Uptake and metabolism of radioactively labeled sphingomyelin in cultured skin fibroblasts from controls and patients with Niemann-Pick disease and other lysosomal storage diseases

The metabolism of [stearoyl-1-14C]- and [choline-methyl-14C]sphingomyelin, [stearoyl-1-14C]ceramide-1-phospho-N,N-dimethylethanolamine (demethylsphingomyelin) and [choline-methyl-14C]phosphatidylcholine was measured 1, 3 and 5 days after uptake from the media of cultured skin fibroblasts. This was done to measure the relative contributions of lysosomal sphingomyelinase and plasma membrane phosphocholine transferase on the metabolism of sphingomyelin, a component of all cell membranes. By using cell lines from controls and from patients with Niemann-Pick disease and other lysosomal storage diseases, it was concluded that a significant portion (10-15%) of the observed degradation of sphingomyelin is due to exchange of the phosphocholine moiety producing phosphatidylcholine. Although cell lines from type A and B Niemann-Pick disease have only 0-2% of lysosomal sphingomyelinase activity measured in vitro, three cell lines from type B Niemann-Pick disease could metabolize 54.4% of the labeled sphingomyelin by day 3 while cell lines from type A Niemann-Pick disease could only metabolize 18.5% by day 3. This compares to 86.7% metabolized in control cells by day 3. Cells from one patient with juvenile Niemann-Pick disease and one with type D Niemann-Pick disease metabolized sphingomyelin normally while cells from two other patients with juvenile or type C Niemann-Pick disease could only metabolize 58.2% by day 3. Cells from patients with I-cell disease and 'lactosylceramidosis' also demonstrated decreased metabolism of sphingomyelin (55.1 and 54.9% by day 3, respectively). Cells from the patient with Farber disease accumulated [14C]stearic acid-labeled ceramide produced from [14C]sphingomyelin. Studies with choline-labeled sphingomyelin and phosphatidylcholine demonstrated that phosphocholine exchange takes place in either direction in the cells, and this is normal in Niemann-Pick disease. Studies in cells from patients with all clinical types of sphingomyelinase deficiency have led to new methods for diagnosis and prognosis and to a better understanding of sphingomyelin metabolism.     

Acid lipase cross-reacting material in Wolman disease and cholesterol ester storage disease

Material cross-reacting with antibodies to acid lipase was demonstrated in fibroblasts of three patients with Wolman disease and three with cholesterol ester storage disease. Quantitation of the immunologically cross-reacting material (CRM) by a single radial immunodiffusion method revealed normal levels in both mutant cell types. CRM specific activity toward triolein and cholesteryl oleate was reduced about 200-fold in the Wolman disease fibroblasts and 50- to 100-fold in the cholesterol ester storage disease cells when compared to normal.     

Lysosomal acid hydrolases in established lymphoblastoid cell lines,transformed by Epstein-Barr virus,from patients with genetic lysosomal storage diseases

Summary Lysosomal acid hydrolases were determined in established lymphoblastoid cell lines, transformed in vitro by Epstein-Barr virus (EBV) from lymphocyte-rich cell populations isolated from the peripheral blood of patients with genetic lysosomal storage diseases—Hurler syndrome, Scheie syndrome, G_M1-gangliosidosis type 1 and type 2, Tay-Sachs disease, and I-cell disease—and from obligate heterozygotes for these diseases.The respective enzyme activity was undectectable in lymphoblastoid cells from the patients, but not from controls. Obligate heterozygotes could not always be distinguished from controls in lymphoblastoid cells as well as in leukocytes. These results suggest that established lymphoblastoid cell lines are useful material for the enzymatic study of genetic lysosomal storage diseases.     

Production and characterization of a monoclonal antibody that binds Reed-Sternberg cells

A murine monoclonal antibody, termed HeFi-1, was produced after immunization with the L428 Hodgkin's disease tissue culture cell line. HeFi-1 selectively stained only the Reed-Sternberg or Hodgkin's cells in 18 of 18 cases of Hodgkin's disease, including the nodular sclerosis, mixed cellularity, and lymphocyte-depleted histologic subtypes. HeFi-1 did not stain any cells in normal lung, brain, salivary gland, thyroid, gall bladder, pancreas, liver, testis, breast, endometrium, or kidney. Rare large cells at the edge of the lymphoid follicles were stained in normal tonsil, colon, and hyperplastic thymus. There was no staining of any cells in 14 cases of B cell non-Hodgkin's lymphoma; however, the malignant cells in three of 11 cases of non-Hodgkin's lymphoma which appeared to express T cell markers were also stained with HeFi-1. Tissue culture cell lines including the T cell acute lymphocytic leukemia lines MOLT4 and CEM, the histiocytic cell line U-937, and the amniotic cell line WISH were not stained. Seven Epstein Barr virus (EBV)-positive lymphoblastoid cell lines were stained with HeFi-1, but there was no staining of three EBV+ African Burkitt's lymphoma cell lines or three EBV- American Burkitt's cell lines. HeFi-1 did not block the ability of the L428 cells to stimulate a mixed lymphocyte reaction or function as accessory cells for mitogen-induced human T cell proliferative responses. Modulation of the HeFi-1 cell surface antigen on the L428 cells was not observed. HeFi-1 specifically immunoprecipitated a cell surface protein of approximately 120,000 daltons from both the L428 and EBV+ lymphoblastoid cell lines. HeFi-1 monoclonal antibody should prove useful not only in the diagnosis, staging, and potential therapy of Hodgkin's disease, but also for determining the cell of origin of the Reed-Sternberg cell.     

