Temporal Variation of the Merozoite Surface Protein-2 Gene of Plasmodium falciparum

Extensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines against Plasmodium falciparum infection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously. MSP2 genes belonging to both major allelic families were observed at both time points. In the case of the FC27 MSP2 family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7 MSP2 family alleles were more heterogeneous. No MSP2 alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.The development of a host-protective immune response against Plasmodium falciparum takes several years and many episodes of infection, at least for children living in areas where malaria is endemic. One of the reasons for this is believed to be the large number of distinct parasite strains circulating within an area of endemicity and the assumption that exposure must occur to a sufficiently large sample of these before lasting immunity is induced. However, the detailed epidemiology of endemic malaria infection remains poorly understood at the molecular level, and there is surprisingly little nucleotide sequence data to support the concept of a large repertoire of antigenically distinct strains.There are at least six antigenically diverse proteins of the asexual stage that are known to be the target of potentially protective host responses. The definition of antigenically distinct strains involves identification of the allelic form expressed at all antigenically diverse loci—the extended antigenic haplotype. The loci would include merozoite surface protein-1 to -3 (3), apical membrane antigen-1 (17), S-antigen (6), and P. falciparum erythrocyte membrane protein-1 (PfEMP-1) (5). Such a complete molecular definition of infecting parasites is a highly ambitious task, particularly in the case of blood samples collected from patients harboring mixed infections. Accordingly, most studies focus on one or other of the antigenically diverse antigens. We have elected to study merozoite surface protein-2 (MSP2) (27), a 45- to 50-kDa glycoprotein anchored in the merozoite surface by a glycosylphosphatidylinositol anchor. This surface protein is a promising candidate for inclusion in a malaria subunit vaccine, as both in vitro and in vivo studies have demonstrated the ability of immune responses to MSP2 to inhibit parasite multiplication (23, 25). However, the efficacy of any subunit vaccine containing a single form of MSP2 may be limited by the presence of antigenically distinct parasite strains within an area of endemicity. We will adopt the recently proposed convention for parasite genes and gene products of denoting the gene sequence as MSP2 and the protein as MSP2.Sequence polymorphism has been described for MSP2 genes of both laboratory-maintained isolates (29) and field isolates (14, 16, 19, 30). Comparison of MSP2 gene sequences from these isolates reveals highly conserved 5′ and 3′ sequences that flank a central variable region. This central region is composed of repeats flanked by nonrepetitive sequences. The nonrepetitive sequences are one or other of two distinct forms that define two allelic families, FC27 and IC-1/3D7 (29). The central repeats are more variable and define the individual alleles of MSP2. There is a correlation between the general form of the central repeat sequence and the allelic family. For example, FC27 family members have variants of a central 96-bp pair sequence that may be present in one to four copies followed by a 21-bp partial repeat and a variably represented 36-bp sequence that may be present in one to five copies. In contrast, alleles belonging to the 3D7 family show a central repeat region made up of variable numbers of 12- to 24-bp repeats separated by repeating 6-bp sequences.Field studies aimed at defining the antigenic diversity of MSP2 have approached the problem by determining MSP2 gene structure by various forms of PCR. The rationale for this is that P. falciparum is haploid and MSP2 has been shown to be present in all laboratory and field isolates examined (8–10, 15). Most studies examining the distribution and frequency of different allelic forms of MSP2 have enumerated the presence of the allelic families (11, 12, 14). Whereas a skewed distribution of predominantly 3D7 family alleles exists among laboratory-adapted strains, in the field a more even distribution of FC27 and 3D7 alleles occurs. Often FC27 family alleles are more prevalent than 3D7 alleles, and novel FC27 and 3D7 family alleles have been found in field malaria strains (14, 16). Some field studies examining recurrent MSP2 infections have been performed, but these have classified MSP2 alleles on the basis of family and length of the central repeat region (7, 11, 14). This makes it difficult to form conclusions about the repertoire of repeat sequences in the circulating pool of parasites and to infer the possible action of immune responses to MSP2 repeats. We were interested to examine the sequences of MSP2 alleles circulating in an area of endemicity over time and to determine the persistence of various MSP2 alleles within a localized area. This study describes MSP2 genotypes from malaria-infected inhabitants of the Oksibil region of Irian Jaya and allows comparison of the variation in MSP2 sequences seen over a 2.5-year period within the region as a whole and in particular individuals.     

Which treatment for whom for ADHD? Moderators of treatment response in the MTA

Using receiver operating characteristics, the authors examined outcome predictors (variables associated with outcome regardless of treatment) and moderators (variables identifying subgroups with differential treatment effectiveness) in the Multimodal Treatment Study of Children with Attention-Deficit/Hyperactivity Disorder (ADHD; MTA). Treatment response was determined using parent- and teacher-reported ADHD and oppositional defiant symptoms, with levels near or within the normal range indicating excellent response. Among 9 baseline child and family characteristics, none predicted but 3 moderated treatment response. In medication management and combined treatments, parental depressive symptoms and severity of child ADHD were associated with decreased rates of excellent response; when these 2 characteristics were present, below-average child IQ was an additional moderator. No predictors or moderators emerged for behavioral and community comparison treatments. The authors discuss conceptual and clinical implications of research on treatment moderators.     

Diagnosis of gastrointestinal stromal tumors: A consensus approach

As a result of major recent advances in understanding the biology of gastrointestinal stromal tumors (GISTs), specifically recognition of the central role of activating KIT mutations and associated KIT protein expression in these lesions, and the development of novel and effective therapy for GISTs using the receptor tyrosine kinase inhibitor STI-571, these tumors have become the focus of considerable attention by pathologists, clinicians, and patients. Stromal/mesenchymal tumors of the gastrointestinal tract have long been a source of confusion and controversy with regard to classification, line(s) of differentiation, and prognostication. Characterization of the KIT pathway and its phenotypic implications has helped to resolve some but not all of these issues. Given the now critical role of accurate and reproducible pathologic diagnosis in ensuring appropriate treatment for patients with GIST, the National Institutes of Health convened a GIST workshop in April 2001 with the goal of developing a consensus approach to diagnosis and morphologic prognostication. Key elements of the consensus, as described herein, are the defining role of KIT immunopositivity in diagnosis and a proposed scheme for estimating metastatic risk in these lesions, based on tumor size and mitotic count, recognizing that it is probably unwise to use the definitive term "benign" for any GIST, at least at the present time.     

Efficiency of the ortho VITROS assay for detection of hepatitis C virus-specific antibodies increased by elimination of supplemental testing of samples with very low sample-to-cutoff ratios

The clinical significance of specimens with low sample-to-cutoff (S/Co) ratios in the Ortho VITROS chemiluminescence assay (CIA) for detection of antibodies to hepatitis C virus (HCV) was evaluated. In one study of 482 CIA-reactive samples, none of the 83 samples with S/Co ratios of < 5 was HCV RNA positive. In a subsequent study, 332 samples with S/Co ratios of between 1 and 20 were tested with the recombinant immunoblot assay (RIBA). None of the 163 samples with S/Co ratios of < 5 was RIBA positive, 83% were RIBA negative, and 28 samples (18%) were RIBA indeterminate. HCV RNA and/or clinical evidence of hepatitis was not found in the 27 indeterminate cases examined. These results show that over 99% of samples with very low S/Co ratios (< or = 5) have no evidence of HCV infection. Therefore, we suggest that the HCV antibody testing algorithm for the VITROS assay might be modified to eliminate supplemental testing of samples with very low S/Co ratios.     

Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle

For most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genesin vivo, suggesting applications to developmental studies and gene therapy.