PHARMACOLOGICAL AND MECHANISTIC STUDIES OF UREQUINONA, A MOLECULE FROM ROOTS OF PENTALINON ANDRIEUXII MUELL-ARG THAT HEALS MURINE LEISHMANIA MEXICANA INFECTION.

In this work we tested both the in vitro and in vivo anti-Leishmania mexicana activity of a molecule we originally identified in the root of Pentalinon andrieuxii Muell-Arg, a plant that is widely used in Mayan traditional medicine. The chemical name of this molecule is 24-methylcholesta-4-24(28)-dien-3-one, but for simplicity's sake, we assigned the short and trivial name of urequinona that will be used throughout this work. It induces necrosis and apoptosis of promastigotes cultured in vitro and extensive ultrastructural damage of amastigotes. It also induces production of Interleukin (IL)-2 and interferon (IFN)-γ by splenic cells from infected and urequinona treated mice stimulated in vitro with parasite antigen (Ag) but inhibits the production of IL-6 and IL-12p70 by bone-marrow-derived macrophages (BMM) infected in vitro and then treated with urequinona. It also induces activation of transcription factors such as NFkB and AP-1 (NFkB/AP-1) in RAW reporter cells. We also developed a novel pharmaceutical preparation of urequinona encapsulated in hydroxyethyl cellulose for dermal application that significantly reduced (P < 0.05) experimentally induced ear lesions of C57BL/6 mice. We conclude the preparation containing this molecule is a good candidate for a novel anti-leishmanial drug's preparation.

Decanethiol functionalized silver nanoparticles are new powerful leishmanicidals in vitro.

We evaluated, for the first time, the leishmanicidal potential of decanethiol functionalized silver nanoparticles (AgNps-SCH) on promastigotes and amastigotes of different strains and species of Leishmania: L. mexicana and L. major isolated from different patients suffering from localized cutaneous leishmaniasis (CL) and L. mexicana isolated from a patient suffering from diffuse cutaneous leishmaniasis (DCL). We recorded the kinetics of promastigote growth by daily parasite counting for 5 days, promastigote mobility, parasite reproduction by CFSE staining's protocol and promastigote killing using the propidium iodide assay. We also recorded IC50's of promastigotes and amastigotes, therapeutic index, and cytotoxicity by co-culturing macrophages with AgNps-SCH or sodium stibogluconate (Sb) used as reference drug. We used Sb as a reference drug since it is used as the first line treatment for all different types of leishmaniasis. At concentrations 10,000 times lower than those used with Sb, AgNps-SCH had a remarkable leishmanicidal effect in all tested strains of parasites and there was no toxicity to J774A.1 macrophages since > 85% were viable at the concentrations used. Therapeutic index was about 20,000 fold greater than the corresponding one for Sb treated cells. AgNps-SCH inhibited > 80% promastigote proliferation in all tested parasites. These results demonstrate there is a high leishmanicidal potential of AgNps-SCH at concentrations of 0.04 µM. Although more studies are needed, including in vivo testing of AgNps-SCH against different types of leishmaniasis, they can be considered a potential new treatment alternative.

Treating murine Kala-azar with a Mayan plant induces immunochemical changes.

Pentalinon andrieuxii Muell Arg is a Mexican-Central American plant anciently used by local people to treat cutaneous leishmaniasis. We evaluated a hexane extract of the root we called PAE for its chemical content and for its immunochemical and in vitro activity against Leishmania donovani and healing of experimental Kala-azar. Chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) identified hexadecanoic acid, hexadecanoic acid ethyl ester, 9, 12-octadecadienoic acid ethyl ester, octadecanoic acid ethyl ester, 9-octadecenoic acid ethyl ester and diethyl phthalate as the main compounds present in PAE. We also demonstrated PAE kills promastigotes and amastigotes in vitro and significantly reduces parasite loads in liver and spleen of infected Balb/c mice. PAE induces expression of NFkB/AP-1 transcription factors and production of IL-2 and IFN-γ by spleen cells of PAE treated but not in the untreated control mice. Furthermore, there were not IL-6, IL-10 nor TNF production in macrophages treated in vitro with PAE. We developed an affordable extract of P. andrieuxii effective to treat experimental Kala-azar in Balb/c mice.

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

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.

17Beta-estradiol increases Leishmania mexicana killing in macrophages from DBA/2 mice by enhancing production of nitric oxide but not pro-inflammatory cytokines.

