Characteristics and risk factors associated with mortality during the first cycle of prone secondary to ARDS due to SARS-CoV-2 pneumonia / Características y factores de riesgo asociados a mortalidad durante el primer ciclo de prono por SDRA secundario a neumonía por SARS-CoV-2

Objective To analyze characteristics, changes in oxygenation, and pulmonary mechanics, in mechanically ventilated patients with ARDS due to SARS-CoV-2 treated with prone position and evaluate the response to this maneuver.Design Cohort study including patients with PaO2/FiO2 <150mmHg requiring prone position over 18 months. We classified patients according to PaO2/FiO2 changes from basal to 24h after the first prone cycle as: 1) no increase 2) increase <25%, 3) 25%–50% increase 4) increase >50%. Setting 33-bed medical-surgical Intensive Care Unit (ICU) in Argentina. Patients 273 patients. Interventions None. Main variables of interest Epidemiological characteristics, respiratory mechanics and oxygenation were compared between survivors and non-survivors. Independent factors associated with in-hospital mortality were identified. Results Baseline PaO2/FiO2 was 116 [97–135]mmHg (115 [94–136] in survivors vs. 117 [98–134] in non-survivors; p=0.50). After prone positioning, 22 patients (8%) had similar PaO2/FiO2 values; 46(16%) increased PaO2/FiO2 ≤25%; 55 (21%) increased it 25%–50%; and 150 (55%), >50%. Mortality was 86%, 87%, 72% and 50% respectively (p<0.001). Baseline PaO2/FiO2, <100mmHg did not imply that patients were refractory to prone position. Factors independently associated with mortality were age, percentage increase in PaO2/FiO2 after 24h being in prone, and number of prone cycles. Conclusions Older patients unable to improve PaO2/FiO2 after 24h in prone position and who require >1 cycle might early receive additional treatments for refractory hypoxemia. After the first 24h in the prone position, a low percentage of PaO2/FiO2 increase over baseline, beyond the initial value, was independently associated with higher mortality. (AU)

Objetivo Analizar las características, cambios en la oxigenación y mecánica pulmonar, en pacientes ventilados mecánicamente con SDRA por SARS-CoV-2 tratados con posición prona, y evaluar la respuesta a esta maniobra. Diseño Estudio de cohorte que incluyó pacientes con PaO2/FiO2 <150mmHg que requirieron posición prona durante 18 meses. Se clasificaron los pacientes según los cambios de PaO2/FiO2 desde el basal y 24horas después del primer ciclo prono como: 1) Sin aumento 2) Aumento <25%, 3) 25–50% de aumento 4) Aumento >50%. Ambito Unidad de Cuidados Intensivos (UCI) médico-quirúrgica de 33 camas en Argentina. Pacientes 273 pacientes. Intervenciones Ninguna. Principales variables de interés Se compararon características epidemiológicas, mecánica respiratoria y oxigenación entre sobrevivientes y no sobrevivientes. Se identificaron factores independientes asociados a la mortalidad hospitalaria. Resultados La PaO2/FiO2 basal fue de 116 [97–135]mmHg (115 [94–136] en sobrevivientes vs. 117 [98–134] en no sobrevivientes; p=0,50). Después de la posición prona, 22 pacientes (8%) tenían valores similares de PaO2/FiO2; 46 (16%) aumentaron PaO2/FiO2 ≤25%; 55 (21%) lo aumentaron 25%–50%; y 150 (55%), >50%. La mortalidad fue de 86%, 87%, 72% y 50% respectivamente (p<0,001). La PaO2/FiO2 basal, <100mmHg no implicó que los pacientes fueran refractarios a la posición prona. Los factores asociados independientemente con la mortalidad fueron la edad, el aumento porcentual de PaO2/FiO2 después de 24horas en prona, y el número de ciclos prono. Conclusiones Los pacientes mayores que no pueden mejorar PaO2/FiO2 después de 24 horas en posición prona y que requieren más de 1 ciclo podrían recibir tratamientos adicionales para la hipoxemia refractaria. Después de las primeras 24horas en decúbito prono, un bajo porcentaje de aumento de PaO2/FiO2 sobre el valor basal, más allá del valor inicial, se asoció de forma independiente con una mayor mortalidad. (AU)

Characteristics and risk factors associated with mortality during the first cycle of prone secondary to ARDS due to SARS-CoV-2 pneumonia.

