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Planar bismuth compounds exhibit tunable Lewis acidity and high catalytic activity for lactone polymerization.

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 ≤ 2 µM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 µM). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

IntroductionWhy are women (not) romantically attracted to dark personalities or villains, which might be a risk factor for intimate partner violence (IPV) victimization? In the current study, it is opted to investigate how adult attachment, maladaptive personality traits, and acceptance of couple violence in women predict romantic attraction to heroic/villainous characters using structural equation modeling (SEM).MethodFirst, a pilot study was conducted in 122 heterosexual women (aged 16–25) to select male TV characters. This resulted in the selection of six villains and 10 heroes for the main study, in which 194 other heterosexual women (aged 16–25) were asked to rate the pictures of TV characters through an online questionnaire. This was combined with self-report measures of maladaptive personality traits, acceptance of couple violence, and adult attachment. These variables were entered into a SEM model to assess model fit.ResultsOverall, women rated heroes higher on physical appearance (pilot study) and romantic attraction (main study) compared to villains. We found different direct effects of avoidant (negative) and anxious (positive) attachment styles on romantic attraction to heroes. Moreover, maladaptive personality traits fully mediated the positive effect of avoidant attachment style on romantic attraction to villains.DiscussionDespite the limitations of the study design (e.g., low N, low notoriety of the TV characters), this study emphasizes that women are generally more romantically attracted to heroes (vs. villains). Besides, there are different predictors of romantic attraction to heroes and villains, which requires further investigation, especially in the context of IPV.

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control., This study presents a barcoding strategy that enables high-throughput phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and applies this to the discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector.

The present research examined whether women’s narcissistic traits, thrill-seeking, impulsivity, and attachment style are associated with their level of attraction towards narcissistic men. Two trait dimensions of narcissism were distinguished: admiration (i.e., promoting oneself to gain admiration) and rivalry (i.e., devaluing others to protect one’s self-view). Participants (195 heterosexual women, Mage = 20.78, SD = 2.40) were asked to rate their level of attraction towards 25 pictures of fictional male characters who score relatively high or low on narcissism. Using multilevel modelling, we found that women were more attracted to men with high levels of narcissistic admiration (vs. low), and less attracted towards men with high scores on narcissistic rivalry (vs. low). We found no evidence that women who scored high on narcissism were more attracted to narcissistic men. Response surface analyses revealed that women who scored high on thrill-seeking (vs. low) were more attracted to narcissistic admiration, and to a lesser extent to narcissistic rivalry. Additionally, we discovered that women who scored high on impulsivity or avoidant attachment (vs. low) were (to a small extent) more attracted to narcissistic rivalry. Identifying women who are attracted to narcissistic men can help to implement and improve prevention or intervention programs related to narcissism in romantic relationships.

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the bloodmeals was achieved through recombinant DNA-taggedAsaiabacteria that successfully colonizedAedesandAnophelesmosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles and neonicotinoids were enriched for compounds with systemic adulticide activity againstAnopheles. Using a luminescentPlasmodium falciparumreporter strain, barcoded screens identified 48 drug-like transmission blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects, and identifies novel candidate small molecules for disease control.

Given the number of global malaria cases and deaths, the need for a vaccine against Plasmodium falciparum (Pf) remains pressing. Administration of live, radiation-attenuated Pf sporozoites can fully protect malaria-naïve individuals. Despite the fact that motility of these attenuated parasites is key to their infectivity and ultimately protective efficacy, sporozoite motility in human tissue (e.g. skin) remains wholly uncharacterized to date. We show that the ability to quantitatively address the complexity of sporozoite motility in human tissue provides an additional tool in the development of attenuated sporozoite vaccines. We imaged Pf movement in the skin of its natural host and compared wild-type and radiation-attenuated GFP-expressing Pf sporozoites. Using custom image analysis software and human skin explants we were able to quantitatively study their key motility features. This head-to-head comparison revealed that radiation attenuation impaired the capacity of sporozoites to vary their movement angle, velocity and direction, promoting less refined movement patterns. Understanding and overcoming these changes in motility will contribute to the development of an efficacious attenuated parasite malaria vaccine.

