Induction of liver-resident memory T cells and protection at liver-stage malaria by mRNA-containing lipid nanoparticles.

Recent studies have suggested that CD8+ liver-resident memory T (TRM) cells are crucial in the protection against liver-stage malaria. We used liver-directed mRNA-containing lipid nanoparticles (mRNA-LNPs) to induce liver TRM cells in a murine model. Single-dose intravenous injections of ovalbumin mRNA-LNPs effectively induced antigen-specific cytotoxic T lymphocytes in a dose-dependent manner in the liver on day 7. TRM cells (CD8+ CD44hi CD62Llo CD69+ KLRG1-) were induced 5 weeks after immunization. To examine the protective efficacy, mice were intramuscularly immunized with two doses of circumsporozoite protein mRNA-LNPs at 3-week intervals and challenged with sporozoites of Plasmodium berghei ANKA. Sterile immunity was observed in some of the mice, and the other mice showed a delay in blood-stage development when compared with the control mice. mRNA-LNPs therefore induce memory CD8+ T cells that can protect against sporozoites during liver-stage malaria and may provide a basis for vaccines against the disease.

Anti-Trypanosoma cruzi activity of Coptis rhizome extract and its constituents.

BACKGROUND: Current therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo. METHODS: As an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging. RESULTS: As a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC50 values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model. CONCLUSIONS: For these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.

Challenges Based on Antiplasmodial and Antiviral Activities of 7-Chloro-4-aminoquinoline Derivatives.

We report the structural functionalization of the terminal amino group of N1 -(7-chloroquinolin-4-yl) butane-1,4-diamine, leading to a series of 7-chloro-4-aminoquinoline derivatives, and their evaluation as potent anti-malarial and anti-viral agents. Some compounds exhibited promising anti-malarial effects against the Plasmodium falciparum 3D7 (chloroquine-sensitive) and Dd2 (chloroquine-resistant) strains. In addition, these compounds were assayed in vitro against influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compound 5 h, bearing an N-mesityl thiourea group, displayed pronounced anti-infectious effects against malaria, IAV, and SARS-CoV-2. These results provide new insights into drug discovery for the prevention or treatment of malaria and virus co-infection.

Effect of Cholesterol Content of Lipid Composition in mRNA-LNPs on the Protein Expression in the Injected Site and Liver After Local Administration in Mice.

Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

A novel coculture system for assessing respiratory sensitizing potential by IL-4 in T cells.

Although several in vitro assays that predict the sensitizing potential of chemicals have been developed, none can distinguish between chemical respiratory and skin sensitizers. Recently, we established a new three-dimensional dendritic cell (DC) coculture system consisting of a human airway epithelial cell line, immature DCs derived from human peripheral monocytes, and a human lung fibroblast cell line. In this coculture system, compared to skin sensitizers, respiratory sensitizers showed enhanced mRNA expression in DCs of the key costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) cell differentiation. Herein, we established a new two-step DC/T cell coculture system by adding peripheral allogeneic naïve CD4+ T cells to the DCs stimulated in the DC coculture system. In this DC/T cell coculture system, model respiratory sensitizers, but not skin sensitizers, enhanced mRNA expression of the predominant Th2 marker interleukin-4 (IL-4). To improve the versatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML were used in the DC coculture system. As in peripheral monocytes, enhanced mRNA expression of OX40L was induced in CD14-ML by respiratory sensitizers compared to skin sensitizers. When these cell lines were applied to the DC/T cell coculture system with peripheral allogeneic naïve CD4+ T cells, respiratory sensitizers but not skin sensitizers enhanced the mRNA expression of IL-4. Thus, this DC/T cell coculture system may be useful for discriminating between respiratory and skin sensitizers by differential mRNA upregulation of IL-4 in T cells.

Stability Study of mRNA-Lipid Nanoparticles Exposed to Various Conditions Based on the Evaluation between Physicochemical Properties and Their Relation with Protein Expression Ability.

