Interleukin-1β and interferon-γ are associated with malaria-induced hypoinsulinemic hypoglycemia in Plasmodium berghei ANKA-infected mice

Malaria-induced hypoglycemia is recognized as a serious complication of malaria and has one of the strongest associations with mortality in children. It has been speculated that oxidative stress and pro-inflammatory response during parasite infection were involved in its pathophysiology. Hence, this study aimed to investigate the development of malaria-induced hypoglycemia during Plasmodium berghei ANKA (PbANKA) infection with particular attention to the involvement of c-peptide, interleukin-1β (IL-1β), and interferon-γ (IFN-γ). ICR mice were infected with 1×107 parasitized erythrocytes of PbANKA, and parasitemia was monitored, and the development of hypoglycemia was assessed by measuring plasma glucose levels. The change of c-peptide level was evaluated. The pro-inflammatory response of IL-1β and IFN-γ were also quantified in plasma. It was found that PbANKA infection resulted in hypoglycemia as indicated by a significantly (P < 0.05) decrease in plasma glucose levels on day 4 post-infection and associated with parasitemia. The c-peptide was slightly increased at day 2 post-infection, and then significantly (P < 0.05) decreased since day 4. Furthermore, we observed a significantly (P < 0.05) increased IL-1β, firstly responded, at day 2 post-infection followed by increasing the IFN-γ level at day 4 in PbANKA-induced hypoglycemia. Our findings support the idea that hypoinsulinemic hypoglycemia in the PbANKA infected mice may be involved in the high IL-1β and IFN-γ against the parasite infection

Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous <it>in vitro </it>culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-<it>P. vivax </it>drugs, bacterial and yeast surrogate models expressing the validated <it>P. vivax </it>target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and <it>in vivo </it>drug metabolism between the surrogate models and <it>P. vivax </it>parasites.</p> <p>Methods</p> <p><it>Plasmodium falciparum </it>and <it>Plasmodium berghei </it>parasites were transfected with DNA constructs bearing <it>P. vivax dhfr-ts </it>pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous <it>dhfr-ts </it>of <it>P. falciparum </it>and <it>P. berghei </it>parasites with <it>P. vivax </it>homologous genes. The integration of <it>Pvdhfr-ts </it>genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.</p> <p>Results</p> <p>Transgenic <it>P. falciparum </it>and <it>P. berghei </it>lines stably expressing <it>Pv</it>DHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type <it>Pv</it>DHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant <it>Pv</it>DHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.</p> <p>Conclusion</p> <p>With the permanent integration of <it>Pvdhfr-ts </it>gene in the genome, the transgenic <it>Plasmodium </it>lines expressing <it>Pv</it>DHFR-TS are genetically stable and will be useful for screening anti-<it>P. vivax </it>compounds targeting <it>Pv</it>DHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-<it>P. vivax </it>drugs in general.</p

Waterborne protozoan pathogens in environmental aquatic biofilms: implications for water quality assessment strategies

Biofilms containing pathogenic organisms from the water supply are a potential source of protozoan parasite outbreaks and a general public health concern. The aim of the present study was to demonstrate the simultaneous and multispatial occurrence of waterborne protozoan pathogens (WBPP) in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottom water (BW) counterparts using manual filtration and elution from low-volume samples. For scenario purposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collected from the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of 69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques for Cryptosporidium, and immunofluorescence staining for the simultaneous detection of Cryptosporidium and Giardia. In the present study, WBPP was found in 43% SAB, 39% SW, and 39% BW samples tested with SAB results reflecting SW and BW results. Further, the potential and advantages of using low-volume sampling for the detection of parasite (oo)cysts in aquatic matrices were also demonstrated. Scanning electron microscopy of OM-SAB revealed a naturally-associated testate amoeba shell, while Cryptosporidium oocysts spiked samples provided a visual profile of what can be expected from naturally contaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens in low-volume environmental aquatic matrices and warrants SAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS)

Evaluation of Antimalarial Potential of Aqueous Crude Gymnema Inodorum Leaf Extract against Plasmodium berghei Infection in Mice

