well as visceral leishmaniasis (VL). There is insufficient data on the treatment, relapse, and secondary prophylaxis in patients coinfected with HIV/Leishmania in Brazil. This study investigated patients with HIV/ATL and HIV/VL to describe the outcome of leishmaniasis in patients assisted at a referral hospital of Brazilian midwestern region. Patients with HIV/ATL (n = 21) mainly presented cutaneous diseases (76.2%) with an overall relapse rate of 28.57% after treatment, whereas HIV/VL (n = 28) patients accounted for 17.5% of the cases. The counts of CD4+ T cells and CD8+ T cells and the CD4+/CD8+ cell ratios at diagnosis or relapses were not significantly different between relapsing and non-relapsing patients. Patients with HIV/ATL or HIV/VL showed high levels of activation markers in CD4+ and CD8+ T cells. The regular use of highly active antiretroviral therapy (HAART) and viral load at the time of diagnosis did not influence the relapse rates. Relapses occurred in 36.4% (4/11) of the patients with HIV/VL receiving secondary prophylaxis and in 5.9% (1/17) of the patients who did not receive secondary prophylaxis (p = 0.06). These data are relevant for the therapeutic management of the patients coinfected with HIV/Leishmania.

Comparative EPR spectroscopy analysis of amphotericin B and miltefosine interactions with Leishmania, erythrocyte and macrophage membranes.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania (L.) amazonensis promastigotes, human erythrocytes and J774.A1 murine macrophages, in comparison with reported and novel data for miltefosine (MIL). One of the objectives of this work is to look for the relationships between the activities of these two drugs in the Leishmania parasite with their changes in the cell membrane. A spin-labeled stearic acid inserted into the cell membranes showed strong interactions with putative AmB/sterol complexes, characterized by reductions in molecular dynamics. The concentration of the drugs in the plasma membrane that reduced the cell population by 50%, and the membrane-water partition coefficient of the drugs, were assessed. These biophysical parameters enabled estimates of possible therapeutic concentrations of these two drugs in the interstitial fluids of the tissues to be made. AmB displayed higher affinity for the plasma membrane of L. amazonensis than for that of the macrophage and erythrocyte, denoting a preference for a membrane that contains ergosterol. AmB also demonstrated higher hemolytic potential than MIL for measurements on erythrocytes in both PBS and whole blood. For MIL, the EPR technique detected membrane changes induced by the drug in the same concentration range that inhibited the growth of parasites, but in the case of AmB, an 8-fold higher concentration of the IC50 was necessary to observe a reduction in membrane fluidity, suggesting a better localized effect of AmB on the membrane. Taken together, the results demonstrate that the antiproliferative and cytotoxic effects of both drugs are associated with changes in cell membranes.

Analysis of the Interactions of Amphotericin B with the Leishmania Plasma Membrane Using EPR Spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania amazonensis promastigotes, erythrocytes, and J774 macrophages. Spin labels embedded into the cell membranes detected strong interactions with putative AmB/sterol complexes that resulted in pronounced changes in the EPR spectra, which can be interpreted as a reduction in membrane fluidity or an increase in the polarity assessed by the spin probe. The EPR spectra of spin-labeled lipids corroborated the findings that AmB does not enter phospholipid membrane-sterol models and probably forms extramembranous aggregates, as predicted by the sterol sponge model. Furthermore, these aggregates were shown to extract the spin probe androstanol from the lipid bilayer. However, in contrast to the results for the model membrane, EPR spectroscopy suggested that AmB easily enters the membranes of the studied cells, implying that the entry process is dependent on interactions with the membrane proteins.

Antileishmanial activity of the chalcone derivative LQFM064 associated with reduced fluidity in the parasite membrane as assessed by EPR spectroscopy.

A novel chalcone derivative, LQFM064, demonstrated antileishmanial activity against Leishmania (L.) amazonensis, with an IC50 value of ~10 µM for the promastigote form. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled stearic acid incorporated in the plasma membrane of L. amazonensis promastigotes revealed that after 2 h of treatment with LQFM064, the parasite showed remarkable reductions in membrane fluidity. The features of the altered EPR spectra were similar to those reported for the erythrocyte membrane, which was suggested to be due to the cross-linking of oxidized hemoglobin with the cytoskeleton spectrin. In comparison to miltefosine (MIL), LQFM064 demonstrated a much lower hemolytic potential against both erythrocytes in PBS and whole blood, less cytotoxicity in J774.A1 macrophages and equivalent ability to kill parasites internalized in J774.A1 macrophages. Measurements of the IC50 values for assays with different cell concentrations enabled the estimation of the membrane-water partition coefficient (KM/W), as well as the concentrations of LQFM064 in membrane (cm50) and aqueous phase (cw50) that reduces the cell population by 50%. From the KM/W and cm50 values it was deduced that LQFM064 has a greater affinity than MIL for the parasite membrane, but the antiproliferative activity of both substances is exerted at a similar concentration in the plasma membrane.

