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1.  Pharmacokinetic/pharmacodynamic modelling of the antimalarial effect of Actelion‐451840 in an induced blood stage malaria study in healthy subjects 
Aims
The aim of this study was to use data from an experimental induced blood stage malaria clinical trial to characterize the antimalarial activity of the new compound Actelion‐451840 using pharmacokinetic/pharmacodynamic (PK/PD) modelling. Then, using simulations from the model, the dose and dosing regimen necessary to achieve cure of infection were derived.
Methods
Eight healthy male subjects were infected with blood stage P. falciparum. After 7 days, a single dose of 500 mg of Actelion‐451840 was administered under fed conditions. Parasite and drug concentrations were sampled frequently. Parasite growth and the relation to drug exposure were estimated using PK/PD modelling. Simulations were then undertaken to derive estimates of the likelihood of achieving cure in different scenarios.
Results
Actelion‐451840 was safe and well tolerated. Single dose treatment markedly reduced the level of P. falciparum parasitaemia, with a weighted average parasite reduction rate of 73.6 (95% CI 56.1, 96.5) and parasite clearance half‐life of 7.7 h (95% CI 7.3, 8.3). A two compartment PK/PD model with a steep concentration−kill effect predicted maximum effect with a sustained concentration of 10–15 ng ml−1 and cure achieved in 90% of subjects with six once daily doses of 300 mg once daily.
Conclusions
Actelion‐451840 shows clinical efficacy against P. falciparum infections. The PK/PD model developed from a single proof‐of‐concept study with eight healthy subjects enabled prediction of therapeutic effects, with cure rates with seven daily doses predicted to be equivalent to artesunate monotherapy. Larger doses or more frequent dosing are not predicted to achieve more rapid cure.
doi:10.1111/bcp.12962
PMCID: PMC4972157  PMID: 27062080
challenge model; malaria; pharmacodynamics; pharmacokinetics; Plasmodium falciparum; proof of concept
2.  Identification, Design and Synthesis of Tubulin-Derived Peptides as Novel Hyaluronan Mimetic Ligands for the Receptor for Hyaluronan-Mediated Motility (RHAMM/HMMR) 
Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2,4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease.
doi:10.1039/c5ib00222b
PMCID: PMC4721638  PMID: 26456171
3.  Plasmodium vivax but Not Plasmodium falciparum Blood-Stage Infection in Humans Is Associated with the Expansion of a CD8+ T Cell Population with Cytotoxic Potential 
PLoS Neglected Tropical Diseases  2016;10(12):e0005031.
P. vivax and P. falciparum parasites display different tropism for host cells and induce very different clinical symptoms and pathology, suggesting that the immune responses required for protection may differ between these two species. However, no study has qualitatively compared the immune responses to P. falciparum or P. vivax in humans following primary exposure and infection. Here, we show that the two species differ in terms of the cellular immune responses elicited following primary infection. Specifically, P. vivax induced the expansion of a subset of CD8+ T cells expressing the activation marker CD38, whereas P. falciparum induced the expansion of CD38+ CD4+ T cells. The CD38+ CD8+ T cell population that expanded following P. vivax infection displayed greater cytotoxic potential compared to CD38- CD8+ T cells, and compared to CD38+ CD8+ T cells circulating during P. falciparum infection. We hypothesize that P. vivax infection leads to a stronger CD38+ CD8+ T cell activation because of its preferred tropism for MHC-I-expressing reticulocytes that, unlike mature red blood cells, can present antigen directly to CD8+ T cells. This study provides the first line of evidence to suggest an effector role for CD8+ T cells in P. vivax blood-stage immunity. It is also the first report of species-specific differences in the subset of T cells that are expanded following primary Plasmodium infection, suggesting that malaria vaccine development may require optimization according to the target parasite.
Trial Registration
anzctr.org.au ACTRN12612000814875; anzctr.org.au ACTRN12613000565741; anzctr.org.au ACTRN12613001040752; ClinicalTrials.gov NCT02281344; anzctr.org.au ACTRN12612001096842; anzctr.org.au ACTRN12613001008718
Author Summary
The specific immune responses that contribute to protective immunity in humans following Plasmodium infection are yet to be fully characterized. The species P. vivax and P. falciparum account for most human infections, yet little is known about P. vivax specific immune responses and whether they are similar to or distinct from P. falciparum. Here, we establish that P. vivax and P. falciparum elicit distinct cellular immune responses following primary infection, with the expansion of a subset of CD38+ CD8+ T cells with a cytotoxic potential in P. vivax but not in P. falciparum infection. This study provides the first evidence for the activation of CD8+ T cells in P. vivax blood-stage infection and demonstrates the existence of species-dependent host immune responses to malaria. These findings have important implications for P. vivax vaccine development, and suggest that future malaria vaccine studies should be adapted according to the target Plasmodium spp.
doi:10.1371/journal.pntd.0005031
PMCID: PMC5145136  PMID: 27930660
4.  Safety and Reproducibility of a Clinical Trial System Using Induced Blood Stage Plasmodium vivax Infection and Its Potential as a Model to Evaluate Malaria Transmission 
PLoS Neglected Tropical Diseases  2016;10(12):e0005139.