OBJECTIVE: To analyze characteristics, changes in oxygenation, and pulmonary mechanics, in mechanically ventilated patients with ARDS due to SARS-CoV-2 treated with prone position and evaluate the response to this maneuver. DESIGN: Cohort study including patients with PaO2/FiO2 <150mmHg requiring prone position over 18 months. We classified patients according to PaO2/FiO2 changes from basal to 24h after the first prone cycle as: 1) no increase 2) increase <25%, 3) 25%-50% increase 4) increase >50%. SETTING: 33-bed medical-surgical Intensive Care Unit (ICU) in Argentina. PATIENTS: 273 patients. INTERVENTIONS: None. MAIN VARIABLES OF INTEREST: Epidemiological characteristics, respiratory mechanics and oxygenation were compared between survivors and non-survivors. Independent factors associated with in-hospital mortality were identified. RESULTS: Baseline PaO2/FiO2 was 116 [97-135]mmHg (115 [94-136] in survivors vs. 117 [98-134] in non-survivors; p=0.50). After prone positioning, 22 patients (8%) had similar PaO2/FiO2 values; 46(16%) increased PaO2/FiO2 ≤25%; 55 (21%) increased it 25%-50%; and 150 (55%), >50%. Mortality was 86%, 87%, 72% and 50% respectively (p<0.001). Baseline PaO2/FiO2, <100mmHg did not imply that patients were refractory to prone position. Factors independently associated with mortality were age, percentage increase in PaO2/FiO2 after 24h being in prone, and number of prone cycles. CONCLUSIONS: Older patients unable to improve PaO2/FiO2 after 24h in prone position and who require >1 cycle might early receive additional treatments for refractory hypoxemia. After the first 24h in the prone position, a low percentage of PaO2/FiO2 increase over baseline, beyond the initial value, was independently associated with higher mortality.

[Survival and decannulation at 90 days after percutaneous dilation tracheostomy in the COVID-19 intensive care unit]. / Supervivencia y decanulación a los 90 días luego de traqueostomía por dilatación percutánea en unidad de cuidados intensivos COVID-19.

INTRODUCTION: Tracheostomy (TCT) is the most frequently performed surgical procedure among COVID-19 patients. In Argentina, survival and decannulation rates are unknown. The main objectives of this study were to evaluate mortality and decannulation rates after 90 days of the percutaneous TCT performance. Secondarily, airway injury rate, days on invasive mechanical ventilation (IMV) and days of hospitalization in the intensive care unit (ICU) were also evaluated. METHODS: This observational analytic prospective cohort study included patients over 18 years old with SARS-CoV-2 who were admitted into the ICU requiring IMV and percutaneous TCT in the period covering from 1 February 2021 to 31 July 2021. RESULTS: the mortality rate in 95 patients was 66.3%. Among the survivors, 67% were decannulated. The youngest patients were the ones who survived [mean 50.6 (SD 10.2) years versus mean 58.9 (SD 13.4) years; p = 0.001] and presented lower Charlson index scores [median 1 (IQR 0-2) versus 2 (1-3) points; p = 0.007]. Patients who were tracheostomized ten days before the start of IMV were fewer days on IMV and had a shorter stay in the ICU, p < 0.01 and p = 0.01, respectively. Charlson Index was identified as an independent factor of mortality for both decannulation mortality at 90 days. DISCUSSION: In our cohort of patients, those who were younger and presented less c omorbidities benefited from TCT. Charlson Index could be used as a prognostic marker among this patient population.

