Flow cytometric analysis of porcine preadipocytes.

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal-vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD-1 cells surface antigen was greater in cultures supplemented with 2% pig serum and 10 nM dexamethasone than in other media. In cultures, GH, TNF-alpha and TGF-beta each inhibited lipid deposition, whereas TNF-alpha and TGF-beta, but not GH, inhibited AD-1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF-alpha and TGF-beta was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.     

The development of adipocytes in primary stromal-vascular culture of fetal pig adipose tissue

Primary cultures of stromal-vascular cells of adipose tissue from fetuses at 70 and 110 days of gestation were evaluated as potential model systems for studies of fetal adipocyte differentiation and proliferation. In the cultures, fat cells developed as very discrete clusters. Fat cell cluster development was dependent on initial cell density and time. Histochemical analysis for NADP-dependent dehydrogenases revealed an age of donor effect. Similar levels of enzymes (malate and glucose-6-phosphate dehydrogenase) were apparent in fat cell clusters and stromal cells in cultures of cells from fetuses at 70 days of gestation. These enzymes were only present in fat cell clusters in cultures of cells from fetuses at 110 days of gestation. The distribution of histochemically detectable esterase activity was dependent on the cell density at time of analysis. In areas of high cell density, esterase was restricted to fat cell clusters whereas, both stromal cells and fat cells were esterase reactive in areas of low cell density. Omitting PMS from the dehydrogenase media revealed differences in enzyme reactions of cells grown on collagen-coated and uncoated glass surfaces. These studies demonstrate that primary cultures of stromal-vascular cells from 110-day-old fetuses would be a useful system to identify factors involved in adipocyte proliferation and differentiation.     

Monoclonal Antibodies against Differentiating Mesenchyme Cells in Larvae of the Ascidian Halocynthia roretzi

Mechanisms of cell specification of mesenchyme during ascidian embryogenesis are poorly understood. This is because no good molecular markers have been available to evaluate differentiation of the mesenchyme cells. To obtain molecular markers of mesenchyme differentiation, we established monoclonal antibodies, Mch-1 and Mch-3, that recognize antigens present in the mesenchyme cells of the larva of Halocynthia roretzi. The antigens recognized by both antibodies start to be detectable in the mesenchyme cells at the late tailbud stage. The Mch-3 antibody specifically recognized all mesenchyme cells of the larva, whereas the Mch-1 antibody stained the cells only in the anterior portions of mesenchyme clusters in the trunk region of the larva. The Mch-1 antibody also stained trunk lateral cells. In addition, both antibodies recognized the mesenchyme cells in the ventro-lateral boundary between endoderm and epidermis that are migrating to the anterior head region of the larva. The partial embryos that originated from the mesenchymelineage cells at the 8-cell stage expressed the Mch-1 and Mch-3 antigens. The Mch-1 and Mch-3 antibodies will be useful as immunological probes for studying the specification mechanisms of mesenchyme cells.     

Induction of proliferation in vitro of resting human natural killer cells: expression of surface activation antigens

In this report, we show that resting human peripheral blood NK cells, positively enriched with antibody B73.1, can be induced to proliferate in vitro in short-term cultures, and newly express membrane activation antigens. B73.1+ NK cells, cultured for 6 days in the presence of conditioned medium containing IL 2 and irradiated B lymphoblastoid cells, show significant [3H]TdR incorporation beginning on day 4. At that time, a large proportion of the cells express HLA-DR and 4F2 antigens, and transferrin and IL 2 receptors, all detectable at high density by indirect immunofluorescence with the use of monoclonal antibodies. These cells maintain their ability to lyse target cells spontaneously or in the presence of antibodies. By two-color immunofluorescence and cell cycle analysis combined with indirect immunofluorescence, we demonstrate directly that the activation antigens are expressed on all NK (B73.1+) cells in the S/G2/M phases of the cell cycle, and on only a proportion of those in the G0/G1 phases.     

