[Measures to improve the health situation of patients with rare diseases in Germany. A comparison with the National Action Plan]. / Maßnahmen zur Verbesserung der gesundheitlichen Situation von Menschen mit seltenen Erkrankungen in Deutschland. Ein Vergleich mit dem Nationalen Aktionsplan.

BACKGROUND: Approximately 4 million patients with a rare disease live in Germany. The medical care of these patients is problematic because of the rarity and heterogeneity of different clinical pictures. The Federal Ministry of Health has therefore published a research report on "Measures to improve the health situation of people with rare diseases in Germany" in 2009. OBJECTIVE: The aim of this paper is to present the main recommendations of this research report and relate it to current developments in the field of medical care for people with rare diseases. METHODOLOGY: The care situation of patients with rare diseases was determined using questionnaires, expert interviews and focus group discussions with representatives of patients, service providers and stakeholders from the health institutions. RESULTS: The main range of actions that have been identified in the research report were centre and network formation, specialized forms of medical care, diagnosis and treatment, information and experience exchange, performance fees and reimbursement of the costs, guidelines and patient pathways, the research, the implementation of a National Action Alliance and the development of a National Action Plan. DISCUSSION: In March 2010 a National Action League for People with Rare Diseases (NAMSE) was founded. The NAMSE created a national plan of action for people with rare diseases for improving medical care in the field of rare diseases which was approved by the Federal Government in August 2013. Thus, two important areas of the research report have already been implemented. In a comparison of the areas of activity of the research report with those of the National Action Plan it becomes clear that priorities will be in the context of health services research in rare diseases, for example the introduction of centres of reference for rare diseases, measures to accelerate the diagnostic process and the promotion of research and information management in the future.

Factors associated with ruminal pH at herd level.

The objective of this study was to evaluate factors associated with ruminal pH at herd level. Four hundred and thirty-two cows of a Thuringian dairy herd were sampled before claw trimming using a rumen fluid scoop. Volume and pH of the rumen sample were measured, and lactation number, percentage of concentrates in the ration, days in milk (DIM), time of day, and daily milk yield were recorded. Rumen sampling was successful in 99.8% of the cows. The average sample volume was 25 mL. Rumen sample pH decreased with increasing percentage of concentrates in the ration. Ruminal pH decreased from calving to 77 DIM, and grew subsequently to 330 DIM. During the day, rumen pH followed a sinus curve, with maxima in the morning (0915 h) and afternoon (1533 h), and a minimum around noon (1227 h). Ruminal pH decreased with increasing daily milk yield. Lactation number interacted with daily milk yield on rumen pH. The percentage of concentrates in the ration, DIM, time of day, and daily milk yield were significant factors affecting ruminal pH at the herd level.

Dual-energy CT for the characterization of urinary calculi: In vitro and in vivo evaluation of a low-dose scanning protocol.

The efficiency and radiation dose of a low-dose dual-energy (DE) CT protocol for the evaluation of urinary calculus disease were evaluated. A low-dose dual-source DE-CT renal calculi protocol (140 kV, 46 mAs; 80 kV, 210 mAs) was derived from the single-energy (SE) CT protocol used in our institution for the detection of renal calculi (120 kV, 75 mAs). An Alderson-Rando phantom was equipped with thermoluminescence dosimeters and examined by CT with both protocols. The effective doses were calculated. Fifty-one patients with suspected or known urinary calculus disease underwent DE-CT. DE analysis was performed if calculi were detected using a dedicated software tool. Results were compared to chemical analysis after invasive calculus extraction. An effective dose of 3.43 mSv (male) and 5.30 mSv (female) was measured in the phantom for the DE protocol (vs. 3.17/4.57 mSv for the SE protocol). Urinary calculi were found in 34 patients; in 28 patients, calculi were removed and analyzed (23 patients with calcified calculi, three with uric acid calculi, one with 2,8-dihyxdroxyadenine-calculi, one patient with a mixed struvite calculus). DE analysis was able to distinguish between calcified and non-calcified calculi in all cases. In conclusion, dual-energy urinary calculus analysis is effective also with a low-dose protocol. The protocol tested in this study reliably identified calcified urinary calculi in vivo.

