DC-SIGN mediates cell-free infection and transmission of human T-cell lymphotropic virus type 1 by dendritic cells.

Despite the susceptibility of dendritic cells (DCs) to human T-cell lymphotropic virus type 1 (HTLV-1) infection and the defined role of these cells in disease pathogenesis, the mechanisms of viral binding to DCs have not been fully delineated. Recently, a glucose transporter, GLUT-1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) were demonstrated to facilitate HTLV-1 entry into T cells. DCs express their own array of antigen receptors, the most important being the DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) with respect to retrovirus binding. Consequently, the role of DC-SIGN and other HTLV-1 attachment factors was analyzed in viral binding, transmission, and productive infection using monocyte-derived DCs (MDDCs), blood myeloid DCs, and B-cell lines expressing DC-SIGN. The relative expression of DC-SIGN, GLUT-1, HSPGs, and NRP-1 first was examined on both DCs and B-cell lines. Although the inhibition of these molecules reduced viral binding, HTLV-1 transmission from DCs to T cells was mediated primarily by DC-SIGN. DC-SIGN also was shown to play a role in the infection of MDDCs as well as model B-cell lines. The HTLV-1 infection of MDDCs also was achieved in blood myeloid DCs following the enhancement of virus-induced interleukin-4 production and subsequent DC-SIGN expression in this cell population. This study represents the first comprehensive analysis of potential HTLV-1 receptors on DCs and strongly suggests that DC-SIGN plays a critical role in HTLV-1 binding, transmission, and infection, thereby providing an attractive target for the development of antiretroviral therapeutics and microbicides.

Evaluating the learning experience of non medical prescribing students with their designated medical practitioners in their period of learning in practice: results of a survey.

AIMS: To evaluate the learning experience of non medical prescribing (NMP) students during their period of learning in practice and to explore strategies for improvement. METHODS: A self-administered questionnaire was used to collect data from two consecutive NMP student cohorts. RESULTS: Of 57 NMP students, the majority (64.9%) worked in primary care setting. In contrast to those from primary care setting, the students working in secondary/tertiary care setting had significantly greater chance of knowing their designated medical practitioner (DMP) prior to starting their course (p=0.044). However, this did not influence whether the student did a learning agreement and time schedule agreement with the DMP at the beginning of practice setting. A learning agreement and time schedule was done by 91.2% and 57.9% students, respectively, at beginning of the course. Prior time schedule agreement was a significant determinant in determining the number of hours that student spent subsequently under direct supervision of DMP: 75.8% of those who did a prior time schedule spent >30% of practice hours under the direct supervision of DMP as compared to only 50% of those who did not. Spending >30% of the practice hours under direct supervision of the DMP was significantly associated with student satisfaction (p=0.025). There was greater likelihood of a student being assessed formatively if a prior learning agreement had been done (p=0.035) resulting in increased student satisfaction. Time and workload constraints, organisational issues and peer support emerged as barriers to student learning. Students commented on difficulties in getting doctors as a DMP; and therefore suggested that learning experience can be enhanced if a qualified practicing Non Medical Prescriber could act as a "co-mentor". There were also suggestions of providing incentives to doctors and giving them more information about the role of NMP to encourage more doctors to act as DMP. CONCLUSIONS: Learning agreement and a time schedule with DMP at the beginning of the supervised period in practice significantly improved the students' learning experience, and was a major determinant of subsequent student satisfaction. Those who spent at least 30% of practice development time under direct supervision of their DMP were likely to be more satisfied with the learning process.

Identification of human T cell leukemia virus type 1 tax amino acid signals and cellular factors involved in secretion of the viral oncoprotein.