Males with adrenoleukodystrophy develop progressive myelopathy causing severe disability later in life. No treatment is currently available, but new disease-modifying therapies are under development. Knowledge of the natural history of the myelopathy is of paramount importance for evaluation of these therapies in clinical trials, but prospective data on disease progression are lacking. We performed a prospective observational cohort study to quantify disease progression over 2 years of follow-up. Signs and symptoms, functional outcome measures and patient-reported outcomes were assessed at baseline, 1 and 2 years of follow-up. We included 46 male adrenoleukodystrophy patients (median age 45.5 years, range 16-71). Frequency of myelopathy at baseline increased with age from 30.8% (50 years). Disease progression was measured in the patients who were symptomatic at baseline (n = 24) or became symptomatic during follow-up (n = 1). Significant progression was detected with the functional outcome measures and quantitative vibration measurements. Over 2 years of follow-up, Expanded Disability Status Score increased by 0.34 points (P = 0.034), Severity Scoring system for Progressive Myelopathy decreased by 2.78 points (P = 0.013), timed up-and-go increased by 0.82 s (P = 0.032) and quantitative vibration measurement at the hallux decreased by 0.57 points (P = 0.040). Changes over 1-year follow-up were not significant, except for the 6-minute walk test that decreased by 19.67 meters over 1 year (P = 0.019). None of the patient-reported outcomes were able to detect disease progression. Our data show that progression of myelopathy in adrenoleukodystrophy can be quantified using practical and clinically relevant outcome measures. These results will help in the design of clinical trials and the development of new biomarkers for the myelopathy of adrenoleukodystrophy.10.1093/brain/awy299_video1awy299media15995811923001.

Background: Epstein-Barr virus positivity (EBV+) and microsatellite instability (MSI-high) have been shown to be positive prognostic factors for long term survival in resectable gastric cancer (GC) in several studies. However, the benefit of perioperative treatment in patients with MSI-high tumors remains topic of discussion. Here, we present the clinicopathological outcome of patients with EBV+ and MSI-high GCs treated with surgery only in the Dutch D1/D2 trial, and treated with chemotherapy or chemoradiotherapy after preoperative chemotherapy and surgery in the CRITICS trial. Patients and methods: EBV was determined in tumor tissue using EBV-encoded RNA in situ hybridization (EBER-ISH). PCR and/or immunohistochemistry were performed to determine MSI status. Results were correlated to histopathological response, morphological tumor characteristics and survival. Results: In the Dutch D1/D2 trial 10.5% (47/447) of tumors were EBV+ and 10.5% (47/447) were MSI-high. In the CRITICS trial 5.5% (25/451) of tumors were EBV+ and 5.5% (25/451) were MSI-high tumors. In the Dutch D1/D2 trial, five-year overall survival probability was 51.1% for EBV+, 46.8% for MSI-high, and 42.5% for EBV-/MSS (P=0.19). In the CRITICS trial, five-year overall survival was 56.0% for EBV+, 47.3% for MSI-high, and 36.5% for EBV-/MSS (P=0.22). In the CRITICS trial, 3 (12.5%) MSI-high tumors showed moderate to complete histopathological response. Interestingly, all three showed a mucinous phenotype. Eight (36.4%) EBV+ and 114 (29.9%) EBV-/MSS tumors showed moderate to complete histopathological response. None of the EBV+ GCs showed mucinous differentiation. Conclusions: The favorable outcome of GC patients with resectable EBV+ or MSI-high tumors compared to EBV-/MSS tumors remains after perioperative chemotherapy. In MSI-high tumors significant histopathological response to neoadjuvant chemotherapy was found only in those with a mucinous phenotype. Citation Format: H.D. Biesma, A.T.T.D. Soeratram, K. Sikorska, I.A. Caspers, H.F. van Essen, J.M.P. Egthuijsen, A. Mookhoek, D.M. Hoek, W. Vos, H.W.M. van Laarhoven, M. Nordsmark, D.L. van der Peet, F.A.R.M. Warmerdam, M.M. Geenen, O.J.L. Loosveld, J.E.A. Portielje, M. Los, E. Meershoek - Klein Kranenbarg, H.H. Hartgrink, J. van Sandick, C.J.H. van de Velde, M. Verheij, A. Cats, B. Ylstra, N.C.T. van Grieken, On behalf of the CRITICS investigators. Mucinous phenotype is associated with response to neoadjuvant chemotherapy in microsatellite instable resectable gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 354.