Background
Interventions to interrupt transmission of malaria from humans to mosquitoes represent an appealing approach to assist malaria elimination. A limitation has been the lack of systems to test the efficacy of such interventions before proceeding to efficacy trials in the field. We have previously demonstrated the feasibility of induced blood stage malaria (IBSM) infection with Plasmodium vivax. In this study, we report further validation of the IBSM model, and its evaluation for assessment of transmission of P. vivax to Anopheles stephensi mosquitoes.
Methods
Six healthy subjects (three cohorts, n = 2 per cohort) were infected with P. vivax by inoculation with parasitized erythrocytes. Parasite growth was monitored by quantitative PCR, and gametocytemia by quantitative reverse transcriptase PCR (qRT-PCR) for the mRNA pvs25. Parasite multiplication rate (PMR) and size of inoculum were calculated by linear regression. Mosquito transmission studies were undertaken by direct and membrane feeding assays over 3 days prior to commencement of antimalarial treatment, and midguts of blood fed mosquitoes dissected and checked for presence of oocysts after 7–9 days.
Results
The clinical course and parasitemia were consistent across cohorts, with all subjects developing mild to moderate symptoms of malaria. No serious adverse events were reported. Asymptomatic elevated liver function tests were detected in four of six subjects; these resolved without treatment. Direct feeding of mosquitoes was well tolerated. The estimated PMR was 9.9 fold per cycle. Low prevalence of mosquito infection was observed (1.8%; n = 32/1801) from both direct (4.5%; n = 20/411) and membrane (0.9%; n = 12/1360) feeds.
Conclusion
The P. vivax IBSM model proved safe and reliable. The clinical course and PMR were reproducible when compared with the previous study using this model. The IBSM model presented in this report shows promise as a system to test transmission-blocking interventions. Further work is required to validate transmission and increase its prevalence.
Trial Registration
Anzctr.org.au ACTRN12613001008718
Author Summary
Blocking the transmission of malaria from infected individuals to mosquitoes is an appealing approach to malaria elimination. However, at present there is no reliable experimental model to test the efficacy of transmission blocking interventions. In this study, we assessed the safety and reproducibility of our clinical trial model, in which we inject blood cells infected with malaria parasites into healthy volunteers. Furthermore, we tested if our clinical trial model could be used as a tool to evaluate malaria transmission. We infected healthy volunteers with Plasmodium vivax parasites and monitored parasite growth by molecular methods. When we detected the parasite stage that is infective to mosquitoes (the sexual stage), blood from infected volunteers was fed to mosquitoes. Then, we investigated the presence of parasites in the midgut of mosquitoes. The results from this study show that our clinical trial model is safe and reproducible. Moreover, we observed low levels of transmission of the malaria parasite from infected volunteers to mosquitoes. We need to validate this finding and to optimize it to increase the rate of malaria transmission. Altogether, our clinical trial model seems to be a reliable system to assess interventions to block malaria transmission, which has enormous public health significance.
doi:10.1371/journal.pntd.0005139
PMCID: PMC5145139  PMID: 27930652
5.  Type I Interferons Regulate Immune Responses in Humans with Blood-Stage Plasmodium falciparum Infection 
Cell reports  2016;17(2):399-412.
Summary
The development of immunoregulatory networks is important to prevent disease. However, these same networks allow pathogens to persist and reduce vaccine efficacy. Here, we identify type I interferons (IFNs) as important regulators in developing anti-parasitic immunity in healthy volunteers infected for the first time with Plasmodium falciparum. Type I IFNs suppressed innate immune cell function and parasitic-specific CD4+ T cell IFNγ production, and they promoted the development of parasitic-specific IL-10-producing Th1 (Tr1) cells. Type I IFN-dependent, parasite-specific IL-10 production was also observed in P. falciparum malaria patients in the field following chemoprophylaxis. Parasite-induced IL-10 suppressed inflammatory cytokine production, and IL-10 levels after drug treatment were positively associated with parasite burdens before anti-parasitic drug administration. These findings have important implications for understanding the development of host immune responses following blood-stage P. falciparum infection, and they identify type I IFNs and related signaling pathways as potential targets for therapies or vaccine efficacy improvement.
doi:10.1016/j.celrep.2016.09.015
PMCID: PMC5082731  PMID: 27705789
6.  Profoundly Reduced CD1c+ Myeloid Dendritic Cell HLA-DR and CD86 Expression and Increased Tumor Necrosis Factor Production in Experimental Human Blood-Stage Malaria Infection 
Infection and Immunity  2016;84(5):1403-1412.
