Casiopeina III-ia: a copper compound with potential use for treatment of infections caused by Leishmania mexicana.

Introduction Casiopeina III-ia (CasIII-ia) is a mixed chelate copper (II) compound capable of interacting with free radicals generated in the respiratory chain through redox reactions, producing toxic reactive oxygen species (ROS) that compromise the viability of cancer cells, bacteria and protozoa. Due to its remarkable effect on protozoa, this study evaluated the effect of CasIII-ia on Leishmania mexicana (L. mexicana) amastigotes and its potential use as a treatment for cutaneous leishmaniasis in the murine model. Methods We analyzed the leishmanicidal effect of CasIII-ia on L. mexicana amastigotes, and on their survival in bone marrow-derived macrophages. Furthermore, we evaluated the production of ROS in treated parasites and the efficacy of CasIII-ia in the treatment of mice infected with L. mexicana. Results Our results show that CasIII-ia reduces parasite viability in a dose-dependent manner that correlates with increased ROS production. A decrease in the size of footpad lesions and in parasite loads was observed in infected mice treated with the intraperitoneal administration of CasIII-ia. Conclusions We propose CasIII-ia as a potential drug for the treatment of cutaneous leishmaniasis.

Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: Differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils.

Eosinophils are mainly associated with parasitic infections and allergic manifestations. They produce many biologically active substances that contribute to the destruction of pathogens through the degranulation of microbicidal components and inflammatory tissue effects. In leishmaniasis, eosinophils have been found within inflammatory infiltrate with protective immunity against the parasite. We analyzed the responses of eosinophils from patients with localized (LCL) and diffuse (DCL) cutaneous leishmaniasis, as well as from healthy subjects, when exposed to Leishmania mexicana. All DCL patients exhibited blood eosinophilia, along with elevated eosinophil counts in non-ulcerated nodules. In contrast, only LCL patients with prolonged disease progression showed eosinophils in their blood and cutaneous ulcers. Eosinophils from DCL patients secreted significantly higher levels of IL-6, IL-8, and IL-13, compared to eosinophils from LCL patients. Additionally, DCL patients displayed higher serum levels of anti-Leishmania IgG antibodies. We also demonstrated that eosinophils from both LCL and DCL patients responded to L. mexicana promastigotes with a robust oxidative burst, which was equally intense in both patient groups and significantly higher than in healthy subjects. Coincubation of eosinophils (from donors with eosinophilia) with L. mexicana promastigotes in vitro revealed various mechanisms of parasite damage associated with different patterns of granule exocytosis: 1) localized degranulation on the parasite surface, 2) the release of cytoplasmic membrane-bound "degranulation sacs" containing granules, 3) release of eosinophil extracellular traps containing DNA and granules with major basic protein. In conclusion, eosinophils damage L. mexicana parasites through the release of granules via diverse mechanisms. However, despite DCL patients having abundant eosinophils in their blood and tissues, their apparent inability to provide protection may be linked to the release of cytokines and chemokines that promote a Th2 immune response and disease progression in these patients.

Leishmania mexicana Lipophosphoglycan Activates Dermal γδ T Cells with Participation of TLR2.

PURPOSE: Leishmania transmission by sand flies is detected by dermal cells that recognize ligands, such as lipophosphoglycan (LPG) on the promastigote glycocalyx. Resident dermal cells include γδ T cells, that recognize antigens by TCR or innate receptors, such as TLRs. We analyzed the response of dermal γδ T cells to Leishmania mexicana infections or inoculation of LPG, and whether parasite LPG activates γδ T cells through TLR2. METHODS: We stimulated γδ T cells with LPG and analyzed colocalization of LPG and TLR2 by confocal microscopy. Activation of TLR2 was evaluated by IκBα phosphorylation. BALB/c mice were inoculated with L. mexicana or LPG in the dermis of earlobes, and LPG+ TLR2+ γδ T cells were analyzed by flow cytometry. TNF+ γδ T cells were examined in earlobe dermis by confocal microscopy. RESULTS: Stimulation with purified LPG showed activation of TLR2 with IκBα phosphorylation in γδ T cells. Inoculation of L. mexicana parasites or LPG into earlobe dermis showed co-expression of LPG+ and TLR2+ in γδ T cells, demonstrating their interaction during infections. A subset of γδ T cells (LPG+ and TLR2-) provided evidence that additional receptors recognize LPG. Inoculation of LPG enhanced overall γδ T cell numbers, including those expressing TNF, whereas infection with the parasite mostly enhanced γδ T cells expressing TNF. CONCLUSION: L. mexicana LPG is a ligand recognized by TLR2 on γδ-T cells leading to their activation, although contribution of other receptors cannot be ruled out and need to be analyzed to elucidate their contribution during Leishmania infections.

