Antimalarial evaluation of alkyl-linked bis-thiadiazine derivatives in murine model infected with two Plasmodium strains.

Background and Purpose: Plasmodium falciparum and P. vivax are responsible for most malaria cases in humans in the African Region and the Americas; these parasites have developed resistance to classic antimalarial drugs. On the other hand, previous investigations of the alkyl-linked bis tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) derivatives compounds show satisfactory results against protozoan parasites such as Trypanosoma cruzi, Trypanosoma vaginalis, Trypanosoma brucei rhodesiense and Leishmania donovani. Therefore, it is possible to see some effect of bis-THTT derivatives on other protozoan parasites, such as Plasmodium. Experimental Approach: This study aimed to perform an in vivo biological evaluation of bis-THTT (JH1 to JH6) derivatives compounds as possible anti-malaria drugs in BALB/c mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL strains. In this work, we evaluated the compounds as potential antimalarial drugs in BALB/c mice infected with Plasmodium strains. Key Results: For each compound, we assess the percentages of parasitemia by smears from tail blood and the humoral response by indirect ELISA test using each compound as an antigen. We also evaluated the B lymphocyte response and the cytotoxicity of the bis-THTT derivatives compounds with MTT cell proliferation assays. Conclusions: Our results show that the bis-THTT derivatives JH2 and JH4 presented effective parasitemia control in mice infected with P. berghei; JH5 and JH6 compounds have similar infection control results as chloroquine in mice infected P. yoelii strain. The evaluation of bis-THTT derivatives compounds in a model of BALB/c mice infected with P. berghei and P. yoelii allowed us to conclude that some of them have an antimalarial effect; however, none of the tested compounds exceeded the efficiency of chloroquine.

Serological Cross-Reaction between Six Thiadiazine by Indirect ELISA Test and Their Antimicrobial Activity.

Malaria is a parasitic infection caused by a protozoon of the genus Plasmodium, transmitted to humans by female biting mosquitoes of the genus Anopheles. Chloroquine and its derivates have caused the parasite to develop drug resistance in endemic areas. For this reason, new anti-malarial drugs as treatments are crucial. This work aimed to evaluate the humoral response. with hyper-immune sera, of mice immunized with six derivatives of tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) by indirect ELISA test. The cross-reactivity between the compounds as antigens and their microbial activity on Gram-positive and Gram-negative bacteria was evaluated. The results of the humoral evaluation by indirect ELISA show that three bis-THTTs react with almost all of the above. Besides, three compounds used as antigens stimulate the BALB/c mice's immune system. The best combination of two antigens as a combined therapy displays similar absorbances between the antigens in the mixture, showing similar recognition by antibodies and their compounds. In addition, our results showed that different bis-THTT presented antimicrobial activity on Gram-positive bacteria, mainly on Staphylococcus aureus strains, and no inhibitory activity was observed on the Gram-negative bacteria tested.

Pregnancy-associated malaria: Effects of cytokine and chemokine expression.

BACKGROUND: Malaria remains a serious public health problem. Malaria caused 409,000 deaths in 2019, and 67% were children under 5 years old. Malaria-infected mothers exhibit several complications, including babies with low birth weight, stillbirth, preterm delivery, poor fetal intrauterine growth and maternal anemia. This review aims to provide an update on the immune response in pregnant women and the role of cytokines and chemokines in modulating immunity after infection by the Plasmodium parasite. METHODS: This review collects information from articles indexed in the main databases associated with malaria in pregnancy and its relationship with the immune response and cytokines. RESULTS: and Discussion: The most influential event in malaria pregnancies for pathology development is placental sequestration. During the gestation period, an imbalance in the immune response due to the over- and under-expression of cytokines promotes high rates of fetal mortality, miscarriage, maternal anemia, and low birth weight. In addition, hormones, parity, gestational age, and age of the mother are risks associated with malaria severity during pregnancy. CONCLUSIONS: The pathology of malaria pregnancy is dependent on factors such as cytokine imbalances, placental sequestration and hormones, parity, gestational age and age of the mother. Thus, a better understanding of immune mechanisms will provide information to improve results for disease treatment.

Incidence of avian malaria in hummingbirds in humid premontane forests of Pichincha Province, Ecuador: A pilot study.

