Desorption-ionization mass spectrometry using deposited nanostructured silicon films.

We present a method for desorption ionization on silicon based on novel column/void-network-deposited silicon thin films. A number of different peptides and proteins in the < or = 6000 Daltons range are analyzed by time-of-flight mass spectrometry in this demonstration of our approach. A variety of sample preparation conditions, including the use of chemical additives, surface treatments, and sample purification are used to show the potential of mass analysis using deposited column/void-network silicon films for high throughput proteomic screening.

Antibody recognition of rodent malaria parasite antigens exposed at the infected erythrocyte surface: specificity of immunity generated in hyperimmune mice.

In regions where malaria is endemic, inhabitants remain susceptible to repeated reinfection as they develop and maintain clinical immunity. This immunity includes responses to surface-exposed antigens on Plasmodium sp.-infected erythrocytes. Some of these parasite-encoded antigens may be diverse and phenotypically variable, and the ability to respond to this diversity and variability is an important component of acquired immunity. Characterizing the relative specificities of antibody responses during the acquisition of immunity and in hyperimmune individuals is thus an important adjunct to vaccine research. This is logistically difficult to do in the field but is relatively easily carried out in animal models. Infections in inbred mice with rodent malaria parasite Plasmodium chabaudi chabaudi AS represent a good model for Plasmodium falciparum in humans. This model has been used in the present study in a comparative analysis of cross-reactive and specific immune responses in rodent malaria. CBA/Ca mice were rendered hyperimmune to P. chabaudi chabaudi (AS or CB lines) or Plasmodium berghei (KSP-11 line) by repeated infection with homologous parasites. Serum from P. chabaudi chabaudi AS hyperimmune mice reacted with antigens released from disrupted P. chabaudi chabaudi AS-infected erythrocytes, but P. chabaudi chabaudi CB and P. berghei KSP-11 hyperimmune serum also contained cross-reactive antibodies to these antigens. However, antibody activity directed against antigens exposed at the surfaces of intact P. chabaudi chabaudi-infected erythrocytes was mainly parasite species specific and, to a lesser extent, parasite line specific. Importantly, this response included opsonizing antibodies, which bound to infected erythrocytes, leading to their phagocytosis and destruction by macrophages. The results are discussed in the context of the role that antibodies to both variable and invariant antigens may play in protective immunity in the face of continuous susceptibility to reinfection.

Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand.

Plasmodium falciparum isolates were obtained from Thai patients attending a malaria clinic on the Thai-Kampuchean border over 4 cross-sectional surveys carried out at 3-monthly intervals. The genetic structure of the parasite populations was determined by nested polymerase chain reaction (PCR) amplification of polymorphic regions of 3 P. falciparum antigen genes: msp1, msp2 and glurp. Although a high degree of diversity characterized these isolates, the overall population structure of the parasites associated with patent malaria infections was observed to remain relatively stable over time. The highest degree of polymorphism was observed with msp2, and the mean number of lines per infection (multiplicity of infection) calculated with this marker was higher than that obtained using msp1 or glurp alone, or combined. Infections with > or = 2 parasite lines were seen in 76% of the samples, and were proportionally more numerous at the start and end of the rainy season. Two interesting exceptions to the random distribution were observed and involved 2 allelic variants which in one case were found dissociated (msp1 MAD20-family) and in the other were associated (msp2 FC27-family). The epidemiological significance of these types of data is discussed.

Acute Plasmodium chabaudi chabaudi malaria infection induces antibodies which bind to the surfaces of parasitized erythrocytes and promote their phagocytosis by macrophages in vitro.

CBA/Ca mice infected with 5 x 10(4) Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks ( approximately 40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P. c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken from P. c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P. c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P. c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P. c. chabaudi CB) did not mediate the same effect against P. c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.

[Genotyping of Plasmodium falciparum isolates by amplification of glutamate-rich protein gene using polymerase chain reaction].

