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Biomedical subjects

Andrea Crisanti

Publications and source records attributed to Andrea Crisanti.

At least 19 recordsLinked to original sources
PubMed2005

An Anopheles transgenic sexing strain for vector control.

Genetic manipulation of mosquito species that serve as vectors for human malaria is a prerequisite to the implementation of gene transfer technologies for the control of vector-borne diseases. Here we report on the development of transgenic sexing lines for the mosquito Anopheles stephensi, the principal vector of human malaria in Asia. Male mosquitoes, expressing enhanced green fluorescent protein (EGFP) under the control of the beta2-tubulin promoter, are identified by their fluorescent gonads in as early as their 3(rd) instar larval stage, and can be efficiently separated from females using both manual methods and automated sorting machines. Importantly, beta2-EGFP males are not impaired in their mating ability and viable fluorescent spermatozoa are also detected in spermathecae of wild-type females mated with transgenic males. The transgenic mosquito lines described here combine most of the features desired and required for a safe application of transgenic methodologies to malaria-control programs.

Animals↗
PubMed2005

Stable solution of the simplest spin model for inverse freezing.

We analyze the Blume-Emery-Griffiths-Capel model with disordered interaction that displays the inverse freezing phenomenon. The behavior of this spin-1 model in crystal field is studied throughout the phase diagram, and the transition lines are computed using the full replica symmetry breaking ansatz. We compare the results both with the formulation of the same model in terms of Ising spins on lattice gas, where no reentrance takes place, and with the model with generalized spin variables recently introduced by Schupper and Shnerb [Phys. Rev. Lett. 93, 037202 (2004)], for which the reentrance is enhanced as the ratio between the degeneracy of full to empty sites increases. The simplest version of all these models, known as the Ghatak-Sherrington model, turns out to hold all the general features characterizing an inverse transition to an amorphous phase.

Journal Article↗
PubMed2005

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein.

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.

Animals↗
PubMed2005

Inverse associations of human leukocyte antigen and malaria parasite types in two West African populations.

Differences in allelic associations between populations continue to cause difficulties in the mapping and identification of susceptibility genes for complex polygenic diseases. Although well recognized, the basis of such interpopulation differences is poorly understood. We present an example of an inverse allelic association of an immune response genotype to an infectious disease in two neighboring West African populations. In this case, both the key environmental contributor, i.e., the malaria parasite, and a major biological mechanism are well defined. We show that this surprising result fits well with the predictions of a mathematical model describing the population genetics and dynamics of this interaction.

Animals↗
PubMed2005

Identification of sex-specific transcripts of the Anopheles gambiae doublesex gene.

The understanding of the molecular mechanisms of sex differentiation in the mosquito Anopheles gambiae could identify important candidate genes for inducing selective male sterility in transgenic lines or for sex-controlled expression of lethal genes. In many insects, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex-determination cascade that determines the differentiation of sexually dimorphic traits. We report here on the identification of the dsx homologue in A. gambiae and on the characterization of its sex-specific transcripts. Agdsx consists of seven exons, distributed over an 85 kb region on chromosome 2R, which are sex-specifically spliced to produce the female and male AgdsxF and AgdsxM transcripts. AgdsxF contains a 795 bp ORF, coding for a protein of 265 amino acids, while AgdsxM comprises a much longer (1866 bp) ORF, coding for a 622 aa protein. Differences in the exon/intron organization suggest that Agdsx sex-specific splicing results from a different mechanism from Drosophila melanogaster dsx. These findings represent an important step towards the understanding of sex differentiation in Anopheles and will facilitate the use of gene transfer technologies to manipulate sex ratios for vector control programs based on the sterile insect technique.

Amino Acid Sequence↗
PubMed2005

Allergen microarrays.

Allergy affects more than 25% of Western populations (1) and is estimated to be the sixth leading cause of chronic disease in the United States and Western Europe. The complexity of the condition is such that hundreds of common allergens have been described, and in order to maximize diagnostic efficiency there is an urgent clinical requirement for assays to provide multiple-allergen determination in a timely and cost-effective manner. Miniaturized immunoassays that utilize protein microarray technology now offer the possibility of circumventing most of the current limitations in the serodiagnosis of allergic disease. The heterogeneous nature of allergens presents many challenges in all aspects of developing such arrays, from immobilization of the capture molecule to detection of the bound ligand. In addition, there is no simple method of protein amplification (such as PCR for nucleic acids), and stabilization is yet a further major consideration. Notwithstanding these challenges, protein microarrays have been developed for the serodiagnosis of allergies and other complex clinical conditions. These assays exhibit good analytical and clinical performance and deliver significant advantages in convenience and cost compared with traditional ELISA test formats. This chapter details the techniques employed in the construction and processing of an allergen array specific for the serodiagnosis of allergic disease. An overview of protein microarray technology is provided and the principles that underpin the suitability for use of this technology in the identification and measurement of particular proteins in patient sera (serum profiling) are discussed.

