Search

The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of an LDL receptor mutation (W556S) found in FH patients and characterised as transport defective. In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-localisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum. Wild type (WT) and W556S LDL receptor GFP fusion proteins were expressed in mouse liver by means of hydrodynamic delivery of naked DNA. Two days after injection liver samples were analysed for GFP fluorescence. The WT LDL receptor GFP protein was located on the cell surface whereas the W556S LDL receptor GFP protein was retained in intracellular compartments. Thus, the GFP-tagged LDL receptor protein allows both detailed time lapse analysis and evaluations in animals for the physiological modelling of mutations. This method should be generally applicable in functional testing of gene products for aberrant processing.

Objective: To determine the effects of porcine mammary secretions on polymorphonuclear (PMN) leukocyte function and to relate concentrations of estradiol-17beta and cortisol in mammary secretions to PMN cell function., Sample Population: Mammary secretions from 10 healthy sows and blood PMN leukocytes from 27 healthy sows., Procedure: Mammary secretions were collected within 24 hours after parturition (colostrum) and 12 to 13 days later (milk). Chemoattractant properties were assessed by use of a cell migration assay. Phagocytic capacity of PMN cells in colostrum and milk was assessed by recording chemiluminescence following phagocytosis of Escherichia coli or zymosan. Estradiol-17beta and cortisol concentrations were determined by use of radioimmunoassays., Results: Chemoattractant properties of colostrum and milk were significantly greater than that of zymosan-activated serum. However, chemoattractant properties did not differ significantly between the 2 types of secretions. The capacity of PMN cells in colostrum to phagocytose either zymosan or E. coli was less, compared with cells in milk, and the ability of cells in either type of mammary secretion to phagocytose E. coli was greater than the ability to phagocytose zymosan. Concentrations of estradiol-17beta and cortisol were greater in colostrum, compared with milk. No clear relation was evident between PMN cell activity and hormone concentrations in mammary secretions., Conclusions and Clinical Relevance: Although chemoattractant properties of colostrum and milk did not differ, the phagocytic capacity of PMN cells in colostrum was significantly less than that of cells in milk. This may predispose sows to coliform mastitis during the early postparturient period.

Objectives: To describe the use of antiasthma drugs among the study patients and to evaluate whether therapeutic outcomes monitoring (TOM) is associated with improved quality of drug therapy., Design: Prospective, controlled, multicenter study. Consumption of antiasthma medications was measured as the number of defined daily doses (DDDs) purchased. Data were collected from the pharmacies' computer systems for a period beginning 6 months before the start of the study (period 1) and during its first and second half-years (periods 2 and 3). Treatment changes for TOM patients were classified on the basis of drug regimens at periods 1 and 3., Setting: Community pharmacies in Denmark (16 intervention, 15 control)., Patients: Five hundred patients with asthma aged 16 to 60 years who were being treated in primary health care; this study used data from 350 patients from this sample., Intervention: TOM., Main Outcome Measures: Changes in the use of individual drugs and changes in therapeutic patterns--distribution of purchased drugs; proportion of corticosteroid users; frequency of drug regimens used; treatment changes for TOM patients., Results: TOM patients' consumption of beta2-agonists decreased by 12% overall from period 1 through period 3, while control patients' consumption of these medications decreased by only 1%. TOM patients' use of inhaled corticosteroids increased by more than 50% compared with 9% among controls. In both groups, about one-half of all purchased DDDs were for inhaled beta2-agonists. The proportion of inhaled corticosteroids increased from 27% to 42% of total DDDs for the TOM group and remained constant for controls. Of patients using beta2-agonists, 68% also used inhaled steroids initially in both the TOM and control groups. The proportion of inhaled steroid users in the TOM group increased to 84%, and to 70% among controls. The most common regimen was inhaled short-acting beta2-agonists and corticosteroids in combination, and the second most common regimen was monotherapy with short-acting beta2-agonists. With time, the regimens changed more toward consensus guidelines among TOM patients. Changes in drug therapy totaled 451, averaging 2.4 changes per TOM patient. The largest number of changes (49%) involved inhaled corticosteroids., Conclusion: Changes in medication use among TOM patients were toward improved asthma treatment. Our results show that community pharmacists, physicians, and patients, working together, can improve prescribing, solve drug therapy problems, and improve outcomes for patients with moderate-to-severe asthma.

The purpose of this study was to evaluate a new automated method for the interpretation of lung perfusion scintigrams using patients from a hospital other than that where the method was developed, and then to compare the performance of the technique against that of experienced physicians. A total of 1,087 scintigrams from patients with suspected pulmonary embolism comprised the training group. The test group consisted of scintigrams from 140 patients collected in a hospital different to that from which the training group had been drawn. An artificial neural network was trained using 18 automatically obtained features from each set of perfusion scintigrams. The image processing techniques included alignment to templates, construction of quotient images based on the perfusion/template images, and finally calculation of features describing segmental perfusion defects in the quotient images. The templates represented lungs of normal size and shape without any pathological changes. The performance of the neural network was compared with that of three experienced physicians who read the same test scintigrams according to the modified PIOPED criteria using, in addition to perfusion images, ventilation images when available and chest radiographs for all patients. Performances were measured as area under the receiver operating characteristic curve. The performance of the neural network evaluated in the test group was 0.88 (95% confidence limits 0.81-0.94). The performance of the three experienced experts was in the range 0.87-0.93 when using the perfusion images, chest radiographs and ventilation images when available. Perfusion scintigrams can be interpreted regarding the diagnosis of pulmonary embolism by the use of an automated method also in a hospital other than that where it was developed. The performance of this method is similar to that of experienced physicians even though the physicians, in addition to perfusion images, also had access to ventilation images for most patients and chest radiographs for all patients. These results show the high potential for the method as a clinical decision support system.

