Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.Many organisms mount specific defense responses to silence invading nucleic acid sequences before these sequences integrate into the host genome and disturb cellular processes. At the core of these sequence-directed immunity mechanisms is dsRNA, 2 which becomes processed and causes gene silencing, referred to as RNA interference (RNAi) (1, 2). In addition to its defense function, RNAi guides endogenous developmental and regulatory processes and is used as a research tool to suppress the expression of cellular genes in numerous model organisms (2).Although species variations exist, the silencing mechanisms in plants, fungi, worms, insects, and mammals share common features and conserved genes. Drosophila S2 cells are widely used to carry out large scale functional screens (3-5) because dsRNA fragments (usually about 500 -700 bp) can be added directly to the cell culture medium, "soaking" the cells rather than transfecting them (6). This makes silencing in these cells easy and efficient, in contrast to mammalian cells that require small interfering RNAs (siRNAs) to be delivered by transfection. In contrast to Caenorhabditis elegans cells, which internalize dsRNA by the channel-forming transmembrane protein SID-1 (7), the mechanism of dsRNA uptake into Drosophila cells is unknown.In the cytosol, dsRNA fragments are processed into short 21-23-nucleotide dsRNA duplexes (siRNAs) by a dsRNA-specific RNase-IIItype endonuclease called Dicer-2 (8 -10). Dicer-2 is stably complexed with the dsRNA binding domain-containing protein R2D2 binding to siRNA and thereby, facilitating siRNA loading onto RNA-induced silencing complexes (RISC) (11). RISC contain a member of the Argonaute (Ago) protein family Ago-2, which has been shown to mediate siRNA-directed mRNA cleavage (12) an...

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.

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The incidence of and mortality from squamous cell carcinoma (SCC) of the tongue have increased during the recent decades in the Western world. Much effort has been made to predict tumour behaviour, but we still lack specific prognostic indicators. The aim of our study was to evaluate the relative importance of the known demographic, clinical and histological factors in a homogeneous population-based group of patients with SCC of the mobile tongue. The demographic and clinical factors were reviewed retrospectively from primary and tertiary care patient files. Histological prognostic factors were determined from pre-treatment biopsies. The TNM stage was found to be the most important prognostic factor. In particular, local spread outside the tongue rather than spread to regional lymph nodes was related to poor prognosis. Several demographic and histopathological factors were closely related to TNM stage. When the cases were divided into stage I–II carcinomas and stage III–IV carcinomas, it appeared that the patient’s older age (> 65 years), a high malignancy score and an absence of overexpressed p53 protein were associated with a poorer prognosis in stage I–II carcinomas. Such cases may require more aggressive treatment. Among patients with stage III–IV carcinomas, heavy use of alcohol was significantly associated with a poor disease-specific survival time. © 2000 Cancer Research Campaign