Restricted genetic and antigenic diversity of Plasmodium falciparum under mesoendemic transmission in the Venezuelan Amazon.

The study of genetic diversity in malaria populations is expected to provide new insights for the deployment of control measures. Plasmodium falciparum diversity in Africa and Asia is thought to reflect endemicity. In comprehensive epidemiological surveys reported here the genetic and antigenic structure of P. falciparum in the Venezuelan Amazon were studied over a 2-year period. DNA polymorphisms in glutamate-rich protein (GLURP), merozoite-surface protein 1 (MSP1) and MSP2 genes, in a multicopy element (PfRRM), all showed low diversity, 1 predominant genotype, and virtually no multi-clonal infections. Moreover, linkage disequilibrium was seen between GLURP, MSP1 and MSP2. Specific antibody responses against MSP1 and MSP2 recombinant antigens reflected the low genetic diversity observed in the parasite population. This is unexpected in a mesoendemic area, and suggests that the low diversity here may not only relate to endemicity but to other influences such as a bottleneck effect. Linkage disequilibrium and a predominant genotype may imply that P. falciparum frequently propagates with an epidemic or clonal population structure in the Venezuelan Amazon.

Differential patterns of human immunoglobulin G subclass responses to distinct regions of a single protein, the merozoite surface protein 1 of Plasmodium falciparum.

Comparisons of immunoglobulin G (IgG) subclass responses to the major polymorphic region and to a conserved region of MSP-1 in three cohorts of African villagers exposed to Plasmodium falciparum revealed that responses to Block 2 are predominantly IgG3 whereas antibodies to MSP-1(19) are mainly IgG1. The striking dominance of IgG3 to Block 2 may explain the short duration of this response and also the requirement for continuous stimulation by malaria infection to maintain clinical immunity.

Issues of aging and adherence to health interventions.

This review article describes several processes and considerations that are important to adherence in aging research and potential strategies that could be used to facilitate adherence among older adults. In many large aging trials participants are purposefully selected to reduce the risk of suboptimal adherence and retention. This selection often involves screening out those with barriers such as transportation needs, sensory deficits, functional dependence, major diseases limiting life expectancy, or apparent psychological distress. However, trends toward extending interventions to the general population of older adults require specific knowledge about the circumstances and processes that support adherence among older adults or the conditions that make them vulnerable to adherence problems. Addressing the diversity of needs, expectations, and capabilities of older adults that promote adherence is a key consideration in aging research. Control Clin Trials 2000;21:171S-183S

A principal target of human immunity to malaria identified by molecular population genetic and immunological analyses.

New strategies are required to identify the most important targets of protective immunity in complex eukaryotic pathogens. Natural selection maintains allelic variation in some antigens of the malaria parasite Plasmodium falciparum. Analysis of allele frequency distributions could identify the loci under most intense selection. The merozoite surface protein 1 (Msp1) is the most-abundant surface component on the erythrocyte-invading stage of P. falciparum. Immunization with whole Msp1 has protected monkeys completely against homologous and partially against non-homologous parasite strains. The single-copy msp1 gene, of about 5 kilobases, has highly divergent alleles with stable frequencies in endemic populations. To identify the region of msp1 under strongest selection to maintain alleles within populations, we studied multiple intragenic sequence loci in populations in different regions of Africa and Southeast Asia. On both continents, the locus with the lowest inter-population variance in allele frequencies was block 2, indicating selection in this part of the gene. To test the hypothesis of immune selection, we undertook a large prospective longitudinal cohort study. This demonstrated that serum IgG antibodies against each of the two most frequent allelic types of block 2 of the protein were strongly associated with protection from P. falciparum malaria.

Using a hand-held computer to collect data in an orthopedic outpatient clinic: a randomized trial of two survey methods.

