Antiparasitic Activity of Two Natural Terpenes from Salvia cuspidata against Leishmania amazonensis.

Leishmaniasis is a neglected disease caused by flagellated parasites of the Leishmania genus affecting more than 10 million people worldwide. Current treatments for leishmaniasis involve the administration of poorly tolerated drugs with toxic side effects in patients. There is an imperative necessity for novel compounds to treat this disease. One of the most used strategies in the search for different antiparasitic compounds is the screening of purified plant molecules. The diterpenes 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (HABTO) and 5-epi-icetexone (ICTX) isolated from Salvia cuspidata were shown to be effective against Leishmania amazonensis in vitro and in vivo. They displayed an antiproliferative effect against L. amazonensis promastigotes. They also induce an increase in ROS levels and affect the mitochondrial activity of parasites. HABTO and ICTX in an in vivo model of cutaneous leishmaniasis decrease footpad swelling, parasite load, and splenic index. Moreover, they induce significant reduction in the O.D. of total anti-Leishmania IgG and IgG1 subtype antibody responses against L. amazonensis compared to the PBS group but maintain high levels of IgG2a. This suggests that in HABTO- or ICTX-treated mice, there is a slowdown in the progression of the disease. These terpenes could be considered as possible novel antileishmanial agents against L. amazonensis and thus treat cutaneous leishmaniasis.

Therapeutic effect of Prosopis strombulifera (LAM) BENTH aqueous extract on a murine model of cutaneous leishmaniasis.

Background and aim: Prosopis strombulifera (Lam.) Benth is a rhizomatous shrub native from different zones of Argentine Republic. P. strombulifera aqueous extract (PsAE) has different effects and several biological activities have been reported. The goal of this study was to analyze the activity of PsAE on a murine model of cutaneous leishmaniasis caused by Leishmania amazonensis. Experimental procedure: PsAE was orally administered at 150 mg/animal/day on BALB/c mice infected in the right footpad (RFP) with 1 × 105 promastigotes of L. amazonensis. As a chemotherapeutic control of treatment, animals receive a commercial form of meglumine antimoniate (MA) (Glucantime®, Aventis, Paris, France). Results and conclusion: We observe that the size of RFP lesions of infected mice without treatment showed a grade of inflammation, ulceration and necrosis at the site of infection much greater than that observed with PsAE or MA treatment. Moreover, PsAE was capable of decreasing parasite burden and splenic index. Furthermore, PsAE treated mice showed a significant decrease in O.D. of total anti-Leishmania IgG antibody responses against L. amazonensis. This decrease was similar to those observed when the reference drug, MA, was used. This would indicate that PsAE treatment inhibits or delays disease progression in mice. In conclusion, our findings suggest that PsAE could be a potential candidate to be used, as a new therapeutic strategy, to treat cutaneous leishmaniasis caused by L. amazonensis.

Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity.

Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis.

An appraisal of the scientific current situation and new perspectives in the treatment of cutaneous leishmaniasis.

Leishmaniasis is a Neglected Tropical Diseases caused by protozoan parasites of the genus Leishmania. It is a major health problem in many tropical and subtropical regions of the world and can produce three different clinical manifestations, among which cutaneous leishmaniasis has a higher incidence in the world than the other clinical forms. There are no recognized and reliable means of chemoprophylaxis or vaccination against infections with different forms of leishmaniasis. In addition, chemotherapy, unfortunately, remains, in many respects, unsatisfactory. Therefore, there is a continuing and urgent need for new therapies against leishmaniasis that are safe and effective in inducing a long-term cure. This review summarizes the latest advances in currently available treatments and improvements in the development of drug administration. In addition, an analysis of the in vivo assays was performed and the challenges facing promising strategies to treat CL are discussed. The treatment of leishmaniasis will most likely evolve into an approach that uses multiple therapies simultaneously to reduce the possibility of developing drug resistance. There is a continuous effort to discover new drugs to improve the treatment of leishmaniasis, but this is mainly at the level of individual researchers. Undoubtedly, more funding is needed in this area, as well as greater participation of the pharmaceutical industry to focus efforts on the development of chemotherapeutic agents and vaccines for this and other neglected tropical diseases.