Long-term culture of human bone marrow macrophages: macrophage development is associated with the production of granulomonopoietic enhancing activity (GM-EA)

We describe a liquid culture system allowing the long-term maintenance and differentiation of human marrow monocytes into well-developed, lipid-containing macrophages, and we show that these cells produce a regulating activity that enhances the colony formation induced by granulocyte-macrophage colony-stimulating factors (GM-CSF). Adherent, low density (less than 1.074 g/cm3) human marrow cells were used as a source of monocytic cells. Of 12 different culture conditions tested, alpha-medium supplemented with 20% horse serum and incubation at 37 degrees C provided the best conditions for macrophage development, adipogenesis, and long-term culture. Neither insulin (1 to 10 micrograms/ml) nor hydrocortisone (10(-6) M) improved the lipid accumulation in cultures containing horse serum. Trypsin was employed to remove fibroblasts without detaching monocytic cells from the marrow-derived adherent cell layers. Marked structural and functional changes characterized the transformation of monocytes into lipid-containing macrophages. Cell enlargement up to seven or eight times by 21 to 28 days of culture was associated with an increase in small and medium-sized lipid granules, as well as in acid phosphatase and nonspecific esterase activities. Ultrastructurally, there was an increase in the number of mitochondria, Golgi apparatus, lysosomes, rough endoplasmic reticulum, and lipid inclusions, which remained of small size and did not coalesce to form larger inclusions. The absolute quantity of Fc receptors, Ia antigens, and antigens recognized by monoclonal antibodies 61D3 and 63D3 also increased as a function of cell size. As marrow monocyte-macrophage differentiation proceeded, a rapid decline in GM-CSF production was accompanied by an increase in an activity that by itself had no capacity to stimulate colony formation in CFU-GM cultures devoid of GM-CSF, but did enhance the colony formation induced by optimal concentrations of GM-CSF. Neither the enhancing activity nor its production was related to the horse serum present in the culture supernatants. The morphology of colonies enhanced by this activity was different from the morphologic spectrum in non-enhanced cultures. This granulomonopoietic enhancing activity (GM-EA) represents another positive feedback regulator of hematopoiesis derived from cells of the monocyte-macrophage system.     

Monoclonal Antibody-Mediated Cytotoxicity of Adipocytes

Monoclonal antibodies (MAbs) raised against porcine adipocyte plasma membranes were used to demonstrate complement-mediated cytotoxicity of adipocytes and preadipocytes in primary stromal-vascular (SV) cultures. Five of the six MAbs tested significantly reduced the number of fat cell clusters in cultures maintained in medium supplemented with pig serum and dexamethasone (PS/DEX) but not in cultures supplemented with insulin, transferrin, and selenium (ITS). Neither MAb nor complement alone affected fat cell cluster number. Treatment of both ITS and PS/DEX cultures with pools of 2 or more MAbs, in combination with complement, eliminated fat cell clusters in all instances. Treatment of cultures prior to appearance of cells containing lipid demonstrated that preadipocytes, or adipose lineage cells, could also be eliminated by MAb/complement treatment. Finally, injection of young rats with a pool of three of the MAbs produced a 30% reduction in inguinal fat pad weight without affecting other tissues. Adipocyte/ preadipocyte-depleted cultures can now be used as a model system to examine progression of cells through the adipose cell lineage at a time not previously possible with primary cells.     

The gene expression of the myocardial lipid droplet protein is highly regulated by PPARγ in adipocytes differentiated from MEFs or SVCs

We demonstrate that expression of the myocardial lipid droplet protein (MLDP) and ERα observed in adipose tissues is undetectable in 3T3-L1 cells but detectable in mouse embryonic fibroblasts (MEFs) and stromal-vascular cells (SVCs) during adipocyte differentiation. MLDP gene expression in MEFs or SVCs is induced by treatment with a PPARγ agonist or forced expression of PPARγ, indicating that PPARγ enhances MLDP expression during adipogenesis. PCR analyses reveal the dual expression of SREBP-1a and SREBP-1c in MEFs and SVCs as well as white adipose tissues unlike the predominant expression of SREBP-1a in 3T3-L1 cells. These results suggest that MEFs and SVCs are useful model cells for examining function of MLDP in lipid droplet formation and adipocyte differentiation.     