Key Points This meta-analysis evaluates the diagnostic accuracy of rapid tests for the detection of respiratory viruses. Sensitivity of tests varied considerably, but specificity was high. Although newly developed tests seem more sensitive, high quality evaluations of these tests are lacking., Rapid diagnosis of respiratory virus infections contributes to patient care. This systematic review evaluates the diagnostic accuracy of rapid tests for the detection of respiratory viruses. We searched Medline and EMBASE for studies evaluating these tests against polymerase chain reaction as the reference standard. Of 179 studies included, 134 evaluated rapid tests for influenza viruses, 32 for respiratory syncytial virus (RSV), and 13 for other respiratory viruses. We used the bivariate random effects model for quantitative meta-analysis of the results. Most tests detected only influenza viruses or RSV. Summary sensitivity and specificity estimates of tests for influenza were 61.1% and 98.9%. For RSV, summary sensitivity was 75.3%, and specificity, 98.7%. We assessed the quality of studies using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) checklist. Because of incomplete reporting, the risk of bias was often unclear. Despite their intended use at the point of care, 26.3% of tests were evaluated in a laboratory setting. Although newly developed tests seem more sensitive, high-quality evaluations of these tests are lacking.

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl-coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl-coenzyme A synthetase and acyl-coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.

Background: Geriatric rehabilitation care (GRC) is short-term and multidisciplinary rehabilitation care for older vulnerable clients. Studies were conducted about its effects. However, elements that influence the quality of GRC have not been studied previously.Methods: In this study realist evaluation is used to find out which are the mechanisms and outcomes and which (groups of) persons are the context for GRC, according to GRC professionals. The mechanisms, outcomes and context of GRC were explored in three consecutive phases of qualitative data gathering, i.e. individual interviews, expert meeting, and focus groups.Results: Eight mechanisms — client centeredness, client satisfaction during rehabilitation, therapeutic climate, information provision to client and informal care givers, consultation about the rehabilitation (process), cooperation within the MultiDisciplinary Team (MDT), professionalism of GRC professionals, and organizational aspects — were found. Four context groups—the client, his family and/or informal care giver(s), the individual GRC professional, and the MDT—were mentioned by the respondents. Last, two outcome factors were determined, i.e. client satisfaction at discharge and rehabilitation goals accomplished.Conclusions: In order to translate these insights into a practical tool that can be used by MDTs in the practice of GRC, identified mechanisms, contexts, and outcomes were visualized in a GRC evaluation tool. A graphic designer developed an interactive PDF which is the GRC evaluation tool. This tool may enable MDTs to discuss, prioritize, evaluate, and improve the quality of their GRC practice.Keywords: Geriatric rehabilitation care (GRC), Quality improvement, Evaluation, Realist evaluation, Mechanisms, Context, Outcomes, GRC evaluation tool

Social networks are sources of support and contribute to the well‐being of older adults who are ageing in place. As social networks change, especially when accompanied by health decline, older adults’ sources of support change and their well‐being is challenged. Previous studies predominantly used quantitative measures to examine how older adults’ social networks change. Alternatively, this study explores the impact of changing social networks on older adults’ lives by examining their personal experiences. We held four focus groups, two with a total of 14 older adults who are ageing in place and receiving home care and two with a total of 20 home‐care nurses from different regions and organisations in the Netherlands. Subsequently, an expert team of home‐care professionals and managers discussed and verified the results. Procedures for grounded theory building were used for analysis. We revealed four themes of high‐impact experiences: (a) struggling with illness/death of the spouse; (b) working out a changing relationship with (grand)children; (c) regretting the loss of people they have known for so long and (d) feeling dependent and stressed when helpers enter the network. Also, network dynamics were found to follow three consecutive stages: (a) awareness of social network change; (b) surprise when social network change actually occurs and (c) acceptance and adjusting to new circumstances. Together, the four themes of experiences and three stages of network change form an integrative model of the role of social network dynamics for older adults’ lives when ageing in place.

Malaria eradication is critically dependent on novel drugs that target resistantPlasmodiumparasites and block transmission of the disease. Here we report the discovery of potent pantothenamide bioisosteres that are active against blood-stageP. falciparumand also block onward mosquito transmission. These compounds are resistant to degradation by serum pantetheinases, show favorable pharmacokinetic properties and clear parasites in a humanized rodent infection model. Metabolomics revealed that CoA biosynthetic enzymes convert pantothenamides into drug-conjugates that interfere with parasite acetyl-CoA anabolism.In vitrogenerated resistant parasites showed mutations in acetyl-CoA synthetase and acyl-CoA synthetase 11, confirming the key roles of these enzymes in the sensitivity to pantothenamides. These new pantothenamides provide a promising class of antimalarial drugs with a unique mode of action.One sentence summaryPantothenamides form antimetabolites that interfere with acetyl-CoA metabolism in the human malaria parasitePlasmodium falciparum

We examined how perceived organizational diversity approaches (colorblindness and multiculturalism) relate to affective and productive work outcomes for cultural majority and minority employees. Using structural equation modeling on data collected in a panel study among 152 native Dutch majority and 77 non-Western minority employees, we found that perceptions of a colorblind approach were most strongly related to work satisfaction and perceived innovation for majority members, while perceptions of a multicultural approach “worked best” for minority members. Moreover, these effects were fully mediated by the extent to which employees felt socially included in the organization. Thus, while inclusion is an important factor for both groups to enhance work outcomes, it is facilitated by different diversity approaches for majority and minority members.