Characterization of coronary atherosclerosis by dual-source computed tomography and HU-based color mapping: a pilot study.

To assess HU-based color mapping for characterization of coronary plaque, using intravascular ultrasound virtual histology (IVUS-VH) as a standard of reference. Dual-source computed tomography and IVUS-VH were prospectively performed in 13 patients. In five lesions, HU thresholds of the color-coding software were calibrated to IVUS-VH. In a 15-lesion verification cohort, volumes of vessel, lumen and plaque or percentages of lipid, fibrous and calcified components were obtained through use of pre-set HU cut-offs as well as through purely visual adjustment of color maps. Calibrated HU ranges for fatty or fibrous plaque, lumen and calcification were -10-69, 70-158, 159-436 and 437+. Using these cut-offs, HU-based analysis achieved good agreement of plaque volume with IVUS (47.0 vs. 51.0 mm(3)). Visual segmentation led to significant overestimation of atheroma (61.6 vs. 51.0 mm(3); P = 0.04) Correlation coefficients for volumes of vessel, lumen and plaque were 0.92, 0.87 and 0.83 with HU-based analysis or 0.92, 0.85 and 0.71 with visual evaluation. With both methods, correlation of percentage plaque composition was poor or insignificant. HU-based plaque analysis showed good reproducibility with intra-class correlation coefficients being 0.90 for plaque volume and 0.81, 0.94 or 0.98 for percentages of fatty, fibrous or calcified components. With use of optimized HU thresholds, color mapping allows for accurate and reproducible quantification of coronary plaque.

Noninvasive coronary angiography using 64-slice spiral computed tomography in an unselected patient collective: effect of heart rate, heart rate variability and coronary calcifications on image quality and diagnostic accuracy.

OBJECTIVE: The aim of this study was to assess the impact of heart rate, heart rate variability and calcification on image quality and diagnostic accuracy in an unselected patient collective. SUBJECTS AND METHODS: One hundred and two consecutive patients with known or suspected coronary artery disease underwent both 64-MSCT and invasive coronary angiography. Image quality (IQ) was assessed by independent observers using a 4-point scale from excellent (1) to non-diagnostic (4). Accuracy of MSCT regarding detection or exclusion of significant stenosis (>50%) was evaluated on a per segment basis in a modified AHA 13-segment model. Effects of heart rate, heart rate variability, calcification and body mass index (BMI) on IQ and accuracy were evaluated by multivariate regression. IQ and accuracy were further analysed in subgroups of significant predictor variables and simple regression performed to calculate thresholds for adequate IQ. RESULTS: Mean heart rate was 68.2+/-13.3 bpm, mean heart rate variability 11.5+/-16.0 beats per CT-examination (bpct) and median Agatston score 226.5. Average IQ score was 2+/-0.6 whilst diagnostic quality was obtained in 89% of segments. Overall sensitivity, specificity, PPV or NPV was 91.2%, 99.2%, 95.3% or 98.3%. According to multivariate regression, overall IQ was significantly related to heart rate and calcification (P=0.0038; P<0.0001). The effect of heart rate variability was limited to IQ of RCA segments (P=0.018); BMI was not related to IQ (P=0.52). Calcification was the only predictor variable with significant effect on the number of non-diagnostic segments (P<0.0001). In a multivariate regression, calcification was also the single factor with impact on diagnostic accuracy (P=0.0049). CONCLUSION: Whilst heart rate, heart rate variability and calcification all show an inverse correlation to IQ, severe calcium burden remains the single factor with translation of such effect into decrease of diagnostic accuracy.

Sixty-four-MSCT in the characterization of porcine acute and subacute myocardial infarction: determination of transmurality in comparison to magnetic resonance imaging and histopathology.