AIM: To develop a genotyping method based on amplifying glutamate-rich protein (GLURP) gene for the diagnosis and identification of Plasmodium falciparum. METHODS: Two pairs of primers specific for GLURP gene of P. falciparum were designed and synthesized. R2 polymorphic domain of GLURP gene was amplified by nested PCR, which was applied to genotyping of P. falciparum isolates obtained from patients attending the malaria clinic at the village of Borai, Thailand. RESULTS: Conspicuous polymorphism of GLURP alleles in natural populations of P. falciparum was found. 290 GLURP alleles were detected in 154 P. falciparum infections. Among the above-mentioned alleles, 12 different GLURP genotypes were distinguished according to different DNA sizes. Of them, the most frequently found allele was a variant of 770 bp, the least allele was that of 1,100 bp. More than 43% of the patients were found to be infected with mixed alleles. No apparent change for frequencies of the 12 different alleles was found in the 9-month longitudinal study. CONCLUSION: A genotyping method is developed for the research of strain taxonomy and pathogenesis of malaria parasites.

Heritability of epileptic seizures in the Belgian tervueren.

Survey data were collected on the incidence of epileptic seizures in 997 Belgian tervuerens. The heritability of this disorder was estimated as 0.77 (with a 95 per cent confidence interval spanning from 0.65 to 0.88) using a Bayesian analysis in an ordered categorical threshold model. Single locus models do not appear adequate as an explanation for this inherited seizure disorder. The high heritability estimate suggests that breeders can wage a successful battle against this disorder by breeding unaffected individuals, particularly those from families with no history of seizures.

Glycosylphosphatidylinositols of Plasmodium chabaudi chabaudi: a basis for the study of malarial glycolipid toxins in a rodent model.

Free and protein-bound glycosylphosphatidylinositols (GPIs) of the blood stages of the rodent malarial parasite Plasmodium chabaudi chabaudi AS were identified and characterized. TLC analysis of material extracted by organic solvents from metabolically labelled parasites revealed a distinct set of glycolipids. These glycolipids were identified as GPIs by specific chemical and enzymic treatments and by structural analysis of their glycan and hydrophobic parts. These analyses revealed that P.c.chabaudi AS synthesizes a set of GPI-biosynthesis intermediates and two potential GPI-anchor precursors exhibiting the following structures: ethanolamine-phosphate [(alpha1-2)mannose]mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine - (acyl) inositol-phosphate-diacylglycerol (P.ch. alpha) and ethanolamine-phosphate - mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine-(acyl)inositol-phosphate-diacylglycerol (P.ch. beta). One of these GPI-anchor precursors (P.ch. alpha) possesses the same carbohydrate structure as the GPI membrane anchor of merozoite surface protein-1 from P.c.chabaudi AS.

A single gene copy merozoite surface antigen and immune evasion?

During the course of chronic malaria infection antigenic variants of a parasite antigen are expressed and exposed on the surface of infected erythrocyte membranes. There also exists a number of apparently invariant single gene copy blood-stage antigens, exposed or non-exposed, which have been shown to afford immunity under experimental conditions. To determine why the host, presented with invariant 'protective' antigens, is unable to control infections effectively, immunity to a representative single gene copy antigen, the merozoite surface protein 1 (MSP1) was investigated in Plasmodium chabaudi chabaudi AS, a murine model of chronic malaria. Immunization with monoclonal antibody affinity purified native MSP1 resulted in enhanced control of parasitaemia on challenge, irrespective of the parasite inoculum size; challenge with a single parasite, however, suggested that expansion of resistant parasite subpopulations was not occurring. Challenge of mice immunized with recombinant fusion proteins encoding N- or C-terminal regions of the P.c. chabaudi AS MSP1 produced inconsistent effects, often parasitaemias were indistinguishable from controls despite significant anti-MSP1 antibody responses. The not unlikely contamination of MSP1 native preparations with erythrocyte (E) components was considered. Immunization with a mixture of the MSP1 C-terminus recombinant polypeptide and a Triton X-100 solubilized lysate of normal E resulted in enhanced control of parasitaemia, however, no effect was seen after administration of either component on its own. Co-immunization of E with the N-terminus polypeptide reversed the inhibition seen, on this occasion with this construct alone.

