Increased differentiation and production of extracellular matrix components of primary human osteoblasts after cocultivation with endothelial cells: A quantitative proteomics approach.

Coculturing of bone-forming and blood vessel-forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue-engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture, revealing 49 significantly upregulated, and 54 significantly downregulated proteins. Amongst the highest regulated proteins, we found the proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor, desmoplakin, galectin-3, and cyclin-dependent kinase 6. The findings were confirmed by enzyme-linked immunosorbent assays. We also investigated whether the mRNA transcripts correlate with the changes in protein levels by quantitative real-time reverse transcription polymerase chain reaction. In addition, the data was compared to our previous microarray analysis of hOB transcriptome. Taken together, this in-depth analysis delivers reliable data suggesting the importance of coculturing of hOBs and HUVECs in tissue engineering.

Health care utilization in persons with spinal cord injury: part 1-outpatient services.

STUDY DESIGN: This was a cross-sectional questionnaire survey. OBJECTIVES: The objective of this study was to identify the care-seeking behavior of persons with spinal cord injury (SCI) with respect to the various health care providers and ascertain circumstances that lead to situations where required care was not received. SETTING: This study was conducted in the entire country of Switzerland. METHODS: Statistical analysis of frequency of annual visits to health care providers by 17 specialties, and description of situations where health care was required but not received, in persons with chronic SCI living in the community. RESULTS: Main medical contact person was the general practitioner (GP; visited by 88% during last 12 months). The physiotherapist (visited by 72%) was the health care provider with the most visits (average of 30 visits in 12 months). GPs, physiotherapists, urologists and spinal medicine specialists were often contacted in combination, by many participants, often for check-up visits. A situation where care was required but not received was reported by 53 (11%) of participants, with a substantially higher rate in migrants (29%). Main problems why care was not received were bladder and bowel problems and main reasons of care not received were regional or temporal unavailability. CONCLUSIONS: Individuals with SCI are frequent users of medical services. There is no group of medical specialists that covers all needs of persons with SCI, what emphasizes health care provision from a comprehensive perspective including a wide array of services. Instances with care required but not received appeared to be rare and more likely in participants with migration background.

Health care utilization in persons with spinal cord injury: part 2-determinants, geographic variation and comparison with the general population.

STUDY DESIGN: Cross-sectional survey. OBJECTIVES: To investigate annual rates and geographic variation of health care utilization in persons with spinal cord injury (SCI), and to identify factors associated with health care utilization. SETTING: Community setting, entire country of Switzerland. METHODS: Annual rates of planned and emergency visits to the general practitioner (GP), planned and emergency outpatient clinic visits and in-patient hospitalizations were compared between individuals with chronic SCI, over 16 years of age residing in Switzerland between late 2011 and early 2013 and a population sample (2012) of the Swiss general population. Risk factors for increased health service utilization were identified by means of regression models adjusted for spatial variation. RESULTS: Of 492 participants (86.2% response rate), 94.1% visited a health care provider in the preceding year, with most persons visiting GPs (88.4%) followed by outpatient clinics (53.1%) and in-patient hospitals (35.9%). The increase in utilization as compared with the general population was 1.3-, 4.0- and 2.9-fold for GP, outpatient clinic and in-patient hospital visit, respectively. GP utilization was highest in persons with low income (incidence rate ratio (IRR) 1.85) and old age (IRR 2.62). In the first 2 years post injury, health service visits were 1.7 (GP visits) to 5.8 times (emergency outpatient clinic visits) more likely compared with those later post injury. CONCLUSIONS: People with SCI more frequently use health services as compared with the general population, across all types of medical service institutions. GP services were used most often in areas where availability of specialized outpatient clinic services was low.

The characteristics of posttraumatic syringomyelia.