Dendritic cells (DCs) are sentinels of the immune system that uniquely prime naive cells and initiate adaptive immune responses. CD1c (BDCA-1) myeloid DCs (CD1c+ mDCs) highly express HLA-DR, have a broad Toll-like receptor (TLR) repertoire, and secrete immune modulatory cytokines. To better understand immune responses to malaria, CD1c+ mDC maturation and cytokine production were examined in healthy volunteers before and after experimental intravenous Plasmodium falciparum infection with 150- or 1,800-parasite-infected red blood cells (pRBCs). After either dose, CD1c+ mDCs significantly reduced HLA-DR expression in prepatent infections. Circulating CD1c+ mDCs did not upregulate HLA-DR after pRBC or TLR ligand stimulation and exhibited reduced CD86 expression. At peak parasitemia, CD1c+ mDCs produced significantly more tumor necrosis factor (TNF), whereas interleukin-12 (IL-12) production was unchanged. Interestingly, only the 1,800-pRBC dose caused a reduction in the circulating CD1c+ mDC count with evidence of apoptosis. The 1,800-pRBC dose produced no change in T cell IFN-γ or IL-2 production at peak parasitemia or at 3 weeks posttreatment. Overall, CD1c+ mDCs are compromised by P. falciparum exposure, with impaired HLA-DR and CD86 expression, and have an increased capacity for TNF but not IL-12 production. A first prepatent P. falciparum infection is sufficient to modulate CD1c+ mDC responsiveness, likely contributing to hampered effector T cell cytokine responses and assisting parasite immune evasion.
doi:10.1128/IAI.01522-15
PMCID: PMC4862702  PMID: 26902728
7.  Prevalence and Predictors of Poor Recovery from Mild Traumatic Brain Injury 
Journal of Neurotrauma  2015;32(19):1488-1496.
Abstract
Although most patients with mild traumatic brain injury (mTBI) recover within 3 months, a subgroup of patients experience persistent symptoms. Yet, the prevalence and predictors of persistent dysfunction in patients with mTBI remain poorly understood. In a longitudinal study, we evaluated predictors of symptomatic and cognitive dysfunction in adolescents and young adults with mTBI, compared with two control groups—patients with orthopedic injuries and healthy uninjured individuals. Outcomes were assessed at 3 months post-injury. Poor symptomatic outcome was defined as exhibiting a symptom score higher than 90% of the orthopedic control (OC) group, and poor cognitive outcome was defined as exhibiting cognitive performance poorer than 90% of the OC group. At 3 months post-injury, more than half of the patients with mTBI (52%) exhibited persistently elevated symptoms, and more than a third (36.4%) exhibited poor cognitive outcome. The rate of high symptom report in mTBI was markedly greater than that of typically developing (13%) and OC (17%) groups; the proportion of those with poor cognitive performance in the mTBI group exceeded that of typically developing controls (15.8%), but was similar to that of the OC group (34.9%). Older age at injury, female sex, and acute symptom report were predictors of poor symptomatic outcome at 3 months. Socioeconomic status was the only significant predictor of poor cognitive outcome at 3 months.
doi:10.1089/neu.2014.3555
PMCID: PMC4702434  PMID: 25970233
cognitive function; human studies; recovery; traumatic brain injury
8.  Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production 
PLoS Pathogens  2016;12(9):e1005839.
Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense.
Trial Registration
ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752
Author Summary
Malaria is one of the three most deadly infectious disease worldwide, together with tuberculosis and HIV. The exact mechanisms underlying effective immunity to malaria remain largely unknown and there is no reliable immune correlate of protection. Here, we take advantage of a unique experimental human infection model to define the immune response to primary exposure of blood-stage malaria parasites in naïve healthy volunteers at the molecular level. We report that Plasmodium parasite levels were inversely correlated to the expansion of a specific subset of CD4+ T cells expressing the activation molecule CD38 and a very unusual phenotype. Although the expansion of CD38+ CD4+ T cells has been described in several viral and bacterial infections, we show for the first time that these cells are associated with a ‘naive-like’ effector phenotype, higher cytolytic potential and a strongly impaired ability to produce IFN-γ and other cytokines. Importantly, this subset of CD38+ CD4+ T cells could be also identified in all healthy volunteers prior to infection, suggesting that these core characteristics of circulating CD38+ CD4+ T cells are independent of active infection and may play an important role in the immune control of other pathogens.
doi:10.1371/journal.ppat.1005839
PMCID: PMC5035011  PMID: 27662621
9.  A Phase II pilot trial to evaluate safety and efficacy of ferroquine against early Plasmodium falciparum in an induced blood-stage malaria infection study 
Malaria Journal  2016;15(1):469.
Background
Ferroquine (SSR97193) is a candidate anti-malarial currently undergoing clinical trials for malaria. To better understand its pharmacokinetic (PK) and pharmacodynamic (PD) parameters the compound was tested in the experimentally induced blood stage malaria infection model in volunteers.
Methods
Male and non-pregnant female aged 18–50 years were screened for this phase II, controlled, single-centre clinical trial. Subjects were inoculated with ~1800 viable Plasmodium falciparum 3D7A-infected human erythrocytes, and treated with a single-dose of 800 mg ferroquine. Blood samples were taken at defined time-points to measure PK and PD parameters. The blood concentration of ferroquine and its active metabolite, SSR97213, were measured on dry blood spot samples by ultra-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Parasitaemia and emergence of gametocytes were monitored by quantitative PCR. Safety was determined by recording adverse events and monitoring clinical laboratory assessments during the course of the study.