Cell surface antigens during submerged development of Myxococcus xanthus examined with monoclonal antibodies

Eighteen monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus were followed by enzyme-linked immunosorbent assay. Three of the monoclonal antibodies were specifically directed against antigens present only on cells undergoing fruiting body development. These cell surface antigens became detectable by the early preaggregation stage (2 to 4 h) of development and increased until early aggregation (9 to 10 h), after which the concentrations of two of the cell surface antigens remained constant and the concentration of the third decreased. The remaining 15 monoclonal antibodies recognized cell surface antigens that were shared by vegetative and developing cells. Based on their relative concentrations during development, these shared antigens can be grouped into three classes. In the first class antigen concentration remained constant, in the second it decreased, and in the third it increased. Western blots of cell surface antigens were probed with monoclonal antibodies. Five monoclonal antibodies reacted with material in distinct bands, five monoclonal antibodies reacted with multiple, diffuse bands, and eight monoclonal antibodies were not reactive in Western blots.     

Retinol-Binding Protein 4 and Its Membrane Receptor STRA6 Control Adipogenesis by Regulating Cellular Retinoid Homeostasis and Retinoic Acid Receptor α Activity

Retinoids are vitamin A (retinol) derivatives and complex regulators of adipogenesis by activating specific nuclear receptors, including the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Circulating retinol-binding protein 4 (RBP4) and its membrane receptor STRA6 coordinate cellular retinol uptake. It is unknown whether retinol levels and the activity of RAR and RXR in adipocyte precursors are linked via RBP4/STRA6. Here, we show that STRA6 is expressed in precursor cells and, dictated by the apo- and holo-RBP4 isoforms, mediates bidirectional retinol transport that controls RARα activity and subsequent adipocyte differentiation. Mobilization of retinoid stores in mice by inducing RBP4 secretion from the liver activated RARα signaling in the precursor cell containing the stromal-vascular fraction of adipose tissue. Retinol-loaded holo-RBP4 blocked adipocyte differentiation of cultured precursors by activating RARα. Remarkably, retinol-free apo-RBP4 triggered retinol efflux that reduced cellular retinoids, RARα activity, and target gene expression and enhanced adipogenesis synergistically with ectopic STRA6. Thus, STRA6 in adipocyte precursor cells links nuclear RARα activity to the circulating RBP4 isoforms, whose ratio in obese mice was shifted toward limiting the adipogenic potential of their precursors. This novel cross talk identifies a retinol-dependent metabolic function of RBP4 that may have important implications for the treatment of obesity.     

Monoclonal Antibodies Specific for Members of the Genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni , M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas. Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni , M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64–200 kD.     

Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system

Four monoclonal antibodies are characterized that have been obtained from a fusion of mouse myeloma P3-NS1/1-Ag4-1 with spleen cells from BALB/c mice immunized with white matter from bovine corpus callosum. The corresponding antigens (O antigens) are designated O1, O2, O3, and O4. The localization of these antigens was investigated by indirect immunofluorescence in cultures of early postnatal mouse cerebellum, cerebrum, spinal cord, optic nerve, and retina. When tested on live cultures none of the O antibodies reacted with the surface of astrocytes, neurons, or fibroblasts, however, all are positive on the surface of oligodendrocytes. The identity of these cells was determined by double-immunolabeling experiments with indpendent cell-type-specific antigenic markers (glial fibrillary acidic protein, tetanus toxin receptors, fibronectin, and galactocerebroside). Antigen O1 is exclusively expressed on galactocerebroside-positive cells, whereas O2, O3, and O4 are expressed on additional cells that are negative for any of the markers tested. None of the O antigens is expressed on the surface of cultured retinal cells. In fresh-frozen sections of adult mouse cerebellum all O antigens are detectable in white matter tracts and in vesicular structures of the granular layer. O2 and O3 antigens are in addition detectable in GFA protein-positive radial fibers in the molecular layer. In fixed cerebellar cultures, where intracellular antigens are accessible, O1, O2, and O3 antibodies label astrocytes in a GFA protein-like pattern. O antigens are expressed in mouse, rat, chicken, and human central nervous systems. O antibodies belong to the IgM immunoglobulin subclass and have been used in complement-dependent cytotoxic elimination of cerebellar oligodendrocytes in culture. At limiting antibody dilutions all processes of oligodendrocytes are preferably lysed over cell bodies.     