Genetic variation in Interleukin-32 influence the immune response against New World Leishmania species and susceptibility to American Tegumentary Leishmaniasis.

Interleukin-32 is a novel inflammatory mediator that has been described to be important in the immunopathogenesis and control of infections caused by Leishmania parasites. By performing experiments with primary human cells in vitro, we demonstrate that the expression of IL-32 isoforms is dependent on the time exposed to L. amazonensis and L. braziliensis antigens. Moreover, for the first time we show the functional consequences of three different genetic variations in the IL32 (rs4786370, rs4349147, rs1555001) modulating IL-32γ expression, influencing innate and adaptive cytokine production after Leishmania exposure. Using a Brazilian cohort of 107 American Tegumentary Leishmaniasis patients and a control cohort of 245 healthy individuals, the IL32 rs4786370 genetic variant was associated with protection against ATL, whereas the IL32 rs4349147 was associated with susceptibility to the development of localized cutaneous and mucosal leishmaniasis. These novel insights may help improve therapeutic strategies and lead to benefits for patients suffering from Leishmania infections.

Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine.

Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.

In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics.

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC50 values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (cm50) and the aqueous phase (cw50) and the membrane-water partition coefficient of nerolidol (KM/W). Via cytotoxicity (CC50) and hemolytic potential (HC50) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a cw50 of 125 µM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean cw50 values of 56 and 74 µM, respectively. The estimated cm50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC50 value.

Alterations in monocyte subsets and cytokine production after TLR activation in American Cutaneous Leishmaniasis.

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 CD14hi 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.

Case Report: Atypical Cutaneous Leishmaniasis in a Patient with Mixed Leishmania guyanensis and Leishmania amazonensis Infection.

The disseminated form of leishmaniasis is a serious and rare disease, being diagnosed in 2% of the cutaneous cases registered per year in Brazil. The main characteristic is the appearance of multiple pleomorphic lesions on the cutaneous surface. A 68-year-old male from the rural area of Tocantins, Brazil, presented atypical disseminated cutaneous leishmaniasis (ACL). The clinical course and histopathological and immunological findings presented a mixed pattern that hindered diagnosis and therapeutic management. Molecular typing revealed a mixed infection with Leishmania (V.) guyanensis and Leishmania (L.) amazonensis. Molecular identification of the agents responsible for ACL is important for adequate therapeutic planning, minimizing the possibility of sequellae that impact the quality of life of the patient.

Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion.

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.

Human Interleukin-32γ Plays a Protective Role in an Experimental Model of Visceral Leishmaniasis in Mice.

Visceral leishmaniasis (VL) is a chronic parasitic disease caused by Leishmania infantum in the Americas. During VL, several proinflammatory cytokines are produced in spleen, liver, and bone marrow. However, the role of interleukin-32 (IL-32) has not been explored in this disease. IL-32 can induce production of proinflammatory cytokines in innate immune cells and polarize the adaptive immune response. Herein, we discovered that L. infantum antigens induced expression of mRNA mainly for the IL-32γ isoform but also induced low levels of the IL-32ß transcript in human peripheral blood mononuclear cells. Furthermore, infection of human IL-32γ transgenic mice (IL-32γTg mice) with L. infantum promastigote forms increased IL-32γ expression in the spleen and liver. Interestingly, IL-32γTg mice harbored less parasitism in the spleen and liver than wild-type (WT) mice. In addition, IL-32γTg mice showed increased granuloma formation in the liver compared to WT mice. The protection against VL was associated with increased production of nitric oxide (NO), interferon gamma (IFN-γ), IL-17A, and tumor necrosis factor alpha by splenic cells restimulated ex vivo with L. infantum antigens. In parallel, there was an increase in the number of Th1 and Th17 T cells in the spleens of IL-32γTg mice infected with L. infantum IL-32γ induction of IFN-γ and IL-17A expression was found to be essential for NO production by splenic cells of infected animals. These data indicate that IL-32γ potentiates the Th1/Th17 immune response during experimental VL, thus contributing to the control of L. infantum infection.

Performance of two immunochromatographic tests for diagnosis of visceral leishmaniasis in patients coinfected with HIV.

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

TLR4 and TLR2 activation is differentially associated with age during Parkinson's disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by loss of dopaminergic neurons associated with neuroinflammation. Toll-like receptors (TLRs) are expressed in peripheral blood leukocytes and also in neurons and glial cells mediating inflammation. This study aimed to investigate the peripheral blood leukocyte response to TLR2 and TLR4 agonists in young and elderly PD patients. Two groups of patients with PD were evaluated (≤ 55 years old and ≥ 65 years old), age-matched with healthy controls (n = 26). Severity of PD was evaluated by Unified Parkinson's Disease Rating Scale (UPDRS). Whole blood cultures were stimulated with lipopolysaccharide (LPS), a TLR4 agonist or Pam3Cys (Pam), a TLR2 agonist. Tumor necrosis factor alpha (TNFα) and interleukin 10 (IL-10) were measured by immunoenzimatic assay. 6 h-TNFα production was increased after TLR4 stimulation, mainly in young PD patients, whereas TLR2-induced TNFα and IL-10 levels were decreased in PD patients independent of age (p < 0.05). A reverse correlation between LPS-induced TNFα production and age was observed in PD patients and controls, but TNFα induced by TLR2 agonist was not associated with age of PD patients or controls. TNFα production induced by TLR4 but not by TLR2 was reversely associated with the age at PD onset and disease duration. No associations between UPDRS scores and cytokine levels were detected. In conclusion, TLR4 and TLR2 responses seem to be differentially affected during PD. Data suggest that TLR2 deficiency in periphery is independent of age of the patients, age at PD onset, or PD duration.

