Thiol Reduction of 3′-Azidothymidine to 3′-Aminothymidine: Kinetics and Biomedical Implications

The ability of thiols to reduce 3-azidothymidine (AZT) to 3-aminothymidine has been investigated. Incubation with glutathione, dithiothreitol (DTT), or mercaptoethanol at pH 7.2 and 37°C leads to quantitative reduction of the azido moiety to an amine. The reaction is first order in AZT and first order in reducing agent (mono- or dithiol). The second-order rate constants are 2.77 × 10^–3, 6.55 × 10^–5, and 6.35 × 10^–6 M ^–l sec^–1 for the dithiothreitol, glutathione, and mercaptoethanol reductions, respectively. The thiol reduction of alkyl azide to amine under mild conditions is a synthetic method particularly suitable for water-soluble azido compounds that are sensitive to catalytic hydrogenation. The potential for the mono- or dithiol-mediated reduction of alkyl azides under biological conditions must be considered when conducting studies of azido drugs.     

Clinical and microbiological effects of daily brushing with either NaF or SnF2 gels in subjects with fixed or removable dental prostheses

61 adults, with fixed or removable dental protheses, completed a 6-month double-blind trial comparing the clinical and microbial effects of brushing twice daily with either 0.22% NaF or 0.4% SnF2. Those subjects brushing with SnF2 had less gingivitis and fewer bleeding sites for both "total teeth" and "abutment teeth". Plaque scores between groups were only statistically different for the "abutment teeth". The microbial parameters, salivary S. mutans and subgingival plaque total CFU, were significantly reduced in the SnF2 group. In both treatment groups, there was a reduction over the course of the study in the number of subjects with recoverable A. actinomycetemcomitans and black pigmented bacteroides, yet there was no difference between groups.     

Septo-optic dysplasia: MR imaging

Septo-optic dysplasia is the diagnosis when optic nerve hypoplasia is seen in conjunction with dysgenesis of the septum pellucidum. Nearly two-thirds of these patients have hypothalamic-pituitary dysfunction, and half have schizencephaly. The disorder is difficult to classify because of the diversity of clinical and pathologic manifestations. Magnetic resonance images of 11 patients with clinical and radiographic evidence of septo-optic dysplasia were reviewed retrospectively. The "syndrome" appears to include two subsets of patients whose abnormalities have different embryogenesis and neuropathologic findings. The existence of these two subsets helps to explain the diversity of the clinical and radiologic findings.     

Extramarital Involvement: A Core Relationship Issue in Need of a Coherent Model, Comprehensive Research, and Empirically Based Intervention

Clinical interventions for extramarital involvement (EMI) have outpaced empirical knowledge about both risk factors for infidelity and effective treatments. Allen et al. (this issue) provide a systematic review of current knowledge organized around stages of the development of EMI and factors concerning the involved partner, the spouse, the couple's relationship, and the interpersonal context. Their review identifies significant gaps in knowledge for which research is needed. Because EMI has multiple determinants, conceptual models and research on it must be multivariate. This comment focuses on priorities for increasing knowledge about EMI, including (a) clarification of variation in definitions and personal standards for EMI through consideration of participants' subjective experiences as well as the views of outsiders (researchers, clinicians); (b) improved qualitative research using interview methods designed to minimize biased questioning by investigators and biased reporting by subjects, to tap individuals' internal experiences with the developmental process of EMI; (c) nonblaming research on characteristics of the noninvolved spouse and the couple's interaction that predict EMI; and (d) adaptation of generic preventive and relationship enrichment interventions for couples involving communication skill-building and psychoeducation, to include information about ways to "inoculate" relationships against commonly unexpected risks of EMI.     

Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus)

TcR alpha, beta, and gamma chain negative cytotoxic NK-like cells were cloned from alloantigen-stimulated PBL obtained from nai;ve channel catfish. Stimulation with allogeneic cells and growth promoting factors are required for their continued in vitro proliferation and cytotoxic activity. These granular cells kill not only the stimulating allogeneic cells, but also unrelated allogeneic targets by a perforin/granzyme-mediated apoptosis pathway. In addition, they are negative for markers that define neutrophils, monocytes/macrophages, and non-specific cytotoxic cells. Although these NK-like clones kill a number of different allogeneic targets, they display interclonal variation in cytotoxicity toward a panel of allogeneic targets, i.e. some clones have no apparent target specificity, while others display a target preference. In addition, flow cytometric analyses revealed that expression of a putative FcmuR, an LFA-1-like molecule, and a putative thymocyte/T cell antigen varies among the different clones, with no clear correlation between surface antigen expression and cytotoxic activity. Although not all clones express a putative FcmuR, it was noted that they all expressed an ITAM containing FcepsilonR gamma chain homolog. This finding suggests that the catfish FcepsilonR gamma chain may potentially be used as an accessory molecule for not only FcmuRs, but also for other unknown activation receptors. These results support the hypothesis that catfish NK-like cells are heterogeneous in terms of target specificities and cell surface phenotype.     

