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Objectives: The aim of this study was to compare post-authorisation measures (PAMs) from the European Medicines Agency (EMA) with data requests in fixed-termed conditional appraisals of early benefit assessments from the German Federal Joint Committee (G-BA)., Methods: Medicinal products with completed benefit assessments during an assessment period of 3.5 years were considered. PAMs extracted from European Public Assessment Reports (EPARs) were compared with data requests issued by the G-BA in the context of conditional appraisals., Results: Twenty conditional appraisals (19 products) and 34 EPARs containing PAMs (33 products) were identified. Data categories (efficacy, safety, etc.), data types (type of study required to address the request) and clarity of requests were determined. Conditional appraisals disproportionately focused on oncology products (13/19 products with conditional appraisals vs. 14/33 products with PAMs). No clear rationale for the G-BA issuing conditional appraisals could be identified in public sources. Both EMA and G-BA requested mainly efficacy and safety data (44/54 and 23/35 categories requested, respectively); however, 28/35 G-BA data requirements went beyond requests made by the EMA. Almost half of the G-BA requests (9/20), but no PAMs, were unclear, and no methodological guidance for fulfilling the data requirements was provided by the G-BA., Conclusions: Better alignment between data requests from regulatory authorities and health technology assessment bodies is strongly recommended.

Free fatty acids (FFA) are released through phospholipase A2 (PLA2), which cleaves the fatty acyl residue at the sn-2 position of phospholipids (PL). During inflammatory diseases, reactive oxygen species (such as HOCl) lead to the formation of oxidatively modified PL (e.g., chlorohydrin generation). It is still widely unknown to which extent the oxidation of PL influences their digestibility by PLA2. Additionally, investigations on the impact of the position of the unsaturated fatty acyl residue (sn-1 versus sn-2 position) and modifications of the headgroup (for instance phosphatidylcholine (PC) versus phosphatidylethanolamine (PE)) are also lacking. Therefore, the aim of this study is the investigation of these aspects using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to elucidate the PL/lysophospholipid (LPL) ratios as measures of the PLA2 digestibility. We will show that oxidative modifications of PL by HOCl have a considerable impact on the PLA2 digestibility, i.e., oxidation of the unsaturated fatty acyl residues leads to a reduced digestibility of both PC and PE. Besides, it will be shown that MALDI MS is a convenient and reliable tool to investigate the related changes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) play important physiological roles and are of particular relevance in the pathogenesis of inflammatory diseases. At inflammatory conditions, the enzyme myeloperoxidase generates hypochlorous acid (HOCl) which adds to the double bonds of fatty acyl residues of (phospho)lipids under the formation of chlorohydrins. This may lead to the development of many inflammatory diseases, such as atherosclerosis or arthritis, if the ROS generation exceeds a certain extent. Using oleic acid as the simplest unsaturated fatty acid which contains just a single double bond, as a model system, we investigated all products - including the chlorohydrin - after its reaction with HOCl by a combination of thin-layer chromatography and electrospray ionization mass spectrometry. Unlike the general acceptance, the reaction of oleic acid and HOCl leads not exclusively to the formation of chlorohydrin (isomers) but is much more complex: there are also considerable amounts of dimeric and (to a minor extent) trimeric products which can be assigned to isomeric ethers and esters. The obtained products after oleic acid chlorination were also compared with the reaction products of 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) and HOCl. The reasons why different products are obtained will be discussed and the involvement of the carboxylic acid emphasized., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dividing the sea-level budget into contributions from ice sheets and glaciers, the water cycle, steric expansion, and crustal movement is challenging, especially on regional scales. Here, Gravity Recovery And Climate Experiment (GRACE) gravity observations and sea-level anomalies from altimetry are used in a joint inversion, ensuring a consistent decomposition of the global and regional sea-level rise budget. Over the years 2002-2014, we find a global mean steric trend of 1.38 ± 0.16 mm/y, compared with a total trend of 2.74 ± 0.58 mm/y. This is significantly larger than steric trends derived from in situ temperature/salinity profiles and models which range from 0.66 ± 0.2 to 0.94 ± 0.1 mm/y. Mass contributions from ice sheets and glaciers (1.37 ± 0.09 mm/y, accelerating with 0.03 ± 0.02 mm/y(2)) are offset by a negative hydrological component (-0.29 ± 0.26 mm/y). The combined mass rate (1.08 ± 0.3 mm/y) is smaller than previous GRACE estimates (up to 2 mm/y), but it is consistent with the sum of individual contributions (ice sheets, glaciers, and hydrology) found in literature. The altimetric sea-level budget is closed by coestimating a remaining component of 0.22 ± 0.26 mm/y. Well above average sea-level rise is found regionally near the Philippines (14.7 ± 4.39 mm/y) and Indonesia (8.3 ± 4.7 mm/y) which is dominated by steric components (11.2 ± 3.58 mm/y and 6.4 ± 3.18 mm/y, respectively). In contrast, in the central and Eastern part of the Pacific, negative steric trends (down to -2.8 ± 1.53 mm/y) are detected. Significant regional components are found, up to 5.3 ± 2.6 mm/y in the northwest Atlantic, which are likely due to ocean bottom pressure variations.

