Antithymidylate resistance enables transgene selection and cell survival for T cells in the presence of 5-fluorouracil and antifolates.

Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers.

The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells.

Pluripotent hematopoietic stem cells (PHSC) are very rare cells whose functional capabilities can only be analyzed indirectly. For a better understanding and possible manipulation of mechanisms that regulate self-renewal and commitment to differentiation of PHSC, it is necessary to purify these cells and to develop assays for their growth in vitro. In the present study, a rapid and simple, widely applicable procedure to highly purify day 14 spleen colony-forming cells (day 14 CFU-S) is described. Low density bone marrow cells (rho less than or equal to 1.078 g/cm3) were enriched by two successive light-activated cell sorting procedures. In the first sort, cells within a predetermined light scatter (blast cell) window that are wheat germ agglutinin/Texas Red (WGA/TxR) positive and mAb 15-1.4.1/fluorescein isothiocyanate negative (granulocyte-monocyte marker) were selected. In the second sort, cells were selected on the basis of retention of the supravital dye rhodamine 123 (Rh123). Cells that take up little Rh123 (Rh123 dull cells) and those that take up more Rh123 (Rh123 bright cells) were 237-fold and 132-fold enriched, respectively, for day 14 CFU-S. Both Rh123 fractions were cultured for various time periods in vitro in the presence of mast cell growth factor (MGF), with or without interleukin 3 (IL-3) or IL-1 alpha. Both Rh123 fractions proliferated in response to MGF alone as determined by a [3H]TdR assay or by counting nucleated cells present in the cultures over time. MGF also acted synergistically with both IL-3 and IL-1 alpha to promote stem cell proliferation. Stimulation of both Rh123 fractions with MGF alone did not result in a net increase of day 14 CFU-S. Stimulation with MGF + IL-3 or MGF + IL-alpha resulted in a 4.4- or 2.6-fold increase of day 14 CFU-S in the Rh123 dull fraction, and an 11.6-fold or 2.6-fold increase of day 14 CFU-S in the Rh123 bright fraction, respectively. The data presented in this paper indicate that in vitro MGF acts on primitive hematopoietic stem cells by itself and also is a potent synergistic factor in combination with IL-3 or IL-1 alpha.

Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody.

The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.

Apolipoprotein C-III-1 activates lysosomal sphingomyelinase in vitro.

Apolipoprotein (apo)C-III-1 from human very low density lipoprotein stimulates 14-fold the activity of lysosomal sphingomyelinase from human fibroblasts. At the sphingomyelin concentrations tested, maximal stimulation was obtained with 5 microM apoC-III-1 or apoC mixture. Apolipoproteins A-I, A-II, B, and C-I conferred little or no stimulation. Sphingomyelinase was stimulated 20-fold by lysophosphatidylcholine with an optimum concentration of 70 microM using 0.3 mM substrate. Sphingomyelinase activity was inhibited by concentrations of apoC-III-1 and lysophosphatidylcholine three- to fivefold above stimulatory levels. Triton X-100 activated sphingomyelinase 300-fold with a pH optimum of 5.0, while the pH optimum with the biological activators was 4.0. These results raise the possibility of an in vivo activity for the biological activators. The proteins that enter lysosomes as part of a lipoprotein complex may activate lysosomal enzymes that degrade the lipid components.

Decreased production of interferon-gamma by human neonatal cells. Intrinsic and regulatory deficiencies.

Human neonatal lymphocytes produced little macrophage activation factor in response to mitogens. This correlated with decreased production of interferon-gamma (IFN gamma): adult lymphokines contained 894.2 +/- 177.1 U/ml, whereas neonatal cord and peripheral lymphokines contained 66.9 +/- 17.0 and 116.7 +/- 29.6 U/ml by bioassay. Results by radioimmunoassay (RIA) for IFN gamma were similar. In contrast, the interleukin 2 content of cord lymphokines was greater (P less than 0.01) and that of neonatal peripheral blood lymphokines similar to that of adults. Interleukin 1 production and interleukin 2 receptor expression and affinity were similar for adult and neonatal cells. Interleukins 1 and 2 in amounts comparable to those in adult lymphokines did not increase production of macrophage activation factor or IFN gamma by neonatal cells. Neonatal cells did not contain intracellular IFN or degrade exogenous IFN. Excess suppressor activity was not found in neonatal cultures. Addition of IFN alpha, 10,000-50,000 U/ml of interleukin 2 or phorbol myristate acetate (PMA) to cord mononuclear cells or of adult monocytes or PMA to cord T cells increased IFN gamma production compared to cells stimulated with concanavalin A (ConA) alone. Nevertheless, under optimal conditions (T cells + PMA + Con A), adult cells produced much more IFN gamma (1,360 +/- 261 U/ml by RIA) than cord cells (122 +/- 37 U/ml). Staphylococcal enterotoxin A (SEA) stimulated cord cell IFN gamma production at low cell densities; nevertheless, adult cells produced more IFN in response to SEA 1,341 +/- 350 U/ml) than cord cells (350 +/- 33 U/ml). Decreased production of IFN gamma by neonatal cells appears to be due both to differences in their intrinsic capacity to produce IFN gamma and to differences in regulatory mechanisms.

Quantitative measurement of human interleukin 2 receptor levels with intact and detergent-solubilized human T-cells.

2A3 monoclonal antibody (gamma 1, kappa) is a novel high-affinity reagent for detecting the human interleukin 2 (IL-2) receptor. The antibody inhibits IL-2 binding to its receptor and is an antagonist of IL-2 action. Detailed analysis of the mechanism of binding of the IgG, (Fab')2 and Fab' of 2A3 antibody shows that the bivalent species cross-link on the cell surface when bound. Measurements of IL-2 receptor expression on digitonin-permeabilized cells suggest that the intracellular pool of receptors is small. The antibody will bind to IL-2 receptors on glutaraldehyde-fixed cells in the presence of Triton X-100. This property is used in designing an assay for quantitative measurements of IL-2 receptor concn in solution. This assay can be used to monitor receptor protein during purification to homogeneity.

High level stable expression of human interleukin-2 receptors in mouse cells generates only low affinity interleukin-2 binding sites.

A bovine papilloma virus-derived vector was used to direct the high level expression in mouse C127 cells of three different cDNAs encoding the human interleukin-2 receptor. These were: the previously described cDNA clone isolated from the T-cell lymphoma, HUT-102; a cDNA clone isolated from mitogen-activated, normal peripheral blood T cells; and an altered version of the HUT-102 receptor in which Ser247, believed to be the site of protein kinase C-mediated phosphorylation, has been changed to an Ala residue. Fluorescence-activated cell-sorting using a monoclonal antibody directed against the human IL-2 receptor was used to derive stable lines of C127 cells expressing from 2-6 X 10(6) IL-2 binding sites per cell. However, all of these receptors bound IL-2 with low affinity.

Amino acid residues of serum and CSF protein in multiple sclerosis. Clinical application of statistical discriminant analysis.

Statistical discriminant analysis of the amino acid compostion of serum and cerebrospinal fluid (CSF) proteins provides an objective method for distinguishing between normal controls and patients with multiple sclerosis (MS). This method also results in a high degree of specificity in separating MS patients from those with other diseases of the nervous system. The CSF protein serine residue is highly correlated with the CSF IgG and holds promise for a more sensitive diagnostic test for MS than the currently used CSF IgG. Finally, the serum/CSF protein serine ratio seems to correlate best with clinically determined degree of activity for the disease, the most active cases having the lowest ratio. These results suggest that investigation of the amino acid composition of serum and CSF protein in multiple sclerosis and, possibly, in other diseases might lead to the development of clinically useful tests of diagnosis and degree of activity of MS.

Multi-level covariance structure analysis of intra-familial substance use.

Conventional covariance structure analysis, such as factor analysis, is often applied to data that are obtained in a hierarchical fashion, such as parents and siblings observed within families. However, multivariate modeling of such data are most frequently done as if the data were obtained as a simple random sample from a single population. An alternative specification is presented which explicitly models the within-level and between-level covariance matrices in familial substance use. Results demonstrate homogeneity in substance use within families but heterogeneity across families which could be accounted for by family-level variables of marital status, economic status, and biological relationships. It is shown that conventional covariance structure software can be easily adapted to handle hierarchical models, providing a large set of new analysis possibilities for multi-level data.

