Functional and immunological mapping of domains of the reticulocyte binding protein, Plasmodium vivax PvRBP2a.

We previously described a novel Plasmodium vivax invasion mechanism into human reticulocytes via the PvRBP2a-CD98 receptor-ligand pair. We assessed the PvRBP2a epitopes involved in CD98 binding and recognised by antibodies from infected patients using linear epitope mapping. We identified two epitope clusters mediating PvRBP2a-CD98 interaction. One cluster named cluster B (PvRBP2a431-448, TAALKEKGKLLANLYNKL) was the target of antibody responses in P. vivax-infected humans. Peptides from each cluster were able to prevent live parasite invasion of human reticulocytes. These results provide new insights for development of a malaria blood stage vaccine against P. vivax.

Can a multi-level intervention approach, combining behavioural disciplines, novel technology and incentives increase physical activity at population-level?

BACKGROUND: Despite a global call for action and growing burden of non-communicable diseases (NCD) associated with physical inactivity, effective interventions to increase community-wide physical activity (PA) remain few. NCDs accounted for 80% of Singapore's disease burden (2015) and yet 40% of Singaporeans did not meet minimum recommended weekly PA despite evidence of the benefits to cardiorespiratory health, diabetes and cancer prevention. METHODS: A large-scale public health intervention was initiated in 2015 to increase population-level PA through incidental daily walking. Intervention components included fitness trackers, redeemable rewards and gamification, implemented in a mutually-reinforcing manner within an eco-system supportive of PA and informed by real-time data analytics. Mean daily step count at baseline and post-intervention were compared across periods, and the influence of participant sub-groups characteristics on overall results, using significance tests. Standards for Reporting on Implementation Studies (StaRI) were adhered to. RESULTS: Intervention reach increased fourfold from 129,677 participants in wave 1 (2015-16) to 690,233 in wave 3 (2017-18) amounting to a total of 1,184,410 Step Challenge participations. Mean days of fitness tracker use increased from 2.4 to 5.0 days/week among participants completing the Challenge in wave 1 and from 5.3 to 6.0 days/week in wave 3. The mean number of daily steps between pre-Challenge and Challenge periods increased by 4163 (sd=1360; p< 0.001) in wave 1, by 2242 (sd=334; p< 0.001) in wave 2 and by 1645 steps/day (sd=54; p< 0.001) in wave 3. Mean daily step increases between wave 1 and 3 also suggest that incidental PA was maintained, a finding supported by a 2017 national population survey showing that incidental PA among adults increased from 5% in 2010 to 14% in 2017 while moderate-intensity PA increased from 5 to 10% over the same period. CONCLUSION: Population-level PA was effectively increased through multi-level interventions integrating technology, behavioural economics, gamification, marketing, communications and community linkages within a supportive context- and climate-appropriate environment. Responsive data analytics were instrumental to strengthen implementation by tailoring modalities that maximise effectiveness at population-level. Further analyses are needed to explore potential barriers, challenges or unmet needs in sub-groups with lower uptake to tailor future interventions for greater reach and impact.

Breadth of humoral response and antigenic targets of sporozoite-inhibitory antibodies associated with sterile protection induced by controlled human malaria infection.

The development of an effective malaria vaccine has remained elusive even until today. This is because of our incomplete understanding of the immune mechanisms that confer and/or correlate with protection. Human volunteers have been protected experimentally from a subsequent challenge by immunization with Plasmodium falciparum sporozoites under drug cover. Here, we demonstrate that sera from the protected individuals contain neutralizing antibodies against the pre-erythrocytic stage. To identify the antigen(s) recognized by these antibodies, a newly developed library of P. falciparum antigens was screened with the neutralizing sera. Antibodies from protected individuals recognized a broad antigenic repertoire of which three antigens, PfMAEBL, PfTRAP and PfSEA1 were recognized by most protected individuals. As a proof of principle, we demonstrated that anti-PfMAEBL antibodies block liver stage development in human hepatocytes. Thus, these antigens identified are promising targets for vaccine development against malaria.

Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance.

OBJECTIVES: Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a renewed place in the pharmacopoeia of modern medicine. The continued spread of drug-resistant Plasmodium vivax and Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on the Thai-Myanmar border where there are increasing rates of failure when treating vivax malaria with chloroquine. METHODS: To do this we used a newly developed ex vivo susceptibility assay utilizing flow cytometry and a portable flow cytometer with a near-UV laser. RESULTS: P. vivax (median methylene blue IC50 3.1 nM, IQR 1.7-4.3 nM) and P. falciparum (median methylene blue IC50 1.8 nM, IQR 1.6-2.3 nM) are susceptible to methylene blue treatment at physiologically relevant levels. Unfortunately, the addition of chloroquine to combination treatments with methylene blue significantly reduces the ex vivo effectiveness of this molecule. CONCLUSIONS: Our data support further efforts to employ methylene blue as a safe, low-cost antimalarial to treat drug-resistant malaria.

An amidation/cyclization approach to the synthesis of N-hydroxyquinolinones and their biological evaluation as potential anti-plasmodial, anti-bacterial, and iron(II)-chelating agents.

A 26-member library of novel N-hydroxyquinolinone derivatives was synthesized by a one-pot Buchwald-type palladium catalyzed amidation and condensation sequence. The design of these rare scaffolds was inspired from N-hydroxypyridones and 2-quinolinones classes of compounds which have been shown to have rich biological activities. The synthesized compounds were evaluated for their anti-plasmodial and anti-bacterial properties. In addition, these compounds were screened for their iron(II)-chelation properties. Notably, four of these compounds exhibited anti-plasmodial activities comparable to that of the natural product cordypyridone B.

Pre-operative indicators for mortality following hip fracture surgery: a systematic review and meta-analysis.

OBJECTIVE: hip fracture is a common and serious condition associated with high mortality. This study aimed to identify pre-operative characteristics which are associated with an increased risk of mortality after hip fracture surgery. DESIGN: systematic search of published and unpublished literature databases, including EMBASE, MEDLINE, AMED, CINAHL, PubMed and the Cochrane Library, was undertaken to identify all clinical studies on pre-operative predictors of mortality after surgery in hip fracture with at least 3-month follow-up. Data pertaining to the study objectives was extracted by two reviewers independently. Where study homogeneity was evidence, a meta-analysis of pooled relative risk and 95% confidence intervals was performed for mortality against pre-admission characteristics. RESULTS: fifty-three studies including 544,733 participants were included. Thirteen characteristics were identified as possible pre-operative indicators for mortality. Following meta-analysis, the four key characteristics associated with the risk of mortality up to 12 months were abnormal ECG (RR: 2.00; 95% CI: 1.45, 2.76), cognitive impairment (RR: 1.91; 95% CI: 1.35, 2.70), age >85 years (RR: 0.42; 95% CI: 0.20, 0.90) and pre-fracture mobility (RR: 0.13; 95% CI: 0.05, 0.34). Other statistically significant pre-fracture predictors of increased mortality were male gender, being resident in a care institution, intra-capsular fracture type, high ASA grade and high Charlson comorbidity score on admission. CONCLUSIONS: this review has identified the characteristics of patients with a high risk of mortality after a hip fracture surgery beyond the peri-operative period who may benefit from comprehensive assessment and appropriate management. PROSPERO REGISTRATION NUMBER: CRD42012002107.

Predictors of orthostatic hypotension in patients attending a transient ischaemic attack clinic: database study.

