Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria.IMPORTANCE Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite Plasmodium falciparum The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.

Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling.

BACKGROUND: Leukocyte-associated immunoglobulin like receptor-1 (LAIR1) is a transmembrane inhibitory receptor that influences susceptibility to a myriad of inflammatory diseases. Our recent investigations of severe malarial anaemia (SMA) pathogenesis in Kenyan children discovered that novel LAIR1 genetic variants which were associated with decreased LAIR1 transcripts enhanced the longitudinal risk of SMA and all-cause mortality. METHODS: To characterize the molecular mechanism(s) responsible for altered LAIR1 signalling in severe malaria, we determined LAIR1 transcripts and protein, sLAIR1, sLAIR2, and complement component 1q (C1q) in children with malarial anaemia, followed by a series of in vitro experiments investigating the LAIR1 signalling cascade. FINDINGS: Kenyan children with SMA had elevated circulating levels of soluble LAIR1 (sLAIR1) relative to non-SMA (1.69-fold P < .0001). The LAIR1 antagonist, sLAIR2, was also elevated in the circulation of children with SMA (1.59 fold-change, P < .0001). There was a positive correlation between sLAIR1 and sLAIR2 (ρ = 0.741, P < .0001). Conversely, circulating levels of complement component 1q (C1q), a LAIR1 natural ligand, were lower in SMA (-1.21-fold P = .048). These in vivo findings suggest that reduced membrane-bound LAIR1 expression in SMA is associated with elevated production of sLAIR1, sLAIR2 (antagonist), and limited C1q (agonist) availability. Since reduced LAIR1 transcripts in SMA were associated with increased acquisition of haemozoin (PfHz) by monocytes (P = .028), we explored the relationship between acquisition of intraleukocytic PfHz, LAIR1 expression, and subsequent impacts on leukocyte signalling in cultured PBMCs from malaria-naïve donors stimulated with physiological concentrations of PfHz (10 µg/mL). Phagocytosis of PfHz reduced LAIR1 transcript and protein expression in a time-dependent manner (P < .050), and inhibited LAIR1 signalling through decreased phosphorylation of LAIR1 (P < .0001) and SH2-domain containing phosphatase-1 (SHP-1) (P < .001). This process was associated with NF-κB activation (P < .0001) and enhanced production of IL-6, IL-1ß, and TNF-α (all P < .0001). INTERPRETATION: Collectively, these findings demonstrate that SMA is characterized by reduced LAIR1 transmembrane expression, reduced C1q, and enhanced production of sLAIR1 and sLAIR2, molecular events which can promote enhanced production of cytokines that contribute to the pathogenesis of SMA. These investigations are important for discovering immune checkpoints that could be future targets of immunotherapy to improve disease outcomes.

Hemozoin is a potent adjuvant for hemagglutinin split vaccine without pyrogenicity in ferrets.

BACKGROUND: Synthetic hemozoin (sHZ, also known as ß-hematin) from monomeric heme is a particle adjuvant which activates antigen-presenting cells (APCs), such as dendritic cells and macrophages, and enhances humoral immune responses to several antigens, including ovalbumin, human serum albumin, and serine repeat antigen 36 of Plasmodium falciparum. In the present study, we evaluated the adjuvanticity and pyrogenicity of sHZ as an adjuvant for seasonal trivalent hemagglutinin split vaccine (SV) for humans using the experimental ferret model. METHOD: Ferrets were twice immunized with trivalent SV, SV with sHZ (SV/sHZ) or Fluad, composed of trivalent SV with MF59. Serum hemagglutination inhibition (HI) titers against three viral hemagglutinin (HA) antigens were measured at every week after the immunization. The pyrogenicity of SV/sHZ was examined by monitoring the body temperature of the immunized ferrets. To evaluate the protective efficacy of SV/sHZ, the immunized ferrets were challenged with influenza virus B infection, followed by measurement of viral titers in the nasal cavity and body temperature. RESULTS: sHZ enhanced HI titers against three viral HA antigens in a dose-dependent manner, to an extent comparable to that of Fluad. The highest dose of sHZ (800 µg) immunized with SV conferred sterile protection against infection with heterologous Influenza B virus, without causing any pyrogenic reaction such as high fever. CONCLUSION: In the present study, sHZ enhanced the protective efficacy of SV against influenza infection without inducing pyrogenic reaction, suggesting sHZ to be a promising adjuvant candidate for human SV.

