Ultrastructure of skin fibroblasts in storage diseases (mucopolysaccharidosis types IV and VI)

Ultrastructure of skin fibroblasts was studied in patients with mucopolysaccharidoses of types IV and VI and their relatives. In MPS VI and keratan-nonexcreting form of MPS IV the cytoplasm of fibroblasts contained numerous vacuoles with material of various electron density. The ultrastructure of cell did not differ from the norm in the keratan-excreting form of MPS IV. Skin fibroblasts from parents and siblings with MPS VI were found to contain a large number of residue bodies. The possible usage of ultrastructural data for early diagnosis of MPS and in medical genetic consultation of families of the patients with MPS is discussed.     

Utilization of electron microscopy in the prenatal diagnosis of genetic disease.

The use of electron microscopy as a further method of diagnosis of disease in cultured skin fibroblasts and cultured amniotic fluid fibroblasts is presented. It was demonstrated that Tay-Sachs disease, Fabry's disease, and metachromatic leukodystrophy had distinctive abnormalities in both cultured skin fibroblasts and cultured amniotic fluid fibroblasts. It was shown that control of culturing conditions made it possible to distinguish normal cell lines from certain cell lines carrying known genetic diseases.     

Cytochemistry of the skin of patients with mucopolysaccharidoses.

The distribution of complex carbohydrates has been investigated at the light and electron microscope levels in sweat glands of normal subjects and patients with Hurler's or Hunter's disease. Normal sweat glands examined with a battery of light microscopic histochemical methods revealed sulphated complex carbohydrate in secretory granules of the dark cells. These granules lacked affinity for dialysed iron (DI) at the light and electron microscope levels. The DI method demonstrated acid complex carbohydrates ultrastructurally on the surface of the intercellular canaliculi and central lumen in normal sweat glands. DI-reactive acidic material, presumably of mucopolysaccharide nature, surrounded and extended between collagen bundles in the stroma of normal skin, but was absent from the band which ensheathed the sweat gland and consisted of individual rather than bundled collagen fibrils. DI-reactive mucopolysaccharide lined and partially filled vacuoles of dark cells showing a laminar distribution in vacuoles of clear cells in sweat glands of a Hunter patient. The DI method also visualized mucopolysaccharide distributed throughout vacuoles in fibroblasts of this patient. DI-reactive acid material covered the luminal surface of the sweat gland, coated collagen bundles in the stroma and spared the periglandular collagenous sheath in skin from Hurler and Hunter patients as in that from normal controls. Acid phosphatase was localized ultrastructually in vacuoles and nearby cytoplasm and on plasmalemmae of clear cells, dark cells and myoepithelial cells of sweat glands from Hurler and Hunter patients. Vacuoles of dermal fibroblasts and Schwann cells in these specimens also exhibited strong acid phosphatase activity.     

Ribonuclease–gold Labels Chondroitin Sulphate in Guinea Pig Basophil Granules

Basophilic leucocytes are metachromatic granule-containing secretory granulocytes that contain a mixture of granular proteoglycans devoid of heparin. In guinea pigs, isolated basophilic leucocyte granules primarily contain chondroitin sulphate. We have recently demonstrated that an enzyme-affinity– gold technique to image RNA, using the reagent RNase gold, also binds specifically to heparin in human mast cell granules. Such binding is based on the known property of heparin as a competitive inhibitor of RNase. Using similar methods, we show here that RNase–gold binds to the chondroitin sulphate in the secretory granules of guinea pig basophils, thus broadening the applicability of this post-embedding affinity–gold method to studies that require imaging of chondroitin sulphate in routinely prepared electron microscopical samples.     

Effect of chloroquine on cultured fibroblasts: release of lysosomal hydrolases and inhibition of their uptake.

Incubation of normal human fibroblasts with 1–5 μM chloroquine at physiological pH for 8 hr produces granular cytoplasmic inclusions, release of lysosomal enzymes into the medium and decrease of intracellular lysosomal enzyme activities. The effects are dose dependent and reversible. The uptake of arylsulfatase A into fibroblasts genetically deficient in arylsulfatase A (grown from skin biopsies of patients with metachromatic leukodystrophy) is completely inhibited by pretreating the cells with 5 μM chloroquine. Arylsulfatase A, which has been taken up as exogenous enzyme from the medium into the cells, is partially released into the culture medium upon incubation with chloroquine. The data suggest that chloroquine competes with the binding of lysosomal enzymes to the cell membrane and to the membranes of pinocytotic vacuoles and causes release of previously internalized exogenous enzyme.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

