Analysis of the Health Product Profile Directory - a new tool to inform priority-setting in global public health.

BACKGROUND: The Health Product Profile Directory (HPPD) is an online database describing 8-10 key characteristics (such as target population, measures of efficacy and dosage) of product profiles for medicines, vaccines, diagnostics and other products that are intended to be accessed by populations in low- and middle-income countries. The HPPD was developed by TDR on behalf of WHO and launched on 15 May 2019. METHODS: The contents of the HPPD were downloaded into an Excel™ spreadsheet via the open access interface and analysed to identify the number of health product profiles by type, disease, year of publication, status, author organization and safety information. RESULTS: The HPPD contains summaries of 215 health product profiles published between 2008 and May 2019, 117 (54%) of which provide a hyperlink to the detailed publication from which the summary was extracted, and the remaining 98 provide an email contact for further information. A total of 55 target disease or health conditions are covered, with 210 profiles describing a product with an infectious disease as the target. Only 5 product profiles in the HPPD describe a product for a non-communicable disease. Four diseases account for 40% of product profiles in the HPPD; these are tuberculosis (33 profiles, 15%), malaria (31 profiles, 14%), HIV (13 profiles, 6%) and Chagas (10 profiles, 5%). CONCLUSION: The HPPD provides a new tool to inform priority-setting in global health - it includes all product profiles authored by WHO (n = 51). There is a need to standardise nomenclature to more clearly distinguish between strategic publications (describing research and development (R&D) priorities or preferred characteristics) compared to target product profiles to guide a specific candidate product undergoing R&D. It is recommended that all profiles published in the HPPD define more clearly what affordability means in the context where the product is intended to be used and all profiles should include a statement of safety. Combining the analysis from HPPD to a mapping of funds available for R&D and those products in the R&D pipeline would create a better overview of global health priorities and how they are supported. Such analysis and increased transparency should take us a step closer to measuring and improving coordination of efforts in global health R&D.

Infectious disease research and the gender gap.

Historically, women have been less likely to be supported through higher degree training programmes, and they continue to hold more junior positions in science. This paper reviews the current gender research and gender capacity-building efforts led by the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR). Created more than 40 years ago as the only United Nations-based Special Programme dedicated to research and research capacity building on infectious diseases, TDR has a longstanding track record both in supporting research into gender-specific questions and in research capacity strengthening among women scientists. We provide an overview of these approaches, then describe a recent pilot programme on Women in Science, designed to understand and remedy the gender gaps in health research. The programme focused on Africa, but it is hoped that the replication of such schemes in TDR and other international funding agencies will lead to more attention being given to women in infectious diseases research in other continents. This article may not be reprinted or reused in any way in order to promote any commercial products or services.

The Structured Operational Research and Training Initiative for public health programmes.

In 2009, the International Union Against Tuberculosis and Lung Disease (The Union) and Médecins sans Frontières Brussels-Luxembourg (MSF) began developing an outcome-oriented model for operational research training. In January 2013, The Union and MSF joined with the Special Programme for Research and Training in Tropical Diseases (TDR) at the World Health Organization (WHO) to form an initiative called the Structured Operational Research and Training Initiative (SORT IT). This integrates the training of public health programme staff with the conduct of operational research prioritised by their programme. SORT IT programmes consist of three one-week workshops over 9 months, with clearly-defined milestones and expected output. This paper describes the vision, objectives and structure of SORT IT programmes, including selection criteria for applicants, the research projects that can be undertaken within the time frame, the programme structure and milestones, mentorship, the monitoring and evaluation of the programmes and what happens beyond the programme in terms of further research, publications and the setting up of additional training programmes. There is a growing national and international need for operational research and related capacity building in public health. SORT IT aims to meet this need by advocating for the output-based model of operational research training for public health programme staff described here. It also aims to secure sustainable funding to expand training at a global and national level. Finally, it could act as an observatory to monitor and evaluate operational research in public health. Criteria for prospective partners wishing to join SORT IT have been drawn up.