STUDY DESIGN: Retrospective cross-sectional study. OBJECTIVES: To investigate the characteristics of posttraumatic symptomatic syringomyelia after spinal cord injury (SCI). SETTING: Swiss Paraplegic Centre, Nottwil, Switzerland. METHODS: The patient database was screened for patients diagnosed with posttraumatic syringomyelia. Syrinx characteristics were determined on T2-weighted magnetic resonance images. Binary logistic regression analysis was used to investigate the effects of age, injury level, injury severity and syrinx location on early syrinx formation, syrinx length and syrinx extending cranial to the lesion. RESULTS: The data of 138 patients were analyzed. The majority of the patients (78.3%) suffered from motor and sensory complete SCI (American Spinal Injury Association Impairment Scale (AIS) A). Syringomyelia was diagnosed a median 15.0 years after SCI at a median age of 42 years. The cervical spine was involved in >57% of the patients, and syringomyelia extended over a median seven vertebral levels. Complete SCI (P=0.035) and age (P=0.001) were significant predictors of early syrinx formation. Syringomyelia occurred significantly earlier in older (>30 years) patients (P⩽0.002) and those with complete SCI (P=0.027) compared with younger patients (⩽30 years) and those with incomplete SCI (AIS B-D), respectively. Age, injury level, injury severity (AIS A) and syrinx location did not have any significant (P>0.9) effect on syrinx extending cranially or syrinx length. CONCLUSIONS: Posttraumatic syringomyelia mainly occurs in patients with complete SCI (AIS A) and involves the cervical spine in 6 of the 10 patients. Patients with complete SCI and those age >30 years have an increased risk of syrinx formation within 5 years after injury.

Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

Propionic acidemia: neonatal versus selective metabolic screening.

BACKGROUND: Whereas propionic acidemia (PA) is a target disease of newborn screening (NBS) in many countries, it is not in others. Data on the benefit of NBS for PA are sparse. STUDY DESIGN: Twenty PA patients diagnosed through NBS were compared to 35 patients diagnosed by selective metabolic screening (SMS) prompted by clinical findings, family history, or routine laboratory test results. Clinical and biochemical data of patients from 16 metabolic centers in Germany, Austria, and Switzerland were evaluated retrospectively. Additionally, assessment of the intelligent quotient (IQ) was performed. In a second step, the number of PA patients who have died within the past 20 years was estimated based on information provided by the participating metabolic centers. RESULTS: Patients diagnosed through NBS had neither a milder clinical course regarding the number of metabolic crises nor a better neurological outcome. Among NBS patients, 63% were already symptomatic at the time of diagnosis, and <10% of all patients remained asymptomatic. Among all PA patients, 76% were found to be at least mildly mentally retarded, with an IQ <69. IQ was negatively correlated with the number of metabolic decompensations, but not simply with the patients' age. Physical development was also impaired in the majority of patients. Mortality rates tended to be lower in NBS patients compared with patients diagnosed by SMS. CONCLUSION: Early diagnosis of PA through NBS seems to be associated with a lower mortality rate. However, no significant benefit could be shown for surviving patients with regard to their clinical course, including the number of metabolic crises, physical and neurocognitive development, and long-term complications.

Treatment recommendations in long-chain fatty acid oxidation defects: consensus from a workshop.

Published data on treatment of fatty acid oxidation defects are scarce. Treatment recommendations have been developed on the basis of observations in 75 patients with long-chain fatty acid oxidation defects from 18 metabolic centres in Central Europe. Recommendations are based on expert practice and are suggested to be the basis for further multicentre prospective studies and the development of approved treatment guidelines. Considering that disease complications and prognosis differ between different disorders of long-chain fatty acid oxidation and also depend on the severity of the underlying enzyme deficiency, treatment recommendations have to be disease-specific and depend on individual disease severity. Disorders of the mitochondrial trifunctional protein are associated with the most severe clinical picture and require a strict fat-reduced and fat-modified (medium-chain triglyceride-supplemented) diet. Many patients still suffer acute life-threatening events or long-term neuropathic symptoms despite adequate treatment, and newborn screening has not significantly changed the prognosis for these severe phenotypes. Very long-chain acyl-CoA dehydrogenase deficiency recognized in neonatal screening, in contrast, frequently has a less severe disease course and dietary restrictions in many patients may be loosened. On the basis of the collected data, recommendations are given with regard to the fat and carbohydrate content of the diet, the maximal length of fasting periods and the use of l-carnitine in long-chain fatty acid oxidation defects.

Management and outcome in 75 individuals with long-chain fatty acid oxidation defects: results from a workshop.

