No change in type 2 diabetes prevalence in children and adolescents over 10 years: Update of a population-based survey in South Germany.

Objective of this study was to analyze prevalence changes in type 2 diabetes (T2D) among children and adolescents over the last 10 years. We performed a cross-sectional survey in Baden-Württemberg (BW), Germany, by using a written questionnaire and comparing these results with T2D prevalence data from the same area retrieved in 2004/2005. In 2016, 50 patients with T2D under 20 years of age were registered in BW, Germany, which corresponds to a prevalence rate of 2.42 per 100 000 (95% confidence interval [CI]: 1.75-3.09). The prevalence rate found in the same geographic area 10 years prior was 2.30 per 100 000 (95% CI: 1.70-2.90). Overall, 70% of T2D patients of this age group were treated by adult diabetologists. Concisely the prevalence of T2D in children and adolescents is still low in South Germany, remaining practically unchanged over the past decade.

Seasonal variation in month of diagnosis in children with type 1 diabetes registered in 23 European centers during 1989-2008: little short-term influence of sunshine hours or average temperature.

BACKGROUND: The month of diagnosis in childhood type 1 diabetes shows seasonal variation. OBJECTIVE: We describe the pattern and investigate if year-to-year irregularities are associated with meteorological factors using data from 50 000 children diagnosed under the age of 15 yr in 23 population-based European registries during 1989-2008. METHODS: Tests for seasonal variation in monthly counts aggregated over the 20 yr period were performed. Time series regression was used to investigate if sunshine hour and average temperature data were predictive of the 240 monthly diagnosis counts after taking account of seasonality and long term trends. RESULTS: Significant sinusoidal pattern was evident in all but two small centers with peaks in November to February and relative amplitudes ranging from ± 11 to ± 38% (median ± 17%). However, most centers showed significant departures from a sinusoidal pattern. Pooling results over centers, there was significant seasonal variation in each age-group at diagnosis, with least seasonal variation in those under 5 yr. Boys showed greater seasonal variation than girls, particularly those aged 10-14 yr. There were no differences in seasonal pattern between four 5-yr sub-periods. Departures from the sinusoidal trend in monthly diagnoses in the period were significantly associated with deviations from the norm in average temperature (0.8% reduction in diagnoses per 1 °C excess) but not with sunshine hours. CONCLUSIONS: Seasonality was consistently apparent throughout the period in all age-groups and both sexes, but girls and the under 5 s showed less marked variation. Neither sunshine hour nor average temperature data contributed in any substantial way to explaining departures from the sinusoidal pattern.

Trends in childhood type 1 diabetes incidence in Europe during 1989-2008: evidence of non-uniformity over time in rates of increase.

AIMS/HYPOTHESIS: The aim of the study was to describe 20-year incidence trends for childhood type 1 diabetes in 23 EURODIAB centres and compare rates of increase in the first (1989-1998) and second (1999-2008) halves of the period. METHODS: All registers operate in geographically defined regions and are based on a clinical diagnosis. Completeness of registration is assessed by capture-recapture methodology. Twenty-three centres in 19 countries registered 49,969 new cases of type 1 diabetes in individuals diagnosed before their 15th birthday during the period studied. RESULTS: Ascertainment exceeded 90% in most registers. During the 20-year period, all but one register showed statistically significant changes in incidence, with rates universally increasing. When estimated separately for the first and second halves of the period, the median rates of increase were similar: 3.4% per annum and 3.3% per annum, respectively. However, rates of increase differed significantly between the first half and the second half for nine of the 21 registers with adequate coverage of both periods; five registers showed significantly higher rates of increase in the first half, and four significantly higher rates in the second half. CONCLUSIONS/INTERPRETATION: The incidence rate of childhood type 1 diabetes continues to rise across Europe by an average of approximately 3-4% per annum, but the increase is not necessarily uniform, showing periods of less rapid and more rapid increase in incidence in some registers. This pattern of change suggests that important risk exposures differ over time in different European countries. Further time trend analysis and comparison of the patterns in defined regions is warranted.

Prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis A and hepatitis E viruses in coagulation factor concentrates.