Lipid nanoparticles (LNPs) are currently in the spotlight as delivery systems for mRNA therapeutics and have been used in the Pfizer/BioNTech and Moderna COVID-19 vaccines. mRNA-LNP formulations have been indicated to require strict control, including maintenance at fairly low temperatures during their transport and storage. Since it is a new pharmaceutical modality, there is a lack of information on the systematic investigation of how storage and handling conditions affect the physicochemical properties of mRNA-LNPs and their protein expression ability. In this study, using the mRNA-LNPs with standard composition, we evaluated the effects of temperature, cryoprotectants, vibration, light exposure, and syringe aspiration from the vials on the physicochemical properties of nanoparticles in relation to their in vitro/in vivo protein expression ability. Among these factors, storage at -80 °C without a cryoprotectant caused a decrease in protein expression, which may be attributed to particle aggregation. Exposure to vibration and light also caused similar changes under certain conditions. Exposure to these factors can occur during laboratory and hospital handling. It is essential to have sufficient knowledge of the stability of mRNA-LNPs in terms of their physical properties and protein expression ability at an early stage to ensure reproducible research and development and medical care.

Focused ultrasound/microbubbles-assisted BBB opening enhances LNP-mediated mRNA delivery to brain.

Messenger RNA (mRNA) medicine has become a new therapeutic approach owing to the progress in mRNA delivery technology, especially with lipid nanoparticles (LNP). However, mRNA encapsulated-LNP (mRNA-LNP) cannot spontaneously cross the blood-brain barrier (BBB) which prevents the expression of foreign proteins in the brain. Microbubble-assisted focused ultrasound (FUS) BBB opening is an emerging technology that can transiently enhance BBB permeability. In this study, FUS/microbubble-assisted BBB opening was investigated for the intravenous delivery of mRNA-LNP to the brain. The intensity of FUS irradiation was optimized to 1.5 kW/cm2, at which BBB opening occurred efficiently without hemorrhage or edema. Exogenous protein (luciferase) expression by mRNA-LNP, specifically at the FUS-irradiated side of the brain, occurred only when FUS and microbubbles were applied. This exogenous protein expression was faster but shorter than that of plasmid DNA delivery. Furthermore, foreign protein expression was observed in the microglia, along with CD31-positive endothelial cells, whereas no expression was observed in astrocytes or neurons. These results support the addition of mRNA-LNP to the lineup of nanoparticles delivered by BBB opening.

Anti-SARS-CoV-2 activity of various PET-bottled Japanese green teas and tea compounds in vitro.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to global public health. The emergence of SARS-CoV-2 variants is a significant concern regarding the continued effectiveness of vaccines and antiviral therapeutics. Thus, natural products such as foods, drinks, and other compounds should be investigated for their potential to treat COVID-19. Here, we examined the in vitro antiviral activity against SARS-CoV-2 of various polyethylene terephthalate (PET)-bottled green Japanese teas and tea compounds. Six types of PET-bottled green tea were shown to inhibit SARS-CoV-2 at half-maximal inhibitory concentrations (IC50) of 121- to 323-fold dilution. Our study revealed for the first time that a variety of PET-bottled Japanese green tea drinks inhibit SARS-CoV-2 infection in a dilution-dependent manner. The tea compounds epigallocatechin gallate (EGCG) and epicatechin gallate showed virucidal activity against SARS-CoV-2, with IC50 values of 6.5 and 12.5 µM, respectively. The investigated teas and tea compounds inactivated SARS-CoV-2 in a dose-dependent manner, as demonstrated by the viral RNA levels and infectious titers. Furthermore, the green teas and EGCG showed significant inhibition at the entry and post-entry stages of the viral life cycle and inhibited the activity of the SARS-CoV-2 3CL-protease. These findings indicate that green tea drinks and tea compounds are potentially useful in prophylaxis and COVID-19 treatment.

