Injection of Plasmodium salivary gland sporozoites into the vertebrate host by Anopheles mosquitoes initiates malaria infection. Sporozoites develop within oocysts in the mosquito midgut and then enter and mature in the salivary glands. Although morphologically similar, oocyst sporozoites and salivary gland sporozoites differ strikingly in their infectivity to the mammalian host, ability to elicit protective immune responses, and cell motility. Here, we show that differential gene expression coincides with these dramatic phenotypic differences. Using suppression subtractive cDNA hybridization we identified highly up-regulated mRNAs transcribed from 30 distinct genes in salivary gland sporozoites. Of those genes, 29 are not significantly expressed in the parasite's blood stages. The most frequently recovered transcript encodes a protein kinase. Developmental up-regulation of specific mRNAs in the infectious transmission stage of Plasmodium indicates that their translation products may have unique roles in hepatocyte infection and/or development of liver stages.Malaria transmission occurs by mosquito bite when Plasmodium sporozoites located in the salivary glands of anopheline mosquitoes enter the vertebrate host. Sporozoites invade hepatocytes and differentiate into exo-erythrocytic forms (EEFs) 1 that after a few days contain several thousand merozoites. After exiting the hepatocyte, merozoites invade erythrocytes and start the blood stage cycle that causes malaria disease. Salivary gland sporozoites and EEFs are rational targets for immunoprophylaxis and drug prophylaxis because they precede the development of the pathogenic blood stages. One important limitation for drug discovery and vaccine development is the lack of candidate target molecules specifically expressed in the pre-erythrocytic stages.To date, only a few sporozoite-expressed proteins have been identified, mainly due to the difficulty of obtaining sporozoites in large quantities. Of those proteins, the sporozoite-specific coat protein CS (1, 2) and the sporozoite-specific invasin TRAP (3, 4) have been identified in a range of different Plasmodium species, and both proteins are lead malaria vaccine candidates either in single formulations or as components of multi-subunit vaccines (5, 6). Sporozoite biology provides a unique opportunity to identify candidate virulence factors that contribute to the successful transmission of Plasmodium. We refer to the observations that oocyst sporozoites and salivary gland sporozoites display dramatically different phenotypes. Sporozoites isolated from the mosquito salivary glands are highly infectious to the mammalian host, migrate in a typical circular gliding pattern, and can elicit strong protective immune responses (7-10). In marked contrast, oocyst sporozoites are ϳ10,000-fold less infective to the mammalian host, do not exhibit circular gliding, and fail to produce protective immunity (Fig. 1). Furthermore, sporozoites that entered the salivary glands are no longer capable of reentering them, suggesting tha...

SummaryInvasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite-red blood cell interactions.

Most studies of gene expression in Plasmodium have been concerned with asexual and͞or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) < 10 ؊4 ), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.Plasmodium yoelii yoelii ͉ expressed sequence tag P rotozoan parasites of the genus Plasmodium are the causative agents of malaria, the most devastating parasitic disease in humans. The parasites occur in distinct morphological and antigenic stages as they progress through a complex life cycle, thwarting decades of efforts to develop an effective malaria vaccine. Plasmodium is transmitted via the bite of an infected Anopheles mosquito, which releases the sporozoite stage into the skin. Sporozoites enter the bloodstream and, on reaching the liver, invade hepatocytes and develop into exo-erythrocytic forms (EEF). After multiple cycles of DNA replication, the EEF contains thousands of merozoites (liver schizont) that are released into the blood stream and initiate the erythrocytic cycle (asexual blood stage) that causes the disease malaria. Changes in life cycle stages are accompanied by major changes in gene expression and therefore by major changes in antigenic composition. The form of the parasite best studied is the asexual blood stage, mainly because of its comparatively easy experimental accessibility. Therefore, most Plasmodium proteins that have been well characterized are expressed during the erythrocytic cycle, among them some major erythrocytic-stage vaccine candidates such as merozoite surface protein-1 (MSP-1) and apical membrane antigen-1 (AMA-1; ref. 1). Erythrocytic-stage vaccines are aimed at inducing an immune response that suppresses or eradicates parasite load in the blood. In contrast, preerythrocytic vaccines are aimed at eliciting an immune response that destroys the sporozoites and the EEF, thereby preventing progression of the parasite to the blood stage. The feasibility of a preerythrocytic v...