Introducción: La traqueostomía (TQT) es el procedimiento quirúrgico más frecuentemente realizado en pacientes COVID-19. La tasa de supervivencia y decanulación en Argentina se desconoce. El objetivo principal de este estudio fue evaluar la mortalidad y la tasa de decanulación al día 90 de realizada la TQT percutánea. Secundariamente se evaluó la tasa de lesión en la vía aérea, días de ventilación mecánica invasiva (VMI) y días de internación en unidad de cuidados intensivos (UCI). Métodos: Estudio observacional analítico de cohorte prospectiva que incluyó 95 pacientes mayores de 18 años infectados por SARS-CoV-2 ingresados de forma consecutiva a la UCI con requerimiento de VMI y TQT percutánea en el periodo comprendido entre 1 de febrero al 31 de julio del 2021. Resultados: La mortalidad fue del 66.3%. De los supervivientes se logró decanular al 67%. Los supervivientes fueron más jóvenes [media 50.6 (DE 10.2) años versus media 58.9 (DE 13.4) años; p = 0.001] y presentaron puntajes más bajos de índice de Charlson [mediana 1 (RIQ 0-2) versus 2 (1-3) puntos; p = 0.007]. Los pacientes TQT antes del día 10 desde el inicio de VMI tuvieron menos días de VMI y menor estadía en UCI, p < 0.01 y p = 0.01 respectivamente. El índice de Charlson se identificó como factor independiente de mortalidad a los 90 días y de decanulación a los 90 días. Discusión: En nuestra cohorte de pacientes fueron los más jóvenes y con menos comorbilidades los que se beneficiaron con la TQT. El índice de Charlson podría utilizarse como marcador pronóstico en esta población de pacientes.

Supervivencia y decanulación a los 90 días luego de traqueostomía por dilatación percutánea en unidad de cuidados intensivos COVID-19 / Survival and decannulation at 90 days after percutaneous dilation tracheostomy in the COVID-19 intensive care unit

Resumen Introducción: La traqueostomía (TQT) es el procedimiento quirúrgico más frecuentemente realizado en pacientes COVID-19. La tasa de supervivencia y decanulación en Argentina se desconoce. El objetivo principal de este estudio fue evaluar la mortalidad y la tasa de decanulación al día 90 de realizada la TQT percutánea. Secundariamente se evaluó la tasa de lesión en la vía aérea, días de ventilación mecánica invasiva (VMI) y días de internación en unidad de cuidados intensivos (UCI). Métodos: Estudio observacional analítico de cohorte prospectiva que incluyó 95 pacientes mayores de 18 años infectados por SARS-CoV-2 ingresados de forma consecutiva a la UCI con requerimiento de VMI y TQT percutánea en el periodo compren dido entre 1 de febrero al 31 de julio del 2021. Resultados: La mortalidad fue del 66.3%. De los supervivientes se logró decanular al 67%. Los supervivientes fueron más jóvenes [media 50.6 (DE 10.2) años versus media 58.9 (DE 13.4) años; p = 0.001] y presentaron puntajes más bajos de índice de Charlson [mediana 1 (RIQ 0-2) versus 2 (1-3) puntos; p = 0.007]. Los pacientes TQT antes del día 10 desde el inicio de VMI tuvieron menos días de VMI y menor estadía en UCI, p < 0.01 y p = 0.01 respectivamente. El índice de Charlson se identificó como factor independiente de mortalidad a los 90 días y de decanulación a los 90 días. Discusión: En nuestra cohorte de pacientes fueron los más jóvenes y con menos comorbilidades los que se beneficiaron con la TQT. El índice de Charlson podría utilizarse como marcador pronóstico en esta población de pacientes.

Abstract Introduction: Tracheostomy (TCT) is the most frequently performed surgical procedure among COVID-19 patients. In Argentina, survival and decannulation rates are unknown. The main objectives of this study were to evaluate mortality and decannulation rates after 90 days of the percutaneous TCT performance. Secondarily, airway injury rate, days on invasive mechanical ventilation (IMV) and days of hospitalization in the intensive care unit (ICU) were also evaluated. Methods: This observational analytic prospective cohort study included patients over 18 years old with SARS-CoV-2 who were admitted into the ICU requiring IMV and percutaneous TCT in the period covering from 1 February 2021 to 31 July 2021. Results: the mortality rate in 95 patients was 66.3%. Among the survivors, 67% were decannulated. The youngest patients were the ones who survived [mean 50.6 (SD 10.2) years versus mean 58.9 (SD 13.4) years; p = 0.001] and presented lower Charlson index scores [median 1 (IQR 0-2) versus 2 (1-3) points; p = 0.007]. Patients who were tracheostomized ten days before the start of IMV were fewer days on IMV and had a shorter stay in the ICU, p < 0.01 and p = 0.01, respectively. Charlson Index was identified as an independent factor of mortality for both decannulation mortality at 90 days. Discussion: In our cohort of patients, those who were younger and presented less c omorbidities benefited from TCT. Charlson Index could be used as a prognostic marker among this patient population.