Stage of disease at diagnosis is a predictor of breast cancer survival. We used data from the Danish Cancer Register and the Danish Breast Cancer Cooperative Group to study stage distribution in 0-69-year-old Danish breast cancer patients diagnosed in 1978-1994. We constructed a modified Nottingham Prognostic Index (NPI) calculated from the number of excised and positive lymph nodes, malignancy grade and tumour diameter. This NPI could be calculated for 63% of the patients, and among these the stage distribution improved during the study period. The proportion of patients with a poor prognostic score decreased from 27% to 20%. Based on a comparison of the crude 3-year survival of patients with an NPI score and those without, it seems probable that the stage of disease at diagnosis on average improved in Danish breast cancer patients below age 70 during the 1980s and the early 1990s.

A Finite Element (FE) model of isolated head and neck complex was developed aiming to investigate the mechanisms of injury from axial impacts, in the sagittal plane, and the injury thresholds from experimental studies reported in the literature. The model was validated on a local and a global level, showing a significant correlation with experimental investigations and thereby having the potential to predict both reported injuries and dynamic buckling modes. The frequently reported Hangmans' fracture was predicted to occur at an axial load of about 3.5 kN and at a local injury threshold of 191 MPa in the compact bone of C2. Also, when analyzing an experimentally designed inner roof of a vehicle, the FE model showed that an induced anterior translation of the head reduced both stress and forces of the cervical spine bone. Moreover, the recent FE model suggests that combined compression/flexion may result in less severe injuries compared to pure compression or compression extension.

The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL receptors are unknown. In the present study we show that the molecular chaperone Grp78/BiP co-immunoprecipitates with both the wild type and two different mutant (W556S and C646Y) LDL receptors in lysates obtained from human liver cells overexpressing wild type or mutant LDL receptors. A pulse-chase study shows that the interaction between the wild type LDL receptor and Grp78 is no longer detectable after 2(1/2) h, whereas it persists for more than 4 h with the mutant receptors. Furthermore, about five times more Grp78 is co-immunoprecipitated with the mutant receptors than with the wild type receptor suggesting that Grp78 is involved in retention of mutant LDL receptors in the ER. Overexpression of Grp78 causes no major alterations on the steady state level of active LDL receptors at the cell surface. However, overexpression of Grp78 decreases the processing rate of newly synthesized wild type LDL receptors. This indicates that the Grp78 interaction is a rate-limiting step in the maturation of the wild type LDL receptor and that Grp78 may be an important factor in the quality control of newly synthesized LDL receptors.

Objective: Clinical databases have been invented to monitor treatment outcomes, therapies or diseases, often in great detail. The traditional population-based cancer registry has been invented to collect a minimum of information about all incident cancers. Do clinical databases render population-based cancer registers obsolete as sources of cancer cases for epidemiological study?, Methods: We compared the study base of first incident breast cancer cases in Denmark in 1978-1994 known from the national cancer register and from the national clinical database on breast cancer patients. The clinical database is used for monitoring protocoled treatment., Results: Combining the two data sources we found 48,522 first primary breast cancers in Denmark 1978-1994. Of these, 37,640 were included in both data sources, 2151 were included only in the clinical database, and 8731 were included only in the cancer register. A major part of the difference between the two data sources was due to treatment-focused data collection in the clinical database, and a minor part due to differences in the registration of second primaries, date of diagnosis and invasiveness., Conclusions: Cancer incidence data are sensitive to registration procedures and definitions. Clinical cancer databases cannot generally replace the traditional cancer register as a reliable data source for incident cancer cases in a national population.

The purpose of this study was to develop a completely automated method for the interpretation of ventilation-perfusion (V-P) lung scintigrams used in the diagnosis of pulmonary embolism. An artificial neural network was trained for the diagnosis of pulmonary embolism using 18 automatically obtained features from each set of V-P scintigrams. The techniques used to process the images included their alignment to templates, the construction of quotient images based on the ventilation and perfusion images, and the calculation of measures describing V-P mismatches in the quotient images. The templates represented lungs of normal size and shape without any pathological changes. Images that could not be properly aligned to the templates were detected and excluded automatically. After exclusion of those V-P scintigrams not properly aligned to the templates, 478 V-P scintigrams remained in a training group of consecutive patients with suspected pulmonary embolism, and a further 87 V-P scintigrams formed a separate test group comprising patients who had undergone pulmonary angiography. The performance of the neural network, measured as the area under the receiver operating characteristic curve, was 0.87 (95% confidence limits 0.82-0.92) in the training group and 0.79 (0.69-0.88) in the test group. It is concluded that a completely automated method can be used for the interpretation of V-P scintigrams. The performance of this method is similar to others previously presented, whereby features were extracted manually.

Aims: The present study analyzed the effects of Saccharomyces boulardii on the biochemical parameters of microbial hindgut metabolism disturbed by clindamycin., Methods: The experiments were carried out under in vitro conditions using the semicontinuous colon-simulation technique. This technique is standardized for quantitatively measuring parameters of microbial hindgut metabolism. The fluid and particle phase of pig hindgut contents were used for the in vitro incubations. The 5-day control period was followed by clindamycin exposure alone (312.5 mg/day for 5 days) or by a combined treatment of clindamycin and S. boulardii (400 mg/day for 5 days)., Results: Clindamycin resulted in significant decreases in production rates of short-chain fatty acids (SCFAs) which were associated with substantial changes in molar SCFA proportions at the expense of butyrate. These effects could at least partly be compensated for by S. boulardii, in particular by enhancements of acetate and propionate fermentation to control levels. In contrast, butyrate fermentation could not be reconstituted. In a second experiment the potential use of S. boulardii as a substrate for hindgut microbial metabolism was studied by comparing living and autoclaved yeast. Propionate and butyrate fermentation rates were unaffected whereas acetate fermentation tended to be higher in the presence of living yeast., Conclusions: S. boulardii can be effective to compensate for changes in microbial fermentation in response to antibiotic treatment. Despite the lack of statistical significance it might be concluded that the increase in fermentation end products can only partly be explained by the utilization of the yeast as a substrate for microbial metabolism., (Copyright 2000 S. Karger AG, Basel)