BACKGROUND AND AIM: Avian malaria is a tropical disease caused by protozoans of the genera Plasmodium and Haemoproteus. As a nonlethal disease, avian malaria can affect the lifespan and reproductive rate of birds. If there is a differential effect depending on bird species, then this disease might have a significant effect on avian biodiversity. The current study aimed to determine the incidence of Plasmodium in hummingbirds in humid premontane forest areas. MATERIALS AND METHODS: Blood samples (n=60) were collected from hummingbirds from two areas (Santuario de Aves Milpe and Hacienda Puyucunapi) of Pichincha Province, Ecuador. Prevalence and parasitemia were determined by microscopic examination of blood smears stained with Giemsa reagent. Both study sites are part of a 1000 m elevational gradient; hence, elevation was used as a predictor variable for prevalence and parasitemia levels in a Mann-Whitney U-test. This test was also used to test for a sex bias. RESULTS: This study reports on a total of 12 bird species that inhabit both study sites. At Milpe, the lower elevation site, a prevalence of 100% was recorded, whereas at Puyucunapi, the prevalence was 96%. The combined prevalence was 97%. Elevation and sex did not influence prevalence nor parasitemia in hummingbirds. CONCLUSION: This study does not suggest a significant elevation or sex bias on prevalence and parasitemia in hummingbirds.

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.

Antimalarial effect of two photo-excitable compounds in a murine model with Plasmodium berghei (Haemosporida: Plasmodiidae) / Efecto antimalárico de dos compuestos foto-excitables en un modelo murino con Plasmodium berghei (Haemosporida: Plasmodiidae)

Abstract Malaria represents a major health problem worldwide, affecting around 198 million people in 2016 according to WHO database. For decades, anti-malarial drug therapy has been used in the battle against this disease and its uncontrolled usage in endemic areas has developed the appearance of the drug resistance. Thus, it has emerged the necessity of finding new treatments that could be used as an alternative cure to malaria infection. The aim of this work was the evaluation of two photo-excitable compounds: Compound 1, which is (2E)-3-(4-dimethylamino-phenyl)-1-(4-imidazol-1-yl-phenyl)prop-2-en-1-one) and Compound 2, (1E,4E)1-[4-(dimethylamino)phenyl]-5-(4-methoxyphenyl)-1,4-pentadiene-3-one) as possible anti-malaria drugs with Plasmodium berghei ANKA strain in BALB/c mice as murine model. Cytotoxicity effect was evaluated by a cell proliferation by colorimetry assay (MTS); and the drug incorporation into the parasite was assessed in vitro with Indirect Immunofluorescence Assay (IFA) to determine the localization of the drugs into the parasitized red blood cells (RBCs). Finally, the curative effect of compounds no-radiation (fundamental state) and ration drugs were evaluated by oral drug administration of this drugs in BALB/c mice and chloroquine was used as positive control. This curative effect was determined daily by the parasitemia percentage. The results showed that both compounds were cytotoxic in fundamental state. Furthermore, cytotoxic effect was increased after radiation into the Solar Simulator, and compound 2 was more cytotoxic than compound 1. Curative assays showed that both compounds in fundamental state were non effective as anti-malarial drug. However, in the curative assays in the mice treated with compound 2, when this was ration showed a survival rate of 33 % and a parasitemia percentage decrease in compare to compound 1. Although the compounds did not show a similar or better anti-malarial effect than Chloroquine, Compound 2 presented certain anti-malarial effect after solar radiation. Rev. Biol. Trop. 66(2): 880-891. Epub 2018 June 01.

Resumen La malaria representa un importante problema de salud en todo el mundo, afectando a alrededor de 198 millones de personas en 2016 según la base de datos de la OMS. Durante décadas, se ha utilizado la terapia con fármacos anti-malpricos en la lucha contra esta enfermedad y su uso incontrolado en las zonas endémicas ha desarrollado la aparición de resistencia a los fármacos. Por lo tanto, se ha surgido la necesidad de encontrar nuevos tratamientos que podrían ser utilizados como una cura alternativa para la infección por el paludismo. El objetivo de este trabajo fue evaluar dos compuestos foto-excitables: El compuesto 1, que es (2E) -3- (4-dimetilamino-fenil) -1- (4-imidazol-1-ilfenil) prop-2 1-ona) y el Compuesto 2, (1E, 4E) -1- [4- (dimetilamino) fenil] -5- (4-metoxifenil) -1,4-pentadieno-3-ona) como posibles drogas antimaláricas con la cepa ANKA de Plasmodium berghei en ratones BALB / c como modelo murino. El efecto de la citotoxicidad se evaluó mediante una proliferación celular con el ensayo de colorimetría (MTS); y la incorporación del fármaco en el parásito se evaluó in vitro con Ensayo de Inmunofluorescencia Indirecta (IFA) para determinar la localización de los fármacos en los glóbulos rojos parasitados (RBCs). Finalmente, se evaluó el efecto curativo de los compuestos sin radiación (estado fundamental) y los fármacos irradiados mediante la administración oral de los fármacos en los ratones BALB / c, y se usó cloroquina como control positivo de cura. Este efecto curativo se determinó diariamente por el porcentaje de parasitemia. Los resultados mostraron que ambos compuestos eran citotóxicos en estado fundamental. Además, el efecto citotóxico se incrementó después de la radiación en el Simulador Solar, y el compuesto 2 fue más citotóxico que el compuesto 1. Los ensayos curativos mostraron que ambos compuestos en estado fundamental no eran eficaces como fármacos antimaláricos. Sin embargo, en los ensayos curativos en los ratones tratados con el compuesto 2, cuando fue irradiado, se observó una tasa de supervivencia del 33 % y una disminución del porcentaje de parasitemia en comparación con el compuesto 1. Aunque los compuestos no mostraron un efecto similar o mejor antimalárico que la cloroquina, el compuesto 2 presentó cierto efecto antimalárico después de la radiación solar.

Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate.

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R(II)-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R(II)α isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-R(II) IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinant T. equiperdum PKA R-like protein recognized the PKA R(II)-subunit purified from porcine heart and the recombinant human PKA R(I)ß-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector in T. equiperdum. Although the function of the S. scrofa PKA R(II)α and the T. equiperdum PKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology.

TNF-alpha in bulls experimentally infected with Trypanosoma vivax: a pilot study.

There are few studies about the immune response during trypanosomosis in cattle. The objective of this research was to evaluate the effect of experimental infection with Trypanosoma vivax (T. vivax) on serum levels of TNF-alpha in bulls and its relationship to hematocrit, body temperature and parasitemia. Two adult crossbred bulls were infected experimentally with T. vivax and two were used as controls. The bulls were evaluated during a 64 day period in terms of temperature, hematocrit, and parasitemia. Serum TNF-alpha levels were determined by ELISA, using an antibody specific for bovine. TNF-alpha in serum began rising on the seventh day after infection and reached a peak on day 40 of post-infection, then dropped. The lowest hematocrit levels corresponded to the upper levels of TNF-alpha, for each animal. In conclusion, the experimental infection of cattle with T. vivax promotes the release of TNF-alpha, demonstrating a pro-inflammatory immune response to this hemotropic parasite. Moreover, the lowest hematocrit levels coincide with high concentrations of TNF-alpha, suggesting that this cytokine can be linked to the observed anemia during the course of infection by T. vivax in cattle.

Suppressive and additive effects in protection mediated by combinations of monoclonal antibodies specific for merozoite surface protein 1 of Plasmodium yoelii.

BACKGROUND: The merozoite surface protein (MSP)-1 is a target antigen of protective immunity and a malaria vaccine candidate. The nature of this protective immune response warrants further investigation: although specific antibody is thought to play a major role, the mechanisms of protection are still unclear. Monoclonal antibodies (mAbs) specific for the C-terminus of MSP-1 from Plasmodium yoelii have been shown previously to provide protection against challenge infection when administered by passive immunization to mice. Three protective mAbs were re-examined and, in particular, the effect of combinations of antibodies on the protection provided was studied. It was found that a combination of two antibodies can either provide additive protective effects or result in a suppression of protection. In this report the importance of antibody subclass and epitope specificity in the outcome of these passive immunization experiments are discussed. METHODS: The minimum protective dose (MPD) for each mAb was determined, and then combinations of antibody at their MPD were investigated for their ability to control parasitaemia and promote survival in groups of mice. Mice were inoculated over three days with the MPD and challenged with a blood stage infection of the virulent P. yoelii 17 XL. The resultant parasitaemia was assessed daily on Giemsa-stained blood films. Following the infection the presence of MSP-1 specific antibodies in the sera was monitored, and the proliferative responses of cells in the spleen of protected mice were measured. RESULTS: Combining antibodies resulted in either an additive effect on protection, with reduced peak parasitaemia and better survival, or resulted in a suppression of protection over that achieved by a single antibody alone. An additive effect was observed when B6 and F5 that have the same isotype and similar fine specificity, were combined. However, a combination of mAb D3, an IgG2a, with either B6 or F5 (both IgG3) suppressed protection, an effect that may have been due to the combination of different isotypes or to the different fine specificity of the antibodies. CONCLUSIONS: These results suggest that a combination of protective antibodies with either the same or different isotypes can produce either an additive or a suppressive effect in passive immunization. This phenomenon may be important in better understanding immunity in this experimental mouse model of malaria.

