A model of pregnancy-associated malaria for inducing adverse pregnancy outcomes in ICR mouse.

BACKGROUND: Based on understanding of placental pathological features and safe medication in pregnancy-associated malaria (PAM), establishment of a stable pregnant mouse infection model with Plasmodium was urgently needed. METHODS: ICR mice with vaginal plugs detected were randomly divided into post-pregnancy infection (Malaria+) and uninfected pregnancy (Malaria-) cohorts. Age-matched mice that had not been mated were infected as pre-pregnancy infection group (Virgin control), which were subsequently mated with ICR males. All mice were inoculated with 1 × 106Plasmodium berghei ANKA-infected RBCs by intraperitoneal injection, and the same amount of saline was given to Malaria- group. We recorded the incidence of adverse pregnancy outcomes and the amounts of offspring in each group. RESULTS: The Virgin group mice were unable to conceive normally, and vaginal bleeding, abortion, or stillbirth appeared in the Malaria+ group. The incidence of adverse pregnancy outcomes was extremely high and statistically significant compared with the control (Malaria-) group (P < 0.05), of which placenta exhibited pathological features associated with human gestational malaria. CONCLUSIONS: The intraperitoneal injection of 1 × 106Plasmodium berghei ANKA-infected RBCs could establish a model of pregnancy-associated malaria in ICR mouse.

CD47 blockade reduces the pathologic features of experimental cerebral malaria and promotes survival of hosts with Plasmodium infection.

CD47 is an antiphagocytic "don't eat me" signal that inhibits programmed cell removal of self. As red blood cells (RBCs) age they lose CD47 expression and become susceptible to programmed cell removal by macrophages. CD47-/- mice infected with Plasmodium yoelii, which exhibits an age-based preference for young RBCs, were previously demonstrated to be highly resistant to malaria infection. Our study sought to test the therapeutic benefit of CD47 blockade on ameliorating the clinical syndromes of experimental cerebral malaria (ECM), using the Plasmodium berghei ANKA (Pb-A) murine model. In vitro we tested the effect of anti-CD47 mAb on Plasmodium-infected RBC phagocytosis and found that anti-CD47 treatment significantly increased clearance of Plasmodium-infected RBCs. Infection of C57BL/6 mice with Pb-A is lethal and mice succumb to the clinical syndromes of CM between days 6 and 10 postinfection. Strikingly, treatment with anti-CD47 resulted in increased survival during the cerebral phase of Pb-A infection. Anti-CD47-treated mice had increased lymphocyte counts in the peripheral blood and increased circulating levels of IFN-γ, TNF-α, and IL-22. Despite increased circulating levels of inflammatory cytokines, anti-CD47-treated mice had reduced pathological features in the brain. Survival of ECM in anti-CD47-treated mice was correlated with reduced cellular accumulation in the cerebral vasculature, improved blood-brain barrier integrity, and reduced cytotoxic activity of infiltrating CD8+ T cells. These results demonstrate the therapeutic benefit of anti-CD47 to reduce morbidity in a lethal model of ECM, which may have implications for preventing mortality in young African children who are the highest casualties of CM.

Increased Neutrophil Percentage and Neutrophil-T Cell Ratio Precedes Clinical Onset of Experimental Cerebral Malaria.

Newly emerging data suggest that several neutrophil defense mechanisms may play a role in both aggravating and protecting against malaria. These exciting findings suggest that the balance of these cells in the host body may have an impact on the pathogenesis of malaria. To fully understand the role of neutrophils in severe forms of malaria, such as cerebral malaria (CM), it is critical to gain a comprehensive understanding of their behavior and functions. This study investigated the dynamics of neutrophil and T cell responses in C57BL/6 and BALB/c mice infected with Plasmodium berghei ANKA, murine models of experimental cerebral malaria (ECM) and non-cerebral experimental malaria, respectively. The results demonstrated an increase in neutrophil percentage and neutrophil-T cell ratios in the spleen and blood before the development of clinical signs of ECM, which is a phenomenon not observed in the non-susceptible model of cerebral malaria. Furthermore, despite the development of distinct forms of malaria in the two strains of infected animals, parasitemia levels showed equivalent increases throughout the infection period evaluated. These findings suggest that the neutrophil percentage and neutrophil-T cell ratios may be valuable predictive tools for assessing the dynamics and composition of immune responses involved in the determinism of ECM development, thus contributing to the advancing of our understanding of its pathogenesis.