The discovery of herbal drugs and natural compounds as inhibitors of SARS-CoV-2 infection in vitro.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2019 has led to a global health crisis. Mutations of the SARS-CoV-2 genome have impeded the development of effective therapeutics and vaccines against SARS-CoV-2. Natural products are important for discovering therapeutics to treat the 2019 coronavirus disease (COVID-19). In the present study, we investigated the antiviral activity of herbal drug extracts from Polygala Root, Areca, and Quercus Bark and natural compounds derived from herbal drug such as baicalin and glabridin, with IC50 values of 9.5 µg/ml, 1.2 µg/ml, 5.4 µg/ml, 8.8 µM, and 2.5 µM, respectively, against SARS CoV-2 infection in vitro. Certain herbal drug extracts and natural compounds were found to inhibit viral RNA levels and infectious titers of SARS-CoV-2 in a dose-dependent manner. Furthermore, viral protein analyses showed that herbal drug extracts and natural compounds effectively inhibited SARS-CoV-2 in the various entry treatments. Our study revealed that three herbal drugs are good candidates for further in vivo and clinical studies.

Phenylpropanoid-conjugated iridoid glucosides from leaves of Morinda morindoides.

Three phenylpropanoid-conjugated iridoid glucosides, acetylgaertneric acid (1), acetyldehydrogaertneroside (2), and dehydrogaertneric acid (10), together with nine known related iridoid glucosides (3-9, 11, and 12), two coumaroyl alkaloids, one benzenoid, and three flavonoid glucosides were isolated from leaves of Morinda morindoides (Rubiaceae). Structures of these isolated compounds were determined using spectroscopic analysis. Compounds 1-18 and previously isolated compounds (19-29) were evaluated for anti-trypanosomal activity against Trypanosoma cruzi Tulahuen strain (trypomastigote and amastigote) together with cytotoxicity against host cells, new-born mouse heart cells. Among them, molucidin (21) and prismatomerin (22) exhibited good anti-trypanosomal activity (IC50 of 4.67 and 5.70 µM, respectively), together with cytotoxicity (CC50 of 2.76 and 3.22 µM, respectively). Compounds 1-18 did not show anti-malarial activity against a chloroquine/mefloquine-sensitive strain of Plasmodium falciparum.

The novel therapeutic target and inhibitory effects of PF-429242 against Zika virus infection.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of African origin that is transmitted by Aedes mosquitoes. ZIKV was historically limited to Africa and Asia, where mild cases were reported. However, ZIKV has recently been responsible for major global outbreaks associated with a wide range of neurological complications. Since no antiviral therapy exists for ZIKV, drug discovery research for ZIKV is crucial. Intracellular lipids regulated by sterol regulatory element-binding proteins (SREBPs) are important in flavivirus pathogenesis. PF-429242 has been reported to inhibit the activity of site-1 protease (S1P), which regulates the expression of SREBP target genes. Our primary objective in this study is to elucidate the mechanism of the antiviral activity of PF-429242 against the African genotype (ZIKVMR-766) and Asian genotypes (ZIKV H/PF 2013 and ZIKV PRVABC59) using several primate-derived cell lines. The virus titer was determined via a focus-forming assay; we used flow cytometry to quantify intracellular lipids in ZIKV-infected and mock-treated cells. The PF-429242 molecule effectively suppressed ZIKV infection in neuronal cell lines; T98G, U-87MG, SK-N-SH and primary monocytes cell, indicating that PF-429242 molecule can be used therapeutically. A strong reduction in ZIKV replication was observed at 12 µM and 30 µM in in neuronal cell lines and primary monocytes, respectively. Interestingly, the inhibitory effects of the PF-429242 molecule were observed when it was tested on various ZIKV-lineage infections. Lipid quantification reveals that ZIKV increases lipogenesis in infected cells, while the exogenous addition of cholesterol effectively blocks ZIKV replication. Furthermore, the supplementation of oleic acid increases the ZIKV titer. Fenofibrate, an inhibitor of lipid droplet formation, reduces the ZIKV titer. Collectively, our results demonstrate that the development of antiviral drugs against ZIKV could be based on key regulators of lipid metabolism. In addition, this study reveals that the mechanism of the PF-429242-mediated suppression among flavivirus infections is not entirely identical. Our results warrant further evaluation of PF-429242 as a prospective antiviral drug, given the multiple advantageous properties of this compound, such as its limited toxicity, neuroprotective properties, and broad spectrum of capabilities.