Formalizing eligibility criteria in a computer-interpretable language would facilitate eligibility determination for study subjects and the identification of studies on similar patient populations. Because such formalization is extremely labor intensive, we transform the problem from one of fully capturing the semantics of criteria directly in a formal expression language to one of annotating free-text criteria in a format called ERGO Annotation. The annotation can be done manually, or it can be partially automated using natural-language processing techniques. We evaluated our approach in three ways. First, we assessed the extent to which ERGO Annotations capture the semantics of 1000 eligibility criteria randomly drawn from ClinicalTrials.gov. Second, we demonstrated the practicality of the annotation process in a feasibility study. Finally, we demonstrate the computability of ERGO Annotation by using it to (1) structure a library of eligibility criteria, (2) search for studies enrolling specified study populations, and (3) screen patients for potential eligibility for a study. We therefore demonstrate a new and practical method for incrementally capturing the semantics of free-text eligibility criteria into computable form.

Because of the high costs associated with ascertainment of families most linkage studies of Bipolar I disorder (BPI) have used relatively small samples. Moreover, the genetic information content reported in most studies has been less than 0.6. While microsatellite markers spaced every 10 centimorgans typically extract most of the genetic information content for larger multiplex families, they can be less informative for smaller pedigrees especially for affected sib pair kindreds. For these reasons we collaborated to pool family resources and carry out higher density genotyping. Approximately 1100 pedigrees of European ancestry were initially selected for study and were genotyped by the Center for Inherited Disease Research using the Illumina Linkage Panel 12 set of 6090 SNPs. Of the ~1100 families, 972 were informative for further analyses and mean information content was 0.86 after pruning for LD. The 972 kindreds include 2284 cases of BPI disorder, 498 individuals with Bipolar II disorder (BPII) and 702 subjects with Recurrent Major Depression. Three affection status models were considered: ASM1 (BPI and schizoaffective disorder, BP cases (SABP) only), ASM2 (ASM1 cases plus BPII) and ASM3 (ASM2 cases plus Recurrent Major Depression). Both parametric and non-parametric linkage methods were carried out. The strongest findings occurred at 6q21 (Nonparametric Pairs Lod 3.4 for rs1046943 at 119 cM) and 9q21 (Nonparametric Pairs Lod 3.4 for rs722642 at 78 cM) using only BPI and SA, BP cases. Both results met genome-wide significant criteria, although neither was significant after correction for multiple analyses. We also inspected parametric scores for the larger multiplex families to identify possible rare susceptibility loci. In this analysis we observed 59 parametric lods of 2 or greater, many of which are likely to be close to maximum possible scores. While some linkage findings may be false positives the results could help prioritize the search for rare variants using whole exome or genome sequencing.

Whole-genome sequencing was performed on 3 bipolar I disorder (BPI) cases from a multiplex pedigree of European ancestry with 7 BPI cases. Within CACNA1D, a gene implicated by genome-wide association studies, a G to C nucleotide transversion at 53,835,340 base pairs (bps) was found predicting the substitution of proline for alanine at amino acid position 1751 (A1751P). Using Sanger sequencing, the DNA variant was shown to co-segregate with the remaining 4 BPI cases within the pedigree. A high-resolution DNA denaturing curve method was then used to screen for the presence of the A1751P change in 4,150 BPI cases from the NIMH Genetics Initiative. The A1751P variant was found in 4 BPI cases. A second variant within exon 43, a C to T nucleotide transition, was found in 1 case at 53,835,355 bps, predicting the substitution of tryptophan for arginine at amino acid position 1771 (R1771W). In the NHLBI Exome Sequencing Project database, the heterozygous A1751P variant was present in 3 of 4,300 subjects of European ancestry, and the R1771W change was not present in any subject. Given the rarity of these variants, large-scale case/control rare variant sequencing studies will be required for definitive conclusions.

We reviewed the clinical, radiographic, and histologic features of nine patients with AIDS and pulmonary disease due to Mycobacterium avium complex (MAC). Pulmonary MAC disease was defined by (1) the isolation of MAC from two or more lower respiratory tract specimens or from a single lung biopsy sample, (2) an infiltrate revealed by chest radiography, and (3) the absence of other identified pulmonary pathogens or malignancies. Pulmonary MAC disease was present in five (2.5%) of 200 patients with disseminated MAC infection and in four additional patients without evidence of dissemination, as assessed by blood culture. The median CD4 cell count at the time of presentation was 90/microL. Pulmonary MAC disease was the initial AIDS-defining infection in five patients and presented within a median of 5 months after the initial infection in four patients. Radiographic patterns for these nine patients included consolidating or nodular infiltrates and cavitation. The histopathology of pulmonary MAC disease was characterized by granulomatous inflammation, often associated with necrosis and few evident organisms. The conditions of all patients treated with multidrug regimens clinically improved.

No abstract