IL-6 dependent expansion of inflammatory MDSCs (CD11b+ Gr-1+) promote Th-17 mediated immune response during experimental cerebral malaria.

Myeloid derived suppressor cells (MDSCs) are a group of heterogeneous cell populations that can suppress T cell responses. Various aspects of MDSCs in regulating immune responses in several cancer and infectious diseases have been reported till date. But the role and regulation of MDSCs have not been systematically studied in the context of malaria. This study depicts the phenotypic and functional characteristics of splenic MDSCs and how they regulate Th-17 mediated immune response during Experimental Cerebral Malaria (ECM). Flow cytometric analysis reveals that MDSCs in the spleen and bone marrow expand at 8 dpi during ECM. Among subtypes of MDSCs, PMN-MDSCs show significant expansion in the spleen but M-MDSCs remain unaltered. Functional analysis of sorted MDSCs from spleens of Plasmodium berghei ANKA (PbA) infected mice shows suppressive nature of these cells and high production of Nitric oxide (NO). Besides, MDSCs were also found to express various inflammatory markers during ECM suggesting the M1 type phenotype of these cells. In-vivo depletion of MDSCs by the use of Anti Gr-1 increases mice survival but doesn't significantly alter the parasitemia. Previously, it has been reported that Treg/Th-17 balance in the spleen is skewed towards Th-17 during ECM. Depletion of MDSCs was found to regulate Th-17 percentages to homeostatic levels and subvert various inflammatory changes in the spleen. Among different factors, IL-6 was found to play an important role in the expansion of MDSCs and expression of inflammatory markers on MDSCs in a STAT3-dependent manner. These findings provide a unique insight into the role of IL-6 in the expansion of the MDSC population which causes inflammatory changes and increased Th-17 responses during ECM.

Characterization and inhibition of four fungi producing citrinin in various culture media.

PURPOSE: This study aimed to investigate the effects of different fermentation conditions (culture medium, temperature, incubation time, pH value and additive) on citrinin production by four fungi. RESULTS: Among the culture media, potato dextrose medium had lowest citrinin production, followed by yeast sucrose medium and monosodium glutamate medium. The lowest citrinin contents were produced by Monascus anka (M. anka) in potato dextrose medium and yeast sucrose medium, Aspergillus oryzae AS3.042 (A. oryzae) produced the lowest citrinin production in monosodium glutamate medium. The optimum fermentation temperatures for citrinin production by Aspergillus niger (A. niger) and Penicillium citrinum (P. citrinum) were at 30 °C, whereas those by M. anka and A. oryzae were at 35 °C. Citrinin synthesis by four fungi were completely inhibited with a pH value of less than 5.4. By adding ethylene diamine tetraacetic acid (EDTA) or triammonium citrate into monosodium glutamate medium, citrinin production by A. oryzae and A. niger were totally inhibited. Ammonium sulfate completely inhibited citrinin production by A. oryzae, M. anka and P. citrinum, and ammonium nitrate completely inhibited citrinin production by A. oryzae. CONCLUSIONS: These results indicated that the suitable fermentation conditions could make considerable contributions to the reduction of citrinin production. This study provided an effective way for decreasing the citrinin production.

Malaria in pregnancy regulates P-glycoprotein (P-gp/Abcb1a) and ABCA1 efflux transporters in the Mouse Visceral Yolk Sac.