TLR9 adjuvants enhance immunogenicity and protective efficacy of the SE36/AHG malaria vaccine in nonhuman primate models.

The SE36 antigen, derived from serine repeat antigen 5 (SERA5) of Plasmodium falciparum, is a promising blood stage malaria vaccine candidate. Ongoing clinical trials suggest the efficacy of the SE36 vaccine could be increased by the incorporation of more effective adjuvants into the vaccine formulation. In this study, we assessed the safety, immunogenicity and protective efficacy of SE36/AHG formulated with TLR9 ligand adjuvants K3 CpG oligodeoxyribonucleotides (CpG ODNs) (K3 ODN), D3 ODN or synthetic hemozoin, in two non-human primate models. SE36/AHG with or without each adjuvant was administrated to cynomolgus monkeys. A combination of TLR9 ligand adjuvant with SE36/AHG induced higher humoral and cellular immune response compared with SE36/AHG alone. Administration of a crude extract of P. falciparum parasite resulted in the induction of more SE36-specific IgG antibodies in monkeys vaccinated with a combination of SE36/AHG and adjuvant, as opposed to vaccination with SE36/AHG alone. The most effective TLR9 ligand, K3 ODN, was chosen for further vaccine trials in squirrel monkeys, in combination with SE36/AHG. All monkeys immunized with the combined SE36/AHG and K3 ODN formulation effectively suppressed parasitemia and symptoms of malaria following challenge infections. Furthermore, no serious adverse events were observed. Our results show that the novel vaccine formulation of K3 ODN with SE36/AHG demonstrates safety, potent immunogenicity and efficacy in nonhuman primates, and this vaccine formulation may form the basis of a more effective malaria vaccine.

Hepcidin is regulated during blood-stage malaria and plays a protective role in malaria infection.

Hepcidin is one of the regulators of iron metabolism. The expression of hepcidin is induced in spleens and livers of mice infected with pathogenic bacteria. Recent studies have indicated that serum hepcidin level is also increased in human subjects infected with Plasmodium falciparum. The mechanism of the regulation of hepcidin expression and its role in the infection of malaria remains unknown. In this study, we determined the expression of hepcidin in livers of mice infected with Plasmodium berghei. The expression of hepcidin in the liver was upregulated and downregulated during the early and late stages of malaria infection, respectively. Inflammation and erythropoietin, rather than the iron-sensing pathway, are involved in the regulation of hepcidin expression in livers of infected mice. Meanwhile, we investigated the effect of hepcidin on the survival of mice infected with P. berghei. Treatment of malaria-infected mice with anti-hepcidin neutralizing Abs promoted the rates of parasitemia and mortality. In contrast, lentiviral vector-mediated overexpression of hepcidin improved the outcome of P. berghei infection in mice. Our data demonstrate an important role of hepcidin in modulating the course and outcome of blood-stage malaria.

Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis.

Nitrophorin 2 (NP2) is a 20 kDa lipocalin identified in the salivary gland of the blood sucking insect, Rhodnius prolixus. It functions as a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. Herein we have investigated the in vivo antithrombotic properties of NP2. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (KD = 43 and 47 nM, respectively), and prolongs the aPTT without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo two distinct models of thrombosis in rats were carried out. In the rose Bengal/laser induced injury model of arterial thrombosis, NP2 increased the carotid artery occlusion time by ≍35 and ≍155%, at doses of 8 and 80 µg/kg, respectively. NP2 also inhibited thrombus formation in an arterio-venous shunt model, showing ≍60% reduction at 400 µg/kg (i.v. administration). The antithrombotic effect lasted for up to 48 hours after a single i.v. dose. Notably, effective doses of NP2 did not increase the blood loss as evaluated by tail-transection model. In conclusion, NP2 is a potent and long-lasting inhibitor of arterial thrombosis with minor effects on haemostasis. It might be regarded as a potential agent for the treatment of human cardiovascular diseases.

Rationale and design of the oral HEMe iron polypeptide Against Treatment with Oral Controlled Release Iron Tablets trial for the correction of anaemia in peritoneal dialysis patients (HEMATOCRIT trial).

