An analysis of the level of evidence behind treatments recommended by the Danish Medicines Council.

OBJECTIVES: We aimed to investigate the quality of evidence and the expected added clinical value of treatments recommended by the Danish Medicines Council (DMC). STUDY DESIGN: This was an observational study. METHODS: The DMC prepares reports on drugs considered for possible new standard treatments in Danish hospitals. These reports evaluate the available evidence on efficacy and safety. The quality of evidence is systematically rated by the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) criteria, and estimates of added clinical value are presented. The recommendations take into account expected economic implications of new treatments. The publicly available reports up until December 29, 2021, were downloaded from the DMC Web page. Reports on drugs marked "recommended" were included. Data on quality of evidence, expected clinical value, and economic implications were imputed in a Microsoft Excel spreadsheet. RESULTS: Seventy-nine reports were included in the analysis. In 79% of these, the quality of evidence was rated low (24%) or very low (55%), whereas no recommendations were based on evidence rated as high quality. Three (5%) of recommended treatments were expected to add large clinical value. CONCLUSIONS: Most recommendations by the DMC are based on evidence formally rated as low or very low quality by GRADE, and no recommendations were based on evidence rated as high quality. The added clinical value of the treatments was often not documented and rarely large. Continued attention to improve the clinical evidence behind national recommendations is necessary.

The role of QT-prolonging medications in a forensic autopsy study from Western Denmark.

Medication-induced prolongation of the QT-interval (miQTP) can lead to cardiac arrhythmia. Our aim was to investigate the prevalence of forensic autopsy cases where fatal cardiac arrhythmia related to treatment with QT-prolonging medications (QT-PMs) could be suspected. We performed a cross-sectional study of 741 forensic autopsies undertaken at our institution in non-drug addicts aged 15 years or above from 2017 to 2019. We defined a high risk of miQTP by one detected QT-PM in a concentration above therapeutic level, or two or more detected QT-PMs in post mortem blood. We reviewed the autopsy reports from cases with a high miQTP-risk to identify cases with no other apparent cause of death. We discarded suicides and cases with lethal levels of QT-PMs. We identified 167 cases (22.5%) with high risk of miQTP, and discarded 36 suicides (4.9%) and 7 (0.9%) with lethal levels of QT-PMs. Apart from a high risk of miQTP, no other apparent explanation of the cause of death was present in seven (0.9%). In 18 cases (2.4%) with high miQTP-risk, the cause of death was primarily attributed to cardiac changes other than acute cardiovascular events. In conclusion, 22.5% had a high risk of miQTP, and fatal cardiac arrhythmia related to treatment with QT-PMs could be suspected in 0.9%. However, a genetic pro-arrhythmic background could not be excluded in our study. Furthermore, it is possible that QT-PMs could have played a role in some of the 2.4% of cases where the cause of death was mainly attributed to cardiac changes and the risk of miQTP was high.

Iridium(iii) hydrido complexes for the catalytic dehydrogenation of hydrazine borane.

The synthesis of 3,5-disubstituted cyclometalated iridium(iii) hydrido complexes of the type [3,5-R2(POCOP)IrHX] (3,5-R2(POCOP) = κ3-C5HR2-2,6-(OPtBu2)2 with R = t-Bu, COOMe; X = Cl, H) is described. All complexes were investigated in the catalytic dehydrogenation of hydrazine borane and compared with the unsubstituted compounds [(POCOP)IrHX] (X = Cl, H). All catalysts are highly active and recyclable, clearly maintaining hydrogen production activity. The dehydrogenation products were structurally characterised by solid state NMR and FTIR spectroscopy. Experimental observations were complemented by a dispersion-corrected DFT study to rationalise the mechanism of hydrazine borane dehydrogenation.

High-risk sun-tanning behaviour: a quantitative study in Denmark, 2008-2011.

OBJECTIVES: The incidences of melanoma and non-melanoma skin cancer have increased markedly over the past 30 years. The main risk factor is ultraviolet radiation from the sun and from sunbeds. The Danish Sun Safety campaign was launched in 2007 to curb this development by reducing the exposure of adolescents and young children. In this study, the characteristics of high-risk sun-tanning behaviour were assessed and the effect of the campaign was determined. STUDY DESIGN: Cross-sectional study. METHODS: Data from annual Internet surveys were compiled in 2008-2011 of 18, 685 15-64-year-old Danes. A tanning index based on sunbed use and intentional tanning in and outside Denmark was the outcome measure in a linear regression model, which included age, gender, skin type, education, income and survey year as exposure variables. RESULTS: High-risk tanning behaviour was associated with female gender, younger age, shorter education, skin type 3 or 4, higher income, smaller household and living in larger cities. The tanning index, where 100 represent high-risk behaviour, increased by 13.45 points for women as compared with men, dropped by 1.35 points for each 5-year increase in age, rose by 20.72 points for skin type 4 as compared with type 1 and increased by 10.33 points with an income >€105, 409 as compared with <€26, 352. High-risk behaviour decreased during the study period, especially among women and younger people. CONCLUSIONS: High-risk sun-tanning behaviour is linked to certain personal and social characteristics. After initiation of the Danish Sun Safety Campaign in 2007, this high-risk behaviour decreased, especially in the groups initially targeted by the campaign. The campaign may thus reduce the future incidence of melanoma and non-melanoma skin cancer.

Advanced magnetic resonance strategies for the elucidation of nanostructured soft matter.

An overview is given on advanced magnetic resonance strategies and techniques, both nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR), as applied to nanostructured soft matter. In addition, the combination of the two forms of spectroscopy to enhance signal intensity in NMR by means of dynamic nuclear polarization (DNP) is described. It is shown how these techniques can provide unique information on the structure of soft matter as well as the local dynamics of the constituents. Examples of recent applications are described, including dendronized and thermoresponsive polymers, hydrogels, synthetic and bio-inspired polymers, as well as polypeptides and biopolymers.

Influence of central glia on spiral ganglion neuron neurite growth.

Spiral ganglion neurons (SGNs) extend processes that interact with Schwann cells (SCs) and with oligodendrocytes (OLs) and astrocytes (ACs). We investigated the ability of these glial cells to support SGN neurite growth. In the presence of cultured ACs, OLs and SCs, SGN neurites tended to follow SCs and OLs and cross-over ACs. Most neurites initially followed the type of glial cell on which the neuronal cell body was found. To determine the influence of homogeneous populations of glia on neurite growth, SG explants were plated on cultured SCs, ACs or OLs. The number of neurites/explant extending onto SCs (463.89±16.25) was significantly greater than the number extending onto ACs (111.38±38.73) or OLs (6.75±2.21), indicating that populations of central glia inhibit SGN neurite growth. Treatment with cell-permeant cpt-cAMP or forskolin (FSK) each significantly increased the number of neurites on OLs (133.54±25.59 and 292.25±83.57, respectively). cpt-cAMP and FSK each also increased the number of neurites on ACs (213.19±36.06 and 208.64±59.25, respectively), however the difference was not significant compared with control. The neurites on ACs and OLs failed to grow radially in a well-fasciculated pattern as on SCs. In explants plated on the borders of cultured OL-SC or AC-SC groups, more neurites extended onto SCs compared with OLs and ACs. Conditioned media (CM) from OL or AC cultures did not reduce neurite length, implying that the inhibition of neurite growth by central glia is not due to soluble factors. Taken together, these results demonstrate that homogeneous populations of central glia inhibit SGN neurite growth.

Fast and slow dynamics in a discotic liquid crystal with regions of columnar order and disorder.

Aromatic disk-shaped molecules tend to self-organize into a herringbone packing where the disks are inclined at angles ±Î¸ with respect to the axis of the column. In discotic liquid crystals this can introduce defects between stacks of limited length. In a C(3)-symmetric hexa-peri-hexabenzocoronene, solid-state NMR, x-ray scattering, and rheology identifies such a packing with θ=43° and stacks of about seven disks. Disordered regions containing defects fill the space in between the ordered stacks. Biaxial intra- and intercolumnar dynamics differing by eight decades are identified.

Reciprocal signaling between spiral ganglion neurons and Schwann cells involves neuregulin and neurotrophins.

To investigate the role of neuron-glial cell interactions in the auditory nerve, we asked whether spiral ganglion neurons (SGNs) express neuregulin and whether neuregulin regulates proliferation and/or neurotrophin expression in spiral ganglion Schwann cells (SGSCs). Using immunocytochemistry, we found that type I and type II SGNs express neuregulin in vivo and in vitro. Cultured SGSCs express the neuregulin receptors ErbB2 and ErbB3, but not ErbB4. Neuregulin activates ErbB2 and ErbB3 in cultured SGSCs, evidenced by increased tyrosine phosphorylation of the receptors following neuregulin treatment. Neuregulin treatment increased the proliferation rate of cultured SGSCs by 2.5-fold. Fibroblast growth factor-2 (FGF-2) and transforming growth factor beta (TGF-beta) also increased SGSC proliferation. The mitogenic effect of neuregulin and FGF-2 was blocked by inhibition of mitogen-activated protein kinase signaling but not by inhibition of phosphatidylinositol-3'-OH kinase. Using RT-PCR, we found that cultured SGSCs express neurotrophins, including brain-derived neurotrophic factor and neurotrophin-3 (NT-3), raising the possibility that SGSCs contribute to the trophic support of SGNs. Treatment with neither neuregulin nor TGF-beta increased neurotrophin expression in cultured SGSCs, as had been observed in developing sympathetic ganglia, but appeared to negatively regulate NT-3 expression. Thus, neuregulin and neurotrophins may mediate reciprocal neuron-glial interactions in the auditory nerve.