Adolescent alcohol use development and young adult outcomes.

This study used latent growth modeling to examine the effects of level of alcohol use and development of alcohol use during adolescence, on young adult outcomes for males and females. Adolescents (N = 480; mean = 13.03 years, S.D. = 1.44; 264 female) were assessed annually over a 4-year period and then 5-6 years later in young adulthood (mean = 22.49 years, S.D. = 1.50). Chronicity of alcohol use in adolescence was related to higher alcohol use, alcohol-related problems, aggressive behavior, theft, and suicide ideation in young adulthood among both males and females. Development of alcohol use during adolescence was related to alcohol-related problems in young adulthood for males and females, and to higher levels of alcohol use and aggressive behavior for males only. The results indicate that development of alcohol use as well as level of alcohol use in adolescence is important for future adjustment outcomes, and that these relationships vary as a function of gender.

Hydrophilic-interaction chromatography of complex carbohydrates.

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

Evaluation of a centrifuge with rapid turnaround time for the preparation of plasma samples for measurement of common STAT markers on the ACS: 180 system.

Reported is the evaluation of a new centrifugation method, Statspin, that addresses both time and sample separation integrity. The method can successfully separate the plasma fraction from the cellular material in 2 minutes as compared to 20 minutes for the conventional centrifuge method. The Statspin, combined with the ACS:180 system, can generate test results in less than 30 minutes, exclusive of transport to the laboratory. This study demonstrated that the combined technologies offer timing-saving improvements for clinical laboratories offering STAT immunoassays for cardiac markers, endocrine molecules, and therapeutic drugs.

Desquamative gingivitis: immunologic findings.

A case of desquamative gingivitis is presented that was a gingival manifestation of cicatricial pemphigoid. Immunologic studies of serum and biopsies were of diagnostic significance. The application of immunofluorescence for the diagnosis of desquamative gingival lesions was discussed. If a definite diagnosis can not be made on the basis of light microscopy, immunofluorescence studies should be performed.

Family support and conflict: prospective relations to adolescent depression.

The relations between family support, family conflict, and adolescent depressive symptomatology were examined longitudinally in a sample of 231 female and 189 male adolescents and their mothers. Structural equation models revealed that less supportive and more conflictual family environments were associated with greater depressive symptomatology both concurrently and prospectively over a 1-year period. Conversely, adolescent depressive symptomatology did not predict deterioration in family relationships. Depressive symptomatology and, to a greater extent, family characteristics showed high levels of stability over the 1-year period. Counter to our expectations, the relations between family variable and depressive symptomatology were similar for boys and girls. The results suggest that the quality of family interactions is relevant for understanding the development of depressive symptoms in adolescents.

A rationale for attached gingiva at the soft-tissue/implant interface: esthetic and functional dictates.

For many years, the ultimate goal of the periodontal/restorative team was to provide an environment for crowns that would provide long-term, successful results. Two essential goals of periodontal therapy were pocket elimination and an adequate zone of keratinized gingiva. The establishment of an adequate zone of keratinized gingiva, which is firmly attached to the underlying periosteum and osseous tissues, is critical for the long-term functional and esthetic results of implant restorative dentistry. Five cases are presented that illustrate the clinical management of soft tissue to achieve functional and esthetic zones of keratinized gingiva.

Hydrophobic interaction chromatography of peptides as an alternative to reversed-phase chromatography.

Hydrophobic interaction chromatography (HIC) was examined as an alternative to reversed-phase chromatography (RPC) for peptide separations by high-performance liquid chromatography. With small peptides, selectivity was similar in both modes. This was the case with commercially available standards and with a set of synthetic peptides having the same amino acid composition but different sequences. Column efficiency was higher in RPC. HIC possesses several other disadvantages, including significant baseline changes during gradient elution and a requirement for non-volatile mobile phases, which complicates peptide isolation. Thus, RPC is still the method of choice for most small peptides. Marked differences in selectivity were noted with small proteins and polypeptides large enough to possess tertiary structure. Good results were also obtained by HIC in the case of some peptides that could not be purified at all by RPC, due to aggregation or poor binding or recovery. Thus, in these cases, HIC is a useful alternative to RPC for peptide purification.