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates.

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates. Paracoccidioidomycosis (PCM) is a progressive systemic mycosis caused by the fungus Paraccocidioides brasiliensis (Pb), endemic to Venezuela and Latin America. In this study, eight different Venezuelan isolates obtained from patients with PCM, were inoculated intraperitoneally in Syrian hamsters (Cricetus auratus) and studied by immune-serum. Each strain was collected by gently scraping the surface of the culture medium (Sabouraud Dextrose Agar) and suspended in 3 ml of 0.15 M phosphate-buffered saline. The antigen obtained was called Paraccocidioides brasiliensis Crude Antigen (CAP). Immunoblotting results showed that the immune-sera from hamsters recognized at least 3 bands: one over 200 kDa, and two of 80 and 15-20 kDa. This study suggests that IgG anti-CAP can reveal a significant variability in the eight Venezuelan isolates. Sera from 88 infected hamsters were evaluated by ELISA with eight different CAPs and Western blot with CAP 37383. ELISA results showed that, the antigen of the virulent isolate 37383 had the highest percentage (38%) of positivity, while the nonvirulent isolate 1458 had the lowest one (13.6%). Furthermore, scanning densitometry revealed that the isolate 37383 had less bands than the non-virulent isolates. These results suggest that the ELISA test with CAP 37383 can detect circulating antibodies, and that this virulent isolate may be useful for the diagnosis of PCM, and to monitor disease responses to treatments.

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates

Humoral response of paracoccidioidomycosis sera in hamsters with different Venezuelan isolates. Paracoccidioidomycosis (PCM) is a progressive systemic mycosis caused by the fungus Paraccocidioides brasiliensis (Pb), endemic to Venezuela and Latin America. In this study, eight different Venezuelan isolates obtained from patients with PCM, were inoculated intraperitoneally in Syrian hamsters (Cricetus auratus) and studied by immune-serum. Each strain was collected by gently scraping the surface of the culture medium (Sabouraud Dextrose Agar) and suspended in 3ml of 0.15 M phosphate-buffered saline. The antigen obtained was called Paraccocidioides brasiliensis Crude Antigen (CAP). Immunoblotting results showed that the immune-sera from hamsters recognized at least 3 bands: one over 200 kDa, and two of 80 and 15-20 kDa. This study suggests that IgG anti-CAP can reveal a significant variability in the eight Venezuelan isolates. Sera from 88 infected hamsters were evaluated by ELISA with eight different CAPs and Western blot with CAP 37383. ELISA results showed that, the antigen of the virulent isolate 37383 had the highest percentage (38%) of positivity, while the non-virulent isolate 1458 had the lowest one (13.6%). Furthermore, scanning densitometry revealed that the isolate 37383 had less bands than the non-virulent isolates. These results suggest that the ELISA test with CAP 37383 can detect circulating antibodies, and that this virulent isolate may be useful for the diagnosis of PCM, and to monitor disease responses to treatments. Rev. Biol. Trop. 57 (3): 505-513. Epub 2009 September 30.

La Paracoccidioidomicosis (PCM), es una micosis sistémica causada por el hongo Paraccocidioides brasiliensis (Pb), endémica en Venezuela y Latino América. En este estudio ocho diferentes aislados venezolanos, obtenidos de pacientes con PCM, fueron inoculados intraperitonealmente en hámsteres y fueron estudiados por ELISA e inmunoblotting. Los antígenos obtenidos de P. brasiliensis fueron llamados, Antígeno Crudo (CAP). Los resultados del immunoblotting mostraron que los sueros inmunes de hámsteres reconocieron al menos tres bandas: una sobre 200, y otras de 80, y 15-20 kDa. Este estudio sugiere que la IgG anti-CAP muestra una variabilidad en los ocho aislados Venezolanos. Sueros de 88 hámsteres infectados fueron evaluados usando ELISA, el antígeno del aislado virulento 37383 mostró el más alto porcentaje de positividad (38%) en los sueros de los hámsteres estudiados. El aislado novirulento 1458 mostró un porcentaje bajo de positividad (13.6%). Además, un escaneo densitométrico reveló que el aislado 37383 tiene menos bandas que el otro aislado no-virulento. Por lo tanto, estos resultados sugieren que el ensayo de ELISA con CAP 37383 puede detectar anticuerpos circulantes y este aislado virulento puede ser útil para el diagnostico de PCM, y para el monitoreo de la respuesta al tratamiento de la enfermedad.