En 2009, L'Union Internationale contre la Tuberculose et les Maladies pulmonaires (L'Union) et Médecins sans Frontières Bruxelles-Luxembourg (MSF) ont commencé à élaborer un modèle orienté par les résultats pour la formation en recherche opérationnelle. En janvier 2013, l'Union et MSF ont rejoint le Programme Spécial de Recherche et de Formation des Maladies Tropicales (TDR) à l'OMS pour former une initiative baptisée « The Structured Operational Research and Training Initiative (SORT IT) ¼ [Initiative structurée de recherche opérationnelle et de formation]. Celle-ci intègre la formation du personnel des programmes de santé publique et la conduite de recherche opérationnelle en fonction des priorités de leur programme. Les programmes SORT IT consistent en trois ateliers d'une semaine, étalés sur 9 mois, avec des étapes bien définies et des résultats attendus. Cet article décrit la vision, les objectifs et la structure des programmes SORT IT, notamment les critères de sélection des candidats, les projets de recherche qui peuvent être entrepris dans le temps imparti, la structure et les étapes du programme, le tutorat, le suivi et l'évaluation des programmes et les suites du programme en termes de recherche ultérieure, de publications et de conception/mise en œuvre de programmes de formation supplémentaire. Il y a un besoin croissant, national et international, de recherche opérationnelle et de renforcement des capacités dans ce domaine en santé publique. SORT IT vise à répondre à ce besoin en plaidant pour un modèle de formation en recherche opérationnelle basé sur les résultats du personnel de santé publique décrit ici. Il vise également à sécuriser un financement pérenne pour la formation des experts au niveau mondial et national. Enfin, il pourrait servir d'observatoire de suivi et d'évaluation de la recherche opérationnelle en santé publique. Les critères de recrutement de nouveaux partenaires potentiels qui souhaitent rejoindre SORT IT ont été élaborés.

En el 2009, La Unión contra la Tuberculosis y las Enfermedades Respiratorias (La Unión) y Médecins sans Frontières de Bruselas y Luxemburgo comenzaron a desarrollar un modelo de capacitación en investigación operativa orientada por los resultados. En enero del 2013, ambas organizaciones se unieron a un Programa Especial de Investigación y Capacitación en Enfermedades Tropicales de la Organización Mundial de la Salud (OMS), con el fin de poner en marcha una iniciativa denominada SORT IT (acrónimo por the Structured Operational Research and Training Initiative, Iniciativa de Capacitación Estructurada en Investigación Operativa). Esta iniciativa articula la capacitación del personal del programa de salud pública con la realización de una investigación operativa a la cual su propio programa atribuye una prioridad. Los programas SORT IT consisten en tres talleres de una semana cada uno, durante un período de nueve meses, cuyos objetivos principales y productos se definen muy claramente. En el presente artículo se describen la visión, los objetivos y la estructura de los programas SORT IT, incluidos los criterios de selección de los solicitantes, los proyectos de investigación que se pueden emprender dentro del tiempo asignado, los objetivos principales y la estructura del programa, la tutoría, el seguimiento y la evaluación de los programas y lo que puede realizarse después del programa, como las futuras investigaciones, las publicaciones y la organización de otros programas de capacitación. Existe una necesidad creciente de investigación operativa y de creación de capacidades conexas en materia de salud pública a escala nacional e internacional. La iniciativa SORT IT busca satisfacer estas necesidades, mediante la promoción del modelo de capacitación en investigación operativa orientada por los resultados que dirige al personal del programa de salud pública descrito aquí. También busca lograr un financiamiento sostenible con el fin de ampliar la capacitación a escala nacional y mundial. Por último, la iniciativa podría tener una función de observatorio encargado de evaluar la investigación operativa en salud pública. Se redactaron asimismo los criterios dirigidos a los futuros asociados que deseen unirse a la iniciativa SORT IT.

Open access for operational research publications from low- and middle-income countries: who pays?

Open-access journal publications aim to ensure that new knowledge is widely disseminated and made freely accessible in a timely manner so that it can be used to improve people's health, particularly those in low- and middle-income countries. In this paper, we briefly explain the differences between closed- and open-access journals, including the evolving idea of the 'open-access spectrum'. We highlight the potential benefits of supporting open access for operational research, and discuss the conundrum and ways forward as regards who pays for open access.

Les articles de journaux en accès libre visent à assurer la dissémination large de nouvelles connaissances et de rendre leur accès libre de façon à pouvoir être utilisées rapidement pour améliorer la santé des populations, surtout dans les pays à revenu faible ou moyen. Dans cet article, nous expliquons briêvement les différences entre les publications à accès limité et à accès libre, notamment l'idée en gestation de « spectre d'accès libre ¼. Nous soulignons les bénéfices potentiels du soutien à l'accès libre pour la recherche opérationnelle et ensuite discutons la question de qui paye pour cet accès et la recherche de solutions.

El propósito de las publicaciones en las revistas de acceso libre es lograr una amplia difusión de los nuevos conocimientos mediante el acceso libre y oportuno, de manera que los avances se puedan aplicar a fin de mejorar la salud de las personas, sobre todo en los países de bajos y medianos ingresos. En el presente artículo se explican brevemente las diferencias entre las revistas de acceso libre y acceso restringido y se analiza además la idea evolutiva del 'espectro del acceso libre'. Se destacan las ventajas que puede ofrecer el respaldo al libre acceso a la investigación operativa y se analiza luego el dilema y las opciones que pueden permitir progresar con respecto a la fuente de financiamiento del libre acceso.

'Because it is a joyful thing to carry a baby': involving men in reproductive, maternal and newborn health in East New Britain, Papua New Guinea.

BACKGROUND: There are many benefits to involving expectant fathers in maternal and newborn health, including reducing vulnerability to HIV (human immunodeficiency virus) and sexually transmitted infections (STIs). Women are at risk of HIV infection and other STIs during pregnancy and breastfeeding and in Papua New Guinea (PNG) a number of complex factors interact to enhance this vulnerability. PNG health policies do support men's involvement in maternal and newborn health, but currently there is limited understanding of appropriate or effective ways by which this could be achieved. AIMS: The aims of this research were to gather information to inform strategies to enable the greater involvement of men in maternal and newborn health services and to explore the factors that contribute to STI and HIV vulnerability among pregnant women in East New Britain Province. METHODS: Between June 2011 and February 2012 we conducted a total of 14 focus group discussions with pregnant women, expectant fathers, older men and older women. Ten in-depth interviews were conducted with health workers and staff within the provincial administration. KEY FINDINGS: Expectant fathers were concerned for the health of their wife and baby both during and after pregnancy. They had many questions about pregnancy, childbirth and the care of their baby and were eager for information. Protecting their family is viewed as an important role for men and could be a useful way of engaging with them. Misconceptions about the safety of sex during pregnancy are one reason that couples are often sexually abstinent for long periods. This may contribute to the likelihood that either partner will seek sex outside marriage during pregnancy or postpartum, and increase a pregnant woman's risk of contracting STIs and HIV. We heard that it is common for men as well as women to have extramarital sex at this time. Currently, male involvement in maternal and child health care is uncommon and community attitudes are mixed. Some significant barriers to involving men relate to traditional customs and feelings of shame and embarrassment. Others can be attributed to health service factors, such as a lack of privacy and the attitudes of health care workers. Various community channels for reaching expectant fathers were suggested.

A neonatal pneumococcal conjugate vaccine trial in Papua New guinea: study population, methods and operational challenges.

Infants in Papua New Guinea (PNG) are at a high risk of invasive pneumococcal disease, and a substantial burden of this falls on children less than six months old. PNG is planning to introduce a pneumococcal conjugate vaccine for infants in the near future, but to make the maximum impact neonatal immunization will have to be considered. To provide evidence on safety and immunogenicity for neonatal and early infant immunization, we undertook an open randomized controlled trial of 7-valent pneumococcal conjugate vaccine (7vPCV). 318 children received 7vPCV at ages 0, 1 and 2 months or at 1, 2 and 3 months or not at all. All children received 23-valent pneumococcal polysaccharide vaccine at age 9 months. This was a large and complex trial: village reporters visited participants weekly during the first year and fortnightly for a further 6 months and nurses monitored self-reported morbidity and collected many thousands of biological samples. The study team was remarkably successful in achieving the study aims, with 18-month follow-up completed on 77% of enrolled children and over 80% of scheduled samples collected. While the results of the trial will be reported elsewhere, this paper discusses the design of the study and dissects out some of the main reasons for its successful completion. Strong community engagement was an essential factor in success and the principles of equitable partnership and service provision led to a strong research partnership. A two-stage consent process, comprising primary assent followed by later informed consent, led to a high drop-out before initial enrolment, but an outstanding retention of those enrolled in the study. We conclude that factors such as strong community participation, reciprocity and a good relationship between the study team and participants are just as important as the technical elements of laboratory testing and data handling in ensuring the success of a vaccine trial in PNG.