2D-quantitative structure-activity relationships model using PLS method for anti-malarial activities of anti-haemozoin compounds.

BACKGROUND: Emergence of cross-resistance to current anti-malarial drugs has led to an urgent need for identification of potential compounds with novel modes of action and anti-malarial activity against the resistant strains. One of the most promising therapeutic targets of anti-malarial agents related to food vacuole of malaria parasite is haemozoin, a product formed by the parasite through haemoglobin degradation. METHODS: With this in mind, this study developed two-dimensional-quantitative structure-activity relationships (QSAR) models of a series of 21 haemozoin inhibitors to explore the useful physicochemical parameters of the active compounds for estimation of anti-malarial activities. The 2D-QSAR model with good statistical quality using partial least square method was generated after removing the outliers. RESULTS: Five two-dimensional descriptors of the training set were selected: atom count (a_ICM); adjacency and distance matrix descriptor (GCUT_SLOGP_2: the third GCUT descriptor using atomic contribution to logP); average total charge sum (h_pavgQ) in pKa prediction (pH = 7); a very low negative partial charge, including aromatic carbons which have a heteroatom-substitution in "ortho" position (PEOE_VSA-0) and molecular descriptor (rsynth: estimating the synthesizability of molecules as the fraction of heavy atoms that can be traced back to starting material fragments resulting from retrosynthetic rules), respectively. The model suggests that the anti-malarial activity of haemozoin inhibitors increases with molecules that have higher average total charge sum in pKa prediction (pH = 7). QSAR model also highlights that the descriptor using atomic contribution to logP or the distance matrix descriptor (GCUT_SLOGP_2), and structural component of the molecules, including topological descriptors does make for better anti-malarial activity. CONCLUSIONS: The model is capable of predicting the anti-malarial activities of anti-haemozoin compounds. In addition, the selected molecular descriptors in this QSAR model are helpful in designing more efficient compounds against the P. falciparum 3D7A strain.

Phenylpropanoid conjugated iridoids with anti-malarial activity from the leaves of Morinda morindoides.

Two phenylpropanoid-conjugated iridoids, deglucosyl gaertneroside (1) and morindoidin (2), were isolated from the leaves of Morinda morindoides (Rubiaceae) by activity-guided fractionation using an anti-malarial activity assay. The known related iridoids molucidin (3) and prismatomerin (4), two lignans, abscisic acid, two megastigmanes, and two flavonol glycosides were also identified. The structures of isolated compounds were elucidated using spectroscopic analysis. The isolated compounds were evaluated for anti-malarial activity against the chloroquine/mefloquine-sensitive strains of Plasmodium falciparum together with cytotoxicity against adult mouse brain cells. Potent anti-malarial activity of 3 and 4 (IC50 of 0.96 and 0.80 µM, CC50 of 1.02 and 0.88 µM, and SI of 1.06 and 1.10, respectively) was shown, while new iridoids 1 and 2 and pinoresinol (5) displayed moderate activity (IC50 of 40.9, 20.6, and 24.2 µM) without cytotoxicity (CC50 > 50 µM). These results indicate that 1-5 may be promising lead compounds for anti-malarial drugs. In addition, our results imply the necessity of the quality control of the extract of M. morindoides leaves based on the contents of 1-5 in terms of the safety and efficacy.

Role of cytokines produced by T helper immune-modulators in dengue pathogenesis: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Modulation of the immune reaction is essential in the development of various diseases, including dengue's "Cytokine Tsunami", an increase in vascular permeability with concomitant severe vascular leakage. We aim to identify the role of T-helper (Th) cells, Th2 and Th7, with their related cytokines in dengue pathogenesis. MATERIAL AND METHODS: Nine electronic databases and manual search were applied to detect available publications. A meta-analysis using a fixed- or random-effect model was performed to measure standardized mean difference (SMD) with 95% confidence interval (CI). The National Institute of Health (NIH) tools for observational cohort, cross-sectional, and case-control studies were used to examine the risk of bias. The protocol was recorded in PROSPERO with CRD42017060230. RESULTS: A total of 38 articles were found including 19 case-control, 11 cross-sectional and 8 prospective cohort studies. We indicated that Th2 cytokines (IL-4, IL-6, IL-8) and Th17 cytokine (IL-17) in dengue patients were notably higher than in a healthy control group in acute phase (SMD = 1.59, 95% CI [0.68, 2.51], p = 0.001; SMD = 1.24, 95% CI [0.41, 2.06], p = 0.003; SMD = 1.13, 95% CI [0.61, 1.66], p<0.0001; SMD = 1.74, 95% CI [0.87, 2.61], p<0.0001), respectively. CONCLUSIONS: This study provides evidence of the significant roles of IL-4, IL-6, IL-8, IL-10 and IL-17 in the pathogenesis of developing a severe reaction in dengue fever. However, to fully determine the association of Th cytokines with dengue, it is necessary to perform further studies to assess kinetic levels during the duration of the illness.

Kinetics of CD4+ T Helper and CD8+ Effector T Cell Responses in Acute Dengue Patients.

Background: The protective or pathogenic role of T lymphocytes during the acute phase of dengue virus (DENV) infection has not been fully understood despite its importance in immunity and vaccine development. Objectives: This study aimed to clarify the kinetics of T lymphocyte subsets during the clinical course of acute dengue patients. Study design: In this hospital-based cohort study, 59 eligible Vietnamese dengue patients were recruited and admitted. They were investigated and monitored for T cell subsets and a panel of clinical and laboratory parameters every day until discharged and at post-discharge from the hospital. Results: We described for the first time the kinetics of T cell response during the clinical course of DENV infection. Severe cases showed significantly lower levels of effector CD8+ T cells compared to mild cases at day -1 (p = 0.017) and day 0 (p = 0.033) of defervescence. After defervescence, these cell counts in severe cases increased rapidly to equalize with the levels of mild cases. Our results also showed a decline in total CD4+ T, Th1, Th1/17 cells during febrile phase of dengue patients compared to normal controls or convalescent phase. On the other hand, Th2 cells increased during DENV infection until convalescent phase. Cytokines such as interferon-γ, IL-12p70, IL-5, IL-23, IL-17A showed tendency to decrease on day 0 and 1 compared with convalescence and only IL-5 showed significance indicating the production during acute phase was not systemic. Conclusion: With a rigorous study design, we uncovered the kinetics of T cells in natural DENV infection. Decreased number of effector CD8+ T cells in the early phase of infection and subsequent increment after defervescence day probably associated with the T cell migration in DENV infection.

Impact of political conflict on tuberculosis notifications in North-east Nigeria, Adamawa State: a 7-year retrospective analysis.

OBJECTIVE: We assessed the impact of political conflict (Boko Haram) on tuberculosis (TB) case notifications in Adamawa State in North-east Nigeria. DESIGN: A retrospective analysis of TB case notifications from TB registers (2010-2016) to describe changes in TB notification, sex and age ratios by the degree of conflict by local government area. SETTING: Adamawa State. PARTICIPANTS: 21 076 TB cases notified. RESULTS: 21 076 cases (62% male) were notified between 2010 and 2016, of which 19 604 (93%) were new TB cases. Areas affected by conflict in 2014 and 2015 had decreased case notification while neighbouring areas reported increased case notifications. The male to female ratio of TB cases changed in areas in conflict with more female cases being notified. The young and elderly (1-14 and >65 years old) had low notifications in all areas, with a small increase in case notifications during the years of conflict. CONCLUSION: TB case notifications decreased in conflict areas and increased in areas without conflict. More males were notified during peace times and more female cases were reported from areas in conflict. Young and elderly populations had decreased case notifications but experienced a slight increase during the conflict years. These changes are likely to reflect population displacement and a dissimilar effect of conflict on the accessibility of services. TB services in conflict areas deserve further study to identify resilient approaches that could reach affected populations.

Knowledge, behaviour and attitudes towards Chagas disease among the Bolivian migrant population living in Japan: a cross-sectional study.

