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Mucopolysaccharidosis type VII (MPS VII) is a rare autosomal recessive lysosomal storage disorder. MPS VII is caused by mutations in the GUSB gene that encodes β-glucuronidase. Adult MPS VII patients present with musculoskeletal abnormalities, coarse features, and corneal clouding. Cardiac and valvular impairment are common; however, severe valvular disease necessitating surgery has not yet been reported. We present a 32-year-old male MPS VII patient admitted to our hospital with decompensated heart failure. We identified aortic valve disease with severe stenosis (valve area 0.69 cm 2 ) and moderate regurgitation. Severe mitral valve stenosis (valve area 1 cm 2 ) with moderate to severe regurgitation was also found in the patient. In addition, an occlusion of the right coronary artery (RCA) was documented. The patient underwent surgical replacement of the mitral and aortic valves with mechanical prostheses and implantation of a venous bypass graft to his RCA. The surgery led to a significant improvement of his clinical symptoms. Six months after the procedure, both mechanical valves function normally. Histopathological assessment identified chronic inflammatory infiltrates, fibrosis and calcifications in both resected valves. Foamy cytoplasmic transformation was most evident in the valvular interstitial cells. The ultrastructural vacuolar abnormality seen in these cells corresponded to storage changes observed in other MPSs. In conclusion, we describe clinical findings and valvular pathology in an MPS VII patient with the first-reported successful combined surgical valve replacement and myocardial revascularization. The histological and ultrastructural analyses revealed that the lysosomal storage predominantly affected the valvular interstitial cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During the COVID-19 pandemics of 2020, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), both adults and children were shown to mount a specific antibody response to the virus. As infected children often exhibit mild symptoms or even remain asymptomatic, they are likely to be under tested for the direct presence of the virus. Mapping the SARS-CoV-2 antibodies frequency informs more accurately on the disease prevalence and helps guide the protective and therapeutic strategies. To date, only few seroprevalence studies included children. In the Czech Republic, in April 2020, the overall SARS-CoV-2 seroprevalence was estimated not to exceed 1.3%. In July and August, 2020, we screened 200 children (0-18 years of age), who attended the pediatric department of a large hospital in Prague for various COVID-19-unrelated reasons, for the presence of SARS-CoV-2 antibodies. Zero seropositive subjects were found. Therefore, we hereby report a low (<0.5%) seroprevalence amongst children in Prague, as of August, 2020., (Copyright © 2020 Bloomfield, Pospisilova, Cabelova, Sediva, Ibrahimova, Borecka and Magner.)

Background: In this retrospective study, we analysed clinical, biochemical and molecular genetic data of 47 Czech patients with Single, Large-Scale Mitochondrial DNA Deletions (SLSMD)., Methods: The diagnosis was based on the long-range PCR (LX-PCR) screening of mtDNA isolated from muscle biopsy in 15 patients, and from the buccal swab, urinary epithelial cells and blood in 32 patients., Results: A total of 57% patients manifested before the age of 16. We did not find any significant difference between paediatric and adult manifestation in either the proportion of patients that would develop extraocular symptoms, or the timespan of its progression. The survival rate in patients with Pearson Syndrome reached 60%. Altogether, five patients manifested with atypical phenotype not fulfilling the latest criteria for SLSMD. No correlation was found between the disease severity and all heteroplasmy levels, lengths of the deletion and respiratory chain activities in muscle., Conclusions: Paediatric manifestation of Progressive External Ophthalmoplegia (PEO) is not associated with a higher risk of multisystemic involvement. Contrary to PEO and Kearns-Sayre Syndrome Spectrum, Pearson Syndrome still contributes to a significant childhood mortality. SLSMD should be considered even in cases with atypical presentation. To successfully identify carriers of SLSMD, a repeated combined analysis of buccal swab and urinary epithelial cells is needed.

Objectives: The mucopolysaccharidoses (MPS) are inherited lysosomal storage disorders with multisystemic and highly variable clinical manifestation. ENT symptoms are common and early signs of MPS. The most common ENT diagnoses are chronic/recurrent rhinosinusitis, acute otitis media, otitis media with effusion, hearing loss and airway obstruction., Methods: A single-centre retrospective chart review of 61 patients (36 M/25F) with different MPS subtypes (MPS I (n = 15), MPS II (n = 10), MPS III (n = 17), MPS IV (n = 15) and MPS VI (n = 4)) was conducted. The age of ENT presentation and frequency of ENT symptoms, surgeries and their distribution among MPS subtypes was studied. The relationship between ENT presentation, first ENT surgery and the age of diagnosis was also evaluated., Results: Median age at the first ENT manifestation was 2.8 years, median age at MPS diagnosis 4.1 years. The great majority of patients (90%) manifested at least one ENT diagnosis; often before the diagnosis of MPS (75%). Chronic/recurrent rhinosinusitis was the most prevalent ENT diagnosis (77%), followed by upper airway obstruction (65%) and hearing loss (53%). Chronic/recurrent rhinosinusitis was the first ENT symptom to appear (median age 2.2 years), followed by otitis media with effusion (3.7 years) and hearing loss (4.5 years). At least one ENT surgery was performed in 57% of patients; in 69% before MPS diagnosis was established. Median age of the first ENT surgery was 4.1 years. ENT symptoms and surgical procedures were earliest present in MPS II., Conclusions: Our study documents high and early occurrence of various otolaryngologic symptoms in MPS and thus highlights the role of ENT specialist in prompt diagnosis of these rare diseases and their long-term management., Competing Interests: Declaration of competing interest MM has received the lecture honoraria from Takeda, Genzyme and Biomarin. LM has received the lecture honoraria from Takeda. PJ has received the lecture honoraria from Genzyme and Takeda. The declared collaboration had no impact on the intention to prepare the study or on its content., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: Evidence has demonstrated that breastfeeding is the optimal nutrition for infants. The present study aims to report possible associations of the duration of full or partial breastfeeding with selected health outcomes during infancy., Methods: Data from 2304 mothers were obtained by online mother-reported questionnaires at the age of 1 year of the child, providing information on full and partial breastfeeding durations, the frequency of infant upper respiratory tract infections and possible antibiotics use, and the occurrence of allergic diseases., Results: Overall breastfeeding initiation rates (i.e. including both partial and full breastfeeding rates counted together) were 97.8%, declined to 95.1% at the age of 3 months, and remained as high as 90.0% at 6 months. At 1 year, 74.7% of children were still partially breastfed. There was no significant benefit of either full or partial breastfeeding over formula feeding for upper respiratory tract infection rates. Fully breastfed children had a significantly lower risk of early exposure to antibiotics when compared with either partially breastfed (odds ratio, OR: 0.74; 95% CI: 0.56, 1.00, P = 0.048) or formula-fed (OR: 0.67; 95% CI: 0.46, 1.0, P = 0.047) children. We found a neutral effect of breastfeeding on the development of allergies., Conclusions: Although no significant association between either full or partial breastfeeding versus formula feeding and the occurrence of respiratory infections during infancy was found, we demonstrated a significantly lower risk of early exposure to antibiotics in fully breastfed children when compared with those either partially breastfed or formula-fed., (© 2019 Dietitians Association of Australia.)