Southeast Asian ovalocytosis is associated with increased expression of Duffy antigen receptor for chemokines (DARC).

The Duffy antigen receptor for chemokines (DARC or Fy glycoprotein) carries antigens that are important in blood transfusion and is the main receptor used by Plasmodium vivax to invade reticulocytes. Southeast Asian ovalocytosis (SAO) results from an alteration in RBC membrane protein band 3 and is thought to mitigate susceptibility to falciparum malaria. Expression of some RBC antigens is suppressed by SAO, and we hypothesized that SAO may also reduce Fy expression, potentiallyleading to reduced susceptibility to vivax malaria. Blood samples were collected from individuals living in the Madang Province of Papua New Guinea. Samples were assayed using a flow cytometry assay for expression of Fy on the surface of RBC and reticulocytes by measuring the attachment of a phycoerythrin-labeled Fy6 antibody. Reticulocytes were detected using thiazole orange. The presence of the SAO mutation was confirmed by PCR. There was a small (approximately 10%) but statistically significant (p=0.049, Mann-Whitney U test) increase in Fy expression on SAO RBC compared with RBC from individuals without this polymorphism: mean Fy expression (mean fluorescence intensity [MFI]) was 10.12 +/- 1.22 for SAO heterozygotes versus an MFI of 8.95 +/- 1.1 for individuals without SAO. For reticulocytes the MFI values were 27.61 +/- 19.12 for SAO heterozygotes and 16.47 +/- 3.81 for controls. SAO is associated with increased and not decreased Fy6 expression so that susceptibility to P. vivax infection is unlikely to be affected.

Muscle cell injury, haemolysis and dark urine in children with falciparum malaria in Papua New Guinea.

During a prospective study of red cell variants and severe malaria in children, a surprising observation was the occurrence of dark urine. Children were grouped according to urine findings: 22 had dark urine that contained a haem protein (Group I), 93 had urine of normal colour that contained a haem protein (Group II) and 236 had normal urine (Group III). To investigate the cause of dark urine, haemolysis and muscle cell injury were assessed. Intravascular haemolysis was greater in Group I than in Groups II and III. However, anaemia was more severe in Group III and is likely to have resulted mainly from extravascular haemolysis. Median plasma myoglobin concentrations were greater in Groups I and II than Group III (P = 0.00060). Plasma myoglobin was greater in children with cerebral malaria, hyperlactataemia and those who died but was not associated with acidosis. Urine myoglobin was greater in Group I than Groups II and III (P = 0.00054). It is likely that both haemoglobin and myoglobin contributed to dark urine. The association between muscle cell injury and coma suggests sequestration of parasitized red cells as a common underlying pathology. In malaria, hyperlactataemia may result directly from breakdown of muscle protein as well as tissue hypoxia.

Adaptive immunity in melioidosis: a possible role for T cells in determining outcome of infection with Burkholderia pseudomallei.

Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei. Individuals with subclinical melioidosis have no apparent clinical signs or symptoms, and are identified only by positive serology. The present study is the first to investigate cell-mediated immune (CMI) responses following in vitro stimulation with B. pseudomallei antigens in peripheral blood mononuclear cells (PBMC), collected under field conditions in Papua New Guinea (PNG) from individuals with exposure to B. pseudomallei (n = 13). While five had a clinical history of melioidosis (C(+)), the remaining individuals (n = 8) were seropositive, yet healthy with no clinical history of melioidosis (S(+)/C(-)). Proliferation and IFN-gamma production were significantly greater in lymphocyte cultures from S(+)/C(-) individuals compared to C(+) individuals (P < 0.001 and P < 0.05, respectively). These findings demonstrate that compared to C(+) patients, individuals with subclinical melioidosis have a stronger CMI response to B. pseudomallei antigens in vitro. Such a response may be essential for protection against disease progression.

Plasmodium falciparum erythrocyte membrane protein 1 functions as a ligand for P-selectin.