Malaria in pregnancy (MiP) induces intrauterine growth restriction (IUGR) and preterm labour (PTL). However, its effects on yolk sac morphology and function are largely unexplored. We hypothesized that MiP modifies yolk sac morphology and efflux transport potential by modulating ABC efflux transporters. C57BL/6 mice injected with Plasmodium berghei ANKA (5 × 105 infected erythrocytes) at gestational day (GD) 13.5 were subjected to yolk sac membrane harvesting at GD 18.5 for histology, qPCR and immunohistochemistry. MiP did not alter the volumetric proportion of the yolk sac's histological components. However, it increased levels of Abcb1a mRNA (encoding P-glycoprotein) and macrophage migration inhibitory factor (Mif chemokine), while decreasing Abcg1 (P < 0.05); without altering Abca1, Abcb1b, Abcg2, Snat1, Snat2, interleukin (Il)-1ß and C-C Motif chemokine ligand 2 (Ccl2). Transcripts of Il-6, chemokine (C-X-C motif) ligand 1 (Cxcl1), Glut1 and Snat4 were not detectible. ABCA1, ABCG1, breast cancer resistance protein (BCRP) and P-gp were primarily immunolocalized to the cell membranes and cytoplasm of endodermic epithelium but also in the mesothelium and in the endothelium of mesodermic blood vessels. Intensity of P-gp labelling was stronger in both endodermic epithelium and mesothelium, whereas ABCA1 labelling increased in the endothelium of the mesodermic blood vessels. The presence of ABC transporters in the yolk sac wall suggests that this fetal membrane acts as an important protective gestational barrier. Changes in ABCA1 and P-gp in MiP may alter the biodistribution of toxic substances, xenobiotics, nutrients and immunological factors within the fetal compartment and participate in the pathogenesis of malaria-induced IUGR and PTL.

Experimental cerebral malaria is associated with profound loss of both glycan and protein components of the endothelial glycocalyx.

Vascular pathology is central to malaria pathogenesis and associated with severity of disease. We have previously documented shedding of the cerebral endothelial glycocalyx in experimental malaria and hypothesized that this action is implicated in the pathogenesis of cerebral malaria (CM). Quantification and characterization of the intraluminal vascular glycocalyx are technically challenging. Here, we used ferritin labeling, computerized image analysis, and biochemical characterization by using in vivo biotinylation and pull down. Image analysis divided mice with CM and uncomplicated malaria and uninfected control mice into 3 non-overlapping groups. Biochemical assessment of the luminal surface revealed malaria-induced alterations in all components of the glycocalyx in CM. This loss was mirrored in increases of the same components in peripheral blood samples. Corticosteroid treatment protected against CM, reduced inflammation, and prevented glycocalyx loss. Adjunctive antithrombin-3 also prevented glycocalyx loss and significantly reduced CM-associated mortality, as well as reduced local inflammation and prevented blood-brain barrier leakage. In contrast, inhibition of matrix metalloproteases with batimastat had limited effects on the glycocalyx and disease progression. Thus, glycocalyx loss may be associated with malaria pathogenesis and could be targeted by adjunctive treatment.-Hempel, C., Sporring, J., Kurtzhals, J. A. L. Experimental cerebral malaria is associated with profound loss of both glycan and protein components of the endothelial glycocalyx.

Host-mediated impairment of parasite maturation during blood-stage Plasmodium infection.

Severe malaria and associated high parasite burdens occur more frequently in humans lacking robust adaptive immunity to Plasmodium falciparum Nevertheless, the host may partly control blood-stage parasite numbers while adaptive immunity is gradually established. Parasite control has typically been attributed to enhanced removal of parasites by the host, although in vivo quantification of this phenomenon remains challenging. We used a unique in vivo approach to determine the fate of a single cohort of semisynchronous, Plasmodium berghei ANKA- or Plasmodium yoelii 17XNL-parasitized red blood cells (pRBCs) after transfusion into naive or acutely infected mice. As previously shown, acutely infected mice, with ongoing splenic and systemic inflammatory responses, controlled parasite population growth more effectively than naive controls. Surprisingly, however, this was not associated with accelerated removal of pRBCs from circulation. Instead, transfused pRBCs remained in circulation longer in acutely infected mice. Flow cytometric assessment and mathematical modeling of intraerythrocytic parasite development revealed an unexpected and substantial slowing of parasite maturation in acutely infected mice, extending the life cycle from 24 h to 40 h. Importantly, impaired parasite maturation was the major contributor to control of parasite growth in acutely infected mice. Moreover, by performing the same experiments in rag1-/- mice, which lack T and B cells and mount weak inflammatory responses, we revealed that impaired parasite maturation is largely dependent upon the host response to infection. Thus, impairment of parasite maturation represents a host-mediated, immune system-dependent mechanism for limiting parasite population growth during the early stages of an acute blood-stage Plasmodium infection.

Effect of Brugia pahangi co-infection with Plasmodium berghei ANKA in gerbils (Meriones unguiculatus).

Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.

Erythrocyte binding ligand region VI specific IgA confers tissue protection in malaria infection.

A direct role for IgA either for elimination of malaria parasite or for improvement in tissue pathology has not been investigated in case of Malaria infection while IgG, IgE and IgM were all implicated in the adverse pathology. In this communication, we delineate further that Malaria specific IgA appears to be significant among individuals who had multiple episodes of infection. Interestingly, the IgA elicited by immunization of the homologous peptides derived from Plasmodium berghei ANKA have also resulted in protection of host from adverse lung pathology, while the parasite load is unaffected. The PfrVI immunized mice and mice infected with repeated cycles of 'infection and recovery', simulating an endemic like situation, have resulted in development of B cell population that secretes the IgA specific to this region VI. Summarily, our results suggest that the IgA specific to the malarial antigen can confer significant advantage to hosts in protecting the overall tissue pathology.

Potential role of arteether on N-methyl-D-aspartate (NMDA) receptor expression in experimental cerebral malaria mice and extension of their survival.

Cerebral malaria (CM) is the severe neurological complication causing acute non-traumatic encephalopathy in tropical countries. The mechanisms underlying the fatal cerebral complications are still not fully understood. Glutamate, a major excitatory neurotransmitter in the central nervous system of the mammalian brain, plays a key role in the development of neuronal cells, motor function, synaptic plasticity, learning and memory processes under normal physiological conditions. The subtypes of ionotropic glutamate receptor are N-methyl-D-aspartate receptors (NMDARs) which are involved in cellular mechanisms of learning and memory, synaptic plasticity and also mediate excitotoxic neuronal injury. In the present study, we found that glutamate level in synaptosomes, as well as expression of NMDAR, was elevated during the extreme condition of CM in C57BL6 mice. Arteether at 50 mg kg-1 × 1, 25 mg kg-1 × 2, days decreased the NMDAR expression and increased the overall survival of the experimental CM mice.

Mice chronically fed a high-fat diet are resistant to malaria induced by Plasmodium berghei ANKA.

C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) develop neurological symptoms and die 6--7-day post-inoculation in the absence of high parasitemia. The effects of chronic intake of a high-fat diet on this process are largely unknown. In this study, we assessed the effect of a high-fat diet on the host-parasite response to malarial infection. Mice were fed ad libitum with either standard or a high-fat diet for 8 weeks and afterwards were infected with PbA. PbA-infected mice feeding a standard diet presented blood parasitemia, hepatic and cerebral histopathological alterations, and hepatic injury with increased hemozoin deposition in the liver. By contrast, these changes were not observed in the malaria high-fat diet group. In addition, mice fed a high-fat diet did not develop the expected neurological symptoms of cerebral malaria and were resistant to death. Taken together, our results indicate that chronic ingestion of high-fat diet prevents the development of experimental malaria induced by PbA injection, suggesting a relationship between a high-fat diet and malaria, which is an interesting subject for further study in humans.

TCRß Combinatorial Immunoreceptor Expression by Neutrophils Correlates with Parasite Burden and Enhanced Phagocytosis during a Plasmodium berghei ANKA Malaria Infection.

Recent studies have demonstrated that a subpopulation of neutrophils express the TCRαß combinatorial immunoreceptor in humans and mice. Here, we report that a Plasmodium berghei ANKA murine malaria infection induces expansion of TCRß expressing CD11b+ Ly6G+ neutrophils in the spleen during the early phase of infection. Measurement of TCRß transcript and protein levels of neutrophils in wild-type versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of nonspecific antibody staining or passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells. Remarkably, on day 3 postinfection, we observed a highly significant correlation between the proportion of neutrophils that express TCRß and peripheral blood parasite burden. In addition, TCRß+ neutrophils phagocytose parasitized erythrocytes with 4-fold greater efficiency than TCRß- neutrophils. Together these results signify that TCR expression by the neutrophil plays an important role in the regulation of parasite burden by enhancing the phagocytic capacity of the neutrophil.

The Glycosylphosphatidylinositol Transamidase Complex Subunit PbGPI16 of Plasmodium berghei Is Important for Inducing Experimental Cerebral Malaria.