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with tyrosine-kinase signaling activity, involved in many cellular functions including cell growth and differentiation. Germ line loss-of-function mutations in EGFR lead to a severe neonatal skin disorder (Online Mendelian Inheritance in Man #131550). We report 18 premature Roma children from 16 families with birthweights ranging 440-1470 g and multisystem diseases due to the homozygous mutation c.1283G˃A (p.Gly428Asp) in EGFR. They presented with thin, translucent, fragile skin (14/15), skin desquamation (10/17), ichthyosis (9/17), recurrent skin infections and sepsis (9/12), nephromegaly (10/16) and congenital heart defects (7/17). Their prognosis was poor, and all died before the age of 6 months except one 13-year-old boy with a severe skin disorder, dentinogenesis imperfecta, Fanconi-like syndrome and secondary hyperaldosteronism. Management of ion and water imbalances and extremely demanding skin care may improve the unfavorable outcome of such patients., (© 2020 Japanese Dermatological Association.)

Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht.

Background: Tay-Sachs disease (TSD) is an inherited neurodegenerative disorder caused by a lysosomal β-hexosaminidase A deficiency due to mutations in the HEXA gene. The late-onset form of disease (LOTS) is considered rare, and only a limited number of cases have been reported. The clinical course of LOTS differs substantially from classic infantile TSD., Methods: Comprehensive data from 14 Czech patients with LOTS were collated, including results of enzyme assays and genetic analyses., Results: 14 patients (9 females, 5 males) with LOTS were diagnosed between 2002 and 2018 in the Czech Republic (a calculated birth prevalence of 1 per 325,175 live births). The median age of first symptoms was 21 years (range 10-33 years), and the median diagnostic delay was 10.5 years (range 0-29 years). The main clinical symptoms at the time of manifestation were stammering or slurred speech, proximal weakness of the lower extremities due to anterior horn cell neuronopathy, signs of neo- and paleocerebellar dysfunction and/or psychiatric disorders. Cerebellar atrophy detected through brain MRI was a common finding. Residual enzyme activity was 1.8-4.1% of controls. All patients carried the typical LOTS-associated c.805G>A (p.Gly269Ser) mutation on at least one allele, while a novel point mutation, c.754C>T (p.Arg252Cys) was found in two siblings., Conclusion: LOTS seems to be an underdiagnosed cause of progressive distal motor neuron disease, with variably expressed cerebellar impairment and psychiatric symptomatology in our group of adolescent and adult patients. The enzyme assay of β-hexosaminidase A in serum/plasma is a rapid and reliable tool to verify clinical suspicions.

Background: Sideroblastic anemia represents a heterogeneous group of inherited or acquired diseases with disrupted erythroblast iron utilization, ineffective erythropoiesis, and variable systemic iron overload. In a cohort of 421 patients with multisystem mitochondrial diseases, refractory anemia was found in 8 children., Results: Five children had sideroblastic anemia with increased numbers of ring sideroblasts >15%. Two of the children had a fatal course of MLASA1 syndrome (mitochondrial myopathy, lactic acidosis, and sideroblastic anemia [SA]) due to a homozygous, 6-kb deletion in the PUS1 gene, part of the six-member family of pseudouridine synthases (pseudouridylases). Large homozygous deletions represent a novel cause of presumed PUS1-loss-of-function phenotype. The other three children with SA had Pearson syndrome (PS) due to mtDNA deletions of 4 to 8 kb; two of these children showed early onset of PS and died due to repeated sepsis; the other child had later onset of PS and survived as the hematological parameters normalized and the disease transitioned to Kearns-Sayre syndrome. In addition, anemia without ring sideroblasts was found in three other patients with mitochondrial disorders, including two children with later onset of PS and one child with failure to thrive, microcephaly, developmental delay, hypertrophic cardiomyopathy, and renal tubular acidosis due to the heterozygous mutations c.610A>G (p.Asn204Asp) and c.674C>T (p.Pro225Leu) in the COX10 gene encoding the cytochrome c oxidase assembly factor., Conclusions: Sideroblastic anemia was found in fewer than 1.2% of patients with multisystem mitochondrial disease, and it was usually associated with an unfavorable prognosis., (© 2018 Wiley Periodicals, Inc.)

Mucopolysaccharidosis type IVB (MPS IVB) is a very rare lysosomal storage disorder characterized by skeletal dysplasia, hearing disorder, and cardiac valvular disease. Herein, we report an extremely rare manifestation of MPS IVB in a 60-year-old female patient who underwent a successful aortic valve replacement. The patient presented with mild coarse facial features, short stature, mild dyspnea, sternal protrusion, mild lumbar hyperlordosis, and waddling gait owing to bilateral femoral head necroses and bilateral arthrosis of the knees. The patient also suffered from dyspnea, NYHA II-III. Echocardiography revealed severe stenosis of a calcified aortic valve (AVA 0.67 cm 2 , AVAi 0.45 cm 2 /m 2 , PG max/mean 130/80 mmHg), left ventricular hypertrophy with predominant septal thickening (18 mm) and mild left ventricle outflow tract obstruction at rest, mild mitral valve regurgitation, and dilated ascending aorta (36 mm, 26.5 mm/m 2 ). Dyspnea resolved after septal myectomy and replacement of the aortic valve with bioprosthesis. Excretion levels and spectrum of glycosaminoglycans (GAGs) in urine were normal in the patient. We confirmed the diagnosis of MPS IVB by identifying decreased beta-galactosidase activity in isolated leukocytes (6 nmol/h/mg; controls 95-272) and by molecular genetic analyses (c.438&#95;440delTCT and c.817&#95;818TG>CT mutations in the GLB1 gene). Primary lysosomal storage of glycosaminoglycans was detected in fibroblasts of the aortic valve. Additional pathologies included valvular fibrosis, calcification, neovascularization, and mild chronic inflammation. In conclusion, the diagnosis of MPS IVB should be considered in older patients with cardiac valvular disease and progressive skeletal abnormality even if urinary excretion levels of GAGs are normal., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hajdu-Cheney syndrome (HCS) is a rare multi-system disease with autosomal dominant inheritance and skeletal involvement, resulting mostly in craniofacial dysmorphy with mid-face hypoplasia, dental anomalies, short stature, scoliosis, shortening of the digits and nail beds, acro-osteolysis and osteoporosis. We report the progression of clinical and radiographic findings in five patients with Hajdu-Cheney syndrome from two families. A custom capture array designed to capture exons and adjacent intron sequences of 230 selected genes were used for molecular analyses, and the pathogenic variants identified were confirmed by PCR and Sanger sequencing. In both families we observed age-dependent changes in the disease, with a progression of pain in older patients, a shortening of digits and nail beds on both the hands and feet, kyphoscoliosis and the persistence of Wormian bones in lambdoid sutures. Molecular analyses performed in two patients revealed that they are heterozygotes for a c.6255T&gt;A (p.Cys2085*) variant in the NOTCH2 gene, resulting in a premature stop-codon. Bone mineral density (Z-score &lt; -2) did not improved in a girl treated with calcium and vitamin D supplementation during childhood and bisphosphonate during adolescence. Hajdu-Cheney syndrome is a slowly progressive disease with a frequently unfavourable prognosis in elderly patients, especially for the development of dental anomalies, osteoporosis and the progression of skeletal complications requiring orthopedic surgeries.