Synergism of in vitro plasmodicidal activity of phospholipase A2 isoforms isolated from panamanian Bothrops asper venom.

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A2 (PLA2s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA2s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA2s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA2-like. Acidic PLA2s catalyzed the degradation of all substrates evaluated; however, for the basic PLA2s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA2s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA2s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA2 and Lys49-PLA2 were associated with one another, denoting synergistic action between these PLA2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA2s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents.

Socially unstable conditions experienced during development prime female Octodon degus to shape the phenotype of their own offspring.

Because residents and immigrants from group living species may experience fitness costs associated with permanent changes in group membership, we examined the hypothesis that females experiencing socially unstable or socially stable conditions during development compensate these costs by shaping the phenotype of their own offspring differently. Groups of adult females experiencing either socially stable or unstable conditions in the early social environment were assigned to either socially stable or unstable conditions in the social environment as adults. We quantified affiliative and agonistic interactions among the females during pregnancy and lactation of the focal female, maternal and allomaternal care, hypothalamic-anterior pituitary-adrenal axis (HPA) acute stress response, and early offspring growth. Social instability during breeding enhanced agonistic interactions among adult females, and offspring that experienced socially unstable conditions exhibited enhanced offspring care, regardless of adult environments. Neither social behavior, offspring care, acute stress physiology, nor early growth was influenced by early or adult social stability conditions. These findings imply that socially unstable conditions prime developing females to shape the phenotype of their offspring to prevent negative effects of socially unstable environments.

Antimalarial activity of basic phospholipases A2 isolated from Paraguayan Bothrops diporus venom against Plasmodium falciparum.

Malaria is a parasitic infectious disease and was responsible for 400.000 deaths in 2018. Plasmodium falciparum represents the species that causes most human deaths due to severe malaria. In addition, studies prove the resistance of P. falciparum to drugs used to treat malaria, making the search for new drugs with antiplasmodial potential necessary. In this context, the literature describes snake venoms as a rich source of molecules with microbicidal potential, including phospholipases A2 (PLA2s). In this sense, the present study aimed to isolate basic PLA2s from Paraguayan Bothrops diporus venom and evaluate their antiplasmodial potential. Basic PLA2s were obtained using two chromatographic steps. Initially, B. diporus venom was subjected to ion exchange chromatography (IEC). The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the PLA2s. The toxins were tested for enzymatic activity using a chromogenic substrate and finally, the antiplasmodial, cytotoxic potential and hemolytic activity of the isolated toxins were evaluated. The electrophoretic profile of the fractions from the IEC permitted the selection of 3 basic fractions, which were subjected to reverse phase chromatography, resulting in the isolation of the two enzymatically active PLA2s, BdTX-I and BdTX-II and the PLA2 homologue BdTX-III. The antiplasmodial potential was evaluated and the toxins showed IC50 values of: 2.44, 0.0153 and 0.59 µg/mL respectively, presenting PLA2 selectivity according to the selectivity index results (SI) calculated against HepG2 cells. The results show that the 3 basic phospholipases isolated in this study have a potent antiparasitic effect against the W2 strain of P. falciparum. In view of the results obtained in this work, further research are necessary to determine the mechanism of action by which these toxins cause cell death in parasites.

Identification of bloodmeal sources of triatomines captured in the Paraguayan Chaco region of South America by means of molecular biology analysis.

The Paraguayan Chaco is an isolated environment with its own unique ecosystem. In this region, Chagas disease remains a health problem. Chagas disease is caused by the parasite Trypanosoma cruzi, and it is primarily transmitted by triatomines. In order to identify the blood meal sources of triatomines, specimens of the vector were collected in domestic and peridomestic areas and the PCR-RFLP method was implemented. Cytochrome b was amplified from the samples and later subjected to digestion with two restriction enzymes: Hae III and Xho I.It was possible to generate distinct restriction patterns on the amplified material to identify several blood meal sources for the vectors. We employed the blood from several species as positive controls: human, chicken, canine, feline, and armadillo blood. However, we identified only 3 sources for the blood meals of the insect vectors: human, chicken and canine blood. In total, 76 triatomines were captured. T. cruzi was not found in any of them. In 61% of the captured specimens, the blood meal sources for the vectors could be identified. In 30% of these cases, the presence of DNA from more than one vertebrate was detected in the same triatomine. The most common blood meal source found was chicken blood. The presence of human and chicken blood in triatomines captured in domestic and peridomestic areas strongly suggests that the parasite can freely move amongst both areas regardless of food availability. Free vector movement in these areas constitutes an epidemiological threat for the inhabitants of the community under study.

First Molecular Report of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) guyanensis in Paraguayan Inhabitants Using High-Resolution Melt-PCR.

American tegumentary leishmaniasis is an endemic anthropozoonosis undergoing expansion on the American continent. The disease is caused by several Leishmania species and it is manifested as cutaneous and mucocutaneous leishmaniasis. In this study, we evaluate the viability of high-resolution melt polymerase chain reaction (HRM-PCR) analysis to differentiate four closely related Leishmania species as a routine tool for the diagnosis of leishmaniasis. For this purpose, biopsy specimens from cutaneous and mucocutaneous lesions were taken from 132 individuals from endemic and non-endemic areas for leishmaniasis. Each sample was processed for parasitological, histopathological, and molecular analysis. Positive biopsy samples were analyzed by HRM-PCR of a 144-bp heat-shock protein (hsp70) gene fragment, and new cases were confirmed by sequencing. Of the 132 samples analyzed, 36 (27%) were positive for Leishmania spp., of which 86% were from cutaneous lesions and 14% from mucocutaneous lesions. We identified Leishmania (Viannia) braziliensis (84%), Leishmania (Leishmania) infantum (13%), and Leishmania (Leishmania) amazonensis (3%) in cutaneous lesions, and L. (V.) braziliensis (40%), L. (L.) infantum (20%), L. (L.) amazonensis (20%), and Leishmania (Viannia) guyanensis (20%) in mucocutaneous lesions. The main purpose of this research was to report for the first time in Paraguay the presence of L. (L.) amazonensis and L. (V.) guyanensis in patients with cutaneous and mucocutaneous lesions, using the HRM-PCR technique. In addition, we report the presence of additional new cases of L. (L.) infantum in cutaneous lesions.

Antiparasitic effect of the Psidium guajava L. (guava) and Psidium brownianum MART. EX DC. (araçá-de-veado) extracts.

In the search for new therapeutic agents against neglected diseases, both aqueous and hydroethanolic extracts from Psidium guajava L. and P. brownianum Mart ex DC leaves were investigated regarding their antiparasitic effect and cytotoxic potential. The extracts were tested at three concentrations (250, 500 and 1000 µg/mL) against Trypanosoma cruzi epimastigote forms (Chagas, 1909), Leishmania braziliensis (Vianna, 1911) and L. infantum promastigotes forms (Nicolle, 1908), as well as against fibroblasts. P. guajava showed no activity against T. cruzi forms, while the hydroethanolic (PBHE), aqueous by decoction (PBAED) and aqueous by infusion (PBAEI) P. browninaum extracts were responsible, respectively, for inhibiting 100, 100 and 92.68% of T. cruzi epimastigote growth at the 1000 µg/mL concentration. The P. brownianum hydroethanolic extract (PBHE) at the highest concentration caused 58.46% death in L. braziliensis, thus demonstrating moderate activity, however when tested against L. infantum, the PBHE inhibited their growth by 37.16%, revealing its low activity. As for the cytotoxicity assays, the P. brownianum aqueous extract by decoction (PBAED) obtained the highest death percentage when compared to the others, causing 90.85% fibroblast mortality at the 1000 µg/mL concentration.

Microstructural Crimp of the Lamina Cribrosa and Peripapillary Sclera Collagen Fibers.

