Angélica Patricia Isaac-Márquez

Role of chemokines in regulation of immunity against leishmaniasis

Successful immunity to Leishmania depends on recruitment of appropriate immune effector cells to the site of infection and chemokines play a crucial role in the process. At the same time, Leishmania parasites possess the ability to modify the chemokine profiles of their host thereby facilitating establishment of progressive infection. Therapeutic and prophylactic strategies targeted at chemokines and their receptors provide a promising area for further research. This review highlights our current knowledge concerning the role of chemokines and their receptors in modulating leishmaniasis in both clinical settings and experimental disease models.

Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii.

A new cholesterol derivative, pentalinonsterol (cholest-4,20,24-trien-3-one, 1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, including 14 sterols (3-16), three coumarins (17-19), and a triterpene (20), were isolated from a n-hexane partition of a methanol extract of the roots of the Mexican medicinal plant Pentalinon andrieuxii. Structure elucidation of compounds 1 and 2 was accomplished by spectroscopic data interpretation. All isolates were evaluated in vitro for their antileishmanial activity. Among these compounds, 6,7-dihydroneridienone (15) was found to be the most potent principle against promastigotes of Leishmania mexicana (L. mexicana). The cholesterol analogue, pentalinonsterol (1), together with two known sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant leishmanicidal activity in this same bioassay. Compounds 1, 3, 15, 16, cholest-4-en-3-one (4), and cholest-5,20,24-trien-3β-ol (7), showed strong antileishmanial activity against amastigotes of L. mexicana, and 4 was found to be the most potent agent with an IC(50) value of 0.03μM. All the isolates were also evaluated for their cytotoxicity in non-infected bone marrow-derived macrophages, but none of these compounds was found active towards this cell line. The intracellular parasites treated with compounds 1, 3, 4, 15, and 16 were further studied by electron microscopy; morphological abnormalities and destruction of the amastigotes were observed, as a result of treatment with these compounds.

Role of phosphatidylinositol-3-kinase-γ (PI3Kγ)-mediated pathway in 17β-estradiol-induced killing of L. mexicana in macrophages from C57BL/6 mice

We recently demonstrated that 17β‐estradiol (E2) enhances killing of Leishmania mexicana in macrophages from both male and female DBA/2 mouse by increasing nitric oxide (NO) production. Here, we analyzed the effect of E2 on leishmanicidal activity and cytokine production by bone marrow‐derived macrophages (BMDMs) from male and female C57BL/6 mice in vitro, specifically examining the role of phosphatidylinositol‐3‐kinase‐γ (PI3Kγ) in E2‐induced parasite killing. Unlike its effect on macrophages from both male and female DBA/2 mice, E2 only increased leishmanicidal activity in macrophages from female C57BL/6 mice, which was evident by a significant reduction in both infection rates and infection levels compared to sham controls. E2‐treated BMDMs from female C57BL/6 mice expressed higher levels of interferon‐γRα, and also produced more interleukin (IL)‐12, IL‐6 and NO than both the sham controls and E2‐treated male‐derived macrophages. Sham‐treated BMDMs from female PI3Kγ−/− C57BL/6 mice displayed lower infection rates and infection levels compared to sham‐treated wild‐type (WT) macrophages. However E2, unlike its effect on macrophages from female WT C57BL/6 mice, failed to reduce infection rates and infection levels in BMDMs from female PI3Kγ−/− mice. Interestingly, E2‐treated BMDMs from female C57BL/6 mice produced significant amounts of inflammatory cytokines and NO in levels comparable to those observed in sham‐treated PI3Kγ‐deficient macrophages as well as E2‐treated macrophages from WT mice. These findings show that E2 exerts a distinct effect on leishmanicidal activity of macrophages from male versus female C57BL/6 mice. In addition, they suggest that PI3Kγ is not required for E2‐induced cytokine and NO production in L. mexicana‐infected macrophages from female C57BL/6 mice but it may be involved in parasite clearance from these cells.

