Biochemical monitoring after haemopoietic stem cell transplant for Hurler syndrome (MPSIH): implications for functional outcome after transplant in metabolic disease.

Hurler Syndrome is corrected by allogeneic BMT by the action of donor enzyme on recipient tissue. In this paper, we describe monitoring of 39 patients transplanted in two centres to determine donor chimerism, enzyme level and residual substrate - expressed as dermatan sulphate to chondroitin sulphate ratio. We show that in fully engrafted recipients, the enzyme level, expressed as mumol/g total protein/h, post-transplant is 24.2 from an unrelated donor and 10.2 from a heterozygote family donor (P<0.0001). There is a tight relationship between mean post-transplant enzyme level and residual substrate - Spearman's rank correlation coefficient (Rho) was -0.76 and -0.80 at 12 and 24 months, respectively (P<0.0001). We propose that these differences affect patient outcome. As unrelated donor transplant outcomes improve and especially given the higher levels of donor cell engraftment following cord transplants, our data might influence donor selection where only heterozygote-matched family members are available.

Enzyme replacement therapy in 12 patients with MPS I-H/S with homozygous p.Leu490Pro mutation.

We describe a cohort of 14 Hurler-Scheie patients homozygous for the p.Leu490Pro missense mutation in the alpha-L-iduronidase gene. Now based in the UK, they are all of Pakistani/Kashmiri descent; 64% were female; 11/14 (79%) had a sibling or cousin with MPS I and the parents are consanguineous in all cases. The median age at diagnosis was 1.8 years (range from antenatal diagnosis to 16.5 years). Twelve were on ERT with recombinant human alpha-L-iduronidase (IDUA; Laronidase, Genzyme) for a median duration of 22.5 months (range 2-71 months) and median age at commencement of ERT was 8.6 years (range 0.4-23.1 years). There was clear improvement in the size of liver and spleen as well as reduction in urine glycosaminoglycans (GAGs). The mean (range) urine GAG levels in mg/mmol creatinine were 63.4 (28.9-105.6), 28.3 (10.9-41.4), 22.8 (12.1-43.1), 15.7 (9.2-24.8) and 16.3 (10.1-21.0) at commencement, 3 months post ERT, 6 months post ERT, 12 months post ERT and 24 months post ERT, respectively. Effects on growth were not clear as there does not seem to be an obvious trend of increase or decrease in height after commencement of ERT and this seems to be the case regardless of the age at which ERT was started.

The nature of the attachment of a regularly arranged surface protein to the outer membrane of an Acinetobacter sp.

Acinetobacter 199A carries on the outer surface of its outer membrane a layer of regularly arranged protein subunits. The isolated surface protein assembles into the same regular array even in the absence of the underlying outer membrane. Cl- minus is required for this self-assembly. Evidence is presented that the interaction of the surface protein with the outer membrane involves the linking of a carboxyl group in the surface protein to a negatively charged group in the outer membrane protein, via a divalent cation. The surface protein could be detached from the outer membrane by the protein perturbant urea, by the chelating agent EDTA and by replacing Mg-2+ with Na+. It could not be detached by treatment with phospholipases A anc D or the detergents Tween 80 and sodium deoxycholate. The conditions favourable for reattachment of surface protein to the cell wall were the presence of divalent cations and a pH of 3-5. Conversion of carboxyl groups in the surface protein to amine with carbodiimide and ethylene diamine interfered with reattachment. The surface protein did not attach to isolated cell wall lipid or lipopolysaccharide.

Donor bone marrow from a sibling with inborn error of metabolism for treatment of acute leukaemia - clinical and biochemical consequences in the non-affected recipient.

Bone marrow transplantation (BMT) is increasingly used in an attempt to correct inborn errors of metabolism (IEM). However, little is known about effects of BMT from patients with IEM donating for non-affected recipients. We present data from a 8.5-year-old girl who underwent BMT in second remission for relapsed acute lymphoblastic leukaemia (ALL) at the age of 7 years from her HLA-identical brother who was severely affected by Hunter syndrome (Mucopolysaccharidosis type II, iduronate-2-sulphatase (IDS) deficiency). After BMT not only leukocyte but also plasma activity of IDS was absent. Mixing experiments and immunoadsorption suggest antibody-mediated enzyme inhibition. However, her urinary glycosaminoglycan excretion has not increased post BMT and clinical signs of mucopolysaccharidosis are absent 20 months after BMT. We conclude that patients with white cell enzyme deficiencies and other IEMs do not have to be excluded from bone marrow donation. Antibody production by the graft may occur and be reflected by a marked reduction in plasma enzyme levels but not tissue activity. Similar antibody responses resulting in enzyme inactivation might also affect other enzyme replacement strategies for individuals with IEM.

