Nuclear Vav3 is required for polycomb repression complex-1 activity in B-cell lymphoblastic leukemogenesis.

Acute B-cell lymphoblastic leukemia (B-ALL) results from oligo-clonal evolution of B-cell progenitors endowed with initiating and propagating leukemia properties. The activation of both the Rac guanine nucleotide exchange factor (Rac GEF) Vav3 and Rac GTPases is required for leukemogenesis mediated by the oncogenic fusion protein BCR-ABL. Vav3 expression becomes predominantly nuclear upon expression of BCR-ABL signature. In the nucleus, Vav3 interacts with BCR-ABL, Rac, and the polycomb repression complex (PRC) proteins Bmi1, Ring1b and Ezh2. The GEF activity of Vav3 is required for the proliferation, Bmi1-dependent B-cell progenitor self-renewal, nuclear Rac activation, protein interaction with Bmi1, mono-ubiquitination of H2A(K119) (H2AK119Ub) and repression of PRC-1 (PRC1) downstream target loci, of leukemic B-cell progenitors. Vav3 deficiency results in de-repression of negative regulators of cell proliferation and repression of oncogenic transcriptional factors. Mechanistically, we show that Vav3 prevents the Phlpp2-sensitive and Akt (S473)-dependent phosphorylation of Bmi1 on the regulatory residue S314 that, in turn, promotes the transcriptional factor reprogramming of leukemic B-cell progenitors. These results highlight the importance of non-canonical nuclear Rho GTPase signaling in leukemogenesis.

The signaling axis atypical protein kinase C λ/ι-Satb2 mediates leukemic transformation of B-cell progenitors.

Epigenetically regulated transcriptional plasticity has been proposed as a mechanism of differentiation arrest and resistance to therapy. BCR-ABL leukemias result from leukemic stem cell/progenitor transformation and represent an opportunity to identify epigenetic progress contributing to lineage leukemogenesis. Primary human and murine BCR-ABL+ leukemic progenitors have increased activation of Cdc42 and the downstream atypical protein kinase C (aPKC). While the isoform aPKCζ behaves as a leukemic suppressor, aPKCλ/ι is critically required for oncogenic progenitor proliferation, survival, and B-cell differentiation arrest, but not for normal B-cell lineage differentiation. In vitro and in vivo B-cell transformation by BCR-ABL requires the downregulation of key genes in the B-cell differentiation program through an aPKC λ/ι-Erk dependent Etv5/Satb2 chromatin repressive signaling complex. Genetic or pharmacological targeting of aPKC impairs human oncogenic addicted leukemias. Therefore, the aPKCλ/ι-SATB2 signaling cascade is required for leukemic BCR-ABL+ B-cell progenitor transformation and is amenable to non-tyrosine kinase inhibition.

Bio-distribution of pharmacologically administered recombinant factor VIIa (rFVIIa).

BACKGROUND: Recent clinical studies suggest that the prophylactic use of recombinant factor VIIa (rFVIIa) markedly reduces the number of bleeding episodes in hemophilic patients with inhibitors. Given the short biological half-life of rFVIIa, it is unclear how rFVIIa could be effective in prophylactic treatment. OBJECTIVES: To examine the extravascular distribution of pharmacologically administered rFVIIa to obtain clues on how rFVIIa could work in prophylaxis. METHODS: Recombinant mouse FVIIa tagged with AF488 fluorophore (AF488-FVIIa) was administered into mice via the tail vein. At different time intervals following the administration, mice were exsanguinated and various tissues were collected. The tissue sections were processed for immunohistochemistry to evaluate distribution of rFVIIa. RESULTS: rFVIIa, immediately following the administration, associated with the endothelium lining of large blood vessels. Within 1 h, rFVIIa bound to endothelial cells was transferred to the perivascular tissue surrounding the blood vessels and thereafter diffused throughout the tissue. In the liver, rFVIIa was localized to sinusoidal capillaries and accumulated in hepatocytes. In bone, rFVIIa was accumulated in the zone of calcified cartilage and some of it was retained there for a week. The common finding of the present study is that rFVIIa in extravascular spaces was mostly localized to regions that contain TF expressing cells. CONCLUSIONS: The present study demonstrates that pharmacologically administered rFVIIa readily associates with the vascular endothelium and subsequently enters into extravascular spaces where it is likely to bind to TF and is retained for extended time periods. This may explain the prolonged pharmacological effect of rFVIIa.