Chlorinated phospholipids are formed by the reaction of hypochlorous acid (HOCl), generated by the enzyme myeloperoxidase under inflammatory conditions, and the unsaturated fatty acyl residues or the head group. In the first case the generated chlorohydrins are both proinflammatory and cytotoxic, thus having a significant impact on the structures of biomembranes. The latter case leads to chloramines, the properties of which are by far less well understood. Since HOCl is also widely used as a disinfecting and antibacterial agent in medicinal, industrial, and domestic applications, it may represent an additional source of danger in the case of abuse or mishandling. This review discusses the reaction behavior of in vivo generated HOCl and biomolecules like DNA, proteins, and carbohydrates but will focus on phospholipids. Not only the beneficial and pathological (toxic) effects of chlorinated lipids but also the importance of these chlorinated species is discussed. Some selected cleavage products of (chlorinated) phospholipids and plasmalogens such as lysophospholipids, (chlorinated) free fatty acids and α -chloro fatty aldehydes, which are all well known to massively contribute to inflammatory diseases associated with oxidative stress, will be also discussed. Finally, common analytical methods to study these compounds will be reviewed with focus on mass spectrometric techniques., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Unlabelled: Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons., Importance: Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: There are currently no uniform standards regarding rehabilitation of patients after lumbar spine surgery. Due to significant improvements in surgical methods in recent years, an increase in postoperative training intensity is now possible. Conservative rehabilitation has yet to adapt to this reality. Earlier initiation of structured rehabilitation after the acute phase is often regarded with skepticism., Objective: To evaluate the effect of structured rehabilitation after lumbar spine surgery in the early phase of treatment (2 weeks after surgery), a group of seven spinal surgery clinics, two inpatient and three outpatient rehabilitation centers in the Rhine-Main area in Germany was formed., Materials and Methods: In this prospective study, 124 patients were divided into groups (A/B/C) by their surgeon, regardless of diagnosis and surgical procedure. For each group of participants, the content of therapy was preplanned., Results: The statistical analysis using the visual analog scale (VAS), Oswestry Disability Index (ODI), and short form-12 health survey (SF-12) to evaluate changes in impairment caused by back pain and in health-related quality of life was evaluated. In all three groups, significant improvements in VAS, ODI, and SF-12 were shown. Re-operation was unnecessary due to the absence of postoperative complications., Conclusion: A structured postoperative rehabilitation program results in significant improvements in the parameters of pain and quality of life, and does not increase the risk of postoperative complications.