Leishmania (L.) amazonensis infection impairs reproductive and fetal parameters in female mice.

Leishmaniasis is a group of parasitic zoonotic diseases caused by intracellular protozoans belonging to the genus Leishmania. Little is known about the effects that this parasitosis may have on the reproductive parameters and pregnancy of infected humans and pets. This study aimed to evaluate the influence of chronic cutaneous leishmaniasis caused by Leishmania (Leishmania) amazonensis on reproductive and fetal parameters using a female murine model. A control group of female BALB/c mice and a group infected with L. (L.) amazonensis were mated with healthy males. Clinical parameters were monitored during the pre-mating and gestational periods. Female mice were euthanized on day 19 of gestation, when the fetuses were weighed and their length measured and embryonic resorptions and fetal death were recorded. We observed five fetal deaths and three embryonic resorptions in the infected group. Furthermore, there was a decrease in fertility in the infected group (26.32%). The weight of the offspring from infected mothers was lower than that in the control group (1.019±0.035g and 1.163±0.032g, p<0.01). Fetal length was reduced in the infected group (3.71±0.05cm in the control group and 3.40±0.06cm in the infected group p<0.001). This study shows that cutaneous leishmaniasis caused by L. (L.) amazonensis impairs reproductive and fetal parameters in mice.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis.

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Leishmaniasis mucocutánea con manifestacion oral: reporte de un caso / Mucocutaneous leishmaniasis with oral manifestation: case report

La Leishmaniasis es un grupo de enfermedades transmitidas por vectores y causada por la Leishmania, un parásito intracelular, que se presenta de preferencia en regiones tropicales y subtropicales. Se manifiesta mediante un amplio rango de formas clínicas como la cutánea, mucocutánea, y visceral, dependiendo de la especie y respuesta inmunológica del paciente. Se presenta el caso de un hombre de 35 años que acudió derivado a Unidad de Estomatología del Hospital Señor del Milagro, Salta, Argentina, presentando en la cavidad oral lesión, granulomatosa, ulcerada, dolorosa a la palpación, única, en paladar blando, de tres meses de evolución. Se realizaron estudios serológicos, parasitológicos y PCR. Los ELISAs lisados, PCRs y cultivos de materiales de lesiones fueron positivos, confirmando diagnóstico de leishmaniasis mucocutánea. El paciente fue derivado al Servicio de Dermatología donde recibió tratamiento con Antimoniato de Meglumina, con repuesta clínica favorable. El conocimiento de las manifestaciones orales puede llevar al diagnóstico clínico de leishmaniasis mucocutánea por parte del odontólogo, pudiendo entregar un tratamiento oportuno y a la vez ayudar al paciente, evitando complicaciones de esta enfermedad.

Leishmaniasis is a group of vector-borne diseases caused by Leishmania, an intracellular parasite, which occurs preferentially in tropical and subtropical regions. It manifests itself through a wide range of clinical forms such as cutaneous, mucocutaneous, and visceral, depending on the species and the patient's immune response. We present a case of a 35-year-old man who was referred to the Stomatology Unit of the Señor del Milagro Hospital, Salta, Argentina, presenting in the oral cavity lesion, granulomatous, ulcerated, painful on palpation, unique, soft palate with three months of evolution. Serological, parasitological and PCR studies were performed. Lysed ELISAs, PCRs and cultures of lesion materials were positive, confirming diagnosis of mucocutaneous leishmaniasis. The patient was referred to the Dermatology Service where he received treatment with Meglumine Antimony, with favorable clinical response. The knowledge of the oral manifestations can lead to the clinical diagnosis of mucocutaneous leishmaniasis by the dentist, being able to provide timely treatment and at the same time help the patient, avoiding complications of this disease.

Immunization with antigenic extracts of Leishmania associated with Montanide ISA 763 adjuvant induces partial protection in BALB/c mice against Leishmania (Leishmania) amazonensis infection.