Malaria is still a serious cause of mortality and morbidity. Moreover, the emergence of malaria parasite resistance to antimalarial drugs has prompted the search for new, effective, and safe antimalarial agents. For this reason, the study of medicinal plants in discovering new antimalarial drugs is important and remains a crucial step in the fight against malaria. Hence, this study is aimed at investigating the antimalarial activity of Gymnema inodorum leaf extract (GIE) in Plasmodium berghei infected mice. Aqueous crude extract of G. inodorum leaves was prepared in distilled water (DW) and acute toxicity in mice was carried out. The antimalarial activity was assessed in the five groups of ICR mice employing the 4-day suppressive and curative tests. Untreated and positive controls were given DW along with 10 mg/kg of chloroquine, respectively. Any signs of toxicity, behavioral changes, and mortality were not observed in mice given GIE up to 5,000 mg/kg. GIE significantly (P < 0.05) suppressed parasitemia by 25.65%, 38.12%, and 58.28% at 10, 50, and 100 mg/kg, respectively, in the 4-day suppressive test. In the curative test, the highest parasitemia inhibition of 66.78% was observed at 100 mg/kg of GIE. Moreover, GIE prevented packed cell volume reduction and body weight loss compared to the untreated control. Additionally, GIE was able to prolong the mean survival time of infected mice significantly. The results obtained in this study confirmed the safety and promise of G. inodorum as an important source of new antimalarial agents and justify its folkloric use for malaria treatment

In Vivo Antimalarial Activity of Annona muricata Leaf Extract in Mice Infected with Plasmodium berghei

Malaria is one of the most important infectious diseases in the world. The choice for the treatment is highly limited due to drug resistance. Hence, finding the new compounds to treat malaria is urgently needed. The present study was attempted to evaluate the antimalarial activity of the Annona muricata aqueous leaf extract in Plasmodium berghei infected mice. Aqueous leaf extract of A. muricata was prepared and tested for acute toxicity in mice. For efficacy test in vivo, standard 4-day suppressive test was carried out. ICR mice were inoculated with 107 parasitized erythrocytes of P. berghei ANKA by intraperitoneal injection. The extracts (100, 500, and 1000 mg/kg) were then given orally by gavage once a day for 4 consecutive days. Parasitemia, percentage of inhibition, and packed cell volume were subsequently calculated. Chloroquine (10 mg/kg) was given to infected mice as positive control while untreated control was given only distilled water. It was found that A. muricata aqueous leaf extract at doses of 100, 500, and 1000 mg/kg resulted in dose dependent parasitemia inhibition of 38.03%, 75.25%, and 85.61%, respectively. Survival time was prolonged in infected mice treated with the extract. Moreover, no mortality to mice was observed with this extract up to a dose of 4000 mg/kg. In conclusion, the A. muricata aqueous leaf extract exerted significant antimalarial activity with no toxicity and prolonged survival time. Therefore, this extract might contain potential lead molecule for the development of a new drug for malaria treatment

Antimalarial Activity of Kaempferol and Its Combination with Chloroquine in Plasmodium berghei Infection in Mice

The search for new antimalarial drugs has become an urgent requirement due to resistance to the available drugs and the lack of an effective vaccine. In this respect, the present study aimed to evaluate the antimalarial activity of kaempferol against Plasmodium berghei infection in mice as an in vivo model. Chronic toxicity and antimalarial activities of kaempferol alone and in combination with chloroquine were investigated in P. berghei ANKA infected ICR mice using standard procedures. The results showed that chronic administration of 2,000 mg/kg of kaempferol resulted in no overt signs of toxicity as well as no hepatotoxicity, nephrotoxicity, or hematotoxicity. Interestingly, kaempferol exerted significant (P < 0.05) chemosuppressive, chemoprophylactic, and curative activities in a dose-dependent manner. The highest antimalarial activity was found at a dose of 20 mg/kg which resulted in a significantly (P < 0.05) prolonged survival of infected mice. Moreover, combination treatment of chloroquine and kaempferol also presented significant (P < 0.05) antimalarial effects, although the effects were not significantly different from the chloroquine treated group. From the results of the present study, it can be concluded that kaempferol possesses acceptable antimalarial activities. However, further investigation should be undertaken on the mechanism responsible for the observed antimalarial activity