In the present study, we have characterized a unique splice donor G to A substitution in the moderately conserved + 5 position in intron 10 of the low-density lipoprotein (LDL) receptor gene. In two Danish families, carriers of the 1592 + 5G --> A mutation display a clinical phenotype ranging from healthy normocholesterolemic persons to classical heterozygous familial hypercholesterolemia (FH) patients. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA from Epstein Barr virus (EBV)-transformed lymphoblasts obtained from members of both families demonstrated abnormal splicing generating two aberrant mRNAs due to either alternative splicing and skipping of exon 10 or activation of a cryptic splice site in intron 10 inserting 66 intronic base pairs. These abnormally spliced mRNAs were predicted to encode two abnormal receptor proteins containing an in-frame deletion of 75 amino acids and an insertion of 22 novel amino acids, respectively. Results obtained by immunofluorescence staining, flow cytometry, and confocal microscopy of transfected Chang and COS-7 cells expressing normal and mutant LDL receptors were compatible with nearly complete retention of the mutant proteins in the endoplasmic reticulum. Quantitative measurements of LDL receptor mRNAs from EBV-transformed lymphoblasts, however, did not reveal any significant differences in variant mRNA contents between mutation carriers in the families that could be related to degree of hypercholesterolemia.

Computer-aided interpretation of electrocardiograms (ECGs) is widespread but many physicians hesitate to rely on the computer, because the advice is presented without information about the confidence of the advice. The purpose of this work was to develop a method to validate the advice of a computer by estimating the error of an artificial neural network output. A total of 1249 ECGs, recorded with computerized electrocardiographs, on patients who had undergone diagnostic cardiac catheterization were studied. The material consisted of two groups, 414 patients with and 835 without anterior myocardial infarction. The material was randomly divided into three data sets. The first set was used to train an artificial neural network for the diagnosis of anterior infarction. The second data set was used to calculate the error of the network outputs. The last data set was used to test the network performance and to estimate the error of the network outputs. The performance of the neural network, measured as the area under the receiver operating characteristic (ROC) curve, was 0.887 (0.845-0.922). The 25% test ECGs with the lowest error estimates had an area under the ROC curve as high as 0.995 (0.982-1.000), i.e. almost all of these ECGs were correctly classified. Neural networks can therefore be trained to diagnose myocardial infarction and to signal when the advice is given with great confidence or when it should be considered more carefully. This method increases the possibility that artificial neural networks will be accepted as reliable decision support systems in clinical practice.

Altered glycosylation is a common feature in tumors of various kind and particular interest has been focused on the expression of tumor-associated gangliosides. We have previously identified some human glioma-associated gangliosides and in this study yet another, not previously described, ganglioside has been isolated. The ganglioside was prepared from human glioma tissue taken at autopsy. The new ganglioside bound cholera-toxin B-subunit and its structure was confirmed by fast atom bombardment-mass spectrometry to be NeuN-GM1 (II3NeuNH2-GgOse4Cer). In the dissected tumor specimen, the concentration of NeuN-GM1 was 0.1 micromol/g wet weight and accounted for approximately 20% of the monosialoganglioside fraction. Normal human brain tissue specimens (n = 10) did not contain detectable (>0.5 nmol/g wet weight of tissue) amounts of NeuN-GM1, indicating that this ganglioside might be associated with human glioma. However, none of the 17 other tumour specimens reveal any detectable amounts of this ganglioside. In conclusion, NeuN GM1 is a glioma-associated ganglioside but its exceptional expression limits its relevance as a molecule involved in general tumor biology.

The aim of the study was to define possible differences between selenite, selenate and selenium yeast on various aspects of selenium status in growing cattle. Twenty-four Swedish Red and White dairy heifers were fed no supplementary selenium for 6 months. The basic diet contained 0.026 mg selenium/kg feed dry matter (DM). After the depletion period the animals were divided into 4 groups; group I-III received 2 mg additional selenium daily as sodium selenite, sodium selenate, and a selenium yeast product, respectively. Group IV, the control group, received no additional selenium. The total dietary selenium content for groups I-III during the supplementation period was 0.25 mg/kg DM. After the depletion period the mean concentration of selenium in blood (640 nmol/l) and plasma (299 nmol/l) and the activity of GSH-Px in erythrocytes (610 mukat/l) were marginal, but after 3 months of supplementation they were adequate in all 3 groups. The concentration of selenium in blood and plasma was significantly higher in group III than in groups I and II, but there was no significant difference between groups I and II. The activity of GSH-Px in erythrocytes did not differ between any of the supplemented groups. The animals in the control group had significantly lower concentrations of selenium in blood and plasma and lower activities of GSH-Px in erythrocytes than those in the supplemented groups. The activity of GSH-Px in platelets was also increased by the increased selenium intake. There was no difference in the concentration of triiodothyronine (T3) between any of the groups, but the concentration of thyroxine (T4) was significantly higher in the unsupplemented control group.

The incidence of congenital clubfoot in Frederiksborg County, Denmark, was studied over a period of 16 years (1979-1994). Altogether 60, 186 live infants were born, and of these 72 had a congenital clubfoot. Twenty-five children (35%) had bilateral clubfoot and 54 (75%) were boys. The overall incidence was 1.20 per thousand children. The incidence increased significantly during the observation period and was 2.41 per thousand in 1994. We cannot explain the rise in incidence.

The objective was to study the effect of storage of blood samples for up to 4.5 h at either room temperature or on ice on granulocyte phagocytosis as assessed by chemiluminscence. The study included three groups of animals: eight pigs, seven cows and eight horses, which were bled at one occasion. The blood samples were divided into two and stored either at room temperature or on ice. Granulocyte phagocytosis in the samples was then assessed every 60 min starting 30 min and finishing 4.5 h after bleeding. The phagocytosis was recorded as the AUC of chemiluminescence following phagocytosis of opsonized zymosan particles. The time of storage of blood samples had a significant (P < 0.001) overall effect on the chemiluminscence following phagocytosis in all species tested, as was the case with storage temperature (P < 0.001). It should be noted, however, that there were considerable species differences in the effect of the storage on the granulocyte phagocytosis. Hence, for valid comparisons of data on granulocyte phagocytosis assessed by chemiluminiscence it is important that the handling procedures of the blood samples have been applied consistently even if the assays have been performed only a few hours after blood collection.

We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease [Bross, Andresen, Knudsen, Kruse and Gregersen (1995) FEBS Lett. 377, 249-252]. In the present study, synthesis, intracellular processing and subcellular localization of hClpP have been analysed in intact cells and in a cell-free system. Using pulse-labelling/immunoprecipitation of Chang cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx. 39 and 37 kDa. A pulse-chase experiment showed that these bands were converted into one mature-enzyme band with a molecular mass of approx. 32 kDa that was stable for at least 24 h. The 37 kDa band co-migrated with a band produced upon expression of full-length hClpP in E. coli, and the 32 kDa band co-migrated with the product of E. coli-expressed hClpP in which the 56 N-terminal residues had been deleted, indicating that the 37 kDa moiety represents the precursor and that approx. 56 residues are cleaved off during maturation. The processing of hClpP in intact cells was dependent on mitochondrial membrane potential. These results were confirmed in an import assay system using in vitro transcription and translation directed by the hClpP cDNA and isolated rat liver mitochondria. No protease activity towards a series of fluorogenic peptides could be observed in extracts of Chang cells overexpressing hClpP, indicating that the protease may not be active without co-factors. Immunofluorescence studies using confocal-laser-scanning microscopy showed co-localization of the hClpP and the mitochondrially located Hsp60 (heat-shock protein 60). Taken together, the results reported here show that hClpP is localized inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochondrial proteins.

The purpose of this study was to explore the feasibility of developing artificial neural networks that are able to provide confidence measures for their diagnostic advice. Computer-aided decision making can improve physician performance, but many physicians hesitate to use these 'black boxes'. If we are to rely upon decision support systems for such tasks as medical diagnosis it is essential that the computers indicate when the advice given is based on experience, i.e. give a confidence measure. An artificial neural network was trained to diagnose healed anterior myocardial infarction and to indicate 'lack of experience' when test electrocardiograms were different from the electrocardiograms of the training set. A database of 1249 electrocardiograms from patients who had undergone cardiac catheterization was used to train and test the neural network. Thereafter, the ability of the network to indicate 'lack of experience' was assessed using 100 left bundle branch block electrocardiograms, an electrocardiographic pattern that was excluded from the training set. The network indicated that 83% of the left bundle branch block electrocardiograms and 1% of the test electrocardiograms from catheterized patients were different from the electrocardiograms of the training set. All but one of the left bundle branch block electrocardiograms would otherwise be falsely classified as anterior myocardial infarction by the network. Artificial neural networks can be trained to indicate 'lack of experience', and this ability increases the possibility for neural networks to be accepted as reliable decision support systems in clinical practice.

Experimental data indicate that certain microbiological degradation experiments have a limited reproducibility. Nine identical batch experiments were carried out on 3 different days to examine reproducibility. A pure culture, isolated from soil, grew with toluene as the only carbon and energy source. Toluene was degraded under aerobic conditions at a constant temperature of 28 degrees C. The experiments were modelled by a Monod model--extended to meet the air/liquid system, and the parameter values were estimated using a statistical nonlinear estimation procedure. Model reduction analysis resulted in a simpler model without the biomass decay term. In order to test for model reduction and reproducibility of parameter estimates, a likelihood ratio test was employed. The limited reproducibility for these experiments implied that all 9 batch experiments could not be described by the same set of parameter values. However, experiments carried out the same day (within the same run) were more uniform than experiments carried out on different days (between runs), and a common set of parameter estimates could be accepted for experiments within runs, but not for experiments from different runs. The limited reproducibility may be caused by variability in the preculture, or more precisely, variations in the physiological state of the bacteria in the precultures just before used as inoculum.

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia-containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia-containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

The study comprises observations in 301 Swedish Yorkshire primiparous sows belonging to a research herd. During the 6 weeks of lactation, they were fed according to a conventional feeding regime based on litter size. The sows were weighed at farrowing and at weaning. After weaning the sows were checked daily for standing reflex and blood samples for determination of plasma progesterone were drawn regularly. Diseases occurring during lactation were recorded in 223 sows. A clinical health examination of these sows was also performed on the day of weaning, including body temperature, condition scoring, appetite, locomotor disorders, remarks on legs and mammary glands. A blood sample was drawn 1 week before weaning in 155 sows for analysis of blood haemoglobin concentration, packed cell volume, total white blood cell count, serum concentration of urea, creatinine, triglycerides, free fatty acids, total protein and electrophoretic separation of proteins. Sows with large weight loss had a higher total incidence of diseases during lactation, mastitis being the most common disease, than sows with small weight loss. Sows with large weight loss seemed to be more catabolic during late lactation than sows with small weight loss, according to the blood analyses. The blood analyses did not show any signs of subclinical infection being more common among sows with large weight loss than among sows with small weight loss. There were neither any significant differences in the total incidence of diseases during lactation nor in the health at weaning between sows returning to oestrus within 10 days of weaning and sows not returning within this time.