Metabolic capacity regulates iron homeostasis in endothelial cells

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.     

Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient

Human lymphoid cell lines established from normal subjects and from a Niemann-Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl)pyrophosphate. The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse3Cer), as visualized by bidimensional thin-layer chromatography. Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease.     

DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients

In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.     

Neutral lipid storage disease: a possible functional defect in phospholipid- linked triacylglycerol metabolism

Neutral lipid storage disease (NLSD) (Chanarin-Dorfman Syndrome) is an autosomal recessive disorder of multisystem triacylglycerol (TAG) storage. Previous work has pointed to a defect in intracellular TAG metabolism. In the studies reported here, the lipid metabolism of three lines of NLSD fibroblasts were compared to normal skin fibroblasts. When pulsed with [3H]oleic acid, the earliest observed abnormality in NLSD cell lines was increased incorporation into phosphatidylethanolamine, followed by accumulation of radiolabel in TAG. Activities of several glycerolipid synthetic enzymes were comparable in NLSD and normal fibroblast lines, excluding oversynthesis of glycerolipid. The proportion of plasmalogen and neutral ether lipid synthesized was normal and alkylglycerols did not accumulate, excluding a defect in ether lipid metabolism. Activities of both acid lipase and Mn2(+)-sensitive lipase within the particulate fractions of NLSD and normal fibroblasts were comparable. These studies are most consistent with functional deficiency of a TAG lipase with activity against a pool of TAG that are normally utilized for phospholipid biosynthesis.     

Definitive prenatal diagnosis for type III glycogen storage disease.

Prenatal diagnosis for type III glycogen storage disease was performed by using (1) immunoblot analysis with a polyclonal antibody prepared against purified porcine-muscle debranching enzyme and (2) a qualitative assay for debranching-enzyme activity. Cultured amniotic fluid cells from three pregnancies (three families in which the proband had absence of debrancher protein) were subjected to immunoblot analysis. Two unaffected and one affected fetus were predicted. In addition, cultured amniotic fluid cells from nine pregnancies (eight families) were screened with a qualitative assay based on the persistence of a polysaccharide that has a structure approaching that of a phosphorylase limit dextrin when the cells were exposed to a glucose-free medium. This qualitative assay predicted six unaffected and three affected fetuses. All predictions by either method were confirmed postnatally except for one spontaneously aborted fetus. Our data indicate that a definitive diagnosis of type III glycogen storage disease can be made prenatally by these methods.     

Neutral lipid storage disease: a possible functional defect in phospholipid-linked triacylglycerol metabolism

Neutral lipid storage disease (NLSD) (Chanarin-Dorfman Syndrome) is an autosomal recessive disorder of multisystem triacyglycerol (TAG) storage. Previous work has pointed to a defect in intracellular TAG metabolism. In the studies reported here, the lipid metabolism of three lines of NLSD fibroblasts were compared to normal skin fibroblasts. When pulsed with [³H]oleic acid, the earliest observed abnormality in NLSD cell lines was increased incorporation into phosphatidylethanolamine, followed by accumulation of radiolabel in TAG. Activities of several glycerolipid synthetic enzymes were comparable in NLSD and normal fibroblast lines, excluding oversynthesis of glycerolipid. The proportion of plasmalogen and neutral ether lipid synthesized was normal and alkylglycerols did not accumulate, excluding a defect in ether lipid metabolism. Activities of both acid lipase and Mn²⁺-sensitive lipase within the particulate fractions of NLSD and normal fibroblasts were comparable. These studies are most consistent with functional deficiency of a TAG lipase with activity against a pool of TAG that are normally utilized for phospholipid biosynthesis.     