Introduction and Objectives FRI has been used to provide a detailed view of overall and local changes in airway resistance and lung volume, in addition to spirometry and body plethysmography. The aim of this study was to assess changes in specific image-based airway volume (siVaw) and resistance (siRaw) in patients with moderate-to-severe COPD after administration of GFF vs placebo. Methods In this double-blind, 2 week dosing, Phase III crossover study (NCT02643082), 20 patients (40–80 years) received twice daily GFF MDI 14.4/10 µg (equivalent to glycopyrrolate/formoterol fumarate 18/9.6 µg) and placebo MDI. Primary endpoints were siVaw and siRaw at Day 15. Additional outcomes included spirometry, plethysmography and safety. Results On Day 15, GFF MDI led to an estimated 75% increase in siVaw and 71% decrease in siRaw relative to placebo MDI (both p 1 and 454 mL in IC in change from baseline for GFF MDI vs placebo MDI (both p Conclusions The dual bronchodilator GFF MDI demonstrated significant benefits on FRI–based airway volume and resistance in the lungs of COPD patients. Benefits were associated with important improvements in FEV 1 , IC and hyperinflation.

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum , the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F . Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii , where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

Contains fulltext : 139106.pdf (Publisher’s version ) (Closed access) BACKGROUND: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. METHODS: Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. RESULTS: Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. CONCLUSIONS: This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.

The glucocorticoid receptor is present in a TCR-associated complex, which includes the Src family tyrosine kinase Lck. Glucocorticoids rapidly dissociate this complex, resulting in the inhibition of canonical Lck-phospholipase C (PLC)γ–dependent TCR signaling. The relative importance of this nongenomic role for the glucocorticoid receptor compared with its direct transcriptional effects is not known. Superantigens induce a state of steroid resistance in activated T cells. It was reported that, in addition to canonical Lck-PLCγ signaling, superantigens can activate a noncanonical G protein–PLCβ–dependent signaling pathway. In this study, we show that staphylococcal enterotoxin B activates a Gαq and PLCβ2–dependent pathway in human T cells. We find that this pathway bypasses the need for canonical Lck-PLCγ signaling in T cell activation and renders superantigen-stimulated T cells insensitive to glucocorticoids in vitro. We show that the PLCβ inhibitor U-73122 sensitizes staphylococcal enterotoxin B–treated mice to dexamethasone in vivo. In conclusion, we find that effects of glucocorticoids on TCR-induced T cell proliferation are mainly nongenomic and can be bypassed by the activation of an Lck-independent signaling pathway.

Background: The repertoire of immunomodulators that can be used for the treatment of inflammatory bowel disease is limited. The use of these drugs is further restricted by the occurrence of side effects in a proportion of patients. Miltefosine (hexadecylphosphocholine) is a lipid drug developed in the 1980s for the treatment of cancer but is nowadays best known for its application in the oral treatment of leishmaniasis. Although the exact mechanism of action of miltefosine has yet to be elucidated, the drug has previously been shown to inhibit phospholipases and protein kinase C, both key components of proproliferative signal transduction in T cells. Methods: Stimulated peripheral blood lymphocyte were treated with miltefosine, and proliferation was measured. We use the CD45RBhighT-cell transfer colitis model to investigate the effect of miltefosine treatment on intestinal inflammation. Effects on the severity of colitis were studied by histochemical and immunohistochemical staining, and cytokine levels were determined using a cytokine bead array. Results: Miltefosine inhibited T-cell proliferation in vitro. In the transfer model, miltefosine significantly ameliorated the severity of colitis as measured by clinical, (immuno)histochemical, and biochemical parameters. Conclusions: Miltefosine inhibits T-cell proliferation and effectively reduces inflammation in the T-cell transfer model. The drug may therefore be a candidate immunomodulator for inflammatory bowel disease. Copyright

Contains fulltext : 167311.pdf (Publisher’s version ) (Open Access) Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.