At present, long-chain fatty acid oxidation (FAO) defects are diagnosed in a number of countries by newborn screening using tandem mass spectrometry. In the majority of cases, affected newborns are asymptomatic at time of diagnosis and acute clinical presentations can be avoided by early preventive measures. Because evidence-based studies on management of long-chain FAO defects are lacking, we carried out a retrospective analysis of 75 patients from 18 metabolic centres in Germany, Switzerland, Austria and the Netherlands with special regard to treatment and disease outcome. Dietary treatment is effective in many patients and can prevent acute metabolic derangements and prevent or reverse severe long-term complications such as cardiomyopathy. However, 38% of patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency had intermittent muscle weakness and pain despite adhering to therapy. Seventy-six per cent of patients with disorders of the mitochondrial trifunctional protein (TFP)-complex including long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, had long-term myopathic symptoms. Of these, 21% had irreversible peripheral neuropathy and 43% had retinopathy. The main principle of treatment was a fat-reduced and fat-modified diet. Fat restriction differed among patients with different enzyme defects and was strictest in disorders of the TFP-complex. Patients with a medium-chain fat-based diet received supplementation of essential long-chain fatty acids. l-Carnitine was supplemented in about half of the patients, but in none of the patients with VLCAD deficiency identified by newborn screening. In summary, in this cohort the treatment regimen was adapted to the severity of the underlying enzyme defect and thus differed among the group of long-chain FAO defects.

[An international comparative legal analysis of the regulation of research with human embryonic stem cells]. / Stammzellforschung aus rechtsvergleichender Sicht.

The article is based on the results of a comparative legal study of the status and protection of extracorporeal embryos in a number of European and non-European countries. In this context, the study also deals with the extent to which in vitro embryos can be created and/or used for research purposes (especially for stem cell research). The results show a considerable divergence with regard to existing solutions. This divergence is not due solely to different concepts of protection but is also an indication of the controversial debate on whether such entities are worthy of protection and, if so, to what degree it should be granted. The discussion indeed begins at the level of terminology where the attribute(s) characterizing an "embryo" in the legal sense are - already and seemingly without deeper meaning - anything but uniform. The reasons for this disparity, such as the time factor (which stages of development must have taken place after fertilization of an egg cell by a sperm cell?) or the method of genesis itself (aside from conception, which other methods are used to generate embryos?), could be particularly relevant. The differences mentioned lead, in turn, to the question of which legal consequences researchers must keep in mind - especially regarding the risk of criminal liability - when engaging in international cooperation efforts with peers from more permissive countries.

Successful control of methicillin-resistant Staphylococcus aureus in a spinal cord injury center: a 10-year prospective study including molecular typing.

STUDY DESIGN: Prospective cohort study with medical record review. OBJECTIVE: To evaluate the clinical utility of an infection control program in a patient cohort at high risk for methicillin-resistant Staphylococcus aureus (MRSA) infection and to identify risk factors interfering with successful decolonization of MRSA. SETTING: All spinal cord injured (SCI) patients hospitalized at the Swiss Paraplegic Center (SPC) Nottwil from April 1991 to April 2001. METHODS: Patients whose medical records indicated laboratory-confirmed MRSA colonization or infection were included. Incidence of MRSA colonization or infection was classified as community acquired, nosocomial or transferred based on standardized criteria. Risk factors for community-acquired MRSA colonization in SCI patients were determined. MRSA subtyping and identification of nosocomial spread was performed through pulse-field gel electrophoresis (PFGE). RESULTS: Of 5992 admissions, 100 episodes of MRSA (colonization 22 cases, infection 78 cases) were identified among 76 patients. Overall incidence (1991-2001) per 1000 patient days was 0.26 cases on admission compared to 0.08 at discharge (P<0.001). Community-acquired MRSA was most frequent (56%) followed by nosocomial acquisition (34%). PFGE subtyping identified two nosocomial clusters with six and three cases, respectively. Most of community-acquired MRSA isolates were genetically unrelated and also distinct from epidemic strains identified in Switzerland during the study period. Decolonization was successful in 60 of 76 (78.9%) MRSA-positive patients. CONCLUSION: In the largest European SCI center, MRSA controlling is feasible if infection control policies are vigorously applied.

Intermediate hyperhomocysteinaemia and compound heterozygosity for the common variant c.677C>T and a MTHFR gene mutation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the regulation of plasma homocysteine levels. MTHFR deficiency, an autosomal recessive disorder, results in homocystinuria and hypomethioninaemia and presents with highly variable symptoms affecting many organs but predominantly the central nervous system. The common polymorphism of the MTHFR gene, c.677C>T, a known risk factor for elevated plasma homocysteine levels, occurs frequently in the caucasian population. In this study we investigated three subjects with moderate hyperhomocysteinaemia (total plasma homocysteine 72 micromol/L in case 1 and 90 micromol/L in case 3, total non-protein-bound homocysteine 144-186 micromol/L in case 2) but different clinical presentation with no symptoms in case 1, muscle weakness at 17 years of age in case 2, and syncopes and cerebral convulsions at 18 years of age in case 3. Each subject was compound heterozygous for the c.677C>T polymorphism and a novel mutation of the MTHFR gene (case 1: c.883G>A [p.D291N]; case 2: c.1552_c.1553delGA [p.E514fsX536]; case 3: c.616C>T [p.P202S]). Moderately decreased fibroblast MTHFR activity was associated with severely reduced affinity for NADPH and increased sensitivity to inhibition by S-adenosylmethionine (AdoMet) in case 2, and with mild FAD responsiveness in case 3. In case 1, fibroblast MTHFR activity was normal but the sensitivity to inhibition by AdoMet was slightly reduced. This study indicates that the sequence alteration c.677C>T combined with severe MTHFR mutations in compound heterozygous state may lead to moderate biochemical and clinical abnormalities exceeding those attributed to the c.677TT genotype and might require in addition to folate substitution further therapy to normalize homocysteine levels.

Misleading diagnosis of partial N-acetylglutamate synthase deficiency based on enzyme measurement corrected by mutation analysis.

UNLABELLED: N-acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder. Most of the patients present in the early neonatal period with severe hyperammonaemia and marked neurological impairment. We report on a Turkish family with an index patient, who died due to hyperammonemia, and another three siblings, who received a prophylactic treatment consisting of arginine hydrochloride, sodium benzoate and phenylbutyrate directly after birth. Enzyme measurement in a liver biopsy suggested a diagnosis of partial NAGS deficiency in all three siblings. Thereafter, N-carbamylglutamate was added to the treatment. None of the patients developed hyperammonaemia. After the human NAGS gene was identified, mutation analysis revealed that the consanguineous parents and two siblings were heterozygous for a private mutation (W484R), whereas the wild-type gene was found in the eldest sibling. Therapy was stopped without any deterioration of urea cycle function. CONCLUSION: Diagnosis of partial NAGS deficiency based on enzyme measurement may be misleading and should be completed by mutation analysis.

Methionine adenosyltransferase (MAT) I/III deficiency with concurrent hyperhomocysteinaemia: two novel cases.

This study reports three novel mutations of the methionine adenosyltransferase (MAT) lA gene and confirms that hyperhomocysteinaemia may be a characteristic finding in MAT I/III deficiency. Thus, MAT I/III deficiency is important in the differential diagnoses of hyperhomocysteinaemia, which may lead to clinical complications of MAT I/III deficiency.

Diagnosis of N-acetylglutamate synthase deficiency by use of cultured fibroblasts and avoidance of nonsense-mediated mRNA decay.

Molecular diagnosis of N-acetylglutamate synthase deficiency (NAGSD) has become possible now that the corresponding gene has been identified. We describe the genetic analysis of a patient with NAGSD using low-level transcripts derived from cultured fibroblasts. One defective allele (c.1306-1307insT) was detected by PCR amplification. However, the transcript from a second mutation (IVS3 - 2A>T), causing aberrant splicing with the generation of a premature termination codon, was not detected until interference of nonsense-mediated mRNA decay was abrogated by the translation inhibitor cycloheximide. We demonstrate that low-level transcripts in cells that do not express significant enzyme activity are a valuable tool for molecular studies of genetic alterations, and suggest routine abrogation of nonsense-mediated mRNA decay using cycloheximide when transcript analysis is performed.

Isolated thrombosis due to the cystathionine beta-synthase mutation c.833T>C (1278T).

Hereditary homocystinuria due to cystathionine beta-synthase (CBS) deficiency is a rare disease (about 1:20000 in Germany) often complicated by thromboembolism. Single mutations, which affect the C-terminal region of the CBS enzyme, lead to isolated thrombosis without further symptoms typical for homocystinuria such as atherosclerosis, psychomotor retardation, and dislocation of the ocular lenses. In this study, DNA samples of patients with stroke (n = 225) and sinus thrombosis (n = 46) were screened for the most common homocystinuria mutation, CBS 1278T. In each group one homozygous patient was identified. Thus, not only C-terminal mutations but also the most common mutation in classical homocystinuria, CBS 1278T, can lead to isolated thrombophilic events. These data support the hypothesis that homocystinuria is an underdiagnosed disease.

Expression of S100A12 (EN-RAGE) in cystic fibrosis.

BACKGROUND: Chronic airway inflammation and recurrent infections are a core phenomenon in cystic fibrosis (CF). Diagnosing acute infectious exacerbations is difficult in the presence of chronic inflammatory processes. S100A12 exhibits proinflammatory functions via interaction with the multiligand receptor for advanced glycation end products. Blocking this interaction inhibits inflammatory processes in mice. METHODS: The expression of S100A12 in lung specimens of patients with end stage lung disease of CF was investigated, and S100A12 levels in the serum of patients with acute infectious exacerbations of CF were measured. RESULTS: Immunohistochemical studies of CF lung biopsy specimens revealed a significant expression of S100A12 by infiltrating neutrophils. High S100A12 levels were found in the sputum of patients with CF, and serum levels of S100A12 during acute infectious exacerbations were significantly increased compared with healthy controls (median 225 ng/ml v 46 ng/ml). After treatment with intravenous antibiotics the mean S100A12 level decreased significantly. There was also a significant difference between S100A12 levels in patients with acute infectious exacerbations and 18 outpatients without exacerbations (median 225 ng/ml v 105 ng/ml). CONCLUSIONS: S100A12 is extensively expressed at local sites of inflammation in CF. It is a serum marker for acute infectious exacerbations. High local expression of S100A12 suggests that this protein has a proinflammatory role during airway inflammation and may serve as a novel target for anti-inflammatory treatments.

Investigation of the cerebral energy status in patients with glutaric aciduria type I by 31P magnetic resonance spectroscopy.

In vivo phosphorus magnetic resonance spectroscopy (MRS) was used to investigate markers of the cerebral energy status in two patients with glutaric aciduria type I (GA-I). Besides an increased concentration of phosphomonoesters in one patient, no other significant alterations from controls were found. This might indicate increased resynthesis of dendritic processes secondary to preceding metabolic crises. In contrast to previous cell-culture studies, no cerebral depletion of phosphocreatine (PCr) was observed. In conclusion, a severe global and permanent depletion of cerebral energy supplies must be ruled out. The benefit of a permanent creatine substitution to stabilize mitochondrial energy metabolism seems thus questionable. However, as MRS was performed during stable clinical conditions, the possibility of a PCr decrease during acute metabolic crises cannot be assessed.

Gene structure of human carbamylphosphate synthetase 1 and novel mutations in patients with neonatal onset.

Carbamylphosphate synthetase 1 (E.C. 6.3.4.16) deficiency is a rare autosomal recessive disorder of the urea cycle that can result in severe neonatal hyperammonemia. Since the genomic structure of the CPS1 gene was not yet elucidated, mutation detection was performed by analysis of transcripts in the past. Here, we present the entire DNA sequence of the human CPS1 gene including all exon-intron boundaries. Moreover, mutation analysis was performed in six patients leading to the detection of 9 novel mutations including the missense mutations c.2528T>C and c.2623A>G, the nonsense mutations c.712C>T and c.2115ins35bp, the splice site mutations c.1263+5G>C, c.3558+1G>C and c.4101+2T>C, and a small deletion c.3036_3038delGGT. The mutations c.2528T>C and c.2623A>G were identified on a double mutated allele. New data on the genomic structure of the CPS1 gene provided in this study are useful to characterize the heterogenous molecular basis of the disease in patients deficient for carbamylphosphate 1 deficiency.