BACKGROUND/PURPOSE: A proper adjuvant has a relevant role in vaccine formulations to generate an effective immune response. In this study, total Leishmania antigen (TLA) formulated with Montanide ISA 763 or R848 as adjuvants were evaluated as a first generation Leishmania vaccine in a murine model. METHODS: Immunization protocols were tested in BALB/c mice with a subcutaneous prime/boost regimen with an interval of 3 weeks. Mice immunized with unadjuvanted TLA and phosphate-buffered saline (PBS) served as control groups. On Day 21 and Day 36 of the protocol, we evaluated the humoral immune response induced by each formulation. Fifteen days after the boost, the immunized mice were challenged with 1 × 10(5) promastigotes of Leishmania (Leishmania) amazonensis in the right footpad (RFP). The progress of the infection was followed for 10 weeks; at the end of this period, histopathological studies were performed in the RFP. RESULTS: Vaccines formulated with Montanide ISA 763 generated an increase in the production of immunoglobulin G (IgG; p < 0.05) compared with the control group. There were no statistically significant differences in IgG1 production between the study groups. However, immunization with TLA-Montanide ISA 763 resulted in an increase in IgG2a compared to the unadjuvanted control (p < 0.001). Also noteworthy was the fact that a significant reduction in swelling and histopathological damage of the RFP was recorded with the Montanide ISA 763 formulation. CONCLUSION: We conclude that the immunization of BALB/c mice with a vaccine formulated with TLA and Montanide ISA 763 generated a protective immune response against L. (L.) amazonensis, characterized by an intense production of IgG2a.

Uso de la reacción en cadena de la polimerasa para el control terapéutico de la infección crónica por trypanosoma cruzi / Use of the polymerase chain reaction (pcr) for the therapeutic control of chronic trypanosoma cruzi infection

En las normas actuales de tratamiento etiológico de la fase crónica tardía de la enfermedad de Chagasen Argentina, Brasil y la Organización Mundial de la Salud, se recomienda controlar la eficaciaterapéutica con pruebas serológicas y parasitológicas convencionales. Sin embargo las primerassuelen continuar positivas 10 años o más luego del tratamiento, y las segundas son, en general, debaja sensibilidad en esta etapa de la enfermedad. La Reacción en Cadena de la Polimerasa (PCR)al ser más sensible que los exámenes parasitológicos convencionales, podría informar con unacobertura mayor si hubo falla terapéutica. Hemos ofrecido tratamiento con benznidazol (5 mg/kg/día, por 60 días) a 138 pacientes de 16 a 35 años de edad, infectados crónicamente con Trypanosomacruzi. La eficacia terapéutica se controló con PCR periódicas, hemocultivo y serología convencionalen dos grupos de pacientes: uno (GT, 57 pacientes) que aceptó y cumplió el tratamiento y otro(GNT, 37 pacientes) que lo rechazó. Antes de la administración de benznidazol la PCR mostró unasensibilidad diagnóstica de 41 por ciento (57/138 pacientes) y el hemocultivo 7,2 por ciento (10/138). Sesenta mesespostratamiento el grupo GT mostró una positividad de PCR acumulada de 28,1 por ciento (16/57) y el grupoGNT 54,1 por ciento (20/37; p=0.0016). A pesar de que la sensibilidad diagnóstica de PCR es limitada, lanegatividad de pruebas repetidas con método normatizado podría evidenciar disminución de laparasitemia o probable curación en 71,9 por ciento de los pacientes tratados, lo que habría que confirmar conel seguimiento serológico...