Background: Inherited arrhythmias were originally considered isolated electrical defects. There is growing evidence that ion channel dysfunction also contributes to myocardial disorders, but genetic overlap has not been reported for sinus node dysfunction (SND) and noncompaction cardiomyopathy (NCCM)., Objectives: The study sought to investigate a familial electromechanical disorder characterized by SND and NCCM, and to identify the underlying genetic basis., Methods: The index family and a cohort of unrelated probands with sinus bradycardia were examined by electrocardiography, Holter recording, exercise stress test, echocardiography, and/or cardiac magnetic resonance imaging. Targeted next-generation and direct sequencing were used for candidate gene analysis and mutation scanning. Ion channels were expressed in HEK293 cells and studied using patch-clamp recordings., Results: SND and biventricular NCCM were diagnosed in multiple members of a German family. Segregation analysis suggested autosomal-dominant inheritance of the combined phenotype. When looking for potentially disease-causing gene variants with cosegregation, a novel hyperpolarization-activated cyclic nucleotide channel 4 (HCN4)-G482R mutation and a common cysteine and glycine-rich protein 3 (CSRP3)-W4R variant were identified. HCN4-G482R is located in the highly conserved channel pore domain. Mutant subunits were nonfunctional and exerted dominant-negative effects on wild-type current. CSRP3-W4R has previously been linked to dilated and hypertrophic cardiomyopathy, but was also found in healthy subjects. Moreover, different truncation (695X) and missense (P883R) HCN4 mutations segregated with a similar combined phenotype in an additional, unrelated family and a single unrelated proband respectively, which both lacked CSRP3-W4R., Conclusions: The symptom complex of SND and NCCM is associated with heritable HCN4 defects. The NCCM phenotype may be aggravated by a common CSRP3 variant in one of the families., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque., Method: In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization., Results: The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5)., Conclusion: This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions., (© 2013. Published by Elsevier B.V. All rights reserved.)

Aims: HCN4 channels are involved in generation, regulation, and stabilization of heart rhythm and channel dysfunction is associated with inherited sinus bradycardia. We asked whether dysfunctional HCN4 channels also contribute to the generation of cardiac tachyarrhythmias., Methods and Results: In a candidate gene approach, we screened 422 patients with atrial and/or ventricular tachyarrhythmias and detected a novel HCN4 gene mutation that replaced the positively charged lysine 530 with an asparagine (HCN4-K530N) in a highly conserved region of the C-linker. The index patient developed tachycardia-bradycardia syndrome and persistent atrial fibrillation (AF) in an age-dependent fashion. Pedigree analysis identified eight affected family members with a similar course of disease. Whole-cell patch clamp electrophysiology of HEK293 cells showed that homomeric mutant channels almost are indistinguishable from wild-type channels. In contrast, heteromeric channels composed of mutant and wild-type subunits displayed a significant hyperpolarizing shift in the half-maximal activation voltage. This may be caused by a shift in the equilibrium between the tonically inhibited nucleotide-free state of the C-terminal domain of HCN4 believed to consist of a 'dimer of dimers' and the activated ligand-bound tetrameric form, leading to an increased inhibition of activity in heteromeric channels., Conclusion: Altered C-linker oligomerization in heteromeric channels is considered to promote familial tachycardia-bradycardia syndrome and persistent AF, indicating that f-channel dysfunction contributes to the development of atrial tachyarrhythmias.