Antimicrobial and anti-inflammatory activity of Cystatin C on human gingival fibroblast incubated with Porphyromonas gingivalis.

Background: Periodontal disease is considered one of the most prevalent chronic infectious diseases, often leading to the disruption of tooth-supporting tissues, including alveolar bone, causing tooth mobility and loss. Porphyromonas gingivalis is considered the major etiological agent of this disease, having a plethora of virulence factors, including, lipopolysaccharides (LPS), hemolysins, and proteinases. Antimicrobial peptides are one of the main components of the innate immune response that inhibit the growth of P. gingivalis. The aim of this study was to analyze the antimicrobial activity of cystatin C and to assess the effect on the inflammatory and anti-inflammatory cytokines, the production of reactive oxygen species, and in the release of nitric oxide by human gingival fibroblasts incubated with P. gingivalis in the presence and absence of cystatin C. Methods: P. gingivalis ATCC 33277 was exposed to cystatin C for 24h and co-cultured with human gingival fibroblasts (HGFs) ATCC CRL-2014. The effect of cystatin on growth of P. gingivalis and HGFs was evaluated. Pro-inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines were determined by ELISA in the supernatants of HGFs incubated with P. gingivalis exposed to cystatin C. Additionally, nitrites and reactive oxygen species (ROS) production were evaluated. Results: Cystatin Cinhibited the growth of P. gingivalis without affecting HGFs. Incubation of HGFs with P. gingivalis led to a significant increase of TNF-α and IL-1ß. In contrast, HGFs incubated with P. gingivalis exposed to cystatin C showed a decreased production of both cytokines, whereas IL-10 was enhanced. Incubation of HGFs with P. gingivalis led to an increase of nitric oxide (NO) and ROS production, which was reduced in the presence of the peptide. Conclusions: Cystatin C inhibits the growth of P. gingivalis and decreases the inflammatory cytokines, ROS, and NO production during infection of HGFs with P. gingivalis. Knowledge on the antimicrobial and immunomodulatory properties of cystatin C could aid in the design of new therapeutic approaches to facilitate the elimination of this bacterium to improve the treatment of periodontal disease.

1H NMR profiling and chemometric analysis as an approach to predict the leishmanicidal activity of dichloromethane extracts from Lantana camara (L.).

The application of 1H NMR spectroscopy and chemometrics for the analysis of extracts of Lantana camara is described. This approach allowed to predict the leishmanicidal activity of samples obtained at different harvest times from their 1H NMR spectra. The anti-leishmanial activity of dichloromethane extracts obtained from the aerial parts of L. camara was measured using an in vitro assay. As the extracts displayed differences in their activity according to a one-way ANOVA analysis, their 1H NMR spectra were subjected to multivariate analysis using exploratory (Principal Component Analysis (PCA) and Anova Simultaneous Component Analysis (ASCA)) and regression, (Partial Least Squares Regression to Latent Structures (PLS)) chemometrics methods. These analyses allowed to establish and characterize a predictive model capable of determining the anti-leishmanial activity of Lantana camara dichloromethane extracts from their 1H NMR spectra. Figures of merit of the developed method are given as well. The identified chemical signals responsible for the iPLS calibration model corresponded to the presence of eicosane, caryophyllene oxide, ß-ionone, tiglic acid, lantanilic acid, camaric acid, and lantadene B; the chemical markers. This study proposed a fast and simple method that avoids the need of using complex biological assays to predict the leishmanicidal activity of L. camara dichloromethane extracts.

Leishmania mexicana: Novel Insights of Immune Modulation through Amastigote Exosomes.