Monoclonal antibodies specific for members of the genus Endotrypanum

Twenty-six monoclonal antibodies were produced against membrane-enriched preparations of Endotrypanum schaudinni or Endotrypanum sp. promastigotes. Fifteen of these monoclonal antibodies (E1-E15) reacted only with the standard strain of E. schaudinni, M6159. Monoclonal antibodies E16-E26 were considered Endotrypanum specific; no cross reactivity was detected with any other genus of the family Trypanosomatidae (Leishmania, Trypanosoma, Leptomonas, Herpetomonas or Crithidia) by dot-blot radioimmune assay. By indirect immunofluorescence assay, the antigens recognized by Endotrypanum specific monoclonal antibodies appear to be associated with the surface of the parasite. Based on Western blot analysis, 4 antigenic molecules ranging in molecular weight from 24 kD to 160 kD were identified by monoclonal antibodies specific for the strain of E. schaudinni, M6159. Monoclonal antibodies specific for the genus Endotrypanum identified an antigen of molecular weight 48 kD as well as a diffuse component migrating with an apparent molecular weight of 64-200 kD.     

Ontogeny and characterization of mesenchyme antigens of the sea urchin embryo

A monoclonal antibody, Sp12, binds to cortical granules, the hyaline layer, and skeletogenic, chromogenic, and blastocoelar mesenchyme of sea urchin eggs and embryos. Adult urchins also express Sp12 antigens in the dermal layer of the test and spines. Antigen is expressed on the surface of primary mesenchyme cells after they have entered the blastocoel, and by two secondary mesenchyme derivatives--the blastocoelar cells after they have been released from the tip of the archenteron, and the pigment cells in prism stage embryos. Immunogold localizations show antigen on the surfaces of mesenchyme, within membrane bounded vesicles, and associated with the Golgi apparatus. Western blots of antigens immunoprecipitated from seven developmental stages reveal twelve antigens ranging in Mr from 35 k to 240 k. Most of these antigens appear, disappear or change Mr over the first five days of development. Characterizations of this complex array of antigens show that the epitope recognized by Sp12 is eliminated by proteolytic enzymes and endoglycosidase F, while immunoreactivity is only reduced by periodate oxidation. As well, calcium magnesium free seawater extracts a subset of antigens different from that retained by crude membrane preparations. It is proposed that the mesenchyme of sea urchin embryos produces a family of developmentally regulated cell surface and extracellular matrix glycoproteins which all exhibit a carbohydrate epitope recognized by Sp12.     

Analysis of glia cell differentiation in the developing chick peripheral nervous system: sensory and sympathetic satellite cells express different cell surface antigens.

To identify and analyse precursor cells of neuronal and glial cell lineages during the early development of the chick peripheral nervous system, monoclonal antibodies were raised against a population of undifferentiated cells of E6 dorsal root ganglia (DRG). Non-neuronal cells of E6 DRG express surface antigens that are recognized by four monoclonal antibodies, G1, G2, GLI 1 and GLI 2. The proportion of non-neuronal cells in DRG that express the GLI 1 antigen is very high during ganglion formation (80% at E4) and decreases during later development (15% at E14). GLI 2 antigen is expressed only on a minority of the cells at E6 and increases with development. The G1 and G2 antigens are expressed on about 60-80% of the cells between E6 and E14. All cells that express the established glia marker O4 are also positive for the new antigens. In addition, it was demonstrated that GLI 1-positive cells from early DRG, which are devoid of O4 antigen, could be induced in vitro to express the O4 antigen. Thus, the antigen-positive cells are considered as glial cells or glial precursor cells. Surprisingly, the antigen expression by satellite cells of peripheral ganglia is dependent on the type of ganglion: antigens G1, G2 and GLI 1 were not detectable on glial cells of lumbosacral sympathetic ganglia and GLI 2 was expressed only by a small subpopulation. These results demonstrate an early immunological difference between satellite cells of sensory DRG and sympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of a panel of monoclonal antibodies to Toxoplasma gondii antigens

A representative collection was obtained containing 68 monoclonal antibodies (MAB) to Toxoplasma gondii antigens, which was characterized by the binding with the below fractions of tochizoites in the immune-enzyme assay (IEA) and immunoblotting (IB): membrane (MEM), somatic (water-soluble, SOM) and excretory-secretory (ES). Most of MABs were produced to MEM antigens (43), 6 MABs reacted with the somatic fraction, and 3 MABs reacted with both fractions. Two MABs to ES antigen were detected in the latter group. An analysis of MABs in concurrent IEA and IB revealed the immune-dominant proteins of the MEM and SOM fractions of antibodies to T. gondii tochizoites (p30 and p27, respectively). The presence of 2 non-overlapping antigenic determinants was shown for p30. Further research would detect MABs that could be used in the diagnosis of toxoplasmosis.     