Cardiac atrial natriuretic peptide (ANP) locally counteracts cardiac hypertrophy via the guanylyl cyclase-A (GC-A) receptor and cGMP production, but the downstream signalling pathways are unknown. Here, we examined the influence of ANP on beta-adrenergic versus Angiotensin II (Ang II)-dependent (G(s) vs. G(alphaq) mediated) modulation of Ca(2+) (i)-handling in cardiomyocytes and of hypertrophy in intact hearts. L-type Ca(2+) currents and Ca(2+) (i) transients in adult isolated murine ventricular myocytes were studied by voltage-clamp recordings and fluorescence microscopy. ANP suppressed Ang II-stimulated Ca(2+) currents and transients, but had no effect on isoproterenol stimulation. Ang II suppression by ANP was abolished in cardiomyocytes of mice deficient in GC-A, in cyclic GMP-dependent protein kinase I (PKG I) or in the regulator of G protein signalling (RGS) 2, a target of PKG I. Cardiac hypertrophy in response to exogenous Ang II was significantly exacerbated in mice with conditional, cardiomyocyte-restricted GC-A deletion (CM GC-A KO). This was concomitant to increased activation of the Ca(2+)/calmodulin-dependent prohypertrophic signal transducer CaMKII. In contrast, beta-adrenoreceptor-induced hypertrophy was not enhanced in CM GC-A KO mice. Lastly, while the stimulatory effects of Ang II on Ca(2+)-handling were absent in myocytes of mice deficient in TRPC3/TRPC6, the effects of isoproterenol were unchanged. Our data demonstrate a direct myocardial role for ANP/GC-A/cGMP to antagonize the Ca(2+) (i)-dependent hypertrophic growth response to Ang II, but not to beta-adrenergic stimulation. The selectivity of this interaction is determined by PKG I and RGS2-dependent modulation of Ang II/AT(1) signalling. Furthermore, they strengthen published observations in neonatal cardiomyocytes showing that TRPC3/TRPC6 channels are essential for Ang II, but not for beta-adrenergic Ca(2+) (i)-stimulation in adult myocytes.

Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3',5'-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.

Atrial (ANP) and B-type natriuretic peptides (BNP) modulate blood pressure and volume through the stimulation of cyclic GMP production by their guanylyl cyclase-A (GC-A) receptor. A novel isoform of GC-A has been identified that is the result of differential splicing of exon 4. The deletion of a 51-bp sequence is predicted to delete 17 amino acids (Lys314-Gln330) in the membrane-distal part of the extracellular domain. Reverse transcription-PCR analyses demonstrated low messenger RNA expression levels of spliced GC-A in all tissues. Homology modeling suggested that the alterations in the protein structure could interfere with ANP binding or signaling. Indeed, functional studies in transfected HEK 293 cells demonstrated that binding of ANP and ANP-induced cyclic GMP formation by GC-ADelta(Lys314-Gln330) were totally abolished. Furthermore, cotransfection studies showed that this GC-A variant forms heterodimers with the wild type receptor and inhibits ligand-inducible cGMP generation. Finally, treatment of mice with angiotensin II (300 ng/kg/min during 7 days) resulted in enhanced pulmonary mRNA expression of spliced GC-A, which was concomitant to diminished GC-A/cGMP responses to ANP. We conclude that alternative splicing can regulate endogenous ANP/GC-A signaling. Angiotensin II-induced alternative splicing of GC-A may represent a novel mechanism for reducing the sensitivity to ANP.

Purpose: To assess the new method of 3-dimensional echocardiography in comparison to the "gold standard" MRI as to its ability to calculate left ventricular volumes in patients with congenital heart disease., Materials and Methods: Eighteen patients between the ages of 3.9 to 37.3 years (mean: 12.8 +/- 9.7) with a geometrically abnormal left ventricle were examined using a 1.5T scanner with a fast gradient-echo sequence (TR = 14 ms, TE = 2.6 - 2.9 ms, FOV = 300 - 400 mm, flip angle = 20 degrees, matrix = 128 : 256, slice thickness = 5 mm, retrospective gating) in multislice-multiphase technique. Transthoracic 3D-echocardiography was performed with a 3.5 MHz transducer and a Tomtec (Munich, Germany) system for 3D reconstruction., Results: Volume calculation was possible in all patients with 3D-echocardiography, but the muscle mass calculation only succeeded in 11 of 18 patients (61 %) due to inadequate visualization of the entire myocardium. Comparing MRI and 3D-echocardiography, the correlation was r = 0.97 for the end-systolic volumes, r = 0.98 for the end-diastolic volumes, r = 0.79 for the end-systolic muscle mass and r = 0.77 for the end-diastolic muscle mass. The agreement between both methods was considered good for the calculated end-diastolic volumes and sufficient for the calculated end-systolic volumes. The muscle mass calculations showed larger differences especially for the end-systolic mass. Mean intraobserver variability was 18.6 % for end-systolic and 8.3 % for end-diastolic volumes., Conclusion: In patients with an abnormal left ventricular configuration due to congenital heart disease, the new method of 3D-echocardiography is sufficient for volume calculations in preselected patients. The high intraobserver variability is still a limitation of transthoracic 3D-echocardiography in comparison to MRI.