Allergens↗
PubMed2004

Disruption of Plasmodium berghei merozoite surface protein 7 gene modulates parasite growth in vivo.

Merozoite invasion of red blood cells is crucial to the development of the parasite that causes malaria. Merozoite surface proteins (MSPs) mediate the first interaction between parasite and erythrocyte. In Plasmodium falciparum, they include a complex of products from at least 3 genes (msp1, msp6, and msp7), one of which, msp7, is part of a gene family containing 3 and 6 adjacent members in Plasmodium yoelii and Plasmodium falciparum, respectively. We have identified and disrupted msp7 in the Plasmodium berghei gene family. The protein is expressed in schizonts and colocalizes with MSP1. The synthesis and processing of MSP1 was unaffected in the parasite with the disrupted gene (MSP7ko). Disruption of msp7 was not lethal but affected blood-stage parasite growth. MSP7ko parasites initially grew more slowly than wild-type parasites. However, when reticulocytes were prevalent, the rate of increase in parasitemia was similar, suggesting that MSP7ko parasites prefer to invade and grow within reticulocytes.

Animals↗
PubMed2004

Antibody response of healthy adults to recombinant thrombospondin-related adhesive protein of cryptosporidium 1 after experimental exposure to cryptosporidium oocysts.

Thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1) belongs to a group of proteins that are also found in Toxoplasma gondii, Eimeria tenella, and Plasmodium species. TRAP-related proteins are needed for gliding motility, host-cell attachment, and invasion. The objective of this study was to characterize the antibody response to recombinant TRAP-C1 (rTRAP-C1) in healthy volunteers exposed to C. parvum and their association with clinical illness. A total of 31 healthy adult volunteers participated. Seven volunteers received the C. parvum TAMU isolate (inocula, 10 to 300 oocysts), and 24 volunteers received the C. parvum UCP isolate (500 to 10(5) oocysts). The total antibody (immunoglobulin M [IgM], IgG, and IgA) response to rTRAP C-1 was measured by enzyme-linked immunosorbent assays prior to and after exposure to Cryptosporidium parvum (days 0 to 45). Results of this study showed that individuals who were uninfected demonstrated higher reactivity at baseline compared to those who became infected. After challenge, increases in antibody reactivity were seen on days 30 and 45 compared to the results seen on days 0 to 5. The increases in antibody reactivity were statistically significant in subjects with diarrhea and with or without detectable oocysts compared to the results seen with those who were uninfected and asymptomatic. These findings suggest that increases in antibody reactivity to rTRAP-C1 occur after recent exposure to C. parvum.

Adult↗
PubMed2004

Protein arrays for serodiagnosis of disease.

Protein microarrays offer the possibility to circumvent most of the current limitations in the serodiagnosis of allergy, autoimmune, and infectious disease by allowing the simultaneous, multiparametric determination of specific subclasses of antibodies directed against many pathogenic antigens. Microarray immunoassays have been developed with these characteristics. A first-generation assay, for the serodiagnosis of infectious disease, allows the determination of IgG and IgM antibodies to various viral and bacterial antigens. In addition, a second-generation assay, designed for the serodiagnosis of allergic disease, permits the determination of IgE antibodies to various allergens implicated in allergic disease. Slides printed with antibody dilution curves and antigen are first incubated with serum samples and then subsequently with secondary antibodies. For detection of human IgG and IgM, fluorescently labeled secondary antibodies are employed. However, because of low-level concentrations of circulating IgE antibodies, a more sensitive protocol is required for human IgE detection. Here, fluorescence is delivered via the coupling of the secondary antibody to tyramide signal-amplification reagentry. Human IgG, IgM, or IgE bound to the printed antigens can then be revealed by confocal scanning microscopy and quantified with internal calibration curves. Generation of analytical and clinical data have demonstrated that the microarray test format provides equivalent performance to enzyme-linked immunosorbent assay (ELISA) tests and offers a significant advantage in convenience and cost when compared to traditional test formats.

Animals↗
PubMed2003

Suppression of gene expression by a cell-permeable Tet repressor.

Engineered transcription factors designed to selectively activate or repress endogenous genes have great potential in medical and biotechnological applications. Ultimately, their success will depend on the development of efficient delivery systems. We show here that a chimeric tetracycline- controlled transcription factor, encompassing the Tet repressor (TetR) from the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and a cell membrane transducing peptide, is able to regulate transcription from a tetracycline responsive promoter (pCMV2xtetO2). When added directly to cultured cells, TetR fused to the full-length Antennapedia homeodomain (AntpHD) from Drosophila (TetRAntp), was able to selectively repress transcription in cells transiently transfected with a tetracycline-regulated reporter transcription unit. Moreover, TetRAntp could repress expression of a tetracycline responsive reporter transcription unit stably integrated into the genome of HeLa cells, demonstrating the possibility of manipulating endogenous gene expression by cell-permeable transcription factors.