OBJECTIVES: In a randomized study, the authors examine how data can be collected at the point of care. Specifically, examining to what extent handheld computer data collection systems introduce bias or increase respondent difficulty. METHODS: Volunteers were randomized to 1 of 2 survey methods: the hand-held computer or a paper and pencil form of similar content. Differences between group scale scores were compared using the Wilcoxon (rank sum) test. RESULTS: The hand-held computer system produced comparable scores to paper and pencil surveys. However, there was evidence of lower internal consistency reliability with the handheld computer. CONCLUSIONS: This study demonstrated the comparability of the hand-held computer methodology to the paper and pencil methodology in obtaining survey information in an ambulatory clinic. The hand-held computer method of survey data collection offers an alternative to paper methods when point-of-care administration is acceptable. Preliminary evidence shows that this method produces comparable results to paper forms.

A longitudinal study of human antibody responses to Plasmodium falciparum rhoptry-associated protein 1 in a region of seasonal and unstable malaria transmission.

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a nonpolymorphic merozoite antigen that is considered a potential candidate for a malaria vaccine against asexual blood stages. In this longitudinal study, recombinant RAP1 (rRAP1) proteins with antigenicity similar to that of P. falciparum-derived RAP1 were used to analyze antibody responses to RAP1 over a period of 4 years (1991 to 1995) of 53 individuals naturally exposed to P. falciparum malaria. In any 1 year during the study, between 23 and 39% of individuals who had malaria developed immunoglobulin G (IgG) antibodies detectable with at least one rRAP1 protein. However, the anti-RAP1 antibody responses were detected only during or shortly after clinical malarial infections. RAP1 antibody levels declined rapidly (within 1 to 2 months) following drug treatment of the infections. No anti-RAP1 antibodies were usually detected a few months after the end of malaria transmission, during the dry season, or by the start of the next malaria season. Thus, RAP1 IgG responses were very short-lived. The short duration of RAP1 antibody response may explain the apparent lack of response in a surprisingly high proportion of individuals after clinical malarial infections. For some individuals who experienced more than one malarial infection, a higher anti-RAP1 antibody response to subsequent infections than to earlier infections was observed. This suggested secondary responses to RAP1 and thus the development of immunological memory for RAP1.

EDI and imaging automate the business office.

By implementing electronic remittance posting and imaging in patient accounting, New York's Memorial Sloan-Kettering Cancer Center was able to eliminate 22 full-time equivalent supervisory and staff positions and save $365,000 in physical space, microfilm and media costs the first year the new system was operational. Memorial Sloan-Kettering currently is participating in a pilot program with its Medicare fiscal intermediary to determine how EDI can be used to manage claim-status information between payer and provider. By combining EDI, fax, and imaging technologies, Memorial Sloan-Kettering is making its patient accounting process more efficient and providing better service to its patients.

Green tobacco sickness.

OBJECTIVE: To describe the health impact of harvesting tobacco and to suggest prevention and risk reduction strategies to avoid contracting green tobacco sickness (GTS). DATA SOURCES: A literature search of Medline, Toxline, and Toxline65 with the terms "green", "tobacco", and "sickness" covering the years 1966-1998. STUDY SELECTION: All studies, reviews, and commentaries that provided information on the health effects of harvesting green tobacco and disease prevention strategies. DATA SYNTHESIS: GTS occurs when tobacco workers hand-harvest, cut, or load tobacco plants, usually in the early morning or after a rainfall when tobacco plants are covered with moisture. GTS occurs through skin exposure to dissolved nicotine from tobacco leaves. Symptoms of GTS include weakness, headache, nausea, vomiting, dizziness, abdominal cramps, breathing difficulty, abnormal temperature, pallor, diarrhoea, chills, fluctuations in blood pressure or heart rate, and increased perspiration and salivation. The onset of the illness is three to 17 hours after exposure and the duration of illness is one to three days. Initial treatment includes cessation of work, change of clothing, showering, fluid intake, and rest. In more extreme cases, intravenous rehydration, anti-emetics, and dimenhydrinate are administered. Protective, water-resistant clothing; chemical-resistant gloves, boots, and socks; working in dry conditions; and dimenhydrinate can reduce the likelihood of contracting GTS. CONCLUSIONS: It is important to provide education to tobacco workers and employers about GTS. An international public awareness campaign about GTS timed to coincide with the tobacco harvest, along with enforced worker safety regulations, should be undertaken to protect the health of individuals working in tobacco production.

A longitudinal study of type-specific antibody responses to Plasmodium falciparum merozoite surface protein-1 in an area of unstable malaria in Sudan.

Merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a malaria vaccine candidate Ag. Immunity to MSP-1 has been implicated in protection against infection in animal models. However, MSP-1 is a polymorphic protein and its immune recognition by humans following infection is not well understood. We have compared the immunogenicity of conserved and polymorphic regions of MSP-1, the specificity of Ab responses to a polymorphic region of the Ag, and the duration of these responses in Sudanese villagers intermittently exposed to P. falciparum infections. Recombinant Ags representing the conserved N terminus (Block 1), the conserved C terminus, and the three main types of the major polymorphic region (Block 2) of MSP-1 were used to determine the specificity and longitudinal patterns of IgG Ab responses to MSP-1 in individuals. Abs from 52 donors were assessed before, during, and after malaria transmission seasons for 4 yr. Ags from the Block 1 region were rarely recognized by any donor. Responses to the C-terminal Ag occurred in the majority of acutely infected individuals and thus were a reliable indicator of recent clinical infection. Ags from the polymorphic Block 2 region of MSP-1 were recognized by many, although not all individuals after clinical malaria infections. Responses to Block 2 were type specific and correlated with PCR typing of parasites present at the time of infection. Responses to all of these Ags declined within a few months of drug treatment and parasite clearance, indicating that naturally induced human Ab responses to MSP-1 are short lived.

Antigenicity of recombinant proteins derived from rhoptry-associated protein 1 of Plasmodium falciparum.

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a potential component of a malaria vaccine. We have expressed in Escherichia coli eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice. Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (PfRAP1), as determined by indirect immunofluorescence and by immunoblotting. These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia. Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature PfRAP1. The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region. Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody. Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1.

Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.

Merozoite surface protein-1 (MSP-1) of the human malaria parasite Plasmodium falciparum undergoes at least two endoproteolytic cleavage events during merozoite maturation and release, and erythrocyte invasion. We have previously demonstrated that mAbs which inhibit erythrocyte invasion and are specific for epitopes within a membrane-proximal, COOH-terminal domain of MSP-1 (MSP-119) prevent the critical secondary processing step which occurs on the surface of the extracellular merozoite at around the time of erythrocyte invasion. Certain other anti-MSP-119 mAbs, which themselves inhibit neither erythrocyte invasion nor MSP-1 secondary processing, block the processing-inhibitory activity of the first group of antibodies and are termed blocking antibodies. We have now directly quantitated antibody-mediated inhibition of MSP-1 secondary processing and invasion, and the effects on this of blocking antibodies. We show that blocking antibodies function by competing with the binding of processing-inhibitory antibodies to their epitopes on the merozoite. Polyclonal rabbit antibodies specific for certain MSP-1 sequences outside of MSP-119 also act as blocking antibodies. Most significantly, affinity-purified, naturally acquired human antibodies specific for epitopes within the NH2-terminal 83-kD domain of MSP-1 very effectively block the processing-inhibitory activity of the anti-MSP-119 mAb 12.8. The presence of these blocking antibodies also completely abrogates the inhibitory effect of mAb 12.8 on erythrocyte invasion by the parasite in vitro. Blocking antibodies therefore (a) are part of the human response to malarial infection; (b) can be induced by MSP-1 structures unrelated to the MSP-119 target of processing-inhibitory antibodies; and (c) have the potential to abolish protection mediated by anti-MSP-119 antibodies. Our results suggest that an effective MSP-119-based falciparum malaria vaccine should aim to induce an antibody response that prevents MSP-1 processing on the merozoite surface.

Antigenicity of recombinant proteins derived from Plasmodium falciparum merozoite surface protein 1.

We have expressed seven recombinant antigens representing two N-terminal regions of the polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum. The antigens include the MAD20 and Palo Alto forms of the relatively conserved Block 1 region, and variants of the Block 2 region from isolates 3D7, Palo Alto FUP, MAD20, Wellcome and RO33, that are representative of a range or amino acid sequence diversity in this most polymorphic section of MSP-1. All recombinant antigens have been able to immunise mice to produce polyclonal antibodies which specifically recognise parasite MSP-1 in indirect immunofluorescence assays and in Western blots. The recombinant antigens also react appropriately in ELISA with murine monoclonal antibodies specific for variant epitopes in Block 2 of MSP-1. These results show that the antigenic structure of the recombinant proteins is similar to that of the native MSP-1 product from parasites. Importantly, human sera from malaria-exposed individuals contain IgG antibodies that recognise very specifically one or another of the Block 2 types, showing that different Block 2 types are immunogenic, antigenically distinct and distinguishable when presented during natural infections. In contrast, the conserved Block 1 is rarely recognised by human antibodies.

Plasmodium falciparum: gp195 tripeptide repeat-specific monoclonal antibody inhibits parasite growth in vitro.

Seven monoclonal antibodies (mAbs) were produced to the precursor of the merozoite surface antigens (MSA-1 or gp195) using the Plasmodium falciparum Uganda-Palo Alto isolate. Three mAbs (CE2, DB8, and EB2) reacted with epitopes on the 83-kDa N-terminal processing fragment by immunoprecipitation of radiolabeled proteins and in immunoblots of native and recombinant proteins. Three other mAbs (BC9, AG5, and AD9) reacted with epitopes on the 42- and 19-kDa C-terminal processing fragments while one remaining mAb (24A1.7) reacted with only 150- and 110-kDa intermediate processing fragments. Epitopes were mapped to either conserved or dimorphic regions of the expressed protein when parasite isolates with known MSA-1 alleles were examined by indirect immunofluorescence. Moreover, one mAb (CE2), specific for the variable tripeptide repeat region SAQ(SGT)5, was growth inhibitory for P. falciparum in vitro. Growth inhibition by the mAb was concentration dependent and its parasite-neutralizing properties were not enhanced when used in combination with other gp195-specific mAbs. These results may be useful in the elucidation of biological variation of field isolates and in the definition of immunologically relevant epitopes in a gp195-based malaria vaccine.

Differential antibody recognition of FC27-like Plasmodium falciparum merozoite surface protein MSP2 antigens which lack 12 amino acid repeats.

Three alleles of the FC27-type allelic family of the MSP2 gene of the malaria parasite Plasmodium falciparum have been sequenced from parasites from the field (The Gambia and Tanzania). These alleles lack the 12 amino acid repeat units which are usual in this family of MSP2 alleles. We have investigated the recognition by sera from an endemic area (The Gambia) of three recombinant MSP2 proteins that have 5, 1 and no copies of this repeat region. Antibody recognition of these recombinant proteins varied according to the number of repeats present. High titre antibody levels were seen with most sera using the recombinant protein with 5 x 12-mer repeats, whereas only low responses were measured using proteins containing 1 or no 12-mer repeats. Several sera entirely failed to recognise the protein which lacked 12-mer repeats. The data suggest that variation in the number of tandem repeat sequences could allow the parasite to avoid high avidity antibody binding and this may allow escape from immune recognition.

Human antibody response to Plasmodium falciparum merozoite surface protein 2 is serogroup specific and predominantly of the immunoglobulin G3 subclass.

MSP2 is a merozoite surface protein of Plasmodium falciparum and, as such, is a potential component of a malaria vaccine. In this study, we have used a panel of recombinant MSP2 antigens in enzyme-linked immunosorbent assays to investigate the recognition of MSP2 by antibodies from malaria-immune human serum. These recombinant antigens include full-length proteins of serogroups A and B and fragments representing the conserved, group-specific, or repeat regions of each serogroup. Ninety-five percent of the serum samples tested contained MSP2-specific antibodies: 81% of serum samples tested responded to serogroup A, and 86% responded to serogroup B. The antibody response is directed almost exclusively towards dimorphic and polymorphic regions of MSP2; the conserved regions are rarely recognized, and antibodies to serogroups A and B do not cross-react. Interestingly, the antibody response is predominately of the cytophilic and complement-fixing subclass immunoglobulin G3.

