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A technique for sample work-up and enrichment using a supported liquid membrane in an automated flow system, connected to a gas chromatograph, was used for the determination of aliphatic amines in human blood plasma. The amines studied were N,N-dimethylethylamine, triethylamine, N-methylmorpholine, cyclohexylamine and N,N-dimethylcyclohexylamine. An efficient clean-up of the complex plasma matrix was achieved, resulting in identical blank chromatograms for plasma samples and aqueous solutions. Different parameters influencing the efficiency and selectivity of the extraction procedure were experimentally studied and theoretically explained. The detection limit depends on the extraction flow-rate and the available sample volume. With 1 ml of sample and a flow-rate giving an extraction time of 16 min, the detection limit was ca. 5 ppb (5 micrograms/l); with 4 ml of sample and a lower flow-rate, sub-ppb detection limits could be reached in ca. 3 h. Linear calibration curves up to 500 ppb were obtained. Blood plasma samples from volunteers exposed to N,N-dimethylethylamine in air were analysed, and the results compared favourably with independent measurements by another method.

The regenerative responses of inlays of lyophilized allogeneic bone of membranous (skull) and enchondral (tibia) origin were studied in an experimental cranioplasty model in rabbits. The lyophilized bone particles were also bioassayed for inductive bone production in an orthotopic critical size defect rat model. Three trephined calvarial defects were evaluated in each of 14 adult rabbits. The experimental materials were implanted into two of the defects and the third was left empty for control purposes. The implants disclosed no major structural divergences as assessed by scanning electron microscopy. Healing was evaluated by light microscopy and contact radiography after periods of four and 15 weeks. The lyophilized bone allografts of both embryonic origins displayed a similar fashion of bone regeneration, bone marrow reappearance, and volumetric density of trabecular bone substance and displayed no obvious differences between experimental groups or intervals. The two materials exhibited low osteoinductive potential.

Studies of amine N-oxides in urine are important for the evaluation of occupational exposure to amines. These thermolabile compounds are difficult to handle by either gas or liquid chromatography, so a device for controlled thermal degradation has therefore been developed. It consists of a short precolumn with shut-off valves at both ends and an aluminum block for heating, and it was connected to the injection port of a gas chromatograph. After injection of amine N-oxides onto the precolumn and thermal degradation, the degradation products were allowed to enter the analytical column. Trimethylamine N-oxide (TMAO) and triethylamine N-oxide (TEAO) were investigated. Their thermal degradation patterns could be used for identification and quantification in aqueous solutions and in urine. Linear calibration graphs based on degradation product peaks (trimethylamine and O,N,N-trimethylhydroxylamine from TMAO and diethylamine and triethylamine from TEAO) were obtained for concentrations up to 500 ppm. Detection limits in aqueous solutions were 0.2 ppm (ca. 1 ng) for TMAO and 1 ppm for TEAO and the precisions were 6% and 9%, respectively. In urine, similar values were obtained for TEAO. The detection limit for TEAO corresponds to the expected concentration in urine after an 8-h exposure to air containing 0.8 mg/m3 of triethylamine.

The positioning of osteotomies in intramembranous cranial bone was studied by exploring the pattern of bone regeneration in growth areas (the sutural region) as compared to that of the bone plate proper. Trephine defects in the left coronal suture area and the right parietal bone were produced in fifty-nine young rabbits. A pilot study to refine operative and analytical methods comprised 22 animals. The experiments were terminated at one, three, and six weeks after surgery. The bone regenerative response was assessed by x-ray planimetry, plain microscopy, enzyme histochemistry, and fluorescent labelling. Only minor divergences in healing capacity between the two defects were found. No adverse effects on the growth process were indicated. As to clinical management, the findings suggest that osteotomies designed to traverse sutural areas will, under normal circumstances, regenerate in a similar manner and rate to adjoining bone plates.

We have studied the effect of T cell receptor (TcR) cross-linking and TcR-CD4 cross-linking on the induction of interleukin (IL) 2 and IL 2 receptor mRNA expression in peripheral T cells. We confirm our previous results using bioassays, suggesting that CD4 is involved in T cell activation by up-regulating the expression of IL 2 receptors. Furthermore, we show that this also concerns other functional T cell genes such as IL 4 and interferon-gamma. In contrast, similar levels of IL 1 mRNA are found in T cells activated with anti-CD3 with and without anti-CD4 antibody, suggesting that the induction of the IL 2 gene is regulated by TcR cross-linking alone and not influences by CD4. However, anti-CD4 appears to accelerate the induction of IL 2 mRNA in the T cell populations, presumably by a more rapid adhesion of the cells to the antibody-coated surface.