Fine structure of the bone marrow of the chicken and pigeon

The structure of bone marrow from chickens and pigeons was studied with light and electron microscopy. Erythropoiesis occurs in the lumen of the medullary sinuses. Immature erythroid cells appear to adhere to the sinus wall and may thus be prevented from entering the peripheral circulation. The wall of the medullary sinuses is formed by elongated lining cells, lacking a basement membrane, which are continous except at sites where blood cells are passing through them. When viewed with the electron microscope, developing heterophil myelocytes, which occur only in the extravascular spaces, possess two populations of granules; one type is globular in content, the other is fibrillar in content. The globular type predominates during all stages of development and appears to be the specific granule. Specific granules originate from material which is formed in the Golgi complex, pinches off, and accumulates in expanded vesicles. The origin of the material in the fibrillar granules was not determined. Like the globular granules of heterophil leucocytes, granules of eosinophil leucocytes arise from material which is formed in the Golgi complex.     

Cytochemical method for staining fish basophils

The occurrence of basophils in peripheral blood of 15 freshwater teleosts was examined using the metachromatic stain, toluidine blue, on blood smears fixed with lead subacetate. Metachromasia was more identifiable through better preservation of the granules. The occurrence of basophils in teleost blood was confirmed as very rare and was not caused by failure to preserve the basophil granules.     

成年草鱼外周血细胞的超微结构

用透视电镜技术研究了成年草鱼外周血细胞的超微结构，结果表明：在外周血细胞中可区分出红细胞，淋巴细胞，单核细胞，嗜中性白细胞，嗜酸性白细胞和血检细胞；嗜碱性白细胞无论在血涂片上或超薄切片上都没有发现。电镜图像显示，在红细胞中，仅能看到少数线粒体和液泡。淋巴细胞以短的伪足，较多的游离核糖体和滑面内质网为其特征。单核细胞的特征是具有较多的伪足和胞质液泡以及偏位核。在外周血液中发现了嗜中性白细胞生长、发育、功能成熟和衰退的全过程，描述了嗜中性白细胞的幼稚型、成熟型和衰退型细胞的超微结构。在成年草鱼外周血中，还发现了一种新型的颗粒细胞，它的特殊颗粒为大的六角形或棒状致密结晶颗粒，对上述特殊颗粒细胞的属性也进行了讨论。     

Identification and Counting of Basophil Leucocytes

A 1% alcoholic solution of toluidine blue is used and recommended for specific staining of the basophil leucocytes in blood smears as well as in tissue sections. The cytoplasmic granules show a metachromatic (purple) stain in contrast to other elements taking an orthochromatic (blue) stain. For direct counting a 0.05% alcoholic solution of toluidine blue adjusted to pH 7.7, and containing an optimal concentration of saponin as a hemolyzing agent, has been successfully used for identifying and counting basophils, eosinophils and all other leucocytes, simultaneously in the same chamber. Blood dilution of 1:10 by means of micropipettes is preferable to that made by the ordinary leucocyte diluting pipettes. The technic described is recommended for routine use in hematological laboratories.     

Identification and Counting of Basophil Leucocytes

A 1% alcoholic solution of toluidine blue is used and recommended for specific staining of the basophil leucocytes in blood smears as well as in tissue sections. The cytoplasmic granules show a metachromatic (purple) stain in contrast to other elements taking an orthochromatic (blue) stain. For direct counting a 0.05% alcoholic solution of toluidine blue adjusted to pH 7.7, and containing an optimal concentration of saponin as a hemolyzing agent, has been successfully used for identifying and counting basophils, eosinophils and all other leucocytes, simultaneously in the same chamber. Blood dilution of 1:10 by means of micropipettes is preferable to that made by the ordinary leucocyte diluting pipettes. The technic described is recommended for routine use in hematological laboratories.     

Occurrence of the periodic acid-Schiff positive granular leucocyte (PAS-GL) in some fishes and its significance

Preparations of peripheral blood of 58 species of fish, mostly teleosts, were examined for the presence of granulocytes. Heterophils are commonly found but eosinophils are rare and metachromatic basophils never seen. Granulocytes (PAS-GL) with periodic acid-Schiff positive granules, were observed in 30 species representing marine and freshwater, and 'primitive' and 'advanced' types. It is postulated that the PAS-GL is the piscine forerunner to the heparin and histamine-containing metachromatic basophil/mast cell of higher vertebrates.     

Identification of peripheral blood leucocytes of the dogfish (Scyliorhinus canicula L.) by electron microscopy

Dogfish peripheral blood leucocytes were examined by electron microscopy after the injection of colloidal carbon. The cells were classified as lymphocytes, plasma cells, monocytes, thrombocytes and granulocytes. The granulocytes were further classified into four types according to the structure of their granules. Monocytes, thrombocytes and two types of the granulocytic cells were phagocytic.     