The malarial protein Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a parasite protein that is exported to the surface of the infected erythrocyte, where it is inserted into the red cell cytoskeleton in the second half of the parasite life cycle. The surface expression of PfEMP1 coincides with the occurrence of the adhesion of infected erythrocytes to vascular endothelium. This protein has been shown to interact with CD36, intercellular adhesion molecule-1 (ICAM-1) and chondroitin sulfate A (CSA). In this study, it is demonstrated by affinity purification and western blot analysis that PfEMP1 also functions as a cell surface ligand for P-selectin, an adhesion molecule that has been shown to mediate the rolling of infected erythrocytes under physiologic flow conditions, leading to a significant increase in adhesion to CD36 on activated platelets and microvascular endothelium.

Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria.

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-1(19) from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-1(19), we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum-immune humans and P. chabaudi-immune mice. In some individual human sera, MSP-1(19) antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-1(19)-based vaccines.

Parasite adhesion and immune evasion in placental malaria.

Parasite sequestration in the placenta is a key feature of infection by Plasmodium falciparum during pregnancy and is associated with severe adverse outcomes for both mother and baby. Here, James Beeson and colleagues draw together the findings of recent studies on parasite mechanisms that mediate this process. They review evidence for novel parasite variants that appear able to evade pre-existing immunity, for the adhesion of P. falciparum-infected erythrocytes to placental glycosaminoglycans (and the molecular basis of these parasite properties) and for the expression of var genes encoding the variant antigen and adhesive ligand P. falciparum-erythrocyte membrane protein 1 (PfEMP1).

Multiple var gene transcripts are expressed in Plasmodium falciparum infected erythrocytes selected for adhesion.

Adherence of Plasmodium falciparum-infected erythrocytes to the post-capillary endothelium is an important characteristic of malaria infection. The adhesion is mediated predominantly by P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a clonally variant protein expressed on the surface of infected red blood cells that appears to be a target of protective immunity. A multi-membered var gene family encodes PfEMP1 and switching expression of different var genes conveys different antigenic and adhesive properties to infected red blood cells. Knowledge about transcriptional control of phenotypic expression, or the mechanisms that allow multiple binding specificities, is very limited. Here, we describe a series of phenotypic selection experiments, which resulted in the expression of different PfEMP1 and the detection of multiple full-length var gene transcripts in the mature trophozoite stage. However, a dominant form of PfEMP1 appeared to be expressed, which suggested that most var transcripts do not lead to a surface expressed PfEMP1 molecule. Parasites bound to specific receptors still expressed multiple full-length var genes and mature trophozoites selected for increased adhesion to a specific receptor retained the ability to bind to multiple receptors. Our findings suggest that a defined adhesive phenotype can be associated with expression of multiple var genes.

Effect of pulmonary stretch receptor feedback and CO(2) on upper airway and respiratory pump muscle activity in the rat.

1. Our purpose was to examine the effects of chemoreceptor stimulation and lung inflation on neural drive to tongue protrudor and retractor muscles in the rat. 2. Inspiratory flow, tidal volume, transpulmonary pressure, compliance and electromyographic (EMG) activity of genioglossus (GG), hyoglossus (HG) and inspiratory intercostal (IIC) muscles were studied in 11 anaesthetized, tracheotomized and spontaneously breathing rats. Mean EMG activity during inspiration was compared with mean EMG activity during an occluded inspiration, at each of five levels of inspired CO(2) (0, 3, 6, 9 and 12 %). 3. Lung inflation suppressed EMG activity in all muscles, with the effect on both tongue muscles exceeding that of the intercostal muscles. Static elevations of end-expiratory lung volume evoked by 2 cmH(2)O positive end-expiratory pressure (PEEP) had no effect on tongue muscle activity. 4. Despite increasing inspiratory flow, tidal volume and transpulmonary pressure, the inhibition of tongue muscle activity by lung inflation diminished as arterial PCO2 (P(a),CO(2)) increased. 5. The onset of tongue muscle activity relative to the onset of IIC muscle activity advanced with increases in P(a),CO(2) but was unaffected by lung inflation. This suggests that hypoglossal and external intercostal motoneuron pools are controlled by different circuits or have different sensitivities to CO(2), lung inflation and/or anaesthetic agents. 6. We conclude that hypoglossal motoneuronal activity is more strongly influenced by chemoreceptor-mediated facilitation than by lung volume-mediated inhibition. Hypoglossal motoneurons driving tongue protrudor and retractor muscles respond identically to these stimuli.

Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in-vivo drug resistance in West Papua, Indonesia.

This study (conducted in 1996-99) examines the association of mutations in pfmdr1, dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum with in-vivo drug resistance in West Papua, Indonesia. Initially, 85 patients infected with P. falciparum were treated with chloroquine, of whom 21 were cleared of parasites, 49 had parasitaemias classified as RI, RII or RIII resistance and 1 patient had recrudescent parasitaemia. Fansidar (pyrimethamine-sulfadoxine) was the second-line treatment and 18 patients were cleared of parasites and 31 had continuing infections classified as RI, RII or RIII resistance and 1 patient had recrudescent parasitaemia. The pfmdr1, dhfr and dhps genes were examined for mutations previously shown to be associated with resistance to these drugs. In this study, mutations in pfmdr1 were associated with chloroquine resistance and mutations in both dhfr and dhps were associated with Fansidar resistance in vivo. Interestingly, Gly-437 in dhps along with Arg-59/Asn-108 in dhfr were associated with RI, RII and RIII resistance whereas Glu-540 was highly associated with only RII and RIII Fansidar resistance. This finding supports the hypothesis that the molecular basis of RI, RII and RIII Fansidar resistance involves an accumulation of mutations in both dhfr and dhps. These results suggest that mutations in both dhfr and dhps genes are a good predictor of potential Fansidar treatment failure.

Towards a malaria vaccine for Papua New Guinea.

Malaria is a major problem in Papua New Guinea, where it accounts for a high proportion of sickness and death. In addition to the human suffering, malaria also puts severe stress on the health services, and may directly hinder economic growth. A malaria vaccine would be the best, most cost-effective and safest public health measure to reduce the burden of malaria. Though considerable technical challenges are present, much natural and scientific evidence suggests a vaccine is achievable. Through the malaria vaccine program at the Papua New Guinea Institute of Medical Research, Papua New Guinea is playing a significant role in the global effort to develop a malaria vaccine, and ensuring that the malaria patterns of the Asia-Pacific region figure strongly in vaccine development strategies. Discussed here are some of the major issues to be considered as we work towards a malaria vaccine for Papua New Guinea.

Identification of glycosaminoglycan binding domains in Plasmodium falciparum erythrocyte membrane protein 1 of a chondroitin sulfate A-adherent parasite.

Accumulation of Plasmodium falciparum-infected erythrocytes in the placenta is a key feature of maternal malaria. This process is mediated in part by the parasite ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1) at the surface of the infected erythrocyte interacting with the host receptor chondroitin sulfate A (CSA) on the placental lining. We have localized CSA binding activity to two adjacent domains in PfEMP1 of an adherent parasite line and shown the presence of at least three active glycosaminoglycan binding sites. A putative CSA binding sequence was identified in one domain, but nonlinear binding motifs are also likely to be present, since binding activity in the region was shown to be dependent on conformation. Characterization of this binding region provides an opportunity to investigate further its potential as a target for antiadhesion therapy.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells.

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.

Evidence for vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes.

Plasmodium falciparum malaria parasites actively remodel the host cell cytosol and plasma membrane during the erythrocytic cycle. The focus of this investigation was to characterize intra-parasitic and -erythrocytic secretory pathways. Electron-dense vesicles, similar in appearance to mammalian secretory vesicles were detected in proximity to smooth tubo-vesicular elements at the periphery of the parasite cytoplasm in mature parasites by transmission electron microscopy. Vesicles (60-100 nm diameter), which appeared to be coated, were visualized on the erythrocytic side of the parasite vacuolar membrane and in the erythrocyte cytosol. The vesicles seemed to bind to and fuse with the erythrocyte membrane, giving rise to cup-shaped electron-dense structures, which might be intermediates in knob structure formation. Treatment of mature parasites with aluminum tetrafluoride, an activator of GTP-binding proteins, resulted in the accumulation of the vesicles with an electron-dense limiting membrane in the erythrocyte cytosol into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminum fluoride treatment. The parasite proteins PfEMP1 and PfEMP3 were found by immunoelectron microscopy to be associated with these vesicles, suggesting they are responsible for transporting these proteins to the erythrocyte membrane.