The Potential Role of Gymnema inodorum Leaf Extract Treatment in Hematological Parameters in Mice Infected with Plasmodium berghei

Malaria remains a significant cause of death in tropical and subtropical regions by serious complications with hematological abnormalities consistent with high parasitemia. Hence, this study aimed to determine the efficacy of the Gymnema inodorum leaf extract (GIE) on hematological alteration in Plasmodium berghei infection in mice. Groups of ICR mice were infected intraperitoneally with parasitized red blood cells of P. berghei ANKA (PbANKA). They were administered orally by gavage of 100, 250, and 500 mg/kg of GIE for 4 consecutive days. Healthy and untreated groups were given distilled water, while 10 mg/kg of chloroquine was treated as the positive control. Hematological parameters including RBC count, hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), RBC distribution width (RDW), white blood cell (WBC) count, and WBC differential count were measured. The results showed that significant decreases of RBC count, Hb, Hct, MCV, MCH, MCHC, and reticulocytes were observed in the untreated group, while RDW was significantly increased compared with the healthy control. Furthermore, the WBC, neutrophil, monocyte, basophil, and eosinophil of untreated mice increased significantly, while the lymphocyte was significantly decreased compared with the healthy control. Interestingly, GIE normalized the hematological alteration induced by PbANKA infection in GIE-treated groups compared with healthy and untreated groups. The highest efficacy of GIE was observed at a dose of 500 mg/kg. Our results confirmed that GIE presented the potential role in the treatment of hematological alteration during malaria infection

Antihemolytic Activities of Green Tea, Safflower, and Mulberry Extracts during Plasmodium berghei Infection in Mice

Malaria-associated hemolysis is associated with mortality in adult patients. It has been speculated that oxidative stress and inflammation induced by malaria parasite are involved in its pathophysiology. Hence, we aimed to investigate the antihemolytic effect of green tea, safflower, and mulberry extracts against Plasmodium berghei infection. Aqueous crude extracts of these plants were prepared using hot water method and used for oral treatment in mice. Groups of ICR mice were infected with 6 × 106 infected red blood cells of P. berghei ANKA by intraperitoneal injection and given the extracts (500, 1500, and 3000 mg/kg) twice a day for 4 consecutive days. To assess hemolysis, hematocrit levels were then evaluated. Malaria infection resulted in hemolysis. However, antihemolytic effects were observed in infected mice treated with these extracts at dose-dependent manners. In conclusion, aqueous crude extracts of green tea, safflower, and mulberry exerted antihemolysis induced by malaria infection. These plants may work as potential source in the development of variety of herbal formulations for malarial treatment

Effects of Gymnema inodorum Leaf Extract on the Alteration of Blood Coagulation Parameters and Platelet Count in Plasmodium berghei-Infected Mice

Malaria remains highly prevalent and one of the major causes of morbidity and mortality in tropical and subtropical regions. Alteration of blood coagulation and platelets has played an important role and attributed to increased morbidity in malaria. Hence, this study was performed to investigate the efficacy of Gymnema inodorum leaf extract on Plasmodium berghei-induced alteration of blood coagulation parameters and platelet numbers in mice. Groups of ICR mice were inoculated with 1×107 parasitized red blood cells of P. berghei ANKA (PbANKA) and given orally by gavage with 100, 250, and 500 mg/kg of G. inodorum leaf extract (GIE). Chloroquine (10 mg/kg) was used as a positive control. Platelet count and blood coagulation parameters were measured. The results showed that PbANKA induced thrombocytopenia in mice as indicated by markedly decreased platelet count. Decreased platelet count had a negative correlation with the degree of parasitemia with R2 value of 0.6668. Moreover, significantly (p<0.05) shortened activated partial thromboplastin time was found in PbANKA-infected group, while prothrombin time and thrombin time were still normal. GIE gave significantly (p<0.05) good results with respect to platelet count, compared with the results obtained from positive and healthy controls. Additionally, GIE reversed the alteration of blood coagulation parameters when compared to untreated mice. The highest efficacy of GIE was observed at a dose of 500 mg/kg. It was concluded that GIE exerted a protective effect on thrombocytopenia and altered blood coagulation parameters induced by PbANKA infection in mice. This plant may be a future candidate for alternative antimalarial development