Identificación de genotipos de cepas aisladas de Plasmodium falciparum mediante la reacción en cadena de la polimerasa y sus posibles usos en estudios epidemiológicos / Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies

La epidemiología del paludismo es el resultado de la interacción que tienen entre sí y con el medio circundante tres acervos genéticos: el del parásito, el del ser humano y el del mosquito vector. Actualmente se están elaborando métodos para caracterizar la genética de las poblaciones humanas en riesgo y de los posibles vectores, y a fin de llegar a conocer más a fondo la epidemiología, mecanismos patógenos y biología de este parásito también sería enormemente útil caracterizar las poblaciones naturales de Plasmodium y su distribución en huéspedes humanos y en insectos de zonas de estudio determinadas, especialmente si este enfoque se combina con estudios simultáneos en seres humanos y con los vectores. En este trabajo se describe un ensayo basado en la reacción en cadena de la polimerasa (RCP), que proporciona un método sensible, práctico y reproducible para caracterizar distintas poblaciones de parásitos de una misma especie. Con el fin de ilustrar la idoneidad de este tipo de ensayo, se escogieron y amplificaron con la RCP cuatro dominios polimórficos de los genes de tres proteínas de P. falciparum (los bloques 2 y 4 de la proteína de superficie del merozoito tipo 1 (PSM1), la tipo 2 (PSM2) y la proteína rica en glutamato (PRGLU) y una región casi enteramente conservada (el bloque 17 de la PSM1). Sirvieron de molde para amplificar con la RCP los ADN derivados de 15 líneas de P. falciparum cultivadas in vitro (siete de las cuales fueron clonadas) y de muestras de sangre de pacientes infectados procedentes de Tailandia. Los productos de la amplificación se analizaron por electroforesis en gel para detectar polimorfismos de longitud. Se detectaron siete variantes alélicas de la PRGLU, cinco del bloque 2 de la PSM1, tres del bloque 4 de la PSM1 y nueve de la PSM2. Este alto grado de polimorfismo se puede usar para caracterizar la composición genética de cualquier población de parásitos en un momento dado. En este trabajo se examina la aplicabilidad de esta forma de identificar genotipos en el campo de la epidemiología y se recomienda la adopción de patrones internacionales para su empleo, de tal modo que se puedan comparar los datos obtenidos en distintos lugares y momentos (AU)

Identificación de genotipos de cepas aisladas de Plasmodium falciparum mediante la reacción en cadena de la polimerasa y sus posibles usos en estudios epidemiológicos / Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies

La epidemiología del paludismo es el resultado de la interacción que tienen entre sí y con el medio circundante tres acervos genéticos: el del parásito, el del ser humano y el del mosquito vector. Actualmente se están elaborando métodos para caracterizar la genética de las poblaciones humanas en riesgo y de los posibles vectores, y a fin de llegar a conocer más a fondo la epidemiología, mecanismos patógenos y biología de este parásito también sería enormemente útil caracterizar las poblaciones naturales de Plasmodium y su distribución en huéspedes humanos y en insectos de zonas de estudio determinadas, especialmente si este enfoque se combina con estudios simultáneos en seres humanos y con los vectores. En este trabajo se describe un ensayo basado en la reacción en cadena de la polimerasa (RCP), que proporciona un método sensible, práctico y reproducible para caracterizar distintas poblaciones de parásitos de una misma especie. Con el fin de ilustrar la idoneidad de este tipo de ensayo, se escogieron y amplificaron con la RCP cuatro dominios polimórficos de los genes de tres proteínas de P. falciparum (los bloques 2 y 4 de la proteína de superficie del merozoito tipo 1 (PSM1), la tipo 2 (PSM2) y la proteína rica en glutamato (PRGLU) y una región casi enteramente conservada (el bloque 17 de la PSM1). Sirvieron de molde para amplificar con la RCP los ADN derivados de 15 líneas de P. falciparum cultivadas in vitro (siete de las cuales fueron clonadas) y de muestras de sangre de pacientes infectados procedentes de Tailandia. Los productos de la amplificación se analizaron por electroforesis en gel para detectar polimorfismos de longitud. Se detectaron siete variantes alélicas de la PRGLU, cinco del bloque 2 de la PSM1, tres del bloque 4 de la PSM1 y nueve de la PSM2. Este alto grado de polimorfismo se puede usar para caracterizar la composición genética de cualquier población de parásitos en un momento dado. En este trabajo se examina la aplicabilidad de esta forma de identificar genotipos en el campo de la epidemiología y se recomienda la adopción de patrones internacionales para su empleo, de tal modo que se puedan comparar los datos obtenidos en distintos lugares y momentos (AU)