BDNF synthesis in spiral ganglion neurons is constitutive and CREB-dependent.

Brain-derived neurotrophic factor (BDNF), which supports spiral ganglion neuron (SGN) survival in vivo and in vitro, is synthesized by SGNs. The BDNF gene generates multiple different transcripts, each from its own promoter region. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we find that SGNs express only the downstream transcripts III and IV in vivo and in vitro. Using RT-PCR assays of BDNF transcripts and transfection of BDNF promoter-reporter constructs, we tested the hypothesis, originally derived from studies of cortical neurons, that depolarization induces BDNF expression via a signaling pathway that includes Ca2+/calmodulin-dependent kinases (CaMKs) and the transcription factor, Ca2+/cyclic AMP response element binding protein (CREB). In contrast, we found that in SGNs in vivo BDNF expression is constitutive and is not increased by electrical activation. Similarly, BDNF expression in vitro is not increased by stimuli that activate CREB, including depolarization, cAMP, or transfection of activated CaMK mutants. However, transfection of dominant-negative CREB mutants did abrogate gene expression driven by BDNF promoters III and IV, indicating that CREB is necessary for constitutive BDNF expression. Thus, BDNF synthesis within SGNs makes possible an autocrine or paracrine mechanism that can contribute to support SGN survival but SGNs are distinctive in that this mechanism is constitutive and not activity-regulated.

Multiple distinct signal pathways, including an autocrine neurotrophic mechanism, contribute to the survival-promoting effect of depolarization on spiral ganglion neurons in vitro.

We have shown previously that BDNF, neurotrophin-3 (NT-3), chlorphenylthio-cAMP (cpt-cAMP) (a permeant cAMP analog), and membrane depolarization promote spiral ganglion neuron (SGN) survival in vitro in an additive manner, depolarization having the greatest efficacy. Expression of both BDNF and of NT-3 is detectable in cultured SGNs after plating in either depolarizing or nondepolarizing medium. These neurotrophins promote survival by an autocrine mechanism; TrkB-IgG or TrkC-IgG, which block neurotrophin binding to, respectively, TrkB and TrkC, partially inhibit the trophic effect of depolarization. The mitogen-activated protein kinase kinase inhibitor PD98059 and the phosphatidylinositol-3-OH kinase inhibitor LY294002 both abolish trophic support by neurotrophins but only partially inhibit support by depolarization. Inhibition by these compounds is not additive with inhibition by Trk-IgGs. The cAMP antagonist Rp-adenosine-3',5'-cyclic-phosphorothioate (Rp-cAMPS) abolishes survival attributable to cpt-cAMP but has no effect on that attributable to neurotrophins, nor do inhibitors of neurotrophin-dependent survival affect survival attributable to cpt-cAMP. However, Rp-cAMPS does partially inhibit depolarization-dependent survival, an inhibition that is additive with that by Trk-IgGs, PD98059, or LY294002. Moreover, Rp-cAMPS prevents depolarization-dependent survival of PC12 cells maintained in subthreshold levels of NGF. Inhibition of Ca(2+)/calmodulin-dependent protein kinases (CaMKs) with KN-62 reduces SGN survival independently of Rp-cAMPS, Trk-IgGs, and LY294002 and additively with them. Combined inhibition of Trk, cAMP, and CaMK signaling prevents depolarization-dependent survival. Thus, survival of SGNs under depolarizing conditions involves additivity among a depolarization-independent autocrine pathway, a cAMP-dependent pathway, and a CaMK-dependent pathway.

Pf1 filamentous phage as an alignment tool for generating local and global structural information in nucleic acids.

Abstract Pf1 filamentous phage represent a simple versatile method for generating partially ordered macromolecules in solution. The phage allow tunable degrees of alignment of macromolecules under a wide range of temperature and solvent conditions. The negatively charged phage are ideal for aligning negatively charged nucleic acids and these phage-nucleic acid solutions are stable indefinitely. We have used Pf1 phage to align various DNA and RNA molecules in solution for measurement of dipolar coupling interactions. These dipolar couplings can be used to improve the local structure of nucleic acids. More importantly they also contain information on the global structure, such as DNA bending, which presently cannot be obtained by standard NMR methods. The principles involved in using Pf1 phage to generate solutions of partially order macromolecules will be discussed. The use of (1)H-(1)H, (1)H-(13)C and (1)H-(15)N dipolar couplings for generating angle constraints for structure refinement of nucleic acids will also be discussed.

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones.

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.

In situ T cell responses against melanoma comprise high numbers of locally expanded T cell clonotypes.

It is well established that melanoma cells express Ags that are recognized by autologous T cells in vitro. Tumor-infiltrating lymphocytes in situ comprise clonotypic T cells, suggesting that their expansion is driven by Ag stimulation. Still, little is known about the detailed characteristics of the in situ T cell response. In the present study, we scrutinized this response by analyzing multiple metastatic lesions for the presence of clonotypic T cells. This approach was chosen to distinguish whether the clonal T cell expansion occurs as a systemic or localized phenomenon. TCR clonotype mapping of six s.c. metastases from two patients revealed the presence of multiple (from 40 to >60) clonotypic T cells in all lesions. Clonotypic T cells were present in TCR beta-variable regions expressed both at high and low levels. Comparison of the T cell clonotypes present in different lesions from individual patients demonstrated that, in general, clonotypes were exclusively detected in a single lesion. Hence, anti-melanoma T cell responses are much more heterogeneous than previously anticipated and accommodate a predominance of strictly localized T cell clonotypes.

An examination of coaxial stacking of helical stems in a pseudoknot motif: the gene 32 messenger RNA pseudoknot of bacteriophage T2.

The RNA pseudoknot located at the 5' end of the gene 32 messenger RNA of bacteriophage T2 contains two A-form helical stems connected by two loops, in an H-type pseudoknot topology. A combination of multidimensional NMR methods and isotope labeling were used to investigate the pseudoknot structure, resulting in a more detailed structural model than provided by earlier homonuclear NMR studies. Of particular significance, the interface between the stacked helical stems within the pseudoknot motif is described in detail. The two stems are stacked in a coaxial manner, with an approximately 18 degrees rotation of stem1 relative to stem2 about an axis that is parallel to the helical axis. This rotation serves to relieve what would otherwise be a relatively close phosphate-phosphate contact at the junction of the two stems, while preserving the stabilizing effects of base stacking. The ability of the NMR data to determine pseudoknot bending was critically assessed. The data were found to be a modestly precise indicator of pseudoknot bending, with the angle between the helical axes of stem1 and stem2 being in the range of 15+/-15 degrees. Pseudoknot models with bend angles within this range are equally consistent with the data, since they differ by only small amounts in the relatively short-range interproton distances from which the structure was derived. The gene 32 messenger RNA pseudoknot was compared with other RNA structures with coaxial or near-coaxial stacked helical stems.

Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions.

Dipolar coupling interactions represent an extremely valuable source of long-range distance and angle information that was previously not available for solution structure determinations of macromolecules. This is because observation of these dipolar coupling data requires creating an anisotropic environment for the macromolecule. Here we introduce a new method for generating tunable degrees of alignment of macromolecules by addition of magnetically aligned Pf1 filamentous bacteriophage as a cosolute. This phage-induced alignment technique has been used to study 1H-1H, 1H-13C, and 1H-15N dipolar coupling interactions in a DNA duplex, an RNA hairpin and several proteins including thioredoxin and apo-calmodulin. The phage allow alignment of macromolecules over a wide range of temperature and solution conditions and thus represent a stable versatile method for generating partially aligned macromolecules in solution.

The immunogenic properties of human melanomas and melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last years significant progress has been achieved in the identification of melanoma-associated antigens (MAA) recognized by cytotoxic T lymphocytes (CTL). These antigens belong to three main groups: tumor-associated testis-specific antigens (MAGE, BAGE, GAGE and PRAME), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2) and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). For the identification of these antigens, CTL cultures from mainly only 4 different melanoma patients have been used. These patients developed a strong anti-melanoma response resulting in long-lasting disease-free periods, pointing to the importance of the identification of highly immunogenic melanomas. In each of these patients, the immune response was observed against a unique set of 4 to 6 individual antigenic epitopes, on one hand suggesting the low immunogenicity of the individual antigens, and on the other pointing to the importance of the identification of additional highly immunogenic melanomas for the discovery of new MAA. The analysis of the available data on the immunogenic and protective properties of individual MAA confirms their low immunogenicity. In our study, we focused on the identification of especially highly immunogenic melanomas among a panel of 40 newly established melanoma cell lines. So far, only two such melanoma cell lines, FM3 and FM57 have been identified in this panel. The immunogenic properties of uncloned FM3 cells and several FM3 clones have been further investigated. It was found that the immunogenic properties of melanoma cells are mainly determined by the expression of progression-associated antigens as well as by ecto-ATPase, a molecule which is able to modulate cell adhesion. Cloning the cultures of PBL, stimulated with uncloned FM3 or with the highly immunogenic FM3 clone, FM3.29, has permitted us to identify the immune response against eight different MAA, five of these probably representing not previously described antigens. (Tab. 2, Fig. 2, Ref. 68.)