Animals↗
PubMed2003

A recombinant H1 histone-based system for efficient delivery of nucleic acids.

We describe here a unique transfer system based on a truncated form of the human linker histone H1F4 for the delivery of nucleic acids to a variety of cells. The efficiency of truncated histone H1.4F was assessed using both primary mammalian and immortalised insect and mammalian cell lines. Our results indicated that recombinant histone H1.4F was able to deliver DNA, dsRNA and siRNA in all cells tested. Quantitative analysis based on reporter gene expression or silencing of target genes revealed that the transfection efficiency of histone H1.4F was comparable to, or better than, liposome-based systems. Notably, the efficiency of histone H1.4F was associated with very low toxicity for transfected cells. The human H1.4F recombinant protein is easily purified in large-scale from bacterial lysates using inexpensive simplified processing. This versatile transfection system represents an important advance in the field of gene delivery and an improvement over earlier nucleic acid delivery methods.

Animals↗
PubMed2003

Stable and heritable gene silencing in the malaria vector Anopheles stephensi.

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles . This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.

Animals↗
PubMed2003

Impact of genetic manipulation on the fitness of Anopheles stephensi mosquitoes.

Genetic modification of mosquitoes offers exciting possibilities for controlling malaria, but success will depend on how transformation affects the fitness of modified insects. The expression of an exogenous gene, the mutations caused by its insertion, and inbreeding while transformed lines are established can all lead to reductions in fitness. Factors influencing fitness were investigated in cage experiments with four lines of transgenic Anopheles stephensi, a vector species of human malaria. The results indicate direct costs of the introduced transgene in at least three out of the four lines, as well as an apparent cost of the inbreeding involved in making transgenic homozygotes.

Animals↗
PubMed2003

Comparative analysis of DNA vectors at mediating RNAi in Anopheles mosquito cells and larvae.

Heritable RNA interference (RNAi) mediated by transgenes exhibiting dyad symmetry represents an important tool to study the function of genes expressed at late developmental stages. In this study, we determined whether the transcriptional machinery of Anopheles mosquitoes is capable of directing suppression of gene expression from DNA constructs designed to transcribe double-stranded RNA (dsRNA) as extended hairpin-loop RNAs. A series of DNA vectors containing sense and antisense regions of the green fluorescent protein EGFP target gene was developed. The effect of these vectors on a transiently expressed or stably integrated EGFP gene was assessed in an Anopheles gambiae cell line and in Anopheles stephensi larvae. Our data indicate that dsRNA-mediated silencing of a target gene from plasmid DNA can be achieved at high levels in Anopheles cell lines and larvae. The region that links the sense and antisense sequences of the target gene plays a determining role in the degree of silencing observed. These results provide important information for the development of heritable RNAi in Anopheles.

Animals↗
PubMed2003

Protein microarray technology for unraveling the antibody specificity repertoire against microbial proteomes.

The genomes of microorganisms responsible for diseases of worldwide medical importance have been sequenced or will be available in the near future. Combinatorial cloning technologies for producing large numbers of proteins have been developed and high-throughput assays such as protein microarrays have been clinically validated for detecting the presence of antibodies directed against microbial antigens in human serum. These scientific and technical achievements offer the opportunity to investigate the natural immune response against the whole proteome of a variety of microorganisms. A powerful combination of genomic information, molecular tools and immunological assays are potentially available to identify the antigens that, either alone or in combination, function as targets of protective immunity, or could be used as markers for serodiagnosis.

Animals↗
PubMed2002

Function of region I and II adhesive motifs of Plasmodium falciparum circumsporozoite protein in sporozoite motility and infectivity.

The circumsporozoite protein of Plasmodium falciparum contains two conserved motifs (regions I and II) that have been proposed to interact with mosquito and vertebrate host molecules in the process of sporozoite invasion of salivary glands and hepatocytes, respectively. To study the function of this protein we have replaced the endogenous circumsporozoite protein gene of Plasmodium berghei with that of P. falciparum and with versions lacking either region I or region II. We show here that P. falciparum circumsporozoite protein functions in rodent parasite and that P. berghei sporozoites carrying the P. falciparum CS gene develop normally, are motile, invade mosquito salivary glands, and infect the vertebrate host. Region I-deficient sporozoites showed no impairment of motility or infectivity in either vector or vertebrate host. Disruption of region II abolished sporozoite motility and dramatically impaired their ability to invade mosquito salivary glands and infect the vertebrate host. These data shed new light on the role of the CS protein in sporozoite motility and infectivity.

Amino Acid Motifs↗
PubMed2002

Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes.

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002) Nature, 417, 452-455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenic Anopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiae carboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at approximately 4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reduced Plasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes.

Animals↗