Upscaling e-mental health in Europe: a six-country qualitative analysis and policy recommendations from the eMEN project.

E-mental health (eMH) encompasses the use of digital technologies to deliver, support, or enhance mental health services. Despite the growing evidence for the effectiveness of eMH interventions, the process of implementation of eMH solutions in healthcare remains slow throughout Europe. To address this issue, the e-Mental Health Innovation and Transnational Implementation Platform North-West Europe (eMEN) project was initiated to increase the dissemination and quality of eMH services in Europe. In this project, status analyses regarding eMH in the six participating countries (i.e., Belgium, France, Germany, Ireland, The Netherlands, and the UK) were conducted and eight recommendations for eMH were developed. Expert teams from the six participating countries conducted status analyses regarding the uptake of eMH based on a narrative literature review and stakeholder interviews. Based on these status analyses, the eMEN consortium developed eight policy recommendations to further support the implementation of eMH in Europe. The status analyses showed that the participating countries are in different stages of implementing eMH into mental healthcare. Some barriers to implementing eMH were common among countries (e.g., a limited legal and regulatory framework), while others were country-specific (e.g., fragmented, federal policies). The policy recommendations included fostering awareness, creating strong political commitment, and setting reliable standards related to ethics and data security. The eMEN project has provided the initial recommendations to guide political and regulatory processes regarding eMH. Further research is needed to establish well-tailored implementation strategies and to assess the generalizability of the recommendations beyond the countries involved in the eMEN project.

Antigen arrays in T cell immunology.

The screening of compound arrays in in vitro bioassays has developed into a powerful tool for the identification of biologically active substances. In the past decade, this technology has increasingly been applied to immunology. As the specificity of the immune system is determined by antigen detection via receptors on B and T cells, targeting the specificity of these immune receptors with random arrays is unique in its ability to generate general and quantitative information on cellular (cross-)reactivity. Synthetic array studies have been useful for identification of epitopes and antigens from databases by defining recognition patterns, isolation of synthetic peptides capable of modulating T cell responsiveness, characterisation of TCR promiscuity, and identification of functionally cross-reacting peptides that are potentially involved in molecular mimicry.

Enantioselective high-performance liquid chromatographic separation of N-methyloxycarbonyl unsaturated amino acids on macrocyclic glycopeptide stationary phases.

This paper describes the enantiomeric resolution of a series of unsaturated N-methyloxycarbonyl-alpha-H-alpha-amino acids (N-MOC-alpha-amino acids) on macrocyclic glycopeptide stationary phases by means of high-performance liquid chromatography (HPLC). Three types of glycopeptide phases, i.e. Chirobiotic T, V and R, were evaluated in both reversed-phase (RP) and polar ionic mode (PIM). The best results in terms of enantioselectivity and resolution were obtained on Chirobiotic R phase, with the PIM mobile phase giving the highest resolution per min. Investigation of the pH of the reversed-phase mobile phase in the pH range 4.1-5.9 showed little effect on enantioselectivity. The method was applied for monitoring the conversion and product enantiomeric excess of an enzymatic hydrolysis reaction using N-MOC-alpha-H-alpha-amino acid esters as substrate.

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells.

Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4+ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCL-specific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.

Enantioselective formal total synthesis of roseophilin.

[formula: see text] An enantioselective formal total synthesis of roseophilin 3 is presented. The 13-membered ring of macrotricycle 1 was formed via an efficient ring-closing metathesis reaction of bicycle 4. A palladium-catalyzed methoxycarbonylation reaction of enol triflate 5 was utilized to functionalize the right-hand ring of bicycle 2. The allyl substituent was introduced by a radical allylation of alpha-bromoketone 17.

Enamide-olefin ring-closing metathesis.

[reaction: see text] The first examples of ring-closing metathesis reactions of olefin-containing enamides using ruthenium-based catalysts have been demonstrated. A preliminary investigation into the scope and limitations, leading to protected five- and six-membered cyclic enamides, will be presented.

Selective azetidine and tetrahydropyridine formation via Pd-catalyzed cyclizations of allene-substituted amines and amino acids.

[reaction: see text] By choosing the right substituents either highly functionalized unusual four-membered ring amino acids or the isomeric pipecolic acid derivatives are obtained in enantiomerically pure form. Starting material is a linear allene-containing amino acid that has been resolved via biocatalysis.

Molecular mimicry in type 1 diabetes: immune cross-reactivity between islet autoantigen and human cytomegalovirus but not Coxsackie virus.

Type 1 diabetes is caused by a T cell-mediated autoimmune destruction of the pancreatic beta cells. Molecular mimicry between viral pathogens and beta cell protein has been a popular theory to explain loss of tolerance in type 1 diabetes. However, functional data in support of this hypothesis have been lacking, presumably because the homologies were defined on the basis of linear similarities in peptide sequences, which ignores the criteria of HLA versus T cell receptor contact residues in peptide epitopes required for T cell recognition. We applied a HLA-binding dedicated peptide microarray analysis using autoreactive T cell clones specific for the autoantigen GAD65 to determine the algorithm of T cell recognition by this given T cell clone. The subsequent database search identified a 100% fit with cytomegalovirus peptide, which was subsequently shown to be recognized by these clonal T cells. However, T cell clones reactive with linear homologies previously described as putative candidates for T cell cross-reactivity between GAD65 and Coxsackie virus peptide were unable to recognize the homologous counterparts.

Synthetic Peptide libraries for T-cell epitope identification.

