Therapeutic targets for malaria_ adjunctive therapies

CONTENTSsummary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .275 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .275 Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .276 references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .281SUMMARYAntimalarial drugs unequivocally reduce mortality in patients with fal-ciparum malaria. However, they have proven ineffective in some patients with severe disease, in whom a tipping point has been breached and a rampant systemic inflammatory response has begun that overwhelms host control mechanisms. Unabated, this immune response triggers a chain of pathological events, compounded by the parasite’s presence and immunomodulatory capacity, which often results in death. Host-directed adjunctive therapies, that is therapies administered in combination with an antiparasitic agent that are designed to prevent these pathological processes, are considered the only way to reduce morbidity and mortality, but none has yet demon-strated absolute efficacy in clinical trials. In this review, we present the key targets of adjunctive therapies for the management of severe cere-bral malaria.Key words:severe malaria – cerebral malaria – Plasmodium falci-parum– Drug targets – Immunomodulation – Immunopathogenesis INTRODUCTIONMalaria is an infectious disease caused by obligate intracellular pro-tozoa of the genus Plasmodium. It is transmitted from person to per-son through the bite of a female Anopheles mosquito. of the five species of plasmodia that are known to infect humans, Plasmodium falciparum is the most highly pathogenic. It causes only about half of all malaria infections, but 91% of its deaths. In 2010 alone, it was responsible for almost 600,000 patients, the vast majority of whom were in sub-saharan africa, where falciparum malaria is directly responsible for 1 in 5 childhood deaths and indirectly contributes to morbidity and mortality from anemia, respiratory infections, diarrhea and malnutrition (1, 2).In individuals without naturally acquired immunity, P. falciparum infection is almost always symptomatic, and clinical symptoms can develop even at very low parasitemias. Without prompt treatment with specific drugs, the disease rapidly progresses to a severe illness that is frequently fatal (mortality is conservatively estimated at 30%) (3). Even with appropriate treatment, mortality rates in naive individ-uals are between 0.6% and 3.8%, and for severe malaria they may exceed 20%, even when managed in intensive care units (4).In areas of intense transmission, children gradually acquire immuni-ty that protects them against high parasitemia and the risk of severe disease. Immunity is generally acquired by the onset of puberty, after which severe disease rarely occurs. sterilizing immunity, however, is never fully achieved and adults remain asymptomatic carriers of par-asites (3).symptoms of falciparum malaria usually develop 11 days after an infectious bite (range: 6-14 days), although parasites can be detect-ed in the blood after 10 days (range: 5-10 days). Patients generally present with fever (92% of cases), chills (79%), headache (70%) and diaphoresis (64%). clinical deterioration to severe malaria usually occurs 3-7 days after the onset of fever (4). children commonly develop severe anemia and hypoglycemia, while adults tend to pres-ent with jaundice and progress to renal failure and respiratory dis-tress due to pulmonary edema (5). about 7% of P. falciparum malar-ia cases develop cerebral malaria (6). cerebral malaria is caused only by P. falciparum and is responsible for up to 40% of the mortal-ity attributable to severe malaria (7). Initial symptoms of brain involvement include acute headache, irritability, agitation and psy-chosis. seizures occur in 80% of children, but only 15-20% of adults. The onset of coma is rapid in children, but only gradual in adults, and can last several hours to several days (8). generally, the deeper the coma, the worse the prognosis (4). If the patient survives, neuro-logical sequelae (e.g., movement disorders, paralysis, difficulty in walking, speech impairment, blindness, deafness, epilepsy, mental disorders and behavioral abnormalities) are present in 6-29% of children and 3-10% of adults (8). Mortality from cerebral malaria ranges from 10-50%, even with treatment (4).since 2011, the World health organization (Who) has advocated intravenous artesunate for the treatment of severe falciparum malaria in both children and adults (9), based on the results of two multicenter randomized trials (10, 11).Drugs of the Future 2014, 39(4): 275-286copyright © 2014 Prous science, s.a.U. or its licensors. all rights reserved.ccc: 0377-8282/20014DoI: 10.1358/dof.2014.39.4.2141023TargETs To WaTchThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEsA.E. Brown and L.A. SorberaThomson reuters, Barcelona, spainCorrespondence:E-mail: lisa.sorbera@.although these trials showed that artesunate offered a significant survival advantage over its predecessor quinine (relative reduction in mortality of 22.5% for african children and 34.7% for southeast asian adults), death rates remained high (8.5% in children and 14.7% in adults), and neither trial reported any impact in preventing the development of neurological sequelae. It is clear then that even using the best available antiparasitic agents under optimum trial conditions, targeting the parasite alone is not sufficient to prevent the high mortality associated with severe disease.severe and cerebral malaria are now thought of as multisystem dis-orders that develop as a result of the host’s response to P. falciparum infection —death occurs as a consequence of infection rather than because of it. adjunctive therapies administered in combination with antimalarial drugs, which are designed to specifically block these pathological events, may be the only means of reducing morbidity and mortality, but none have demonstrated unequivocal efficacy in clinical trials (reviewed in references 8and 12).The pathogenesis of malaria depends on various host and parasite factors, and on interactions between the parasite and its host’s immune system throughout its life cycle (Figure 1). The ultimate out-come of the disease, and whether severe and cerebral malaria devel-ops, is a fine balance between the beneficial and harmful effects of these immune responses.Infection starts with a bite of an infected mosquito, when the motile sporozoite is introduced into the skin together with mosquito saliva. The sporozoite enters the bloodstream and is rapidly carried to the liver, where it invades a hepatocyte. Parasite development in the liver goes largely unnoticed and causes little or no pathology. During this phase of its life cycle, the parasite actively exports proteins across the vacuolar membrane into the cytosol and nucleus of the hepatocyte, which modulate host gene expression and create a favorable environment for its development. Within the hepatocyte, merozoites develop and are released into the bloodstream within a host cell-derived cloak, the merosome, which largely hides them from immune recognition (13). P. falciparum is unique among human malaria parasites because it uses multiple, often functionally redun-dant, host cell receptors to invade red blood cells (rBcs) of any age (14). Plasmodium parasites export hundreds of remodeling and viru-lence proteins into the erythrocyte in order to establish and maintain infection (15, 16). over the course of 48 hours, each parasite actively consumes intracellular proteins to produce 8-32 daughter mero-zoites (average of 10) that develop within a membrane-derived com-partment, the parasitophorous vacuole, which is established during the invasion process. These merozoites must destroy their host cell in order to gain access to fresh rBcs and perpetuate the infection. Lysis of the rBc releases hundreds of parasite products, some of which (i.e., the glycosylphosphatidylinositol [gPI] anchor or hemo-zoin [hZ; malaria toxin], a detoxified crystalline form of heme pro-duced by the parasite following the catabolism of hemoglobin) orchestrate the release of proinflammatory and pyrogenic cytokines, (i.e., TNF-α, interleukin-6 [IL-6] and IL-1) (17-21). These cytokines ini-tially serve to control parasitemia and are responsible for the symp-toms of mild malaria: headache, chills, fever and lethargy (22). subsequent activation of cD4+T cells and natural killer (NK) cells triggers further cytokine production (i.e., interferon-γ[IFN-γ]), which activates macrophages and induces the infiltration of immune cells (i.e., neutrophils, monocytes and cD8+T cells) that precipitate para-site clearance and permit the development of immunological mem-ory for subsequent infection (23, 24). however, during a severe malarial infection, the enormous parasite load can overwhelm the capacity of the mononuclear phagocyte system to eliminate it and circulating immune cells become hyperactivated and release large amounts of proinflammatory cytokines (i.e., TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-12 and IL-18). Macrophages and dendritic cells also become laden with indigestible material (i.e., hemozoin), which impinges upon their ability to act as antigen-presenting cells (aPcs) (25). Thus, the excessive production of cytokines without appropriate reg-ulatory controls (i.e., well-developed antibody responses, downreg-ulation of cytotoxic immune mechanisms and regulatory cytokine production) induces multiple pathological changes in host tissues that Plasmodium exploits to perpetuate the development of severe pathology (26-31).Proinflammatory cytokines (i.e., TNF-α, IFN-γand IL-6) induce the upregulation of cell adhesion molecules (i.e., intercellular adhesion molecule 1 [IcaM-1], cD36, vascular cell adhesion protein 1 [VcaM-1] and E- and P-selectin [32-36]) on endothelial cells that mediate the binding and sequestration of rBcs via PfEMP1(37-41). PfEMP1 is inserted into the erythrocyte membrane by the parasite during this stage of its life cycle as a survival mechanism to prevent the removal of parasitized erythrocytes (PEs) by the spleen (42, 43). It can also induce the formation of clusters of uninfected rBcs (termed rosettes) within the bloodstream. The sequestration of rBcs in the microvasculature of organs is one of the key pathogenic drivers of severe, cerebral and pregnancy-associated malaria (44-53). Parasite sequestration and local inflammation lead to endothelial activation and dysfunction, and cause disruption to the blood−brain barrier (54), that may be sufficient to perturb vital organ function and allow metabolites to impair consciousness or induce seizures (8).here, we review the therapeutic targets of adjunctive therapies for the treatment of severe and cerebral malaria (including targets under active development, as well as candidate and investigational targets). Figure 2 depicts these targets in the context of disease pathology. Table I provides a selection of patents for selected tar-gets.TARGETSToll-like receptor 9 (TLR9)TLr9 is an intracellular pattern recognition receptor that plays a key role in innate immune responses to Plasmodium infection. It specifi-cally recognizes non-methylated cytosine-guanosine (cpg) motifs that are common to viral and bacterial genomes but largely absent from those of vertebrates, in whom cpg dinucleotides are highly methylated (55). Despite being extremely aT rich, the P. falciparum genome comprises multiple cpg B-class motifs in subtelomeric regions (56). It also contains a number of non-cpg motifs (5’-aTTTTTac-3’) that have been shown to activate TLr9 signaling (56, 57). Both hemozoin (19, 56, 58) and parasite nucleosomes have been implicated as sources of these signals (59, 60).TLr9 is expressed only on B cells and plasmacytoid dendritic cells (pDcs) (61). In resting cells, it resides within the endoplasmic reticu-ThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEs A.E. Brown and L.A. SorberaA.E. Brown and L.A. Sorbera ThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEsFigure 1.The life cycle of Plasmodium falciparum and its interplay with the host immune system. Plasmodium parasites are transmitted to a vertebrate host following the bite of an infected female Anopheles mosquito. The majority of sporozoites remain in the dermis trapped by antibodies; however, a small propor-tion are able to enter blood capillaries or lymph vessels, from which they are carried to lymph nodes and prime T and B cells, or to the liver. sporozoites typi-cally take 15-30 minutes to migrate from the skin to the liver, and after initially passing through Kupffer cells, they enter hepatocytes. here, the parasite devel-ops and multiplies within a host cell-derived vacuole the merosome that protects them from immune recognition. During this period the parasite also releases proteins that interfere with host cell ribosome function and inhibit hepatocyte death and prevent killing by cD4+and cD8+T cells, thereby promoting mero-some survival. after ~8 days, the parasite induces its host hepatocyte death and merozoites are released into the bloodstream. Each merozoite invades an ery-throcyte within 30 seconds of being released and undergoes a period of growth and multiplication to produce a schizont. after ~48 hours, the red blood cell (rBc) ruptures and releases 8-32 merozoites, which are freed to invade new erythrocytes and repeat the erythrocytic cycle. rBc lysis also releases parasite protein, DNa and metabolites (i.e., glycosylphosphatidylinositol [gPI], hemozoin, nucleosome) that are recognized by Toll-like receptors (TLrs) (i.e., TLr9 and TLr2) on dendritic cells, and stimulates the production of proinflammatory and pyrogenic cytokines. These cytokines direct Th1 cD4+T cell differentiation and promote the development of antibody responses to merozoite surface proteins or parasite proteins (i.e., PfEMP1) that are expressed on the surface of erythro-cytes during their intracellular development. IFN-γexpression by cD4+cells also activates macrophages that phagocytose and kill opsonized merozoites or parasitized erythrocytes. Local cytokine production also induces the expression of adhesion molecules (e.g., IcaM-1) on endothelial cells to which PfEMP1 expressed on parasitized erythrocytes (PEs) binds in order to prevent their clearance by the spleen. after a number of intraerythrocytic cycles, a proportion of merozoites terminally differentiate into gametocytes and are acquired by the mosquito upon blood-feeding. Within the mosquito they undergo their sexual developmental, which produces fresh sporozoites ready to initiate a new cycle of infection after approximately 9-12 days.ThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEs A.E. Brown and L.A. Sorbera Figure 2.Malaria: adjunctive Therapy Targetscape. a diagram showing an overall cellular and molecular landscape or comprehensive network of connections among the current therapeutic targets for malarial adjunctive therapy and their biological actions. gray or lighter symbols are targets that are not validated (i.e., targets not associated with a product that is currently under active development for malarial adjunctive therapy). abbreviations: BBB, blood-brain barri-er; gPI, glycosylphosphatidylinositol; 15-hETE, 15-hydroxyeicosatetraenoic acid; 13-hoDE, 13-(S)-hydroxyoctadecadienoic acid; hMgB1, high mobility group box protein 1; IcaM-1, intercellular adhesion molecule 1; IL, interleukin; NF-κB, nuclear factor NF-kappa-B; PParγ,peroxisome proliferator-activated receptor gamma; rhoa, ras homolog gene family, member a; rocK, rho-associated protein kinase (nonspecified subtype); ros, reactive oxygen species; sIL-6r, sol-uble interleukin 6 receptor; TLr, Toll-like receptor.lum, but upon stimulation translocates to the site of cellular cpg-DNa uptake, the endosome, where signal transduction is initiated via myeloid differentiation factor 88 (MyD88), an adaptor molecule shared by all TLrs (62). TLr9 stimulation induces the expression of type I IFNs (i.e., IFN-αand IFN-β) (63, 64) and cytokines and chemokines (i.e., IL-1β, IL-6 and TNF-α) with a predominately T helper 1 (Th1) profile (65). a number of single nucleotide polymor-phisms (sNPs) in the promoter region of TLr9 have been identified that modulate disease outcome and cytokine expression profiles during severe malarial infections (66-70). It also upregulates the expression of co-stimulatory molecules (i.e., cD40, B7-1 [cD80], B7-2 [cD86]) and Mhc class II on pDcs and B cells that promote the expansion of cD8+cytotoxic T lymphocytes (cTLs) and Th1 cD4+T cells (71), and induces the differentiation of pDcs into aPcs capable of inducing effector/memory cD8+T-cell responses (72); TLr9 is central to the development of protective immunity to malaria in ani-mal models (73) and naive humans (74).activation of TLr9 on pDcs induces the production of cD4+cD25+T regulatory (Treg) cells that are capable of suppressing naive T cell differentiation (75). It is a mechanism Plasmodium exploits for immune evasion (76-78), where rapid parasite growth correlates with the upregulation of Tregs during infection in humans (79).TLr9 has been implicated in both the initiation (73,80,81) and the perpetuation of the immune response to Plasmodium(82,83); the cytokine milieu (i.e., IFN-γand IL-12) released during Plasmodium infection is proposed to prime TLr immune responses that can lead to deleterious hyperinflammation upon their subsequent reactivation. consistent with this, TLR9−/−mice are partially protect-ed from lethal lipopolysaccharide-induced shock during infection with the rodent malaria parasite Plasmodium chabaudi (80). The small-molecule antagonist E-6446 confers even greater protection (86% survival vs. 38% for TLR9−/−mice) and can significantly improve survival of mice following infection with the murine malaria parasite Plasmodium berghei. It can also prevent the development of cerebral malaria, even when administered to mice with established disease (i.e., 3-6 days post-infection) but not in those already exhibiting cerebral pathology (i.e., > 6 days post-infection) (84).A.E. Brown and L.A. Sorbera ThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEsanother potential TLr9 antagonist is chloroquine. chloroquine is an inhibitor of endosomal acidification and blocks the TLr9-cpg-DNa interaction and attenuates TLr9-mediated signal transduction (85),albeit with an eightfold lower Ic50than E-6446 (mean Ic50= 0.01 vs.0.08 mM) (84). It has successfully been used to treat rheumatoid arthritis (ra) and systemic lupus erythromatosus (sLE) and canTable II. selected patents for targets being pursued or explored as adjunctive therapies for malaria (from Thomson reuters Integrity sM).ThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEs A.E. Brown and L.A. Sorberaattenuate proinflammatory cytokine release in mice with sepsis (86). It may be worth reconsidering chloroquine for the treatment of severe malaria, not for its anti-parasitic activity, which is compro-mised anyway because of resistance, but for its adjuvant effect. It has, like E-664, however, proven ineffective in preventing the devel-opment of cerebral malaria in late-stage disease (87).Interleukin-6 (IL-6)IL-6 is a multifunctional cytokine and one of the earliest secreted by peripheral blood mononuclear cells (PBMcs) (88, 89) following TLr-mediated recognition of Plasmodium-specific patterns like gPI and parasite DNa (84, 89). IL-6 plays a key role in controlling parasite density (90) by promoting monocyte recruitment (91,92). hyperparasitemia is associated with significantly lower levels of IL-6 (27), and polymorphisms in the promoter of IL-6 have been associat-ed with the relative resistance and significantly lower parasite rates of the Fulani people of Mali and Burkina Faso (93, 94), compared to other sympatric ethnic groups (95). IL-6 also induces IL-21 produc-tion, which promotes the differentiation of B helper cD4+T cells (96, 97), which are necessary for the proper development of anti-Plasmodium antibody responses (98-100).IL-6 exerts its biological activity through two molecules: IL-6r (IL-6 receptor) and membrane glycoprotein 130 (gp130). mIL-6r (mem-brane-bound IL-6r) is expressed on the surface of hepatocytes and leukocytes (i.e., neutrophils, macrophages and some T cells) and mediates IL-6 classic signaling (101). a soluble form of IL-6r (sIL-6r) is also found in serum and mediates IL-6 trans-signaling. sIL-6r is produced by proteolytic cleavage (“shedding”) of the ectodomain of mIL-6r by aDaM 10 and aDaM 17 (102-104) and can bind free IL-6r and activate any gp130 (gp130 is expressed on the surface of most cells). When IL-6 binds to IL-6r, it stimulates the homodimerization of gp130 and induces the JaK/sTaT3 and ras/ErK/c/EBP path-ways (105). a soluble form of gp130, sgp130, is also found in serum and is believed to antagonize the bioactivity of IL-6 by blocking the soluble IL-6–IL-6r complex from attaching to membrane-bound gp130 (106).Levels of IL-6 and sIL-6r are increased in patients with severe and cerebral malaria (107) and IL-6 levels correlate with disease severity and mortality (27, 108). Those of sgp130, however, remain unchanged (107), suggesting that aberrant IL-6 trans-signaling may be responsible for the pathogenesis of malaria, as is observed in a number of inflammatory diseases, including inflammatory bowel disease (109), ra (110) and sepsis (111). consistent with this, inhibi-tion of IL-6 using sgp130Fc, a recombinant fusion protein comprising the extracellular domain of gp130 (domains 1-6) fused to human Igg Fc (112), significantly increased survival of mice following lethal P. chabaudi infection (113). IL-6 trans-signaling is presumably initiat-ed following the proteolytic processing of IL-6r from neutrophils recruited to endothelial cells by chemokines (i.e., cXcL1) (83) released following pleural effusion (PE) sequestration. IL-6 trans-signaling would subsequently stimulate endothelial cells to express monocytes (i.e., ccL2/McP-1, ccL8/McP-2, cXcL5/ENa-78 and cXcL6/gcP-2), T cell-attracting chemokines (i.e., ccL4/MIP-1-beta, ccL5/raNTEs, ccL17/Tarc and cXcL10/IP-10) and cell adhesion molecules (i.e., IcaM-1) (114, 115) that would perpetuate the inflam-matory response and amplify PE sequestration, ultimately leading to endothelial dysfunction (116, 117).Peroxisome proliferator-activated receptor gamma (PPAR-γ) PPar-γis a ligand-activated transcription factor of the nuclear hor-mone receptor superfamily that modulates metabolic and immune functions. During malaria infection it is probably activated as a homeostatic response in an attempt to attenuate (hyper)inflamma-tion and oxidative stress and prevent its pathological consequences. In a genetic screen in mice to identify genes involved in susceptibili-ty and resistance to P. berghei infection, Pparg was one of only two genes identified in a locus on chromosome 6 that modifies survival and prevents the development of cerebral malaria (118).PPar-γis activated by the hydroxy-polyunsaturated fatty acids 15-hydroxyecosatetraenoic acid (15-hETE) and 13-hydroxyoctadeca-dienoic acid (13-hoDE), which are produced following the interac-tion of Plasmodium hemozoin with membrane phospholipids (119-121). Upon ligand binding, PPar-γforms a heterodimeric complex with the retinoid X receptor that binds to cis-acting peroxisome pro-liferator response elements on DNa to regulate the transcription of target genes (122).PPar-γnormally acts as a transcriptional activator of genes involved in lipid and carbohydrate metabolism (123). however, it can also sequester transcription factors, such as nuclear factor NF-κB, aP-1, c/EBP beta, sTaT1 and NFaT (122), and thereby inhibits the expres-sion of proinflammatory cytokines (124, 125) and prevents the pro-duction of ros by monocytes and macrophages (126). It can also block the expression of IcaM-1 and VcaM-1 (127), and attenuate the expression of inducible proinflammatory proteins in endothelial cells, including cyclooxygenase-2 (coX-2), cytosolic phospholipase a2 (cPLa2) and nitric oxide synthase, inducible (iNos) (126, 128, 129). PPar-γplays a critical role in protecting blood vessels, and interference with PPar-γsignaling produces cerebral arteriolar endothelial dysfunction via a mechanism involving oxidative stress (130). Furthermore, PPar-γagonists have been shown to prevent inflammation and neuronal death after focal cerebral ischemia in rodents (131-135). consistent with this, mice treated with rosiglita-zone were protected from developing cerebral malaria, even when it was administered just prior to the development of cerebral patholo-gy (i.e., 5 days post-infection) (136). rosligitazone also offers bene-fits in addition to its immunomodulatory properties. a randomized, double-blind, placebo-controlled phase I/IIa trial testing the safety, tolerability and efficacy of rosiglitazone adjunctive therapy in Thai patients with uncomplicated falciparum malaria, showed that rosiglitazone in combination with atovaquone/proguanil significant-ly reduced parasite clearance times compared to atovaquone/ proguanil alone (137), probably because of increased phagocytosis as a direct result of PPar-γ-mediated upregulation of the scavenger receptor cD36 on macrophages (136, 138).Nuclear factor NF-kappa-B (NF-κB)NF-κB is a dimeric protein composed of members of the rel family of transcription factors that regulate the expression of many inflam-matory genes. a gene expression analysis of human brain endothe-lium after interaction with P. falciparum revealed that the NF-κB pathway was central to the host response (83).humans express 5 rel/NF-κB proteins (rela/p65, c-rel, rel-B, p50 [NF-κB1] and p52 [NF-κB2]), the specific combination of whichdetermine the specificity of transcriptional activation (139-141). For example, E-selectin (142-144) and VcaM-1 (145, 146) DNa κB sites preferentially bind p50/p65 heterodimers, while the IcaM-1 ele-ment preferentially binds p50/crel heterodimers or c-rel homod-imers (147). In unstimulated cells, the NF-κB complex is sequestered in the cytoplasm by an inhibitory protein, I-κB that masks its C-ter-minal transactivation domain (148, 149). Upon stimulation, NF-κB is activated in a ubiquitin-dependent process which requires the phosphorylation of IκB by IkappaB kinase (IKK) (150, 151). Following degradation of IκB by the 26s proteosome, NF-κB is freed to enter the nucleus and activate the expression of inflammatory mediators (152, 153).as a class of drugs, antioxidants have demonstrated efficacy in pre-venting experimental cerebral malaria (154). (–)-Epigallocatechin gallate (Egcg) is a green tea-derived polyphenol that can regulate NF-κB activation by inhibiting IKK activity (155, 156). The (+)-Egcg enantiomer acts as an inhibitor of cytoadherence. It has a similar structure to the DE loop (Leu42-arg49) of IcaM-1, which is likely to be a key domain in its interaction with PfEMP1 (157, 158), and can block the binding of PEs of field-derived Plasmodium isolates to IcaM-1-Fc in vitro by up to 80% at micromolar concentrations (158)(Ic50= 5-10 mM) (157). Egcg also possesses intrinsic anti-Plasmodium activity and can attenuate sporozoite gliding motility(Ic50= 137 mM) and induce sporozoite death (Ic50= 1,095 mM at 6hours; 118 mM at 12 hours) (159).curcumin, a polyphenol derived from turmeric, binds p50 and inhibits IκBαdegradation following stimulation by IL-1 and TNF-α(160). It can prevent the development of cerebral malaria in mice fol-lowing lethal P. berghei challenge (161).Pyrrolidine dithiocarbamate (PDTc) is an inhibitor of p50, p52, c-rel and rel-B (162) that can block gPI-induced upregulation of IcaM-1, VcaM-1 and E-selectin on human umbilical vein endothelial cells (hUVEcs) and attenuate parasite cytoadherence (163).N-acetyl-L-cysteine (Nac) is a suppressor of IKKαand IKKβactiva-tion (164) that can protect the brain from free radical injury, apopto-sis and inflammation after focal cerebral ischemia in rodents (165, 166). clinical trials testing its potential as an adjunctive treatment for falciparum malaria, however, have failed to demonstrate any sur-vival benefit (167, 168).Rho-associated protein kinase (ROCK)rhoa gTPase is activated upon adherence of PEs to human lung endothelial cells in vitro (169). rhoa is the major activator of actin-myosin contraction in endothelial cells and thereby a key determi-nant of increased endothelial cell permeability (170-172). Endothelial cells line the intima of microvesicles and form a semi-permeable barrier that controls the exchange of macromolecules and fluids between the blood and interstitial space. Precise regula-tion of this barrier is necessary to maintain circulatory homeostasis and proper organ function (173). Postmortem histological studies of adults with cerebral malaria show widespread vascular endothelial cell activation, as evidenced by higher blood levels of angiopoietin-2 (aNg-2) (174) and circulating Weibel-Palade bodies (WPBs) (175, 176) and disruption of cell junction proteins, particularly in vessels containing PEs (177).Upon rhoa activation, its effector kinases rocK-I and rocK-II phosphorylate and inhibit myosin light chain phosphatase (MLcP), resulting in a net increase in phosphorylated myosin light chain (MLc) that induces actin–myosin contraction and intracellular gap formation (178). rhoa is activated by some inflammatory agonists released during Plasmodium infection. ros are released by activat-ed neutrophils bound to the vascular endothelium and induce endothelial contraction by activating rhoa directly (178-181). TNF-αis believed to increase barrier permeability directly by activating rhoa (178, 182) and indirectly by upregulating the expression of endothelial adhesion molecules (i.e., IcaM-1 and E-selectin), there-by promoting neutrophil adhesion and ros production at the endothelium (183-185). The rocK inhibitor fasudil (ha-1077) can restore endothelial monolayer integrity and prevent apoptosis in vitro following exposure to PEs or TNF-α(169, 186). It has also been shown to arrest the development of cerebral malaria in mice (161). DISCLOSURESThe authors state no conflicts of interest.Submitted: March 21, 2014. Accepted: April 3, 2014.REFERENCESglobal health observatory Data repository: Malaria 2010. World health 1.organization, geneva, switzerland. available online. accessed 26 January 26, 2013.World Malaria report 2008: World health organization, geneva, 2.switzerland 2008. IsBN: 9789241563697. available online. accessed 26 January 26, 2013.Doolan, D.L., Dobaño, c., Baird, J.K. Acquired immunity to malaria. clin 3.Microbiol rev 2009, 22(1): 13-36.Trampuz, a., Jereb, M., Muzlovic, I., Prabhu, r.M. Clinical review: Severe 4.malaria. crit care 2003, 7(4): 315-23.Perkins, D.J., Were, T., Davenport, g.c., Kempaiah, P., hittner, J.B., 5.ong’echa, J.M. Severe malarial anemia: Innate immunity and pathogene-sis. Int J Biol sci 2011, 7(9): 1427-42.Maitland, K., Newton, c.r. Acidosis of severe falciparum malaria: Heading 6.for a shock?Trends Parasitol 2005, 21(1): 11-6.Severe falciparum malaria. World Health Organization, Communicable 7.Diseases Cluster. Trans r soc Trop Med hyg 2000, 94(suppl. 1): s1-90.Mishra, s.K., Newton, c.r. Diagnosis and management of the neurologi-8.cal complications of falciparum malaria. Nat rev Neurol 2009, 5(4): 189-98.World health organization, guidelines for the Treatment of Malaria, 2nd 9.edition, IsBN: 9789241547925, World health organization geneva 2011.available online. accessed January 27, 2013.Dondorp, a.M., Fanello, c.I., hendriksen, I.c. et al. Artesunate versus qui-10.nine in the treatment of severe falciparum malaria in African children (AQUAMAT): An open-label, randomised ncet 2010, 376(9753): 1647-57.Dondorp, a., Nosten, F., stepniewska K. et al. South East Asian Quinine 11.Artesunate Malaria Trial (SEAQUAMAT) group. Artesunate versus quinine for treatment of severe falciparum malaria: A randomised ncet 2005, 366(9487): 717-25.John, c.c., Kutamba, E., Mugarura, K., opoka, r.o. Adjunctive therapy for 12.cerebral malaria and other severe forms of Plasmodium falciparum malar-ia. Expert rev anti Infect Ther 2010, 8(9): 997-1008.A.E. Brown and L.A. Sorbera ThEraPEUTIc TargETs For MaLarIa: aDJUNcTIVE ThEraPIEs。