Response involvement in brain stimulation reward

The hypothesis that responding contributes to the reward value of brain stimulation was tested in two novel experimental paradigms. In the first experiment rats lever-presssed for rewarding brain stimulation during 90 sec periods. After each period the lever automatically retracted and experimenter-administered stimulation (EAS) was presented at the same rate and current parameters as during the self-stimulation (SS). The rats could demonstrate a preference for SS (vs EAS) by pressing a reset lever on the opposite wall of the test chamber. This action terminated the EAS and reinstated the SS-lever for an additional 90 sec. Results showed that the rats preferred to respond for stimulation than to have that same stimulation administered by the experimenter. This was true even when a signal preceded each train of EAS or when subjects had a great deal of previous EAS experience. In the second experiment conditioned taste preferences were observed following novel taste/SS pairings but not following novel taste/EAS pairings. The data from these two experiments suggest that responding contributes to the rewarding value of brain stimulation.     

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the ³H-labeled natural isomers of nicotine or cotinine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10⁸ M⁻¹ were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly- -lysine werecoated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5–10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and ³H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.     

A framework for modeling health behavior protocols and their linkage to behavioral theory

With the rise in chronic, behavior-related disease, computerized behavioral protocols (CBPs) that help individuals improve behaviors have the potential to play an increasing role in the future health of society. To be effective and widely used CBPs should be based on accepted behavioral theory. However, designing CBPs while at the same time specifying their linkages to behavioral theory and developing reusable CBP components (interventions) are challenges to developers of CBPs. Having an ontology with which to describe CBPs could help with these issues. As a first step towards creating such an ontology, we modeled PACE-Adolescent, a theory-based behavioral protocol that uses the Stages of Change Model and Social Cognitive Theory, using PROTEGE-2000, an ontology editor and knowledge acquisition system. We created a three-part knowledge model. Two sub-ontologies described behavioral interventions and psychological theories. The third component, implemented using Guideline Interchange Format (GLIF3), provided a way to describe the structure of a protocol and to link intervention resources and groups of actions to elements of psychological theory. Using this framework, we formally described the PACE-Adolescent protocol. Creating knowledge models such as this may lead to improvements in the design and evaluation of computerized health behavior protocols.     

Adoptive immunotherapy of feline immunodeficiency virus with autologous ex vivo-stimulated lymphoid cells modulates virus and T-cell subsets in blood

The potential of immunotherapy with autologous virus-specific T cells to affect the course of feline immunodeficiency virus (FIV) infection was explored in a group of specific-pathogen-free cats infected with FIV a minimum of 10 months earlier. Popliteal lymph node cells were stimulated by cocultivation with UV-inactivated autologous fibroblasts infected with recombinant vaccinia viruses expressing either FIV gag or env gene products, followed by expansion in interleukin-2. One or two infusions of both Gag- and Env-stimulated cells resulted in a slow increase in FIV-specific gamma interferon-secreting T cells in the circulation of cats. In the same animals, viral set points fluctuated widely during the first 2 to 3 weeks after adoptive transfer and then returned to pretreatment levels. The preexisting viral quasispecies was also found to be modulated, whereas no novel viral variants were detected. Circulating CD4(+) counts underwent a dramatic decline early after treatment. CD4/CD8 ratios remained instead essentially unchanged and eventually improved in some animals. In contrast, a single infusion of Gag-stimulated cells alone produced no apparent modulations of infection.     

Clinical utility of an enhanced human immunodeficiency virus type 1 p24 antigen capture assay

The presence of p24 core antigen in the serum of individuals with human acquired immunodeficiency syndrome has been used as one of the important prognostic markers of HIV-1 infection and also as an end point in evaluating antiviral drugs and vaccines. Unfortunately the majority of p24 antigen present in serum exists as an antigenantibody complex and is not detected with the commercial kits currently available to measure p24 antigen. In this study, we report a simple procedure utilizing treatment of serum samples with glycine buffer (pH 1.85) to dissociate antigen-antibody complexes prior to assaying for p24 antigen. A 300% increase in the number of p24-reactive samples and a 3- to 12-fold increase in the quantity of antigen detected were observed when samples were pretreated with 1.5M glycine buffer (pH 1.85) for 1 hr. Glycine treatment of samples did not result in nonspecific positive tests and samples previously shown to be reactive remained positive. In reconstruction experiments the release of antigen was found to be inversely proportional to the amount of p24 antibody present in the serum. The percentage of HIV-1-infected patients positive for p24 antigen was clearly a function of CD4 count. Forty-nine percent of patients with more than 500 CD4 cells and 100% of patients with less than 200 CD4 were p24 positive. The improved sensitivity for detection of p24 provided by this procedure enhances our understanding of the pathogenesis of AIDS by showing that the majority of patients with HIV-1 infection is p24 positive and facilitates the analysis of data obtained in clinical trials involving anti-HIV compounds.