Results
Eight subjects were enrolled into the study, inoculated with infected erythrocytes and treated with 800 mg ferroquine. Ferroquine was rapidly absorbed with maximal exposure after 4–8 and 4–12 h exposure for SSR97213. Non-compartmental PK analysis resulted in estimates for half-lives of 10.9 and 23.8 days for ferroquine and SSR97213, respectively. Parasite clearance as reported by parasite reduction ratio was 162.9 (95 % CI 141–188) corresponding to a parasite clearance half-life of 6.5 h (95 % CI: 6.4–6.7 h). PK/PD modelling resulted in a predicted minimal parasiticidal concentration of 20 ng/mL, and the single dosing tested in this study was predicted to maintain an exposure above this threshold for 454 h (37.8 days). Although ferroquine was overall well tolerated, transient elevated transaminase levels were observed in three subjects. Paracetamol was the only concomitant treatment among the two out of these three subjects that may have played a role in the elevated transaminases levels. No clinically significant ECG abnormalities were observed.
Conclusions
The parameters and PK/PD model derived from this study pave the way to the further rational development of ferroquine as an anti-malarial partner drug. The safety of ferroquine has to be further explored in controlled human trials.
Trial registration anzctr.org.au (registration number: ACTRN12613001040752), registered 18/09/2013
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-016-1511-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12936-016-1511-3
PMCID: PMC5022189  PMID: 27624471
Induced blood-stage malaria (IBSM); Drug discovery; Ferroquine; Malaria; Plasmodium falciparum; Pharmacokinetic/pharmacodynamic modelling; Translational models; SSR97193; Ferrochloroquine
10.  Systematic Review and Operative Technique of Recalcitrant Pressure Ulcers Using a Fillet Flap Technique 
Background:
The purpose of this article is to describe the indications, operative technique, outcomes, and systematic review of the literature on the reconstruction of patients with end-stage pressure ulcers using a fillet flap technique. In this technique, the femur, tibia, and fibula are removed from the thigh and leg, and the soft tissue is used as a pedicled, or free, myocutaneous flap for reconstruction. Long-term outcomes, salient surgical technique of flap elevation, and design are detailed for patients who had a fillet of leg flap for reconstruction of extensive pressure ulcers.
Methods:
The indications, surgical technique, and postoperative outcomes of 5 patients who had pedicled fillet flaps are reviewed including patient age, sex, underlying comorbidities, duration of paraplegia, operative technique, and complications. A systematic review of the literature was performed searching PubMed, Cochrane Database, and Medline with the following MeSH terms: pressure ulcer, pressure sore, decubitus ulcer, fillet flap, and fillet flap. Inclusion criteria were use of a fillet technique, article data on the number of reconstructions before fillet flap, complications, and English language.
Results:
Most of our patients were male 75% (n = 3) with an average age of 47.5 years, had been paralyzed for an average of 16 years, and had few medical comorbidities. Two patients (3 flaps) required hip disarticulation, 1 patient had a bilateral fillet flaps, and 3 patients had resection of tibia/fibula. After following patients for an average of 1.4 years (4 mo to 2 yr), complications were limited to 1 patient who had partial-thickness flap loss at the distal skin flap that healed by secondary intention and 1 patient who had ulcer recurrence because of noncompliance. Four articles met inclusion criteria for systematic review and 3 were excluded.
Conclusions:
The fillet of leg flap remains a useful and reliable method of reconstructing end-stage pressure ulcers.
doi:10.1097/GOX.0000000000001001
PMCID: PMC5010346  PMID: 27622082
11.  Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons 
Nature protocols  2016;11(3):499-524.
A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.
doi:10.1038/nprot.2016.015
PMCID: PMC4941947  PMID: 26890679
12.  Hyaluronan stimulates ex vivo B lymphocyte chemotaxis and cytokine production in a murine model of fungal allergic asthma 
Immunobiology  2015;220(7):899-909.
Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive eosinophilic and lymphocytic inflammation with associated changes in the extracellular matrix (ECM) resulting in airway wall remodeling. Hyaluronan (HA) is a nonsulfated glycosaminoglycan ECM component that functions as a structural cushion in its high molecular mass (HMM) but has been implicated in metastasis and other disease processes when it is degraded to smaller fragments. However, relatively little is known about the role HA in mediating inflammatory responses in allergy and asthma. In the present study, we used a murine Aspergillus fumigatus inhalational model to mimic human disease. After observing in vivo that a robust B cell recruitment followed a massive eosinophilic egress to the lumen of the allergic lung and corresponded with the detection of low molecular mass HA (LMM HA), we examined the effect of HA on B cell chemotaxis and cytokine production in the ex vivo studies. We found that LMM HA functioned through a CD44-mediated mechanism to elicit chemotaxis of B lymphocytes, while high molecular mass HA (HMM HA) had little effect. LMM HA, but not HMM HA, also elicited the production of IL-10 and TGF-β1 in these cells. Taken together, these findings demonstrate a critical role for ECM components in mediating leukocyte migration and function which are critical to the maintenance of allergic inflammatory responses.
doi:10.1016/j.imbio.2015.01.011
PMCID: PMC4441586  PMID: 25698348
Hyaluronan; Aspergillus fumigatus; B lymphocytes
13.  Endangered Right Whales Enhance Primary Productivity in the Bay of Fundy 
PLoS ONE  2016;11(6):e0156553.
