Alpha-mannosidosis: characterization of CNS pathology and correlation between CNS pathology and cognitive function.

Alpha-mannosidosis (AM) (OMIM 248500) is a rare lysosomal storage disease. The understanding of the central nervous system (CNS) pathology is limited. This study is the first describing the CNS pathology and the correlation between the CNS pathology and intellectual disabilities in human AM. Thirty-four patients, aged 6-35 years, with AM were included. Data from 13 healthy controls were included in the analysis of the magnetic resonance spectroscopy (MRS). Measurements of CNS neurodegeneration biomarkers in cerebrospinal fluid (CSF), CSF-oligosaccharides, and performance of cerebral magnetic resonance imaging (MRI) and MRS were carried out. On MRI, 5 of 10 patients had occipital white matter (WM) signal abnormalities, and 6 of 10 patients had age-inappropriate myelination. MRS demonstrated significantly elevated mannose complex in gray matter and WM. We found elevated concentrations of tau-protein, glial fibrillary acidic protein and neurofilament light protein in 97 patients, 74% and 41% of CSF samples, respectively. A negative correlation between CSF-biomarkers and cognitive function and CSF-oligosaccharides and cognitive function was found. The combination of MRS/MRI changes, elevated concentrations of CSF-biomarkers and CSF-oligosaccharides suggests gliosis and reduced myelination, as part of the CNS pathology in AM. Our data demonstrate early neuropathological changes, which may be taken into consideration when planning initiation of treatment.

Cognitive profile and activities of daily living: 35 patients with alpha-mannosidosis.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) (AM) is a rare lysosomal storage disease caused by a deficiency of the alpha-mannosidase enzyme. The typical signs consist of hearing impairment, intellectual disabilities, coarse facial features and motor function disturbances. We report on the cognitive function and activities of daily living in patients with AM. METHODS: Thirty five AM patients, age 6-35 years, were included in the study. As a cognitive function test, we used the Leiter international performance scale-revised (Leiter-R), which consists of two batteries: the visual function and reasoning battery and the memory and attention battery, the latter including a memory screening. Additional two questionnaires, The Childhood Health Assessment Questionnaire (CHAQ) and EQ-5D-5 L, were filled out. RESULTS: We found IQ in the range of 30-81 in our cohort. The total equivalent age (mental age) was significantly reduced, between 3-9 years old for the visual function and reasoning battery, between 2.3-10.2 years for the memory screening. Data suggested a specific developmental profile for AM with a positive intellectual development until the chronological age 10-12 years, followed by a static or slightly increasing intellectual level. All patients were to varying degrees socially and practically dependent and unable to take care of themselves in daily life. CONCLUSIONS: Intellectual disability is a consistent finding in patients with alpha-mannosidosis but with extensive variation. We assess that this group of patients has, despite their intellectual disabilities, a potential for continuous cognitive development, especially during childhood and early teenage years. This should be included and supported in the individual educational planning.

Enzyme replacement therapy for alpha-mannosidosis: 12 months follow-up of a single centre, randomised, multiple dose study.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disease (LSD) caused by alpha-mannosidase deficiency. Manifestations include intellectual disabilities, facial characteristics and hearing impairment. A recombinant human alpha-mannosidase (rhLAMAN) has been developed for weekly intravenous enzyme replacement therapy (ERT). We present the preliminary data after 12 months of treatment. METHODS: This is a phase I-II study to evaluate safety and efficacy of rhLAMAN. Ten patients (7-17 y) were treated. We investigated efficacy by testing motor function (6-minutes-Walk-Test (6-MWT), 3-min-Stair-Climb-Test (3-MSCT), The Bruininks-Oseretsky Test of Motor Proficiency (BOT2), cognitive function (Leiter-R), oligosaccharides in serum, urine and CSF and Tau- and GFA-protein in CSF. RESULTS: Oligosaccharides: S-, U- and CSF-oligosaccharides decreased 88.6% (CI -92.0 -85.2, p < 0.001), 54.1% (CI -69.5- -38.7, p < 0,001), and 25.7% (CI -44.3- -7.1, p < 0.05), respectively. Biomarkers: CSF-Tau- and GFA-protein decreased 15%, p < 0.009) and 32.5, p < 0.001 respectively. Motor function: Improvements in 3MSCT (31 steps (CI 6.8-40.5, p < 0.01) and in 6MWT (60.4 m (CI -8.9 -51.1, NS) were achieved. Cognitive function: Improvement in the total Equivalence Age of 4 months (0.34) was achieved in the Leiter R test (CI -0.2-0.8, NS). CONCLUSIONS: These data suggest that rhLAMAN may be an encouraging new treatment for patients with alpha-mannosidosis.The study is designed to continue for a total of 18 months. Longer-term follow-up of patients in this study and the future placebo-controlled phase 3 trial are needed to provide greater support for the findings in this study.