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols. In contrast with P10 and P12, P4 was not incorporated into neutral lipids. When the cells were incubated for 24 h with the pyrene fatty acids, the amount of fluorescent lipids synthesized by the cells was proportional to the fatty acid concentration in the culture medium. After a 24 h incubation in the presence of P10 or P12, at any concentration, the fluorescent triacylglycerol content of MLSM cells was 2-5-fold higher than that of control cells. Concentrations of pyrene fatty acids higher than 40 microM seemed to be more toxic for mutant cells than for control cells. This cytotoxicity was dependent on the fluorescent-fatty-acid chain length (P12 greater than P10 greater than P4). Pulse-chase experiments permitted one to demonstrate the defect in the degradation of endogenously biosynthesized triacylglycerols in MLSM cells (residual activity was around 10-25% of controls on the basis of half-lives and initial rates of P10- or P12-labelled-triacylglycerol catabolism); MLSM lymphoid cells exhibited a mild phenotypic expression of the lipid storage (less severe than that observed in fibroblasts). P4 was not utilized in the synthesis of triacylglycerols, and thus did not accumulate in MLSM cells: this suggests that natural short-chain fatty acids might induce a lesser lipid storage in this disease.     

Development and characterization of three new diploid cell lines from Labeo rohita (Ham.)

Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28°C in Leibovitz‐15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Detection of T-cell surface determinants in three Marek's disease lymphoblastoid cell lines.

The T-cell surface antigens on three lymphoblastoid cell lines, MOB1, MOB-2 and MSB-1, derived from Marek's disease lymphomas were examined by the cytotoxicity test and the indirect membrane immunofluorescent test. These cell lines reacted specifically with antisera prepared against chicken thymus cells, although their reactivities were less than that of typical thymus cells. These three cell lines were of thymus origin.     

Ratliver cells in culture: Effect of storage,long-term culture,and transformation on some enzyme levels

Summary Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.     

Clinical and genetic characterization of Chanarin-Dorfman syndrome

We describe the clinical features, muscle pathology features, and molecular studies of seven patients with Chanarin-Dorfman syndrome (CDS) or neutral lipid storage disease and ichthyosis (NLSDI), a multisystem triglyceride storage disease with massive accumulation of lipid droplets in muscle fibers.All patients presented with congenital ichthyosiform erythroderma, cytoplasmic lipid droplets in blood cells, mild to severe hepatomegaly, and increased serum CK levels and liver enzymes. Three patients showed muscle symptoms and three had steathorrea. Molecular analysis identified five mutations, three of which are novel.These findings expand the clinical and mutational spectrum and underline the genetic heterogeneity of this disease.     

Cellular resistance to Evans blue toxicity involves an up-regulation of a phosphate transporter implicated in vesicular glutamate storage

It has recently been suggested that the brain-specific Na+-dependent phosphate inorganic co-transporter (BNPI) is able to support glutamate transport and storage in synaptic vesicles. A procedure for measuring the vesicular pool of glutamate is described and was used to select cell lines according to their glutamate storage capacity. Two cell lines were selected: C6BU-1, with a large intracellular glutamate storage capacity, and NG108-15, devoid of it. Their contents in BNPI mRNA were compared by RT-PCR. We found that both cell lines had BNPI, but in addition C6BU-1 alone expresses the other isoform, DNPI. We also carried out a clonal selection of NG108-15 cells in the presence of the dye Evans blue, a competitive inhibitor of vesicular glutamate transport, very toxic for cells in culture. It was assumed that only those that sequester and eliminate the drug by overexpressing a vesicular glutamate transporter would survive. We found that the NG108-15 clones resistant to Evans blue had an increased storage capacity for glutamate. These cells also up-regulated the BNPI isoform of the phosphate transporter as shown by RT-PCR and northern blot.     

Immortalization of murine muscle cells from lysosomal α-glucosidase deficient mice: A new tool to study pathophysiology and assess therapeutic strategies for Pompe disease

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder caused by defects in the acid α-glucosidase (GAA) gene leading to lysosomal glycogen accumulation, mainly in cardiac and muscle tissues. In order to facilitate biological investigation on this disease and to avoid time-consuming direct cell isolation and culture, we have established murine myogenic GSDII cell lines. Lentiviral/retroviral expression of SV40 T antigen, Bmi-1 or cyclin-dependent kinase 4 (CDK4) genes was used to induce the immortalization of primary satellite cells from GSDII mice. The resulting immortalized myoblasts exhibit phenotypic characteristics of their parental cells, including profound GAA deficiency, glycogen accumulation and the ability to fully differentiate into myotubes when placed in proper culture conditions. These cell lines will constitute a powerful tool for both basic and applied studies focused on a better understanding of the pathophysiological mechanisms involved in GSDII and for assessing putative therapeutic strategies.     

Rat liver cells in culture: effect of storage, long-term culture, and transformation on some enzyme levels.

Aryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.