Lysergic acid diethylamide: radioimmunoassay.

A radioimmnunoassay for d-lysergic acid diethylamide (LSD) is described. Antibodies to LSD were obtained by immunizing rabbits with a conjugate of LSD and human serum albumin. The specificity of the antibody was shown by competitive binding studies. The method has been used to detect the presence of LSD in human urines. Picogram amounts can be measured by this assay.

Structural integration of skin equivalents grafted to Lewis and Sprague-Dawley rats.

Bilayered skin equivalents, composed of a sheet of epidermal cells overlying a collagen lattice populated with fibroblasts, quickly become structurally integrated with the surrounding host skin after grafting to Lewis rats. Three days after transplantation, the skin equivalent lies on a bed of host granulation tissue and is loosely attached to the adjoining host dermis. Blood vessels begin to invade the collagen lattice by 5 days after grafting. By the 7th day a fully keratinized, hypertrophic epidermis covers the surface of the graft and blood vessels penetrate the lattice to the base of the epidermis. Vascularization of the graft is accompanied by activation and proliferation of the fibroblasts and by a condensation of the collagen matrix. During the 2nd week after grafting, the collagen fibrils become organized into thin fibers that show a basketweave pattern of birefringence when examined using polarized light. By 1 month the structure of the skin equivalent has become stabilized. The fibroblasts now resemble the quiescent fibrocytes of normal, resting dermis and the epidermis remains moderately hypertrophic. One to two years after grafting to Sprague-Dawley rats, the skin equivalents do not appear hypertrophic. The graft lacks secondary derivatives such as hair follicles and sweat glands, presumably because the stem cells are lost during the isolation of the epidermal cells. Grafts that are prepared using epidermal cells overlying a collagen gel without fibroblasts give rise to raised, linear scars within 2 weeks.

Fibroblasts in isogeneic skin equivalents persist for long periods after grafting.

We have fabricated skin equivalents by combining fibroblasts from female Fischer rats with collagen to form a lattice and overlaying the lattice with a suspension of epidermal cells. The epidermal cells attach and form a sheet which differentiates. These skin equivalents were then grafted to male Fischer rats in order to follow the fate of the fibroblasts after implantation. Biopsies of the skin equivalent were taken between 9 days and 13 months after grafting and examined histologically or placed in tissue culture to permit karyotyping of the resident fibroblasts. Approximately 82% of the fibroblasts from the graft biopsied at 9 days were female, with this proportion decreasing sharply to 50% at 2 weeks and 60-64% at 1 month. At 1 month, this initial sharp drop is followed by a slow, linear decline which continues through the 13th month when 42% of the fibroblasts are female. We conclude that fibroblasts of the grafted skin equivalent become permanent residents of the skin of the host rat.

Acceptance of allogeneic fibroblasts in skin equivalent transplants.

Living skin equivalents (SE) were prepared by combining cultured fibroblasts with a collagen matrix and overlaying this lattice with keratinocytes. SEs prepared using allogeneic female rat fibroblasts or xenogeneic rabbit or human fibroblasts and keratinocytes isogeneic to the graft recipient were transplanted to recipient male rats. Biopsies of some of these SE grafts were examined histologically at intervals ranging from 5 days to 2 months. Biopsies of other grafts were done, and fibroblasts grown from them were karyotyped to determine the percentage of donor fibroblasts remaining in the graft. SEs containing xenogeneic fibroblasts were rejected. Allografted fibroblasts in SEs were accepted by recipient rats after a transient mononuclear cell response. A second SE allograft from the same donor strain did not provoke rejection either in the original allograft or in the challenge allograft. A secondary graft of allogeneic skin did not provoke rejection in the original SE graft, although the skin graft was rejected. Grafting the recipient first with allogeneic skin and then with the SE allograft led to rejection of the skin but not of the SE graft, ruling out the possibility that suppressor T cells were responsible for SE allograft acceptance. Allografted fibroblasts in SEs do not provoke a rejection response, even in presensitized animals, do not render the recipient tolerant to allogeneic skin, and do not act as targets when active rejection is taking place. We propose that cells bearing class I antigens may be acceptable graft constitutents if incorporated in a tissue equivalent excluding cells with class II antigens.

An immunocytochemical approach to cell kinetics automation.

Antibodies specific for 5-bromodeoxyuridine (BrdUrd) can be used to measure labeling indices in an automated system by image analysis. The antibody, used with an indirect immunoperoxidase technique, will detect de novo DNA synthesis subsequent to growing the cells for various time intervals in 5-bromodeoxyuridine-containing medium. Asynchronously growing CHO cells were pulsed with 3H-5-bromodeoxyuridine, fixed, denatured and then stained with anti-bromouridine antiserum. Peroxidase-coupled goat anti-rabbit IgG was used as the secondary antibody, and slides were stained with diaminobenzidine. Cells which are positive display a reticular pattern indicative of replicating chromatin. "Labeling indices" were generated by scanning the nuclei by TV image analysis. The percentage of labeled cells by the immunocytochemical technique correlates well with that found by autoradiography. Some of the applications of this automated method include cell kinetics and analysis of S-phase by pattern recognition technique.

Transformation of nuclear morphology during cellular maturation.

Using in vivo labeling of mouse leukocytes with tritiated thymidine, cells in the neutrophilic series were studied to determine the change in their nuclear shape as a function of maturation level. Several morphologic shape parameters including perimeter and bending energy were used to quantify the distribution of the nuclear morphology in a given age cohort. The change in these distributions as a function of calender age level was determined. The two parameters named above were used to test the possibility of inferring the age from the quantitative morphology.

A measurement technique for cell adhesiveness.

Determination of the percentage of cells in clumps on a stained smear of human peripheral blood porvided a useful, accurate technique for measuring cell adhesiveness. Smears of human peripheral blood drawn with EDTA were prepared on a blood slide centrifuge, stained, and examined under a light microscope. Statistical analysis showed that the method resulted in a Poisson distribution of particles on the slide, where a particle was considered to be a simple cell, or two or more cells which appeared to be touching, Analysis of the distributions of erythrocytes and leukocytes showed that clumps were formed before the cells were deposited on the slide. When adhesiveness of erythrocytes or leukocytes was increased by incubation with antiserum to the corresponding cell type, the percentage of that cell type in clumps increased proportionately, Preliminary results using the method showed that normal human donors had similar to 1% of their erythrocytes and 1-5% of their leukocytes in clumps. In chronic myelocytic leukemia, as many as 60% of the leukocytes were in clumps.