Issues in methodological research: perspectives from researchers and commissioners.

OBJECTIVES: (1) Methodological research has few well-defined tools and processes analogous to those available for reviews and data collection in substantive health technology assessment. (2) This project was set up to obtain researchers' and others' views on the innovative projects on research methodology under the NHS Health Technology Assessment Programme and the usefulness of the research. (3) The study was intended to span both epistemological and management issues. (4) The following issues were explored: (a) the degree to which researchers would feel constrained by the "Cochrane" approach to systematic reviews when undertaking reviews of a methodological nature; (b) whether methodological projects may require exceptional design and management arrangements, in view of their novelty, subjectivity and complexity; (c) whether researchers would seek out other methods, in addition to undertaking reviews of argument, as a means of extending their understanding of methodological issues (there may be three categories of research methods in methodology: reviews of methodological argument, studies that use the literature as a source of data, and research that collects new primary data); (d) whether the Methodology Programme overall can be considered a "success". METHODS: (1) Telephone interviews were carried out on researchers (one senior and one junior per project), resulting in 35 interviews from 19 of the 20 target projects. (2) A qualitative postal survey was sent to 12 people who had played a key role in the development of the Methodology Programme; replies were received from six of them. (3) Analysis was undertaken of the hit rates for 29 projects on the NCCHTA website by the end of February and the end of May 1999, comparing those concerned with methodology (n = 10) and those concerned with other issues (n = 19). RESULTS: UNDERTAKING METHODOLOGICAL RESEARCH: VIEWS OF RESEARCHERS: This section summarises the views of 35 researchers who were interviewed by telephone. RESULTS: UNDERTAKING METHODOLOGICAL RESEARCH: VIEWS OF RESEARCHERS: (THE NATURE OF METHODOLOGICAL REVIEWS): (1) There was a reluctance among researchers to use the term "systematic review" in the methodological context. (2) Practical problems in undertaking methodological reviews were found at every stage of the research process. (a) In the initial search stage, preplanned strategies were difficult to maintain, owing to the need to respond to the problems of too few or too many references. (b) At the analysis stage, most studies were not formally weighted, but there was implicit weighting in researchers' views of their merits or relevance. (c) It was often only at the synthesis stage that researchers could see clearly what their study was able to do; iteration was frequently necessary at this point. (d) It was difficult to form simple conclusions and recommendations beyond summaries of what was known in the field. (e) Dissemination activities were most often directed to other health service researchers, with some attention to NHS policy makers and research commissioners. RESULTS: UNDERTAKING METHODOLOGICAL RESEARCH: VIEWS OF RESEARCHERS (THE NEED FOR FLEXIBILITY): (1) Few researchers had amended their topic or methods once their research was under way, although some had made minor changes to their original plan, generally to refine the topic to fit the time or data available. (2) Changing a topic was seen as inappropriate unless checked with funders, but changes in research methods were viewed as reasonable because questions might be refined in the light of information gained or early thinking. RESULTS: UNDERTAKING METHODOLOGICAL RESEARCH: VIEWS OF RESEARCHERS (THE QUESTION OF BIAS): (1) Few researchers considered that this kind of research could be undertaken or presented in a wholly unbiased way because of the need to assess the research studied. (2) Objectivity was nonetheless seen as something that researchers should strive towards. Efforts to do so included presenting data clearly, separating findings from discussion, covering all points of view, setting out their own assumptions and values, and testing their ideas on others known to have differing views. (3) The formal peer-review process was not seen to have made a difference here, primarily because of the stage at which referees become involved. RESULTS: UNDERTAKING METHODOLOGICAL RESEARCH: VIEWS OF RESEARCHERS (PROJECT MANAGEMENT--TIMING AND TIME MANAGEMENT): (1) A majority of projects were completed within 3 months of their due date. Those studies completed roughly on time were considered to have efficient junior researchers and good project management, including clear deadlines for different stages of the research. (2) Some studies had severe problems of time management. Too much time tended to be spent on collecting and reading the literature and the writing stage was not always well planned. Referees' comments were also slow in coming. (ABSTRACT TRUNCATED)

A family of human receptors structurally related to Drosophila Toll.

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors--named TLRs 1-5--are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role--akin to Toll in the dorsoventralization of the Drosophila embryo--as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, we also show that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel beta/alpha fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.

Molecular characterization and modular analysis of human MyD88.

MyD88 was first characterized as a myeloid differentiation primary response gene in mice, activated in M1 myeloleukemic cells following interleukin-6 (IL-6) induced growth arrest and terminal differentiation. Analysis of expressed sequence tags (ESTs) from activated dendritic cell libraries led to the indentification of cDNAs encoding the human homolog (hMyD88). The original description of MyD88 as a 243 aa protein may reflect a truncated mouse cDNA since the 2682 nt hMyD88 cDNA predicts a 296 aa cytoplasmic protein. Consistent with this proposal is the detection of a 33 kDa protein in human heart, kidney and liver tissue. The expression pattern of MyD88 is also more widespread than originally believed: a 2.6 kb hMyD88 mRNA species was found to be constitutively expressed in many adult human tissues; in addition MyD88 expression was observed in monocyte, T, B, NK and dendritic cells. The MyD88 protein has a modular structure composed of an N-terminal 'death domain' (DD) similar to the intracellular segments of TNF receptor 1 (TNFR1) and FAS and a C-terminal region related to the signaling domains of vertebrate interleukin-1 receptors (IL-1R) and the Drosophila morphogen Toll. This intriguing structural framework may endow MyD88 with unique signaling capabilities.