A solid phase assay with radioactively-labelled antibody for the detection of Brucella abortus.

An assay for the detection of Brucella abortus is described. IgG from rabbit antisera against live brucellae or brucella extracts was chemically linked to cellulose to form a solid phase reagent capable of binding brucella antigens present in buffer solutions or serum. After washing away any unbound material the presence of bound antigen was revealed by incubation with radioactively-labelled anti-brucella antibodies. The assay was capable of detecting less than 100 pg brucella antigen in a 20 microliter sample. Experiments in which IgG of non-related specificity was used in place of anti-brucella IgG showed that the test was specific. Normal human serum had only a slight inhibitory effect but anti-brucella antibodies were strongly inhibitory if present in the test sample. The extent of this effect and its relationship to antibody titre was investigated in 12 sera from brucellosis patients.

Detection of gonococcal antigens in urine by radioimmunoassay.

A method of detecting gonococcal antigens by solid-phase radioimmunoassay with radioactively labelled antibody is described. A specificity test has been developed that enables this method to be used to detect gonococcal antigens in urine sediments. When sediments from samples of urine from male patients with gonorrhoea were tested, 31 (74%) of 42 gave positive results, clearly distinguishing them from sediments from urine samples from men with non-specific urethritis, none of which was positive. Ten of 14 urine sediments from urine samples from women with gonorrhoea gave positive results, as did 3 of 18 sediments from urine samples from women patients without gonorrhoea. These experiments demonstrate that gonococcal antigens can be detected in urine by radioimmunoassay; the method could be useful in diagnosis if, after refinement, its sensitivity and specificity were to be increased.

Inhibitors in urine of radioimmunoassay for the detection of gonococcal antigens.

Several substances in urine were found to inhibit the radioimmunoassay of added gonococcal antigens. The supernatants of two-thirds of urine samples from male patients with either gonorrhoea or non-specific urethritis (NSU) were inhibitory. The inhibition caused by many, but not all, samples was reduced or completely abolished by the addition of soybean trypsin inhibitor (STI); STI-sensitive inhibition is thought to be due to proteolytic enzymes, probably from pus cells. Their inhibitory effect was shown to be due to their action on gonoccocal antigens and not on antibodies in the assay system. Some supernatants contained other inhibitors unaffected by STI; some of these were dialysable and others were not. Sediments from the urine of patients with NSU or gonorrhoea were often strongly inhibitory, but treatment with STI annulled all but very slight inhibition. STI-treated sediments could, therefore, be used in an assay designed to detect gonococcal antigens.

Detection of Chlamydia psittaci from various animal species by reverse passive haemagglutination.

A reverse passive haemagglutination test (RPH) has previously been developed to detect the genus-specific antigen of Chlamydia. Clinical samples were obtained from various sites of different animal species. The RPH test detected chlamydial antigen from clinical cases of conjunctivitis in cats, abortion in sheep and psittacosis in birds. Although not as sensitive as cell culture isolation, this test has the advantages of rapidity and of detecting antigen from dead chlamydiae.

Application of low dose rate pulsed fluoroscopy in cardiac pacing and electrophysiology: patient dose and image quality implications.

The performance of a low dose rate pulsed fluoroscopy option and its successful application to cardiac pacing and electrophysiology is reported. Low dose rate 6.25 frames per second pulsed fluoroscopy was made available in two catheter laboratories at a specialist cardiac centre in February 2003, and was adopted as the standard imaging technique for cardiac pacing procedures. The image quality was found to be considerably poorer than conventional modern units, being very similar to that which would have been accepted as adequate performance 20 years ago, but at less than one-tenth of the dose rate. No problems with the clinical acceptance of this imaging mode for cardiac pacing and electrophysiology have been reported. The already low median patient dose-area product for pacing at this cardiac centre was further reduced by 50% with the introduction of this fluoroscopy option.

Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

Heparan sulfate levels in mucopolysaccharidoses and mucolipidoses.

Glycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.

Freeze-etching of the cell envelope of an Acinetobacter species which carries a regular array of surface subunits.

Freeze-etching was applied to preparations, with and without glycerol, of Acinetobacter sp. strain MJT/F5/199A, consisting of intact cells after normal growth or after incubation with chloramphenicol, spheroplasts, and isolated cell walls and outer membranes. Etched preparations show that a regular array of subunits forms the surface of normal cells. Near the zones of constriction in dividing cells, blebs and irregularities are seen, and some blebs, consisting of both surface subunits and outer membrane, are released from the cells. The cross-fractured cell envelope shows four layers which are related to the structures seen in section as follows: cw1, which is not visible in section, contains the surface subunits; cw2 consists of all or part of the outer membrane; cw3 includes the intermediate and dense, peptidoglycan-containing layers; within these cell wall layers is the plasma membrane. Internal fracture of the plasma membrane occurs under all conditions tested, but the fracture plane in the cell wall is only revealed in chloramphenicol-treated cells or normal cells freeze-fractured with glycerol present; the characteristic fracture faces are not seen in spheroplasts or isolated outer membranes. The concave fracture face cw2 consists of densely packed granules, while the convex face cw3 is fibrillar. The probable location of this fracture plane is discussed. After incubation with chloramphenicol, the outer surface of the cells is obscured by extracellular material, the dense peptidoglycan-containing layer is increased in thickness, and the cytoplasm contains rounded bodies bounded by one or more unit membranes.

Human beta-mannosidase deficiency: biochemical findings in plasma, fibroblasts, white cells and urine.

Marked deficiencies of beta-mannosidase activity were demonstrated in plasma, leukocytes, fibroblasts and urine of a patient with beta-mannosidosis, similar deficiencies were observed in the proband's sibling. All other lysosomal enzymes measured, including sulphamidase, exhibited normal activity. Both parents showed reduced plasma and leukocyte beta-mannosidase activity. Urinary glycosaminoglycan excretion was normal but TLC of urinary oligosaccharides revealed an abnormal band with the mobility of a disaccharide. This finding was confirmed by Bio-Gel P2 column chromatography. Further purification of this compound revealed two disaccharides, both of which yielded mannose and glucosamine following acid hydrolysis and mannose and N-acetylglucosamine following enzymic digestion. These two compounds are thought to be structural isomers of the disaccharide Man beta-GlcNAc.

A study by high-resolution two-dimensional polyacrylamide gel electrophoresis of relationships between Neisseria gonorrhoeae and other bacteria.

High-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.

Comparison of red-cell linked anti-IgE and 125I-labelled anti-IgE in a solid-phase system for the measurement of IgE specific for castor bean allergen.

The report described the development of a mixed reverse (solid-phase) passive antiglobulin haemadsorption (MRsPAH) test for specific IgE antibody to castor bean allergen. The allergen is immobilised by formalin fixation in the wells of polyvinyl chloride microtitre plates. After allowing allergen-specific antibodies in the test serum to bind to the allergen, plates are washed thoroughly, and red cells coupled by chromic chloride to sheep IgG anti-human IgE are used to detect specifically bound IgE. This system was compared with a solid-phase radioimmunoassay in which 125I-labelled anti-IgE was substituted for the antiglobulin-linked red cells of the MRsPAH test; the earlier stages of both tests being the same. 12 sera, 10 from patients with allergic asthma to castor bean allergen and two from non-allergic controls, were tested for castor-bean-specific serum IgE by both methods and the results showed high correlation. The MRsPAH tst for allergen-specific serum IgE provides a useful alternative to the RAST system, being free of the disadvantages inherent in the use of radio-labelled materials.

First-trimester diagnosis of Hunter syndrome: very low iduronate sulphatase activity in chorionic villi from a heterozygous female fetus.

Iduronate sulphatase activity was determined in uncultured chorionic villi from four fetuses at risk for Hunter syndrome. All fetuses were shown to be female by chromosome analysis. Biopsy material from three fetuses showed iduronate sulphatase activity within normal limits whilst the fourth fetus showed activity reduced to 7 per cent of our control mean. The importance of fetal sexing in prenatal diagnosis of this condition is emphasized as female carrier fetuses may show iduronate sulphatase activity reduced to levels observed in affected males.

Detachment and chemical characterization of the regularly arranged subunits from the surface of an Acinetobacter.

Acinetobacter sp. strain MJT/F5/199A carries an array of tetragonally arranged subunits on its outer surface. The subunits can be detached from isolated cell walls by incubation with 1 M urea or by washing with water after treatment with 10 mM ethylenediaminetetraacetic acid or ethyleneglycol-bis(beta-aminoethylether)N,N'-tetraacetic acid. After removal of the urea, they reaggregate into the same ordered array at air-water interfaces in the presence of MgCl(2). The detached subunits were characterized as an acidic protein of molecular weight 65,000. They represent one-fifth of the total cell wall protein.

Location of the fracture faces within the cell envelope of Acinetobacter species strain MJT-F5-5.

The cell wall of the gram-negative bacterium Acinetobacter species strain MJT/F5/5 shows in thin section an external "additional" layer, an outer membrane, an intermediate layer, and a dense layer. Negatively stained preparations showed that the additional layer is composed of hexagonally arranged subunits. In glycerol-treated preparations, freeze-etching revealed that the cell walls consist of four layers, with the main plane of fracture between layers cw 2 and cw 3. The surface of [Formula: see text] 2 consisted of densely packed particles, whereas [Formula: see text] 3 appeared to be fibrillar. In cell envelopes treated with lysozyme by various methods, the removal of the dense layer has detached the outer membrane and additional layer from the underlying layers, as shown in thin sections. When freeze-etched in the absence of glycerol, these detached outer membranes with additional layers fractured to reveal both the faces [Formula: see text] 2 and [Formula: see text] 3 with their characteristic surface structures, and, in addition, both the external and internal etched surfaces were revealed. This experiment provided conclusive evidence that the main fracture plane in the cell wall lies within the interior of the outer membrane. This and other evidence showed that the corresponding layers in thin sections and freeze-etched preparations are: the additional layer, cw 1; the outer membrane, cw (2 + 3); and the intermediate and dense layers together from cw 4. Because of similarities in structure between this Acinetobacter and other gram-negative bacteria, it seemed probable that the interior of the outer membrane is the plane most liable to fracture in the cell walls of most gram-negative bacteria.

Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp.

Chemical analysis of fractions of the cell envelope of Acinetobacter sp. strain MJT/F5/199A, prepared by breakage in the French press and removal of plasma membranes, followed by sequential treatment with lysozyme and with papain, confirmed the existence of layers previously identified by electron microscopy. Outside the plasma membrane and periplasmic space, the envelope is composed of (i) a peptidoglycan-containing dense layer, (ii) an intermediate layer, (iii) a lipopolysaccharide-containing outer membrane, and (iv) an ordered array of protein subunits. A small amount of carbohydrate (3%) is found associated with protein in the fraction containing both the surface subunits and the intermediate layer. The papain-treated outer membranes contain 67% protein, 24% lipid, together with 11% lipopolysaccharide, and about 6% of non-lipopolysaccharide hexosamine. Lipid is located only in the papain-treated outer-membrane and is mainly phospholipid: 29% phosphatidyl glycerol, 30% phosphatidyl ethanolamine, and 40% cardiolipin. The principal fatty acid is C(18:1). Significant amounts of alcohols(16:1) and alcohols(18:1), which are found in Acinetobacter waxes, were recovered from the outer membrane.

Surface antigens of gonococci: correlation with virulence and serum resistance.

Encapsulated and non-encapsulated variants of one strain of gonococcus were compared for their capacity to produce infection in chambers implanted subcutaneously in mice, for their reactions with specific antibody and for their precipitation with wheat germ agglutinin. Only the encapsulated variant could infect implanted chambers. Specific rabbit antiserum raised against the non-encapsulated variant killed both variants. Saline extracts and lipopolysaccharide preparations of the encapsulated variant differed from those of the non-encapsulated one in their reactions with wheat germ agglutinin and antibody in diffusion and electrophoresis tests. Preparations from infective encapsulated gonococci reacted with wheat germ agglutinin while those from the non-encapsulated variant did not. Immunodiffusion tests showed that lipopolysaccharides from both variants share a common antigenic determinant, but saline extracts and lipopolysaccharides from encapsulated gonococci possess an additional determinant. The significance of these findings is discussed.