A skin suction blister model in hairless rats: application to the study of anti-inflammatory and immunomodulatory drugs.

A suction blister model was developed in the hairless rat, in order to study the effects of various agents on the migration of polymorphonuclear leukocytes (PMN). A standardized abrasion, a suction blister, was formed by applying negative pressure to the skin and then separating the epidermis from the dermis. A migration chamber containing serum as the chemoattractant was placed over the wound. After 6 h of migration, the cells in the chamber were harvested, counted and identified. We evaluated PMN migration after treating the animals with active compounds: niflumic acid, and anti-inflammatory drug, and RU 41740, an immunomodulator. This in vivo model provided reproducible data and could be used to study further the functional properties of PMN. In addition, because this assay can also be used in man, a drug found to be effective in the animal system could then be tested for its activity in man.

Endocytosis of MHC molecules by B cell-B lymphoma and B cell-T lymphoma hybrids.

Different cells and different cell surface determinants of the same cells take up liposomes, bound to them via monoclonal antibodies, with variable efficiency. We have previously reported that T and B lymphocytes differ in the extent to which they take up liposomes bound to MHC class I molecules; T cells endocytose class I molecules rapidly, but B cells endocytose class I molecules much less efficiently, although their endocytosis of class II molecules is rapid. An approach toward understanding the molecular basis for this difference was made by evaluating the internalization patterns of somatic cell hybrids of B and T cells. Hybrid cells were constructed between LPS-stimulated purified B cell blasts from C57BL/6 mice (H-2b) and the HAT-sensitive AKR T lymphoma BW 5147 (H-2k). Hybrids between the BALB/c B lymphoma M12.4.1 (H-2d) and B cell LPS blasts from B10.BR (H-2k) mice were also evaluated. In all cases, for hybrid tumor cells, liposomes that were bound to class I molecules encoded by genes from the B cell donor were endocytosed as efficiently as liposomes bound to the class I molecules of the recipient lymphoid cell. T cell tumors efficiently internalized their own class I molecules and those donated by B cells; B cell tumors internalized liposomes that were bound to their own and the donor B cell class I molecules poorly. Thus, our results suggest that the internalization of an MHC molecule is not an intrinsic property of the molecule, but rather of the cell in which it is found.

Human insulin prepared by recombinant DNA techniques and native human insulin interact identically with insulin receptors.

Human insulin synthesized from A and B chains separately produced in Escherichia coli from cloned synthetic genes (prepared by the Eli Lilly Research Laboratories, Indianapolis, IN) was characterized by examining its interaction with human cultured lymphocytes, human circulating erythrocytes in vitro, and isolated rat fat cells. The binding behavior of the biosynthetic insulin with human cells was indistinguishable from that of native human or porcine insulins, with respect to affinity, association and dissociation kinetics, negative cooperativity, and the down-regulation of lymphocyte receptors. Similarly, the biosynthetic insulin was as potent as the native insulins in stimulating lipogenesis in isolated rat fat cells. We also examined the receptor binding characteristics of 125I-labeled human and porcine insulins monoiodinated solely at Tyr-A14, which were obtained by means of high-performance liquid chromatography of the iodination reaction mixture (this material was prepared by B. Frank, Eli Lilly Research Laboratories). In all aspects studied, the pure [TyrA14-125I]iodoinsulins were superior as tracers to the monoiodoinsulin purified by the more conventional method of gel filtration.

Receptor binding properties and biologic activity in vitro of biosynthetic human insulin.

The interaction of biosynthetic human insulin with human cultured lymphocytes, human circulating erythrocytes, and isolated rat fat cells was examined. The binding of the biosynthetic insulin to human cells was identical to that of native human or pork insulins, with respect to affinity, kinetics of association and dissociation, negative cooperativity, and downregulation of lymphocyte receptors. The biosynthetic insulin also had equal potency in stimulating the incorporation of [3H]-glucose into the lipids of isolated rat fat cells. These data suggest that the structure of the biosynthetic insulin has been integrally reconstituted.