This chapter describes a methodology for elucidating immunogenic epitopes stimulatory for CD4(+) T-cell clones (Fig. 1). The methodology makes use of synthetic peptide libraries and must be regarded as an alternative to other approaches, such as peptide elution or the application of genetic libraries. The methodology only requires knowledge about the restriction element of the T-cell clone. The restriction element determines which major histocompatibility complex (MHC)-binding anchor motif must be built into the library peptides. A synthetic peptide library is prepared comprising approx 8 million peptides. The synthesis proceeds via a mix-and-split protocol using a solidphase approach on a hybrid resin (1,2). On a hybrid resin, most of the peptide material (84%) is attached via an acid-labile linker whereas the remaining part of the peptide material is acid-stable attached (3). During synthesis, resinbound peptides comprising 14 amino acid residues are produced, with each resin bead containing one unique peptide (4,5). The beads are split into 384 pools, with each pool containing 20,000 beads. From each pool, about 28% of the peptide material is cleaved from every bead. Subsequently, in the first screening round, the 384 pools, each containing 20,000 solubilized peptides, are tested in a proliferation assay with the T-cell clone. Fig. 1. Flow diagram of the complete procedure for the identification of T-cell epitopes using synthetic peptide libraries (1).

Total Synthesis of (+)-Gelsedine.

A novel iodide-promoted, allene-terminated cyclization of an N-acyliminium ion, a stereoselective Heck spirocyclization, and a chemoselective demethylation at the nitrogen atom of an oxindole are the key transformations in the first total synthesis of the indole alkaloid (+)-gelsedine (1). This dextrorotatory form of natural gelsedine was formed as a single enantiomer in 21 steps from (S)-malic acid.

Fermentation kinetics of Zymomonas mobilis near zero growth rate.

The fermentation kinetics Zymomonas mobilis were studied near zero growth rate in fed-batch cultures and continuous cultures with complete cell recycle. The results show the ethanol enhances that specific substrate conversion rate under these conditions. The maximum achievable ethanol concentration in continuous cultures with cell recycle (66 g/L) was significantly lower than in fed-batch cultures (100 g/L). The results indicate that growth-rate-independent metabolism is not instantaneous and can lag behind steadily increasing ethanol concentrations in fed-batch fermentations. A model is proposed to account for this slow adaptation.

Non-polio enterovirus activity in Wisconsin based on a 20-year experience in a diagnostic virology laboratory.

Laboratory test results, demographic information, and clinical data on 1364 patients who had specimens submitted to the Wisconsin State Laboratory of Hygiene and from whom non-polio enteroviruses were isolated during 1957--1976 were examined. Echoviruses (echo) and Coxsackievirus group B (CB) and group A (CA) were isolated from 719, 389, and 256 of the patients, respectively. Thirty-five different serotypes were identified in the 20-year period. The six most frequently occurring serotypes (echo 9, CB 5, echo 18, CB 2, CB 4, and echo 6) were associated with 57 per cent of all illnesses. The first three serotypes occurred in an epidemic-like manner whereas the next three appeared to be endemic. Eighty-eight per cent of all isolations were from patients whose illnesses began in July through October. The percentage of CA isolates obtained from adults was much smaller than the corresponding percentages seen with CB or echo. A majority of the non-polio enteroviruses were obtained from males. Central nervous system and gastrointestinal tract involvement was greatest with echo and CB 5 tract and lowest with CA and CB 1--4 illnesses. The highest frequency of respiratory tract involvement was with CA and echo 9 illnesses and rash was most frequent with CA illnesses. The highest frequency of hospitalization was with CB 5 patients.

First total synthesis of ent-gelsedine via a novel iodide-promoted allene N-acyliminium ion cyclization.

The first total synthesis of the oxindole alkaloid gelsedine (1) starting from (S)-malic acid is described. The key step is a novel iodide-promoted intramolecular reaction of an allene with an N-acyliminium ion intermediate which provided in a single step the bicyclic vinyl iodide 11. Other important steps are the highly stereoselective Pd-catalyzed Heck cyclization of N-methylanilide 23a which led to the desired spiro-oxindole 24a, the fully regioselective intramolecular oxymercuration of 25a to the desired cyclic ether, and the remarkable oxindole N-demethylation of 29 via a radical mechanism by using dibenzoyl peroxide. The total synthesis was concluded by the stereoselective introduction of the ethyl group from the bis-Boc compound 41 followed by methoxylation of the oxindole nitrogen. This total synthesis leads to the unnatural (+)-enantiomer of gelsedine in 21 steps and 0.10% overall yield.

Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface.

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing.

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead-one-peptide library synthesis, using a Tentagel S-NH2 solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out.

Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors.

The inactivation of HIV by gamma-radiation was studied in frozen and liquid plasma; a reduction of the virus titer of 5 to 6 logs was achieved at doses of 5 to 10 Mrad at -80 degrees C and 2.5 Mrad at 15 degrees C. The effect of irradiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII (FVIII) and prothrombin complex was examined. A recovery of 85 percent of the biologic activity of therapeutic components present in frozen plasma and in lyophilized coagulation factor concentrates was reached at radiation doses as low as 1.5 and 0.5 Mrad, respectively. As derived from the first-order radiation inactivation curves, the radiosensitive target size of HIV was estimated to be 1 to 3 MDa; the target size of FVIII was estimated to be 130 to 160 kDa. Gamma radiation must be disregarded as a method for the sterilization of plasma and plasma-derived products, because of the low reduction of virus infectivity at radiation doses that still give acceptable recovery of biologic activity of plasma components.

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium.

The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a uncompetitive [corrected] manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity.