Enzyme replacement therapy by fibroblast transplantation: long-term biochemical study in three cases of Hunter's syndrome.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.

Survival of a five-strain cocktail of Escherichia coli O157:H7 during the 60-day aging period of cheddar cheese made from unpasteurized milk.

The U.S. Food and Drug Administration Standard of Identity for Cheddar cheeses requires pasteurization of the milk, or as an alternative treatment, a minimum 60-day aging at > or =2 degrees C for cheeses made from unpasteurized milk, to reduce the number of viable pathogens that may be present to an acceptable risk. The objective of this study was to investigate the adequacy of the 60-day minimum aging to reduce the numbers of viable pathogens and evaluate milk subpasteurization heat treatment as a process to improve the safety of Cheddar cheeses made from unpasteurized milk. Cheddar cheese was made from unpasteurized milk inoculated with 10(1) to 10(5) CFU/ml of a five-strain cocktail of acid-tolerant Escherichia coli O157:H7. Samples were collected during the cheese manufacturing process. After pressing, the cheese blocks were packaged into plastic bags, vacuum sealed, and aged at 7 degrees C. After 1 week, the cheese blocks were cut into smaller-size uniform pieces and then vacuum sealed in clear plastic pouches. Samples were plated and enumerated for E. coli O157:H7. Populations of E. coli O157:H7 increased during the cheese-making operations. Population of E. coli O157:H7 in cheese aged for 60 and 120 days at 7 degrees C decreased less than 1 and 2 log, respectively. These studies confirm previous reports that show 60-day aging is inadequate to eliminate E. coli O157:H7 during cheese ripening. Subpasteurization heat-treatment runs were conducted at 148 degrees F (64.4 degrees C) for 17.5 s on milk inoculated with E. coli O157:H7 at 10(5) CFU/ml. These heat-treatment runs resulted in a 5-log E. coli O157: H7 reduction.

The comparative content of the mitochondrial phosphoglyceroyl-ATP-containing polymer, purinogen, in rat tissues.

Here, we describe an assay for the tissue content of purinogen, a highly phosphorylated labile polymer containing ATP and phosphoglycerate found in the mitochondrial intermembrane space. We report the purinogen content (as adenine nucleotide equivalents) of rat heart and, for the first time, of rat liver, kidney, brain and mixed skeletal muscle. The findings show that purinogen contains very significant proportions of cell adenine nucleotides ranging from 25% of the free pool in brain and skeletal muscle to 135% of it in kidney. The evidence that purinogen may form a controlled intracellular reservoir of inorganic phosphate is briefly discussed.

Evidence against the incorporation into protein of amino acids directly from the membrane transport system in rat heart.

The possibility suggested recently [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433; and Adamson, L.F., Herington, A.C. and Bornstein, J. (1972) Biochim. Biophys. Acta 282, 352-365] that protein synthesis takes place using amino acids directly from the membrane transport system and not from an intracellular pool has been investigated in rat heart. The tissue was perfused first for 30 min with either [14C]glycine or [14C]leucine and then for a further 30 min with identical medium containing [3H]glycine or [3H]leucine, respectively. After an initial lag, [14C]glycine was incorporated into protein at a linear rate up to 60 min. The [3H]glycine was accumulated into tissue water and incorporated just as readily as the [14C]glycine had been. The rate of total protein synthesis agrees with literature values only if intracellular and not extracellular specific activity values are used in the calculation. Some glycine was converted to serine or threonine. Leucine influx and efflux were very rapid in contrast to the relatively slow exchange reported for incubated tissues [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433]. The results are consistent with the existence of an intracellular precursor pool for glycine. Some possible reasons for the discrepancies between this and the other studies are discussed.

A comparison of rapid adenine nucleotide incorporation into phosphoglyceroyl-ATP and into RNA-like species in perfused rat heart.