Background and Aims: Anti-tumour necrosis factor [TNF] antibodies induce regulatory macrophages which display a phenotype resembling M2 type macrophages. Anti-TNF induced macrophages [Mϕind] have immunosuppressive and wound healing properties. The factors that contribute to the induction of Mϕind remain to be explored. Autophagy has been described as a factor that is important for the induction and function of M2 type macrophages. We studied the contribution of autophagy to the induction of Mϕind. Methods: We studied the effect of autophagy on Mϕind in vitro using peripheral blood mononuclear cells. Interferon gamma [IFN-γ] induced macrophages [Mφ1] were generated by culturing monocytes in the presence of IFN-γ. Mϕind were generated by performing mixed lymphocyte reactions [MLR] in the presence of anti-TNF antibodies; 28 healthy donors were genotyped for rs_2241880 [ATG16L1]. Cells were analysed by autophagy gene array, immunofluorescence, western blot, flowcytometry, 3H-thymidine incorporation and MTS assay. Results: Mϕind had a different expression profile of autophagy related transcripts with increased expression of 33/40 altered genes compared with Mφ1. In addition, autophagic activity was increased in Mϕind compared with Mφ1. Induction of Mϕind was positively correlated to the number of wild-type alleles for the ATG16L1 T300A risk allele present in the culture. Finally, the autophagy-related protein cathepsin S was highly expressed in Mφind and inhibition resulted in decreased viability as well as decreased expression of CD206. Conclusions: Mϕind have increased levels of autophagy compared with inflammatory Mφ1, and the induction of these macrophages is impaired in donors carrying the T300A risk allele for the ATG16L1 . Given the association between Mϕind and clinical response, this suggests that an intact autophagy pathway may be important for an optimal response to anti-TNF therapy in inflammatory bowel disease.

Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3–5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites.

Background and aims Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I–III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays Methods The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied Results MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment Conclusions This study demonstrates that, in vitro , MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy.

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant's final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March-April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.

Contains fulltext : 69323.pdf (Publisher’s version ) (Closed access) The amphiphilic dye FM4-64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4-64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4-64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4-64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4-64 interacts with.

Current first-line treatments for uncomplicated falciparum malaria rapidly clear the asexual stages of the parasite, but do not fully prevent parasite transmission by mosquitoes. The standard membrane feeding assay (SMFA) is the biological gold standard assessment of transmission reducing activity (TRA), but its throughput is limited by the need to determine mosquito infection status by dissection and microscopy. Here we present a novel dissection-free luminescence based SMFA format using a transgenic Plasmodium falciparum reporter parasite without resistance to known antimalarials and therefore unrestricted in its utility in compound screening. Analyses of sixty-five compounds from the Medicines for Malaria Venture validation and malaria boxes identified 37 compounds with high levels of TRA (>80%); different assay modes allowed discrimination between gametocytocidal and downstream modes of action. Comparison of SMFA data to published assay formats for predicting parasite infectivity indicated that individual in vitro screens show substantial numbers of false negatives. These results highlight the importance of the SMFA in the screening pipeline for transmission reducing compounds and present a rapid and objective method. In addition we present sixteen diverse chemical scaffolds from the malaria box that may serve as a starting point for further discovery and development of malaria transmission blocking drugs.

Measles remains endemic in many East African countries, where it is often associated with high morbidity and mortality. We collected clinical specimens from Sudanese measles patients between July 1997 and July 2000. Sequencing of the 3′ 456 nucleotides of the nucleoprotein gene from 33 measles virus (MV) isolates and 8 RNA samples extracted from clinical specimens demonstrated the presence of a single endemic MV strain with little sequence variation over time (overall nucleotide divergence of 0 to 1·3%). This was confirmed by sequencing of the complete H gene of two isolates from 1997 and two from 2000, in which the overall divergence ranged between 0 and 0·5%. Comparison with MV reference strains demonstrated that the viruses belonged to clade B, genotype B3, and were most closely related to a set of viruses recently isolated in Nigeria. Our study demonstrates a remarkable genetic stability of an endemically circulating MV strain.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.