Glioma-associated gangliosides, in particular the expression of foetal gangliosides 3'-isoLM1 and 3'6'-isoLD1, have been investigated in biopsies from 44 patients with astrocytoma grade II, anaplastic astrocytoma, anaplastic oligodendrogliomas, oligodendrogliomas, and glioblastoma multiforme. The total ganglioside content decreased in proportion to the estimated number of tumour cells present in the biopsy. Ganglioside GD3 was increased in 17 of the tumour tissues and in 6 of the surrounding area specimens. In agreement with our previous studies, tumour specimens contained the lactoseries ganglioside 3'-isoLM1 and, as demonstrated for the first time, the disialylated form of the ganglioside, 3'6'-isoLD1. These gangliosides are in normal brain tissue restricted to the developmental period. Most frequent was the expression of ganglioside 3'-isoLM1 which was detected in 92% of the tumour specimens in concentrations varying between just detectable amounts to 13 nmol/g wet weight. In the area surrounding the macroscopic tumour tissue 78% of the specimens expressed 3'-isoLM1 and the values varied between just detectable amounts to 24 nmol/g wet weight. Ganglioside 3'6'-isoLD1 was found in 47% of the tumour biopsies and in only 17% of the specimens taken outside macroscopic tumour tissue. The values varied between non-detectable amounts to 40 nmol/g wet weight of tissue. The expression of 3'-isoLM1 correlated significantly (p < 0.01) to malignancy grade. For 3'6'-isoLD1 a significant (p < 0.05) difference was found between tumour and surrounding tissue. Likewise, 3'6'-isoLD1 correlated significantly (p < 0.05) to malignancy grade. The correlation between 3'-isoLM1 and 3'6'-isoLD1 to malignancy grade and the frequent expression of these gangliosides both in the tumour itself and in its surrounding area should encourage additional studies concerning their biological role in tumour disease.

Increased metabolic activity represented by an increase in both anabolism and catabolism in tumours, including gliomas, is a well known phenomenon and utilised in positron emission tomography imaging of tumours. In this study lysosomal enzyme activities of some glycohydrolases were investigated in glioma tissue from human brain. Tumour tissue (ten cases) and brain tissue surrounding the tumour tissue (seven cases) from patients with a histopathological diagnosis of glioblastoma multiforme or anaplastic astrocytoma were analysed for activity of the lysosomal enzymes galactosylceramidase, glucosylceramidase, beta-galactosidase, beta-N-acetyl-glucosaminidase, beta-glucuronidase and acid phosphatase. All of the investigated lysosomal enzymes except galactosylceramidase showed increased activity compared with that in normal brain tissue. Moreover, despite sparsity of tumour cells the specimens taken from surrounding areas showed elevated activities of the same enzymes. The findings indicate an upregulation of the activity not only in tumour but also in normal cells of the surrounding area.

The aim of the present study was to examine the glial cell response and the possible involvement of the complement cascade following a cerebral cortical contusion. The lesion was produced using a standardized weight-drop technique in adult rats. The blood-brain barrier was damaged, as demonstrated by a decrease of immunoreactivity for a tight junction protein normally expressed by endothelial cells of small vessels in the central nervous system. Increased immunoreactivity for microglial (OX42) and astroglial cells (glial fibrillary acidic protein), as well as macrophages expressing ED1-immunoreactivity (IR) were found in the vicinity of the lesion at all postoperative survival times (2-14 days). In the present study complement factor C3d- and C9-IR was found around the lesion, indicating that activation of the complement cascade had taken place. Furthermore, immunoreactivity for the putative complement inhibitor clusterin (sulfated glycoprotein-2) was found in some of the injured neurons. The contralateral hemisphere showed no evidence of the reaction found in the ipsilateral hemisphere. The balance between complement activation and complement inhibitors may have an impact on the degenerative components in the brain following traumatic injury and in particular on the events leading to nerve cell death.

Interleukin-1 (IL-1) is a key mediator in inflammatory processes. There are several immunoassays for human, but not porcine, IL-1 commercially available. Four of these were tested on porcine specimens. IL-1 was not detected, by any of the immunoassays, in cell culture supernatants with confirmed IL-1 activity in a bioassay. The results indicate that the tested antibodies to human IL-1 do not crossreact with porcine IL-1.

The insulin-like growth factor (IGF) system has a role in repair following hypoxic-ischemic injury in many tissues including the brain. To study the involvement of the IGF system following head trauma, we used a rat contusion model, which produces a focal lesion of the cerebral cortex. Molecules in the IGF system were analyzed using in situ hybridization at different times following impact. We observed a dramatic up-regulation of insulin-like growth factor binding protein-2 (IGFBP-2) mRNA in cortical areas adjacent to the injury 24 h after impact, with a peak 10-fold increase engaging most of the ipsilateral cortex 2 and 3 days post-contusion. Seven days after the contusion, IGFBP-2 expression was only moderately up-regulated and again concentrated around the injury. IGFBP-4 mRNA levels increased 4-fold ipsilateral to the site of injury, with retained pattern of cortical expression. IGFBP-3, IGFBP-5 and IGFBP-6 mRNA all displayed distinct expression patterns in the brain but no significant changes were observed following injury. In contrast, IGF-1 mRNA levels were very low prior to contusion, but increased markedly at the site of injury with a peak at day 3. We were unable to detect any changes in the type 1 IGF-receptor or IGF-2 mRNA following contusion. The neuropeptide cholecystokinin (CCK) mRNA was clearly up-regulated following contusion, with an even distribution over the ipsilateral cortex. The expression pattern of molecules in the IGF system post-contusion differs in part to changes observed following hypoxic-ischemia or ischemia alone, perhaps reflecting different regulatory mechanisms depending on the type of injury.

Saccharomyces boulardii (S.b.) is used for the prevention and treatment of diarrhea of different etiologies. We prospectively investigated the effects of S.b. on lymphocytes and duodenal mucosa. Before and after oral administration of S.b. for 3 weeks, circulating and intestinal lymphocytes were isolated and characterized by flow cytometry. Trophic effects on duodenal mucosa were investigated by morphometry and determination of brush border enzyme activity. Results were compared intraindividually before and after S.b. In intestinal lymphocytes no phenotypic changes were observed. CD4+ cells of the peripheral blood had a significantly increased expression of CD25 (p < 0.02). None of twelve volunteers had an increase in villous surface area (n.s.). Immunoglobulin A content in small intestine secretion was unaltered. An increase in brush border enzyme activity of lactase, alpha-glucosidase, and alkaline phosphatase was observed (p < 0.01). Our findings indicate that S.b. has a positive effect on the maturation of enterocytes and only a minor influence on lymphocytes.

Between 1968 and 1988, 2298 head-injured patients of all grades of severity were registered in the data bank of a single clinic. The majority of patients were admitted to a community hospital and transferred later to the neurosurgical clinic. The data included mechanism of injury and clinical status at admission, including the level of consciousness according to the Glasgow Coma Score (GCS) before and after resuscitation. After admission, nearly all patients below a score of 8 were intubated and treated with controlled ventilation until the patient awakened, died, or had remained in a steady state for approximately 3 weeks. Assessment of the final outcome was made according to the Glasgow Outcome Scale (GOS) both at discharge and again after the patient's status was stabilized. The results show a stable yearly outcome during the 21 years of recording but a significantly improved good recovery and mortality rate when compared with our previous results. Outcome was significantly correlated to age and type and severity of lesion, as judged by the postresuscitation GCS. The outcome of the 1264 most severely injured, comatose patients (GCS < 9) shows a good recovery rate of 55%, a severely disabled rate of 14%, a vegetative rate of 7%, and a mortality rate of 24%. We attribute these results, which compare favorably with others, to prompt airway control and controlled ventilation in unconscious patients.

An increased referral of patients with anticoagulation related haemorrhages necessitated an analysis of causes and outcome of these complications in a patient group that reflected Swedish therapeutic traditions of anticoagulation treatment. Prospectively, all patients from Stockholm evaluated for warfarin related intracranial haemorrhage occurring during 1987 were analysed and their 6 month outcome recorded. Sixty-eight patients were included. The results of intracranial haemorrhagic complications were catastrophic with a 77% mortality rate. Their incidence was much higher than expected. Forty-one patients had non-traumatic intracerebral haematomas, 1 had a non-traumatic subarachnoid haemorrhage and 26 had traumatic injuries. In the 42 patients with non-traumatic haemorrhages, the indications for anticoagulation were cerebral ischaemic events in a majority (27/42). The remaining 15 patients had different indications for anticoagulation. They also had an increased frequency of hypertension (p < 0.05). In the 26 patients with traumatic haematomas, only 6/26 patients had previous cerebral ischaemic injuries (p < 0.01). Valvular heart prosthesis was their most common indication (11/26) for anticoagulation. Caution in instituting anticoagulation therapy in patients with hypertension or cerebrovascular disease, which is an important indication for anticoagulation in Sweden, is mandatory. Adherence to strict treatment regimens and their continuous reevaluation may help to avoid complications. The finding of more patients than expected with haemorrhagic complications is not compatible with the risk evaluations used to justify anticoagulation therapy in the patient groups studied. Clinical practice must have changed with time, showing that risk evaluations from controlled trials or retrospectively collected clinical data from selected patients are not necessarily applicable for long-term clinical practice.

The effect of mimicking prepartum concentration of estradiol-17 beta on the inflammatory response to endotoxin in gilts was studied. The study was performed in a split-litter design and comprised 5 pairs of littermates. A catheter was inserted into the jugular vein 2 days prior to the start of the study. In each pair, 1 littermate was treated IM with 2.5 mg of estradiol-17 beta/75 kg of body weight, and the other littermate was given peanut oil IM as a control. The day after treatment, all gilts were challenge-exposed with a Salmonella typhimurium-derived endotoxin (1 microgram/kg, IV) and the inflammatory response to challenge exposure was monitored. There was no effect of estradiol treatment on the transient clinical signs of endotoxemia or on the increase in rectal temperature. The increase in blood concentrations of prostaglandin F2 alpha metabolite and cortisol after endotoxin challenge exposure was not affected by estradiol. Decrease in number of circulating blood mononuclear cells and polymorphonuclear leukocytes was not changed by estradiol treatment. Taken together, mimicking prepartum concentration of estradiol did not affect either the magnitude or the kinetics of the inflammatory response to endotoxin in gilts. Relevance of these findings to development of endotoxin-mediated diseases, such as the postpartum agalactia syndrome, needs further study.

The effect of oral intake of endotoxin (ET) combined with inhalation of ET-contaminated feed-dust was studied in 6 prepubertal gilts. The animals were given 100 mg of ET each in their ordinary feed ration and meanwhile stayed for 4 h in a continuous flow exposure chamber where the endotoxin concentration was approximately 9 micrograms/m3. Blood samples were collected during the exposure and for 8 h afterwards. Bronchoalveolar lavage (BAL) was performed 24-26 h after the exposure. Six additional animals received ordinary feed and were exposed to feed-dust without added ET. Blood samples were analysed for total white blood cell count (B-WBC) and differential count: polymorphonuclear (PMN) and mononuclear (MN); total red blood cell count (B-RBC); platelets (B-PLT); total serum bile acids (S-BA); glutamate dehydrogenase (S-GLDH); and a blood plasma prostaglandin F2 alpha metabolite (15-ketodihydro-PGF2 alpha; P-PG). The major findings were decreased numbers of MN, PMN and B-RBC 3 to 6 h after the start of the ET exposure and increased numbers of neutrophilic granulocytes in the BAL fluid of the ET-exposed animals. It is concluded that a combined oral and airborne ET challenge induces hematological and cytological changes that may contribute to the development of respiratory disorders.

Insulin-like growth factor 1 (IGF-1) is involved in the regulation of brain development and has been suggested as an autocrine stimulator of brain tumor cell proliferation. This study demonstrates the expression of IGF-1 in tumor tissue from human gliomas and one esthesioneuroblastoma. Using immunohistochemistry, expression of an IGF-1-like peptide was localized in tumor cells of 6 of the 9 gliomas examined as well as the esthesioneuroblastoma. From one anaplastic oligodendroglioma (which showed strong IGF-1 immunostaining) the IGF-1 transcripts were characterized after isolation of mRNA followed by amplification using the reverse transcriptase-polymerase chain reaction. Two IGF-1 complementary DNAs resulting from alternative splicing of the IGF-1 primary transcript were identified. These transcripts encode two different precursor proteins which correspond to Ea IGF-1 and Eb IGF-1. The significance of IGF-1 alternative mRNA splicing pathways remains to be determined.

D-Glucosamine is a structural unit of many biologically interesting macromolecules. To investigate the feasibility of using labelled D-glucosamine as a tracer for anabolic processes, a two-step synthetic procedure for specifically labelling D-glucosamine in position 1 with carbon-11 was developed. [11C]Cyanide was reacted with an imine precursor, N-benzyl-D-arabinosylamine, to generate the [1-11]alpha-amino nitrile. Reduction to [1-11C]D-glucosamine was accomplished by catalytic hydrogenation using PdCl2 and the N-benzyl group was simultaneously removed. The total synthesis time from end-of-trapping of [11C]cyanide was 40-45 min and the decay-corrected radiochemical yield was 5-10% after HPLC isolation. The biodistribution of [1-11C]D-glucosamine in rat following i.v. bolus injection was investigated using positron emission tomography and showed that the availability of this substance for CNS anabolism is low with the primary limitation being the intact blood-brain barrier.

Sixteen patients treated between 1969 and 1989 for a colloid cyst of the foramen of Monro by stereotactically guided aspiration (not stereotactic extirpation) were evaluated to assess the long-term outcome of the procedure. Thirteen of these patients required reoperation due to an acute comatose state, failure to achieve permanent reduction of the cyst, or symptomatic hydrocephalus. Of these 13, six were treated twice and two were treated three times by stereotactic aspiration. Five patients underwent microsurgical extirpation and three had a shunt placed following a failed aspiration. Failure of the first procedure was detected within the first 2 months after treatment in eight patients and after more than 6 years in seven. Following stereotactic aspiration, three patients experienced a temporary memory deficit and confusion and one patient suffered a central pain syndrome. Eleven of the 26 procedures were followed by a recurrence 6 to 15 years after treatment; seven recurrent cysts were detected after more than 8 years. Of the patients with recurrences, three did not undergo repeat surgery but showed an increase in cyst size at the latest follow-up examination. It is suggested that radical removal by open or stereotactically guided microsurgery is the method of choice since stereotactic aspiration fails to offer a radical or permanent treatment for colloid cysts of the third ventricle.

Serum and cerebrospinal fluid (CSF) samples from 12 patients were analyzed for interleukin (IL)-6, soluble IL-2 receptor (IL-2R), and soluble CD8 levels in order to determine the immune activation profile following subarachnoid hemorrhage (SAH). Dramatically increased levels of IL-6 and moderate increases of soluble IL-2R were detected in the CSF in 11 of the 12 patients; slightly elevated levels of soluble CD8 were observed in six patients. The IL-6 levels were higher on Day 6 than on Days 3 and 9. The increases in IL-6, soluble IL-2R, and soluble CD8 levels in the CSF samples were not paralleled by increased values in the serum samples, and thus probably reflected an intrathecal synthesis of the cytokine. Passive transfer of IL-6 across the blood-brain barrier seemed not to occur since the serum and CSF levels of IL-6 showed a negative correlation. The findings suggest a severe inflammatory affection of the central nervous system that could be of importance in understanding the clinical course in patients following SAH.

Basic Remarks: Among travellers to distant countries with a low socioeconomic status and poor hygiene, traveller's diarrhea is a major problem. Once this epidemiological fact had been recognized, intensive efforts were made to reduce the incidence of this illness by prophylactic medication. Among non-antibiotic substances investigated, Saccharomyces boulardii (SB) appeared to show promising results in earlier studies., Method: In a placebo-controlled, double-blind study, various dosages (250 mg and 1,000 mg SB) were administered prophylactically to 3,000 Austrian travellers to distant regions., Results: A significant reduction in the incidence of diarrhea was observed, with success depending directly on the rigorous use of the preparation. A tendency was noted for SB to have a varying regional effect, which was particularly marked in North Africa and in the Near-east (Turkey!); in addition, the effect also proved to be dose-dependent. The medication can be classified as low on side effects.

The effect of oral intake of endotoxins was studied in 12 prepubertal gilts. The animals were given 30 or 100 mg of ET each in their regular morning feed ration. Blood samples were collected periodically during 24 h and the clinical status, including rectal temperature, was recorded at the same time. Hematological and clinical chemical analyses that included serum bile acids, glutamate dehydrogenase, alkaline phosphatase, calcium, iron, zinc and a blood plasma metabolite of prostaglandin F2 alpha, were done. The animals showed no obvious clinical symptoms following endotoxin feeding. The major findings were increased bile acid and glutamate dehydrogenase values with the most prominent rises being recorded 10-12 h after endotoxin intake. In a later experiment, 6 animals were injected i.v. with endotoxin in doses in the range 0.1-0.5 micrograms/kg b.w. Blood samples were taken and analysed as in the endotoxin-feeding experiment. Within 1 h of injection, all animals showed symptoms such as vomiting, fever and dyspnea. The clinical signs disappeared within 2-5 h. The injections were followed by increases in bile acids, glutamate dehydrogenase and prostaglandin F2 alpha metabolite. To conclude, this study indicates that clinically healthy prepubertal gilts react to ingested endotoxin in feed but that no apparent clinical disturbances ensue.

Based on animal research suggesting that nerve growth factor (NGF) can stimulate central cholinergic neurons, the known losses of cholinergic innervation of the cortices in Alzheimer's disease (AD), and our experience of infusing NGF to support adrenal grafts in parkinsonian patients, we have initiated clinical trials of NGF infusions into the brain of patients with AD. Here we report a follow-up of our first case, a 69-year-old woman, with symptoms of dementia since 8 years. Intraventricular infusion of 6.6 mg NGF during three months resulted in a marked transient increase in uptake and binding of 11C-nicotine in frontal and temporal cortex and a persistent increase in cortical blood flow as measured by PET as well as progressive decreases of slow wave EEG activity. After one month of NGF, tests of verbal episodic memory were improved whereas other cognitive tests were not. No adverse effects could be ascribed to the NGF infusion. Taken together, the results of this case study indicate that NGF may counteract cholinergic deficits in AD, and suggest that further clinical trials of NGF infusion in AD are warranted.

Maintaining adequate medical staffing in rural hospitals remains a serious problem. A study was undertaken to discover why doctors came to work in rural hospitals, why they stay, and why they leave. Doctors in rural hospitals in KwaZulu and Natal were surveyed using a postal questionnaire. A total response rate of 74% was obtained. Based on the results, specific recommendations are made for improving working conditions and training and the support needs of doctors in rural hospitals.

Neuropeptide Y (NPY) was measured in central and peripheral cerebrospinal fluid (CSF) in patients suffering from various intracranial disorders. The central NPY-like immunoreactivity (LI) level showed a concentration of 129 +/- 19 pmol.l-1 and was significantly increased (p less than 0.05) compared to peripheral CSF (73 +/- 9 pmol.l-1). From five patients with subarachnoid haemorrhage the CSF NPY-LI levels reached 154 +/- 47 pmol.l-1. In five patients peripheral and central CSF was collected at the same occasion and the CSF NPY-LI concentration was 76 +/- 17 pmol.l-1 in peripheral and 142 +/- 23 pmol.l-1 in central CSF (p less than 0.01), respectively. In a reference group of 9 patients, who were examined by lumbar myelography because of suspected intervertebral herniated discs, the peripheral CSF NPY-LI concentration was 59 +/- 5 pmol.l-1 a value which was also significantly lower compared to NPY-LI levels in central CSF. Thus it is obvious that NPY is present in human CSF with a relatively higher concentration in central than in peripheral CSF at least in patients with disorders of the central nervous system, suggesting a central origin of the NPY.

The ganglioside composition of six human medulloblastomas was analyzed. The characterization was performed by thin-layer chromatography, sialidase hydrolysis, and immunological staining with a panel of characterized antiganglioside monoclonal antibodies. The total ganglioside content ranged from 60 to 1,130 nmol of ganglioside sialic acid/g wet weight. Neuronal gangliosides (gangliotetraose series) were found in varying amounts in all medulloblastomas. Gangliosides of the neolactotetraose series (3'-LM1 and LD1) were present in all specimens, and the lactotetraose series ganglioside 3'-isoLM1 was found in all cases showing astrocytic differentiation. This supports our previous findings that 3'-isoLM1 is a marker for proliferating astroglial cells.

Neopterin concentrations, reflecting T-cell macrophage activation, were analyzed in serum and cerebrospinal fluid (CSF) obtained from 14 patients with subarachnoid hemorrhage (SAH). Neopterin concentrations were elevated in both the serum and CSF. The increase in neopterin concentrations was most marked in the CSF, rising from Days 1 to 3 through Days 6 to 9; levels were highest in patient suffering from delayed cerebral ischemia. The present data were interpreted as signs of an ongoing T cell activation both systemically and in the CSF compartment following SAH.

Eight patients with malignant gliomas verified on CT scan, received an intravenous injection of 50 mg of Adriamycin R, 24 hours prior to surgical removal of the tumour. Peroperatively, both tumour and surrounding tissue specimens were obtained for determination of the tissue concentrations of Adriamycin and its reduced metabolite Adriamycinol. It was found that Adriamycin could be detected in tumour tissue from all patients. The concentration varied between 0.9 and 4.6 nmol/g tissue. In contrast, Adriamycin could only be detected in surrounding brain tissue from one patient. In an in vitro study a human malignant glioma cell line (U-251 MG) was exposed to various concentrations of Adriamycin for 24 hours. It was found that an intracellular drug concentration above 30 nmol/g cells caused a concentration dependent inhibition of cell growth. Thus, it is likely that the poor effect of Adriamycin on patients with malignant gliomas is due to an ineffective drug accumulation in the tumour tissue.

A patient with an anaplastic (malignant) astrocytoma was examined with computed tomography (CT) and with positron emission tomography (PET), in the latter case using [68Ga]EDTA, [11C]glucose, and [11C]methionine. The CT examination as well as the [68Ga]EDTA study showed a small tumor located in the region of the head of the left caudate nucleus. The [11C]glucose examination showed increased uptake on the same region, as did the [11C]methionine examination, but the latter also showed a considerable uptake in the entire left thalamic region. The patient died 15 days after the [11C]methionine study and a histologic evaluation of thin sections obtained at autopsy showed excellent agreement between tumor extent and activity distribution after [11C]methionine administration. The tumor tissue seen only with [11C]methionine was histologically different from that part of the tumor observed with the other tracers. Although cytologically similar, the latter showed large necrotic areas and an ability to induce marked endothelial proliferation, whereas in the former neither necroses nor notable endothelial proliferation was seen. In this case more than 50% of the tumor would have remained radiologically imperceptible without the [11C]methionine PET examination.

The ganglioside composition in meningioma specimens from 20 patients was analyzed to find potential meningioma-associated structures. The characterization was performed by immunological staining with specific monoclonal antibodies to ganglioside antigens and fast atom bombardment-mass spectrometry. The major gangliosides were GM3 and GD3, and most of the meningioma specimens could be divided into a "GM3-rich" or a "GD3-rich" group. Gangliosides of the gangliotetraose series were represented by GM1, GD1a, GD1b, and GT1b, which were found in minor amounts in all the specimens. The ratios of GM1/GD1a and GD1a/GD1b differed from that in normal brain, and therefore existence of this series could not be explained by contamination with brain material. Ganglioside 3'-isoLM1, found in human malignant glioma, could not be detected in any meningioma specimen.

Expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) genes in the human brain and in glioma was studied by slot blot and Northern analysis following hybridization of IGF-I and IGF-II cDNA probes to isolated mRNA. In the fetal brain there were major IGF-I transcripts of 7.5 and 4.4 kilobases (kb) and one major IGF-II transcript of 6.0 kb. IGF-II mRNA was less abundant in the adult brain, with IGF-II mRNA being scarcely detectable. All glioma tissue examined displayed a marked enhancement of both IGF-I and IGF-II gene expression when compared to normal brain. IGF-I transcripts of 4.4 and 2.2-1.8 kb were identified. Similar to fetal brain a major IGF-II transcript of 6.0 kb was found in the glioblastoma examined.