US National Institutes of Health Intramural Research Program, Sanaria.

. 18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription–polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2–3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1–8.1 days) than blood smears (11.0 days; 95% CI: 10.3–11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6–13.3 days). Discrepant analysis showed that the risk of a blood smear–positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.

Establishment of the vertebrate body plan requires a variety of signaling molecules. In a search for tyrosine kinases expressed in early zebrafish embryos, a model system for the study of vertebrate development, we discovered Jak1 kinase to be maternally encoded and the mRNA evenly distributed among the cells of blastula-stage embryos. Injection of RNA-encoding dominant-negative Jak1 kinases reduces a specific cell migration, epiboly, and results in the reduction of goosecoid expression and of anterior structures. This work establishes that, in addition to its role in signal transduction of cytokines in adult tissues, Jak1 kinase has a role in early vertebrate development.One of the earliest events of vertebrate development is the specification of the primordial germ layers, ectoderm, endoderm, and mesoderm, and the extensive cell migrations that immediately follow. These processes are likely to require a variety of signal transduction cascades, and several peptide growth factors have been implicated in these processes (1-3). In this study, we show that Jak1 kinase also is required for these processes.In a model vertebrate, the zebrafish, developmental events are easily visualized and studied. In a search for signal transducers that might regulate early development, we used a degenerate PCR approach to identify tyrosine kinases expressed during zebrafish gastrulation. Other than the FGF receptor, no other tyrosine kinase has been implicated in the earliest events of vertebrate development. Using degenerate primers to conserved residues in the catalytic domain of tyrosine kinases, we have isolated a homologue of the Jak1 kinase from early gastrula embryos. This is surprising, as Jak1 kinase has been implicated thus far only in signaling for adult tissues in vertebrates.Jak1 is one of four members of a tyrosine kinase family that possesses several features in common (4, 5). The Jak family members, Jak1, Jak2, Jak3, and Tyk2, lack SH2 and SH3 domains, are cytoplasmic, and possess two kinase domains. The N-terminal kinase domain has only limited homology to the phosphotransferase domains of other tyrosine kinases and the isolated domain, expressed as a glutathione S-transferase (GST) fusion, does not possess kinase activity (6). The Cterminal domain, however, possesses all the motifs found in tyrosine kinase domains and this domain, fused to GST, has phosphotransferase activity (6).Jak1 kinase is ubiquitously expressed in adult mouse tissues (6) and has been shown to transduce signals from a variety of cytokines (4, 5). Its developmental expression, however, has never been analyzed. Jak1 has been thought to function exclusively in adults, primarily in the modulation of immune function. We show that during early development, Jak1 kinase is exclusively of maternal origin, and through the use of dominant negative Jak1 kinases, that this kinase is required for cell migrations, goosecoid expression, and anterior shield formation. In agreement with our findings, recent studies have shown that Jak kinase plays a ro...