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a number of pathologic abnormalities, including adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The viral oncoprotein Tax has been implicated in the pathogenesis of these diseases. Recently, cell-free Tax was detected in the cerebrospinal fluid of HAM/TSP patients, implying that extracellular Tax may be relevant to neurologic disease. Additionally, the presence of a nuclear export signal within Tax and its active secretion has been demonstrated in vitro. However, the mechanism of Tax secretion remains to be established. Studies reported herein elucidate the process of Tax secretion and identify domains of Tax critical to its subcellular localization and secretion. Tax was shown to interact with a number of cellular secretory pathway proteins in both the model cell line BHK (baby hamster kidney)-21 and an HTLV-1-infected T cell line, C8166, physiologically relevant to HTLV-1-induced disease. Silencing of selected components of the secretory pathway affected Tax secretion, further confirming regulated secretion of Tax. Additionally, mutations in two putative secretory signals within Tax DHE and YTNI resulted in aberrant subcellular localization of Tax and significantly altered protein secretion. Together, these studies demonstrate that Tax secretion is a regulated event facilitated by its interactions with proteins of the cellular secretory pathway and the presence of secretory signals within the carboxyl-terminal domain of the protein.

Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1.

Human T cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense CTL cell response directed against the viral transactivator protein Tax. In addition, patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DC), likely contributing to the robust, Tax-specific CTL response. In this study, extracellular Tax has been shown to induce maturation and functional alterations in human monocyte-derived DC, critical observations being confirmed in freshly isolated myeloid DC. Tax was shown to promote the production of proinflammatory cytokines and chemokines involved in the DC activation process in a dose- and time-dependent manner. Furthermore, Tax induced the expression of DC activation (CD40, CD80, and CD86) and maturation (CD83) markers and enhanced the T cell proliferation capability of DC. Heat inactivation of Tax resulted in abrogation of these effects, indicating a requirement for the native structure of Tax, which was found to bind efficiently to the DC membrane and was internalized within a few hours, suggesting that extracellular Tax may possess an intracellular mechanism of action subsequent to entry. Finally, inhibitors of cellular signaling pathways, NF-kappaB, protein kinase, tyrosine kinase, and phospholipase C, were shown to inhibit Tax-mediated DC activation. This is the first study reporting the immunomodulatory effects of extracellular Tax in the DC compartment. These results suggest that DC, once exposed to Tax by uptake from the extracellular environment, can undergo activation, providing constant antigen presentation and costimulation to T cells, leading to the intense T cell proliferation and inflammatory responses underlying HAM/TSP.

Induction of pro-inflammatory cytokines by human T-cell leukemia virus type-1 Tax protein as determined by multiplexed cytokine protein array analyses of human dendritic cells.

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by a hyperstimulated immune response, including elevated levels of inflammatory cytokines/chemokines and oligoclonal expansion of virus-specific CD8(+) T cells in the cerebrospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immune recognition by antigen presenting cells (APCs), such as dendritic cells (DCs). DCs are relevant to the pathogenesis of HAM/TSP because the presentation of Tax peptides by activated DCs to naïve CD8(+) T cells may play an important role in the induction of the Tax-specific immune response that is observed in HAM/TSP. In this study, a human cytokine protein array was used to study the secretion of cytokines by monocyte-derived DCs (MDDCs) exposed to Tax. Of the 16 cytokines analyzed, 6 cytokines were secreted in significantly high amounts (> or =2-fold), including Th1 cytokines (IFN-gamma, IL-12, and TNF-alpha) and C-C chemokines (Eotaxin, MCP-1, and MCP-3). Selected cytokines were further examined at two concentrations of Tax and at two time periods. Furthermore, a transient exposure to Tax did not result in any cytokine production when examined at three different time points after exposure, indicating that a prolonged presence of Tax is required for its activity. Finally, inhibition of the NF-kappaB signaling pathway by specific inhibitors, abrogated Tax-mediated cytokine secretion. Collectively, these findings suggest a role for Tax-induced cytokine secretion from MDDCs, which may be critical for the cellular activation and tissue damage that has been observed in HAM/TSP.

A novel high throughput quantum dot-based fluorescence assay for quantitation of virus binding and attachment.

Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated QDots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4(+) and CD8(+) T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry.

AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation.

