Efficient Treatment of Experimental Cerebral Malaria by an Artemisone-SMEDDS System: Impact of Application Route and Dosing Frequency.

Artemisone (ART) has been successfully tested in vitro and in animal models against several diseases. However, its poor aqueous solubility and limited chemical stability are serious challenges. We developed a self-microemulsifying drug delivery system (SMEDDS) that overcomes these limitations. Here, we demonstrate the efficacy of this formulation against experimental cerebral malaria in mice and the impact of its administration using different routes (gavage, intranasal delivery, and parenteral injections) and frequency on the efficacy of the treatment. The minimal effective daily oral dose was 20 mg/kg. We found that splitting a dose of 20 mg/kg ART given every 24 h, by administering two doses of 10 mg/kg each every 12 h, was highly effective and gave far superior results compared to 20 mg/kg once daily. We obtained the best results with nasal treatment; oral treatment was ranked second, and the least effective route of administration was intraperitoneal injection. A complete cure of experimental cerebral malaria could be achieved through choosing the optimal route of application, dose, and dosing interval. Altogether, the developed formulation combines easy manufacturing with high stability and could be a successful and very versatile carrier for the delivery of ART in the treatment of human severe malaria.

Blood‒Brain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis.

Streptococcus pneumoniae is a major meningitis-causing pathogen globally, bringing about significant morbidity and mortality, as well as long-term neurological sequelae in almost half of the survivors. Subsequent to nasopharyngeal colonisation and systemic invasion, translocation across the blood‒brain barrier (BBB) by S. pneumoniae is a crucial early step in the pathogenesis of meningitis. The BBB, which normally protects the central nervous system (CNS) from deleterious molecules within the circulation, becomes dysfunctional in S. pneumoniae invasion due to the effects of pneumococcal toxins and a heightened host inflammatory environment of cytokines, chemokines and reactive oxygen species intracranially. The bacteria‒host interplay within the CNS likely determines not only the degree of BBB pathological changes, but also host survival and the extent of neurological damage. This review explores the relationship between S. pneumoniae bacteria and the host inflammatory response, with an emphasis on the BBB and its roles in CNS protection, as well as both the acute and long-term pathogenesis of meningitis.

Benzo[b]quinolizinium Derivatives Have a Strong Antimalarial Activity and Inhibit Indoleamine Dioxygenase.

The heme-containing enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and IDO-2 catalyze the conversion of the essential amino acid tryptophan into kynurenine. Metabolites of the kynurenine pathway and IDO itself are involved in immunity and the pathology of several diseases, having either immunoregulatory or antimicrobial effects. IDO-1 plays a central role in the pathogenesis of cerebral malaria, which is the most severe and often fatal neurological complication of infection with Plasmodium falciparum. Mouse models are usually used to study the underlying pathophysiology. In this study, we screened a natural compound library against mouse IDO-1 and identified 8-aminobenzo[b]quinolizinium (compound 2c) to be an inhibitor of IDO-1 with potency at nanomolar concentrations (50% inhibitory concentration, 164 nM). Twenty-one structurally modified derivatives of compound 2c were synthesized for structure-activity relationship analyses. The compounds were found to be selective for IDO-1 over IDO-2. We therefore compared the roles of prominent amino acids in the catalytic mechanisms of the two isoenzymes via homology modeling, site-directed mutagenesis, and kinetic analyses. Notably, methionine 385 of IDO-2 was identified to interfere with the entrance of l-tryptophan to the active site of the enzyme, which explains the selectivity of the inhibitors. Most interestingly, several benzo[b]quinolizinium derivatives (6 compounds with 50% effective concentration values between 2.1 and 6.7 nM) were found to be highly effective against P. falciparum 3D7 blood stages in cell culture with a mechanism independent of IDO-1 inhibition. We believe that the class of compounds presented here has unique characteristics; it combines the inhibition of mammalian IDO-1 with strong antiparasitic activity, two features that offer potential for drug development.

Production, fate and pathogenicity of plasma microparticles in murine cerebral malaria.

In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.

Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C(hi) monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8(+) T lymphocytes. A critical number of CD8(+) T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8(+) T lymphocytes in regulating vascular pathology in this disease.

Synergistic induction of CXCL10 by interferon-gamma and lymphotoxin-alpha in astrocytes: Possible role in cerebral malaria.

Cerebral malaria (CM) has a high mortality rate and incidence of neurological sequelae in survivors. Hypoxia and cytokine expression in the brain are two mechanisms thought to contribute to the pathogenesis of CM. The cytokines interferon (IFN)-γ and lymphotoxin (LT)-α and the chemokine CXCL10 are essential for the development of CM in a mouse model. Furthermore, serum IFN-γ protein levels are higher in human CM than in controls, and CXCL10 is elevated in both serum and cerebrospinal fluid in Ghanaian paediatric CM cases. Astrocytes actively participate in CNS pathologies, becoming activated in response to various stimuli including cytokines. Astrocyte activation also occurs in murine and human CM. We here determined the responsiveness of mouse and human astrocytes to IFN-γ and LT-α, with the aim of further elucidating the role of astrocytes in CM pathogenesis. Initially we confirmed that Ifn-γ and Cxcl10 are expressed in the brain in murine CM, and that the increased Cxcl10 expression is IFN-γ-dependant. IFN-γ induced CXCL10 production in human and murine astrocytes in vitro. The degree of induction was increased synergistically in the presence of LT-α. IFN-γ induced the expression of receptors for LT-α, while LT-α increased the expression of the receptor for IFN-γ, in the astrocytes. This cross-induction may explain the synergistic effect of the two cytokines on CXCL10 production. Expression of these receptors also was upregulated in the brain in murine CM. The results suggest that astrocytes contribute to CM pathogenesis by producing CXCL10 in response to IFN-γ and LT-α.

The kynurenine pathway of tryptophan degradation is activated during osteoblastogenesis.

The mechanisms involved in the anabolic effect of interferon gamma (IFNγ) on bone have not been carefully examined. Using microarray expression analysis, we found that IFNγ upregulates a set of genes associated with a tryptophan degradation pathway, known as the kynurenine pathway, in osteogenic differentiating human mesenchymal stem cells (hMSC). We, therefore, hypothesized that activation of the kynurenine pathway plays a role in osteoblastogenesis even in the absence of IFNγ. Initially, we observed a strong increase in tryptophan degradation during osteoblastogenesis with and without IFNγ in the media. We next blocked indoleamine 2,3-dioxygenase-1 (IDO1), the most important enzyme in the kynurenine pathway, using a siRNA and pharmacological approach and observed a strong inhibition of osteoblastogenesis with a concomitant decrease in osteogenic factors. We next examined the bone phenotype of Ido1 knockout (Ido1(-/-)) mice. Compared to their wild-type littermates, Ido1(-/-) mice exhibited osteopenia associated with low osteoblast and high osteoclast numbers. Finally, we tested whether the end products of the kynurenine pathway have an osteogenic effect on hMSC. We identified that picolinic acid had a strong and dose-dependent osteogenic effect in vitro. In summary, we demonstrate that the activation of the kynurenine pathway plays an important role during the commitment of hMSC into the osteoblast lineage in vitro, and that this process can be accelerated by exogenous addition of IFNγ. In addition, we found that mice lacking IDO1 activity are osteopenic. These data therefore support a new role for the kynurenine pathway and picolinic acid as essential regulators of osteoblastogenesis and as potential new targets of bone-forming cells in vivo.

Antibody-induced neutrophil depletion prior to the onset of pneumococcal meningitis influences long-term neurological complications in mice.

During pneumococcal meningitis, clearance of bacteria by recruited neutrophils is crucial for host protection. However, these innate immune mechanisms are often insufficient and treatment with antibiotics is necessary to prevent death. Despite this antibiotic treatment, approximately half of all survivors suffer lifelong neurological problems. There is growing evidence indicating the harmful effects of neutrophils on CNS integrity. Therefore, the present study investigated the roles of neutrophils in the acute inflammatory response and the resulting long-term neuropsychological effects in murine pneumococcal meningitis. Long-term behavioural and cognitive functions in mice were measured using an automated IntelliCage system. Neutrophil depletion with antibody 1A8 as adjunctive therapy was shown to remarkably impair survival in meningitic C57BL/6J mice despite antibiotic (ceftriaxone) treatment. This was accompanied by increased bacterial load in the cerebrospinal fluid (CSF) and an increase in IL-1ß, but decrease in TNF, within the CSF at 20h after bacterial inoculation. In the longer term, the surviving neutrophil-depleted post-meningitic (PM) mice displayed reduced diurnal hypolocomotion compared to PM mice treated with an isotype antibody. However, they showed nocturnal hyperactivity, and greater learning impairment in a patrolling task that is believed to depend upon an intact hippocampus. The data thus demonstrate two important mechanisms: 1. Neutrophil extravasation into the CNS during pneumococcal meningitis influences the pro-inflammatory response and is central to control of the bacterial load, an increase in which may lead to death. 2. Neutrophil-mediated changes in the acute inflammatory response modulate the neuropsychological sequelae in mice that survive pneumococcal meningitis.

IRGM3 contributes to immunopathology and is required for differentiation of antigen-specific effector CD8+ T cells in experimental cerebral malaria.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

Treatment of murine cerebral malaria by artemisone in combination with conventional antimalarial drugs: antiplasmodial effects and immune responses.

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.

The Fe-heme structure of met-indoleamine 2,3-dioxygenase-2 determined by X-ray absorption fine structure.

Multiple-scattering (MS) analysis of EXAFS data on met-indoleamine 2,3-dioxygenase-2 (IDO2) and analysis of XANES have provided the first direct structural information about the axial donor ligands of the iron center for this recently discovered protein. At 10K, it exists in a low-spin bis(His) form with Fe-Np(av)=1.97Å, the Fe-NIm bond lengths of 2.11Å and 2.05Å, which is in equilibrium with a high-spin form at room temperature. The bond distances in the low-spin form are consistent with other low-spin hemeproteins, as is the XANES spectrum, which is closer to that of the low-spin met-Lb than that of the high-spin met-Mb. The potential physiological role of this spin equilibrium is discussed.

The pro-inflammatory cytokine interferon-gamma is an important driver of neuropathology and behavioural sequelae in experimental pneumococcal meningitis.

Interferon-gamma is known to play a complex modulatory role in immune defence during microbial infections. Its actions in pneumococcal meningitis, however, remain ill-defined. Here, a pathological role for IFN-γ was demonstrated using a murine model of pneumococcal meningitis, in that C57BL/6J mice deficient in this pro-inflammatory cytokine (IFN-γ(-/-)) showed less severe acute and long-term neuropathology following intracerebral challenge with Streptococcus pneumoniae. The absence of IFN-γ significantly lengthened the survival of mice that otherwise would have developed fatal clinical signs within two days of CNS infection. Compared to their wild-type counterparts, IFN-γ(-/-) mice showed a diminished inflammatory response (attenuated levels of pro-inflammatory cytokines in the cerebrospinal fluid) and milder brain pathologies (less BBB permeability to protein and brain haemorrhage) during the acute phase of disease. Following a full regime of antibiotic treatment, we found substantial brain injuries in the wild-type mice 10days after infection. IFN-γ(-/-) mice, however, showed decreased neuronal damage in both hippocampus and cortex. In the longer term (≈10weeks p.i.), the wild-type mice that had survived meningitis due to antibiotic treatment had neurobehavioural abnormalities including diurnal hypoactivity, nocturnal hyperactivity and impaired performance in a discrimination reversal task. IFN-γ(-/-) mice, concomitantly tested in the automated IntelliCage platform, had reduced behavioural and cognitive disorders compared to wild-type mice. Both IFN-γ(-/-) and wild-type survivors of pneumococcal meningitis showed impaired working memory in the IntelliCage-based complex patrolling task. These observations indicate an association between IFN-γ-driven acute brain pathology and the long-term neurological sequelae resulting from pneumococcal meningitis.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis.

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Indoleamine 2,3-dioxygenase 2 (IDO2) and the kynurenine pathway: characteristics and potential roles in health and disease.

The kynurenine pathway is the major route for the oxidative degradation of the amino acid tryptophan. Activity of the pathway is involved in several disease conditions, both in the periphery and the central nervous system, including cancer, inflammatory disorders, neurological conditions, psychiatric disorders and neurodegenerative diseases. Three enzymes are now known to catalyze the first and rate-limiting step in the catabolism of tryptophan along this pathway: tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO, subsequently named IDO1), both of which have been extensively studied, and a third enzyme, indoleamine 2,3-dioxygenase 2 (IDO2), a relative newcomer to the kynurenine pathway field. The adjuvant chemotherapeutic agent, 1-methyl-D-tryptophan, was intially suggested to target IDO2, implying involvement of IDO2 in tumorigenesis. Subsequently this compound has been suggested to have alternative actions and the physiological and pathophysiological roles of IDO2 are unclear. Targeted genetic interventions and selective inhibitors provide approaches for investigating the biology of IDO2. This review focuses on the current knowledge of IDO2 biology and discusses tools that will assist in further characterizing the enzymes of the kynurenine pathway.

Applicability of Redirecting Artemisinins for New Targets.

Employing new therapeutic indications for drugs that are already approved for human use has obvious advantages, including reduced costs and timelines, because some routine steps of drug development and regulation are not required. This work concentrates on the redirection of artemisinins (ARTS) that already are approved for clinical use, or investigated, for malaria treatment. Several mechanisms of action are suggested for ARTS, among which only a few have been successfully examined in vivo, mainly the induction of oxidant stress and anti-inflammatory effects. Despite these seemingly contradictory effects, ARTS are proposed for repurposing in treatment of inflammatory disorders and diverse types of diseases caused by viral, bacterial, fungal, and parasitic infections. When pathogens are treated the expected outcome is diminution of the causative agents and/or their inflammatory damage. In general, repurposing ARTS is successful in only a very few cases, specifically when a valid mechanism can be targeted using an additional therapeutic agent and appropriate drug delivery. Investigation of repurposing should include optimization of drug combinations followed by examination in relevant cell lines, organoids, and animal models, before moving to clinical trials.

Synthesis of artemiside and its effects in combination with conventional drugs against severe murine malaria.

This research describes the use of novel antimalarial combinations of the new artemisinin derivative artemiside, a 10-alkylamino artemisinin. It is a stable, highly crystalline compound that is economically prepared from dihydroartemisinin in a one-step process. Artemiside activity was more pronounced than that of any antimalarial drug in use, both in Plasmodium falciparum culture and in vivo in a murine malaria model depicting cerebral malaria (CM). In vitro high-throughput testing of artemiside combinations revealed a large number of conventional antimalarial drugs with which it was additive. Following monotherapy in mice, individual drugs reduced parasitemias to nondetectable levels. However, after a period of latency, parasites again were seen and eventually all mice became terminally ill. Treatment with individual drugs did not prevent CM in mice with recrudescent malaria, except for piperaquine at high concentrations. Even when CM was prevented, the mice developed later of severe anemia. In contrast, most of the mice treated with drug combinations survived. A combination of artemiside and mefloquine or piperaquine may confer an optimal result because of the longer half life of both conventional drugs. The use of artemiside combinations revealed a significant safety margin of the effective artemiside doses. Likewise, a combination of 1.3 mg/kg of body weight artemiside and 10 mg/kg piperaquine administered for 3 days from the seventh day postinfection was completely curative. It appears possible to increase drug concentrations in the combination therapy without reaching toxic levels. Using the drug combinations as little as 1 day before the expected death of control animals, we could prevent further parasite development and death due to CM or anemic malaria. Earlier treatment may prevent cognitive dysfunctions which might occur after recovery from CM.

Identification of selective inhibitors of indoleamine 2,3-dioxygenase 2.

The kynurenine pathway is responsible for the breakdown of the majority of the essential amino acid, tryptophan (Trp). The first and rate-limiting step of the kynurenine pathway can be independently catalysed by tryptophan 2,3-dioxygenase (Tdo2), indoleamine 2,3-dioxygenase 1 (Ido1) or indoleamine 2,3-dioxygenase 2 (Ido2). Tdo2 or Ido1 enzymatic activity has been implicated in a number of actions of the kynurenine pathway, including immune evasion by tumors. IDO2 is expressed in several human pancreatic cancer cell lines, suggesting it also may play a role in tumorigenesis. Although Ido2 was originally suggested to be a target of the chemotherapeutic agent dextro-1-methyl-tryptophan, subsequent studies suggest this compound does not inhibit Ido2 activity. The development of selective Ido2 inhibitors could provide valuable tools for investigating its activity in tumor development and normal physiology. In this study, a library of Food and Drug Administration-approved drugs was screened for inhibition of mouse Ido2 enzymatic activity. A number of candidates were identified and IC(50) values of each compound for Ido1 and Ido2 were estimated. The Ido2 inhibitors were also tested for inhibition of Tdo2 activity. Our results showed that compounds from a class of drugs used to inhibit proton pumps were the most potent and selective Ido2 inhibitors identified in the library screen. These included tenatoprazole, which exhibited an IC(50) value of 1.8µM for Ido2 with no inhibition of Ido1 or Tdo2 activity detected at a concentration of 100µM tenatoprazole. These highly-selective Ido2 inhibitors will be useful for defining the distinct biological roles of the three Trp-catabolizing enzymes.

Differential microRNA expression in experimental cerebral and noncerebral malaria.

MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ(-/-)) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ(-/-) mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

Gp91(phox) contributes to the development of experimental inflammatory bowel disease.

Inflammatory bowel disease (IBD) is related to dysfunction of intestinal immunity. Neutrophils have an important role in innate immunity via the oxidative burst, using the p47phox- and gp91(phox)-containing NAD(P)H oxidase known as Nox2. In dextran sulphate sodium (DSS)-induced colitis, no significant difference in inflammation between p47(phox-/-) and wild-type (WT) mice was reported, but there was improved endothelium-dependent arteriolar dilation in gp91(phox-/-) mice, compared with that in WT mice. Gp91(phox) and p47 (phox) are not only essential components of phagocyte Nox2, but also have roles in other enzymes. Thus the differences in response of their respective gene knockout mice to DSS challenge are not completely unexpected, but need further investigation. The clinicopathological changes and immunological responses to DSS challenge have not been fully described in gp91(phox-/-) mice. Thus we treated WT and gp91(phox-/-) mice with 2.5% DSS for 7 days. The gp91(phox-/-) mice developed less severe colitis than WT mice following DSS treatment, reflected by a smaller body weight loss, less rectal bleeding and fewer histopathological changes. Less colonic myeloperoxidase was observed in gp91(phox-/-), compared with WT mice, following DSS challenge, correlating with interleukin (IL)-6 production. IL-10 was upregulated in both gp91(phox-/-) and WT mice, but was significantly higher in the latter, following 7 days DSS challenge. These results suggest that gp91(phox-/-) mice are less susceptible to acute DSS-induced colitis, possibly because of a reduced oxidative burst in the intestine and, consequently, less tissue damage.

Total synthesis, stereochemical assignment, and antimalarial activity of gallinamide A.

The total synthesis and stereochemical assignment of gallinamide A, an antimalarial depsipeptide of cyanobacterial origin, is described. Synthesis of the four possible N-terminal diastereoisomers of gallinamide A (including the natural product symplostatin 4) was achieved using a divergent strategy from a common imide fragment. The natural product and corresponding diastereoisomers were synthesized in 30-33% overall yield in a longest linear sequence of 8 steps. Comparative NMR spectroscopic studies of the four synthetic diastereoisomers with the isolated natural product demonstrated that gallinamide A possesses a dimethylated L-isoleucyl residue at the N-terminus. As such, we have shown that gallinamide A is structurally and stereochemically identical to symplostatin 4. Gallinamide A and its N-terminal diastereoisomers were also shown to possess significant antimalarial activity with IC(50) values in the nanomolar range against the 3D7 strain of Plasmodium falciparum.