Current norms for the etiological treatment of chronic Chagas disease, recommended by WHOor currently in force for Argentina and Brazil, advise the control of therapeutic efficacy usingconventional serological and parasitological tests. However, serology usually remains positive 10 ormore years after treatment and parasitological tests are insensitive in the chronic stage. PolymeraseChain Reaction (PCR) is more sensitive than parasitological tests and could provide earlier evidenceof therapeutic failure. We offered benznidazole treatment (5 mg/kg/day, 60 days) to 138 patients(age=16 to 35 years old), chronically infected with Trypanosoma cruzi. Therapeutic efficacy waschecked with periodic PCR, haemoculture and conventional serology in two groups of patients: One(TG) accepting and complying with treatment and the other (NTG) rejecting it. Before benznidazoleadministration, PCR displayed a diagnostic sensitivity of 41.3 percent (57/138) and haemoculture 7.2 percent(10/138). Sixty months after treatment, TG displayed a cumulated PCR positivity of 28.1 percent (16/57)and NTG 54.1 percent (20/37; p=0.0166). Even though the sensitivity of PCR is limited, repeated negativeresults of a standardized method may reveal lower parasitaemia or probable cure, in 71.9 percent of treatedpatients, to be confirmed with serological follow up...

Immunological correlates of cure in the first American Cutaneous Leishmaniasis patient treated by immunotherapy in Argentina: A case report. / Correlatos inmunológicos de curación en el primer paciente con Leishmaniasis Cutánea Americana tratado con inmunoterapia en Argentina: Reporte de un caso

A patient with localized cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis infection was treated with an antigen containing heat-killed L. (L.) amazonensis promastigotes plus BCG. Expression of T-cell differentiation, memory and senescence receptors markers were analyzed on T cell subpopulations, in order to establish the correlation between the percentages of expression of these receptors and his clinical status, at different stages of his follow up. The following case reports on the achievement of a successful clinical outcome with complete resolution after receiving immunotherapy. A thorough clinical and immunological follow up supporting the healing process of this patient’s lesion is presented in detail.

Un paciente con leishmaniasis cutánea localizada producida por Leishmania (Leishmania) amazonensis fue tratado con un antígeno compuesto por promastigotes de L. (L.) amazonensis muertos por calor combinado con BCG. Se analizó la expresión de distintos receptores de diferenciación, de memoria y de senescencia en las subpoblaciones de células T, con el fin de establecer una relación entre los porcentajes de expresión de dichos receptores y la clínica del paciente en diferentes momentos del seguimiento. Se reporta en este caso un resultado exitoso, con resolución completa de la lesión después de recibir la inmunoterapia, y se presenta en detalle un seguimiento clínico e inmunológico completo durante el proceso de curación.

Immunological correlates of cure in the first American cutaneous leishmaniasis patient treated by immunotherapy in Argentina. A case report.

A patient with localized cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis infection was treated with an antigen containing heat-killed L. (L.) amazonensis promastigotes plus BCG. Expression of T-cell differentiation, memory and senescence receptors markers were analyzed on T cell subpopulations, in order to establish the correlation between the percentages of expression of these receptors and his clinical status, at different stages of his follow up. The following case reports on the achievement of a successful clinical outcome with complete resolution after receiving immunotherapy. A thorough clinical and immunological follow up supporting the healing process of this patient's lesion is presented in detail.

Serological evaluation of specific-antibody levels in patients treated for chronic Chagas' disease.

Serological tests are the main laboratory procedures used for diagnosis during the indeterminate and chronic stages of Chagas' disease. A serological regression to negativity is the main criterion used to define parasitological cure in treated patients. The aim of this work was to monitor the individual specificities of antibody levels for 3 years posttreatment in 18 adult patients. Conventional serological techniques (hemagglutination assays and enzyme-linked immunosorbent assay [ELISA]) were modified by using recombinant antigens to detect early markers of treatment effectiveness. For this purpose, serum samples were taken before and during treatment and every 6 months after treatment for at least 3 years. When hemagglutination assays were used, a decrease in antibody levels was observed in only one patient. When ELISA with serum dilutions was used, antibody clearance became much more apparent: in 77.7% (14/18) of the patients, antibody titers became negative with time. This was observed at serum dilutions of 1/320 and occurred between the 6th and the 30th months posttreatment. The immune response and the interval for a serological regression to negativity were different for each patient. For some of the recombinant antigens, only 50% (9/18) of the patients reached the serological regression to negativity. Recombinant antigen 13 might be a good marker of treatment effectiveness, since 66.6% (six of nine) of the patients presented with an early regression to negativity for specific antibodies to this antigen (P = 0.002).