Exosomes are extracellular microvesicles of endosomal origin (multivesicular bodies, MVBs) constitutively released by eukaryotic cells by fusion of MVBs to the plasma membrane. The exosomes from Leishmania parasites contain an array of parasite molecules such as virulence factors and survival messengers, capable of modulating the host immune response and thereby favoring the infection of the host. We here show that exosomes of L. mexicana amastigotes (aExo) contain the virulence proteins gp63 and PP2C. The incubation of aExo with bone marrow-derived macrophages (BMMs) infected with L. mexicana led to their internalization and were found to colocalize with the cellular tetraspanin CD63. Furthermore, aExo inhibited nitric oxide production of infected BMMs, permitting enhanced intracellular parasite survival. Expressions of antigen-presenting (major histocompatibility complex class I, MHC-I, and CD1d) and costimulatory (CD86 and PD-L1) molecules were modulated in a dose-dependent fashion. Whereas MHC-I, CD86 and PD-L1 expressions were diminished by exosomes, CD1d was enhanced. We conclude that aExo of L. mexicana are capable of decreasing microbicidal mechanisms of infected macrophages by inhibiting nitric oxide production, thereby enabling parasite survival. They also hamper the cellular immune response by diminishing MHC-I and CD86 on an important antigen-presenting cell, which potentially interferes with CD8 T cell activation. The enhanced CD1d expression in combination with reduction of PD-L1 on BMMs point to a potential shift of the activation route towards lipid presentations, yet the effectivity of this immune activation is not evident, since in the absence of costimulatory molecules, cellular anergy and tolerance would be expected.

Chemical Constituents with Leishmanicidal Activity from a Pink-Yellow Cultivar of Lantana camara var. aculeata (L.) Collected in Central Mexico.

Lantana camara (L.) is employed by several ethnical groups to treat numerous diseases. Although there are no ethnomedical reports on its use against leishmaniasis, organic extracts prepared from L. camara were shown to display leishmanicidal activity. In the present study, we carried out a bioassay-guided fractionation of the dichloromethane extract from Mexican L. camara in order to identify the compounds responsible for the leishmanicidal activity. Eighteen chromatographic fractions (FI⁻FXVIII) were evaluated in vitro against Leishmania mexicana and L. amazonensis. FII, FX, FXI, FXV, and FXVI showed significant activity against both Leishmania strains, the most potent of which was FXV. Eicosane (1), squalene (2), ß-ionone (3), caryophyllene oxide (4), ß-caryophyllene (5), hexanoic acid (6), tiglic acid (7), a mixture of lantanilic (8) and camaric (9) acids, and lantadene B (10) were identified and obtained from the active fractions and evaluated for their leishmanicidal activity. The mixture of lantanilic (8) and camaric (9) acids (79%/21%) was the most potent one (half maximal inhibitory concentration (IC50) = 12.02 ± 0.36 µM). This study indicates that this cultivar of L. camara has high potential for the development of phytomedicines or as a source of natural products, which might represent lead compounds for the design of new drugs against leishmaniasis.

Leishmania mexicana cell death achieved by Cleoserrata serrata (Jacq.) Iltis: Learning from Maya healers.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Cleoserrata serrata (Jacq.) Iltis are widely used in South-Central Mexico to treat wounds and bacterial skin infections and in Panama by Kuna, Ngöbe-Buglé, and Teribe Indians for tropical warm baths and by Kunas in the form of "Ina kuamakalet" for snakebites. AIMS OF THE STUDY: To evaluate the effect of Cleoserrata serrata extract on growth and viability of L. mexicana amastigotes and promastigotes in vitro, as well as on bacteria that usually co-infect skin ulcers. MATERIALS AND METHODS: Cleoserrata serrata was collected in La Chontalpa, Tabasco, Mexico. The antiproliferative effect of the extract was tested on growth of Leishmania mexicana amastigotes and promastigotes in vitro, as well as on bacteria that usually co-infect skin ulcers. RESULTS: Our data show that Cleoserrata serrata significantly inhibits parasite growth (which was more important in infective amastigotes) and additionally inhibits growth of the co-infective bacteria Staphylococcus aureus and Pseudomonas aeruginosa. Confocal microscopy showed a leishmanicidal effect. CONCLUSION: We conclude that Cleoserrata serrata extract is potentially an optimal treatment alternative for patients with cutaneous leishmaniasis infected with Leishmania mexicana, since it controls both the parasite as well as bacterial co-infections. Furthermore, it can be applied topically. The precise metabolites responsible for the anti-Leishmania and anti-bacterial effects remain to be established.

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway.

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes. We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains.