The spectrum of canine cutaneous perivascular wall tumors: morphologic, phenotypic and clinical characterization.

Perivascular wall tumors (PWTs) are defined as neoplasms deriving from mural cells of blood vessels, excluding the endothelial lining. The spectrum of human cutaneous PWT includes glomus tumor, hemangiopericytoma (HEP), myopericytoma, angioleiomyoma/sarcoma, angiomyofibroblastoma, and angiofibroma. The purpose of this study was to revise clinical presentation, cytology, histopathology, and immunohistology of canine cutaneous PWT with cytology typical of canine HEP. Diagnosis was established on the basis of vascular growth patterns (staghorn, placentoid, perivascular whorling, bundles from media) and immunohistology, including 7 smooth muscle markers and the cell membrane ganglioside of unknown origin recognized by the antibody 3G5 (CMG-3G5). Twenty cases were included. Ages ranged from 6 to 13 years; 12 dogs were males and 8 were females, and there was a prevalence of crossbreeds. Tumors arose from a single site with preferential acral location (10/20). Cytology revealed moderate to high cellularity in all cases, cohesive groups of cells (19/20), capillaries (18/20), and bi- to multinucleated cells (18/20). Six myopericytomas, 5 angioleiomyomas, 2 angioleiomyosarcomas, 2 HEP, 1 angiofibroma, and 1 adventitial tumor were identified. A definitive diagnosis was not possible in 3 cases. Smoothelin, heavy caldesmon, desmin, myosin, calponin, and CMG-3G5 were the most valuable markers to differentially diagnose canine PWT. Similar to reports in humans, canine HEP embodied a spectrum of neoplastic entities arising from different vascular mural cells. Before canine PWTs are assimilated into one prognostic category, a consistent classification and characterization of their biology is necessary. As proposed in humans, HEP should also be considered a diagnosis of exclusion in dogs.

Circulating anti-pericyte autoantibodies are present in Type 2 diabetic patients and are associated with non-proliferative retinopathy.

AIMS/HYPOTHESIS: This study aims to determine the prevalence of anti-pericyte autoantibodies in Type 2 diabetes and to characterize these autoantibodies as new markers of disease activity in diabetic retinopathy. METHODS: A total of 299 patients with Type 2 diabetes participated in this study. Retinopathy was assessed by 7-field stereo fundus photography and was graded according to the ETDRS scale. Serum anti-pericyte autoantibodies were detected by immunofluorescence on tissue cultured bovine retinal pericytes. RESULTS: The prevalence of anti-pericyte autoantibodies in Type 2 diabetic patients was 54% and was approximately equal in men and women. The prevalence was approximately 55% with retinopathy at grades from 10 to 53. At grades above 53 the prevalence declined to 23% ( p<0.0001). The highest prevalence by duration of diabetes, 70%, was found at 0 to 5 years and the lowest, 25% at more than 25 years duration ( p<0.0001). CONCLUSION/INTERPRETATION: Anti-pericyte autoantibodies are present at high prevalence in Type 2 diabetes. Their presence during earlier stages of retinopathy could be due to a reaction with antigens expressed by "activated" pericytes. The decline in antibody prevalence in advanced retinopathy could mark pericyte loss and progression to an angiogenic retinal milieu.

Human dermal pericytes express 3G5 ganglioside--a new approach for microvessel histology in the skin.

BACKGROUND: Pericytes cover the abluminal surface of microvessels and play an important role in capillary regulation and pathology. Studies on pericytes have been hindered by the lack of specific markers with which to facilitate microscopic identification of this cell type. Expression of the cell surface 3G5 ganglioside antigen has been reported in cultured retinal and cardiac pericytes. The objective of this study was to determine the usefulness of monoclonal antibody 3G5 as a pericyte marker in human skin. METHODS: Cryosections of 21 skin biopsies were examined by direct fluorescence technique with anti-3G5, anti-von Willebrand factor, anti-alpha-smooth muscle actin or DNA fluorochrome. RESULTS: In human dermis, 3G5 expression is limited to pericytes discriminating this cell type from all other cells including smooth muscle cells, myofibroblasts and myoepithelial cells. We found a pericyte: endothelial cell ratio of 1:12.4 (+/-7.1), and a difference of alpha-smooth muscle actin expression between the subpapillary plexus and the microvessels of the Stratum reticulare. CONCLUSIONS: 3G5 mAB is an excellent and so far the only reported tool for identification of dermal pericytes by fluorescent light microscopy. Moreover, this is the first report of the application of 3G5 technique to the microvasculature in tissue sections at the light microscopic level.