Purpose: Although collagen microstructural crimp is a major determinant of ocular biomechanics, no direct measurements of optic nerve head (ONH) crimp have been reported. Our goal was to characterize the crimp period of the lamina cribrosa (LC) and peripapillary sclera (PPS) at low and normal IOPs. Methods: ONHs from 11 sheep eyes were fixed at 10-, 5-, or 0-mm Hg IOP and crimp periods measured manually from coronal cryosections imaged with polarized light microscopy (PLM). Using linear mixed-effect models, we characterized the LC and PPS periods, and how they varied with distance from the scleral canal edge. Results: A total of 17,374 manual collagen crimp period measurements were obtained with high repeatability (1.9 µm) and reproducibility (4.7 µm). The periods were smaller (P < 0.001) and less variable in the LC than in the PPS: average (SD) of 13.8 (3.1) µm in the LC, and 31.0 (10.4) µm in the PPS. LC crimp period did not vary with distance from the scleral canal wall (P > 0.1). PPS period increased with the square root of the distance to the canal (P < 0.0001). Conclusions: Small, uniform crimp periods within the sheep LC and immediately adjacent PPS may indicate that these tissues are setup to prevent large or heterogeneous deformations that insult the neural tissues within the canal. An increasing more variable period with distance from the canal provides a smooth transition of mechanical properties that minimizes stress and strain concentrations.

BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential.

Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100µg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64µg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150µg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

The Paraguayan Rhinella toad venom: Implications in the traditional medicine and proliferation of breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Toads belonging to genus Rhinella are used in Paraguayan traditional medicine to treat cancer and skin infections. AIM OF THE STUDY: The objective of the study was to determine the composition of venoms obtained from three different Paraguayan Rhinella species, to establish the constituents of a preparation sold in the capital city of Paraguay to treat cancer as containing the toad as ingredient, to establish the effect of the most active Rhinella schneideri venom on the cell cycle using human breast cancer cells and to assess the antiprotozoal activity of the venoms. METHODS: The venom obtained from the toads parotid glands was analyzed by HPLC-MS-MS. The preparation sold in the capital city of Paraguay to treat cancer that is advertised as made using the toad was analyzed by HPLC-MS-MS. The effect of the R. schneideri venom and the preparation was investigated on human breast cancer cells. The antiprotozoal activity was evaluated on Leishmania braziliensis, L. infantum and murine macrophages. RESULTS: From the venoms of R. ornata, R. schneideri and R. scitula, some 40 compounds were identified by spectroscopic and spectrometric means. Several minor constituents are reported for the first time. The preparation sold as made from the toad did not contained bufadienolides or compounds that can be associated with the toad but plant compounds, mainly phenolics and flavonoids. The venom showed activity on human breast cancer cells and modified the cell cycle proliferation. The antiprotozoal effect was higher for the R. schneideri venom and can be related to the composition and relative ratio of constituents compared with R. ornata and R. scitula. CONCLUSIONS: The preparation sold in the capital city of Paraguay as containing the toad venom, used popularly to treat cancer did not contain the toad venom constituents. Consistent with this, this preparation was inactive on proliferation of human breast cancer cells. In contrast, the toad venoms of Rhinella species altered the cell cycle progression, affecting the proliferation of malignant cells. The findings suggest that care should be taken with the providers of the preparation and that the crude drug present a strong activity towards human breast cancer cell lines. The antiprotozoal effect of the R. schneideri venom was moderate while the venom of R. ornata was devoid of activity and that of R. scitula was active at very high concentration.

BmajPLA(2)-II, a basic Lys49-phospholipase A(2) homologue from Bothrops marajoensis snake venom with parasiticidal potential

Snake venoms contain various proteins, especially phospholipases A(2) (PLA(2)s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA(2) from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA(2)-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA(2)-II as a basic Lys49 PLA(2) homologue, compatible with other basic snake venom PLA(2)s (svPLA(2)), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA(2)-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100 mu g/mL. The venom and BmajPLA(2)-II presented IC50 of 0.14 +/- 0.08 and 6.41 +/- 0.64 mu g/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64 +/- 1 7.94 and >150 mu g/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

Phenolic composition and antiparasitic activity of plants from the Brazilian Northeast "Cerrado".