Preeclampsia is associated with lower production of vascular endothelial growth factor by peripheral blood mononuclear cells.

BACKGROUND: Recent studies show that vascular endothelial growth factor (VEGF) downregulation is implicated in preeclampsia (PE) pathophysiology. This study assessed the relationship between PE and VEGF levels produced by peripheral blood mononuclear cells (PBMCs) and their serum levels. METHODS: A cross-sectional design was performed in 36 patients who had hypertensive disorders during pregnancy. We also used a longitudinal design with 12 pregnant women with risk factors for PE development and/or abnormal uterine arteries by Doppler study. VEGF and soluble fms-like tyrosine kinase-1 (sFlt-1) levels were measured for all patients in both designs. RESULTS: sFlt-1 serum was higher in preeclamptic patients (n = 26), whereas VEGF produced by stimulated PBMCs was lower than in healthy pregnant women and VEGF levels produced by stimulated PBMCs were even lower (p <0.003) in severe PE (n = 16). The receiver-operating characteristic curve analysis allowed establishing a cut-off value to identify patients with PE. VEGF production by PBMCs was 339.87 pg/mL. In addition, a robust linear regression model was performed to adjust the variance in VEGF levels. The patients' age decreased VEGF levels and was adjusted by weeks of gestation (WG) in our model. In the longitudinal study, 7/12 patients developed PE. VEGF produced by PBMCs cells was significantly lower in PE at 24-26 WG. CONCLUSIONS: VEGF production by PBMCs is inhibited during PE, creating a downregulation of the microenvironment; this deficiency may contribute to the pathogenesis of disease.

Attenuated Salmonella enterica serovar typhi live vector with inducible chromosomal expression of the T7 RNA polymerase and its evaluation with reporter genes.

Attenuated Salmonella strains with defined gene deletions have been extensively evaluated as suitable live carriers of passenger antigens. A number of strategies for antigen delivery by these strains have been attempted, ranging from plasmid-based to chromosomal integration systems. We report here the chromosomal integration of the T7 RNA polymerase gene (T7pol) in the attenuated strain Salmonella enterica serovar Typhi (Salmonella typhi) CVD908 (aroC(-), aroD(-)). The T7pol gene was amplified by PCR from Escherichia coli BL21(DE3) and cloned in the pNir3 plasmid under the control of the anaerobically inducible nirB promoter. Then it was subcloned in a pKTN701 derivative, suicide plasmid with the R6K ori, and flanked by the aroC gene. After evaluation of its functionality in E. coli SY327, the aroC-T7pol-aroC cassette was integrated into the aroC locus of S. typhi CVD908 by homologous recombination. The resulting strain, S. typhi CVD908-T7pol, was able to transcomplement two plasmids bearing the luc or the lacZ reporter genes controlled by the T7 promoter and produce luciferase and beta-galactosidase under anaerobic culture conditions. Therefore, an inducible system for recombinant antigen production in attenuated S. typhi was achieved.

Impacto de nuevas metodologías en el estudio de parásitos: III. Variación genética intraespecie de Leishmania mexicana mexicana y su repercusión sobre la fisiopatogenia de la enfermedad / Impact of new methodologies in the study of parasites: III

Las formas más frecuentes de leishmaniosis en México son la leishmaniosis cutánea localizada (LCL), un padecimiento relativamente benigno, y la leishmaniosis cutánea diseminada (LCD), de evolución generalmente mortal. La caracterización fenotípica de parásitos aislados de pacientes con LCL y LCD ha revelado, que el agente casual de ambos cuadros clínicos de Leishmania mexicana mexicana. Sin embargo la resistencia a medicamentos y la virulencia inesperada en algunos pacientes hacen sospechar una posible introducción de nuevas especies en México o bien mutaciones intraespecie. En este trabajo realizamos un análisis genotípico del kADN de leishmanias aisladas de pacientes con LCL y LCD, mediante endonucleasas de restricción (Hae II.I y Hpa II) y RAPD (amplificacion aleatoria de ADN polimórfico con oligonucleótidos no específicos). Encontramos polimorfismo en las digestiones sugestivas de la introducción de nuevas especies en México, lo cual se tendrá que confirmar con PCR especie-especificos. Mediante el RAPD detectamos una ligera diferencia entre un paciente con LCD y otros con LCL. Esta variación intraespecie pudiera ser una de las posibles causas de diseminación del parásito.