Isolation and in vitro characterization of human dermal microvascular pericytes.

Pericytes cover the abluminal surface of capillaries and venules and are thought to play an important role in microvascular regulation and pathology. The purpose of this study was to isolate and characterize human dermal microvascular pericytes (HDMPC), a minor cell type in the skin but a relatively easily obtainable human source of tissue. We developed and compared two procedures that differed in the preselection method. Isolation of dermal microvessel fragments from neonatal foreskins by trypsin digestion was followed by mechanical release of subepidermal tissue, collagenase treatment, and sieving through 100- and 30-microm meshes. After subcultivation, pericytes were preselected either by isolation of outgrowing capillary fragments or by 3G5-coupled magnetic beads. Pericytes were selected finally by cultivation of single cells in endothelial cell-conditioned media. Cultured HDMPC were seen to be large and well spread with irregular edges and prominent stress fibers. They lack contact inhibition, are positive for 3G5 antigen, alpha-smooth muscle actin, and vimentin, and are negative for the endothelial cell marker CD31, diI-acetylated low-density lipoprotein uptake, cytokeratin 5, 6, and 18, and S100 protein. Using both preselection methods, we could establish purified cell cultures of HDMPC. The results of these studies represent the first report of HDMPC isolation.

Bovine retinal microvascular pericytes : isolation, propagation, and identification.

The growth of new capillaries from existing vessels (angiogenesis) is of fundamental importance in wound healing and in pathological situations such as proliferative diabetic retinopathy (1), rheumatoid arthritis (2), and tumor growth. Consequently, considerable interest in vascular cell biology has arisen in apparently disparate clinical and experimental fields. Held in common, however, is the hope that an understanding of the cellular and molecular mechanisms that regulate angiogenesis will lead to novel therapeutic agents and targets.

Immunoelectron microscopic detection of tissue ganglioside antigens.

Glycolipid antigens are emerging as important markers of differentiated cells in vitro and in vivo. The study of the expression of these antigens in whole tissues by immunoelectron microscopy, using standard techniques, does not give acceptable results. We have established conditions for the specific demonstration of antibody binding to tissue glycolipid antigens by immunoelectron microscopy. Dehydration of tissues with alcohol is to be avoided as it extracts the glycolipid antigen out of the tissue. Dehydration in acetone provides good results. Embedding of the tissue in Araldite 512 results in high non-specific binding of the primary antibody and a decreased effective titre of the primary antibody. Embedding of tissues in Lowicryl HM20 resin resulted in low non-specific binding. We also describe a method of curing the Lowicryl resin that does not require a purpose built curing chamber. Quantitative analysis of immunogold binding reveals that acetone dehydration of tissues and embedding in Lowicryl gives greatly superior results in comparison with dehydration in alcohol and embedding in Araldite.

Circulating antipericyte autoantibodies in diabetic retinopathy.

PURPOSE: A number of reports suggest that autoimmune mechanisms may play a role in diabetic microangiopathy, and the existence of circulating antiendothelial cell autoantibodies in diabetic retinopathy has been reported. Because capillary pericyte injury/dysfunction is considered to be an early event in the pathogenesis of diabetic retinopathy and pericyte "drop out" or loss is considered pathognomonic in diabetic retinopathy, we screened diabetic sera for the presence of circulating antipericyte autoantibodies. METHODS: Diabetic sera were screened for the presence of antipericyte autoantibodies by indirect immunofluorescence on tissue cultured bovine retinal pericytes. Analysis of autoantibody prevalence data was performed with 2x2 contingency tables analyzed using Fisher's exact test. RESULTS: Diabetic subjects were found to have autoantibodies to microvascular pericytes in their circulation. The prevalence of these antibodies declines with increasing severity of retinopathy. A peak of antibody prevalence was seen at 5-10 years' duration of diabetes. These autoantibodies were found in both Type I and Type II diabetics and in pred iabetics. CONCLUSIONS: The finding of antipericyte autoantibodies in the circulation of a subpopulation of diabetic subjects suggests that the immune system may play a role in the early pathophysiology of diabetic retinopathy in some patients. These results may contribute to understanding why retinopathy progresses in some patients despite consistent reduction of blood sugar.

Measurement of glucose consumption by hybridoma cells growing in hollow fiber cartridge bioreactors: use of blood glucose self-monitoring devices.

Two different types of blood glucose self-monitoring device, designed for use by diabetics (Accu-Chek Advantage* and Accu-Chek III*), were evaluated for the purpose of monitoring glucose concentration in tissue culture media. The Accu-Chek Advantage* meter was found to systematically overestimate the glucose concentration in a variety of commonly used tissue culture media by 50-90%, in comparison with their formulated glucose concentration. The Accu-Chek III* meter reliably estimated glucose concentrations from 100 to 300 mg/dl and overestimated glucose concentrations above 300 mg/dl. The systematic overestimation of glucose concentration in tissue culture fluids by the Accu-Chek Advantage* meter was further investigated. A standard curve was constructed and the meter reading was found to be linearly related to the actual glucose concentration and the best linear fit was given by the formula y = -43.504 + 1.9246x, where y is the meter reading and x is the actual glucose concentration. Rearranging the equation to make x the subject gave the following algorithm x = (y + 43.504) divided by 1.9246 which could be used to correct the 'raw' meter reading. The mean corrected glucose concentrations deviated from the formulated glucose concentration by less than 3.5% in five media tested, indicating that this meter is more than adequate for monitoring glucose consumption by cells growing in hollow fiber cartridge bioreactors, when used in conjunction with this correction factor.

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique.

BACKGROUND: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. METHODS: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2-3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures -30 degrees C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. RESULTS: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intracellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. CONCLUSIONS: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions.

Characterization of type I IGF receptor and IGF-I mRNA expression in cultured human and bovine glomerular cells.

Glomerular hypertrophy is reported in several endocrine disorders such as acromegaly and diabetes mellitus, where abnormalities of growth hormone and insulin-like growth factor (IGF-I) have been reported. In the present report, we have cultured bovine and human glomerular endothelial cells, and bovine glomerular epithelial and mesangial cells, and characterized the expression of IGF-I mRNA and its receptor in these cells. High affinity, specific receptors for IGF-I were identified in all three types of cells by radioreceptor assays. Receptor number (Ro) derived by Scatchard analysis revealed an unusually high number of Type I IGF receptors, approx. 1.2 x 10(5) receptors/cell in glomerular endothelial cells. Affinity crosslinking studies and immunoprecipitation with antibodies against the Type I IGF receptor identified the alpha-subunit of the IGF-I receptor as having a molecular mass of 140 kDa. Biologically, IGF-I was more potent than insulin or IGF-II in stimulating DNA synthesis in glomerular endothelial cells. Northern blot analysis showed that glomerular and aortic endothelial cells expressed IGF-1 mRNA of 1.7 kb. In contrast, renal glomeruli showed several IGF-1 mRNAs of 7.5, 1.7 and 1.2 kb. Thus, the demonstration of both a prepondence of Type I IGF receptors coupled with the growth promoting effects of IGF-I in glomerular endothelial and epithelial cells, as well as the local production of IGF-I mRNA suggests that IGF-I serves an important role as an autocrine or paracrine regulator of the growth of renal glomeruli.

Role of epicardial mesothelial cells in the modification of phenotype and function of adult rat ventricular myocytes in primary coculture.

Adult rat ventricular myocytes undergo a well-documented sequence of phenotypic changes during adaptation to primary culture. However, we observed that coculture of myocytes with a specific subset of nonmyocyte cardiac cells could slow and even reverse the process of adaptation. These nonmyocyte cells were isolated and identified by immunohistochemical and ultrastructural criteria as being of epicardial mesothelial origin. When added to long-term primary cultures of adult ventricular myocytes, epicardial mesothelial cells appeared to induce myofibrillar arrays that were more organized than those seen in noncocultured myocytes; these changes that occurred were concurrent with the appearance of large amplitude contractions and multicellular synchronous beating that was facilitated by gap junctions between myocytes and epicardial mesothelial cells. The changes in morphology and function were accompanied by a marked increase in beta-myosin heavy chain isoform transcription in cocultured myocytes, a return to the ratio of cardiac to skeletal alpha-actin expected in adult rat myocardium, and a much reduced expression of smooth muscle alpha-actin. These changes in myocyte phenotype and function appeared to require epicardial cell-myocyte contact, or close apposition, because media conditioned by epicardial mesothelial cells alone or in coculture had no effect. Thus, these rapid and reversible changes in myocyte ultrastructure, function, and gene expression may provide a useful in vitro model with which to study the mechanism responsible for regulating the plasticity of ventricular myocyte phenotype and the role of specific cell-cell interactions.

Expression of a monoclonal antibody (3G5) defined ganglioside antigen in the renal cortex.