BACKGROUND: The main hypothesis of this study is that oral heme iron polypeptide (HIP; Proferrin ES) administration will more effectively augment iron stores in erythropoietic stimulatory agent (ESA)-treated peritoneal dialysis (PD) patients than conventional oral iron supplementation (Ferrogradumet). METHODS: Inclusion criteria are peritoneal dialysis patients treated with darbepoietin alpha (DPO; Aranesp(R), Amgen) for >or= 1 month. Patients will be randomized 1:1 to receive either slow-release ferrous sulphate (1 tablet twice daily; control) or HIP (1 tablet twice daily) for a period of 6 months. The study will follow an open-label design but outcome assessors will be blinded to study treatment. During the 6-month study period, haemoglobin levels will be measured monthly and iron studies (including transferring saturation [TSAT] measurements) will be performed bi-monthly. The primary outcome measure will be the difference in TSAT levels between the 2 groups at the end of the 6 month study period, adjusted for baseline values using analysis of covariance (ANCOVA). Secondary outcome measures will include serum ferritin concentration, haemoglobin level, DPO dosage, Key's index (DPO dosage divided by haemoglobin concentration), and occurrence of adverse events (especially gastrointestinal adverse events). DISCUSSION: This investigator-initiated multicentre study has been designed to provide evidence to help nephrologists and their peritoneal dialysis patients determine whether HIP administration more effectively augments iron stores in ESP-treated PD patients than conventional oral iron supplementation. TRIAL REGISTRATION: Australia New Zealand Clinical Trials Registry number ACTRN12609000432213.

F2L, a peptide derived from heme-binding protein, inhibits formyl peptide receptor-mediated signaling.

F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of (125)I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits FPR- and FPRL1-mediated signaling, and is expected to be useful in the study of FPR and FPRL1 signaling and in the development of drugs to treat diseases involving the FPR family of receptors.

Targeting cancer cells by novel engineered modular transporters.

A major problem in the treatment of cancer is the specific targeting of drugs to these abnormal cells. Ideally, such a drug should act over short distances to minimize damage to healthy cells and target subcellular compartments that have the highest sensitivity to the drug. We describe the novel approach of using modular recombinant transporters to target photosensitizers to the nucleus, where their action is most pronounced, of cancer cells overexpressing ErbB1 receptors. We have produced a new generation of the transporters consisting of (a) epidermal growth factor as the internalizable ligand module to ErbB1 receptors, (b) the optimized nuclear localization sequence of SV40 large T-antigen, (c) a translocation domain of diphtheria toxin as an endosomolytic module, and (d) the Escherichia coli hemoglobin-like protein HMP as a carrier module. The modules retained their functions within the transporter chimera: they showed high-affinity interactions with ErbB1 receptors and alpha/beta-importin dimers and formed holes in lipid bilayers at endosomal pH. A photosensitizer conjugated with the transporter produced singlet oxygen and (*)OH radicals similar to the free photosensitizer. Photosensitizers-transporter conjugates have >3,000 times greater efficacy than free photosensitizers for target cells and were not photocytotoxic at these concentrations for cells expressing a few ErbB1 receptors per cell, in contrast to free photosensitizers. The different modules of the transporters, which are highly expressed and easily purified to retain full activity of each of the modules, are interchangeable, meaning that they can be tailored for particular applications.

Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection.

During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), beta-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1beta and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria.

Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP-1 beta) in vitro, and altered thermoregulation in vivo.

A characteristic feature of malaria infection is the occurrence of periodic bouts of fever. Experimental and clinical studies have strongly implicated inflammatory cytokines, like tumour necrosis factor (TNF), in the induction of these intermittent fevers [Clark et al., Infect Immunol 32:1058-1066, 1981; Clark et al., Am J Pathol 129:192-199, 1987; Karunaweera et al., Proc Natl Acad Sci USA 89:3200-3203, 1992], but the malaria-specific metabolite(s) which induce the production of such endogenous pyrogens have not yet been fully characterized. It is well known that during the course of malaria infection, a unique schizont component, alternatively referred to as "malaria pigment" or hemozoin, is released along with merozoites as the host erythrocyte bursts [Urquhart, Clin Infect Dis 19:117-131, 1994]. We have recently determined that the core structure of hemozoin comprises a novel insoluble polymer of heme units linked by iron-carboxylate bonds [Slater et al., Proc Natl Acad Sci USA 88:325-329, 1991; Slater et al., Nature 355:167-169, 1992]. We now report that purified native, as well as chemically synthesized, hemozoin crystals potently induce the release of several pyrogenic cytokines, including TNF, MIP-1 alpha, and MIP-1 beta, from murine macrophages and human peripheral blood monocytes in vitro. Also, intravenous administration of chemically synthesized preparations of hemozoin to anaesthetized rats results in a marked drop in body temperature. A similar drop in body temperature is observed following the intravenous injection of other well-characterized pyrogenic cytokines (e.g., TNF) which are known to induce a fever response in awake animals, and is thought to reflect the inability of rats to appropriately regulate their body temperature while anaesthetized. As a consequence of its ability to induce pyrogenic cytokines in vitro, and thermal dysregulation in vivo, we propose that this unique parasite metabolite is an important pyrogen released by malaria parasites at schizogomy, which acts by eliciting the production of a group of potent endogenous pyrogens, which include MIP-1 alpha and MIP-1 beta, as well as TNF, in macrophages.

A model for long-term intracochlear administration of pharmacologic agents.

A technique is described that provides a method to study the effects of intracochlear pharmacologic agents on the degenerating auditory nerve. The model involves implanting a guinea pig with an osmotic minipump that allows slow perfusion of the cochlea over a period of hours to days with up to two water soluble substances. Neomycin and horseradish peroxidase, whose histopathologic effects on the cochlea are well known, are used in the present study to validate the efficacy of the system.

Effect of ingestion of hemoproteins on fecal excretion of hemes and porphyrins.

Stools from asymptomatic volunteers on diets containing red meat, whole blood, or high fiber were analyzed for their content of hemes and dicarboxylic (heme-derived) porphyrins by the "HemoQuant" assay, the "Hemoccult" test, and "high-performance" liquid chromatography (HPLC). In 49 subjects, ingestion of red meat increased HemoQuant-determined combined fecal heme plus dicarboxylic porphyrins by an average 375%; the contribution of heme-derived porphyrins to total fecal porphyrins increased from 37% to 78%. Of subjects on a red-meat diet, 27% passed stools with a porphyrin content suggestive of a porphyria, compared with only 4% on a red-meat-free diet. These increases were due largely to protoporphyrin and its derivatives pemptoporphyrin and deuteroporphyrin, all of which were present in feces as iron-free porphyrins and iron-ligated (heme) forms. Ingestion of blood had an effect similar to that of red meat, but ingestion of fiber had no effect. These effects of dietary and endogenous hemoproteins must be considered when such methods are used to test feces for occult blood or to test for excess fecal porphyrins as an indicator of a porphyria.

Modes of protein and peptide uptake in the pineal gland of the Mongolian gerbil: an ultrastructural study.

The possible existence of either a blood-brain barrier or a CSF-brain barrier was examined in the pineal gland of the Mongolian gerbil using the ultrastructural tracers, horseradish peroxidase (HRP) and microperoxidase (MP). The mechanism of protein and peptide transport within the pineal gland and its possible relationship to pineal concretions was also considered. Gerbils were injected with either MP or HRP intravenously (IV), or they received intraventricular (IVT) injections of HRP. The IV injections resulted in both MP and HRP movement through the endothelial cells of the gland by vesicular transport and by diffusion through the endothelial cells of the gland by vesicular transport and by diffusion through the endothelial intercellular junctions. Following the IVT injections, the tracer was demonstrated in the subarachnoid space as well as in the glial cells associated with the periphery of the gland. In addition, after the IVT injections, rounded enlargements of the intercellular space that resembled canaliculi were filled with reaction product. In both cases (IVT and IV), the reaction product was localized to the perivascular space, to the glial cells and pinealocytes, and to the intercellular spaces. More importantly, there was specific localization of the tracers in the vacuolated pinealocytes and in the pineal concretions. The results of this study demonstrate several significant findings: 1) neither a blood-brain barrier nor a CSF-brain barrier exists in the pineal gland of the gerbil, 2) localization of the tracers in pineal concretions indicates a relationship between these structures and protein and peptide storage within the gland, and 3) the presence of the tracers within canaliculi after the IVT injections suggests a possible mode of secretion of pineal substances into the CSF.