Marine mammals have recently been documented as important facilitators of rapid and efficient nutrient recycling in coastal and offshore waters. Whales enhance phytoplankton nutrition by releasing fecal plumes near the surface after feeding and by migrating from highly productive, high-latitude feeding areas to low-latitude nutrient-poor calving areas. In this study, we measured NH4+ and PO43- release rates from the feces of North Atlantic right whales (Eubalaena glacialis), a highly endangered baleen whale. Samples for this species were primarily collected by locating aggregations of whales in surface-active groups (SAGs), which typically consist of a central female surrounded by males competing for sexual activity. When freshly collected feces were incubated in seawater, high initial rates of N release were generally observed, which decreased to near zero within 24 hours of sampling, a pattern that is consistent with the active role of gut microflora on fecal particles. We estimate that at least 10% of particulate N in whale feces becomes available as NH4+ within 24 hours of defecation. Phosphorous was also abundant in fecal samples: initial release rates of PO43- were higher than for NH4+, yielding low N/P nutrient ratios over the course of our experiments. The rate of PO43- release was thus more than sufficient to preclude the possibility that nitrogenous nutrients supplied by whales would lead to phytoplankton production limited by P availability. Phytoplankton growth experiments indicated that NH4+ released from whale feces enhance productivity, as would be expected, with no evidence that fecal metabolites suppress growth. Although North Atlantic right whales are currently rare (approximately 450 individuals), they once numbered about 14,000 and likely played a substantial role in recycling nutrients in areas where they gathered to feed and mate. Even though the NH4+ released from fresh whale fecal material is a small fraction of total whale fecal nitrogen, and recognizing the fact that the additional nitrogen released in whale urine would be difficult to measure in a field study, the results of this study support the idea that the distinctive isotopic signature of the released NH4+ could be used to provide a conservative estimate of the contribution of the whale pump to primary productivity in coastal regions where whales congregate.
doi:10.1371/journal.pone.0156553
PMCID: PMC4917091  PMID: 27331902
14.  Efficacy of OZ439 (artefenomel) against early Plasmodium falciparum blood-stage malaria infection in healthy volunteers 
Objectives
OZ439, or artefenomel, is an investigational synthetic ozonide antimalarial with similar potency, but a significantly improved pharmacokinetic profile, compared with artemisinins. We wished to measure key pharmacokinetic and pharmacodynamic parameters and the pharmacokinetic/pharmacodynamic relationship of artefenomel in humans to guide the drug's further development as combination therapy in patients.
Patients and methods
We tested artefenomel in the human induced blood-stage malaria (IBSM) model. Plasmodium infection was monitored by quantitative PCR (qPCR) and upon reaching 1000 parasites/mL single doses of 100, 200 and 500 mg of artefenomel were administered orally with evaluation of drug exposure and parasitaemia until rescue treatment after 16 days or earlier, if required.
Results
A single 100 mg dose had only a transient effect, while the 200 mg dose resulted in a significant reduction in parasitaemia before early recrudescence. At the highest (500 mg) dose, initial clearance of parasites below the limit of detection of qPCR was observed, with a 48 h parasite reduction ratio (PRR48) >10 000 and a parasite clearance half-life of 3.6 h (95% CI 3.4–3.8 h). However, at this dose, recrudescence was seen in four of eight subjects 6–10 days after treatment. Pharmacokinetic/pharmacodynamic modelling predicted an MIC of 4.1 ng/mL.
Conclusions
These results confirm the antimalarial potential of artefenomel for use in a single-exposure combination therapy. The observations from this study support and will assist further clinical development of artefenomel.
doi:10.1093/jac/dkw174
PMCID: PMC4992851  PMID: 27272721
15.  Linking Murine and Human Plasmodium falciparum Challenge Models in a Translational Path for Antimalarial Drug Development 
Effective progression of candidate antimalarials is dependent on optimal dosing in clinical studies, which is determined by a sound understanding of pharmacokinetics and pharmacodynamics (PK/PD). Recently, two important translational models for antimalarials have been developed: the NOD/SCID/IL2Rγ−/− (NSG) model, whereby mice are engrafted with noninfected and Plasmodium falciparum-infected human erythrocytes, and the induced blood-stage malaria (IBSM) model in human volunteers. The antimalarial mefloquine was used to directly measure the PK/PD in both models, which were compared to previously published trial data for malaria patients. The clinical part was a single-center, controlled study using a blood-stage Plasmodium falciparum challenge inoculum in volunteers to characterize the effectiveness of mefloquine against early malaria. The study was conducted in three cohorts (n = 8 each) using different doses of mefloquine. The characteristic delay in onset of action of about 24 h was seen in both NSG and IBSM systems. In vivo 50% inhibitory concentrations (IC50s) were estimated at 2.0 μg/ml and 1.8 μg/ml in the NSG and IBSM models, respectively, aligning with 1.8 μg/ml reported previously for patients. In the IBSM model, the parasite reduction ratios were 157 and 195 for the 10- and 15-mg/kg doses, within the range of previously reported clinical data for patients but significantly lower than observed in the mouse model. Linking mouse and human challenge models to clinical trial data can accelerate the accrual of critical data on antimalarial drug activity. Such data can guide large clinical trials required for development of urgently needed novel antimalarial combinations. (This trial was registered at the Australian New Zealand Clinical Trials Registry [http://anzctr.org.au] under registration number ACTRN12612000323820.)
doi:10.1128/AAC.02883-15
PMCID: PMC4879391  PMID: 27044554
16.  Complexities and Perplexities: A Critical Appraisal of the Evidence for Soil-Transmitted Helminth Infection-Related Morbidity 
PLoS Neglected Tropical Diseases  2016;10(5):e0004566.