The monoclonal antibody (mAb) 3G5 was found, by indirect immunofluorescence, to bind to renal cortical structures in frozen sections of human, rat and calf kidneys. Double indirect immunofluorescence studies on frozen sections of rat kidneys showed that 3G5 stained only the glomerulus and the distribution of the 3G5 antigen on the glomerulus was more extensive than the staining observed with antibodies to Factor VIII antigen. 3G5 stained the proximal convoluted tubules and collecting tubules in bovine renal sections but glomeruli did not stain with 3G5. The 3G5 mAb did not stain tissue cultured bovine glomerular endothelial cells or mesangial cells, but did stain bovine glomerular epithelial cell cultures. 3G5 did not stain MDCK cell cultures. The binding of mAb 3G5 to glomeruli was investigated by immunoelectron microscopy of rat renal tissue. In contrast to the podocyte specificity on bovine glomerular cells in vitro, it was found that the specificity of 3G5 expression on rat glomerular cells in vivo was broader. No binding of mAb 3G5 was found outside the glomerulus in the rat renal cortex. Podocytes, endothelial cells and capsular epithelial cells expressed the 3G5 antigen most strongly. A lesser amount of binding was found in the glomerular basement membrane. The mesangium showed a little binding of mAb 3G5 and no binding at all was found to other cortical structures. The 3G5 antigen in rat renal tissue was found to be a glycolipid that migrated between the ganglioside markers GM2 and GM1 by immunostaining of thin layer chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Capping and internalization of a monoclonal antibody-surface antigen complex: a possible mode of interaction of monoclonal antibodies and tumor cells.

Our results demonstrate that upon incubation of 125I-3G5 (a monoclonal IgM against a membrane ganglioside antigen on RINm5F cells) with rat insulinoma RINm5F cell monolayers at 37 degrees C, the IgM is rapidly internalized. Cell-bound radioactivity, detectable within 10 to 15 minutes, reaches a peak at 4 hours. By 24 hours the intracellular radioactivity has decreased to about 37.5% of the 4-hour value, accompanied by an increase in free 125I in the incubation medium. The incubation of 125I-3G5 with RINm5F cell monolayers at 4 degrees C shows that this series of events is inhibited by low temperature. Microautoradiography confirms these events indicating the presence of radiolabeled antibody on the plasma membrane as well as distinct capping processes and diffuse radioactive deposits within the cells as early as 5 to 10 minutes after initiating incubation at 37 degrees C. Electron microscopy autoradiography provides a detailed demonstration of the capping phenomenon and of endocytic vacuoles, followed at later times by the distribution of radioactive deposits throughout the cell. This model constituted by the capping of the 125I-3G5-ganglioside complex on rat insulinoma RINm5F cells may be useful in elucidating a possible mode of interaction of monoclonal antibodies and tumor cells.

Ganglioside expression in human pancreatic islets.

Recent biochemical studies have shown that the cytoplasmic islet cell-antibody autoantigen has properties of a monosialoganglioside (GM). To characterize islet glycolipids and ascertain whether islets express unique gangliosides, we determined the pattern of ganglioside expression in whole human pancreas and isolated human islets using high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of whole human pancreas were GM3, GD3 (disialoganglioside), and in a lesser amount, a GD1a-comigrating ganglioside. In contrast to whole human pancreas, isolated human islets were found to predominantly express GM3, an acidic glycolipid comigrating with GM2, and a ganglioside with mobility between GM2 and GM1 by both HPLC and HPTLC. Quantitation of the major ganglioside UV peaks seen on HPLC gave the following results. In whole pancreas, GM3 represented 66.7% of total gangliosides detected; an asialoglycolipid comigrating with GM2, 2.0%; a ganglioside migrating between GM2 and GM1, 2.6%; GD3, 22.6%; and a GD1a-comigrating ganglioside, 6.1%. In isolated islets, these components were found at the following levels: GM3, 14.9%; GM2-comigrating glycolipid, 74.2%; a ganglioside migrating between GM2 and GM1, 9.8%; GD3, 1.1%; and the GD1a-comigrating ganglioside, not detectable.

Modulation by sodium butyrate of the differentiated status of a clonal pancreatic B-cell line (RIN).

