Parasite maturation and host serum iron influence the labile iron pool of erythrocyte stage Plasmodium falciparum.

Iron is a critical and tightly regulated nutrient for both the malaria parasite and its human host. The importance of the relationship between host iron and the parasite has been underscored recently by studies showing that host iron supplementation may increase the risk of falciparum malaria. It is unclear what host iron sources the parasite is able to access. We developed a flow cytometry-based method for measuring the labile iron pool (LIP) of parasitized erythrocytes using the nucleic acid dye STYO 61 and the iron sensitive dye, calcein acetoxymethyl ester (CA-AM). This new approach enabled us to measure the LIP of P. falciparum through the course of its erythrocytic life cycle and in response to the addition of host serum iron sources. We found that the LIP increases as the malaria parasite develops from early ring to late schizont stage, and that the addition of either transferrin or ferric citrate to culture media increases the LIP of trophozoites. Our method for detecting the LIP within malaria parasitized RBCs provides evidence that the parasite is able to access serum iron sources as part of the host vs. parasite arms race for iron.

Quantification of the burden and consequences of pregnancy-associated malaria in the Democratic Republic of the Congo.

BACKGROUND: Pregnancy-associated malaria (PAM) produces poor birth outcomes, but its prevalence is commonly estimated in convenience samples. METHODS: We assessed the prevalence of malaria using real-time polymerase chain reaction (PCR) and estimated the consequences of infection on birth outcomes, using specimens from a nationally representative sample of 4570 women of childbearing age (WOCBA) responding to the 2007 Demographic and Health Survey in Democratic Republic of the Congo (DRC). RESULTS: Overall, 31.2% (95% confidence interval [CI], 29.2-33.1) of WOCBA were parasitemic, which was significantly more common in pregnant (37.2% [31.0-43.5]) than nonpregnant women (30.4% [CI, 28.4-32.5], prevalence ratio [PR] 1.22 [1.02-1.47]). Plasmodium falciparum was highest among pregnant women (36.6% vs 28.8%, PR 1.27 [1.05-1.53]). By contrast, P malariae was less common in pregnant (0.6%) compared with nonpregnant women (2.7%, PR 0.23 [0.09-0.56]). Extrapolation of the prevalence estimate to the population at risk of malaria in DRC suggests 1.015 million births are affected by P falciparum infection annually, and that adherence to preventive measures could prevent up to 549 000 episodes of pregnancy-associated malaria and 47 000 low-birth-weight births. CONCLUSIONS: Pregnancy-associated malaria and its consequences are highly prevalent in the DRC. Increasing the uptake of malaria preventive measures represents a significant opportunity to improve birth outcomes and neonatal health.

Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin.

Erythropoietin (EPO), a member of the type 1 cytokine superfamily, plays a critical hormonal role regulating erythrocyte production as well as a paracrine/autocrine role in which locally produced EPO protects a wide variety of tissues from diverse injuries. Significantly, these functions are mediated by distinct receptors: hematopoiesis via the EPO receptor homodimer and tissue protection via a heterocomplex composed of the EPO receptor and CD131, the beta common receptor. In the present work, we have delimited tissue-protective domains within EPO to short peptide sequences. We demonstrate that helix B (amino acid residues 58-82) of EPO, which faces the aqueous medium when EPO is bound to the receptor homodimer, is both neuroprotective in vitro and tissue protective in vivo in a variety of models, including ischemic stroke, diabetes-induced retinal edema, and peripheral nerve trauma. Remarkably, an 11-aa peptide composed of adjacent amino acids forming the aqueous face of helix B is also tissue protective, as confirmed by its therapeutic benefit in models of ischemic stroke and renal ischemia-reperfusion. Further, this peptide simulating the aqueous surface of helix B also exhibits EPO's trophic effects by accelerating wound healing and augmenting cognitive function in rodents. As anticipated, neither helix B nor the 11-aa peptide is erythropoietic in vitro or in vivo. Thus, the tissue-protective activities of EPO are mimicked by small, nonerythropoietic peptides that simulate a portion of EPO's three-dimensional structure.

DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis.