Cardiomyopathy is a common manifestation in neonates and infants with mitochondrial disorders. In this study, we report two cases manifesting with fatal mitochondrial hypertrophic cardiomyopathy, which include the third known patient with thymidine kinase 2 deficiency and the ninth patient with alanyl-tRNA synthetase 2 deficiency. The girl with thymidine kinase 2 deficiency had hypertrophic cardiomyopathy together with regression of gross motor development at the age of 13 months. Neurological symptoms and cardiac involvement progressed into severe myopathy, psychomotor arrest, and cardiorespiratory failure at the age of 22 months. The imaging methods and autoptic studies proved that she suffered from unique findings of leucoencephalopathy, severe, mainly cerebellar neuronal degeneration, and hepatic steatosis. The girl with alanyl-tRNA synthetase 2 deficiency presented with cardiac failure and underlying hypertrophic cardiomyopathy within 12 hours of life and subsequently died at 9 weeks of age. Muscle biopsy analyses demonstrated respiratory chain complex I and IV deficiencies, and histological evaluation revealed massive mitochondrial accumulation and cytochrome c oxidase-negative fibres in both cases. Exome sequencing in the first case revealed compound heterozygozity for one novel c.209T>C and one previously published c.416C>T mutation in the TK2 gene, whereas in the second case homozygozity for the previously described mutation c.1774C>T in the AARS2 gene was determined. The thymidine kinase 2 mutations resulted in severe mitochondrial DNA depletion (to 12% of controls) in the muscle. We present, for the first time, severe leucoencephalopathy and hepatic steatosis in a patient with thymidine kinase 2 deficiency and the finding of a ragged red fibre-like image in the muscle biopsy in a patient with alanyl-tRNA synthetase 2 deficiency.

Proper cell size is essential for cellular function. Nonetheless, despite more than 100 years of work on the subject, the mechanisms that maintain cell-size homeostasis are largely mysterious [1]. Cells in growing populations maintain cell size within a narrow range by coordinating growth and division. Bacterial and eukaryotic cells both demonstrate homeostatic size control, which maintains population-level variation in cell size within a certain range and returns the population average to that range if it is perturbed [1, 2]. Recent work has proposed two different strategies for size control: budding yeast has been proposed to use an inhibitor-dilution strategy to regulate size at the G1/S transition [3], whereas bacteria appear to use an adder strategy, in which a fixed amount of growth each generation causes cell size to converge on a stable average [4-6]. Here we present evidence that cell size in the fission yeast Schizosaccharomyces pombe is regulated by a third strategy: the size-dependent expression of the mitotic activator Cdc25. cdc25 transcript levels are regulated such that smaller cells express less Cdc25 and larger cells express more Cdc25, creating an increasing concentration of Cdc25 as cells grow and providing a mechanism for cells to trigger cell division when they reach a threshold concentration of Cdc25. Because regulation of mitotic entry by Cdc25 is well conserved, this mechanism may provide a widespread solution to the problem of size control in eukaryotes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Patients who undergo clipping of cerebral aneurysms face an inherent risk for new postoperative neurologic deficits. Intraoperative neuromonitoring (IONM) is used often for early detection of ischemic changes, while it is still potentially reversible. However, the value, safety, and efficacy of temporary clipping and multimodal IONM to minimize risks are debated. Our retrospective series examined the sensitivity and specificity of IONM using transcranial motor evoked potentials and somatosensory evoked potentials and quantified the safety of temporary clipping by duration and vascular territory., Methods: Our prospectively collected database (2010-2013) included 123 consecutive patients who underwent clipping of 133 cerebral aneurysms with use of IONM. We determined postoperative deficit rate and sensitivity and specificity of monitoring to predict these changes intraoperatively. The rate of permanent deficit after temporary clipping was correlated with duration, vascular territory, and IONM findings., Results: Of 133 clipped aneurysms, 15 instances of IONM changes occurred, including 12 temporary without new postoperative deficit and 3 permanent with new postoperative deficit. Somatosensory evoked potential monitoring predicted one of the permanent deficits and transcranial motor evoked potentials predicted the other 2 deficits., Conclusions: Multimodal IONM was highly specific and sensitive for detecting new deficits. Three patients with new deficits had temporary clipping, including 2 patients with IONM changes not temporally associated with clip placement. Our 1.1% rate of permanent neurologic deficit attributed to temporary clipping support its safety. Differences in patterns of IONM changes among vascular territories warrant further investigation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by developmental regression with loss of motor, communication and social skills, onset of stereotypic hand movements and often seizures. RTT is primarily caused by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). We established a high-resolution melting (HRM) technique for mutation scanning of the MECP2 gene and performed analyses in Czech patients with RTT, autism spectrum conditions and intellectual disability with Rett-like features. In the cases with confirmed MECP2 mutations, we determined X-chromosome inactivation (XCI), examined the relationships between genotype and clinical severity and evaluated the modifying influence of XCI. Our results demonstrate that HRM analysis is a reliable method for the detection of point mutations, small deletions and duplications in the MECP2 gene. We identified 29 pathogenic mutations in 75 girls, including four novel mutations: c.155&#95;1189del1035;909&#95;932inv;insC, c.573delC, c.857&#95;858dupAA and c.1163&#95;1200del38. Skewed XCI (ratio >75%) was found in 19.3% of the girls, but no gross divergence in clinical severity was observed. Our findings confirm a high mutation frequency in classic RTT (92%) and a correlation between the MECP2 mutation type and clinical severity. We also demonstrate limitations of XCI in explaining all of the phenotypic differences in RTT.

Background: Impairment of cerebrovascular autoregulation is a risk factor for ischemic damage following severe brain injury. Autoregulation can be assessed indirectly using intracranial pressure monitoring as a surrogate of cerebral blood volume, but this measure may not be applicable to patients following decompressive craniectomy. Here, we describe assessment of autoregulation using regional cerebral blood flow (rCBF)., Methods: In seven patients with severe brain trauma who underwent neurological surgery, a Hemedex® rCBF probe was placed intraoperatively in peri-lesional tissue. Autoregulation was assessed as a moving Pearson correlation between CPP and rCBF (rCBFx)., Results: Composite data from all patients showed relatively constant perfusion over a wide CPP range (50-90 mmHg) and a U-shaped autoregulation curve with maximal autoregulation (CPPopt) at 55-60 mmHg. All rCBF values fell below the ischemic threshold (<18 ml/100 g/min) when CPPs were <50 mmHg compared with 11 % ischemia when CPPs >50 mmHg (P < 0.05). We examined the percent time during which both autoregulation was intact and rCBF exceeded the ischemic threshold. In the composite data, this variable was maximal in the CPP range of 75-80 mmHg (CPPideal). In individual patients, the range of CPPs with intact autoregulation varied widely. Individual CPPopt values ranged between 60 and 100 mmHg and CPPideal ranged between 65 and 105 mmHg., Conclusions: Assessment of autoregulation with Hemedex® rCBF monitor is feasible and could be used to guide CPP management strategies to optimize both autoregulation and perfusion. Autoregulatory impairment and CPPopt vary considerably between patients, and the addition of rCBF monitoring could help guide CPP targeting decisions.