Background: Soil-transmitted helminths (STH) have acute and chronic manifestations, and can result in lifetime morbidity. Disease burden is difficult to quantify, yet quantitative evidence is required to justify large-scale deworming programmes. A recent Cochrane systematic review, which influences Global Burden of Disease (GBD) estimates for STH, has again called into question the evidence for deworming benefit on morbidity due to STH. In this narrative review, we investigate in detail what the shortfalls in evidence are. Methodology/Principal Findings: We systematically reviewed recent literature that used direct measures to investigate morbidity from STH and we critically appraised systematic reviews, particularly the most recent Cochrane systematic review investigating deworming impact on morbidity. We included six systematic reviews and meta-analyses, 36 literature reviews, 44 experimental or observational studies, and five case series. We highlight where evidence is insufficient and where research needs to be directed to strengthen morbidity evidence, ideally to prove benefits of deworming. Conclusions/Significance: Overall, the Cochrane systematic review and recent studies indicate major shortfalls in evidence for direct morbidity. However, it is questionable whether the systematic review methodology should be applied to STH due to heterogeneity of the prevalence of different species in each setting. Urgent investment in studies powered to detect direct morbidity effects due to STH is required.
doi:10.1371/journal.pntd.0004566
PMCID: PMC4873196  PMID: 27196100
17.  Pharmacokinetic/pharmacodynamic modelling of the antimalarial effect of Actelion‐451840 in an induced blood stage malaria study in healthy subjects 
Aims
The aim of this study was to use data from an experimental induced blood stage malaria clinical trial to characterize the antimalarial activity of the new compound Actelion‐451840 using pharmacokinetic/pharmacodynamic (PK/PD) modelling. Then, using simulations from the model, the dose and dosing regimen necessary to achieve cure of infection were derived.
Methods
Eight healthy male subjects were infected with blood stage P. falciparum. After 7 days, a single dose of 500 mg of Actelion‐451840 was administered under fed conditions. Parasite and drug concentrations were sampled frequently. Parasite growth and the relation to drug exposure were estimated using PK/PD modelling. Simulations were then undertaken to derive estimates of the likelihood of achieving cure in different scenarios.
Results
Actelion‐451840 was safe and well tolerated. Single dose treatment markedly reduced the level of P. falciparum parasitaemia, with a weighted average parasite reduction rate of 73.6 (95% CI 56.1, 96.5) and parasite clearance half‐life of 7.7 h (95% CI 7.3, 8.3). A two compartment PK/PD model with a steep concentration−kill effect predicted maximum effect with a sustained concentration of 10–15 ng ml−1 and cure achieved in 90% of subjects with six once daily doses of 300 mg once daily.
Conclusions
Actelion‐451840 shows clinical efficacy against P. falciparum infections. The PK/PD model developed from a single proof‐of‐concept study with eight healthy subjects enabled prediction of therapeutic effects, with cure rates with seven daily doses predicted to be equivalent to artesunate monotherapy. Larger doses or more frequent dosing are not predicted to achieve more rapid cure.
doi:10.1111/bcp.12962
PMCID: PMC4972157  PMID: 27062080
challenge model; malaria; pharmacodynamics; pharmacokinetics; Plasmodium falciparum; proof of concept
18.  Piperaquine Monotherapy of Drug-Susceptible Plasmodium falciparum Infection Results in Rapid Clearance of Parasitemia but Is Followed by the Appearance of Gametocytemia 
The Journal of Infectious Diseases  2016;214(1):105-113.
Background. Piperaquine, coformulated with dihydroartemisinin, is a component of a widely used artemisinin combination therapy. There is a paucity of data on its antimalarial activity as a single agent. Such data, if available, would inform selection of new coformulations.
Methods. We undertook a study in healthy subjects, using the induced blood stage malaria (IBSM) model to test the antimalarial activity of single doses of piperaquine (960, 640, and 480 mg) in 3 cohorts. In a pilot study in the third cohort, gametocyte clearance following administration of 15 mg, or 45 mg or no primaquine was investigated.
Results. Parasite clearance over the 48-hour period after piperaquine administration was more rapid in the 960 mg cohort, compared with the 640 mg cohort (parasite reduction ratio, 2951 [95% confidence interval {CI}, 1520–5728] vs 586 [95% CI, 351–978]; P < .001). All 24 subjects developed gametocytemia as determined by pfs25 transcripts. Clearance of pfs25 was significantly faster in those receiving primaquine than in those not receiving primaquine (P < .001).
Conclusions. Piperaquine possesses rapid parasite-clearing activity, but monotherapy is followed by the appearance of gametocytemia, which could facilitate the spread of malaria. This new information should be taken into account when developing future antimalarial coformulations.