This work describes the antiparasitic and cytotoxic activities of three plant species from the Cerrado biome, Northeastern Brazil. Significant antiparasitic inhibition was observed against Trypanosoma cruzi (63.86%), Leishmania brasiliensis (92.20%) and Leishmania infantum (95.23%) when using ethanol extract from leaves of Guazuma ulmifolia Lam. (Malvaceae), at a concentration of 500 µg/mL. However, low levels of inhibition were observed when assessing leishmanicidal and trypanocidal (Clone CL-B5) activities of crude ethanol extracts from leaves and bast tissue of Luehea paniculata (Malvaceae) and leaves and bark of Prockia crucis (Salicaceae) at a concentration of 500 µg/mL. The extracts revealed the presence of phenolic acids such as gallic acid, chlorogenic acid, caffeic acid and rosmarinic acid, as well as flavonoids such as rutin, luteolin, apigenin and quercetin - the latter detected only in G. ulmifolia. G. ulmifolia extract displayed higher leishmanicidal activity probably due to the presence of quercetin, a potent known leishmanicidal compound. A cytotoxicity test indicated values over 50% at the highest concentration (1000 µg/mL) for all natural products, which were considered cytotoxic. This points out the need for further tests to enable future in vivo trials, including antineoplastic activity on human tumor cells.

Zanthoxylum chiloperone leaves extract: first sustainable Chagas disease treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum chiloperone var. angustifolium Engl. (Rutaceae) stem bark is used traditionally in Paraguay for its antiparasitic properties. Canthin-6-one is main compound isolated from Zanthoxylum chiloperone var angustifolium with broad spectrum antifungal, leishmanicidal and trypanocidal activities. AIM OF THE STUDY: The qualitative and quantitative characterization and the isolation of main alkaloidal components of different organs of Zanthoxylum chiloperone are investigated by HPLC-UV-MS. The in vitro biological activity of each extract against trypomastigote and amastigote forms of Trypanosoma cruzi parasites were evaluated, then comparison the in vivo efficacy of the ethanolic leaves extract of Zanthoxylum chiloperone with reference drug, benznidazole, in acute Trypanosoma cruzi infected mice when administered by oral route. We have also evaluated the mutagenic and cytotoxic activity of the main component of Zanthoxylum chiloperone, i.e. canthin-6-one, by mouse bone marrow micronucleus test. MATERIALS AND METHODS: The compositions of the ethanol extracts obtained after the maceration process were studied by HPLC-UV-MS methods. The quantitation analysis was performed by external standard method, using a calibration curve constructed utilizing solutions containing different concentrations of the reference samples. The anti-trypomastigote activity was evaluated by the lysis effect on mouse blood trypomastigotes (Y strain Trypanosoma cruzi). The anti-amastigote Trypanosoma cruzi activity was evaluated by a modified colorimetric method with chlorophenol red-ß-d-galactopyranoside (CPRG). The cytotoxicity of extracts and compounds was performed on NCTC 929 cells. The in vivo efficacy of the ethanolic leaves extract of Zanthoxylum chiloperone and benznidazole, in acute Trypanosoma cruzi (two different strains) was evaluated in Trypanosoma cruzi infected mice; the drugs were administered by oral route. The mortality rates were recorded and parasitaemias in control and treated mice were determined once weekly for 70 days. The mutagenic and cytotoxic activity of the main component of Zanthoxylum chiloperone, canthin-6-one, by mouse bone marrow micronucleus test. RESULTS: Canthin-6-one was the main compound of stem and root bark and 5-methoxy-canthin-6-one in leaves and fruits. The ethanolic leaves extract, canthin-6-one and benznidazole presented, approximately, the same level of in vitro activity against trypomastigote and amastigote forms of Trypanosoma cruzi. We have also evaluated the mutagenic and cytotoxic effects of canthin-6-one by micronucleus test in mice. This test showed any mutagenic and cytotoxic damages. The effects of oral or subcutaneous treatments at 10 mg/kg daily for 2 weeks with the ethanolic extract of leaves of Zanthoxylum chiloperone were examined in Balb/c mice infected acutely with Trypanosoma cruzi (CL or Y strain) and compared with benznidazole at 50 mg/kg for 2 weeks. In these experiments, 70 days after infection, parasitaemia and serological response were significantly reduced with the oral ethanolic extract treatment compared with reference drug. CONCLUSIONS: This study have shown the efficacy of the leaves extract of Zanthoxylum chiloperone in reducing Trypanosoma cruzi parasitaemia in vivo assays and could be welcomed by scientific and rural communities of Paraguay because it could help them towards the use of local resources to treat an endemic infection, Chagas disease, affecting 20% of the population of this country.