Ehlers-Danlos syndrome type VI (EDSVI) is an autosomal recessively inherited connective tissue disease, characterized by kyphoscoliosis, muscular hypotonia and ocular manifestations. The cause of the syndrome is a deficiency in the activity of lysyl hydroxylase (LH), one of the enzymes involved in the post-translational modification of collagens. We describe here an unusual compound heterozygote British patient with EDSVI. Our investigations indicate that a maternally inherited nonsense mutation (Y511X) in exon 14 of the LH gene (PLOD1) results in a reduction of the mRNA level as well as a skipping of exon 14 sequences in the mRNA that produces a protein shortened by 38 amino acids. The transcription of the other allele of the LH gene is considerably reduced from the normal for reasons that are not yet known. As a consequence, the LH activity of the skin fibroblasts of the patient is markedly reduced.

Background: Rotationplasty is considered to be a treatment option for patients who have had a primary malignant bone tumor of the distal part of the femur or the proximal part of the tibia. The present study was performed to evaluate the muscle activity, the kinetics (range of motion of the hip and knee joints), and the kinematics (joint moments) after rotationplasty and to determine whether there was an association between these parameters and the functional outcome., Methods: Forty-three patients who had been managed with rotationplasty for the treatment of a femoral or tibial bone tumor were evaluated clinically and functionally. The mean age (and standard deviation) at the time of follow-up was 24.4 +/- 10.7 years (range, eight to sixty-eight years), the mean age at the time of the procedure was 17.8 +/- 10.2 years (range, seven to sixty-three years), and the mean duration of follow-up was 6.7 +/- 4.9 years (range, 0.7 to eighteen years). Instrumented gait and electromyographic analyses were performed. The qualitative data were compared with the functional outcome, which was determined with the functional evaluation score of the Musculoskeletal Tumor Society., Results: Gait analysis revealed a fairly normal walking pattern with a slight limp and a lateral lean of the trunk over the ipsilateral limb that led to a reduced joint moment in the hip (moment on involved side, 68 percent [compared with a control group]; moment on uninvolved side, 81 percent). The ranges of motion of the hips (uninvolved side, 42.0 +/- 8.2 degrees; involved side, 42.4 +/- 8.0 degrees) and the knees (uninvolved side, 59.7 +/- 5.0 degrees; involved side [former ankle joint], 58.1 +/- 11.6 degrees) were symmetrical even though the knee-motion pattern of the involved limb indicated a slightly reduced extensor mechanism in 51 percent (twenty-two) and a markedly reduced extensor mechanism in 35 percent (fifteen) of the forty-three patients. Electromyography revealed function of the muscles of the involved limb, with comparable amplitudes in the involved and uninvolved limbs. The leg muscles of the involved limb were active in the stance phase (the soleus and the lateral and medial heads of the gastrocnemius) and the swing phase (the peroneus longus and the tibialis anterior) according to their function in relation to the new knee joint. The patients had a good functional result, with a mean score of 23.9 +/- 2.7 of 30 points. With the numbers available for study, we could not show the duration of follow-up to be related to the overall outcome, but the age at the time of the operation was related to the total functional score as well as to gait and walking ability (p < 0.05)., Conclusions: The results of the electromyographic and gait analyses demonstrated good functional restoration of gait following rotationplasty.

We report the first deletion mutation and the first splicing defect in the lysyl hydroxylase gene in a compound heterozygote patient with Ehlers-Danlos syndrome type VI with markedly reduced lysyl hydroxylase activity. Northern analysis of the RNA isolated from skin fibroblasts of the patient demonstrated the presence of a truncated lysyl hydroxylase mRNA. PCR and sequence analysis confirmed the truncation and indicated that the cells contain two types of shortened mRNAs, one lacking the sequences corresponding to exon 16 and the other lacking that corresponding to exon 17 of the lysyl hydroxylase gene. Analysis of genomic DNA revealed deletion of the penultimate adenosine from the 3' end of intron 15 from one allele. This defect was probably responsible for the skipping of exon 16 sequences from the transcript. The other allele, inherited from the mother, contains an Alu-Alu recombination with a deletion of about 3,000 nucleotides from the gene; this abnormality explains the lack of exon 17 sequences. The identified mutations in exon 16 and exon 17 do not alter the reading frame of the transcripts.

Purpose: We report the first case of abscess formation after combined spinal-epidural block (CSE). Penetration of the dura in CSE may constitute an additional risk of subarachnoid spread of bacteria when post-puncture epidural infection is present., Clinical Features: The combination of a spinal and a continuous epidural block (CSE) using a needle through needle technique was used in a 72-yr-old man for reconstructive vascular surgery of the lower limb. On the fourth postoperative day the patient demonstrated back pain, fever, and exudation of pus from the CSE-puncture site. An epidural abscess was diagnosed by magnetic resonance imaging and subsequently an emergency hemiaminectomy was performed. Physical examination and surgery did not show evidence of bacterial spread into the subarachnoid space., Conclusion: Epidural abscess formation after CSE may increase the risk of bacterial spread into the subarachnoid space. In this case spontaneous exudation and surgical drainage of abscess material may have prevented intrathecal infection. Rapid diagnosis and treatment of an epidural abscess appears particularly essential after CSE to prevent neurological sequelae.

Hyperprostaglandin E-syndrome (HPS), a recently described variant of Bartter's syndrome (BS), resembles BS in a number of symptoms but is distinct from BS in others. Similar to BS, HPS is characterized by congenital hypokalemic alkalosis, hypertrophy of the juxtaglomerular apparatus, hyperreninemia, secondary hyperaldosteronism, normal blood pressure and renal diabetes insipidus. Other than BS, HPS is constantly associated with chronic hypercalciuria and nephrocalcinosis as well as both renal and systemic PGE2 overproduction. Correction of most of the symptoms in HPS is achieved by permanent inhibition of prostaglandin synthesis with indomethacin. Among the causes leading to HPS, a selective damage of the distal tubule in HPS has been suggested. Therefore, synthesis of Tamm-Horsfall protein (THP), a glycoprotein exclusively produced in the thick ascending limb of the loop of Henle, was measured by ELISA in the urine of seven infant HPS patients (aged 3 to 8 years). Patients were investigated both under constant indomethacin treatment and after a one week period without indomethacin. Nine healthy children (aged 5 months to 10 years) served as controls. In controls mean daily THP excretion was 54.2 +/- 13.9 (median 46.0) mg/24 hr/1.73 m2 whereas in HPS, THP levels were strongly diminished. During withdrawal of indomethacin treatment, mean THP level was 12.7 +/- 10.1 (median 7.2) mg/24 hr/1.73 m2 and 10.3 +/- 10.1 (median 3.5) mg/24 hr/1.73 m2 under indomethacin treatment, respectively. THP excretion values both without indomethacin and under indomethacin treatment were significantly different from controls (P < or = 0.005); however, there was no significant difference between the THP levels during or after cessation of indomethacin treatment. Creatinine clearance in HPS patients was 75.1 +/- 15.9 (median 76.2) ml/min/1.73 m2 without indomethacin and 81.9 +/- 15.1 (median 83.0) ml/min/1.73 m2 under indomethacin treatment. Control values were not obtained. Comparative measurements of THP excretion in six classical BS-patients (aged 3 months to 17 years) revealed normal THP values in two individuals and intermediate levels in the others: the mean level of six BS patients was 30.8 +/- 13.5 (median 25.0) mg/24 hr/1.73 m2 and was thus significantly higher than in HPS both with and without indomethacin treatment (P < or = 0.05). Immunohistochemistry in renal biopsies of three of the HPS patients showed a strong reduction of cortical tubular THP immunoreactivity in two cases and a less pronounced reduction in the third. In situ hybridization using a THP-riboprobe in these three biopsies revealed significantly reduced or absent THP-mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

The following parameters were tested in clinically intact dairy cows and those afflicted with parturitional paresis, over a period from four weeks before to 16 weeks after parturition: calcium, inorganic phosphate (PA), magnesium, alkaline phosphatase (AP), glucose, total protein, mineral mass/fat-free dry matter (A/FFT), mineral mass/volume of fresh skeletal bioptate (A/V). Literature data regarding the behaviour patterns of Ca, PA, Mg, AP, and glucose were confirmed. The dynamics of physiological mineral catabolism and mineral anabolism were expressed by the A/V changes more clearly than they had been reflected in A/FFT. The criterion A/V should be included as a function of age in the spectrum of parameters of metabolic monitoring.

Hypocalcaemia was experimentally induced to cattle of different age groups, and the resulting model showed that intraplasmic parathormone-induced calcium rise due to activation of calcium from the apatite crystals of the skeletal depot was a function of age and a sufficiently plausible explanation for age-dependent growing morbidity rates in the context of hypocalcaemic hysteroparalysis in concomitance with parturition. High vitamine D3 doses are recommended for systematic prophylaxis.

In a retrospective study the sensitivity of SPECT and CT in detecting changes in cerebrovascular disease is compared, based on a group of one hundred patients. The higher sensitivity of SPECT with regard to right-positive results, especially in TIA and PRIND and the markedly lower number of false-negative results are underlined and discussed. The fact that both methods reveal only a slight sensitivity regarding the detection of changes in the basal regions of the brain is also pointed out, with SPECT proving to be the more sensitive of the two methods.

This is a report on a massive thickening of the gallbladder wall accompanying an acute cholecystitis with cholelithiasis. The sonographic criteria for the diagnosis of cholecystitis are discussed. It is pointed out that a thickening of the gallbladder wall is not a specific sign for cholecystitis.

Strong correlations between electric systole (QT time) and calcium-serum concentration can be statistically secured. In this context, all-transistor direct-printing electrocardiographs were used for indirect determination of calcium concentrations with the view to assessing QT time. The approach is described with reference to the need for safe and independent diagnosis of hypocalcaemic hysteroparalysis in concomitance with parturition against the background of industrialised cattle farming.

DNA distribution patterns were flow cytometrically recorded in gastric biopsy specimens from patients with chronic gastritis (CG) and chronic atrophic gastritis (CAG). DNA aneuploidy was found in 3 of 58 patients with CG and in 7 of 82 patients with CAG. Cell cycle analysis disclosed significantly higher percentages of cells in S-phase and G2M-phase, respectively, in CAG than in CG. With regard to the proliferative activity the total CAG group could be partially differentiated by the degree of severity. CAG with total atrophy showed significantly higher percentages of cells in S-phase than CAG with mild and moderate atrophy. CAG without dysplasia showed lower percentages of cells in S-phase and G2M-phase than CAG with severe dysplasia (p greater than 0.05). The occurrence of intestinal metaplasia was correlated to a significantly higher percentage of cells in G2M-phase.