The leptin haemopoietic cytokine fold is stabilized by an intrachain disulfide bond.

Structure prediction algorithms have tagged leptin as the newest member of the haemopoietic cytokine family, a diverse class of secreted hormone-like factors with pleiotropic effects in immunity and haemopoietic development. While haemopoietic cytokines typically lack sequence similarity, they conserve a distinctive three-dimensional fold, a four-alpha-helix bundle structure that is recognized by the cognate family of haemopoietic cellular receptors. We have constructed a detailed molecular model of the human leptin helical fold that places the two cysteine residues of the leptin chain, Cys96 and Cys146, in close spatial proximity to each other. In this report, we present evidence that these cysteines are involved in an intrachain disulfide bridge that is critical for the structural integrity and stability of leptin. A leptin variant that is unable to form the disulfide link shows a reduced biological response when administered to leptin-deficient, ob/ob mice.

Expression and purification of a synthetic human obese gene product.

Human obese (hOB) protein was recently identified as a secreted hormone-like factor that is exclusively produced by adipose tissue and appears to regulate the size of the body's fat stores. We describe a rapid and efficient repetitive extension PCR method for the construction of a 453-bp synthetic hOB gene with high-frequency codons to optimize expression in Escherichia coli. The use of a bacterial signal sequence fused to hOB together with expression of bacteriocin release protein resulted in efficient release of hOB into the culture medium. The protein was readily purified to homogeneity from the culture medium using a two-step procedure.

Roles of disulfide bonds in recombinant human interleukin 6 conformation.

Human IL-6 has two disulfide bonds linking Cys45 to Cys51 and Cys74 to Cys84, respectively. Previous site-directed mutagenesis studies have demonstrated that the Cys74-Cys84 bond is essential for full biological and receptor binding activities. To address the structural importance of these disulfide bonds in the formation and stabilization of IL-6 secondary and tertiary structures, we have generated a panel of disulfide bond-deficient rIL-6 analogs both by chemical reduction and alkylation as well as by site-directed mutagenesis. Conformational changes affecting these rIL-6 analogs were probed by circular dichroism spectroscopy, as well as reactivity with monoclonal antibodies, and correlated with changes in biological activities. We have shown that the first disulfide bridge (Cys45-Cys51) is highly sensitive to reduction and, therefore, more solvent-exposed or less thermodynamically stable. Contrary to previous reports, this bridge contributes, although minimally, to the full biological activity of the cytokine. However, no significant changes in secondary or tertiary structures were observed upon removal of this bond. In marked contrast, analogs lacking the disulfide bridge between Cys74 and Cys84 exhibited as little as 0.5% and 0.05% wild-type biological and receptor binding activities, respectively. These dramatic changes correlated with a slight reduction in alpha-helical content and a decreased reactivity with the neutralizing monoclonal antibody mAb8 which recognizes a conformational epitope associated with the active site. Our results suggest that the second disulfide bridge plays a critical role in maintaining the spatial relationship between the putative IL-6 A and D helices.

Structure-function analysis of the C-terminal segment of human interleukin-6.

It has been hypothesized that interleukin-6 (IL-6) and granulocyte-colony-stimulating factor (G-CSF) may fold as four-alpha-helix bundle proteins. To probe the functional role of the putative fourth helical segment of IL-6 (D-helix), a chimeric IL-6/G-CSF analog containing the predicted D-helix of G-CSF as well as a panel of IL-6 D-helix point mutants were analyzed for their respective secondary structure, antigenicity, and receptor binding and biological activities. The putative D-helix of IL-6 could not be replaced by its G-CSF counterpart in spite of their high degree of similarity and thus is indispensable for the antigenic and functional integrity of the IL-6 receptor binding site. Conversely, the grafting of the G-CSF D-helix did not confer any G-CSF activity to IL-6. A synthetic helical peptide containing the IL-6 D-helix was inactive, even when mixed with or linked to a peptide from the A-helix known to be involved in the active site. However, the conserved residues F173, R179, and R182 found in the D-helices of both IL-6 and G-CSF critically contribute to the architecture of the IL-6 active site. Indeed, mutation of F173 or R179 markedly affected IL-6 receptor binding and biological activities, but not the conformation of a major neutralization epitope. Furthermore, substitution of R182 resulted in a significant unfolding of the D-helix accompanied by a drastic loss in IL-6 antigenicity and functional activities. Nevertheless, residues other than F173, R179, and R182 also contribute to IL-6 specificity.

Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine.

A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli. Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera. A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion. After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts. Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines. The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay. It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes. Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.

[Age-related and sex differentiated behavior of serum uric acid levels]. / Zum altersabhängigen und geschlechtsdifferenten Verhalten des Serum-harnsäurespiegels.

In the GDR, the definition of hyperuricaemia is based on sex different reference spheres, independent of age. Our examinations (1872 diabetics and other persons) and the study of literature showed, that it would be better to define the hyperuricaemia only in fertile women different from men.