The immune response of wild rainbow trout, Salmo gairdneri Richardson, to naturally acquired plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825)

Natural infections of rainbow trout with two species of Diphyllobothrium result in a host inflammatory response encapsulating the plerocercoid. The encapsulating cyst, observed by light and electron microscopy, comprises leucocytes, fibroblasts and collagenous connective tissue and is infiltrated with a blood vascular network. An indirect immunofluorescence technique and enzyme-linked immunosorbant assay (ELISA) have shown that specific antibodies are elicited by the fish host to these Diphyllobothrium spp. These antibodies have been semi-quantitatively measured by ELISA and correlated with worm burdens in individual fish.     

Pancreatic damage induced by injecting a large dose of arginine

Male Wistar rats were injected intraperitoneally with a large dose of arginine (500 mg/100 g body weight) and were sacrificed 24, 48 and 72 h later. Pancreatic tissue was examined by electron microscopy to study the resulting process of degeneration. Degeneration started with disorganization of the rough endoplasmic reticulum into whorls with a concomitant decrease in the numbers of zymogen granules. The main changes in acinar cells after 24 h were partial distension of the endoplasmic reticulum, whorls of agranular membranes encircling zymogen granules and perinuclear vacuoles. At this time large sequestered areas in the cytoplasm contained disarranged rough endoplasmic reticulum and degraded zymogen granules. The mitochondria showed only slight changes. After 48 h, dissociation and necrosis of acinar cells were noted. Subsequently, the necrotic cells were replaced by interstitial tissue composed of leucocytes and fibroblasts. It was concluded that a large dose of arginine is toxic to the rat pancreas when injected intraperitoneally. The early morphological changes of the acinar cells may be related to metabolic alterations associated with the endoplasmic reticulum. The disorganization of the endoplasmic reticulum and the reduced number of zymogen granules may indicate disturbance of protein synthesis. The focal sequestration and degradation of the cytoplasm seemed to represent changes of the acinar cells associated with removal of damaged organelles.     

A major histocompatibility locus in fish: serological identification and segregation of transplantation antigens in the common carp (Cyprinus carpio L.)

In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic carp showing either slow of fast rejection of skin transplants, the latter were chosen for alloantiserum production by hyperimmunization with peripheral blood leucocytes. The resulting alloantisera were analyzed for hemagglutinating reactivity with gynogenetic siblings and proved to be operationally monoscpecific in absorption experiments. The serologically determined carp erythrocyte specificites were shown to correspond to two codominantly expressed allelic products of a single locus and were designated K1 and K2, respectively. Flow cytometer analysis revealed that the same products are also present on leucocytes from peripheral blood, thymus, spleen, and pronephros.K1-andK2-homozygous second-generation gynogenetic siblings were used to study the histocompatibiligy nature of the K locus products. Skin transplants between K-allogeneic gynogenetic siblings were rejected significantly faster than within K-syngeneic combinations. Taken together, these data suggest that theK locus incorporates MHC class I-like characteristics.     

Mucopolysaccharidosis 3 A (Sanfilippo A disease): deficiency of a heparin sulfamidase in skin fibroblasts and leucocytes

Cultured skin fibroblasts and peripheral leucocytes from patients with Sanfilippo A disease are strikingly deficient in sulfamidase activity (sulfamatase, EC 3.1.6.?), as measured with heparin - N³⁵SO₄. A partial sulfamidase deficiency was found in the cells of the heterozygote carriers. Since Sanfilippo A fibroblasts have normal sulfate ester hydrolase activities towards oligosaccharides prepared from ³⁵SO₄-labelled heparan sulfate by nitrous acid treatment, the basic defect in Sanfilippo A disease is considered to be the inactivity of a heparin (heparan sulfate) sulfamidase.     

Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.

A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of the beta 1 subgroup of the integrins in human cells and tissues

We studied the distribution of the beta 1 integrin subfamily in human tissues and cells by light microscopy, electron microscopy, and immunoblotting, using monoclonal antibody DH12, previously shown to react with the beta 1 subunit of the human fibronectin receptor. Crossreaction with the other beta subunits of the integrin family, which have 45% and 47% primary amino acid sequence identity with the beta 1 subunit, was excluded, as MAb DH12 did not react with the beta 2 subunit in granulocytes and the beta 3 subunit in thrombocytes. Reactivity with the anti-beta 1 antibody was found in skin, lung, heart, striated and smooth muscle, blood cells, liver, kidney, intestine, spleen and placenta. Thus, cells of mesodermal, ectodermal, and entodermal origin express the beta 1 subunit. In skin fibroblasts cultured in vitro, beta 1 subunit was also detected intracellularly. The wide distribution of the beta 1 family, originally detected in activated T-lymphocytes after prolonged culture in vitro, contrast with the restricted distribution of the beta 2 integrins on leucocytes.