In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model.

A new three-dimensional human skin model consisting of several layers of actively dividing and metabolically active human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown on nylon mesh has been used to assess the in vitro toxicity of test agents from various classes. Utilizing a slight modification of the published neutral red viability assay for endpoint determination, we have assayed and obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n = 15), alcohols (n = 5), metal chlorides (n = 10), perfumes and colognes (n = 5), shampoos (n = 4), conditioners (n = 3), moisturizers (n = 3), pesticides (n = 3), and antimicrobial preservatives (n = 4). Limited comparisons to in vivo ocular irritancy data with alcohols and detergents are encouraging. We have demonstrated the utility of this metabolically active dermal substrate containing naturally secreted collagen and other extracellular matrix proteins along with the neutral red viability assay for assessing the toxicity of a number of test agents from a variety of different classes with broad industrial applications.

Rat bone-marrow cell proliferation and differentiation as an index of the effects of xenobiotics in vitro.

A three-dimensional bone-marrow culture system was utilized for assessing the toxicity of various chemotherapeutic agents. This model, which exhibits multilineage haematopoiesis and promotes the growth of progenitor cells for extended periods, was treated with various concentrations of beta-d cytosine arabinofuranoside (Ara-C), cyclophosphamide (CP), methotrexate (MTX) or 5-fluorouracil (5-FU). The effects of these agents on the phenotypes of adherent zone cells was ascertained by labelling with monoclonal antibodies against rat leucocytes and evaluating by flow cytometry. Adherent zones also were assayed for content of colony-forming unit culture (CFU-C) and cellular viability after drug exposure was measured using the neutral red (NR) assay. The results indicated that Ara-C, CP, MTX and 5-FU treatment caused dose-dependent decreases in the CFU-C concentration of adherent zones in cultures of various ages. These agents also altered the phenotypic distribution of adherent zone cells and displayed differential lineage specificities. A dose-related decrease in viability also was observed with the NR assay, albeit at higher drug doses than those which induced measurable CFU-C and phenotypic alterations. These three-dimensional cultures may prove to be ideal substrates for toxicity testing as they contain all of the cell types present in vivo, are physiological with respect to their growth patterns, are easily manipulable, and can be maintained for extended periods.

Cytotoxicity testing using neutral red and MTT assays on a three-dimensional human skin substrate.

The use of a three-dimensional dermal culture system as a substrate in cytotoxicity assays is described. This substrate consists of several layers of dermal fibroblasts, derived from human foreskin, grown on pretreated nylon mesh. This physiological model of the human dermis has been used in conjunction with the neutral red assay and the MTT assay to assess the in vitro toxicity of a panel of 15 test agents from several different classes. NR(50) and MTT(50) endpoints (test agent concentrations yielding 50% viability) were obtained for compounds/formulations from the following groups: surfactants, alcohols, antimicrobial preservatives, metal chlorides and pesticides. In addition, the carboxylic ionophore, monensin, was tested in both assays. Limited comparisons of the in vitro neutral red and MTT results, using the three-dimensional culture system, with existing in vivo rabbit ocular irritancy data look promising. This three-dimensional model may afford several advantages over monolayer cultures.

Anti-HIV antibodies in patients receiving intravenous immunoglobulin.

Until recently, donors of serum for intravenous immunoglobulin (IVIG) were not screened for the presence of antibodies to the human immunodeficiency virus (HIV). We detected anti-HIV antibodies (Ab) in the sera of seven patients following IVIG infusions. In addition, enzyme immunoassay values increased and/or Western blot converted from negative to positive in all patients following infusions and declined again over time confirming passive acquisition. Patients who received IVIG manufactured after the screening of donors was initiated may still test positive for anti-HIV Ab due to antibodies to human T-lymphocyte cell lines in which viral antigen was prepared. Such false positives must be excluded when testing IVIG recipients. Despite the presence of antibodies to HIV, IVIG has never been documented to transmit the virus.

Human T-cell lymphotropic virus type-I antibodies in Falashas and other ethnic groups in Israel.

Epidemiological studies of the human T-cell leukaemia/lymphoma virus type I (HTLV-I), a type-C retrovirus of the human T-lymphotropic virus family, have used serological surveys to identify population subgroups possessing a high prevalence of naturally occurring HTLV-I-specific antibodies. Studies carried out to delineate the global distribution of the virus have demonstrated natural antibodies to HTLV-I in the serum of healthy donors from specific geographical areas, and have defined viral endemic areas in Japan, the Caribbean basin, Africa and the southeastern United States. Such studies have suggested that the prevalence of HTLV-I antibodies is directly correlated with age, is associated with the clinical syndrome of adult T-cell lymphoma, and is associated with transmission from mother to child. A separate subtype of the human retrovirus, HTLV-II (refs 21, 22), has also been identified. The population of Israel in part comprises groups of immigrants of various ethnic and geographical origins. Because of this, and the fact that Israel has a highly developed public health system, we surmised that the ethnic groups in Israel could be used in a seroepidemiological survey of HTLV infection. The serological survey reported here demonstrates a high prevalence of HTLV-I antibodies in new immigrants from Ethiopia. This previously ethnically and geographically isolated group, the 'Black Jews' or 'Falashas', from the Gondar region in the northern rural highlands of Ethiopia, has the highest endemic rate of HTLV-I yet reported outside Japan.

Clonal proliferation unlinked to terminal deoxynucleotidyl transferase synthesis in thymocytes of young mice.

Terminal deoxynucleotidyl transferase (TdT) is a specialized DNA polymerase that is potentially mutagenic. It is expressed only in the thymus and in some bone marrow cells, suggesting that it acts selectively in immature lymphoid cells, especially T cells. We investigated the extent to which de novo synthesis of TdT is correlated with the clonal expansion that T cell precursors undergo in the thymus. Using biosynthetic pulse-labeling and immune precipitation, we found that thymocytes from late-fetal and neonatal mice make TdT at considerably lower levels than weanling or adult mouse thymocytes. Adult levels of TdT synthesis are only reached a week after birth. Thus, the high proportion of proliferating cells in the perinatal thymus does not entail a correspondingly high production' of TdT synthesis is blocked by cytolytic treatment with anti-Lyt-2 and complement, suggesting that the inducible cells are already Lyt-2+ like most adult TdT+ thymocytes. These observations imply that the stroma of the perinatal thymus either suppresses or fails to trigger high-level TdT synthesis. At the same time, it is in this microenvironment that maximum proliferation and export of functional T cell precursors take place. In contrast, the conditions that stimulate high TdT synthesis in vitro are not associated with mitogenesis but rather with growth arrest. High-level TdT synthesis in general is not regulated so as to precede or coincide with clonal expansion, either in perinatal or in adult thymocyte populations. The possibility remains that in many cells it is linked with a commitment to die.

Lyt-2 glycoprotein is synthesized as a single molecular species.

We investigated the possibility that the Lyt-2 molecules made by uncloned mouse T lymphocytes would show variable primary structures like those of immunoglobulins. Newly synthesized Lyt-2/3 complexes were found to include only two major components, both discrete glycoproteins with apparent molecular weights of 31,000 (31 K) and 35,000 (35 K). When products of Lyt-2.1 and Lyt-2.2 thymocytes were compared by two-dimensional nonequilibrium pH gradient electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the isoelectric points of the 35 K molecules were different; thus, the 35 K component was likely to be encoded by the Lyt-2 locus itself. However, the 35 K molecules made by any one genotype were homogeneous in charge as well as in size. The homogeneity was obscured rapidly by post-translational modification. Most strikingly, within 30 min of initial synthesis, these processing events generated the conspicuous array of microheterogeneous products that form the "38 K" component of cell-surface Lyt-2/3.

Structure and expression of glycoproteins controlled by the Qa-1a allele.

The alloantigen controlled by the Qa-1a allele is a glycoprotein that exists in two forms. The first, an intracellular molecule of apparent Mr 44 000 daltons, appears to be a kinetic precursor of the second, a cell-surface molecule with an apparent size of 47 000 daltons. The intracellular form of Qa-1 is distinct from that of the TL glycoprotein in two ways: (1) its polypeptide backbone is approximately 5000 daltons shorter, and (2) it possesses three sites of high-mannose carbohydrate attachment, while TL has only one. In the cell-surface form of Qa-1, all three carbohydrate chains are processed to structures that resist endoglycosidase H digestion, presumably complex-type oligosaccharides. Concomitant with these late carbohydrate-processing steps is the formation of stable complexes between Qa-1 and beta 2-microglobulin. The timing of this association provides a further contrast between Qa-1 and TL, which is associated with beta 2-microglobulin shortly after its synthesis. The Qa-1 glycoproteins have been identified genetically by their synthesis in B6-TL+ (Qa-1a/Tlaa) splenocytes but not in splenocytes of congenic B6.K1 and B6.K2 (Qa-1b/Tlab) mice, and by their absence from the products of BALB/c (Qa-1b/Tlac) splenocytes. The cells synthesizing Qa-1 are at least as prevalent in Ig+ spleen cell populations as in T-cell-enriched splenic Ig- populations. Thus, active Qa-1 synthesis appears to take place at a high rate in normal splenic B cells without mitogenic stimulation.

"Mature" thymocytes are not glucocorticoid-resistant in vitro.

Dexamethasone, administered in vitro either continuously or for a 30-min pulse period followed by a chase of 18 to 24 hr, is shown to decrease protein synthetic rates as well as cellular viabilities in a dose-dependent manner in murine thymocytes. Differential effects on cortical and medullary thymocyte subpopulations should be detectable by alterations in the rates of synthesis of specific molecules which are stable in vitro, relative to total protein synthesis, in the absence of steroids. However, terminal deoxynucleotidyl transferase (TdT), a marker for cortical cells, and Qa-1 and H-2, preferentially expressed in medullary cells, continue to be produced as constant fractions of total synthesis even after treatment with up to 10(-6) M dexamethasone. Furthermore, thymocytes obtained from normal and in vivo cortisone-treated mice show little, if any, difference in their intrinsic sensitivities to dexamethasone in vitro. The results reported here suggest that a corticoresistant thymocyte per se does not exist in vitro and that the thymus may produce factor(S) in vivo that protect the medullary subpopulation from in vivo glucocorticoid-induced lysis.

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes.

Mouse thymocyte populations enriched in functionally incompetent, "immature" cells on the one hand, or in competent "mature" cell on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of "immature" and "mature" thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells. TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.