The vapor pressures of decyl acetate, five decenyl acetate isomers, (Z)-7-dodecenyl acetate, and (Z)-9-tetradecenyl acetate have been determined at three to six temperatures in the interval 25-45 °C by a gas Chromatographic method suitable for accurate measurements of the low vapor pressures of moth sex pheromone components at biologically relevant temperatures. The vapor pressure values at 30.5 °C are 3.80 Pa for decyl acetate, 4.08-5.40 Pa for the decenyl acetate isomers, 0.562 Pa for (Z)-7-dodecenyl acetate, and 0.094 Pa for (Z)-9-tetradecenyl acetate. The vapor pressures of the decenyl acetates show a small but significant dependence on the double bond position. Four of the compounds in this study, 10∶Ac,Z5-10∶Ac,Z7-12∶Ac, andZ9-14∶Ac have recently been identified as sex pheromone components of the turnip moth,Agrotis segetum. Large differences between the mole percentages of the component as found in liquid extracts of female abdominal tips and the corresponding mole percentages in the vapor phase are predicted.

The activation of T lymphocytes by an antigenic challenge requires that the CD3 T cell receptor alpha/beta (TcR) is bound to an appropriate ligand, i.e. major histocompatibility complex antigen, on an antigen-presenting cell. In addition, numerous studies suggest that the accessory molecules CD4 and CD8 participate in the recognition process, and may have inhibitory as well as augmenting effects depending on the ways in which they participate. In the present study we attempt to define the conditions by which CD8 exerts enhancing and inhibitory effects on resting CD8+ T cell activation, and which parameters of activation are regulated through participation of CD8/CD4. We find that experimental procedures leading to TcR-CD8 aggregation induce T cell activation whereas experimental procedures preventing TcR-CD8 aggregation inhibit T cell activation. CD8/CD4-induced variations in the extent of T cell activation are apparent as variations in interleukin 2 (IL 2)-dependent growth and in the number of blastoid cells bearing IL 2 receptors. Inhibition of CD8+ T cell activation is successful only if the majority, if not all CD8 molecules are occupied by soluble antibody. This latter finding argues against the suggestion of other groups that CD8 may be a receptor for negative signaling. Rather, the results support the alternative notion that a basal level of TcR-CD8 aggregation, existing in the resting state or induced by TcR-ligand interaction, is an essential prerequisite for CD8+ T cell activation. Enhancement or depression of this basal level of aggregation causes facilitation or inhibition, respectively, of activation. This may be a mechanism for the regulation of IL 2-dependent clonal expansion of T cells in immune responses.

We studied the activation of small resting mouse T lymphocytes by antibodies to the T cell antigen receptor in combination with antibodies to other T cell surface antigens. Solid-phase but not soluble antibodies KJ16-133 and F23.1, both directed to beta chains of the V beta 8 family, activate T cells to proliferate in the presence of growth factors, in a dose-dependent fashion. Antibodies to Lyt-2 and to L3T4 had no activating effect at any concentration. However, submitogenic concentrations of KJ16-133 and of F23.1 synergized with a wide range of concentrations of anti-Lyt-2 and anti-L3T4 to cause T cell proliferation similar or greater in magnitude to that caused by high concentrations of anti-T cell receptor antibody. Synergistic activation was also observed with antibodies to Lyt-1, LFA-1 and H-2 class I antigens but to a significantly lower degree. This was particularly clear in limiting dilution experiments in which the corrected frequencies of T cells proliferating in response to low amounts of anti-T cell receptor antibody together with anti-Lyt-2 were 1/4 to 1/7 for BALB/c T cells. The frequencies of BALB/c T cells responding to high concentrations of anti-T cell receptor antibody alone were between 1/14 and 1/126 and still lower frequencies of T cells proliferated in synergistic responses with anti-LFA-1 or anti-Lyt-1. Synergistic activation leads to the induction of functional cytotoxic cells. We interpret these data as suggestive that cross-linking of the T cell antigen receptor with either Lyt-2 (CD8) or L3T4 (CD4) represents an optimal activating signal for resting T cells. We think that, in physiological T cell activation, cross-linking of the T cell receptor to CD8 or CD4 is induced by their simultaneous binding to major histocompatibility complex (MHC) class I (for CD8) or MHC class II (for CD4) molecules on stimulator cells. We consider the possibility that similar cross-linking requirements may also exist during T cell repertoire selection in ontogeny, thus accounting for the strict coexpression of MHC class I and class II-restricted T cell receptors with CD8 and CD4 molecules, respectively.