We have shown recently that cDNA vaccination, using a virtual lymph node, ameliorates experimental allergic encephalomyelitis. Successful cure from mammary tumor requires resolution of local tumor growth and metastases. We have examined whether targeting of CD44 cell surface adhesion molecule by cDNA vaccination plays a role in resolving mammary tumor development. We show here that CD44 cDNA vaccination decreases the tumor mass and metastatic potential in experimental mammary tumor of BALB/c mice. Vaccination of mice, inoculated with the mammary tumors, by cDNA of CD44 variant (CD44v) but not by cDNA of standard CD44, markedly reduced local tumor development and lung metastasis. Concomitantly, transfection of CD44 antisense into a highly metastatic mammary tumor cell line disrupted the CD44 expression of the cells and reduced their ability to establish local tumors as well as metastatic colonies in the lung. Moreover, when CD44v, but not standard CD44 sense cDNA, was transfected into the poorly metastatic cell line, tumor development was markedly enhanced. It is possible therefore that DNA vaccination with a specific CD44v construct could induce an immune resistance to mammary tumor progression.

CD44 gene vaccination for insulin-dependent diabetes mellitus in non-obese diabetic mice.

BACKGROUND: Standard CD44 and its alternatively spliced variants were found to be associated with the metastatic potential of tumor cells and with cell migration of autoimmune inflammatory cells, including cells involved in experimental insulin-dependent diabetes mellitus. OBJECTIVES: To investigate whether induction of anti-CD44 immune reactivity, through cDNA vaccination, could attenuate IDDM in a transfer model of NOD mice. METHODS: Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with a 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of male NOD recipients reconstituted with diabetogenic spleen cells of female NOD donors. The VLN were implanted 20 days before and 3 days after cell transfer. RESULTS: In contrast to control groups of recipient mice, recipients vaccinated with VLN loaded with CD44v or CD44s cDNAs developed resistance to IDDM almost to the same extent. Our results suggest that the gene vaccination effect was mediated by anti-CD44 antibody rather than by cellular immunity. Histopathological examinations revealed a significant protection of pancreatic islets in the DNA-vaccinated recipients, whereas the islets of control recipients of diabetogenic cells were almost totally destroyed. CONCLUSIONS: These findings may open new opportunities for IDDM therapy in the future.

CD44 variant DNA vaccination with virtual lymph node ameliorates experimental autoimmune encephalomyelitis through the induction of apoptosis.

Standard CD44 (CD44s) and its alternatively spliced variants (CD44v) were found to be associated with the metastatic potential of tumor cells, and with cell migration of autoimmune inflammatory cells, including cells involved in experimental autoimmune encephalomyelitis (EAE). The aim of the present study was to evaluate whether induction of anti-CD44 immune reactivity, through cDNA vaccination could down-regulate EAE. Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of SJL/J mice immunized with myelin antigens for EAE induction. Animals vaccinated with CD44v cDNA developed significantly less severe EAE when compared with sham vaccinated animals or animals vaccinated with CD44s cDNA. The in vitro proliferation of lymphocytes was preserved regarding myelin antigens and mitogens. Histopathological examinations revealed a significant reduction of EAE lesions and enhanced apoptosis in central nervous system (CNS)-infiltrating cells of the successfully vaccinated animals. Such methods of cDNA vaccination with CD44 could be applicable in inflammatory CNS diseases, like multiple sclerosis.

Treatment of mild traumatic brain injury with an erythropoietin-mimetic peptide.

Mild traumatic brain injury (mTBI) results in an estimated 75-90% of the 1.7 million TBI-related emergency room visits each year. Post-concussion symptoms, which can include impaired memory problems, may persist for prolonged periods of time in a fraction of these cases. The purpose of this study was to determine if an erythropoietin-mimetic peptide, pyroglutamate helix B surface peptide (pHBSP), would improve neurological outcomes following mTBI. Sixty-four rats were randomly assigned to pHBSP or control (inactive peptide) 30 µg/kg IP every 12 h for 3 days, starting at either 1 hour (early treatment) or 24 h (delayed treatment), after mTBI (cortical impact injury 3 m/sec, 2.5 mm deformation). Treatment with pHBSP resulted in significantly improved performance on the Morris water maze task. Rats that received pHBSP required 22.3±1.3 sec to find the platform, compared to 26.3±1.3 sec in control rats (p=0.022). The rats that received pHBSP also traveled a significantly shorter distance to get to the platform, 5.0±0.3 meters, compared to 6.1±0.3 meters in control rats (p=0.019). Motor tasks were only transiently impaired in this mTBI model, and no treatment effect on motor performance was observed with pHBSP. Despite the minimal tissue injury with this mTBI model, there was significant activation of inflammatory cells identified by labeling with CD68, which was reduced in the pHBSP-treated animals. The results suggest that pHBSP may improve cognitive function following mTBI.