Four main species of rapidly synthesised trichloroacetic acid-insoluble derivatives of adenylate can be separated in extracts from rat hearts. The major species, accounting for more than 70% of the total, is phosphoglyceroyl-ATP; two others (16% of the total) are closely related to it. Around 10% of incorporated radioactivity is in a high-molecular-weight form with a phosphate/purine ratio of 0.8; comparison of [14C]uridylate and [14C]adenylate incorporation supports the suggestion that this is a rapidly synthesised species of RNA which represents about 4% of total RNA in heart. Values are given for the contents of UTP, UDP and UDPglucose in adult rat hearts.

Substantial quantities of the high energy derivative oligophosphoglyceroyl-ATP are located in mitochondria in rat liver.

Perfusion with [8-14C]adenosine demonstrated the likely existence in rat liver of oligophosphoglyceroyl-ATP (OPG-ATP). Purification followed by assay with a new specific 3' phosphodiesterase confirmed this. The quantities present were 5-10-fold those found previously and comparable to total soluble nucleotides. OPG-ATP was also purified from the mitochondrial fraction, shown to co-distribute with succinate dehydrogenase and can be co-purified with an enzyme confined to intermembrane space.

Biliary tract cancers: SEOM clinical guidelines.

Biliary tract cancer (BTC) is an uncommon and highly fatal malignancy. It is composed of three main different entities; Gall bladder carcinoma (GBC), intrahepatic cholangiocarcinoma (iCC) and extrahepatic cholangiocarcinoma (eCC) sharing different genetic, risk factors and clinical presentation. Multidetector-row computed tomography (MDCT) and magnetic resonance cholangio-pancreatography (MRCP) are the more important diagnostic techniques. Surgery is the only potentially curative therapy but disease recurrence is frequent. Treatment with chemotherapy, radiotherapy or both has not demonstrated survival benefit in the adjuvant setting. Cisplatin plus gemcitabine constitutes the gold standard in metastatic disease. New ongoing studies mainly in the adjuvant and neoadjuvant setting along with molecular research will hopefully help to improve survival and quality of life of this disease.

Evidence for ADP-ribosylation in the mechanism of rapid thyroid hormone control of mitochondria.

Triiodothyronine in vitro at concentrations between 10(-13) and 10(-11) M very rapidly activates oxidative phosphorylation in hypothyroid rat liver mitochondria. Comparing the concentrations of hormone with estimates of the amounts of respiratory chain components present suggests that this activation may involve an amplification mechanism. Here we present evidence that while no changes in phosphorylation were detected following hormone administration, nicotinamide, an inhibitor of mono ADP-ribosylation reported to occur rapidly and reversibly in mitochondria, prevented activation by hormone. Moreover incubation with nicotinamide of euthyroid mitochondria and derived intact inner membrane vesicles revealed lowered ADP/O ratios under the same conditions as shown by hypothyroid preparations. While this lesion could be reversed simply by washing the intact mitochondria, the membrane vesicles required triiodothyronine addition.

Direct thyroid hormone signalling via ADP-ribosylation controls mitochondrial nucleotide transport and membrane leakiness by changing the conformation of the adenine nucleotide transporter.

Addition of triiodothyronine at 10 pM in vitro to hypothyroid rat liver mitochondria doubles the rate of the adenine nucleotide transporter at low ADP concentrations. Nicotinamide abolishes this effect in parallel with its inhibition of the ADP-ribosylation of an inner membrane protein identical in size to the transporter. Nicotinamide also renders euthyroid preparations indistinguishable from hypothyroid ones. A mechanism is offered to explain these findings in which it is proposed that the adenine nucleotide transporter is a true allosteric protein and that its covalent modification by ADP-ribosylation increases the stability of the less favoured externally-facing C-conformation and thus increases the proportion of transporters in this orientation: although the C-conformation is significantly more leaky to cations than the tight matrix-facing M-conformation, this enhances ADP import. This model is shown to offer an explanation not only for the transport effects of T3 but also for those of oxidative stress and ADP-ribosylation inhibitors on Ca2+, H+ and K+ transfer across the mitochondrial inner membrane. Ca2+ at 30 nM appears to stabilize the M-conformation of the transporter by a mechanism other than ADP-ribosylation.

The influence of thyroid hormone on the degree of control of oxidative phosphorylation exerted by the adenine nucleotide translocator.

Impaired phosphorylation efficiency in liver mitochondria from hypothyroid rats is paralleled by a defect in adenine nucleotide transport. Both of these lesions can be corrected within 15 min by a near-physiological dose of triiodo-L-thyronine. Measurement of the control strength of the translocator shows, however, that this step has a smaller share of the control for oxidative phosphorylation after thyroidectomy and that this is unaltered after 15 min by replacement therapy. Rapid control by triiodothyronine is thus exerted elsewhere than at this transfer and the effects of hormone on the translocator are likely to be indirect.

The use of the preparation of F(ab')2 antibody from soluble immune complexes to determine the complexed antigens.

An autoradiographic technique for the characterization of antibody specificity in soluble antigen--antibody complexes has been developed. The circulating antigen--antibody complexes are precipitated by polyethylene glycol (PEG). The antibodies are liberated as F(ab')2 from the complexes by pepsin digestion. The antibody specificity against the putative antigen was revealed by radioimmunoelectrophoresis using [125I]-F(ab')2 reagents. The method was developed by using an experimental model of C3/anti-C3 complexes.

Desquamative interstitial pneumonia and antigen-antibody complexes in two infants with congenital rubella.

Desquamative interstitial pneumonia was observed in two infants with the late-onset congenital rubella syndrome. In both infants this unusual lung disease was associated with circulating immunoglobulin M complexes and interstitial pulmonary deposits of IgM by immunofluorescence. Both infants had IgG deficiency. The first child recovered with a reduction in IgM complex levels and synthesis of rubella-specific IgG. The second infant died during the acute phase of his illness at which time there were high serum concentrations of IgM complexes and slightly increased levels of IgG complexes. Delayed maturation of the immune response in congenital rubella may predispose to persistent antigenemia and pulmonary deposition of rubella antigen-containing IgM complexes resulting in an acute form of interstitial pneumonia.

Abnormal responses to rubella infection.

Two cases of rubella are described which caused initial problems in laboratory diagnosis due to abnormal features in the immune response. One patient presented with thrombocytopenic purpura and associated circulating immune complexes. The other patient, who was in early pregnancy, had an unusually prolonged rash and a delayed humoral immune response. The possible reasons for the difficulties in serological confirmation are discussed.

Nature of circulating immune complexes in infective endocarditis.

Two percent polyethylene glycol (PEG) precipitation was found to be a useful method for detecting circulating immune complexes (CIC) and could be used diagnostically to implicate infective endocarditis. Complexes consisting of a least Clq, IgG, and IgA were typically detected in sera from patients with infective endocarditis. Serial studies showed that CIC detection and measurement could be used to monitor clinical progress. Successful clinical improvement was reflected by decreasing CIC levels and the disappearance of rheumatoid factor, and, where increasing amounts of CIC were found, this may indicate situations where treatment was insufficient or inappropriate. There was specific free antibody demonstrable in the serum of six out of nine patients against their own infecting organisms, but attempts to identify the specificity of the complexed antibody as being directed against these organisms or their extracellular products failed. We could not detect any radioactive precipitin arcs, indicative of the free antibody also being in the complex, between the F(ab')2 preparation from the complex and the electrophoresed bacterial antigens in a radio-immunoelectrophoresis system. Eleven out of 13 sera that contained Clq, IgG, and IgA in their complexes also contained rheumatoid factor. Immunisation against the patient's own persisting CIC may explain this phenomenon.

Circulating and deposited immune complexes in renal disease and their clinical correlation.

In 48 patients undergoing renal biopsy there was a strong correlation (chi 2 11.45 (P less than 0.01)) between the demonstration of circulating and deposited immune complexes. Serial studies of circulating immune complex levels have shown fluctuations which only sometimes appear to coincide with clinical changes in individual patients.

Human papovavirus in Papanicolaou smears of urinary sediment detected by transmission electron microscopy.

A technique is described whereby inclusion-bearing cells identified by light microscopy in stained smears of urinary sediment were reprocessed for examination in the electron microscope. The nuclei of the abnormal cells were found to contain numerous virus particles, 35 nm in diameter, which morphologically resembled papovaviruses. The technique was applied in this case to identify further the virus producing the cytopathic changes in the Papanicolaou smear. It could be particularly valuable for retrospective studies of mounted cytological or histological material when suitable specimens are no longer available for virological investigation.

The control of cellular adenine nucleotides: the discovery and metabolism of an oligomeric derivative of ATP and phosphoglyceric acid in mitochondrial intermembrane space.

The observation of large systematic variations in the purine nucleotides in perfused rat hearts inexplicable by known metabolic transformations led us to propose the existence of some unknown major form of nucleotide. Radiolabelling studies located a rapidly metabolized TCA/methanol-insoluble species in heart, kidney and liver which selective digestion and chromatography allied to 31P-nmr and mass spectrometry suggested to be a tetraphosphate derivative of adenosine and phosphoglyceric acid of the form p3Gri1p[pp5'p3Gri1p]npp5'A, oligo-phosphoglyceroyl-ATP. Partial chemical synthesis has confirmed this basic structure. We have shown that substantial amounts of oPG-ATP are located in liver mitochondrial intermembrane space tightly bound to a specific 3' phosphodiesterase which releases the monomer p3Gri1ppp5'A. Here we show that oPG-ATP is remarkably stabilized by Mg2+ ions and describe the 300-fold purification of the stable substrate-free form of the 3' phosphodiesterase; it has an apparent M(r) = 165k with possibly up to 4 subunits of M(r) = 42.5k, 41.9k, 40.5k and 38.9k. The potential function of oPG-ATP is briefly discussed.

Genetic and immunological factors in human recurrent abortion.

The theoretical causes of recurrent spontaneous abortion may be several. Gill and others have suggested that the human disease may be mimicked by the rodent genetic diseases, as exemplified by the T locus in mice. He would suggest that there may be lethal recessive gene phenomena which may cause repeated abortion, or if the abortion is prevented by immunological means the birth of genetically defective individuals. The evidence presented here shows that prevention of abortion by immunological treatment, that is immunisation of the wife with paternal lymphocytes, is associated with a very high success rate, and the children born do not have more than the normal incidence of birth defects. Thus far, over the 7 years of study, the children also appear to develop normally. Probably the reason for this is that the methods of selection for treatment tend to exclude quite effectively those couples with pure genetic problems. In particular, the the exclusion of women who have had more than one live child removes a group of women who might have lethal recessive problems which would only affect 25-50% of the offspring. The interactive gene products that may produce abortion of all pregnancies, suggested as a theoretical concept by Gill, would be expected to show an incidence no more than the square root of the frequency of the recessive disease, and are probably too few to be seen even in the more than 1,500 couples we have studied.

Histopathologic alterations in the decidua in human spontaneous abortion: loss of cells with large cytoplasmic granules.

The histopathology of decidua obtained from the placental bed was evaluated by phloxine-tartrazine staining, which allows clear definition of cells with cytoplasmic granules. Mononuclear cells with large granules were seen in biopsy specimens taken from women at 8-31 weeks of normal pregnancy. In contrast, cells with large granules were missing in sections taken from the decidua of five women who were aborting or were destined to abort. Since the presence of suppressor cell activity in murine decidua correlates with the success of pregnancy and since this suppression is associated with small lymphocytes with cytoplasmic granules, the observations made using human placental bed biopsy material suggest that a possible suppressor cell deficiency might occur in the early stages of spontaneous abortion in human females.

The rapid transient stimulation of both cytoplasmic and mitochondrial ornithine decarboxylases in the liver of thyroidectomized rats by 3,5,3'-triiodo-L-thyronine.

Evidence is presented that liver from thyroidectomized rats has ornithine decarboxylase (ODC) in both mitochondrial and soluble fractions. The cytosolic activity was stimulated 7-fold and the mitochondrial activity 3-fold 15 min after the administration of triiodothyronine (T3). While the rapid transient stimulation of ODC could represent a direct intracellular response to T3, an incidental effect on a very-rapidly-turning-over enzyme seems more likely; this suggests that in vivo ODC may be controlled by a demand for polyamines.