OBJECTIVE: The aim of this study was to assess the accuracy of MSCT in characterizing myocardial infarction (MI) and, thereby, determine the extent of early perfusion defect (ED), microvascular obstruction (MO) and transmural depth of late enhancement (LE) in comparison to MRI and histology. MATERIALS AND METHODS: Seven pigs were studied with MSCT (Somatom Sensation 64) and MRI (Magnetom Sonata) a median 1 and 21 days following temporary occlusion of a diagonal branch and creation of small reperfused infarction. For depiction of ED, CT images were acquired in the early arterial phase and following 35 s; LE and MO were evaluated on images obtained at 3, 5, 10 and 15 min. Thereby, a bolus/low-flow contrast injection protocol was used. Triphenyltetrazolium-chloride (TTC) stain and histology were obtained. Volumes of enhancement patterns were assessed as percentage of the ventricle and compared by Bland-Altman analysis. Segmental co-localization and graded transmurality was evaluated with weighted-kappa-test. RESULTS: Close spatial agreement was observed for MRI-MO and MSCT-MO (bias=0.55; CI=-1.49 to 2.60 at 5 min MSCT), TTC and MSCT-LE (bias=-1.28; CI=-3.76 to 1.19) or MRI-LE and MSCT-LE (bias=-0.79; CI=-4.19 to 2.60). There was good segmental co-localization for MO (weighted kappa=0.93) and high agreement for transmural extent of TTC, MRI-LE and MSCT-LE (weighted kappa=0.84 TTC versus MSCT; 0.86 MRI versus MSCT). Arterial and 35s ED significantly underestimated infarct size and showed poor segmental or transmural agreement (weighted kappa=0.33; 0.44). CONCLUSIONS: MSCT late-scans not only reliably depict size of MO and LE in acute or subacute infarct phases but, moreover, allow for accurate determination of LE transmurality.

Stoichiometric binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate to bovine heart cytochrome c oxidase.

The binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP-ATP per monomer COX with a Kd of 1.6 microM.

A comparative assessment of the roles of CD8 and CD2 in the functions of activated murine CD8+ T lymphocytes.

Both CD8 and CD2 are T cell surface receptors involved in physical cell interaction and in transmembrane signalling. The present paper addresses their role in the induction of two different functions of the cloned murine cytotoxic T cell C196: target cell lysis and IFN-gamma production. These functions were induced in C196 either by stimulation with the specific stimulator/target cell P815 or, bypassing specific recognition, by the aCD3 hybridoma 145-2C11 or by solid phase aTCR antibodies. These responses were tested for their susceptibility to inhibition/enhancement by a panel of aCD8 and aCD2 mAb. In addition, CD8 deficient and CD8/CD2 double-deficient variants of C196 were transfected with the CD8 and CD2 genes and the resulting cell lines were analysed for their functional capacities. The following results were obtained: (i) CD8 is primarily important in the specific recognition process of activated CTL; (ii) transmembrane signalling of activated CTL through the TCR does not require CD8, nor is it sensitive to modification through CD8; (iii) CTL can nevertheless be directly activated through CD8; however, this is restricted to induction of cytotoxicity but does not result in IFN-gamma production; (iv) CD2 does not seem to be important in any of these responses.

Age associated changes of AGE-receptor expression: RAGE upregulation is associated with human heart dysfunction.

The binding of advanced glycation endproducts (AGEs) to their receptors is known to cause changes in cell function during normal ageing and is implicated in the pathogenesis of cardiovascular disease. In this study, expression of the AGE-receptor 3 (AGE-R3) and the receptor for AGEs (RAGE) was compared on the mRNA and protein level in the ageing human heart. Western blot and RT-PCR analysis of the AGE receptors from the cardiac auricles in senescent and adult patients was performed and compared with young controls. Whereas the expressions of AGE-R3 as well as RAGE protein were significantly upregulated in the senescent population, only the upregulation of RAGE is associated with reduced heart function. Therefore, our results support a pathophysiological function for RAGE in the ageing human heart.

Regulation of 5-HT release from enterochromaffin cells.