Brain N-acetylaspartate levels correlate with motor function in metachromatic leukodystrophy.

BACKGROUND: Late infantile metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder that causes severe demyelination of the nervous system. The neuronal metabolite N-acetylaspartate (NAA) serves as a source of acetyl groups for myelin lipid synthesis in oligodendrocytes and is known as a marker for neuronal and axonal loss. NAA and other metabolite levels measured by proton magnetic resonance spectroscopy (MRS) correlate with performance of the brain in normal children. There is a need for sensitive measures of disease progression in patients with MLD to enable development of future treatments. METHODS: A cross-section of 13 children with late infantile MLD were examined by proton MRS. Signals from NAA, total choline, and total creatine in the deep white matter were measured and correlated with the results of cognitive and motor function tests. RESULTS: The NAA signal decreased as the disease process advanced. Motor function, measured by the Gross Motor Function Measure-88, varied from 13 (only head movement in the supine position) to 180 (able to walk) across the study cohort, demonstrating a wide range in functional status. Similarly, varied decreases were observed in cognitive function. We report strong positive correlations between standardized measures of motor and cognitive function and NAA levels in the deep white matter. CONCLUSIONS: We suggest that NAA levels could serve as a sensitive biomarker in children with MLD. Proton MRS may provide a valuable tool for measuring the effects of treatment interventions in this disorder.

Weight loss and delayed gastric emptying following a South American herbal preparation in overweight patients.

BACKGROUND: Obesity and overweight may soon affect more than half of the population in some regions of the world and are associated with diabetes, hypertension and other diseases that cause morbidity, mortality and high health-care expenditure. No one approach, whether dietetic management, medication, or commercial weight loss programme, can alone solve the problem--all potential treatments need to be investigated and exploited. Among the herbal preparations known to non-western cultures are materials which may have applications in modulating physiological processes which influence gut motility, food intake and energy balance. One such mixed herbal preparation is 'YGD' containing Yerbe Maté (leaves of Ilex paraguayenis), Guarana (seeds of Paullinia cupana) and Damiana (leaves of Turnera diffusa var. aphrodisiaca). AIMS: This study had two distinct aims: to determine the effect of a herbal preparation 'YGD' containing Yerbe Maté, Guarana and Damiana on gastric emptying; to determine the effect of the same preparation on weight loss over 10 days and 45 days and weight maintenance over 12 months. METHODS: Gastric emptying was observed using ultrasound scanning in seven healthy volunteers following YGD and placebo capsules taken with 420 mL apple juice. Body weight was observed before and after 10 days of treatment with three YGD capsules or three placebo capsules before each meal for 10 days in 44 healthy overweight patients attending a primary health care centre. Forty-seven healthy overweight patients entered a double-blind placebo-controlled parallel trial of three capsules of YGD capsules before each main meal for 45 days compared with three placebo capsules on body weight. Body weight was monitored in 22 patients who continued active (YGD capsules) treatment for 12 months. RESULTS: The herb preparation YGD was followed by a prolonged gastric emptying time of 58 +/- 15 min compared to 38 +/- 7.6 min after placebo (P = 0.025). Body weight reductions were 0.8 +/- 0.05 kg after YGD capsules compared to 0.3 +/- 0.03 kg after placebo capsules over 10 days, and 5.1 +/- 0.5 kg after PGD capsules compared to 0.3 +/- 0.08 kg after placebo over 45 days. Active treatment with YGD capsules resulted in weight maintenance of the group (73 kg at the beginning and 72.5 kg at the end of 12 months). CONCLUSIONS: The herbal preparation, YGD capsules, significantly delayed gastric emptying, reduced the time to perceived gastric fullness and induced significant weight loss over 45 days in overweight patients treated in a primary health care context. Maintenance treatment given in an uncontrolled context resulted in no further weight loss, nor weight regain in the group as a whole. The herbal preparation is thus shown to be one that significantly modulates gastric emptying. Further clinical studies with dietetic monitoring of energy intake, dietary quality, satiety ratings, body weight and body composition are now indicated, and examination of the active principles contained in the three herbal components may prove rewarding.