A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine. DOI: http://dx.doi.org/10.7554/eLife.03582.001, eLife digest Vaccines commonly contain a weakened or dead version of a disease-causing microorganism, or its toxins, or surface proteins. These prime the immune system to rapidly recognize, respond to, and eliminate the actual infectious pathogen if later encountered. While vaccines are currently available to help prevent a large number of diseases, vaccines for many deadly diseases, including malaria, do not yet exist. Malaria is caused by a group of parasites called Plasmodium, which are transferred to humans by mosquitoes. While measures to control mosquito populations and prevent mosquito bites have helped to reduce the incidence of malaria in some countries, the number of people—and especially children—that die of malaria every year remains very high. When a mosquito carrying Plasmodium in its salivary glands bites a human, the parasite is injected into the human's bloodstream and travels to the liver. The parasite reproduces in the liver cells until there are so many of them that the cells rupture, and the parasites are released back into the bloodstream. Any mosquito that then feeds on the blood of the infected individual may also suck up the parasite. The parasite then goes through a further stage of development in the mosquito, eventually migrating to the salivary glands, from where the parasite can be transmitted into a new human host. Recent work in rodents suggests that genetically altered or weakened Plasmodium falciparum sporozoites—the form of the parasite found in mosquito saliva—could be used to vaccinate humans against malaria caused by this parasite species. Now, van Schaijk, Ploemen et al. evaluate whether a safe and effective vaccine could be made from sporozoites that lack two genes, called b9 and slarp, which are critical for the parasites to develop inside liver cells. When mice were injected with the modified sporozoites, their immune cells were able to detect the parasites and respond against them. The mice subsequently did not develop malaria when they were infected with normal, unmodified parasites. Furthermore, none of the mice contracted malaria from the modified sporozoites. The modified sporozoites behaved similarly in human liver cells: after invading these cells, the parasites were unable to develop. Clinical testing and further development are now needed to see if a successful malaria vaccine can be made from these sporozoites. DOI: http://dx.doi.org/10.7554/eLife.03582.002

Contains fulltext : 137134.pdf (Publisher’s version ) (Closed access) The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

Background/Aims —Killer inhibitory receptors (KIR) have a modulating effect on the cytotoxic functions of natural killer (NK) cells and T cells. Because lymphoma cells often have the same receptors as their non-neoplastic counterparts, this study investigated the expression of KIR on well defined groups of NK and T cell lymphomas, with and without a cytotoxic phenotype, from different sites of origin. Methods —Nine CD56 + /CD3 − NK cell lymphomas, 29 CD3 + /CD56 − T cell lymphomas with a cytotoxic phenotype, and 19 T cell lymphomas without a cytotoxic phenotype were stained for KIR using monoclonal antibodies specific for CD94, CD158a, and CD158b. In addition, the expression of KIR was studied on normal lymphoid tissues. Results —KIR expression was seen in five of nine true NK cell lymphomas including three of four nasal, one of four cutaneous, and one of one intestinal lymphoma nasal type. Double staining for CD56 and CD94 in normal lymphoid tissues revealed that KIR was predominantly expressed by CD56 + NK cells and sporadically on CD8 + T cells. Moreover, enteropathy-type T cell lymphomas with a cytotoxic phenotype showed KIR expression (three cases expressing CD94 and one case expressing CD158a). All nodal and extranodal non-intestinal T cell lymphomas with or without a cytotoxic phenotype lacked expression of KIR. Conclusions —These results show that KIR expression is restricted to CD56 + /CD3 − true NK cell lymphomas originating from the nose, gut, and skin, as well as in a subset of extranodal T cell lymphomas originating from the small intestine, which possessed a cytotoxic phenotype. Thus, the presence of KIR on NK/T cell lymphomas seems to mimic the distribution of KIR found on NK and T cells in normal lymphoid tissue.

Objective and design: A systematic literature review to compare mobility scales used for lower limb amputees. A literature search was carried out by computerized search of biomedical literature including Medline and Embase. The studies included were published between 1978 and 1998 and including the following keywords: amputation, artificial limbs, prosthesis, lower limb, activities of daily living, mobility. Results: Thirty-five studies were identified; 19 had a measurement of separate levels of mobility comparable to each other. Sixteen studies used ordinal and ratio scales without separate levels of mobility. The widest range of measurement found was the scale from ‘walking with prosthesis without a walking aid’ to ‘totally confined to bed’. The Stanmore Harold Wood mobility scale was published most frequently. None of the 35 studies presented give a continuous measurement of mobility. Conclusion: A multitude of measurement scales and questionnaires are available for differ in methods and measuring range. Measuring mobility by a scale has been shown to have limitations. Several authors did extensive research but they all measure only a number of aspects of mobility. Consensus about the measurement of mobility of lower limb amputees is not available in the recent literature.

Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.