Human T cell leukemia virus type 1 (HTLV-1) has previously been shown to infect antigen-presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV-1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV-1 viral gene expression in established monocytic cell lines was examined. U-937 promonocytic cells transiently transfected with a HTLV-1 long-terminal repeat (LTR) luciferase construct were treated with phorbol 12-myristate 13-acetate (PMA) to induce cellular differentiation. PMA-induced cellular differentiation resulted in activation of basal and Tax-mediated transactivation of the HTLV-1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA-induced cellular differentiation induced DNA-binding activity of cellular transcription factors to Tax-responsive element 1 (TRE-1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP-1) family of basic region/leucine zipper proteins (Fra-1, Fra-2, JunB, and JunD) were induced to bind to TRE-1 repeat II during cellular differentiation. Inhibition of AP-1 DNA-binding activity by overexpression of a dominant-negative c-Fos mutant (A-Fos) in transient expression analyses resulted in severely decreased levels of HTLV-1 LTR activation in PMA-induced U-937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV-1 viral gene expression may become up-regulated by AP-1 during differentiation into macrophages or dendritic cells.

Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells.

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

HTLV-1 Tax nucleocytoplasmic shuttling, interaction with the secretory pathway, extracellular signaling, and implications for neurologic disease.

The human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax interacts with numerous cellular pathways promoting both the survival and pathogenesis of the virus in the human population. Tax has been studied extensively with respect to its role in transcriptional transactivation and its involvement in the up-regulation of a number of cellular genes during the process of oncogenic transformation. These processes are dependent on Tax localization to the nucleus where it interacts with a number of cellular transcription factors during its course of nuclear action. However, there is mounting evidence suggesting that Tax may shuttle between the nucleus and cytoplasm, localize to several cytoplasmic organelles with subsequent secretion from both Tax-transfected cells as well as HTLV-1-infected cells. In addition, the presence of cell-free Tax in cerebral spinal fluid (CSF) was recently demonstrated to occur during all stages of HAM/TSP. This has brought about an increased interest in the cytoplasmic localization of Tax and the implications this localization may have with respect to the progression of HTLV-1-associated disease processes. This review addresses the functional implications relevant to the localization and accumulation of Tax in the cytoplasm including the Tax amino acid signals and cellular protein interactions that may regulate this process. Specifically, we have discussed three important processes associated with the cytoplasmic localization of Tax. First, the process of Tax shuttling between the nucleus and cytoplasm will be described and how this process may be involved in regulating different transcriptional activation pathways. Second, cytoplasmic localization of Tax will be discussed with relevance to Tax secretion and the interaction of Tax with proteins in the cellular secretory pathway. Finally, the secretion of Tax and the effects of extracellular Tax on HTLV-1 pathogenesis will be addressed.

Pharmacoeconomics.

Pharmacoeconomics focuses on the costs and benefits of drug therapy and pharmacoeconomic evaluations provide a basis for resource allocation and utilization. It is increasingly becoming important for health policy decision-making. A pharmacoeconomic evaluation may be conducted as an economic assessment incorporated into clinical trials. Such trials should compare the new drug/procedure with an older drug or existing intervention. Four techniques are used for economic evaluation, namely, cost-minimization analysis, cost-effectiveness analysis, cost-utility analysis and cost-benefit analysis. The choice of the evaluation method depends on the nature of outcomes and the context in which the choices need to be made. Pharmacoeconomics is a young science that will improve with application. Its need is undeniable, especially in developing countries.

Nodular myofibroblastic stromal hyperplasia of the mammary gland as an accurate name for pseudoangiomatous stromal hyperplasia of the mammary gland.

Pseudoangiomatous stromal hyperplasia (PASH) of the mammary gland is a well-known benign localized form of stromal overgrowth with probable hormonal etiology. We describe the histologic findings and immunohistochemistry of two cases. Two women, 16 and 58 years old, presented with a breast mass and underwent surgical excision. Grossly, they consisted of a well-circumscribed, rubbery tissue with a solid white-tan homogeneous cut surface. One of the cases showed focal cystic areas. Histologically the lesion showed a proliferation of the collagenous stroma with varying degrees of density, and hyalinization with many pseudovascular slit-like anastomosing spaces lined by spindle cells with scant cytoplasm and bland chromatin. The spindle cells lining the spaces were strongly reactive for vimentin and weakly reactive for CD34, actin, and desmin. They were negative for factor VIII, S-100, and pankeratin. In PASH, the "pseudoangiomatous" term describes a recognizable pattern but does not describe the tumor's histologic nature. We propose the name nodular myofibroblastic hyperplasia of the mammary stroma as a more accurate name.