BACKGROUND AND OBJECTIVES: Due to their high resistance to inactivation procedures, nonenveloped viruses such as parvovirus B19, human bocavirus (HBoV), human parvovirus 4 (PARV4), hepatitis A (HAV) and hepatitis E virus (HEV) pose a particular threat to blood products. Virus transmission to patients treated with blood products presents an additional burden to disease. We determined the frequency and the amount of nucleic acid specific for nonenveloped viruses in recently manufactured preparations of commercial coagulation factor concentrates. MATERIALS AND METHODS: At least three different batches of each of 13 different plasma-derived and recombinant coagulation factor products were tested for the presence and the amount of nucleic acid for parvovirus B19, HBoV, human parvovirus 4, hepatitis A virus and HEV by using quantitative polymerase chain reaction. RESULTS: Whereas none of the recombinant products tested positive for any of these viruses, parvovirus B19 DNA with amounts ranging between 2×10(1) and 1.3×10(3) genome equivalents/ml was detected in five plasma-derived products. In addition to parvovirus B19 genotype 1, genotypes 2 and 3 were observed in two batches of a factor VIII/von-Willebrand factor product. In two products (one factor VIII concentrate and one activated prothrombin complex concentrate), a combination of both genotypes 1 and 2 of parvovirus B19 was detected. CONCLUSION: The data show that nucleic acids from several relevant nonenveloped viruses are not found at detectable levels in coagulation factor concentrates. In some cases, parvovirus B19 DNA was detectable at low levels. Testing of the plasma pools for the full range of parvovirus genotypes is advocated for ensuring product safety.

Selectin-blocking semisynthetic sulfated polysaccharides as promising anti-inflammatory agents.

Selectin-induced leucocytes rolling along the endothelial surface of blood vessels initiate a complex adhesion cascade, which is an essential step in the cellular immune response. Consequently, blocking the binding between the selectins and their ligands represents a promising strategy for suppressing pathological inflammatory reactions. This study describes the effects of an unfractionated heparin and a low-molecular-weight heparin and a series of structurally well-defined semisynthetic glucan sulfates on selectin-mediated cell-rolling with respect to inhibition. To simulate the blood flow characteristics of postcapillary venules, the rolling experiments were performed in a dynamic-flow-chamber system with immobilized selectins and selectin ligand-carrying U937 cells. The influence of the test compounds on cell rolling was measured by the percentage of adherent cells after a certain flow time and the velocity of the rolling cells. Whereas the test compounds displayed no inhibitory effect on E-selectin-mediated cell rolling, they efficiently blocked the rolling induced by P-selectin. The glucan sulfates were much more active than either unfractionated heparin or low-molecular-weight heparin, or the standard inhibitor Sialyl Lewis(X). Their inhibitory potency turned out to be strongly dependent on various structural parameters, such as sulfation pattern and molecular weight. In conclusion, the semisysnthetic glucan sulfates represent promising candidates in the development of selectin blocking agents.

[Improvement of care for diabetics using the care model from Saxony]. / Verbesserung der Versorgung von Diabetikern durch das sächsische Betreuungsmodell.

BACKGROUND AND OBJECTIVE: By promoting the networking of all those involved in caring for diabetics in Saxony, through agreement between those who provide help to them and the organizations which pay the costs, the intention is to improve the overall quality of care of diabetics. It was the aim of this study to evaluate the effectiveness of this integrated health model. PATIENTS AND METHODS: As part of the 3rd Diabetes Agreement in Saxony, a total of 275,804 diabetics were registered, treated and their management costed in the first quarter of 2000 and the fourth quarter of 2001 (56.3% females, 43.7% males; median age 68,7 years). They were patients of 2800 general practitioners and 88 specialist practices. RESULTS: Nearly 80% of all diabetics were included. Taking the level of HbA1c as the criterion of quality achieved, it had decreased from 7,1 +/- 1,3 % in the first 3 months of 2000 to 6,8 +/- 1,3 % in the last 3 months of 2001, and regional differences had been reduced. There was an obvious correlation between early referral to specialist practices and good treatment results, as measured by HbA1c and blood pressure levels. While in 1996 patients were referred when the HbA1c level was 8.8% (median 8.5%), referrals in the last quarter of 2001 were made when the mean was 8,0% (median 7.7%). After two years the risk of inadequate treatment (HbA1c > 7.5% and blood pressure > 140/90 mmHg) had been clearly reduced in about half the cases. CONCLUSIONS: Diabetes agreements, as promulgated in Saxony, have provided effective disease management programs (DMP) for efficacious and efficient integrated diabetic care, so that with continuing effectiveness and further development the St. Vincent targets can be reached. Successful regional diabetes agreements must therefore be maintained within the new, politically centralized, DMPs.

Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells.

In the present study the cellular uptake of targeted immunoliposomes by interleukin-1 activated human endothelial cells has been analysed by several spectroscopical and microscopical fluorescence techniques. Previous in vitro experiments demonstrated that the targeting of immunoliposomes to vascular selectins is a potential way for a selective drug delivery at inflammatory sites. In attempts to further adapt the targeting experiments to physiological conditions, we demonstrate that E-Selectin-directed immunoliposomes are able to bind their target cells under the simulated shear force conditions of capillary blood flow cumulatively for up to 18 h. In order to consequently follow the fate of liposomes after target binding, we analysed the route and degree of liposome internalization of the cells concentrating on cell activation state or various liposomal parameters, e.g., sterical stabilization, type of antibody or antibody coupling strategy. The use of NBD-labelled liposomes and subsequent fluorescence quenching outside the cells with dithionite show that circa 25% of the targeted immunoliposomes were internalized. According to inhibition experiments with agents that interfered with the endocytotic pathway, we found out that the major mechanism of liposome entry is endocytic. The entry involves, at least in part, receptor-mediated endocytosis via E-Selectin, a liposome accumulation in the endosomes and their acidification was proved by pyranine spectroscopic results. Furthermore, microscopical investigations demonstrate that also a fusion of liposomes with the cell membrane occurs followed by a release of entrapped calcein into the cytoplasm. These observations gain insight into the behaviour of E-Selectin-targeted immunoliposomes and indicate that these immunoliposomes have great potential to be used as drug carriers for intracellular drug delivery at inflammatory sites.

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.

Synthesis of the sialyl Lewis X epitope attached to glycolipids with different core structures and their selectin-binding characteristics in a dynamic test system.

Sialyl Lewis X (sLeX)/selectin-mediated leukocyte rolling along endothelial cells has recently gained wide interest. In this paper the influence of the spacer length of laterally clustered neoglycolipids 1a-d on cell rolling in a dynamic test system is investigated. The required di-O-hexadecyl glycerols with none, and with three, six, or nine ethylene glycol units as spacer groups (compounds 4a-d) could be readily obtained. The synthesis of 1-O-thexyldimethylsilyl-protected sLeX 24 was based on sialylation of 2,3,4-O-unprotected galactose derivative 11 with sialyl phosphite 8 as donor; this afforded the desired disaccharide 12, which was transformed into trichloroacetimidate 14 as disaccharide donor. Reaction of 3-O-unprotected glucosamine derivative 18 with fucosyl donor 20 afforded disaccharide 21, which was transformed into the 4-O-unprotected derivative 23. Reaction of 14 with 23 furnished the desired tetrasaccharide 24 in good yield. Transformation of 24 into the trichloroacetimidate 26 as donor, followed by the reaction with 4a-d as acceptor gave, after deprotection, the target molecules 1a-d. For comparison, 4d was also connected with a sialyl residue (-->31) and with an N-acetylglucosamine residue (-->34). Compounds 1c and 1d with a hexaethylene glycol and a nonaethylene glycol spacer, respectively, were much more efficient in mediating selectin-dependent cell rolling in the dynamic test system than compounds 1a and 1b, which had no spacer (1a), or only a triethylene glycol spacer (1b).

Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at the water-air interface.

The monomolecular organization of the main tetraether phospholipid from the archaeon Thermoplasma acidophilum was studied by means of a Langmuir film balance integrated into a fluorescence microscope. After transfer to solid surfaces at different pressures the films were further investigated by ellipsometry, small angle X-ray scattering and atomic force microscopy. In order to complete former results about the main tetraether phospholipid of T. acidophilum [Strobl, C., Six, L., Heckmann, K., Henkel, B., Ring, K., 1985. Z. Naturforsch. 40c, 219-222], the thickness and the two-dimensional organization of the monomolecular films were investigated. Two mean heights values were determined, one of 1.5-1.8 nm and another one of 4-5 nm, indicative for two different molecular arrangements. The former one is interpreted as a 'horseshoe' organization with two polar endings in the aqueous subphase, whereas the latter appears to represent the upright population of molecules with one polar end in the subphase and the other one in the air. In freshly spread and compressed films small domains of the upright lipid population are initially observed, which enlarge with increasing pressure. These domains are no longer existent after 12 h of spreading without compression.

Surface-modification of polystyrene-microtitre plates via grafting of glycidylmethacrylate and coating of poly-glycidylmethacrylate.

Photografting of glycidylmethacrylate (GMA) onto commercially available polystyrene-microtitre plates was carried out in methanol as well as butanol with benzophenone (BP) as photoinitiator. Alternatively, prepolymers of polyglycidylmethacrylate (PGMA) were synthesized in methanol with azo-iso-butyrodinitrile (AIBN) as initiator and dip-coated onto polystyrene-microtitre plates. Both modification methods were tested in order to reach a high binding capacity for proteins. Peroxidase was used as model protein. In addition, the immobilization of myelin basic protein (MBP) to epoxy-modified microtitre plates is shown and a MBP-based ELISA has been developed.

Quantification of lipids, liposomes, amino acids, and proteins by thermal ultramicrodigestion and an ultramicrocoulometric assay, based on the reaction of hypobromite with ammonia.

The quantification of nitrogen in organic material is described. It is based on a novel thermal ultramicrodigestion in combination with an ultramicrocoulometric quantification. The lower detection limit of the coulometric measurement is 0.5 microg nitrogen, which corresponds to 20 microg lipid, 3 microg glycine, or 4 microg protein. Therefore it is as sensitive as the frequently used Lowry method. In contrast to the Lowry protein determination it is not disturbed by detergents and most other interfering substances.

Targetability of novel immunoliposomes prepared by a new antibody conjugation technique.

In order to develop long-circulating immunoliposomes (IL), which combine sterical stabilization with a superior targetability, we have introduced a new methodology for attaching monoclonal antibodies directly onto the distal ends of liposome-grafted polyethylene glycol (PEG) chains. Therefore, we have synthesized a new PEG-PE derivative, which had been endgroup-functionalized with cyanuric chloride. Antibodies can simply be coupled to this membrane anchor in mild basic conditions (pH 8.8) without the need for previous antibody derivatizations. The coupling results have been determined with consideration to various liposome parameters and have been compared to several established antibody coupling procedures, where antibodies had been linked directly to the liposome surface in the presence of PEG (conventional IL). To investigate the targetability of the resulting new IL, anti E-selectin mAb have been coupled and the degree of binding selectin-containing cells has been analyzed. The terminal coupled antibodies show a 1.8-fold higher degree of in vitro cell binding compared to conventional IL, which has been attributed to the antibody position being more easy accessible at the PEG termini. Furthermore, we have illustrated the liposome surface topology and the coupled antibodies by atomic force microscopy, which for such fluid IL has been used first. These images have finely corresponded to the cell binding results, and have been discussed in terms of antibody position and flexibility at the liposome surface.

Investigation of the orientation of purple membrane sheets in Langmuir-Blodgett films by a quartz crystal microbalance.

By means of the quartz crystal microbalance (QCM), a convenient method was developed to determine the degree of orientation of purple membrane (PM) sheets on the air/water interface. Langmuir-Blodgett films from both wild-type and SH-mutant PM (bR D36C) were vertically deposited on the surface of gold-sputtered AT-cut quartz crystals. The shift of resonance frequency of the QCM during a special washing protocol allowed us to differentiate between physically adsorbed PM fragments and any PM attached to the gold surface via chemical bonds. By washing with organic solvents, complete desorption of the wild-type PM was achieved, whereas for the SH-mutant, approximately 60% of the PM fragments could not be detached from the surface. These PM sheets should be oriented with the cytoplasmic side facing the water subphase to that their SH-groups can chemically bind to the gold surface of the quartz plate.

[Medical guidelines for diabetes mellitus in Saxony. An instrument for interdisciplinary quality management for optimizing patient care]. / Arztliche Leitlinien Diabetes mellitus in Sachsen. Instrument interdisziplinären Qualitätsmanagements zur Optimierung der Patientenversorgung.

Patients with diabetes still have a life expectancy of 5-10 years less and a markedly reduced quality of life than non-diabetic persons. Concepts, models, and contracts aiming at an efficient co-operative care for chronically ill patients have been developed in the new German states to overcome shortage of care. The dual care of motivated diabetic patients by family physicians and experts for metabolic diseases has proven to be efficient both in pilot studies as well as in country-wide investigations. A representative commission for diabetes has developed guidelines for such a structure of dual care. Design and content of these regional developed guidelines about an cooperative evidence based care for diabetic patients fulfills the criteria suggested by international bodies of experts and the medical society for quality assurance. The Saxonian guidelines for diabetes have been successfully implemented step by step in medical offices and hospitals. We are sure that the further implementation of the shared care system diabetes will further improve quality of care.

Selectins as new targets for immunoliposome-mediated drug delivery. A potential way of anti-inflammatory therapy.

Endothelial cell adhesion molecules, which are expressed in response to inflammatory signals to mediate recruitment of leukocytes to sites of inflammation, appear to be excellent targets for drug delivery systems to open new perspectives of antiinflammatory therapies. In this study we describe the preparation and characterization of antibody-coupled liposomes (immunoliposomes) as directed against endothelial (E)-selectins. We have examined the factors affecting the covalent coupling of antibodies to the membrane anchor N-glutaryl-phosphatidylethanolamine via amide bound and have compared them to other coupling procedures. The target sensitivity has been demonstrated in a cell-containing in-vitro model, where liposome binding to selectins under either static, or simulated blood flow conditions was illustrated by using fluorescence microscopical means. It could be shown that even under shear force conditions liposomes selectively accumulate at selectin-containing cells when a specific lipid composition and a certain balance in the lipid/antibody ratio was maintained. Furthermore, the need for polyethylene glycol-derived lipids to sterically stabilize the liposomes for preventing unspecific liposome attachment to cells has been demonstrated.

The role of glycolipids in mediating cell adhesion: a flow chamber study.

Selectins constitute a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium by recognizing various carbohydrate ligands in response to inflammation. To test the hypothesis that multivalent binding of selectins to their ligands is the molecular basis for achieving sufficient binding forces, we have performed this flow chamber study. Selectin-containing Chinese hamster ovarial cells (CHO-E) bind and roll along a support-fixed phospholipid membrane containing a defined concentration of a synthetic Sialyl Lewisx (sLex) glycolipid ligand. Ligands are either homogeneously distributed, or arranged in defined lateral clusters, as illustrated here for the first time. The lateral glycolipid clusters which appear as recognition motifs are essential for mediating cell rolling. Furthermore, the transition from firm cell adhesion to cell rolling depends on the site density of ligands. Rolling velocity shows little dependence on shear forces within a broad range. As we found out that cells do not roll along the model membranes with homogeneous ligand distribution, our results therefore support the hypothesis of multivalent binding events. Since these investigations suggest that lipid-anchored sLex, functionally embedded in a lipid matrix, can mediate cell rolling, this study demonstrates the relationship between dynamic glycolipid binding to selectins with the hypothesis of multivalency of binding for the first time.

Quantitative determination of thiolipids in organic solution, in membranes, and on HPTLC plates.

In the presence of ortho-phthalaldehyde and glucosamine, thiolipids form fluorescent isoindole derivatives. This reaction can be used to quantify single- and double-chain mercaptans in membranes (liposomes) and micellar solutions. The lower detection limit is 100 pmol. In addition, the assay allows the detection of 1.9 nmol thiolipids on HPTLC plates and the fluorescence signal is stable for days. A minor modification of the commonly used DTNB (Ellman's) assay allows the quantification of thiolipids in organic solutions at a concentration down to 3 nmol.

A liposome-based model system for the simulation of lectin-induced cell adhesion.

A parallel plate flow chamber with defined wall shear rates was developed in order to study and simulate cellular adhesion to biological membranes as mediated by lectin/carbohydrate interactions. Planar bilayers containing clustered areas of various long-chain alkyl mannosides as carbohydrate ligands and supported on transparent materials were used as model membranes. Their interaction with liposomes bearing Concanavalin A as model cells was observed fluorimetrically by confocal laser scanning microscopy. The use of supported membranes made it possible to study the dependence of adhesion upon different physicochemical parameters of membranes. The liposomes of this model were able to simulate the lectin-mediated adhesion of cells in a shear flow. Once specific receptor-mediated adhesion had taken place, liposomes tended to attach irreversibly to the membrane. This could be avoided by employing lipid compositions which represent a special balance between charged and polyethylene glycol-coupled lipids. This is discussed in term of the interplay between the various attractive and repulsive forces at membrane surfaces. The dependence of liposome adhesion upon the shear rate could be detected. These results were used to evaluate binding forces between lectin-bearing liposomes and ligand-containing planar bilayers.