In vitro Leishmanicidal and Cytotoxic Activities of the Glycoalkaloids from Solanum lycocarpum (Solanaceae) Fruits

Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania and is the second most prevalent parasitic protozoal disease after malaria in the world. We report the in vitro leishmanicidal activity on promastigote forms of Leishmania amazonensis and cytotoxicity, using LLCMK2 cells, of the glycoalkaloids from the fruits of Solanum lycocarpum, determined by colorimetric methods. The alkaloidic extract was obtained by acid‐base extraction; solamargine and solasonine were isolated by silica‐gel chromatography, followed by reversed‐phase HPLC final purification. The alkaloidic extract, solamargine, solasonine, as well as the equimolar mixture of the glycoalkaloids solamargine and solasonine displayed leishmanicidal activity against promastigote forms of L. amazonensis, whereas the aglycone solasodine was inactive. After 24 and 72 h of incubation, most of the samples showed lower cytotoxicities (IC50 6.5 to 124 μM) as compared to leishmanicidal activity (IC50 1.1 to 23.6 μM). The equimolar mixture solamargine/solasonine was the most active with an IC50 value of 1.1 μM, after 72 h. Likewise, solamargine was the most active after 24 h with an IC50 value of 14.4 μM, both in comparison with the positive control amphotericin B.

Serotypes of Vibrio cholerae Non-O1 Isolated from Water Supplies for Human Consumption in Campeche, México and their Antibiotic Susceptibility Pattern

The presence of Vibrio cholerae non-O1 in water supplies for human consumption in the city of Campeche and rural locality of Bécal was investigated. V. cholerae non-O1 was detected in 5.9% of the samples obtained in deep pools of Campeche. Studies conducted in Bécal and neighbourhood of Morelos in Campeche indicated that collected samples harbored V. cholerae non-O1 in 31.5% and 8.7% respectively. There was a particular pattern of distribution of V. cholerae non-O1 serotypes among different studied regions. Accordingly, V. cholerae non-O1 serotype O14 predominated in the deep pools of Campeche and together with V. cholerae non-O1, O155 were preferentially founds in samples taken from intradomiciliary faucets in the neighbourhood of Morelos. Samples from Bécal predominantly presented the serotype O112. 60% and 53.8% of all studied strains of V. cholerae non-O1 proved to be resistant to ampicillin and carbenicillin. 3.1%, 7.7% and 6.2% presented resistant to doxycycline, trimethoprim-sulfamethoxazole and erythromycin respectively. The study showed the necessity of performing a strong epidemiologic surveillance for emergence and distribution of V. cholerae non-O1.

Pentalinon andrieuxii root extract is effective in the topical treatment of cutaneous leishmaniasis caused by Leishmania mexicana.

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

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer: reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chicleros ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chicleros ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chicleros ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chicleros ulcer.

Sex-associated Susceptibility in Humans with Chiclero's Ulcer: Resistance in Females is Associated with Increased Serum-levels of GM-CSF