Large amounts of 5-HT are present in the mammalian intestine where the amine is concentrated in the enterochromaffin cells (ECs) of the mucosa. ECs have the enzymes to synthesize 5-HT, are endowed with a specific, imipramine-sensitive 5-HT uptake mechanism and can store 5-HT in specific secretory vesicles. ECs can secrete 5-HT in a calcium-dependent manner. In particular, calcium influx through voltage-regulated channels and receptor-mediated liberation of intracellular calcium can evoke 5-HT release. 5-HT secretion from ECs occurs predominantly at the interstitial side and is controlled by a complex pattern of receptor-mediated mechanisms. Stimulatory receptors (beta-adrenoceptors, muscarine, nicotine and 5-HT3 receptors) and inhibitory receptors (alpha 2-adrenoceptors, histamine H3, GABAA- and GABAB-, A2 and P2y alpha purine and 5-HT4 receptors as well as receptors for vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase stimulating peptide (PACAP) and somatostatin) have been shown to be involved in the control of 5-HT release from the ECs.

Characterization of histamine H3 receptors inhibiting 5-HT release from porcine enterochromaffin cells: further evidence for H3 receptor heterogeneity.

The nature of the histamine receptor mediating inhibition of 5-HT release was investigated in strips of the porcine small intestine by investigating the effects of histamine ligands on the overflow of endogenous 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). The overflow was measured by HPLC, combined with electrochemical detection and represents calcium-sensitive 5-HT release from enterochromaffin cells, as reported previously. The histamine H3 receptor selective agonists (R)-alpha-methyl-histamine and imetit inhibited the overflow of 5-HT maximally by 50-60%, with EC50 values of 48 and 3.2 nmol/l, respectively. Effects on 5-HT overflow were always accompanied by similar effects on the overflow of 5-HIAA. Thioperamide (100 nmol/l) shifted the concentration response curve of (R)-alpha-methyl-histamine to the right (pKB value 8.38). The inhibitory effect of 1 mumol/l (R)-alpha-methyl-histamine was antagonized in a concentration-dependent manner by thioperamide (IC50: 65 nmol/l) and dimaprit (IC50: 8.6 mumol/l); however, the effect of (R)-alpha-methyl-histamine was weakly antagonized by burimamide (by 38% at 100 mumol/l) and not significantly affected by other H3 receptor antagonists, such as impromidine, betahistine and phenyl-butanoyl-histamine (each up to 100 mumol/l). In conclusion, H3 receptors mediating inhibition of 5-HT release from porcine enterochromaffin cells have a particular pharmacological profile indicating that heterogeneity of H3 receptors may exist. The data suggest that histamine H3 receptors modulating 5-HT release in pig small intestine do not belong to either H3A or H3B receptors as defined in rat tissue.

Modulation of noradrenaline release in rat isolated stomach by prostanoids, but not by histaminergic mechanisms.

Several gastric functions are modulated by the sympathetic nervous system, but local mechanisms involved in the control of noradrenaline release are largely unknown. Overflow of endogenous noradrenaline was studied from isolated rat stomach incubated in Ussing chambers allowing the separate determination of mucosal and serosal overflow. Spontaneous noradrenaline overflow was similar at the mucosal and serosal side, but electrical field stimulation caused a frequency-dependent increase in noradrenaline overflow selectively at the serosal side. Evoked noradrenaline overflow was blocked by tetrodotoxin, not affected by indometacin and markedly enhanced (by about 250%) by yohimbine. In the presence of indometacin and yohimbine, sulprostone (an agonist at EP1/EP3 receptors) and misoprostol (an agonist at EP2/EP3 receptors) reduced the noradrenaline overflow evoked by stimulation at 3 Hz maximally by about 80% (EC50: 6 nmol/l and 11 nmol/l, respectively). The EP1 receptor selective antagonist AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) did not antagonize the inhibition by sulprostone. Noradrenaline overflow evoked by stimulation at 1 Hz and 3 Hz was increased by scopolamine by about 50% and almost completely inhibited by oxotremorine. Neither, histamine nor the H3 receptor selective agonist (R)-alpha-methyl-histamine, nor the H1, H2 and H3 selective receptor antagonists mepyramine, cimetidine and thioperamide significantly affected noradrenaline overflow evoked by stimulation at 1 Hz or 3 Hz. In conclusion, impulse-induced noradrenaline release in the rat stomach is controlled by multiple presynaptic mechanisms involving alpha 2-adrenergic autoreceptors, EP3 prostanoid and muscarine heteroreceptors, whereas histaminergic mechanisms do not appear to be significant.