Se publica también en inglés en el Bull. WHO. Vol. 73(1), 1995

Processing of the Plasmodium chabaudi chabaudi AS merozoite surface protein 1 in vivo and in vitro.

Processing of the Plasmodium merozoite surface protein 1 (MSP-1) has been described for parasites maintained under in vitro conditions. We have now demonstrated, using CBA/Ca mice infected with Plasmodium chabaudi chabaudi AS, that MSP-1 processing also occurs in vivo. The major proteolytic cleavage sites and a processing scheme were deduced from N-terminal amino-acid sequences of the MSP-1 breakdown products. Comparison of MSP-1 processing in P. falciparum and P.c. chabaudi indicates a degree of conservation and in two cases the position of protease cleavage appears identical. Significant amounts of MSP-1 polypeptides are found in plasma during schizogony. Various aspects of MSP-1 processing including immunological and physiological reactions in the host during the critical period of schizogony can now be examined in vivo.

Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies.

The epidemiology of malaria results from the interactions of three gene pools--parasite, human, and mosquito vector--with one another and with their environment. Methods are being developed for characterizing the genetics of human populations at risk and of potential vectors. The characterization of natural populations of Plasmodium and knowledge of their distribution within the human and insect hosts in any given area under study would also greatly enhance understanding of the epidemiology, pathology and biology of this parasite, particularly when combined with simultaneous human and vector studies. This paper describes a polymerase chain reaction (PCR)-based assay which provides a sensitive, reproducible and practical method by which parasite populations within species can be characterized. In order to illustrate the suitability of the PCR assay, four polymorphic domains on the genes of three P. falciparum proteins (MSP1 blocks 2 and 4, MSP2, and GLURP) and one largely conserved region (MSP1 block 17) were chosen for amplification by PCR. DNA derived from 15 in-vitro cultured lines of P. falciparum (7 of which were cloned) and from blood samples obtained from infected patients in Thailand were used as templates for PCR amplification. The amplification products were analysed by gel electrophoresis for length polymorphisms. Seven allelic variants of GLURP, five of MSP1 block 2, three of MSP1 block 4, and nine of MSP2 were detected. This high degree of polymorphism can be used to characterize the genetic composition of any parasite population, at a given time. The paper discusses the applicability of this type of genotyping to epidemiology and urges the adoption of international standards for its use so that data from different areas and different times can be compared.

Plasmodium falciparum: selective growth of subpopulations from field samples following in vitro culture, as detected by the polymerase chain reaction.

Analysis of the Plasmodium falciparum parasites circulating in the blood of infected persons frequently reveals the presence of two or more genetically distinct parasite populations. P. falciparum parasites cultured in vitro, from blood specimens collected in the field, are often used for biological, immunological, and drug-resistance investigations relating to the epidemiology in the area concerned or on the assumption that the parasites which grow in vitro are in general representative of all P. falciparum parasites. By using the polymerase chain reaction to detect and characterize a number of parasite polymorphic genes with great sensitivity, the composition of P. falciparum populations from 51 isolates were compared on the day of collection and following 2 months of in vitro culture. It was found that substantial changes in the parasite population profile could be detected in ca. 70% of the samples analyzed. The implications of this observation for studies using parasite isolates cultured in vitro are discussed.