BACKGROUND: Orthostatic hypotension (OH) is common amongst the older population and is associated with morbidity and mortality. We sought to investigate predictors of OH to assist the clinician in identifying patients at risk. METHODS AND RESULTS: Database of 2696 patients attending a transient ischaemic attack (TIA) clinic between January 2006 and May 2009 was examined. Logistic regression models were constructed to determine clinical associates of OH. Demographics, co-morbidities, cardiovascular risk factors and medications were included in the multivariate models. Simple data mining models in the form of rule sets were developed for each component and they were assessed for predictive accuracy. The best models were validated on a smaller sample. Prevalence of OH was 22.3% in the TIA clinic population (50.6% men, mean 72 years; 49.4% women, mean 75 years). A significant postural drop in systolic blood pressure (BP) (≥ 20 mmHg) was more prevalent than a significant diastolic BP drop (≥ 10 mmHg). Isolated systolic hypertension was common (52.4%). Common factors predicting a significant systolic and diastolic BP fall were older age, previous TIA, being a current smoker, having diabetes and the use of beta-blockers. Both mean arterial and pulse pressure (MAP and PP) derived from supine BP were significantly associated with OH. CONCLUSIONS: OH should be assessed routinely in TIA clinics. MAP and PP may provide information on the predictability of OH.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

Pharmacological treatment of postprandial reductions in blood pressure: a systematic review.

OBJECTIVES: To systematically review the current literature on the pharmacological treatment of postmeal reductions in blood pressure (BP). DESIGN: A systematic literature search and standardized data collection of randomized controlled trials on the pharmacological prevention of postprandial reductions in BP in adults using MEDLINE (1950-), EMBASE (1980-), and CINAHL databases was conducted up to July 2013. Bibliographies of relevant reports were also hand-searched to identify all potentially eligible studies. SETTING: Systematic review of randomized controlled trials using PRISMA guidelines. MEASUREMENTS: Articles were assessed using the Critical Appraisal Skills Programme for randomized controlled trials. RESULTS: Thirteen articles reporting 12 studies (1 study was reported in 2 articles) demonstrated that caffeine (5 studies); acarbose; 3,4-DL-threo-dihydroxyphenylserine; guar gum (3 studies); and octreotide (2 studies) statistically attenuated the postprandial reduction in BP. One caffeine study did not show this. Most studies did not include individuals with symptomatic postprandial hypotension (PPH), so interpretation and application of these findings to this patient group should be made with caution. For symptomatic participants, there was improvement with acarbose but none with caffeine. Differences in the way the data were presented in the studies did not allow for quantification of treatment effects using meta-analysis. CONCLUSION: Drug interventions can attenuate postprandial reductions in BP, but they may not necessarily be effective in people with symptomatic PPH.

A practical approach to immunotherapy of hepatocellular carcinoma using T cells redirected against hepatitis B virus.

Hepatocellular carcinoma (HCC) cells often have hepatitis B virus (HBV)-DNA integration and can be targeted by HBV-specific T cells. The use of viral vectors to introduce exogenous HBV-specific T-cell receptors (TCR) on T cells to redirect their specificity is complex and expensive to implement in clinical trials. Moreover, it raises safety concerns related to insertional mutagenesis and potential toxicity of long-lived HBV-specific T cells in patients with persistent infection. To develop a more practical and safer approach to cell therapy of HCC, we used electroporation of mRNA encoding anti-HBV TCR. Approximately 80% of CD8(+) T cells expressed functional HBV TCR 24 hours postelectroporation, an expression efficiency much higher than that obtained by retroviral transduction (~18%). Antigen-specific cytokine production of electroporated T cells was efficient within 72-hour period, after which the redirected T cells lost their HBV-specific function. Despite this transient functionality, the TCR-electroporated T cells efficiently prevented tumor seeding and suppressed the growth of established tumors in a xenograft model of HCC. Finally, we established a method for large-scale TCR mRNA electroporation that yielded large numbers of highly functional clinical-grade anti-HBV T cells. This method represents a practical approach to cell therapy of HCC and its inherently self-limiting toxicity suggests potential for application in other HBV-related pathologies.Molecular Therapy-Nucleic Acids (2013) 2, e114; doi:10.1038/mtna.2013.43; published online 13 August 2013.

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.