Cultivation of recombinant baby hamster kidney cells in a fluidized bed bioreactor system with porous borosilicate glass.

Dense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers. Experiments were carried out with different medium formulations for a period of 48 days. Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform. Maximal cell densities and product yields could be maintained, even when protein-free medium was perfused exceeding 2 reactor volumes per day. Final cell densities of magnitude 7.1 x 10(7) mL-1 intrasphere volume were reached, while the interleukin-2 production rate was 0.70 mg day-1. The cell specific productivity reached a value of 1.3 x 10(-10) mg day-1. The first results were presented with a cell line that grows under glutamine-free medium conditions. The use of a glutamine-free medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism. Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro. However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant. The metabolism of threonine has been detected to be directly dependent on the presence of glutamine. Cells grown in glutamine-free culture medium produced glycine yields 6 times higher than those grown in glutamine-containing medium. A bead-to-bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.

Detoxification of lignocellulose hydrolysates with ion-exchange resins.

Lignocellulose hydrolysates contain fermentation inhibitors causing decreased ethanol production. The inhibitors include phenolic compounds, furan aldehydes, and aliphatic acids. One of the most efficient methods for removing inhibiting compounds prior to fermentation is treatment of the hydrolysate with ion-exchange resins. The performance and detoxification mechanism of three different resins were examined: an anion exchanger, a cation exchanger, and a resin without charged groups (XAD-8). A dilute acid hydrolysate of spruce was treated with the resins at pH 5.5 and 10.0 prior to ethanolic fermentation with Saccharomyces cerevisiae. In addition to the experiments with hydrolysate, the effect of the resins on selected model compounds, three phenolics (vanillin, guaiacol, and coniferyl aldehyde) and two furan aldehydes (furfural and hydroxymethyl furfural), was determined. The cation exchanger increased ethanol production, but to a lesser extent than XAD-8, which in turn was less effective than the anion exchanger. Treatment at pH 10.0 was more effective than at pH 5.5. At pH 10.0, the anion exchanger efficiently removed both anionic and uncharged inhibitors, the latter by hydrophobic interactions. The importance of hydrophobic interactions was further indicated by a substantial decrease in the concentration of model compounds, such as guaiacol and furfural, after treatment with XAD-8.

Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae.

Phenolic compounds released and generated during hydrolysis inhibit fermentation of lignocellulose hydrolysates to ethanol by Saccharomyces cerevisiae. A wide variety of aromatic compounds form from lignin, which is partially degraded during acid hydrolysis of the lignocellulosic raw material. Aromatic compounds may also form as a result of sugar degradation and are present in wood as extractives. The influence of hydroxy-methoxy-benzaldehydes, diphenols/quinones, and phenylpropane derivatives on S. cerevisiae cell growth and ethanol formation was assayed using a defined medium and oxygen-limited conditions. The inhibition effected by the hydroxy-methoxy-benzaldehydes was highly dependent on the positions of the substituents. A major difference in inhibition by the oxidized and reduced form of a diphenol/quinone was observed, the oxidized form being the more inhibitory. The phenylpropane derivatives were examined with respect to difference in toxicity depending on the oxidation-reduction state of the gamma-carbon, the presence and position of unsaturated bonds in the aliphatic side chain, and the number and identity of hydroxyl and methoxyl substituents. Transformations of aromatic compounds occurring during the fermentation included aldehyde reduction, quinone reduction, and double bond saturation. Aromatic alcohols were detected as products of reductions of the corresponding aldehydes, namely hydroxy-methoxy-benzaldehydes and coniferyl aldehyde. High molecular mass compounds and the corresponding diphenol were detected as products of quinone reduction. Together with coniferyl alcohol, dihydroconiferyl alcohol was identified as a major transformation product of coniferyl aldehyde.

Respiratory chain proteins.