Clinical Trials Registration ACTRN12613000565741.
doi:10.1093/infdis/jiw128
PMCID: PMC4907420  PMID: 27056954
P. falciparum; malaria; piperaquine clinical trial; gametocytemia
19.  Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design 
PLoS Neglected Tropical Diseases  2016;10(3):e0004578.
Background
The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.
Methodology/Principal Findings
Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay.
Conclusions/Significance
The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.
Author Summary
With a growing emphasis on the mapping and elimination of soil transmitted helminth (STH) infections, the need for optimal and specific diagnostic methods is increasing. While PCR-based diagnostic methods for the detection of these parasitic organisms exist, these assays make use of sub-optimal target sequences. By designing assays that target non-coding, high copy-number repetitive sequences, both the limit of detection and species-specificity of detection improve. Using next-generation sequencing technology, we have identified high copy-number repeats for a series of STH species responsible for the greatest burden of disease. Using these repetitive sequences as targets in the design of novel real-time PCR assays, we have improved both the limits of detection and species-specificity of detection, and we have demonstrated this improved detection by testing these assays against an established PCR-based diagnostic methodology. Accordingly, these assays should facilitate mapping and monitoring efforts, and the generalized application of this approach to assay design should improve detection efforts for other eukaryotic pathogens.
doi:10.1371/journal.pntd.0004578
PMCID: PMC4814118  PMID: 27027771
20.  Significance of the NOR1-FOXA1/HDAC2-Slug regulatory network in epithelial-mesenchymal transition of tumor cells 
Oncotarget  2016;7(13):16745-16759.
The epithelial-mesenchymal transition (EMT) process is believed to play a crucial role in nasopharyngeal carcinoma (NPC) progression, a squamous cell carcinoma of the head and neck with the tendency to metastasize early. At present, much attention has been given to the inducer of EMT involved in NPC progression, while antagonists have been less intensively characterized. In this study, unbiased analysis of EMT-associated gene expression patterns was performed using data mining of global gene expression profiles derived from NPC samples, leading to the successful identification of NOR1, FOXA1, and Slug, all of which showed aberrant expression during NPC progression. The effect of tumor suppressor NOR1 on Slug-induced NPC cells during the EMT process was investigated by use of ectopic expression and RNA interference methods. The molecular mechanisms underlying the tumor-suppressing effect of NOR1 on Slug-induced EMT were thought to be dependent on the cooperation of NOR1 with the FOXA1-HDAC2 complex. We also showed that FOXA1 and HDAC2 bind the slug promoter and directly repress its transcription. Our data revealed a previously unrecognized role of the NOR1-FOXA1/HDAC2-Slug network in the regulation of the EMT process and aggressiveness of NPC.
doi:10.18632/oncotarget.7778
PMCID: PMC4941348  PMID: 26934447
epithelial-mesenchymal transition; nasopharyngeal carcinoma; FOXA1; NOR1
22.  Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings 
Malaria Journal  2016;15:88.
As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral flow rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination.
doi:10.1186/s12936-016-1158-0
PMCID: PMC4754967  PMID: 26879936
Malaria elimination; High-throughput; Molecular diagnostics; LAMP; PCR; Field application
23.  Artesunate–mefloquine versus chloroquine for treatment of uncomplicated Plasmodium knowlesi malaria in Malaysia (ACT KNOW): an open-label, randomised controlled trial 
The Lancet. Infectious diseases  2015;16(2):180-188.
Summary
Background
The zoonotic parasite Plasmodium knowlesi has become the most common cause of human malaria in Malaysia and is present throughout much of southeast Asia. No randomised controlled trials have been done to identify the optimum treatment for this emerging infection. We aimed to compare artesunate–mefloquine with chloroquine to define the optimum treatment for uncomplicated P knowlesi malaria in adults and children.
Methods
We did this open-label, randomised controlled trial at three district hospitals in Sabah, Malaysia. Patients aged 1 year or older with uncomplicated P knowlesi malaria were randomly assigned, via computer-generated block randomisation (block sizes of 20), to receive oral artesunate–mefloquine (target dose 12 mg/kg artesunate and 25 mg/kg mefloquine) or chloroquine (target dose 25 mg/kg). Research nursing staff were aware of group allocation, but allocation was concealed from the microscopists responsible for determination of the primary endpoint, and study participants were not aware of drug allocation. The primary endpoint was parasite clearance at 24 h. Analysis was by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT01708876.