Identification of Monoclonal Antibody At5 as a New Member of HNK-1 Antibody Family: The Reactivity with Myelin-Associated Glycoprotein and with Two Brain-Specific Proteoglycans,Phosphacan and Neurocan

Monoclonal antibody At5 was primarily developed against chordin, a notochord-specific antigen of Acipenseridae (sturgeon fishes). In higher vertebrates the antibody reacted mainly with neural tissue antigens. In this study we have shown that the specificity of monoclonal antibody At5 is similar to that of antibodies of HNK-1 family which react with two glycolipids and with several high molecular weight glycoconjugates of neural tissue. We have demonstrated by protein sequencing and immunoblotting that one of At5 target antigens of human brain is dMAG, a derivative of myelin-associated glycoprotein. In the preparations of At5 antigens proteoglycans phosphacan and neurocan were identified by immunoblotting with specific monoclonal antibodies 6B4 and 1G2, respectively. The distribution of At5 and 6B4 immunoreactivity was studied on sections of mixed oligoastrocytoma. Oligodendroglioma area of this tumor was intensely stained with both antibodies, whereas astrocytoma area did not exhibit any At5 or 6B4 immunoreactivity.     

Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue

We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro. This work was supported by grants from the Japan Ministry of Education, Science, Sports, and Culture (no. 19580348) and from MEXT. HAITEKU (2007–2011).     

Detection of avian hematopoietic cell surface antigens with monoclonal antibodies to myeloid cells : Their distribution on normal and leukemic cells of various lineages

The isolation and characterization of monoclonal antibodies reacting with cell surface antigenic determinants of normal and leukemic avian hematopoietic cells is described. The antibodies were produced by immunizing mice with normal macrophages, as well as with myeloid cells transformed with the avian acute leukemia viruses MC29, AMV and E26. Eleven antibodies were characterized for their reactivity with a variety of normal and leukemic cells of the myeloid, B- and T-lymphoid and of the erythroid cell lineage. Using several methods, they could be subdivided into five distinct types: I. Four antibodies were specific for the myeloid lineage, predominantly reacting with immature myeloid cells. II. One antibody reacted with mature and immature myeloid cells as well as with T-lymphoid cells. III. Four antibodies reacted with myeloid, erythroid and T-lymphoid cells. IV. One antibody reacted with myeloid as well as with T- and B-lymphoid cells. V. One antibody reacted with all kinds of chicken hematopoietic cells except erythrocytes. The first type of antibodies detected glycoproteins with MWs of 170 and 130 kD. The pattern of antigens precipitated varied with the different monoclonal antibodies of this group. The antibody of the fourth type precipitated a 30 kD polypeptide from extracts of myeloid and lymphoid cells. None of the other antibodies precipitated any detectable proteins.     

Monoclonal antibodies that distinguish between active and inactive forms of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified from human postheparin plasma. Specific monoclonal antibodies (MAbs) were produced that discriminate between active (native) and inactive (denatured) forms of the enzyme. Mice immunized with native H-TGL resulted in MAbs that recognized only the native protein. The antibodies did not react with H-TGL treated with 1% sodium dodecyl sulfate or heated at 60 degrees C. The loss of immunoreactivity with heating correlated directly with the loss of enzyme activity and there was a corresponding increase in immunoreactivity with the MAbs prepared against the denatured enzyme. Western blot analysis of postheparin plasma with the MAbs against denatured H-TGL gave a single protein band of 65 kD; preheparin plasma showed no detectable immunoreactivity with either MAb. These immunochemical studies suggest that there are no circulating active or inactive forms of H-TGL in man. Furthermore, the MAbs provide the necessary reagents for development of immunoassays for H-TGL.     

Monoclonal antibodies against defined Leishmania antigens protect mice against infection by different species of Leishmania

Mice monoclonal antibodies (IgG) have been raised against Leishmania infantum promastigotes by fusing SP 2/0 myeloma cells and immunized mice splenic cells. The monoclonal antibodies have been detected by indirect immunofluorescence. In vivo tests showed that some of them could inhibit the life cycle of several Leishmania species from the Old and the New World. Studies of these protective monoclonals by the western blot technique showed the presence of three constant antigens (40 kD, 70 kD and 113 kD) amongst the Leishmania species studied.