American tegumentary leishmaniasis: epidemiological and molecular characterization of prevalent Leishmania species in the State of Tocantins, Brazil, 2011-2015.

Determination of the epidemiological profile of the American tegumentary leishmaniasis (ATL) and identification of Leishmania species that are prevalent in the State of Tocantins were carried out through a retrospective and descriptive study based on data reported in SINAN, in the period from 2011 to 2015. Molecular techniques such as PCR-RFLP and PCR-G6PD to amplify Leishmania DNA were performed on stored on Giemsa-stained slides from lesion scarifications of ATL patients who were amastigote-positive by the direct microscopic examination. There were 1,434 ATL cases in Tocantins reported in this period. The highest incidence was reported in men aged over 60 years, rural residents, the most affected ethnic group was mixed ethnicity (mixed black and white) and the ones with lower education. The predominant clinical form was cutaneous, being diagnosed mainly by laboratory methods. Pentavalent antimonial was effective in resolving cases. The predominant species found in 271 analyzed samples from 32 municipalities located in 8 different health regions of Tocantins was Leishmania (Viannia) braziliensis. Identifying the epidemiological profile and characterizing the Leishmania spp species on regional level is essential to establish control and prevention behaviors, minimizing the number of cases and treatment resistance, recurrence and evolution to mucosal forms.

Platelet-activating factor increases reactive oxygen species-mediated microbicidal activity of human macrophages infected with Leishmania (Viannia) braziliensis.

Platelet-activating factor (PAF) is produced by macrophages during inflammation and infections. We evaluated whether PAF is able to modulate the infection of human macrophages by Leishmania braziliensis, the main Leishmania sp. in Brazil. Monocyte-derived macrophages were incubated with promastigote forms in absence or presence of exogenous PAF. We observed that the treatment of macrophages with low concentrations of PAF prior to infection increased the phagocytosis of L. braziliensis. More importantly, exogenous PAF reduced the parasitism when it was added before, during or after infection. In addition, treatment with a PAF antagonist (PCA 4248) resulted in a significant increase of macrophage infection in a concentration-dependent manner, suggesting that endogenous PAF is important to control L. braziliensis infection. Mechanistically, while exogenous PAF increased production of reactive oxygen species (ROS) treatment with PCA 4248 reduced oxidative burst during L. braziliensis infection. The microbicidal effects of exogenous PAF were abolished when macrophages were treated with apocynin, an NADPH oxidase inhibitor. The data show that PAF promotes the production of ROS induced by L. braziliensis, suggesting that this lipid mediator may be relevant to control L. braziliensis infection in human macrophages.

IL-32γ promotes the healing of murine cutaneous lesions caused by Leishmania braziliensis infection in contrast to Leishmania amazonensis.

BACKGROUND: Interleukin 32 (IL-32) is a pro-inflammatory cytokine induced in patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. Here, we investigated whether IL-32 is also expressed in patient lesions caused by L. amazonensis. In addition, we evaluated experimental L. amazonensis and L. braziliensis infections in C57BL/6 transgenic mice for human IL-32γ (IL-32γTg) in comparison with wild-type (WT) mice that do not express the IL-32 gene. RESULTS: Human cutaneous lesions caused by L. amazonensis express higher levels of IL-32 than healthy control skin. In mice, the presence of IL-32γ promoted the control of cutaneous lesions caused by L. braziliensis, but not lesions caused by L. amazonensis in an ear dermis infection model. In addition, IL-32γTg mice displayed less tissue parasitism and inflammation in IL-32γTg than WT mice during the healing phase of L. braziliensis infection. Production of antigen-specific pro-inflammatory cytokines was higher in IL-32γTg mice than in WT mice during L. braziliensis infection but not during L. amazonensis infection. CONCLUSIONS: Human cutaneous lesions caused by L. amazonensis express high levels of IL-32. In mice, the presence of IL-32γ contributes to the lesion healing caused by L. braziliensis but not by L. amazonensis. Data suggest that despite the ability for both species to induce IL-32 in humans, the connections between this cytokine and other immune players induced by related species of parasites can lead to distinct outcomes of the murine infections.

The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.

In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (cw50) and the cell membrane (cm50) and the membrane-aqueous medium partition coefficient of MT (K). With a cw50 of 8.7µM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had cw50 values of 2.4-3.1µM. The estimated cm50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays.