Aspartylglucosaminuria in a Canadian family.

Aspartylglucosaminuria (McKusick 208400) is a lysosomopathy associated with aspartylglucosaminidase (L-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) deficiency. It has been most frequently encountered in Finland, where the regional incidence may be as high as 1 in 3600 births. In North America it is very rare, having been reported in only 8 patients. We encountered 4 patients with aspartylglucosaminuria in a Canadian family of 12 siblings. The 4 siblings affected--2 brothers and 2 sisters--were apparently normal at birth; however, their developmental milestones, particularly speech, were slow, and they acquired only a simple vocabulary. Throughout life, there was a progressive coarsening of facial features; 3 had inguinal hernia and recurrent diarrhea; all became severely retarded and by the 4th decade showed evident deterioration of both cognitive and motor skills; 2 exhibited cyclical behavioural changes. Three of the siblings have died, at 33, 39 and 44 years of age. Two died of bronchopneumonia and 1 of asphyxiation following aspiration. In the urine of all 4 siblings, and in the 1 liver examined, we found 2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta-D-glucosamine (GlcNAc-Asn) and alpha-D-mannose-(1,6)-beta-D-mannose-(1,4)-2-acetamido- 2-deoxy-beta-D-glucose-(1,4)-2-acetamido-1-N-(4-L-aspartyl)-2-deoxy-beta - D-glucosamine (Man2-GlcNAc2-Asn). Compared with the level of activity in controls, aspartylglucosaminidase activity was less than 2% in fibroblasts from 3 of the siblings, less than 0.5% in leukocytes from 1 sibling, and less than 1% in the liver of 1 sibling, whereas other acid hydrolase activities in these tissues were normal. Ultrastructural studies of skin showed that fibroblasts, endothelial cells and pericytes contained vacuoles with fine reticulo-floccular material. Glial and neuronal cells of the central nervous system showed similar inclusions as well as others composed of concentric or parallel membranous arrays intermingled with lipid droplets.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)-syndrome. Ultrastructural changes of mitochondria in cultured dermal fibroblasts of three patients.

Mitochondria of fibroblasts cultured from the skin obtained at biopsy from three patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH)-syndrome, one of the autosomal recessive, heritable urea cycle disorders, were studied with appropriate controls ultrastructurally. The patients were two severely retarded 10- and 12-year-old boys, and a 22-year-old sister of the former whose mental status was at the low normal range; she never had motor impairments or seizures. The mitochondria, similar in all three patients, were increased in number, very long, branching and/or "looping," and tortuous. "Spurs" or "buddings" extended from their lateral surfaces and the terminal segments were often bulbous. Other unusual configurations were also present. In addition, giant forms with large diameter contained innumerable closely-packed and parallel cristae which traversed the entire width of these mitochondria; at times they assumed a "whirled" pattern. The mitochondrial matrix was usually of high electron density. These changes were not a feature of fibroblastic mitochondria of controls. Several changes resembled those of hepatic mitochondria in this disorder. All features are interpreted as an attempt at expanding the mitochondrial volume (via structural substratum) to compensate for the metabolic incompetence of these organelles (a block in transmembranous transfer of ornithine from hyaloplasm into mitochondria for conversion to citrulline).

Ciliated cultured dermal fibroblasts in a patient with hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome.

Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH)-syndrome is a rare autosomal recessive disorder of the urea cycle, probably caused by a defect in ornithine transport across the hepatic inner mitochondrial membrane. Single rudimentary cilia were present in approximately ten percent of post-divisional or dividing fibroblasts cultured from the skin of a patient with the HHH-syndrome, whereas no such organelles were observed in dermal fibroblasts cultured from normal controls. Since single rudimentary ("primary," "oligo," "solitary") cilia have been observed in a variety of cells in animals and men but the stimuli for their formation and their significance remain controversial, a brief report on their presence in the as yet unreported condition (HHH-syndrome) was considered of interest; hopefully, it might contribute to the ultimate unravelling of some of the unresolved problems. It is of note that unlike the author's previous findings of these unusual organelles in cells affected by a pathological process (atherosclerosis), the rudimentary cilia were observed in the present instance in dividing or postdivisional cells. The implications of these (and other) observations must await further work.

Cellular localization of metallothionein in human term placenta.

Cellular localization of metallothionein (MT) in placenta may provide information on its function as a metal binding protein. Rabbit antibodies to rat liver MT cross-reacted with human MT and were used to localize MT in human term placenta by avidin-biotin peroxidase technique. Serial sections (5 microns) were cut from paraffin-embedded placentae obtained at term from five normal women and incubated with rabbit antibodies to MT. Normal rabbit serum was used as a negative control. The slides were incubated with biotinylated swine anti-rabbit IgG (linking antibody) then with avidin-biotin horseradish peroxidase complex and developed with diaminobenzidine in hydrogen peroxide (0.03 per cent) substrate. The optimum staining of MT was obtained at a 1:800 antibody dilution. MT was identified in fetal amniotic cells, syncytial trophoblasts and villous interstitial cells, and in maternal decidual cells. The presence of MT at specific cellular sites suggests that it may regulate the transplacental transport of metals such as zinc, copper and cadmium. Since the level of cadmium is lower and that of zinc and copper higher in fetal than in maternal blood, this may suggest that placental MT may restrict cadmium while enhancing zinc and copper transport.

Aortic features in Tangier disease and pathogenetic considerations--Part I. Fatty dots and streaks.

Morphological studies of aortic fatty dots and streaks, and the adjacent normally appearing intima, in a 5 3/4-year-old boy who died of pneumonia, showed several hitherto unreported features. In lesions, lipid vacuoles and/or other cytoplasmic "inclusions" (ultrastructurally considered to present complex forms of lipids) were present on occasion in the endothelium but consistently involved the (intimal) smooth muscle cells (SMC). Similar changes were present in the adjacent intima, but were here less prominent and "tapered off" distally. A moderate number of macrophages also contained cytoplasmic lipids but such cells entirely free of lipid inclusions were observed, too. Most surprisingly, dilated and many cisternae of rough-surfaced endoplasmic reticulum (ER) in the SMC of lesions were associated spatially with cytoplasmic droplets and other forms of lipids. The results of these studies question the generally accepted central role of macrophages as being primarily involved in the pathogenesis of tissue changes in Tangier disease. It is possible that in view of the absence or paucity of high-density lipoproteins (HDL) and alterations of (their) apo A-I and apo A-II (as well as of other lipids), the arterial SMC may be in some way involved in the metabolism of the above substances in this disorder. Support of this tentative (and highly speculative) assumption must await further work utilizing tissues and cells other than those containing macrophages and other derivatives of reticulo-endothelial system.

The Canadian Atherosclerosis Society--history and present status.

Since its inception in 1983 the Canadian Atherosclerosis Society (CAS) has established itself firmly on the national and international scene as a forceful scientific voice. Its presence and activities have had their dominant expression at annual meetings held jointly with the Royal College of Physicians and Surgeons of Canada (RCPSC) and the Canadian Society for Clinical Investigation (CSCI) and in sponsoring other scientific and educational events, the most important of which was the Canadian Consensus Conference on Cholesterol (Ottawa, March 1988). It provided a forum for interaction between the scientific community, government, funding agencies, industry and the general public, and culminated in concrete recommendations for the populace of Canada. It also 'induced' a continuum in governmental and public concern for health with respect to atherosclerosis, and beyond it, the field of cardiovascular diseases. This dialogue continues. As a member (Constituent Society) of the International Atherosclerosis Society (IAS), the CAS has a voice in the international community, its policies and activities. The membership increase from 69 in 1983 to 175 in 1991 reflects steady growth of the CAS. The Society has been active in other areas (publications, awards for young investigators, and common educational endeavours with other groups) and will be host to the 1994 International Symposium on Atherosclerosis. Over a short period of only eight years, all of the above attests to sufficient progress (or achievement) for any scientific society. And yet, there remain quite a few areas not addressed as yet and some sad experiences (eg, that with the Long Term Planning Committee) that must be quickly remedied, if the Society is to keep pace with the everchanging emphasis in research that in the final analysis aims at improving the overall well-being and health of all Canadians. Inherent in the definition of history is the premise that accounts be provided of facts only. Historians who research their subjects derive these facts from studying the necessary accounts relating to these 'facts', using different and preferably controversial resources, so as to present the facts as objectively as possible. It is impossible, however, to fulfill all the above criteria for a historian who lived through every phase of 'life' of the subject of his or her account, because no matter how objective one wishes to remain (and bends backwards to achieve this) there will be always an element of a personal prism through which the historian lived the 'life' with his subject. For being human and thus unable to eliminate entirely that personal component, this writer asks humbly for the reader's understanding.

The genesis of atherosclerosis in pediatric age-group.

The three forms of origin of the atherosclerotic plaque of adults, that is, the fatty streaks, gelatinous elevations, and microthrombi, all occur in arteries of normal infants and children. Some of these may become arrested or regress, but many progress to the prominent lesions that precipitate various clinical catastrophies. The aim of modern medicine is to modify or eliminate many of the factors known to advance the atherosclerotic process and thus decrease the incidence of this disease, which ranks highest on the list of causes of morbidity and mortality in the Western world. Of these factors, some may be controlled by dietary means (low salt; low total fat and cholesterol; appropriate ratios of saturated to mono- and polyunsaturated fatty acids; high content of complex carbohydrates and fiber); controlling hypertension, diabetes, and obesity; abstaining from cigarette smoking; and vigorous physical activities. Because patterns of life-style are determined in childhood and adolescence, and because it is only during that period of life that measures to prevent progression of atherosclerosis may be predictably effective, it becomes increasingly apparent that atherosclerosis is, indeed, a pediatric problem.

Placental weight in diabetic pregnancies.

The placenta from 30 women with diabetes mellitus were examined and weighed at delivery. Nineteen of these were from women with overt and eleven from women with gestational diabetes. Eleven placentae from normal pregnancies served as controls. There was no difference between the mean +/- s.d. placental weight for the diabetic group and the control group (609 +/- 148 versus 591 +/- 93 g, NS). The mean placental weight ratios for the diabetic group and the control group were also similar (0.98 +/- 0.23 versus 0.89 +/- 0.15, NS). Moreover, there was no difference between the weights and weight ratios of placentae from women with overt (622 +/- 173 g, 1.02 +/- 0.27) and those with gestational diabetes (586 +/- 90 g, versus 0.90 +/- 0.13). Placental weights correlated with birthweights (r = 0.70, P less than 0.01) and with skinfold thickness measurements fo the infants (r = 0.40, P less than 0.05), but neither with gestational ages (r = 0.15, NS) nor with maternal glycosylated haemoglobin levels in the third trimester (r = 0.24, NS). Among the women with overt diabetes, placental weights were greater in those in White's class B and C than those in class D and R (689 +/- 143 versus 530 +/- 177 g; P less than 0.05). In general, placentae from well controlled diabetic patients were not heavier than those from normal pregnant women, although there was an increase in placental weight in White's class B and C, as compared with those in class D and R.

The hyperornithinemia, hyperammonemia, homocitrullinuria syndrome: an ornithine transport defect remediable with ornithine supplements.

The primary defect in patients presenting with a history of protein intolerance, mental retardation, and epilepsy of variable degree, with the unique triad of hyperornithinemia, hyperammonemia, and homocitrullinuria (the HHH syndrome) has been postulated to be a defect in translocation of ornithine into the mitochondria. In a 12-year-old boy with the HHH syndrome, the hyperammonemia observed following a protein load was prevented when the same load was given orally with a 1 mmol/kg of ornithine-HCl. At a dosage level of 0.5 to 1.0 mmol/kg/day of ornithine HCl, administered in 3 divided doses with meals, the patient's protein tolerance improved. As pretreatment hyperammonemia reverted to normal levels, the patient was able to cope with increased dietary protein and his growth accelerated. During the 2-year interval of the study, the ornithine HCl supplements were withdrawn on 2 occasions, and within a week the hyperammonemia recurred. Whereas cultured fibroblasts from the HHH patient were capable of oxidizing U-14C-glutamate to 14CO2 as rapidly as normal cells. 1-14C-ornithine or 5-14C-ornithine were oxidized at only 1/28 or 1/49 of the normal rate. Ultrastructural studies of the HHH cultured fibroblast mitochondria revealed distinctive alterations in size and shape; unusually long, branching, and "curling," HHH mitochondria also showed accelerated regressive changes.

Endothelial cilia in human aortic atherosclerotic lesions.

"Primary" cilia were present in the endothelial cells of human aortic fatty dots and streaks but not in those of normal intima. They had the features of cilia of the "9 + 0" axonemal configuration observed in many other cells. A lateral foot process and transitional fibers "anchored" the ciliary basal body in the cytoplasm, but rootlets were not identified in material examine. Ladder-like configurations interconnected the two centrioles (= diplosome) of control endothelium. The "primary" cilia of endothelium differed from those of the rudimentary type observed in smooth muscle cells in similar lesions of man, but shared many features with cilia of those present in experimental atherosclerosis in rabbit. Cilia were rarely described in vascular endothelium. It is believed that, to date, they were not reported to occur in normal or pathological arteries in man. It is being stressed that whereas the significance of these unusual organelles remains uncertain, their widespread occurrence may indicate that their role is more important than was believed previously, and they should cease being a curiosity only.

Ciliated smooth muscle cells in atherosclerotic lesions of human aorta.

One or two rudimentary cilia were observed by electron microscopy in smooth muscle cells (SMCs) of fatty dots and streaks, but not in normal intima of human aorta. Similar organelles are known to occur in many cell types and various species, but to the best of the author's knowledge, have never been found in the SMCs of arterial or other tissues of man in health and disease; recently, they were reported to be present in the SMCs of experimental atherosclerosis in rabbits. The rudimentary cilia observed in this study had a "9 + 0" axoneme (microtubular complex) and differed also in other aspects from the classical cilia. Semiserial sections of SMCs containing a diplosome disclosed that on several occasions both of its constituent centrioles gave rise to rudimentary cilia. SMCs containing cilia or their basal bodies were observed more often in the human than in experimental atherosclerotic lesions. Whereas the function and significance of the rudimentary cilia remain largely unknown, the current theory proposes that a sudden transformation from a mitotic replicative to a nonmitotic structured tissue "diverts" centrioles to the formation of these unusual organelles. It is conceivable that rudimentary cilia could serve as morphological indicators of aborted mitosis in human atherosclerotic lesions.

Arterial involvement in genetic diseases.

Whereas the information on the subject of arterial status is sketchy and haphazard with respect to any one genetic disorder, the number of these diseases would have precluded the provision of a critical review within the scope of this presentation. Thus, it was deemed more meaningful to approach the subject selectively. A brief summary was provided on the nature of the arterial wall and its involvement in genetic diseases either as a primary target or a secondarily affected organ, and on "affinity" of various genetic disorders for a type (elastic, muscular, or smallest), segment (proximal, distal), and layer (intimal, medial, adventitial) of the arterial tree or the arterial wall, respectively. Genetic diseases may affect arteries by "causing" (a) congenital malformations, (b) alteration of the arterial "makeup" without necessarily producing definable lesions, and (c) modification of a nongenetic arterial disease (e.g., atherosclerosis), or by "producing" (d) arterial lesions that are characteristic of (even specific for?) a given genetic disorder. A few examples were selected to illustrate (b) (tuberous sclerosis; infantile GM1-gangliosidosis), (c) Wolman's disease; familial hyperlipoproteinemias), and (d) [Hurler's disease, neurofibromatosis; Ehlers-Danlos syndrome (type IV)]. Whenever available, the results of electron microscopic studies carried out in our laboratories were included. Some of these have not been reported in the literature to date. The need for a coordinated multidisciplinary approach to the study of genetic diseases in general is stressed in closing.

Congenital leukemia with placental involvement. Report of a case with ultrastructural study.

The study of a stillborn infant and the placenta, showing extensive infiltration of fetal organs and chorionic villi by leukemic cells, is reported. The nature of the malignant cells was not apparent by light microscopic examination. Ultrastructural examination showed that these were immature myeloblasts, stressing the usefulness of electron microscopic study in establishing the diagnosis. The current case study seems to be the first in the literature in which both placenta and fetus were studied in extenso.

The effect of ascorbate on the synthesis of minor (non-interstitial) collagens by cultured bovine aortic smooth muscle cells.

Ultrastructural and biochemical studies were carried out on bovine aortic smooth muscle cells cultured in the presence or absence of ascorbate. In its absence, electron microscopic examination of cultures revealed that the extracellular components consisted primarily of microfibrils. Morphologically identifiable collagen fibrils were only observed in the matrix upon ascorbate supplementation. Smooth muscle cells grown in ascorbate-free media synthesized large amounts of type VI collagen. The identity of the latter was confirmed by ion exchange chromatography, slab gel electrophoresis, and amino acid analysis. Addition of ascorbate resulted in a stimulation of type I collagen production, levels of the type III remained constant, and types V and VI were decreased. Since, in the absence of ascorbate, smooth muscle cells are known to synthesize predominantly elastin, the present data support the contention that the type VI collagen and the microfibrillar component of elastic tissue are either identical or similar.