A gene coding for a high-molecular mass rhoptry protein of Plasmodium yoelii.

We describe the deduced amino acid sequence for a gene encoding a high molecular mass rhoptry protein of Plasmodium yoelii. The sequence was obtained from an EcoRI genomic clone that overlaps a short DraI fragment isolated previously. The open reading frame consists of 2294 codons and putative hydrophobic signal and membrane anchor sequences were identified. Similarity with sequence from a clone coding for part of a Plasmodium vivax reticulocyte-binding protein was noted. Based on the sequence and location of the protein and the biological properties of antibodies that react with it, we propose that this may be an erythrocyte-binding protein.

Nucleotide sequence analysis and epitope mapping of the merozoite surface protein 1 from Plasmodium chabaudi chabaudi AS.

The complete nucleotide sequence of the gene encoding the merozoite surface protein 1 (MSP-1) from the rodent malaria parasite Plasmodium chabaudi chabaudi AS has been determined by direct sequencing of overlapping PCR derived fragments. Comparison of the P. c. chabaudi AS nucleotide sequence with the previously published P. c. chabaudi IP-PC1 sequence indicates that although the MSP-1 gene of these two P. c. chabaudi strains is highly conserved, with sequence identity often approaching 100%, interspersed throughout the molecule are 5 regions of divergence. This is at variance with published data which suggested that the P. c. chabaudi AS and P. c. chabaudi IP-PC1 MSP-1 sequences are largely identical. Epitope mapping studies with a panel of anti-P. c. chabaudi AS MSP-1 monoclonal antibodies demonstrate that whilst most of these mAbs recognise epitopes at the N-terminus of the MSP-1 molecule, two mAbs, including one capable of inhibiting challenge infections in mice in an in vivo passive transfer assay, recognise epitopes which map to the C-terminus.

A single amino acid determines the specificity of a monoclonal antibody which inhibits Plasmodium chabaudi AS in vivo.

The in vivo inhibitory action of NIMP23, a monoclonal antibody raised against the rodent parasite Plasmodium chabaudi chabaudi AS, has previously been shown to be strain-specific, capable of delaying significantly the onset of P. c. chabaudi AS but not a P. c. chabaudi CB challenge parasitaemia. The epitope to which this mAb binds has been mapped to the second of two epidermal growth factor-like domains located at the C-terminus of the merozoite surface protein 1 (MSP-1) of P. c. chabaudi AS. The C-terminus region of the MSP-1 of P. c. chabaudi is a region of heterogeneity with AS and CB strain parasites showing only 78% identity at the amino acid level. The critical amino acid substitution which accounts for the strain specificity of the NIMP23 monoclonal antibody has now been identified. Polymerase chain reaction directed mutagenesis experiments demonstrate that a single proline to asparagine substitution at position 1722 in the primary amino acid sequence is sufficient to convert NIMP23-negative P. c. chabaudi CB expression constructs into NIMP23-positive clones whilst the converse substitution of an asparagine for a proline residue converts P. c. chabaudi AS expression constructs into NIMP23-negative clones.

The importance of sensitive detection of malaria parasites in the human and insect hosts in epidemiological studies, as shown by the analysis of field samples from Guinea Bissau.

A method based on the polymerase chain reaction (PCR) for highly sensitive detection and identification of human malaria parasites was applied to blood and mosquito samples obtained from a village in Guinea Bissau. The prevalence of parasites in the human population was shown to be greatly underestimated by microscopical examination. In particular, a high incidence of Plasmodium malariae and P. ovale parasites was revealed only by the PCR assay. Preliminary evidence was obtained to show that the distribution of P. malariae infections within the village was non-random. This was supported by analysis of the parasite species infecting the mosquito vector. The implication of these results for the design and interpretation of epidemiological surveys is discussed.