Role of NKT cells in the digestive system. II. NKT cells and diabetes

Natural killer T (NKT) cells are a subset of regulatory T lymphocytes that recognize glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. NKT cells have been implicated in regulating the progression of Type 1 diabetes (T1D) in human patients and in an animal model for T1D. In addition, glycolipid agonists of NKT cells have been successful in preventing diabetes in mice, raising enthusiasm for the development of NKT cell-based therapies for T1D.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Avian antigen-binding cells. II. Enrichment of antigen-binding B and T cells.

Antigen-binding B and T cells from chicken spleens were selected on plates of antigen-derived gelatin. Hapten-specific B cells from DNP-immune normal chicken spleens were selected on DNP-gelatin. As much as 165-fold functional enrichment of precursors of anti-DNP antibody-producing cells, as measured in an adoptive transfer system, was achieved. However, the enrichment of DNP-binding cells assessed by rosette formation and autoradiography was no more than 25-fold. HGG-specific T cells from bursectomized agammaglobulinemic chickens immunized with deaggregated HGG were selected on HGG-gelatin. In the fraction adherent to HGG-gelatin, at least a 20-fold enrichment of suppressors of the antibody response to TNP-HGG, as measured by adoptive transfer, was accomplished. In contrast, no more than 6-fold enrichment of HGG-binding cells was detected by autoradiography. Antigen-specific depletion and enrichment of suppressor T cells and of HGG-binding cells occurred in parallel, suggesting that suppressor cells can bind soluble antigen and can be isolated on antigen coupled to a solid support.     

Isolation of mouse isometallothioneins. A comparison of isometallothioneins in growing cells and post-mitotic cells.

Mouse metallothioneins (MTs) were separated into three isoforms by an anion-exchange h.p.l.c. column; conventionally isolated MT-1 and MT-2 showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2), respectively. In growing cells, developing hepatocytes and growing tumour cells, MT-1/MT-2 ratios were less than 0.6, irrespective of the type of MT inducer, whereas adult liver post-mitotic cells had a ratio of more than 1.0. A large amount of the MT-2-2 subfraction was found in dexamethasone-treated FM3A cells; 90% of MTs was MT-2-2, suggesting that glucocorticoid hormone mainly induces MT-2-2 in tumour cells.     

Automaticity in cardiac cells.

Automaticity is the result of dynamic changes in transmembrane currents during electrical diastole. It is readily demonstrated in most cardiac cell types. In all four cardiac cell types studied by the voltage clamp technique (Purkinje, ventricular, atrial, and sino-atrial node fibers), the major change detected during diastolic depolarization is a decrease in outward current. This decrease in a repolarizing current (largely potassium mediated) permits an inward current (sodium and/or calcium mediated) to depolarize the cell.All four cardiac cell types appear to possesess a time-dependent potassium conductance which controls the decrease in outward current over the ?70 to ?30 mV potential range. Purkinje fibers manifest an additional conductance which is responsible for automaticity in this type of cell at potentials between ?100 and ?70 mV.     

Fucosyl-glycoprotein and precursor polls in HeLa cells.

An enzymatic-radioactive isotope method has been developed for the direct quantitation of L-fucose in amounts as low at 0.5 plus or minus 0.05 nmol. Fucose kinase is used to transfer [32-P]phosphate from ATP to [3-H]fucose. The labeled enzymatic products are then separated electrophoretically and the amount and specific activity of the fucose are determined from the known specific activity of the phosphate donor. This assay has been used to measure the GDP-L-fucose and macromolecualar fucose in HeLa cells after extraction and purification of the sugar. It has been determined there are 0.5 nmol of GDP-L-fucose in 10-7 cells with a nine- to tenfold dilution of specific activity in converting L-[3-H] fucose to GDP-L-[3-H]fucose. After 2 to 3 days of labeling, the GDP-L-[3-H]fucose pool is essentially at equilibrium with the macromolecular pool, and hence it can be concluded that the dilution of label is due to a nine- to tenfold contribution to GDP-L-fucose from an endogenous source, as compared to exogenously supplied fucose. The fucosyl-glycoprotein pool has been shown to be much larger containing 6 to 8 nmol of fucose in 10-7 cells. It has further been shown that GDP-fucose is the only soluble fucose intermediate present in significant amount.     

Homologous and nonhomologous recombination in monkey cells.

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.     

Hyperosmotic injury in mammalian cells. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures.

Correlated studies on volume distributions and cation (Na⁺ and K⁺) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 mOsm in both the presence and absence of DMSO (5–20%; $\text{w}\text{v}$). The effects superimposed by ouabain (10^?2–10^?4m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the

TABLE 2. Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension

     

Osmolality mOsm	Exposure (min)								Hypertonic agent
					NaCl				Sucrose
		$\text{V}{}^{\mathrm{a}}$	Na+	K+	$\text{S}{}^{\mathrm{b}}$	V	Na+	K+	S
1000	60 or less	Small	High	High	High	Normal	Low	High	High
1500–2000	60 or less	Small	High	Low	Low	Normal	Low	High	High
2000 or over	60 or more	Small or largec	High	Very low	Very low	Small	Very low	Very low	Very low