Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation.

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 µM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Aryl hydrocarbon receptor antagonism mitigates cytokine-mediated inflammatory signalling in primary human fibroblast-like synoviocytes.

OBJECTIVES: Rheumatoid Arthritis (RA) is a chronic inflammatory disease of unclear aetiology, which is associated with inflamed human fibroblast-like synoviocytes (HFLS). Epidemiological studies have identified a positive correlation between tobacco smoking (a rich source of aryl hydrocarbon receptor (AHR) agonists) and aggressive RA phenotype. Thus, we hypothesise that antagonism of AHR activity by a potent AHR antagonist GNF351 can attenuate the inflammatory phenotype of HFLS-RA cells. METHODS: Quantitative PCR was used to examine IL1B-induced mRNA expression in primary HFLS-RA cells. A structurally diverse AHR antagonist CH223191 and transient AHR repression using AHR small interfering RNA (siRNA) in primary HFLS-RA cells were used to demonstrate that effects observed by GNF351 are AHR-mediated. The levels of PTGS2 were determined by western blot and secretory cytokines such as IL1B and IL6 by ELISA. Chromatin-immunoprecipitation was used to assess occupancy of the AHR on the promoters of IL1B and IL6. RESULTS: Many of the chemokine and cytokine genes induced by IL1B in HFLS-RA cells are repressed by co-treatment with GNF351 at both the mRNA and protein level. Pretreatment of HLFS-RA cells with CH223191 or transient gene ablation of AHR by siRNA confirmed that the effects of GNF351 are AHR-mediated. GNF351 inhibited the recruitment of AHR to the promoters of IL1B and IL6 confirming occupancy of AHR at these promoters is required for enhanced inflammatory signalling. CONCLUSIONS: These data suggest that AHR antagonism may represent a viable adjuvant therapeutic strategy for the amelioration of inflammation associated with RA.

Selective aryl hydrocarbon receptor modulator-mediated repression of CD55 expression induced by cytokine exposure.

Modulation of aryl hydrocarbon receptor (AHR) activity by a class of ligands termed selective AHR modulators (SAhRMs) has been demonstrated to attenuate proinflammatory gene expression and signaling, including repression of cytokine-mediated induction of acute-phase genes (e.g., Saa1). These effects are observed to occur through an AHR-dependent mechanism that does not require canonical signaling through dioxin response elements. Previously, we have demonstrated that the SAhRM 3',4'-dimethoxy-α-naphthoflavone (DiMNF) can repress the cytokine-mediated induction of complement factor genes. Here, we report that the activation of the AHR with DiMNF can suppress cytokine-mediated induction of the membrane complement regulatory protein CD55. When CD55 is expressed on host cells, it facilitates the decay of the complement component 3 (C3) convertase, thereby protecting the cell from complement-mediated lysis. Tumor cells often exhibit elevated CD55 expression on the cell surface in the inflammatory microenvironment of the tumor, and such enhanced expression could represent a means of escaping immune surveillance. DiMNF can repress the cytokine-mediated induction of CD55 mRNA and protein. Luciferase reporter analysis has identified possible response elements on the CD55 promoter, which may be targets for this repression. A C3 deposition assay with [(125)I]C3 revealed that repression of cytokine-mediated CD55 expression by DiMNF led to an increase of C3 deposition on the surface of Huh7 cells, which would likely stimulate the formation of the membrane attack complex. These results suggest that SAhRMs such as DiMNF have therapeutic potential in regulating the immune response to tumor formation.

Low fidelity bypass of O(2)-(3-pyridyl)-4-oxobutylthymine, the most persistent bulky adduct produced by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by model DNA polymerases.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O(2)-POB-dT and O(2)-methylthymidine (O(2)-Me-dT) by two model polymerases, E. coli DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affected the reactivity and that the specificity of adduct bypass was very low. The k(cat)/K(m) for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ∼10(6)-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 10(3)-fold more slowly than undamaged DNA. The dNTP specificity was less for Dpo4 than for Kf. In general, dA was the preferred base pair partner for O(2)-Me-dT and dT the preferred base pair partner for O(2)-POB-dT. With enzyme in excess over DNA, the time courses of the reactions showed a biphasic kinetics that indicates the formation inactive binary and ternary complexes.

Preliminary physiologically based pharmacokinetic models for benzo[a]pyrene and dibenzo[def,p]chrysene in rodents.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated as byproducts of natural and anthropogenic combustion processes. Despite significant public health concern, physiologically based pharmacokinetic (PBPK) modeling efforts for PAHs have so far been limited to naphthalene, plus simpler PK models for pyrene, nitropyrene, and benzo[a]pyrene (B[a]P). The dearth of published models is due in part to the high lipophilicity, low volatility, and myriad metabolic pathways for PAHs, all of which present analytical and experimental challenges. Our research efforts have focused upon experimental approaches and initial development of PBPK models for the prototypic PAH, B[a]P, and the more potent, albeit less studied transplacental carcinogen, dibenzo[def,p]chrysene (DBC). For both compounds, model compartments included arterial and venous blood, flow limited lung, liver, richly perfused and poorly perfused tissues, diffusion limited fat, and a two compartment theoretical gut (for oral exposures). Hepatic and pulmonary metabolism was described for both compounds, as were fractional binding in blood and fecal clearance. Partition coefficients for parent PAH along with their diol and tetraol metabolites were estimated using published algorithms and verified experimentally for the hydroxylated metabolites. The preliminary PBPK models were able to describe many, but not all, of the available data sets, comprising multiple routes of exposure (oral, intravenous) and nominal doses spanning several orders of magnitude.

Facile syntheses of O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine, the major adduct formed by tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in vivo, and its site-specifically adducted oligodeoxynucleotides.

O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) is the most persistent adduct detected in the lung and liver of rats treated with tobacco specific nitrosamines: N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). It is an important biomarker to assess the human exposure to these carcinogens. The only synthetic method reported for O(2)-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.4%). We have developed for the first time a regioselective and efficient method for the total synthesis of O(2)-POB-dThd and its site-specifically adducted oligonucleotides. The main step in the synthesis of O(2)-POB-dThd was achieved by a novel method. The treatment of O(2)-5'-anhydrothymidine with the sodium salt of 4-(1,3-dithian-2-yl)-4-(3-pyridyl)butan-1-ol gave exclusively the O(2)-alkylated adduct, which was deprotected in one step to furnish the desired O(2)-POB-dThd in excellent yield. The product was characterized by NMR ((1)H and (13)C), high-resolution MS, and HPLC analysis. This work provided for the first time a reliable method for large scale total synthesis of O(2)-POB-dThd that allowed for solid state site-specifically adducted oligomer synthesis. The O(2)-POB-dThd was converted to its phosphoramidite and subsequently used for the synthesis of oligodeoxynucleotides by standard methods. The oligomers were characterized by MS and HPLC analysis. These oligomers will facilitate the elucidation of the mutagenic potential of the O(2)-POB-dThd adduct, which will provide further insight into the role of tobacco-specific nitrosamines in inducing cancers in smokers.

Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway.

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 µM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 µM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 µM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.

Proinflammatory responses by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum are mainly mediated through the recognition of TLR2/TLR1.

The glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum have been shown to activate macrophages and produce inflammatory responses. The activation of macrophages by malarial GPIs involves engagement of Toll like receptor 2 (TLR2) resulting in the intracellular signaling and production of cytokines. In the present study, we investigated the requirement of TLR1 and TLR6 for the TLR2 mediated cell signaling and proinflammatory cytokine production by macrophages. The data demonstrate that malarial GPIs, which contain three fatty acid substituents, preferentially engage TLR2-TLR1 dimeric pair than TLR2-TLR6, whereas their derivatives, sn-2 lyso GPIs, that contain two fatty acid substituents recognize TLR2-TLR6 with slightly higher selectivity as compared to TLR2-TLR1 heteromeric pair. These results are analogous to the recognition of triacylated bacterial and diacylated mycoplasmal lipoproteins, respectively, by TLR2-TLR1 and TLR2-TLR6 dimers, suggesting that the lipid portions of the microbial GPI ligands play essential role in determining their TLR recognition specificity.

Development of a selective modulator of aryl hydrocarbon (Ah) receptor activity that exhibits anti-inflammatory properties.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, the role of the AHR in normal physiology is still an area of intense investigation. For example, this receptor plays an important role in certain immune responses. We have previously determined that the AHR can mediate repression of acute-phase genes in the liver. For this observation to be therapeutically useful, selective activation of the AHR would likely be necessary. Recently, the selective estrogen receptor ligand WAY-169916 has also been shown to be a selective AHR ligand. WAY-169916 can efficiently repress cytokine-mediated acute-phase gene expression (e.g., SAA1) yet fail to mediate a dioxin response element-driven increase in transcriptional activity. The goals of this study were to structurally modify WAY-169916 to block binding to the estrogen receptor and increase its affinity for the AHR. A number of WAY-169916 derivatives were synthesized and subjected to characterization as AHR ligands. The substitution of a key hydroxy group for a methoxy group ablates binding to the estrogen receptor and increases its affinity for the AHR. The compound 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA 360), in particular, exhibited essentially no AHR agonist activity yet was able to repress cytokine-mediated SAA1 gene expression in Huh7 cells. SGA 360 was tested in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated ear inflammatory edema model using C57BL6/J and Ahr(-/-) mice. Our findings indicate that SGA 360 significantly inhibits TPA-mediated ear swelling and induction of a number of inflammatory genes (e.g., Saa3, Cox2, and Il6) in C57BL6/J mice. In contrast, SGA 360 had no effect on TPA-mediated ear swelling or inflammatory gene expression in Ahr(-/-) mice. Collectively, these results indicate that SGA 360 is a selective Ah receptor modulator (SAhRM) that exhibits anti-inflammatory properties in vivo.

Plasmodium falciparum GPI mannosyltransferase-III has novel signature sequence and is functional.

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum are indispensable for parasite survival since merozoite surface proteins-1, -2, -4, -5, and -10, crucial for erythrocyte invasion, are GPI-anchored. Therefore, the GPI biosynthetic pathway can offer potential targets for novel anti-malarial drugs. Here, we characterized the putative P. falciparum PIG-B gene (PfPIGB) that encodes mannosyltransferase-III of GPI biosynthesis. PfPIGB mRNA is transcribed in a developmental stage specific manner. A protein corresponding to the expected size of PfPIG-B is expressed by the parasite and is localized in the endoplasmic reticulum. Treatment of parasites with PfPIG-B specific siRNA caused reduction in GPI synthesis, affecting the PIG-B specific GPI intermediate. These data demonstrate that PfPIG-B is functional and encodes mannosyltransferase-III of the parasite GPI biosynthesis. The parasite PfPIG-B is novel in that its signature sequence HKEHKI is unique and is only partially conserved as compared to HKEXRF signature motif of mammalian PIG-B enzymes.

Serum antibody levels to glycosylphosphatidylinositols in specimens derived from matched Malian children with severe or uncomplicated Plasmodium falciparum malaria and healthy controls.

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.

Differential antibody responses to Plasmodium falciparum glycosylphosphatidylinositol anchors in patients with cerebral and mild malaria.

Glycosylphosphatidylinositol (GPI) membrane anchors of Plasmodium falciparum surface proteins are thought to be important factors contributing to malaria pathogenesis, and anti-GPI antibodies have been suggested to provide protection by neutralizing the toxic activity of GPIs. In this study, IgG responses against P. falciparum GPIs and a baculovirus recombinant MSP1p19 antigen were evaluated in two distinct groups of 70 patients each, who were hospitalized with malaria. Anti-GPI IgGs were significantly lower in patients hospitalized with confirmed cerebral malaria compared to those with mild malaria (P < 0.01) but did not discriminate for fatal outcome. In contrast, a specific marker of the anti-parasite immunity, as monitored by the anti-MSP1p19 IgG response, was similar in both cerebral and mild malaria individuals, although it was significantly lower in a subgroup with fatal outcomes. These results are consistent with a potential anti-toxin role for anti-GPI antibodies associated with protection against cerebral malaria.

Age-dependent impairment of IgG responses to glycosylphosphatidylinositol with equal exposure to Plasmodium falciparum among Javanese migrants to Papua, Indonesia.

Immune responses directed at glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum may offer protection against symptomatic malaria. To independently explore the effect of age on generation of the anti-GPI IgG response, we measured serum anti-GPI IgGs in a longitudinal cohort of migrant Javanese children (6-12 years old) and adults (> or = 20 years old) with equivalent numbers of exposures to P. falciparum in Papua, Indonesia. While the peak response in adults was achieved after a single infection, comparable responses in children required > or = 3-4 infections. Significantly fewer children (16%) than adults (41%) showed a high (optical density > 0.44) anti-GPI IgG response (odds ratio [OR] = 3.8, 95% confidence interval [CI] = 2.3-6.3, P < 0.0001), and adults were more likely to show a persistently high response (OR = 5.5, 95% CI = 1.0-56.8, P = 0.03). However, the minority of children showing a strong response were significantly less likely to experience symptoms with subsequent parasitemia compared with those with a weak response (OR = 4.0, 95% CI = 1.1-13.8, P = 0.02). This effect was not seen among high- and low-responding adults (OR = 1.2, 95% CI = 0.5-2.8, P = 0.60). Host age, independent of cumulative exposure, apparently represents a key determinant of the quantitative and qualitative nature of the IgG response to P. falciparum GPI.

Anti-calmodulin acridone derivatives modulate vinblastine resistance in multidrug resistant (MDR) cancer cells.

Multidrug resistance (MDR) is one of the main obstacles limiting the efficacy of chemotherapy treatment of tumors. Parent acridones 1A and 1B were prepared by the Ullmann reaction followed by cyclization and N-alkylation. N-(omega-Chloroalkyl) analogues were subjected to iodide catalyzed nucleophilic substitution reaction with secondary amines to get the compounds 3A-13A and 3B-13B, which enhanced the uptake of vinblastine in KBChR-8-5 cells to a greater extent (2.6-13.1-fold relative to control) than verapamil. The study on the structure-activity relationship revealed that substitution of -H at position C-4 in acridone nucleus by -OCH3 increased the cytotoxic and anti-MDR activities. The ability of acridones to inhibit calmodulin dependent cyclic AMP phosphodiesterase has been determined and the results have shown a strong positive correlation between anti-calmodulin activity and cytotoxicity in KBChR-8-5 cells or anti-MDR activity.

Ah receptor antagonism represses head and neck tumor cell aggressive phenotype.

The aryl hydrocarbon receptor (AhR) has been shown to play a role in an increasing number of cellular processes. Recent reports have linked the AhR to cell proliferation, cytoskeletal arrangement, and tumor invasiveness in various tumor cell types. The AhR plays a role in the de-repression of the interleukin (IL)6 promoter in certain tumor cell lines, allowing for increased transcriptional activation by cytokines. Here, we show that there is a significant level of constitutive activation of the AhR in cells isolated from patients with head and neck squamous cell carcinoma (HNSCC). Constitutive activation of the AhR in HNSCCs was blocked by antagonist treatment, leading to a reduction in IL6 expression. In addition, the AhR exhibits a high level of expression in HNSCCs than in normal keratinocytes. These findings led to the hypothesis that the basal AhR activity in HNSCCs plays a role in the aggressive phenotype of these tumors and that antagonist treatment could mitigate this phenotype. This study provides evidence that antagonism of the AhR in HNSCC tumor cells, in the absence of exogenous receptor ligands, has a significant effect on tumor cell phenotype. Treatment of these cell lines with the AhR antagonists 6, 2', 4'-trimethoxyflavone, or the more potent GNF351, decreased migration and invasion of HNSCC cells and prevented benzo[a]pyrene-mediated induction of the chemotherapy efflux protein ABCG2. Thus, an AhR antagonist treatment has been shown to have therapeutic potential in HNSCCs through a reduction in aggressive cell phenotype.

MAPK-activated protein kinase 2 differentially regulates plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

Src-family kinase dependent disruption of endothelial barrier function by Plasmodium falciparum merozoite proteins.

Pulmonary complication in severe Plasmodium falciparum malaria is manifested as a prolonged impairment of gas transfer or the more severe acute respiratory distress syndrome (ARDS). In either clinical presentation, vascular permeability is a major component of the pathologic process. In this report, we examined the effect of clinical P falciparum isolates on barrier function of primary dermal and lung microvascular endothelium in vitro. We showed that parasite sonicates but not intact infected erythrocytes disrupted endothelial barrier function in a Src-family kinase-dependent manner. The abnormalities were manifested both as discontinuous immunofluorescence staining of the junctional proteins ZO-1, claudin 5, and VE-cadherin and the formation of interendothelial gaps in monolayers. These changes were associated with a loss in total protein content of claudin 5 and redistribution of ZO-1 from the cytoskeleton to the membrane and the cytosolic and nuclear fractions. There was minimal evidence of a proinflammatory response or direct cellular cytotoxicity or cell death. The active component in sonicates appeared to be a merozoite-associated protein. Increased permeability was also induced by P falciparum glycophosphatidylinositols (GPIs) and food vacuoles. These results demonstrate that parasite components can alter endothelial barrier function and thus contribute to the pathogenesis of severe falciparum malaria.

Naturally elicited antibodies to glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum require intact GPI structures for binding and are directed primarily against the conserved glycan moiety.

Immunization with a synthetic glycan corresponding to Plasmodium falciparum glycosylphosphatidylinositols (GPIs) has been proposed as a vaccination strategy against malaria. We investigated the structural requirements for binding of naturally elicited anti-GPI antibodies to parasite GPIs. The data show that anti-GPI antibody binding requires intact GPI structures and that the antibodies are directed predominantly against GPIs with a conserved glycan structure with three mannoses and marginally against the terminal fourth mannose. The results provide valuable insight for exploiting GPIs for the development of malaria vaccines.

Induction of nitric oxide synthase in Anopheles stephensi by Plasmodium falciparum: mechanism of signaling and the role of parasite glycosylphosphatidylinositols.

Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h postinfection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that adversely impact parasite development. In mammals, P. falciparum glycosylphosphatidylinositols (PfGPIs) can induce NOS expression in immune and endothelial cells and are sufficient to reproduce the major effects of parasite infection. These effects are mediated in part by mimicry of insulin signaling by PfGPIs. In this study, we demonstrate that PfGPIs can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi Akt/protein kinase B and a pathway involving DSOR1, a mitogen-activated protein kinase kinase, and an extracellular signal-regulated kinase. However, despite the involvement of kinases that are also associated with insulin signaling in A. stephensi cells, signaling by P. falciparum and by PfGPIs is distinctively different from signaling by insulin. Therefore, although mimicry of insulin by PfGPIs appears to be restricted to mammalian hosts of P. falciparum, the conservation of PfGPIs as a prominent parasite-derived signal of innate immunity can now be extended to include Anopheles mosquitoes, indicating that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.