Findings
Between Oct 16, 2012, and Dec 13, 2014, we randomly assigned 252 patients to receive either artesunate–mefloquine (n=127) or chloroquine (n=125); 226 (90%) patients comprised the modified intention-to-treat population. 24 h after treatment, we recorded parasite clearance in 97 (84% [95% CI 76–91]) of 115 patients in the artesunate–mefloquine group versus 61 (55% [45–64]) of 111 patients in the chloroquine group (difference in proportion 29% [95% CI 18·0–40·8]; p<0·0001). Parasite clearance was faster in patients given artesunate–mefloquine than in those given chloroquine (18·0 h [range 6·0–48·0] vs 24·0 h [6·0–60·0]; p<0·0001), with faster clearance of ring stages in the artesunate–mefloquine group (mean time to 50% clearance of baseline parasites 8·6 h [95% CI 7·9–9·4] vs 13·8 h [12·1–15·4]; p<0·0001). Risk of anaemia within 28 days was lower in patients in the artesunate–mefloquine group (71 [62%; 95% CI 52·2–70·6]) than in those in the chloroquine group (83 [75%; 65·6–82·5]; p=0·035). Gametocytaemia as detected by PCR for pks25 was present in 44 (86%) of 51 patients in the artesunate–mefloquine group and 41 (84%) of 49 patients in the chloroquine group at baseline, and in three (6%) of 49 patients and two (4%) of 48 patients, respectively, at day 7. Fever clearance was faster in the artesunate–mefloquine group (mean 11·5 h [95% CI 8·3–14·6]) than in the chloroquine group (14·8 h [11·7–17·8]; p=0·034). Bed occupancy was 2426 days per 1000 patients in the artesunate–mefloquine group versus 2828 days per 1000 patients in the chloroquine group (incidence rate ratio 0·858 [95% CI 0·812–0·906]; p<0·0001). One (<1%) patient in the artesunate–mefloquine group had a serious neuropsychiatric event regarded as probably related to study drug.
Interpretation
Artesunate–mefloquine is highly efficacious for treatment of uncomplicated P knowlesi malaria. The rapid therapeutic response of the drug offers significant advantages compared with chloroquine monotherapy and supports a unified treatment policy for artemisinin-based combination therapy for all Plasmodium species in co-endemic areas.
Funding
Malaysian Ministry of Health, Australian National Health and Medical Research Council, and Asia Pacific Malaria Elimination Network.
doi:10.1016/S1473-3099(15)00415-6
PMCID: PMC4753673  PMID: 26603174
24.  Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination 
PLoS Neglected Tropical Diseases  2016;10(2):e0004443.
Introduction
Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority.
Methods
A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.
Results
The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87–99%); 61/64), and specificity of 100% (95% CI 86–100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29–96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).
Conclusion
This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.
Author Summary
Plasmodium vivax has a worldwide distribution and is the second most common causative agent of human malaria. The dormant liver stage of P. vivax allows the infection to recur unless diagnosed and treated appropriately, which poses a significant challenge to the goals of malaria elimination and eradication as outlined by the WHO. Although highly sensitive molecular diagnostic tools are available in reference laboratory settings, the currently available diagnostic tools outside referral settings for the detection of P. vivax are limited to microscopy and rapid diagnostic tests, which are insufficiently sensitive for the detection of low level parasitemia particularly in asymptomatic individuals. Based on a DNA amplification technology called loop-mediated isothermal amplification (LAMP), this study describes the development and validation of a colourimetric, high throughput assay (HtLAMP) suitable for the detection of P. vivax infection in non-referral settings. The sensitivity of the assay combined with its field applicability offers the potential for it to play an important role as a diagnostic tool for the purpose of malaria elimination.
doi:10.1371/journal.pntd.0004443
PMCID: PMC4752294  PMID: 26870958
25.  Thromboelastography in Patients with Acute Ischemic Stroke 
Background
Thromboelastography (TEG) measures the dynamics of coagulation. There are limited data about TEG in acute ischemic stroke other than a single study from 1974 suggesting that acute ischemic stroke patients are hypercoagulable. There have been no studies of TEG in the thrombolytic era despite its potential usefulness as a measure of clot lysis. This study was designed to provide initial TEG data in stroke patients before and after tissue plasminogen activator (tPA) therapy, and to provide the necessary preliminary data for further study of TEG’s ability to identify clot subtype and predict response to tPA therapy.
Methods
All acute ischemic stroke patients presenting between 11/2009 and 2/2011 eligible for tPA therapy were screened and 56 enrolled. Blood was drawn before (52 patients) and 10 minutes after tPA bolus (30 patients). Demographics, vitals, labs, 24hr National Institutes of Health Stroke Scale (NIHSS) and computed tomography (CT) scan results were collected. Patients were compared to normal controls.
Results
Acute ischemic stroke patients had shorter R (4.8±1.5 vs 6.0 ±1.7 min, p =0.0004), greater a-Angle (65.0±7.6 vs 61.5 ± 5.9°, p =0.01), and shorter K (1.7 ±0.7 vs 2.1 ±0.7 min, p =0.002) indicating faster clotting. Additionally, a subset formed clots with stronger platelet-fibrin matrices. Treatment with tPA resulted in reduction in all indices of clot strength (LY30=0(0–0.4) vs 94.4 (15.2–95.3) p<0.0001), however there was considerable variability in response.
Conclusions
TEG demonstrates that many acute ischemic stroke patients are hypercoaguable. TEG values reflect variable clot subtype and response to tPA. Further study based on these data will determine if TEG is useful for measuring the dynamic aspects of clot formation and monitoring lytic therapy.
doi:10.1111/j.1747-4949.2012.00919.x
PMCID: PMC3535538  PMID: 23017088
cerebral infarction; ischaemic stroke; thrombolysis; stroke; tPA; haemocoagulation

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