We studied a randomly selected population of 90 patients with chicleros’s ulcer (localized cutaneous leishmaniasis) in Campeche, Mexico, and found that 81 (90%) of infected individuals were males and nine of them (10%) were females. We also found significantly higher serum levels of GM-CSF in females than in males. The leishmaniases comprise a group of diseases that display a wide range of biological manifestations in the host depending on both the parasite species initiating infection and the host’s immune response [1]. There have been numerous observations on ageand sexdetermined patterns of infection in different parasitic diseases, both in animals and humans [2, 3]. With some exceptions, it has been widely accepted that males are more susceptible to parasitic infections than females [4]. Moreover, it has also been reported that gender can influence the course of infection with Leishmania spp. in mice, and that female or male hormones can mediate sex-determined resistance or susceptibility to infection [2, 5]. In this work, we report that chiclero’s ulcer, an infection predominantly provoked by Leishmania mexicana, is a sex-associated disease partly regulated by GM-CSF. Leishmania mexicana promastigotes (MHOM/MX/84/ ISET GS) were obtained by serial passages of amastigotes previously inoculated subcutaneously into the shaven rumps of BALB/c mice and then cultured in RPMI-1640 supplemented with fetal calf serum (10%), penicillin (100 U) and streptomicin (100 lg/ml). Parasites preparations treated with 2% formaldehyde were used for Montenegro reactions. This study was conducted according to the ethical guidelines of the 1975 Helsinki Declaration and all patients provided informed consent to participate in the study. Patients included in this study lived, during the period of study, in sylvatic areas of Campeche, which is endemic for chiclero’s ulcer. Field activities within the studied population were performed from 06:00 to 11:00 hours. It is well established that sand fly bites occur in afternoon or late at night when the studied individuals (males and females) stayed together outdoors or at home. In addition, patients’ dwellings were located near the fields (<100 m) and is also shown that sand flies can present a dispersal far from the breeding place of up to 1 km [6]. Clinical evaluation of 90 patients with a typical, single lesion with 3–12 months of evolution was performed, followed by laboratory tests, such as IgE determination, lesion smear observation and delayed hypersensitivity response evaluation as indicated in detail in previous work [7]. For cytokine determination (TNF-a, IFN-c, GM-CSF and IL-4), only 18 patients (nine males and nine females), with nodular lesions and abundant parasites in lesion smears, but not ulcerated lesions and free of bacterial or fungal contamination (that could affect specific cytokine production) and with no other associated pathologies (specially intestinal parasites) were included in this study as previously described [7]. After blood collection and positive diagnosis, patients were treated with one round of 20 daily intramuscular injections of 1.5 g of antimoniate of meglumine administered by physicians appointed by The Health Services of Campeche to completely heal skin-lesions in the population under study. Results of this study showed that 81 of 90 patients (90%) were males and only nine of 90 patients (10%) were females, suggesting the existence of a sex-related resistance in humans with chiclero’s ulcer, as all patients were exposed to sand fly bites in an endemic region. Moreover, other exposed females did not develop cutaneous leishmaniasis. All patients presented a positive DTH response and there were no differences between males and females. All but 10 males presented a lesion-smear with

A New Antileishmanial Preparation of Combined Solamargine and Solasonine Heals Cutaneous Leishmaniasis through Different Immunochemical Pathways

ABSTRACT Little has been done during the past 100 years to develop new antileishmanial drugs. Most infected individuals live in poor countries and have a low cash income to be attractive targets to pharmaceutical corporations. Two heterosidic steroids, solamargine and solasonine, initially identified as major components of the Brazilian plant Solanum lycocarpum, were tested for leishmanicidal activity. Both alkaloids killed intracellular and extracellular Leishmania mexicana parasites more efficiently than the reference drug sodium stibogluconate. A total of 10 μM each individual alkaloid significantly reduced parasite counts in infected macrophages and dendritic cells. In vivo treatment of C57BL/6 mice with a standardized topical preparation containing solamargine (45.1%) and solasonine (44.4%) gave significant reductions in lesion sizes and parasite counts recovered from lesions. Alkaloids present different immunochemical pathways in macrophages and dendritic cells. We conclude that this topical preparation is effective and a potential new and inexpensive treatment for cutaneous leishmaniasis.

General Mechanisms of Tissue Injury in Parasitic Infections

Parasitic infections represent a serious health problem recognized by the World Health Organization. Most mechanisms of tissue injury during these types of infections are directly related to immunological processes. There are several immunologically based responses of tissue damage triggered by chronic parasite infections. Apoptosis, cytokines, and nitric oxide production constitute some of the most important pathways of protection against infection, but when unproperly regulated, they can induce tissue damage to infected hosts. In this review, we analyze different mechanisms of tissue damage triggered for some of the most important parasitic diseases recognized by the World Health Organization (WHO).