Mammalian mitochondrial DNA codes for 13 proteins, which are all components of energy transducing enzyme complexes of the respiratory chain, i.e. the complexes which translocate protons across the inner mitochondrial membrane. The number of subunits of these enzyme complexes increase with increasing evolutionary stage of the organism. The additional nuclear coded subunits of the enzyme complexes from higher organisms are involved in the regulation of respiration, as demonstrated by the influence of intraliposomal ATP and ADP on the reconstituted cytochrome c oxidase (COX) from bovine heart. This regulation is not found with the reconstituted enzyme from P. denitrificans, which lacks the nuclear coded subunits. Some of the nuclear coded subunits occur in tissue-specific isoforms, as reported for COX and NADH dehydrogenase. Tissue-specific regulation of COX activity is also demonstrated by the differential effects of intraliposomal ADP on the kinetics of reconstituted COX from bovine liver and heart, which differ in subunits VIa, VIIa and VIII. At least 3 different COX isozymes occur in bovine liver, heart or skeletal muscle and smooth muscle. An evolutionary relationship between COX subunits VIa and VIc and between VIIa and VIIb is suggested based on the crossreactivity of monoclonal antibodies, amino acid sequence homology and hybridization at low stringency of PCR-amplified cDNAs for subunits VIa-1, VIa-h and VIc from the rat.

Is mesenteric panniculitis truely a paraneoplastic phenomenon? A matched pair analysis.

PURPOSE: Mesenteric panniculitis (MP) is an underdiagnosed inflammatory condition of mesenteric adipose tissue. Prior studies suggested an association of MP with malignancy. To reassess this hypothesis, we performed the first matched case-control study comparing prevalence of malignancy and other disease in patients with and without MP. MATERIAL AND METHODS: With a keyword search we identified CT examinations of MP patients between 2010 and 2012. Each MP patient was matched with two control patients for age, gender, abdominal diameter and CT protocol. Manifestation and extent of mesenteric panniculitis was classified independently by two investigators according to established criteria. Concomitant disease, laboratory parameters and follow up CTs were recorded and analyzed for all patients. RESULTS: 77 of 13485 CT patients were diagnosed with MP (prevalence 0.58%). 50.6% of MP patients suffered from malignancy vs. 60.2% in the control group (p=0.157). Over up to 4 years of follow up in 35 of these 77 MP patients no association between development of MP and the course of tumor diseases could be identified. There was also no significant difference in the rate of frequent concomitant diseases such as hypertension, diabetes or previous surgery between the two groups. CONCLUSION: In this first case-control-study we could show that, contrary to previous reports, mesenteric panniculitis is neither paraneoplastic nor is it associated with other diseases.

Automatic bone and plaque removal using dual energy CT for head and neck angiography: feasibility and initial performance evaluation.

PURPOSE: We sought to evaluate the feasibility and efficiency of dual energy (DE) bone and plaque removal in head and neck CT angiography. MATERIALS AND METHODS: 20 patients with suspected carotid stenoses received head and neck DE-CTA as part of their pre-interventional workup. Visual grading using multiplanar reformations (MPR), thick slab maximum intensity projections (MIP) and quantitative vessel analysis (QVA) of stenoses was performed prior and after DE bone removal. Results were evaluated for the detection of relevant stenoses (vessel area reduction >70%). Vessel segmentation errors were analyzed. RESULTS: Segmentation errors occurred in 19% of all vessel segments. Nevertheless, most post-bone removal artifacts could be recognized using the MPR technique for reading. Compared to MPR reading prior to bone removal, sensitivity, specificity, positive and negative predictive values after bone removal were 100%, 98%, 88% and 100% for MPR reading and 100%, 91%, 63% and 100% for exclusive MIP reading, respectively. There was a good agreement between the QVA results prior and post-DE plaque removal (r(2)=0.8858). CONCLUSION: DE bone and plaque removal for head and neck angiography is feasible and offers a rapid and highly sensitive overview over vascular head and neck studies. Due to a slightly limited specificity of the MIP technique due to segmentation errors, possible stenoses should be verified and graded using MPR techniques.