We have previously shown that female DBA/2 mice are significantly more resistant to Leishmania mexicana compared with males. Here, we have analyzed the effect of 17beta-estradiol (E(2)) on function and cytokine production in male and female DBA/2 macrophages in vitro. We show that E(2) increases NO production and parasite killing in L. mexicana-infected male and female DBA/2 macrophages without increasing production of pro-inflammatory cytokines. These data indicate that E(2) may enhance leishmanicidal activity in macrophages by directly regulating production of NO.

Systemic cytokine response in humans with chiclero's ulcers.

In this work, we report the existence of enhanced concentrations of IL-2 (Th1 cytokine), TNF-alpha (mainly a Th1 cytokine), IL-12p40, a strong delayed type hypersensitivity response, and the absence of a serum-specific antibody response (IgG or IgE) in patients with active chiclero's ulcers. There was a low serum concentration of IL-6 (a Th2 cytokine) that was further reduced once patients healed following antimonial therapy (i.e., 4 months after treatment started). There was an absence of serum IL-4 (a Th2 cytokine) in patients before and after healing. The serum content of nitric oxide, transforming growth factor-beta1, and IL-12p70 in patients with progressive chiclero's ulcers was not different from that of controls. Patients had an alteration in blood-cell counts and showed a poor T cell proliferation after in vitro stimulation with Leishmania mexicana gp63. We concluded that patients with progressive chiclero's ulcers develop a Th1 type of response at this stage of the disease which correlates with increases in circulating eosinophils and B cells and a reduction of circulating CD8(+) T lymphocytes.

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.

Immunomodulation of Chiclero's ulcer. Role of eosinophils, T cells, tumour necrosis factor and interleukin-2.

The progression of cutaneous leishmaniasis is controlled largely by cell-mediated immunity. Two subpopulations of CD4+ T cells exist that control healing or immunopathology of murine and, perhaps, human leishmaniasis. To better understand the immunological pathways controlling outcome of the human disease, we analysed the pattern of tumour necrosis factor (TNF) and interleukin-2 (IL-2), both of which were present in the sera of humans with active or healed chiclero's ulcer, in relation to the development of delayed-type hypersensitivity (DTH) responses and leucocyte counts in peripheral blood. Increased serum levels of IL-2 and TNF-alpha were apparent only in individuals with active lesions. All individuals with localized cutaneous leishmaniasis developed a strong DTH. The number of T cells was lower in the blood of diseased individuals and the CD4/CD8 ratio was reduced (from 1.5 to 1.0) when compared with the control group. However, diseased and recently cured individuals developed eosinophilia. We conclude that important alterations of the immune response exists in humans suffering from this normally self-healing infection.

[Sanitary quality of water for human consumption a in rural community of Mexico]. / Calidad sanitaria del agua para consumo humano en una comunidad rural de México.

The purpose of this study was to evaluate the quality of water for human consumption in Bécal, rural community of México. The presence of total and fecal coliforms, as well as aerobic plate counts in differents places of the water distribution network, in intradomiciliary deep pools and rainwater cistern system were investigated. Results from this set of experiments suggest the existance of local social and environmental factors that open the way for water contamination with organic material in the water sources studied. We demonstrated the necessity of performing a careful sanitary control and proper handling of water for human consumption through programs of systematic surveillance of the water supplies in order to provide good quality water to the population of this rural community. Additionally, it is necessary to perform educational programs about procedures for preservation of hygiene with special reference to prevention of gastrointestinal diseases.

Vaccination of different strains of mice against cutaneous leishmaniosis: usefulness of membrane antigens encapsulated into liposomes by intraperitoneal and subcutaneous administration.

The objective of this study was to assess the usefulness of parasite-surface molecules reconstituted into liposomes to vaccinate four different strains of mice (C57BL/10, CBA/ca, C57BL/6 and NZB) with different levels of susceptibility to L. m. mexicana infection and to find out possible increases in specific antibody response after vaccination, but before infection with virulent promastigotes. Mice were vaccinated with parasite membrane antigens incorporated into liposomes and antibody levels were recorded. Vaccination was effective to protect CBA/ca and C57BL/6 but not C57BL/10 mice and NZB animals were naturally resistant. Intraperitoneal (ip) was more effective than the subcutaneous (sc) route of inoculation, and the induction of disease-resistance correlated with the production of IgG anti-Leishmania in CBA/ca, C57BL/6 and C57BL/10 mice.

Vaccination of C57BL/6 mice against cutaneous leishmaniosis. Kinetics of antibody synthesis and induction of cellular responses.

In this report, the effectiveness of vaccination of C57BL/6 mice and its association to long term production of IgG anti-Leishmania, delayed type hypersensitivity response (DTH) and the enlargement of the spleen associated to hyperplasia or parasite visceralization were studied. Mice were vaccinated with Leishmania-membrane antigens incorporated into liposomes, and two weeks later, animals received a homologous challenge with living amastigotes. Antibody levels were weekly recorded during 18 weeks after immunization. It was concluded that this vaccine induces disease-resistance in C57BL/6 mice and this effect correlates with the production of IgG anti-Leishmania, but not with the induction of a DTH or lymphoid hyperplasia.

CD4+, CD8+ and CD4- CD8- T cell-subsets can confer protection against Leishmania m. mexicana infection

We studied the role of CD4+, CD8+, CD4- CD8- T cells and IgG anti-Leishmania after infection or vaccination in the CBA/ca mouse. Mice were either infected with L. m. mexicana promastigotes or vaccinated with parasite-membrane antigens incorporated into liposomes. Successfully vaccinated mice were used as cell-donors in adoptive transfer experiments. Naive, syngeneic recipients received highly-enriched CD4+, CD8+ or CD4- CD8- T cells from those two set of donors and challenged with live parasites. Our results showed that, both CD4+ and CD8+ T cells from infected or vaccinated donors conferred significant disease-resistance to naive recipients. In addition, adoptive transfer of CD4- CD8- T cells from vaccinated donors significantly delayed lesion growth in recipient mice. We concluded that vaccination of CBA mice correlates with the induction of protective CD4+, CD8+ and CD4- CD8- T cells and the synthesis of IgG anti-Leishmania.

CD4+, CD8+ and CD4- CD8- T cell-subsets can confer protection against Leishmania m. mexicana infection.

We studied the role of CD4+, CD8+, CD4- CD8- T cells and IgG anti-Leishmania after infection or vaccination in the CBA/ca mouse. Mice were either infected with L. m. mexicana promastigotes or vaccinated with parasite-membrane antigens incorporated into liposomes. Successfully vaccinated mice were used as cell-donors in adoptive transfer experiments. Naive, syngeneic recipients received highly-enriched CD4+, CD8+ or CD4- CD8- T cells from those two set of donors and challenged with live parasites. Our results showed that, both CD4+ and CD8+ T cells from infected or vaccinated donors conferred significant disease-resistance to naive recipients. In addition, adoptive transfer of CD4- CD8- T cells from vaccinated donors significantly delayed lesion growth in recipient mice. We concluded that vaccination of CBA mice correlates with the induction of protective CD4+, CD8+ and CD4- CD8- T cells and the synthesis of IgG anti-Leishmania.

[Sanitary quality of water supply for human consumption in Campeche]. / Calidad sanitaria de los suministros de agua para consumo humano en Campeche.

This paper presents data of a study undertaken to know the sanitary features of water supply (deep pools) for human consumption in the city of Campeche, Mexico. Levels of intestinal bacteria (total and fecal coliforms) were monitored, as well as heterotrophic plate counts and the surroundings of each deep pool were inspected. Each water supply was monitored three times from January to July, 1993 and presented unacceptable levels of heterotrophic plate counts and coliforms which is a strong evidence of fecal contamination of animal or human origin. These findings are a clear indication of unacceptable contamination of water supply for human consumption which requires an improvement and systematic inspection in order to provide good quality water to the population of Campeche.

Cytokine control of Leishmania infection in the BALB/c mouse: enhancement and inhibition of parasite growth by local administration of IL-2 or IL-4 is species and time dependent.

The therapeutic potential of locally injected interleukin-2 (IL-2) or interleukin-4 (IL-4) was studied in the footpads of Leishmania mexicana or Leishmania major infected BALB/c mice. The disease state was measured both pathologically, by measuring lesion size, and parasitologically, by counting total parasite numbers from infected footpads. IL-2 (0.5 microgram/dose) or IL-4 (0.1 microgram/dose) was administered either early, 1 day and/or 15 days after infection, or late, after palpable lesions had developed. Results differed markedly depending on which Leishmania species was used and at what time during the course of disease that therapy commenced. Both L. major and L. mexicana infections, as measured by footpad thickness and parasite number, were exacerbated if IL-4 was injected into the infected footpads early, during the first two weeks of infection. Paradoxically, late intralesional injection (i.e. after measurable lesions had developed) of IL-4 markedly inhibited both lesion size and parasite growth in L. major, though not L. mexicana, infected mice. IL-2 had no measurable effect on the course of L. major infections no matter when or how often, the infected footpads of mice were treated. However, early administration of IL-2 did exacerbate L. mexicana lesion and parasite growth while late treatment had no effect. Generally, but not always, increases in footpad size correlated with increases in parasite number.