Serum antibodies from malaria-exposed people recognize conserved epitopes formed by the two epidermal growth factor motifs of MSP1(19), the carboxy-terminal fragment of the major merozoite surface protein of Plasmodium falciparum.

The major merozoite surface protein of Plasmodium falciparum (PfMSP1) is a candidate antigen for a malaria vaccine. A 19-kDa C-terminal processing product of PfMSP1 (PfMSP1(19)) is composed of two domains sharing a cysteine-rich motif with epidermal growth factor (EGF) and is the target of monoclonal antibodies which block erythrocyte invasion in vitro. We have evaluated human antibody responses to PfMSP1(19) by using recombinant proteins representing the EGF motifs encoded by the two main alleles of the MSP1 gene. We find that both EGF motifs are antigenic but that only 10 to 20% of malaria-exposed individuals have serum antibodies that recognized either of the motifs. When both EGF motifs were expressed together as a single protein, they were recognized by more than 40% of sera from malaria-exposed individuals. Major epitopes recognized by human antibodies are dependent upon the correct tertiary structure of the protein and are cross-reactive between the different allelic sequences of PfMSP1(19). This suggests that antibodies induced by vaccination with one or the other allelic forms of the protein could recognize all strains of P. falciparum. Immunoglobulin G (IgG) subclass-specific enzyme immunoassays indicate that PfMSP1(19) antibodies are predominantly of the IgG1 subclass.

Memorial Sloan-Kettering cures paperwork problems with document imaging.

The world's largest cancer center, New York's Memorial Sloan-Kettering, is finding concrete efficiencies--and real savings--by implementing "OSCAR" the Optical System Controlling Administrative Records. The system now handles more than 2.7 million documents, including patient accounts and medical records.

Tumour necrosis factor and interleukin-6 production induced by components associated with merozoite proteins of Plasmodium falciparum.

P. falciparum merozoite antigens, merozoite surface protein-1 (MSP-1) and rhoptry associated protein-1 (RAP-1), were shown to be liberated into the supernatant of in vitro parasite cultures and to be included in the endotoxin-like exoantigen complex, previously designated Ag7. Material affinity purified from culture supernatants, using immobilized monoclonal antibodies specific for RAP-1 or MSP-1, stimulated normal human mononuclear cells to produce TNF and IL-6 in vitro. However, stimulation of TNF was absent, and that of IL-6 was reduced, when the antigens were purified from detergent extracts of infected erythrocytes. These results indicate that the RAP-1 and MSP-1 proteins themselves do not stimulate the production of TNF. Instead, other components associating with these exoantigens may be responsible for the TNF production. Mouse antisera blocking TNF production stimulated by P. yoelii exoantigens also blocked TNF production stimulated by material affinity purified from P. falciparum culture supernatants using RAP-1 specific monoclonal antibody, indicating the conserved structure of the TNF inducing component.

Inhibitory monoclonal antibodies recognise epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum.

The low-molecular-weight rhoptry-associated protein (RAP) complex of Plasmodium falciparum consists of at least two gene products, RAP-1 and RAP-2, and has the ability to immunise Saimiri monkeys against experimental P. falciparum infection. Several monoclonal antibodies specifically recognise this complex and in this study we show that purified immunoglobulin derived from these monoclonals is capable of inhibiting parasite growth in vitro. It has previously been shown that RAP-1 initially appears as an 80-kDa protein (p80) in early schizogony and is processed to a 65-kDa protein (p65) in late schizogony. Several of the inhibitory monoclonals recognise both the 80- and 65-kDa proteins by Western blot analysis suggesting that they recognise linear epitopes on RAP-1. We have mapped these epitopes by testing the reactivity of the monoclonals against fragments of the rap-1 gene expressed as beta-galactosidase fusion proteins and subsequently against synthetic peptides. All of the epitopes map to a region 10-20 amino acids C-terminal to the proteolytic cleavage site for the processing of p80 to p65 at amino acid 190. We also show that the 65-kDa protein is not present in purified merozoites, suggesting that its generation is associated with merozoite release rather than erythrocyte invasion. These results are discussed with respect to possible inhibitory mechanisms for the monoclonals.