RINmRH cells are a cloned cell line derived from a transplantable rat insulinoma. These cells display only some of the differentiated structure/function features of native pancreatic B-cells. In particular, they do not efficiently or reproducibly express islet B-cell surface antigens, which would otherwise render them useful for screening for the presence of anti-islet cell surface antibodies in the serum of suspected diabetic patients or their relatives. This study examines whether sodium butyrate can enhance expression of B-cell differentiation antigens on RIN cells. RIN cells were exposed to 1,2 or 4 mM butyrate for nine days, and cell growth followed. At 1 mM, butyrate inhibited cell growth by 90%. At the higher concentrations, there was a net loss in the number of cells per culture dish. Exposing the cells to 1 mM or 2 mM butyrate for two days, resulted in a 50% increase in cellular insulin content at the expense of a partial (1 mM) or complete (2 mM) loss of stimulated insulin release in response to glyceraldehyde or serine. A concentration of 1 mM butyrate was therefore used for subsequent studies. The binding to RIN cells of a panel of monoclonal antibodies (mAb's) known to bind native islet cells (R2D6, A2B5, A1D2, 3G5) as well as of serum from a diabetic patient known to carry anti-islet cell antibodies, was screened by cytofluorography or by a radio-binding assay. The relative binding affinity of the mAb's was 3G5 greater than A1D2 greater than A2B5 greater than R2D6. Only 2-3% of the cells were bound by the diabetic patient serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate regulation of glucose transporter function, number, and gene expression by insulin and sulfonylureas in L6 rat skeletal muscle cells.

The extrapancreatic actions of sulfonylureas on the glucose transport system were studied in the L6 line of cultured rat skeletal muscle cells. Insulin (10(-7) M) increased 2-deoxyglucose uptake in differentiated L6 myotubes by 30-40% after 8 h of incubation. The sulfonylurea tolazamide (0.6 mg/ml, 22 h) had no effect on glucose uptake in the absence of insulin, but increased insulin-stimulated 2-deoxyglucose uptake twofold. The total cellular content of glucose transporters was assessed with a monoclonal anti-transporter antibody by a solid-phase ELISA method. Insulin (8 h) increased the quantity of glucose transporters, with a maximal twofold increase at 10(-7) M and a dose-response curve similar to that for insulin stimulation of glucose uptake. In spite of its lack of effect on glucose uptake, tolazamide alone (0.6 mg/ml) increased the cellular content of transporters by 70%. The effects of insulin and tolazamide on transporter gene expression were studied with probes derived from Hep G2 glucose transporter cDNA. Insulin increased the transporter mRNA level 1.7-fold, tolazamide increased it 1.5-fold, and the combination of insulin and tolazamide increased transporter mRNA 3-fold. It is concluded that sulfonylureas, together with insulin, enhance glucose uptake in L6 skeletal muscle cells by increasing the number of functioning glucose transport molecules. The long-term regulation of the glucose transport system in skeletal muscle by insulin and sulfonylureas in vivo may involve similar changes in transporter function, number, and gene expression.

Binding of cytoplasmic islet cell antibodies is blocked by human pancreatic glycolipid extracts.

With biochemical and enzymatic treatment of frozen sections of pancreas, we have previously shown that cytoplasmic islet cell antibodies (ICAs) react with carbohydrate determinants of islet cell glycoconjugates. As a first step toward purifying these glycoconjugates, human pancreas tissue was extracted in a mixture of chloroform and methanol, and the glycolipids were obtained by effecting a Folch partition. The protein pellet, lipid fraction, and glycolipid fraction so obtained were assessed for their ability to block the binding of ICAs to frozen sections of human pancreas, the effect being quantitated with a photometer. Only the glycolipid extract could block ICA binding, and blocking was dose dependent. Subfractionation of the glycolipid extract by hydrophobic interaction on C18 cartridges demonstrated that blocking activity resided in the fraction bound and eluted with methanol, consistent with the autoantigen being a glycolipid. Furthermore, the binding of an anti-islet cell ganglioside monoclonal antibody, 3G5, could be blocked with these extracts, whereas the binding of an anti-islet cell protein monoclonal antibody, 4F2, was unaffected. The major gangliosides of the pancreas were seen to be GM3 and GD3 by thin-layer chromatography (TLC). Fractions scraped and eluted from TLC plates were tested for their ability to block ICA binding to pancreatic sections. Neither GM3- nor GD3-containing fractions could block ICA binding; however, a fraction containing minor pancreatic gangliosides (including GM2) of monosialoganglioside mobility was a potent inhibitor of ICA binding to pancreas sections. TLC of a chloroform-methanol extract of human islets demonstrated that islets differentially express monosialogangliosides (especially GM2).