Mitochondrial disorders (MD) represent a clinically, biochemically and genetically heterogeneous group of diseases associated with dysfunction of the oxidative phosphorylation system and pyruvate dehydrogenase complex. Our aim was to illustrate the most common clinical presentation of MD on the example of selected diseases and syndromes. The minimal prevalence of MD is estimated as 1 to 5,000. MD may manifest at any age since birth until late-adulthood with acute manifestation or as a chronic progressive disease. Virtually any organ may be impaired, but the organs with the highest energetic demands are most frequently involved, including brain, muscle, heart and liver. Some MD may manifest as a characteristic cluster of clinical features (e.g. MELAS syndrome, Kearns-Sayre syndrome). Diagnostics includes detailed history, the comprehensive clinical examination, results of specialized examinations (especially cardiology, visual fundus examination, brain imaging, EMG), laboratory testing of body fluids (lactate, aminoacids, organic acids), and analysis of bioptic samples of muscle, skin, and liver, eventually. Normal lactate level in blood does not exclude the possibility of MD. Although the aimed molecular genetic analyses may be indicated in some of mitochondrial diseases, the methods of next generation sequencing come into focus. Examples of treatment are arginine supplementation in MELAS syndrome, ketogenic diet in pyruvate oxidation disorders or quinone analogs in patients with LHON. Conclusion: The clinical suspicion of a mitochondrial disorder is often delayed, or the disease remains undiagnosed. The correct diagnosis and adequate treatment can improve prognosis of the patient. Access to genetic counseling is also of great importance.

Objectives: TMEM70 deficiency is the most common nuclear-encoded defect affecting the ATP synthase. In this multicentre retrospective study we characterise the natural history of the disease, treatment and outcome in 48 patients with mutations in TMEM70. Eleven centers from eight European countries, Turkey and Israel participated., Results: All 27 Roma and eight non-Roma patients were homozygous for the common mutation c.317-2A > G. Five patients were compound heterozygotes for the common mutation and mutations c.470 T > A, c.628A > C, c.118&#95;119insGT or c.251delC. Six Arab Muslims and two Turkish patients were homozygous for mutations c.238C > T, c.316 + 1G > T, c.336 T > A, c.578&#95;579delCA, c.535C > T, c.359delC. Age of onset was neonatal in 41 patients, infantile in six cases and two years in one child. The most frequent symptoms at onset were poor feeding, hypotonia, lethargy, respiratory and heart failure, accompanied by lactic acidosis, 3-methylglutaconic aciduria and hyperammonaemia. Symptoms further included: developmental delay (98%), hypotonia (95%), faltering growth (94%), short stature (89%), non-progressive cardiomyopathy (89%), microcephaly (71%), facial dysmorphism (66%), hypospadias (50% of the males), persistent pulmonary hypertension of the newborn (22%) and Wolff-Parkinson-White syndrome (13%). One or more acute metabolic crises occurred in 24 surviving children, frequently followed by developmental regression. Hyperammonaemic episodes responded well to infusion with glucose and lipid emulsion, and ammonia scavengers or haemodiafiltration. Ten-year survival was 63%, importantly for prognostication, no child died after the age of five years., Conclusion: TMEM70 deficiency is a panethnic, multisystemic disease with variable outcome depending mainly on adequate management of hyperammonaemic crises in the neonatal period and early childhood.

Objectives: Mitochondrial disorders of energetic metabolism (MD) represent a heterogeneous group of diseases manifesting at any age with a broad spectrum of clinical symptoms, including psychiatric disorders., Methods: The aim of the study was to characterize psychiatric symptoms and diagnoses in five patients with MELAS syndrome between the ages of 17 and 53 years., Results: Four of MELAS patients them harbored the prevalent mitochondrial DNA (mtDNA) mutation 3243A>G, and one patient had the mtDNA mutation 12706T>C. Three patients had positive family histories for MELAS syndrome. In one patient, depression was diagnosedas the first symptom ofMELAS syndrome. Depression also preceded a stroke-like episode in one patient. Four patients had disturbed cognitive functions, confusional states occurred in three patients. One patient manifested psychotic (schizophrenia-like) symptoms., Conclusion: Mitochondrial disorders deserve consideration as part of the differential diagnosis, especially, if there is suspected involvement of other organ groups or positive family history of MD.

Objective: Shortcomings created by the lack of both a uniform definition of ventriculostomy-associated infection (VAI) and reporting standards have led to widely ranging infections rates (2-24%) whose significance is uncertain. We propose a standardized definition of VAI and a consistent reporting format compliant with Centers for Disease Control and Prevention (CDC) for device-related infections. Using those parameters to establish an infection-control surveillance program, we report our 4-year institutional VAI rates., Methods: In this prospective study covering ventriculostomy utilization (October 2006-December 2010), 498 patients had a total of 4,673 ventriculostomy days. By review of the literature and our institutional analysis, we defined VAI as a positive CSF culture in a patient with ventriculostomy catheter, plus one or more of the following (1) fever recorded >101.5 °F or (2) cerebrospinal fluid (CSF) glucose level, either <50 mg/dL or <50% of a serum glucose level drawn within 24 h of the CSF glucose. In a report format that is CDC compliant, rates of VAI are reported., Results: Among our patients, the CDC-compliant infection rate was 2.14 per 1,000 ventriculostomy days. Of the 10 VAIs occurring in 498 patients during 4,673 ventriculostomy days, this 2.0% infection rate was lower than the previously reported 8.8% composite rates of VAI. Average duration of ventriculostomy was 9.4 days. Neither antibiotic-impregnated catheters nor periprocedural or prophylactic antibiotics were used., Conclusions: Our standardized VAI definition and CDC format seems promising toward facilitating future study and guideline development. Given our strict protocol of sterile catheter placement and care, and our institution's low 2.0% infection rates, we propose an infection-rate target of ≤5 per 1,000 device days. Our results suggest that the use of antibiotics or antibiotic-impregnated catheters is unwarranted--a positive given concerns of evolving anti-microbial resistance.

Marfan syndrome (MFS) is an autosomal dominant disorder caused by mutations in the fibrillin 1 gene (FBN1). Neonatal form of MFS is rare and is associated with severe phenotype and a poor prognosis. We report on a newborn girl with neonatal MFS who displayed cyanosis and dyspnea on the first day of life. The main clinical features included mitral and tricuspid valve insufficiency, aortic root dilatation, arachnodactyly, and loose skin. Despite the presence of severe and inoperable heart anomalies, the girl was quite stable on symptomatic treatment and lived up to the 7th month of age when she died due to cardiorespiratory failure. Molecular-genetic studies revealed a novel intronic c.4211-32&#95;-13del mutation in the FBN1 gene. Subsequent in vitro splicing analysis showed this mutation led to exon 35 skipping, presumably resulting in a deletion of 42 amino acids (p.Leu1405&#95;Asp1446del). Interestingly, this mutation is localized outside the region of exons 24-32, whose mutation is responsible for the substantial majority of cases of neonatal MFS. Although the family history of MFS was negative, the subsequent molecular genetic examination documented a mosaicism of the same mutation in the maternal blood cells (10-25% of genomic DNA) and the detailed clinical examination showed unilateral lens ectopy., (© 2014 Wiley Periodicals, Inc.)

We report the second known family with a very rare, maternally inherited missense m.8851T>C mutation in the mitochondrial MTATP6 gene. A failure to thrive, microcephaly, psychomotor retardation and hypotonia were present in a 3-year-old girl with a high mtDNA mutation load (87-97%). Ataxia and Leigh syndrome were subsequently documented in a neurological examination and brain MRI. A muscle biopsy demonstrated decreased ATP synthase and an accumulation of succinate dehydrogenase products, indicating mitochondrial myopathy. Her 36-year-old mother (68% blood heteroplasmy) developed peripheral neuropathy and muscle weakness at the age of 22 years. Our findings extend the clinical and laboratory phenotype associated with the m.8851T>C mutation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RFT1-CDG is a rare N-glycosylation disorder. Only 6 children with RFT1-CDG have been described, all with failure to thrive, feeding problems, hypotonia, developmental delay, epilepsy, decreased vision, deafness and thrombotic complications. We report on two young adult siblings with RFT1-CDG, compound heterozygotes for the novel missense mutations c.1222A>G (p.M408V) and c.1325G>A (p.R442Q) in RFT1 gene. Similar to the previously described patients, these siblings have profound intellectual disability but no feeding problems or failure to thrive. Their epilepsy is well controlled and coagulopathy is mild without clinical consequences. In addition, visual acuity is normal in both patients and hearing impairment is present only in one. Our findings extend the phenotype associated with RFT1-CDG., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We report on the seventh known patient with S-adenosylhomocysteine hydrolase (SAHH) deficiency presenting at birth with features resembling phosphomannomutase 2 (PMM2-CDG Ia) deficiency. Plasma methionine and total homocysteine levels were normal at 2 months and increased only after the 8th month of age. SAHH deficiency was confirmed at 4.5 years of age by showing decreased SAHH activity (11% in both erythrocytes and fibroblasts), and compound heterozygosity for a known mutation c.145C>T (p.R49C) and a novel variant c.211G>A (p.G71S) in the AHCY gene. Retrospective analysis of clinical features revealed striking similarities between SAHH deficiency and the PMM2-CDG Ia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Mitochondrial disorders (MD) may manifest in neonates, but early diagnosis is difficult. In this study, clinical and laboratory data were analyzed in 129 patients with neonatal onset of MD to identify any association between specific mitochondrial diseases and their symptoms with the aim of optimizing diagnosis., Materials and Methods: Retrospective clinical and laboratory data were evaluated in 461 patients (331 families) with confirmed MD., Results: The neonatal onset of MD was reported in 28% of the patients. Prematurity, intrauterine growth retardation and hypotonia necessitating ventilatory support were present in one-third, cardiomyopathy in 40%, neonatal seizures in 16%, Leigh syndrome in 15%, and elevated lactate level in 87%. Hyperammonemia was observed in 22 out of 52 neonates. Complex I deficiency was identified in 15, complex III in one, complex IV in 23, complex V in 31, combined deficiency of several complexes in 53, and PDH complex deficiency was identified in six patients. Molecular diagnosis was confirmed in 49 cases, including a newborn with a 9134A>G mutation in the MTATP6 gene, which has not been described previously., Conclusion: The most significant finding is the high incidence of neonatal cardiomyopathy and hyperammonemia. Based on our experience, we propose a diagnostic flowchart applicable to critically ill neonates suspicious for MD. This tool will allow for the use of direct molecular genetic analyses without the need for muscle biopsies in neonates with Alpers, Barth, MILS and Pearson syndromes, SCO1, SCO2, TMEM70, ATP5E, SUCLG1 gene mutations and PDH complex deficiency.

Background: Increased lactate is an important biochemical marker in diagnosis of children with suspicion of mitochondrial disorders. A diagnostic dilemma may originate if analyses are performed after seizures, when the increased lactate levels may be considered to result from the seizures. To address this problem, we ascertained the diagnostic value of lactate and alanine in blood (B) and cerebrospinal fluid (CSF) in children with mitochondrial disorders (n = 24), epilepsy (n = 32), psychomotor retardation (n = 23), meningitis (n = 12) and meningism (n = 16)., Methods: Lactate concentration was measured using a spectrophotometric method. Amino acids in serum and CSF were analyzed by ion exchange chromatography with ninhydrin detection., Results: Average blood and CSF-lactate levels were significantly higher in children with mitochondrial disorders (3.87 ± 0.48 and 4.43 ± 0.55 mmol/l) and meningitis (2.77 ± 0.45 and 8.58 ± 1.08 mmol/l) than in children with epilepsy (1.72 ± 0.13 and 1.62 ± 0.04 mmol/l), psychomotor retardation (1.79 ± 1.40 and 1.68 ± 0.06 mmol/l) or meningism (1.70 ± 0.13 and 1.64 ± 0.07 mmol/l). Blood and CSF-alanine levels were also higher in children with mitochondrial disorders (558 ± 44 and 51 ± 8 μmol/l) than in children with epilepsy (327 ± 23 and 27 ± 3 μmol/l) or psychomotor retardation (323 ± 27 and 26 ± 3 μmol/l). The CSF-lactate levels of children with epilepsy were similar whether the samples were obtained 3 ± 0.6 h after an attack of brief seizures or from children without history of recent seizures., Conclusion: Elevated cerebrospinal fluid lactate level is a reliable marker pointing to mitochondrial origin of disease, even in children who have recently suffered short-lasting seizures. Some children with mitochondrial disorders manifest only mild or intermittent elevation of lactate levels., (Copyright © 2010 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Several recent studies describing a solely vascular presentation of cystathionine beta-synthase (CBS) deficiency in adulthood prompted us to analyze the frequency of patients manifesting with vascular complications in the Czech Republic. Between 1980 and 2009, a total of 20 Czech patients with CBS deficiency have been diagnosed yielding an incidence of 1:311,000. These patients were divided into three groups based on symptoms leading to diagnosis: those with vascular complications, with connective tissue manifestation and with neurological presentation. A vascular event such as a clinical feature leading to diagnosis of homocystinuria was present in five patients, while two of them had no other symptoms typical for CBS deficiency at the time of diagnosis. All patients with the vascular manifestation were diagnosed only during the past decade. The median age of diagnosis was 29 years in the vascular, 11.5 years in the connective tissue and 4.5 years in the neurological group. The ratio of pyridoxine responsive to nonresponsive patients was higher in the vascular (4 of 5 patients) and connective tissue groups (6 of 7 patients) than in the neurological group (2 of 8 patients). Mutation c.833T>C (p.I278T) was frequent in patients with vascular (6/10 alleles) and connective tissue presentation (8/14 alleles), while it was not present in patients with neurological involvement (0/16 alleles). During the last decade, we have observed patients with homocystinuria diagnosed solely due to vascular events; this milder form of homocystinuria usually manifests at greater ages, has a high ratio of pyridoxine responsiveness/nonresponsiveness, and the mutation c.833T>C (p.I278T) is often present.

The most common cause of pyruvate dehydrogenase complex (PDHc) deficiency is the deficit of the E1α-subunit. The aim of this study was to describe distinct course of the disease in two boys with mutations in PDHA1 gene and illustrate the possible obstacles in measurement of PDHc activity. Clinical data and metabolic profiles were collected and evaluated. PDHc and E1α-subunit activities were measured using radiometric assay. Subunits of PDHc were detected by Western blot. PDHA1 gene was analysed by direct sequencing. In patient 1, the initial hypotonia with psychomotor retardation was observed since early infancy. The child gradually showed symptoms of spasticity and arrest of psychomotor development. In patient 2, the disease manifested by seizures and hyporeflexia in the toddler age. The diagnosis was confirmed at the age of seven years after attacks of dystonia and clinical manifestation of myopathy with normal mental development. Brain MRI of both patients revealed lesions typical of Leigh syndrome. Enzymatic analyses revealed PDHc deficiency in isolated lymphocytes in the first but not in the second patient. The direct measurement of PDH E1-subunit revealed deficiency in this individual. In patient 1, a novel hemizigous mutation c.857C>T (Pro250Leu) was detected in the X-linked PDHA1 gene. Mutation c.367C>T (Arg88Cys) was found in patient 2. We present first two patients with PDHc deficit due to mutations in PDHA1 gene in the Czech Republic. We document the broad variability of clinical symptoms of this disease. We proved that normal PDHc activity may not exclude the disease.

Background: Maternal vitamin B(12) (Cbl) deficiency causes nutritional Cbl deficiency in breastfed infants., Aims: To analyse clinical presentation and metabolic consequences in 40 breastfed infants with Cbl deficiency., Methods: Cbl levels in serum and breast milk were determined by an electrochemiluminescence immunoassay, methylmalonic acid level by GC/MS, plasma homocysteine by HPLC and propionylcarnitine by MS/MS. Profound Cbl deficiency was found in 17 children (69 ± 17 ng/l, controls 200-900), and milder Cbl deficiency in 23 children (167 ± 40 ng/l). Maternal Cbl deficiency was mostly caused by insufficient Cbl absorption. Only six mothers were vegetarian., Results: The average age at diagnosis was 4.4 ± 2.5 months. Clinical symptoms included failure to thrive (48% of children), hypotonia (40%), developmental delay (38%) and microcephaly (23%). 63% of children had anaemia (megaloblastic in 28% of all children). All but one patient had methylmalonic aciduria, 80% of patients had hyperhomocysteinemia and 87% had increased aminotransferases. Propionylcarnitine was elevated in two out of 25 infants. Comparing groups with severe and mild Cbl deficiency, a marked difference was found in severity of clinical and laboratory changes., Conclusion: Maternal Cbl status and diagnostic delay are the major factors influencing severity and progression of Cbl deficiency in breastfed infants. In our cohort, propionylcarnitine was not sufficiently sensitive marker of Cbl deficiency. Although symptoms are reversible on Cbl substitution, permanent neurological damage can result. Selective screening for Cbl deficiency is indicated in all breastfed infants with failure to thrive, hypotonia, developmental delay, microcephaly or megaloblastic anaemia. The best prevention in future could be the screening of all pregnant women., (Copyright © 2009 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Background: Mutations in the alpha-l-iduronidase A (IDUA) gene cause mucopolysaccharidosis type I (MPS I), a progressive multisystem disorder with features ranging over a continuum from mild to severe which is inherited in an autosomal recessive manner. To date over 100 mutations are known, nonetheless genotype-phenotype prediction is complicated and hampered due to attenuating polymorphisms, rare sequence variants, varied genetic backgrounds and environmental effects., Methods: In this study we report the first development of a denaturating high performance liquid chromatography (dHPLC) protocol for the rapid and accurate detection of recently described mutations in the IDUA gene. Optimal PCR running and dHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to 14 IDUA exons and their adjacent intronic/flanking sequences., Results: A total of 12 different mutations, 5 nonsense, 4 missense, 1 deletion, and 2 splice site (intron), in 10 MPS I patients were screened. All mutations revealed a distinct dHPLC pattern thus enabling their accurate detection., Conclusions: A dHPLC screening method was developed for the detection of mutations and sequence variants in the IDUA gene and the results presented in this study revealed that this promising method proved to be robust, automated, economical and above all, highly sensitive. Costs for the detection of mutations causing MPS I disease should be reduced by using this method as a pre-analytical tool followed by sequencing of aberrant heteroduplex-forming amplicons., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background: Hereditary tyrosinemia type 1 (HT1) is a rare autosomal recessive inborn error of metabolism caused by deficiency of fumarylacetoacetate hydrolase. HT1 manifests with severe liver and kidney impairment and associates with an increased risk of liver cancer development. The aim of our study is to present a detailed clinical picture and results of biochemical and molecular genetic analyses in 11 Czech patients with HT1 diagnosed in our clinic within 1982-2006., Methods and Results: In 9 patients the disease manifested between 1.5-7 months of age with refusal to eat, failure to thrive and vomiting. In 4 children HT1 progressed to acute liver failure. One clinically healthy boy was diagnosed because of affected sister. In one boy with liver cirrhosis the diagnosis was delayed until the age of 5.5 years. In all children the biochemical investigation showed elevated liver enzymes, alpha1-fetoprotein and hypophosphatemic rickets. Metabolic investigation revealed increased plasma tyrosine level, urinary excretion of succinylacetone and in 8 measured patients also increased urinary delta-aminolevulinic acid concentration. Three patients born before 1988 died due to liver cancer development (two of them) or liver failure. The average age of our 8 living patients is 10.7 +/- 8.3 years. Mutation analysis of FAH gene confirmed the HT1 in these patients and three novel mutations were found in FAH gene: c.579C>A, c.680G>T and c.1210G>A. Clinical status in six patients is favourable on strict low protein diet combined with Orfadin therapy. However, in two children despite of the maximal available therapy lasting 2 and 10 years resp., the disease progressed towards liver cancer development and necessity of liver transplantation., Conclusions: Early diagnostics of HT1 as a part of extended newborn screening is the only possibility to further improve the prognosis of the patients. Moreover, available molecular-genetic analysis of the FAH gene enables prenatal diagnostics in affected families.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918&#95;1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein.

The analysis of mtDNA haplogroup frequency in various populations is a tool for studying human history and population dynamics. The aim of this study is to map the frequency of major mtDNA haplogroups in 300 maternally unrelated individuals representing the Czech population of the central part of the Czech Republic. Eighteen polymorphic sites in the coding region of mtDNA were screened by PCR-RFLP to determine 11 mtDNA haplogroups and 5 subhaplogroups. The most frequent haplogroups were H (41%) and U (21%). Less frequent haplogroups were J and T, each with a frequency of 8%. Frequencies of other haplogroups (V, K, HV, W, preV, X, and I) did not exceed 5%. The results of our study reveal that the frequency of mtDNA haplogroups in the Czech population is similar to the frequencies obtained in other European countries, especially Poland, Germany, and Russia. On the contrary, significant differences in haplogroup frequency were found between the Czech and Finnish populations (haplogroups U, T, W) and populations from Bulgaria and Turkey (haplogroups H).

We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in individuals with isolated mitochondrial ATP synthase deficiency and identified disease-causing mutations in TMEM70. Complementation of the cell lines of these individuals with wild-type TMEM70 restored biogenesis and metabolic function of the enzyme complex. Our results show that TMEM70 is involved in mitochondrial ATP synthase biogenesis in higher eukaryotes.

Background: Zinc is an essential trace element for the immune system. The zinc deficiency diminishes antibody- and cell-mediated responses in man. Lymphopenia and thymic atrophy are usually the early hallmarks of zinc deficiency. Surprisingly, only scarce data are available about polymorphonuclear cells (PMNs) functions in infants with zinc deficiency. We present the results of immunological analyses in one infant with transient zinc deficiency due to decreased zinc concentration in mother milk resulting in severe lactogenic acrodermatitis enteropathica., Material/methods: Nine repeated examination of oxidative burst of PMNs and immunoglobulin levels using nitroblue tetrazolium dye test, chemiluminescence, flow cytometry and nephelometry were performed in the infant with severe zinc deficiency during 28 months period., Results: The unusual prolonged but transient impairment of PMNs respiratory burst accompanied with hypogammaglobulinaemia developed since the age of 2.5 months. Dramatic improvement of the skin was observed within days with total resolution of skin lesions on the 9th day of zinc therapy, but decreased PMNs respiratory burst persisted until the age of 23 months., Conclusions: We conclude that zinc deficiency may lead to prolonged impairment of polymorphonuclear cells functions and hypogammaglobulinaemia.

Chondrodysplasia punctata represents clinically and genetically a heterogeneous group of disorders characterized by the presence of multiple congenital anomalies and stippled epiphyses. We present clinical course of the disease and the results of metabolic, X-ray and molecular analyses in 19-months old girl with X-linked dominant chondrodysplasia punctata with intrauterine growth retardation, craniofacial dysmorphy, cataracts, cutaneous anomalies including ichthyosis, asymmetric rhizomesomelic shortness of the limbs, deformity of the spine, club foot, polydactyly, syndactyly, epiphyseal stippling and low cholesterol (2.29 mmol/l). Spectrophotometric analysis revealed the presence of abnormal pattern of cholesterol precursors in blood. The increased level of 8-dehydrocholesterol (42.2 micromol/l, controls < 1) and 7-dehydrocholesterol (25.5 micromol/l, controls < 1) recognised with GC/MS suggested an endogenous defect of cholesterol biosynthesis. The diagnosis of X-linked dominant chondrodysplasia punctata (CDPX2) was confirmed by the molecular analysis. Sequencing of the EBP gene encoding for 3beta-hydroxysteroid-delta8,delta7-isomerase revealed the presence of "de novo" heterozygous mutation c.327C>T (p.Arg110Stop). High cholesterol diet normalized cholesterol level (3.28 mmol/l) but it had no influence on the unfavourable prognosis of the disease. Low level of cholesterol with abnormal sterol profile in a child with congenital development anomalies represent an important laboratory marker suggesting an inherited defect of cholesterol biosynthesis.

Genotype polymorphism studies at the 13 loci STRs included in the combined DNA index system [CODIS and PCR-based short tandem repeat loci, in: Proceedings of the Second European Symposium on Human Identification, Promega Corporation, Madison, WI, 1998, pp. 73-88; J. Forensic Sci. 46 (2001) 453] (CODIS: D3S1358, HUMvWA31, HUMFGA, D8S1179 D21S11, D18S51, D5S818, D13S317, D7S820, HUMTH01, HUMTPOX, HUMCSF1PO and D16S539) were carried out in a sample of 1429 unrelated Colombian individuals belonging to 25 different departments. As many other countries in Latino-America, Colombia shows an important admixture component, basically integrated by Amerindians, European-descendants and African-descendants. Due to the fact that only partial population analyses have been carried out in the country, the main aim of the present analysis is to establish a database of forensic interest based on the widely used CODIS systems covering the main Colombian regions.

An acute inpatient psychiatric ward suffered from low morale, high staff turnover and a lack of leadership. Taking part in a Clinical Governance Development Programme helped the ward team to examine the problems. They were then able to work in groups to identify solutions with the multidisciplinary team, patients and carers and set short- and long-term goals.

Objective: This study aimed to measure the outcomes of a harmonised, structured pharmaceutical care programme provided to elderly patients (> or =65 years of age) by community pharmacists in a multicentre international study performed in 7 European countries., Design and Setting: The study was a randomised, controlled, longitudinal, clinical trial with repeated measures performed over an 18-month period. A total of 104 intervention and 86 control pharmacy sites participated in the research and 1290 intervention patients and 1164 control patients were recruited into the study., Main Outcome Measures and Results: A general decline in health-related quality of life over time was observed in the pooled data; however, significant improvements were achieved in patients involved in the pharmaceutical care programme in some countries. Intervention patients reported better control of their medical conditions as a result of the study and cost savings associated with pharmaceutical care provision were observed in most countries. The new structured service was well accepted by intervention patients and patient satisfaction with the services improved during the study. The pharmacists involved in providing pharmaceutical care had a positive opinion on the new approach, as did the majority of general practitioners surveyed. The positive effects appear to have been achieved via social and psychosocial aspects of the intervention, such as the increased support provided by community pharmacists, rather than via biomedical mechanisms., Conclusions: This study is the first large-scale, multicentre study to investigate the effects of pharmaceutical care provision by community pharmacists to elderly patients. Future research methodology and implementation will be informed by the experience gained from this challenging trial.

Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy.

Both VEGF protein and VEGF DNA in combination with an adenoviral vector have been shown to enhance collateral formation in a porcine model of chronic myocardial ischemia. We sought to determine whether direct intramyocardial injection of naked DNA encoding for VEGF could similarly improve myocardial perfusion. Initially, 23 nonischemic pigs received either 200 microg of plasmid DNA encoding beta-galactosidase (pCMVbeta, n = 11) or 500 microg of phVEGF165 (n = 12) into four separate sites in the myocardium via a small anterolateral thoracotomy incision in the fourth intercostal space. Two additional groups of pigs received an intramyocardial injection of either phVEGF165 (n = 6) or pCMVbeta (n = 7) 3 to 4 weeks after implantation of an ameroid constrictor around the left circumflex coronary artery. The injections caused no change in heart rate or blood pressure, and no ventricular arrhythmias or histologic evidence of inflammation. VEGF protein was detected by Western blot in VEGF-treated animals, with the strongest bands closest to the injection site. Plasma VEGF concentration (ELISA) increased from 3+/-2 to 27+/-13 pg/ml (p = 0.035) by day 4 after treatment. No increase in VEGF protein was noted in pCMVbeta-treated animals whereas these did stain positive for beta-Gal. Resting myocardial blood flow (colored microspheres) was significantly reduced in the ischemic versus nonischemic territory in control animals (1.07+/-0.05 versus 1.32+/-0.05; p < 0.05) but not VEGF-treated pigs (1.32+/-0.24 versus 1.13+/-0.12; p = NS). Maximal vasodilatation with adenosine significantly increased flow to the ischemic region in VEGF-treated pigs (2.16+/-0.57 versus 1.32+/-0.24; p < 0.05) but not controls (1.31+/-0.05 versus 1.17+/-0.06;p = NS). Collateral filling of the occluded circumflex artery improved in five of six VEGF-treated pigs (mean change in Rentrop score, +1.5). We conclude that direct intramyocardial transfection phVEGF165 is safe and capable of producing sufficient VEGF protein to enhance collateral formation and myocardial perfusion. This approach may offer an alternative therapy for patients with intractable myocardial ischemia not amenable to PTCA or CABG.

Purpose: This study examined whether patients insured by a managed dental care plan receive lower quality dental hygiene care than those not enrolled in a managed dental care plan., Methods: Questionnaire data were gathered from 193 dental hygienists in the Chicago, Illinois area. Managed care was measured by a questionnaire item that assessed the proportion of managed care patients treated by the subject; quality of dental hygiene care was measured by items that assessed the extent to which the subject performed each of 23 dental hygiene tasks., Results: A factor analysis of the 23 items assessing the quality of dental hygiene care indicated four meaningful factors named: periodontal procedures, appointment time, visual examinations, and oral examinations. Measures based on these factors were the dependent variables in regression analysis that included managed care as the primary independent variable and demographic characteristics of the subjects and their practices as control variables. Managed care had a significant (p < .05) negative relationship with appointment time, but did not have a significant relationship with periodontal procedures, visual examinations, or oral examinations., Conclusion: This study suggests that managed dental care program patients may have inadequate appointment time with a dental hygienist, which may affect whether they receive important services, such as oral health education. Dental hygienists in managed-care environments should be certain they are making effective use of the scheduled appointment time and procedures to ensure managed-care patients receive adequate time for dental hygiene care.

Background: Remodeling of the extracellular matrix plays an important role during the pathogenesis of atherosclerosis and restenosis. The matrix glycoprotein thrombospondin-1 (TSP1) inhibits endothelial cell proliferation and migration in vitro. In contrast, TSP1 facilitates the growth and migration of cultured vascular smooth muscle cells. Accordingly, we investigated the hypothesis that administration of anti-TSP1 antibody could facilitate reendothelialization and inhibit neointimal thickening in balloon-injured rat carotid artery., Methods and Results: Sprague-Dawley rats were subjected to left common carotid artery denudation, after which arteries were treated with C6.7 anti-TSP1 or control antibody. Evans blue dye staining 2 weeks after injury disclosed significantly increased reendothelialization in arteries treated with C6.7 antibody compared with the control group, and this effect was associated with increased number of proliferating cell nuclear antigen-positive endothelial cells. In contrast, treatment with C6.7 antibody decreased the number of proliferating cell nuclear antigen-positive vascular smooth muscle cells in the injured arterial wall. Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries receiving C6.7 antibody versus the control group at both the 2-week and 4-week time points., Conclusions: Intra-arterial delivery of antibody against TSP1 facilitated reendothelialization and reduced neointimal lesion formation after balloon denudation.

Circulating endothelial progenitor cells (EPCs) have been isolated in peripheral blood of adult species. To determine the origin and role of EPCs contributing to postnatal vasculogenesis, transgenic mice constitutively expressing beta-galactosidase under the transcriptional regulation of an endothelial cell-specific promoter (Flk-1/LZ or Tie-2/LZ) were used as transplant donors. Localization of EPCs, indicated by flk-1 or tie-2/lacZ fusion transcripts, were identified in corpus luteal and endometrial neovasculature after inductive ovulation. Mouse syngeneic colon cancer cells (MCA38) were implanted subcutaneously into Flk-1/LZ/BMT (bone marrow transplantation) and Tie-2/LZ/BMT mice; tumor samples harvested at 1 week disclosed abundant flk-1/lacZ and tie-2/lacZ fusion transcripts, and sections stained with X-gal demonstrated that the neovasculature of the developing tumor frequently comprised Flk-1- or Tie-2-expressing EPCs. Cutaneous wounds examined at 4 days and 7 days after skin removal by punch biopsy disclosed EPCs incorporated into foci of neovascularization at high frequency. One week after the onset of hindlimb ischemia, lacZ-positive EPCs were identified incorporated into capillaries among skeletal myocytes. After permanent ligation of the left anterior descending coronary artery, histological samples from sites of myocardial infarction demonstrated incorporation of EPCs into foci of neovascularization at the border of the infarct. These findings indicate that postnatal neovascularization does not rely exclusively on sprouting from preexisting blood vessels (angiogenesis); instead, EPCs circulate from bone marrow to incorporate into and thus contribute to postnatal physiological and pathological neovascularization, which is consistent with postnatal vasculogenesis.

Background: The impact of disordered lipid metabolism on collateral vessel development was studied in apolipoprotein (apo)E-/- mice with unilateral hindlimb ischemia., Methods and Results: Hindlimb blood flow and capillary density were markedly reduced in apoE-/- mice versus C57 controls. This was associated with reduced expression of vascular endothelial growth factor (VEGF) in the ischemic limbs of apoE-/- mice. Cell-specific immunostaining localized VEGF protein expression to skeletal myocytes and infiltrating T cells in the ischemic limbs of C57 mice; in contrast, T-cell infiltrates in ischemic limbs of apoE-/- mice were severely reduced. The critical contribution of T cells to VEGF expression and collateral vessel growth was reinforced by the finding of accelerated limb necrosis in athymic nude mice with operatively induced hindlimb ischemia. Adenoviral VEGF gene transfer to apoE-/- mice resulted in marked augmentation of hindlimb blood flow and capillary density., Conclusions: These findings thus underscore the extent to which hyperlipidemia adversely affects native collateral development but does not preclude augmented collateral vessel growth in response to exogenous cytokines. Moreover, results obtained in the apoE-/- and athymic nude mice imply a critical role for infiltrating T cells as a source of VEGF in neovascularization of ischemic tissues.

Background: Endothelial cells (ECs) represent the critical cellular element responsible for postnatal angiogenesis. Because ACE inhibitors may favorably affect endothelial function, we investigated the hypothesis that administration of the ACE inhibitor quinaprilat could enhance angiogenesis in vivo., Methods and Results: Ten days after resection of 1 femoral artery, New Zealand White (NZW) rabbits were randomly assigned to receive recombinant human vascular endothelial growth factor (rhVEGF) administered as a single intra-arterial injection (n=6), quinaprilat (n=8) or captopril (n=7) administered as a daily subcutaneous injection, or no treatment (controls, n=6). Angiogenesis was monitored in vivo by measurement of blood pressure, vasoreactivity, and resistance in ischemic versus normal limbs at day 10 (D10) and D40; angiographic studies to identify sites of neovascularization were performed at D10 and D40, and morphometric analysis of capillary density in the ischemic limb was performed at necropsy (D40). Both functional and morphological outcomes documented augmented angiogenesis in quinaprilat-treated rabbits similar to that observed for rhVEGF and superior to that observed with either captopril or no drug (controls). Residual ACE activity was equivalent for the captopril and quinaprilat groups in plasma (42.54+/-0.03% versus 41.53+/-0.02%, P=NS) but not in tissue, where quinaprilat lowered ACE activity significantly (P<0.01) compared with captopril (13% versus 61%)., Conclusions: ACE inhibition with quinaprilat promotes angiogenesis in a rabbit model of hindlimb ischemia. Thus, nonsulfhydryl ACE inhibitors with high tissue affinity may be potentially useful for therapeutic angiogenesis in ischemic tissues. Moreover, previous evidence that ACE inhibition benefits patients with myocardial ischemia may be due in part to augmented collateral development.

Endothelial progenitor cells (EPCs) have been isolated from circulating mononuclear cells in human peripheral blood and shown to be incorporated into foci of neovascularization, consistent with postnatal vasculogenesis. We determined whether endogenous stimuli (tissue ischemia) and exogenous cytokine therapy (granulocyte macrophage-colony stimulating factor, GM-CSF) mobilize EPCs and thereby contribute to neovascularization of ischemic tissues. The development of regional ischemia in both mice and rabbits increased the frequency of circulating EPCs. In mice, the effect of ischemia-induced EPC mobilization was demonstrated by enhanced ocular neovascularization after cornea micropocket surgery in mice with hindlimb ischemia compared with that in non-ischemic control mice. In rabbits with hindlimb ischemia, circulating EPCs were further augmented after pretreatment with GM-CSF, with a corresponding improvement in hindlimb neovascularization. There was direct evidence that EPCs that contributed to enhanced corneal neovascularization were specifically mobilized from the bone marrow in response to ischemia and GM-CSF in mice transplanted with bone marrow from transgenic donors expressing beta-galactosidase transcriptionally regulated by the endothelial cell-specific Tie-2 promoter. These findings indicate that circulating EPCs are mobilized endogenously in response to tissue ischemia or exogenously by cytokine therapy and thereby augment neovascularization of ischemic tissues.