Carcino-embryonic antigen in monitoring the growth of human colon adenocarcinoma tumour cells SK-CO-1 and HT-29 in vitro and in nude mice.

A set of experimental model systems were designed to investigate (a) the inter-relationship between growth of two human cancer cell lines (SK-CO-1, HT-29) and carcino-embryonic antigen (CEA) kinetics; and (b) whether neoplastic growth or CEA concentration is modulated by human growth hormone (hGH). We found that increasing CEA concentration depended on tumour burden. SK-CO-1 cells had the lowest growth rates but the highest rates of CEA production. The rate of CEA increase exceeded the growth rate of both SK-CO-1 and HT-29. hGH modulated neither neoplastic growth nor CEA production. In conclusion, our results suggest that experimental models may be useful for investigating the role of serological markers as monitors of increasing tumour burden. It will be of interest to investigate the performance of those model systems in examining the effect of cytotoxic agents in neoplastic growth.

The potential immunogenicity of human insulin and insulin analogues evaluated in a transgenic mouse model.

Transgenic mice with tissue-specific expression of the human insulin gene in the beta cells of the pancreas do not produce insulin-specific antibodies when injected with human insulin. Tolerant transgenic mice injected with human or porcine insulin reflect the clinical situation. When injected with bovine insulin the transgenic mice produce antibodies. The potential immunogenicity of 12 recombinant human insulin analogues has been tested in this transgenic model. The analogues were designed either to prevent hexamer formation or to improve chemical stability or both. The analogues have amino acid substitutions or deletions at residue 8, 10 and 21 in the A-chain and residue 3, 9, 27 and 28 in the B-chain. The results show that substitution of single amino acids in the A-chain loop of human insulin for the corresponding amino acids in bovine insulin at residues A8 or A10 is sufficient to elicit an antibody response in responder mice. Only human insulin analogues with substitutions at residues 8 or 10 in the A-chain elicit antibody formation in the transgenic mice, whereas non-transgenic control groups respond to insulin and all analogues. Antibodies developed against the human insulin analogues are cross reactive with recombinant human insulin. Antibodies developed against an immunogenic analogue could therefore neutralize both the analogue and the native insulin and thereby aggravate the patient's condition. This transgenic mouse immunogenicity model should be useful as an in vivo model to map immunogenic areas of recombinant proteins.

Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy.

To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.

Properties of the scrapie prion protein: quantitative analysis of protease resistance.