Ketamine does not produce relief of neuropathic pain in mice lacking the ß-common receptor (CD131).

Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the ß-common receptor (ßcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the ßcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine's analgesic and side effects.

Antimalarial iron chelator, FBS0701, shows asexual and gametocyte Plasmodium falciparum activity and single oral dose cure in a murine malaria model.

Iron chelators for the treatment of malaria have proven therapeutic activity in vitro and in vivo in both humans and mice, but their clinical use is limited by the unsuitable absorption and pharmacokinetic properties of the few available iron chelators. FBS0701, (S)3"-(HO)-desazadesferrithiocin-polyether [DADFT-PE], is an oral iron chelator currently in Phase 2 human studies for the treatment of transfusional iron overload. The drug has very favorable absorption and pharmacokinetic properties allowing for once-daily use to deplete circulating free iron with human plasma concentrations in the high µM range. Here we show that FBS0701 has inhibition concentration 50% (IC(50)) of 6 µM for Plasmodium falciparum in contrast to the IC(50) for deferiprone and deferoxamine at 15 and 30 µM respectively. In combination, FBS0701 interfered with artemisinin parasite inhibition and was additive with chloroquine or quinine parasite inhibition. FBS0701 killed early stage P. falciparum gametocytes. In the P. berghei Thompson suppression test, a single dose of 100 mg/kg reduced day three parasitemia and prolonged survival, but did not cure mice. Treatment with a single oral dose of 100 mg/kg one day after infection with 10 million lethal P. yoelii 17XL cured all the mice. Pretreatment of mice with a single oral dose of FBS0701 seven days or one day before resulted in the cure of some mice. Plasma exposures and other pharmacokinetics parameters in mice of the 100 mg/kg dose are similar to a 3 mg/kg dose in humans. In conclusion, FBS0701 demonstrates a single oral dose cure of the lethal P. yoelii model. Significantly, this effect persists after the chelator has cleared from plasma. FBS0701 was demonstrated to remove labile iron from erythrocytes as well as enter erythrocytes to chelate iron. FBS0701 may find clinically utility as monotherapy, a malarial prophylactic or, more likely, in combination with other antimalarials.

Neuroprotection with an erythropoietin mimetic peptide (pHBSP) in a model of mild traumatic brain injury complicated by hemorrhagic shock.

Pyroglutamate helix B surface peptide (pHBSP) is an 11 amino acid peptide, designed to interact with a novel cell surface receptor, composed of the classical erythropoietin (EPO) receptor disulfide linked to the beta common receptor. pHBSP has the cytoprotective effects of EPO without stimulating erythropoiesis. Effects on early cerebral hemodynamics and neurological outcome at 2 weeks post-injury were compared in a rat model of mild cortical impact injury (3m/sec, 2.5 mm deformation) followed by 50 min of hemorrhagic hypotension (MAP 40 mm Hg for 50 min). Rats were randomly assigned to receive 5000 U/kg of EPO, 30 µg/kg of pHBSP, or an inactive substance every 12 h for 3 days, starting at the end of resuscitation from the hemorrhagic hypotension, which was 110 min post-injury. Both treatments reduced contusion volume at 2 weeks post-injury, from 20.8±2.8 mm(3) in the control groups to 7.7±2.0 mm(3) in the EPO-treated group and 5.9±1.5 mm(3) in the pHBSP-treated group (p=0.001). Both agents improved recovery of cerebral blood flow in the injured brain following resuscitation, and resulted in more rapid recovery of performance on beam balancing and beam walking tests. These studies suggest that pHBSP has neuroprotective effects similar to EPO in this model of combined brain injury and hypotension. pHBSP may be more useful in the clinical situation because there is less risk of thrombotic adverse effects.