In animal models of experimental cerebral malaria (ECM), the glycosylphosphatidylinositols (GPIs) and GPI anchors are the major factors that induce nuclear factor kappa B (NF-κB) activation and proinflammatory responses, which contribute to malaria pathogenesis. GPIs and GPI anchors are transported to the cell surface via a process called GPI transamidation, which involves the GPI transamidase (GPI-T) complex. In this study, we showed that GPI16, one of the GPI-T subunits, is highly conserved among Plasmodium species. Genetic knockout of pbgpi16 (Δpbgpi16) in the rodent malaria parasite Plasmodium berghei strain ANKA led to a significant reduction of the amounts of GPIs in the membranes of merozoites, as well as surface display of several GPI-anchored merozoite surface proteins. Compared with the wild-type parasites, Δpbgpi16 parasites in C57BL/6 mice caused much less NF-κB activation and elicited a substantially attenuated T helper type 1 response. As a result, Δpbgpi16 mutant-infected mice displayed much less severe brain pathology, and considerably fewer Δpbgpi16 mutant-infected mice died from ECM. This study corroborated the GPI toxin as a significant inducer of ECM and further suggested that vaccines against parasite GPIs may be a promising strategy to limit the severity of malaria.

Blood-Stage Plasmodium Berghei ANKA Infection Promotes Hepatic Fibrosis by Enhancing Hedgehog Signaling in Mice.

BACKGROUND/AIMS: Malaria is the most deadly parasitic infection in the world, resulting in damage to various organs, including the liver, of the infected organism; however, the mechanism causing this damage in the liver remains unclear. Liver fibrosis, a major characteristic of liver diseases, occurs in response to liver injury and is regulated by a complex network of signaling pathways. Hedgehog (Hh) signaling orchestrates a number of hepatic responses including hepatic fibrogenesis. Therefore, we investigated whether Hh signaling influenced the liver's response to malarial infection. METHODS: Eight-week-old male C57BL/6 mice inoculated with blood containing Plasmodium berghei ANKA (PbA)-infected erythrocytes were sacrificed when the level of parasitemia in the blood reached 10% or 30%, and the livers were collected for biochemical analysis. Liver responses to PbA infection were examined by hematoxylin and eosin staining, real-time polymerase chain reaction, immunohistochemistry and western blot. RESULTS: Severe hepatic injury, such as ballooned hepatocytes, sinusoidal dilatation, and infiltrated leukocytes, was evident in the livers of the malaria-infected mice. Hypoxia was also induced in 30% parasitemia group. With the accumulation of Kupffer cells, inflammation markers, TNF-α, interleukin-1ß, and chemokine (C-X-C motif) ligand 1, were significantly upregulated in the infected group compared with the control group. Expression of fibrotic markers, including transforming growth factor-ß, α-smooth muscle actin (α-SMA), collagen 1a1, thymosin ß4, and vimentin, were significantly higher in the infected groups than in the control group. With increased collagen deposition, hepatic stellate cells expressing α-SMA accumulated in the liver of the PbA-infected mice, whereas those cells were rarely detected in the livers of the control mice. The levels of Hh signaling and Yes-associated protein (YAP), two key regulators for hepatic fibrogenesis, were significantly elevated in the infected groups compared with the control group. Treatment of mice with Hh inhibitor, GDC-0449, reduced hepatic inflammation and fibrogenesis with Hh suppression in PbA-infected mice. CONCLUSION: Our results demonstrate that HSCs are activated in and Hh and YAP signaling are associated with this process, contributing to increased hepatic fibrosis in malaria-infected livers.

Neuregulin-1 attenuates experimental cerebral malaria (ECM) pathogenesis by regulating ErbB4/AKT/STAT3 signaling.

BACKGROUND: Human cerebral malaria (HCM) is a severe form of malaria characterized by sequestration of infected erythrocytes (IRBCs) in brain microvessels, increased levels of circulating free heme and pro-inflammatory cytokines and chemokines, brain swelling, vascular dysfunction, coma, and increased mortality. Neuregulin-1ß (NRG-1) encoded by the gene NRG1, is a member of a family of polypeptide growth factors required for normal development of the nervous system and the heart. Utilizing an experimental cerebral malaria (ECM) model (Plasmodium berghei ANKA in C57BL/6), we reported that NRG-1 played a cytoprotective role in ECM and that circulating levels were inversely correlated with ECM severity. Intravenous infusion of NRG-1 reduced ECM mortality in mice by promoting a robust anti-inflammatory response coupled with reduction in accumulation of IRBCs in microvessels and reduced tissue damage. METHODS: In the current study, we examined how NRG-1 treatment attenuates pathogenesis and mortality associated with ECM. We examined whether NRG-1 protects against CXCL10- and heme-induced apoptosis using human brain microvascular endothelial (hCMEC/D3) cells and M059K neuroglial cells. hCMEC/D3 cells grown in a monolayer and a co-culture system with 30 µM heme and NRG-1 (100 ng/ml) were used to examine the role of NRG-1 on blood brain barrier (BBB) integrity. Using the in vivo ECM model, we examined whether the reduction of mortality was associated with the activation of ErbB4 and AKT and inactivation of STAT3 signaling pathways. For data analysis, unpaired t test or one-way ANOVA with Dunnett's or Bonferroni's post test was applied. RESULTS: We determined that NRG-1 protects against cell death/apoptosis of human brain microvascular endothelial cells and neroglial cells, the two major components of BBB. NRG-1 treatment improved heme-induced disruption of the in vitro BBB model consisting of hCMEC/D3 and human M059K cells. In the ECM murine model, NRG-1 treatment stimulated ErbB4 phosphorylation (pErbB4) followed by activation of AKT and inactivation of STAT3, which attenuated ECM mortality. CONCLUSIONS: Our results indicate a potential pathway by which NRG-1 treatment maintains BBB integrity in vitro, attenuates ECM-induced tissue injury, and reduces mortality. Furthermore, we postulate that augmenting NRG-1 during ECM therapy may be an effective adjunctive therapy to reduce CNS tissue injury and potentially increase the effectiveness of current anti-malaria therapy against human cerebral malaria (HCM).

Elevated IL-17 levels in semi-immune anaemic mice infected with Plasmodium berghei ANKA.

BACKGROUND: Alterations in inflammatory cytokines and genetic background of the host contribute to the outcome of malaria infection. Despite the promising protective role of IL-17 in infections, little attention is given to further understand its importance in the pathogenesis of severe malaria anaemia in chronic/endemic situations. The objective of this study, therefore, was to evaluate IL-17 levels in anaemic condition and its association with host genetic factors. METHODS: Two mice strains (Balb/c and CBA) were crossed to get the F1 progeny, and were (F1, Balb/c, CBA) taken through 6 cycles of Plasmodium berghei (ANKA strain) infection and chloroquine/pyrimethamine treatment to generate semi-immune status. Cytokine levels and kinetics of antibody production, CD4+CD25+T regulatory cells were evaluated by bead-based multiplex assay kit, ELISA and FACs, respectively. RESULTS: High survival with high Hb loss at significantly low parasitaemia was observed in Balb/c and F1. Furthermore, IgG levels were two times higher in Balb/c, F1 than CBA. While CD4+CD25+ Treg cells were lower in CBA; IL-4, IFN-γ, IL-12α and IL-17 were significantly higher (p < 0.05) in Balb/c, F1. CONCLUSIONS: In conclusion, elevated IL-17 levels together with high IL-4, IL-12α and IFN-γ levels may be a marker of protection, and the mechanism may be controlled by host factor (s). Further studies of F2 between the F1 and Balb/c will be informative in evaluating if these genes are segregated or further apart.

In vivo validation of anti-malarial activity of crude extracts of Terminalia macroptera, a Malian medicinal plant.

BACKGROUND: Plasmodium falciparum malaria is still one of the most deadly pathology worldwide. Efficient treatment is jeopardized by parasite resistance to artemisinin and its derivatives, and by poor access to treatment in endemic regions. Anti-malarial traditional remedies still offer new tracks for identifying promising antiplasmodial molecules, and a way to ensure that all people have access to care. The present study aims to validate the traditional use of Terminalia macroptera, a Malian plant used in traditional medicine. METHODS: Terminalia macroptera was collected in Mali. Leaves (TML) and roots ethanolic extracts (TMR) were prepared and tested at 2000 mg/kg for in vivo acute toxicity in Albino Swiss mice. Antiplasmodial activity of the extracts was assessed against a chloroquine resistant strain P. falciparum (FcB1) in vitro. In vivo, anti-malarial efficacy was assessed by a 4-day suppressive test at 100 mg/kg in two malaria murine models of uncomplicated malaria (Plasmodium chabaudi chabaudi infection) and cerebral malaria (Plasmodium berghei strain ANKA infection). Constituents of TMR were characterized by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation pattern. RESULTS: Lethal dose of TML and TMR were greater than 2000 mg/kg in Albino Swiss mice. According to the OECD's Globally Harmonized System of Classification, both extracts are non-toxic orally. Antiplasmodial activity of T. macroptera extracts was confirmed in vitro against P. falciparum FcB1 strain with IC50 values of 1.2 and 1.6 µg/mL for TML and TMR, respectively. In vivo, oral administration of TML and TMR induced significant reduction of parasitaemia (37.2 and 46.4% respectively) in P. chabaudi chabaudi infected mice at the 7th day of infection compared to untreated mice. In the cerebral malaria experimental model, mice treated with TMR and TML presented respectively 50 and 66.7% survival rates at day 9 post-infection when all untreated mice died. Eleven major compounds were found in TMR. Among them, several molecules already known could be responsible for the antiplasmodial activity of the roots extract of T. macroptera. CONCLUSIONS: This study confirms both safety and anti-malarial activity of T. macroptera, thus validating its traditional use.

S-Nitrosoglutathione Reductase Deficiency Confers Improved Survival and Neurological Outcome in Experimental Cerebral Malaria.

Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by S-nitrosothiols (SNOs). Based on these observations, we hypothesized that animals deficient in the SNO-metabolizing enzyme, S-nitrosoglutathione reductase (GSNOR), which exhibit enhanced S-nitrosylation, would have improved outcomes in a preclinical model of cerebral malaria. GSNOR knockout (KO) mice infected with Plasmodium berghei ANKA had significantly delayed mortality compared to WT animals (P < 0.0001), despite higher parasite burdens (P < 0.01), and displayed markedly enhanced survival versus the wild type (WT) when treated with the antimalarial drug artesunate (77% versus 38%; P < 0.001). Improved survival was associated with higher levels of protein-bound NO, decreased levels of CD4+ and CD8+ T cells in the brain, improved blood-brain barrier integrity, and improved coma scores, as well as higher levels of gamma interferon. GSNOR KO animals receiving WT bone marrow had significantly reduced survival following P. berghei ANKA infection compared to those receiving KO bone barrow (P < 0.001). Reciprocal transplants established that survival benefits of GSNOR deletion were attributable primarily to the T cell compartment. These data indicate a role for GSNOR in the host response to malaria infection and suggest that strategies to disrupt its activity will improve clinical outcomes by enhancing microvascular integrity and modulating T cell tissue tropism.

Pigment fingerprint profile during extractive fermentation with Monascus anka GIM 3.592.

BACKGROUND: Traditional submerged fermentation mainly accumulates intracellular orange pigments with absorption maxima at 470 nm, whereas extractive fermentation of Monascus spp. with Triton X-100 can promote the export of intracellular pigments to extracellular broth, mainly obtaining extracellular yellow pigments with absorption maxima at approximately 410 nm. In this study, a strain of Monascus (M. anka GIM 3.592) that produces high yields of pigments was employed to investigate the differences in pigment fingerprint profiles between submerged and extractive fermentations. RESULTS: Using extractive fermentation with this high-yield strain, the extracellular pigments exhibited an absorption maximum at 430 nm, not 410 nm, as previously observed. By comparing the pigment fingerprint profiles between submerged and extractive fermentations, extractive fermentation was found to not only export intracellular pigments to the extracellular broth, but also to form four other yellow pigments (Y1-Y4) that accounted for a large proportion of the extracellular pigments and that were not produced in submerged fermentation. The yields of Y1-Y4 were closely related to the concentration and feeding time point of Triton X-100. Y1-Y4 presented identical UV-Vis spectra with absorption maxima at 430 nm and fluorescence spectra with absorption maxima (emission) at 565 nm. HPLC-MS and the spectral analysis showed that the four pigments (Y1-Y4) had not been previously reported. The results indicated that these pigments may rely on the bioconversion of orange pigments (rubropunctatin and monascorubrin). CONCLUSIONS: Using extractive fermentation with M. anka led to a high yield of extracellular yellow pigments (AU410 nm = 114), and the pigment fingerprint profile significantly differed compared to the results of traditional submerged fermentation. These results provide information and a detailed view of the composition and variation of pigments in extractive fermentation and could also contribute to characterizing pigment metabolism during extractive fermentation.