[Efficacy of agreements within the Enchede Stroke Service to refer patients with a stroke from the stroke unit in the hospital to a nursing home for short-term rehabilitation]. / Effectiviteit van afspraken binnen de Enschedese stroke-service om patiënten met een beroerte adequaat te verwijzen van de stroke-unit in het ziekenhuis naar een verpleeghuis voor kortdurende reactivering.

OBJECTIVE: To assess the efficacy of agreements within the Enschede Stroke Service to refer patients with a stroke from the stroke unit in the hospital to a nursing home for short-term rehabilitation. DESIGN: Prospective, partly retrospective. METHOD: All patients who were referred from the stroke unit at Medisch Spectrum Twente to the CVA Rehabilitation Unit (CRU) in the period 1 July 1999-31 July 2003 were included. Referral took place via an active multidisciplinary approach and specific referral agreements. The primary outcome was the number of patients that could be discharged home after rehabilitation. In addition, we assessed the influence on final discharge destination of age, the Barthel and Rankin scores at the time of admission to the CRU and the medical complications during the period of rehabilitation. RESULTS: 232 patients were included (133 women and 99 men, mean age 76.4 years). Within 3 months, 63% of the patients were discharged home. After 6 months, 82% had returned home. 8% of the patients died within 6 months and 9% had to stay in a nursing home permanently. Of the patient aged 80 years or older, 75% could return home within 6 months. Patients with poor Barthel and Rankin scores and medical complications had a smaller chance of being discharged home. CONCLUSION: Effective referral of patients from the stroke unit to a nursing home for short-term rehabilitation is possible. With adequate patient selection, the use of good referral agreements and multidisciplinary consultations, most patients could finally return home.

Accurate differentiation of parkinsonism and essential tremor using visual assessment of [123I]-FP-CIT SPECT imaging: the [123I]-FP-CIT study group.

OBJECTIVE: To evaluate whether visual assessment of [123I]-FP-CIT (DaTSCAN, Nycomed Amersham, plc) single photon emission computerized tomography (SPECT) images can differentiate between parkinsonism and essential tremor (ET). METHODS: [123I]-FP-CIT SPECT imaging was conducted in a six-center study of 158 patients with a clinical diagnosis of parkinsonism compared with 27 ET cases and 35 healthy volunteers. Striatal uptake of the radioligand was graded normal or abnormal, and abnormal images were further graded to three levels of severity. An institutional read whereby each center visually assessed the images blinded to the clinical data and a consensus blinded read by a panel of five was undertaken. RESULTS: The institutional reading scored 154 of 158 cases of parkinsonism abnormal, all 27 cases of ET as normal, and 34 of 35 healthy volunteers as normal compared with the consensus blinded read scoring 150 cases of parkinsonism as abnormal, 25 ET cases as normal, and 33 healthy volunteers as normal. Sensitivity for the clinical diagnosis of parkinsonism was 97% and specificity for ET was 100% for the institutional read, whereas sensitivity was 95% and specificity 93% for the consensus blinded read. Semiquantitative analysis of specific: nonspecific caudate and putamen uptake were consistent with the results of visual inspection. CONCLUSION: Visual assessment of [123I]-FP-CIT SPECT images is an easily applied diagnostic test which is helpful in the differential diagnosis of tremor disorders and in confirming a clinical diagnosis of a hypokinetic-rigid syndrome.

Measuring right-hemisphere dysfunction in children: validity of two new computer tests.

The validity of two new computer-mediated tests for the detection of right-cerebral hemisphere lesions in children--the Right-hemisphere Dysfunction Test and the Visual Perception Test--was evaluated. Normative data were drawn from a group of 91 children (aged five to 14 years) and 14 young adults. The tests were also administered to 14 children with acquired lesions of either right- or left-cerebral hemisphere. The results demonstrate that the Right-hemisphere Dysfunction Test and the Visual Perception Test, with predictive values of 71 per cent and 88 per cent, respectively, are useful in clinical practice for detection of right-hemisphere dysfunction in children.

Ultrastructural localization of glucocerebrosidase in cultured Gaucher's disease fibroblasts by immunocytochemistry.

The subcellular localization of glucocerebrosidase was studied in cultured skin fibroblasts from eight patients with Gaucher's disease. The enzyme, in situ, was visualized under the electron microscope by incubating ultrathin frozen sections of fibroblasts with antibodies against glucocerebrosidase, followed by a second incubation with goat anti- (rabbit IgG) coupled to colloidal gold. In control cells, most of the gold label was found in lysosomes, associated with the membrane. Labelling of the rough endoplasmic reticulum (RER) and Golgi complex was also observed. In fibroblasts from three Gaucher's disease patients without neurological symptoms (type 1 disease), a near normal amount of cross-reactive material (CRM) was detected in lysosomes, but in a fourth such patient, the lysosomal CRM was reduced. Little lysosomal glucocerebrosidase was detected in cells from patients with the acute neuronopathic form (type 2) or the subacute neuronopathic form (type 3) of Gaucher's disease. CRM in lysosomes correlates with amount of mature, 59 kDA glucocerebrosidase which is undetectable in type 2 and type 3 Gaucher's disease cell lines. These findings demonstrate that different mutations in the gene for glucocerebrosidase result in mutant proteins that have different intracellular localizations. They also suggest that there is a relationship between the amount of cross-reactive material in the lysosomes and the phenotypic expression of the disease.

Prenatal detection of cystic fibrosis; comparative study of maltase and alkaline phosphatase activities in amniotic fluid.

The potential value of microvillar enzymes in the prenatal diagnosis of cystic fibrosis (CF) has previously been demonstrated and is corroborated in the present comparative study. Maltase and alkaline phosphatase (ALP) activities were studied in the amniotic fluids of 57 pregnancies with a 1 in 4 risk for CF or with a known CF outcome and in 489 controls. A simple assay for maltase activity (MU-maltase) with the fluorogenic substate 4-methylumbelliferyl alpha-glucoside, offers great technical advantages and an at least equal detection rate of CF, when compared to the previously used test with maltose as substrate. Intestinal ALP was estimated either as phenylalanine inhibitable activity (PI-ALP) or as the proportions of residual activity in the presence of the inhibitors phenylalanine or homoarginine. MU-maltase and PI-ALP appeared the most successful methods: both tests were able to detect 14 of the 16 (88 per cent) pregnancies with fetal CF. Each of the two tests alone also allowed a correct prediction in 24 of the 25 pregnancies at risk but with normal outcome; however all 25 cases could be correctly predicted by a combined evaluation. It is suggested that more than one intestinal enzyme activity should be evaluated to allow optimal results in the prenatal monitoring of pregnancies at high risk for CF.

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts.

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.

Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase").

The culture fluid of Hep G2 human hepatoma cells contains triglyceridase activity resistant to high-salt concentrations. The lipase binds to Sepharose-heparin columns from which it can be eluted by 0.8 to 0.9 M NaCl. The nature of this lipase was studied using antibodies raised against "liver" lipases from human and rat origin. The anti-rat liver lipase inhibits both the postheparin human and rat plasma enzyme while the anti-human liver lipase has no effect on the rat enzyme. The lipase of the Hep G2 cultures showed affinity to the antibodies raised against rat as well as human "liver" lipase as shown by inhibition experiments. These results show that Hep G2 cells secrete "liver" lipase and that there seems to exist a structural homology between the lipases from rat and human origin.

Monoclonal antibodies against salt-resistant rat liver lipase. Cross-reactivity with lipases from rat adrenals and ovaries.

To obtain monoclonal antibodies against rat salt-resistant liver lipase, mice were immunized with enzyme purified from heparin-containing rat liver perfusates. Hybridomas were screened for antibody production by means of an enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay. Five hybridoma cell lines secreting antibodies against rat liver lipase indicated as A, B, C, D and E, have been obtained. All antibodies possess gamma one (gamma 1) heavy chains and kappa (kappa) light chains. The antibodies precipitate salt-resistant lipase from rat post-heparin plasma, are positive in ELISA, inhibit liver lipase activity and bind monospecifically with the enzyme as shown by immunoblotting. The monoclonal antibodies showed no significant reactivity with human liver lipase. The salt-resistant lipases of rat adrenals and ovaries are also precipitated by the monoclonal antibodies directed against the liver enzyme. Therefore, the heparin-releasable lipases of the liver, adrenals and ovaries possess identical epitopes.

Synthesis and intracellular localization of chick acid alpha-glucosidase in chick erythrocyte-human fibroblast heterokaryons. A model system for the study of lysosomal enzyme synthesis.

The synthesis and localization of chick acid alpha-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid alpha-glucosidase were used. It was found that the acid alpha-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.

The subcellular localization of soluble and membrane-bound lysosomal enzymes in I-cell fibroblasts: a comparative immunocytochemical study.

Using electron microscopic immunocytochemistry with gold probes, we have studied the localization of acid alpha-glucosidase, N-acetyl-beta-hexosaminidase and beta-glucocerebrosidase in cultured skin fibroblasts from control subjects and patients with mucolipidosis II (I-cell disease). In control fibroblasts, a random distribution of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase within the lysosomes was observed, whereas beta-glucocerebrosidase was found to be localized on or near the lysosomal membrane. The observations confirm the soluble character of acid alpha-glucosidase and N-acetyl-beta-hexosaminidase and the membrane-bound character of beta-glucocerebrosidase. In I-cell fibroblasts an abnormal localization of the two soluble enzymes was found. Labeling in lysosomes was very weak, but instead, small 'presumptive' vesicles containing both enzymes were detected throughout the cytoplasm and close to the plasma membrane. These vesicles could be involved in the secretion of the two enzymes. In contrast, a normal membrane-bound lysosomal localization was observed for beta-glucocerebrosidase. It is concluded that the intracellular transport of beta-glucocerebrosidase to the lysosomes can occur even when the mannose-6-phosphate recognition system is defective. This explains the normal activity of beta-glucocerebrosidase in I-cells in contrast to the deficiency of most other lysosomal enzymes.

Monoclonal antibody to human lysosomal alpha-glucosidase in immunocytochemistry: unexpected reactivity with cytoskeletal structures.

A well-characterized monoclonal antibody against human lysosomal alpha-glucosidase has been used for the immunohistochemical localization of the enzyme in cultured human skin fibroblasts. Under conditions that are routinely used for the preparation of cells for immunocytochemistry, this monoclonal antibody does not react with acid alpha-glucosidase but in contrast with components of the cytoskeleton. Double-labelling experiments with the monoclonal antibody and rabbit anti-vimentin antiserum identified the cytoskeletal components as intermediate filaments. The implications of this observation for the use of monoclonal antibodies in immunocytochemistry in general are discussed.

Characteristics of maltase activity in amniotic fluid.

The nature and origin of maltase activity present in amniotic fluid, and used as a marker enzyme in the prenatal monitoring of cystic fibrosis, has been studied. Using monoclonal antibodies against human intestinal disaccharidases and via heat inactivation experiments it is shown that the maltase activity found in amniotic fluids from pregnancies of 16-24 wk of gestational age originates completely from sucrase-isomaltase; no maltase-glucoamylase could be detected. With various monospecific antibodies the possible contribution of non-intestinal brush border enzymes to the total maltase pool could be excluded: neither renal nor lysosomal maltase appeared to be present.

Sucrase-isomaltase and cystic fibrosis.

The intestinal microvillar enzyme complex sucrase-isomaltase has been studied in cystic fibrosis and control ileum. A number of biochemical parameters of the enzyme in ileum homogenates have been determined. Both solubilized as well as membrane-bound sucrase-isomaltase were analyzed with respect to their reaction with monoclonal antibodies against human sucrase-isomaltase. Finally the subcellular localization of sucrase-isomaltase was verified by immunoelectronmicroscopy or via the analysis of purified brush-border membrane preparations. At all levels no significant differences could be detected between sucrase-isomaltase of cystic fibrosis and control ileum. It is concluded that an abnormal subcellular localization and/or abnormal enzymatic activity of sucrase-isomaltase in cystic fibrosis intestine cannot explain the markedly decreased disaccharidase activities in amniotic fluids from pregnancies resulting in a child affected with cystic fibrosis.

The chromosomal localization of human beta-galactosidase revisited: a locus for beta-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22.

A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme beta-galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental beta-galactosidase, it was observed that the structural locus for the beta-galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human beta-galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against beta-galactosidase, followed by analysis via gel electrophoresis and fluorography. The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the "protective protein" was previously shown to be intimately associated with human beta-galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of beta-galactosidase by aggregating beta-galactosidase monomers into high molecular weight multimers. Both chromosome 3 and 22 are therefore necessary to obtain normal levels of beta-galactosidase activity in human cells.

Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli.

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D-galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta-galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta-galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

Orientation of rat-liver plasma membrane vesicles. A biochemical and ultrastructural study.

Using both biochemical and morphological methods, the membrane orientation of plasma membrane vesicles from rat liver which are capable of catalysing the active transport of amino acids was investigated. In intact vesicles, the plasma membrane enzyme (Na+ + K+)-ATPase displays only a minor portion of its total activity which is greatly increased upon vesicle disruption. The same intact vesicles show an almost maximal binding of ouabain, which binds only to the extracellular side of the plasma membrane. A freeze-fracture analysis of the vesicles shows that a distinct population of relatively large vesicles have predominantly the in vivo membrane orientation. These large vesicles are labelled with numerous filipin-sterol complexes following exposure to the cholesterol probe, filipin, and are therefore assumed to be plasma membrane vesicles. A population of smaller vesicles with mainly an inside-out orientation were not labelled with filipin and are probably microsomes. The data obtained with both biochemical and ultrastructural techniques indicate that the plasma membrane vesicles isolated from rat liver for transport studies are mostly (at least 70%) orientated as in vivo, i.e. inside-in.