Promises and pitfalls in erythopoietin-mediated tissue protection: are nonerythropoietic derivatives a way forward?

The essential biological role of erythropoietin (EPO) in maintaining erythrocyte mass has been well understood for many years. Although EPO is required for the maturation of red cells, it also has strong procoagulant effects on the vascular endothelium and platelets, which limit erythrocyte losses after hemorrhage. Like other members of the type 1 cytokine superfamily, EPO has multiple biological activities. For the past 10 years, multiple investigators have shown that EPO acts as a locally produced antagonist of proinflammatory cytokines that are generated by the innate immune response in response to infection, trauma, or metabolic stress. Specifically, EPO inhibits apoptosis of cells surrounding a locus of injury, reduces the influx of inflammatory cells, and recruits tissue-specific stem cells and endothelial progenitor cells. Available evidence suggests that these multiple, nonerythropoietic effects of EPO are mediated by a tissue protective receptor (TPR) that is distinct from the homodimeric receptor responsible for erythropoiesis. Notably, activation of the TPR requires a higher concentration of EPO than is needed for maximal erythropoiesis. Unfortunately, these higher concentrations of EPO also stimulate hematopoietic and procoagulant pathways, which can cause adverse effects and, therefore, potentially limit the clinical use of EPO for tissue protection. To circumvent these problems, the EPO molecule has been successfully modified in a variety of ways to interact only with the TPR. Early clinical experience has shown that these compounds appear to be safe, and proof of concept trials are ready to begin.

Molecular malaria epidemiology: mapping and burden estimates for the Democratic Republic of the Congo, 2007.

BACKGROUND: Epidemiologic data on malaria are scant in many high-burden countries including the Democratic Republic of the Congo (DRC), which suffers the second-highest global burden of malaria. Malaria control efforts in regions with challenging infrastructure require reproducible and efficient surveillance. We employed new high-throughput molecular testing to characterize the state of malaria control in the DRC and estimate childhood mortality attributable to excess malaria transmission. METHODS AND FINDINGS: The Demographic and Health Survey was a cross-sectional, population-based cluster household survey of adults aged 15-59 years in 2007 employing structured questionnaires and dried blood spot collection. Parasitemia was detected by real-time PCR, and survey responses measured adoption of malaria control measures and under-5 health indices. The response rate was 99% at the household level, and 8,886 households were surveyed in 300 clusters; from 8,838 respondents molecular results were available. The overall prevalence of parasitemia was 33.5% (95% confidence interval [C.I.] 32-34.9); P. falciparum was the most prevalent species, either as monoinfection (90.4%; 95% C.I. 88.8-92.1) or combined with P. malariae (4.9%; 95% C.I. 3.7-5.9) or P. ovale (0.6%; 95% C.I. 0.1-0.9). Only 7.7% (95% CI 6.8-8.6) of households with children under 5 owned an insecticide-treated bednet (ITN), and only 6.8% (95% CI 6.1-7.5) of under-fives slept under an ITN the preceding night. The overall under-5 mortality rate was 147 deaths per 1,000 live births (95% C.I. 141-153) and between clusters was associated with increased P. falciparum prevalence; based on the population attributable fraction, 26,488 yearly under-5 deaths were attributable to excess malaria transmission. CONCLUSIONS: Adult P. falciparum prevalence is substantial in the DRC and is associated with under-5 mortality. Molecular testing offers a new, generalizable, and efficient approach to characterizing malaria endemicity in underserved countries.

Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor.

The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common beta receptor (betacR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because betacR knockout mice exhibit normal erythrocyte maturation, betacR is not required for erythropoiesis. We hypothesized that betacR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with betacR. Further, antibodies against EpoR coimmunoprecipitated betacR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo betacR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the betacR knockout mouse. These data support the concept that EpoR and betacR comprise a tissue-protective heteroreceptor.

Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo.

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood-brain barrier and provide neuroprotection.

Derivatives of erythropoietin that are tissue protective but not erythropoietic.

Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype-selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.