The disease-specific isoform of the prion protein (PrPSc) is an essential part of the infectious particle which causes spongiform degeneration in various mammalian species. PrPSc differs from PrP of normal animals (PrPc) by its relative protease resistance. The physical nature of this difference is still unknown. We analyzed the protease resistance of PrPSc quantitatively using an enzyme-linked immunofiltration assay. PrPSc was rendered completely protease-sensitive at alkaline pH or in > 1.5 M guanidinium thiocyanate (GdnSCN). Denaturation in 4 M GdnSCN completely abolished the protease resistance of PrPSc within 15 min, while denaturation in 7.2 M urea showed a slower time course. In the presence of ethanol, PrPSc was protected from denaturation by GdnSCN or alkaline pH. Denaturation curves were used to calculate the free energy (delta GD) as a function of different denaturant concentrations. Linear regression of delta GD values was used to extrapolate the free energy in the absence of denaturants (delta GH2O), yielding similar values (delta GH2O,GdnSCN = -2.3 kcal/mol; delta GH2O,urea = -3.1 kcal/mol). The linear relationship between delta GD and the denaturant concentration is suggestive of a two-state model involving the conformational change of a single protein domain. This is also reflected in the small number of side chains (11.6) additionally exposed to the solvent upon conversion of PrPSc to its protease-sensitive isoform. Our results suggest that only minor rearrangements of the structure of PrP are needed to abolish the protease resistance of PrPSc.

[Bone scintigraphy in assessment of risk of complications after medial femoral neck fractures]. / Knogleskintigrafi til vurdering af risiko for komplikationer efter medial collum femoris fraktur.

The value of visually evaluated bone scintigraphy in predicting postoperative non-union and avascular necrosis was examined in a prospective, consecutive series of patients with intracapsular fracture of the femoral neck. 49 patients had scintigraphy carried out preoperatively, and 37 postoperatively. In 18 scintigraphy was done preoperatively as well as postoperatively. The patients were followed clinically and radiographically for at least two years after operation. In the preoperative group 19 patients had been excluded at follow up and 13 patients in the postoperative group. The results indicate a high correlation between a reduced uptake of isotope in preoperative scintigraphy and development of late complications in these patients. It is concluded that visually evaluated preoperative planar scintigraphy is of value in selecting those patients with intracapsular femoral neck fractures, who are at risk of developing late complications.

A specific assay for leukotriene B4 in human whole blood.

Leukotrienes (LTs) are potent mediators of inflammatory and allergic responses, and are present in biological fluids in minute amounts, that is, in the picogram range. The aim of this study was to develop and validate a method for determination of LTB4 synthesized in vitro in human whole blood. Heparinized blood was stimulated with calcium-ionophore A23187 at 37 degrees C. After 30 min cells were separated by centrifugation. LTB4 was analyzed by radioimmunoassay (RIA). When sample preparation was restricted to protein precipitation with acetone, interference was demonstrated by lack of parallelism between standard and sample dilution curves. Purification was, therefore, extended by combinations of the following steps: 1) protein precipitation, 2) lipid extractions, and 3) high-performance liquid chromatography (HPLC). One of two commercially available LTB4 standards was found to contain multiple components, several of which were immunoreactive in RIA. Even for the standard containing pure LTB4, interference was demonstrated by lack of parallelism between sample and standard dilution curves. Testing eight combinations of varying purification steps, we found that only a three-step purification procedure, including 1) solid-phase extraction, 2) protein precipitation at -20 degrees C, and 3) HPLC, was able to eliminate interference in RIA. Using this procedure, the recovery was 78%. Stimulation of whole blood from normal subjects with calcium-ionophore showed optimal LTB4 production at 10 microM ionophore, yielding 6.6 ng LTB4/mL blood.

Rapid estimation of left ventricular ejection fraction in acute myocardial infarction by echocardiographic wall motion analysis.

Echocardiographic estimates of left ventricular ejection fraction (ECHO-LVEF) in acute myocardial infarction (AMI) were obtained by a new approach, using visual analysis of left ventricular wall motion in a nine-segment model. The method was validated in 41 patients using radionuclide ventriculography (RNV) and contrast ventriculography measurements of LVEF for comparison. ECHO-LVEF from the 41 patients correlated well with the reference methods (y = 1.5x - 14.7, r = 0.93; linear regression analysis; 95% confidence limit for a single determination of ECHO-LVEF was 17.2). Interobserver variability by linear regression was r = 0.89, SEE = 7.1 with a mean difference between paired observations of -1.5 +/- 6.9 (SD). In a random sample of 18 patients (45 observations), ECHO-LVEF allowed separation between RNV-LVEF values greater than or equal to 40 and less than 40, representing low and high risk groups following AMI. Thus, the results showed that simple, readily available wall motion-derived estimates of LVEF were as closely associated with LVEF measured by standard reference methods as were previously published, more cumbersome, planimetric echocardiographic methods. Reporting on global LVEF function in LVEF units rather than in nonstandardized wall motion scores of index values may facilitate intra- and interhospital communication and the use of optimized echocardiographic risk stratification after AMI.

Prediction of late complications of femoral neck fractures by scintigraphy.

A prospective study was undertaken to determine the value of scintigraphy in predicting nonunion or necrosis of the femoral head following femoral neck fractures. Scintigraphy was carried out in 49 patients before operation and in 37 one week after operation; in 18 it was done before and after operation. The uptake of isotope was estimated visually as either normal or reduced compared to the opposite side. All the patients were followed up for at least 2 years. The results indicate that pre-operative bone scintigraphy is useful in elderly patients to predict nonunion and necrosis, and therefore is recommended to select those patients who will benefit from primary arthroplasty instead of fixation of the fracture.

Epirubicin cardiotoxicity: a study of 135 patients with advanced breast cancer.

The cardiotoxicity of epirubicin (EPI) was evaluated clinically, radiologically, with ECG, and with multiple ECG-gated radionuclide determination of the left ventricular ejection fraction (LVEF) during rest in 135 patients with advanced breast cancer. The EPI doses were 60 mg/m2 on days 1 and 8 every 4 weeks or 45 mg/m2 plus vindesine 3 mg/m2 on the same schedule. The median cumulative dose of EPI was 500 mg/m2 (range, 47 to 1,563). Eight of the 135 patients developed congestive heart failure (CHF). Of 67 patients treated with EPI less than 500 mg/m2, none developed CHF. Among 48 patients treated with doses between 500 and 1,000 mg/m2, one had CHF (2%; 95% confidence limits, 0.1 to 11.1). Among 20 patients who received EPI from 1,000 to 1,563 mg/m2, seven developed CHF (35%; 95% confidence limits, 15.4 to 59.2). Four patients died due to cardiotoxicity. The risk of EPI cardiotoxicity at the present schedule is considerable at doses above 1,000 mg/m2. At doses between 500 and 1,000 mg/m2 the risk of CHF decreases, and at doses below 500 mg/m2, it is negligible. For all patients, the prevalence of CHF was 6% and the sensitivity of LVEF high (95%), mainly due to the low incidence of CHF. Among the 20 patients who received EPI at more than 1,000 mg/m2, the prevalence of CHF was 35% and the sensitivity only 64%. The specificity was maximally 62%. Our results suggest that LVEF is of no value as a predictor for CHF.

Abnormal oesophageal function in patients with non-toxic goiter or enlarged left atrium, demonstrated by radionuclide transit measurements.

In 10 patients with enlarged atrium and in 29 patients with goiters and neck discomfort dynamic oesophageal scintigraphy was performed. The passage of water and capsules, containing 99mTc-pertechnetate, was studied with the patients in the supine and in the sitting positions. As a reference group we examined 35 healthy, age-matched volunteers. Mean transit time (MTT) was calculated, residual activity was expressed as a percentage of maximum activity, and the number of spikes in the curves was defined by visual analysis. Both in patients with enlarged left atrium and in those with large goiters the studies showed significantly prolonged MTT, increased residual activity, and a higher frequency of spikes, compared with healthy volunteers and with patients with small goiters. There was no relationship between symptoms and abnormal scintigraphic results. The passage of capsules was impaired only in cardiac patients. It is concluded that abnormal oesophageal function is often present in patients with enlarged left atrium and in patients with large, but not with small, goiters. Inhibition of oesophageal transit appears to be dependent on mechanical compression, but the nature of oesophageal impairment may vary with the level of compression. The frequent complaints of neck sensations in patients with goiters are probably not of oesophageal origin.

Reproducibility in the analysis of multigated radionuclide studies of left ventricular ejection fraction.

The authors determined the reproducibility (the standard deviation [SD]) in the analysis of multigated radionuclide studies of left ventricular ejection fraction (LVEF). Radionuclide studies from a consecutive series of 38 patients suspected of ischemic heart disease were analyzed independently by four nuclear medicine physiologists and four laboratory technicians. Each study was analyzed three times by each of the observers. Based on the analyses of the eight observers, the SD could be estimated by the use of a variance component model for LVEF determinations calculated as the average of the analyses of an arbitrary number of observers making an arbitrary number of analyses. This study presents the SDs for LVEF determinations based on the analyses of one to five observers making one to five analyses each. The SD of a LVEF determination decreased from 3.96% to 2.98% when an observer increased his number of analyses from one to five. A more pronounced decrease in the SD from 3.96% to 1.77% was obtained when the LVEF determinations were based on the average of a single analysis made by one to five observers. However, when dealing with the difference between LVEF determinations from two studies, the highest reproducibility was obtained if the LVEF determinations at both studies were based on the analyses made by the same observer. No significant difference was found in the reproducibility of analyses made by nuclear medicine physicians and laboratory technicians. Our study revealed that to increase the reproducibility of LVEF determinations, special efforts should be made to standardize the outlining of the end-systolic region interest.

Persistent impairment of liver function caused by a pendulated accessory liver lobe.

A large pendulated accessory liver lobe and complete absence of the left lobe were discovered by liver scintigraphy and ultrasound scanning in a patient suspected of having an ovarian tumor. The patient had a history of fluctuating impaired liver function tests for many years, probably caused by intermittent torsion of the peduncle. Pedunculated accessory liver lobes are extremely rare and seldom diagnosed in vivo. Our patient seems to be the 11th published case, and the only 1 in which longstanding fluctuating impaired liver function tests have been observed.

Reliability of scintigraphic diagnosis of liver metastases.

Three experienced specialists in nuclear medicine independently read 52 liver scans for the presence or absence of metastases. The accuracy of the assessments was, for each of the observers and for the observers' agreed-upon assessments, determined against findings at laparotomy or autopsy. Overall accuracy, predictive values of a positive and negative test result, sensitivity, and specificity were 0.71-0.79, 0.80-0.90, 0.63-0.72, 0.63-0.77, and 0.77-0.91. The accuracy was not higher for the agreed-upon assessment than for each of the observers' assessments. The interobserver variation was calculated as overall agreement between pairs of observers and ranged from 0.85 to 0.94. After adjustment of overall agreement for chance agreement, kappa values from 0.70 to 0.88 were obtained. In the determination of intraobserver variation overall agreement rates and kappa values ranged from 0.90 to 0.94 and 0.84 to 0.88. The scintigraphic diagnosis of hepatic metastases is reliable but, to increase the reliability further, it should be examined whether observer error reflects error in the primary detection of findings in the scans or error in the overall interpretation.

Inherited coagulation disorders.

Inherited coagulation disorders have been diagnosed in many breeds of dogs as well as in mongrels and cats. This article presents the different coagulation factor deficiencies that are known to exist in small animals. A description is given of each coagulation factor along with the relevant clinical signs, inheritance, and the breeds affected. Suggestions are also given for the diagnosis and therapy of these deficiencies.

A study of hemophilia A in German shepherd dogs in Denmark.

Classical hemophilia, hemophilia A, is the most common coagulation disorder in dogs. This article presents a comprehensive study of this disease in German Shepherd dogs in Denmark. Emphasis is placed on pathophysiology, diagnosis, genetics, therapy, and prophylaxis. Furthermore, a scheme for a rational elimination of the causative gene from the population is presented.