OBJECTIVES: This study aimed to investigate the knowledge, behaviour and attitudes towards Chagas disease (CD) among Latin American migrants in Japan and to evaluate the effectiveness of an educational activity (EA) in increasing knowledge of CD. DESIGN: A cross-sectional, mixed-methods study employing a preknowledge and postknowledge test and focus group discussion, conducted from March 2018 to June 2018. PARTICIPANTS: Seventy-two participants were included, all born in Bolivia and residents in four Japanese cities. Fifty-nine of them participated in the EA. INTERVENTIONS: The EA comprised showing three videos about CD and a group discussion covering different dimensions of CD and was evaluated with questionnaires to analyse the knowledge of the participants before and after. RESULTS: Seventy-two participants were enrolled, predominantly from highly endemic CD areas of Bolivia. Though most participants were familiar with vector-borne transmission, epidemiology and symptomatology of CD, the baseline knowledge of CD was low. Less than 10% of them had been tested prior for CD. The dominant factors associated with better knowledge were living in Japan for more than 10 years (OR=8.42, 95% CI 1.56 to 48.62) and previously testing for CD (OR=11.32; 95% CI 1.52 to 105.9). The EA significantly improved the CD knowledge of the participants (p value <0.0001; 95% CI 2.32 to 3.84). The participants associated the term 'Chagas' mostly with fear and concern. The level of stigmatisation was low, in contrast to the results of other studies. The barriers encountered in care-seeking behaviour were language, the migration process and difficulties to access the healthcare system. CONCLUSION: EA with an integrative approach is useful to increase the knowledge of CD within the Bolivian migrant population living in Japan. The activity brings the possibility to explore not only the level of knowledge but also to reveal experiences and to understand the needs of the people at risk. Considering them as actors towards healthcare solutions could lead to better outcomes for the success of future policies and interventions aimed to decrease the global burden.

Anti-malarial activity of traditional Kampo medicine Coptis rhizome extract and its major active compounds.

BACKGROUND: Herbal medicine has been a rich source of new drugs exemplified by quinine and artemisinin. In this study, a variety of Japanese traditional herbal medicine ('Kampo') were examined for their potential anti-malarial activities. METHODS: A comprehensive screening methods were designed to identify novel anti-malarial drugs from a library of Kampo herbal extracts (n = 120) and related compounds (n = 96). The anti-malarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) strains of Plasmodium falciparum. The cytotoxicity was also evaluated using primary adult mouse brain cells. After being selected through the first in vitro assay, positive extracts and compounds were examined for possible in vivo anti-malarial activity. RESULTS: Out of 120 herbal extracts, Coptis rhizome showed the highest anti-malarial activity (IC50 1.9 µg/mL of 3D7 and 4.85 µg/mL of Dd2) with a high selectivity index (SI) > 263 (3D7) and > 103 (Dd2). Three major chlorinated compounds (coptisine, berberine, and palmatine) related to Coptis rhizome also showed anti-malarial activities with IC50 1.1, 2.6, and 6.0 µM (against 3D7) and 3.1, 6.3, and 11.8 µM (against Dd2), respectively. Among them, coptisine chloride exhibited the highest anti-malarial activity (IC50 1.1 µM against 3D7 and 3.1 µM against Dd2) with SI of 37.8 and 13.2, respectively. Finally, the herbal extract of Coptis rhizome and its major active compound coptisine chloride exhibited significant anti-malarial activity in mice infected with Plasmodium yoelii 17X strain with respect to its activity on parasite suppression consistently from day 3 to day 7 post-challenge. The effect ranged from 50.38 to 72.13% (P < 0.05) for Coptis rhizome and from 81 to 89% (P < 0.01) for coptisine chloride. CONCLUSION: Coptis rhizome and its major active compound coptisine chloride showed promising anti-malarial activity against chloroquine-sensitive (3D7) and -resistant (Dd2) strains in vitro as well as in vivo mouse malaria model. Thus, Kampo herbal medicine is a potential natural resource for novel anti-malarial agents.

IL-17A, a possible biomarker for the evaluation of treatment response in Trypanosoma cruzi infected children: A 12-months follow-up study in Bolivia.

BACKGROUND: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. METHODS AND FINDINGS: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual's treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months' time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. CONCLUSIONS: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation.