NEUDOSE: A CubeSat Mission for Dosimetry of Charged Particles and Neutrons in Low-Earth Orbit.

During space missions, astronauts are exposed to a stream of energetic and highly ionizing radiation particles that can suppress immune system function, increase cancer risks and even induce acute radiation syndrome if the exposure is large enough. As human exploration goals shift from missions in low-Earth orbit (LEO) to long-duration interplanetary missions, radiation protection remains one of the key technological issues that must be resolved. In this work, we introduce the NEUtron DOSimetry & Exploration (NEUDOSE) CubeSat mission, which will provide new measurements of dose and space radiation quality factors to improve the accuracy of cancer risk projections for current and future space missions. The primary objective of the NEUDOSE CubeSat is to map the in situ lineal energy spectra produced by charged particles and neutrons in LEO where most of the preparatory activities for future interplanetary missions are currently taking place. To perform these measurements, the NEUDOSE CubeSat is equipped with the Charged & Neutral Particle Tissue Equivalent Proportional Counter (CNP-TEPC), an advanced radiation monitoring instrument that uses active coincidence techniques to separate the interactions of charged particles and neutrons in real time. The NEUDOSE CubeSat, currently under development at McMaster University, provides a modern approach to test the CNP-TEPC instrument directly in the unique environment of outer space while simultaneously collecting new georeferenced lineal energy spectra of the radiation environment in LEO.

Specificity of binding of HIV-1 anti-p24 antibodies to CD4+ lymphocytes from HIV-infected subjects.

The clinical utility of a flow cytometric assay (FCA) for intracellular HIV p24 antigen was evaluated in a group of HIV-1-infected subjects. A previously described method, p24-FCA (1), was modified to minimize nonspecific staining and to include irrelevant isotype-matched control antibodies. Blood mononuclear cells from 32 HIV-1 seropositive subjects and 14 HIV-1 seronegative controls were examined. In HIV-1 seropositive individuals, the proportion of CD4+ lymphocytes that bound the p24 monoclonal and the isotype control antibodies were not different. The frequency of cells binding p24 antibodies increased with declining CD4 counts and was highest in patients with low CD4+ lymphocyte counts. Although results of p24-FCA do reflect disease progression, the high nonspecific binding of monoclonal antibodies in infected subjects obscures specific p24 binding and precludes its use as an accurate measure of infection within single cells.

Quantifying phagocytosis of Mycobacterium avium complex by human monocytes in whole blood.

Studies of phagocytic efficiency in cells of the macrophage lineage have assumed additional importance since the discovery that HIV infection of these cells impairs their immune function. A rapid method has been developed for measuring phagocytosis of the opportunistic pathogen Mycobacterium avium complex by human monocytes. Fluoresceinated M. avium complex (F-MAC) was incubated with whole blood at 37 degrees C and the fluorescence of extracellular F-MAC was quenched using a vital blue stain. Monocytes were then stained with a monoclonal antibody (mAb) to human CD14 conjugated to phycoerythrin (PE) red cells were lysed, and the percentage of monocytes which had phagocytosed F-MAC was measured by flow cytometry. The results were reproducible in samples of blood taken from individual donors over a period of 1 or 2 weeks, and optimum F-MAC concentrations and an optimum incubation time were determined by experiment. This method has the advantages of requiring only a small volume of blood, not necessitating manipulation of cells before testing, and using a phagocytic target relevant to the pathogenesis of HIV infection.

A simple method for the propagation of cervical lymphocytes.

Local immune function is most likely a key influence in the establishment of human papillomavirus infections and its subsequent disease. Unfortunately, little information is known about local cervical immunity, and even less is known about human papillomavirus immunoreactivity. In addition, studies of local immunoreactivity have been hampered by the technical difficulty in obtaining cervical lymphocytes. The objective of the present study was to develop a simple method for the propagation of cervical lymphocytes from biopsy-size specimens. Cervical tissue was obtained from women undergoing a hysterectomy. Cervical samples measuring approximately 3 by 5 by 2 mm were minced and divided into two portions. One portion was digested by standard digestion methods and density gradient lymphocyte separation. The sample was then immunocharacterized for CD4 and CD8 cells by flow cytometry. The other portion was minced into 1-mm3 sections, and each section was placed into a separate well with tissue culture medium and interleukin-2. Lymphocyte counts and immunophenotypic analysis were performed after 18 to 20 days in culture. After 18 to 20 days in culture, the analysis demonstrated that this method of direct lymphocyte culture from a biopsy specimen yielded approximately 1 x 10(6) to 5 x 10(6) lymphocytes. Immunophenotypic studies of the digested sample at day 0 revealed CD4-to-CD8 ratios of between 0.7:1 and 3.5:1, and at days 18 to 20 they revealed ratios of between 2.3:1 and 98:1. In summary, we developed a simple technique for propagating cervical lymphocytes from small tissue samples for the study of the local immune response. Studies are under way to optimize lymphocyte growth and to preserve CD8 populations.

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression.

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.

Lymphocyte subset analysis by Boolean algebra: a phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence.

Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated monoclonal antibodies. We describe a rapid method whereby total CD3+ T-cells, CD4+ T-cells (CD3+ CD4+), CD8+ T-cells (CD3+ CD8+), putative gamma delta-receptor-T-cells (CD3+ CD4- CD8-), and T-cells that are CD3+ CD4+ CD8+ as well as B-lymphocytes and NK-cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.

Isolation and analysis of primitive hemopoietic progenitor cells on the basis of differential expression of Qa-m7 antigen.

In the presence of the hemopoietic growth factor CSF-1, the later committed cells of the macrophage lineage can be detected by their ability to form small colonies in clonal agar culture (CFCCSF-1). Synergistic factors have been described that in combination with CSF-1 stimulate developmentally early hemopoietic progenitor cells of high proliferative potential (HPP-CFC). By using a monoclonal antibody to the Qa-m7 antigenic determinant, we investigated and compared the expression of Qa-m7 on CFCCSF-1 and on HPP-CFC of two types that grow in response to either 1) CSF-1 plus synergistic factor from human placenta-conditioned medium (HPP-CFCHplac+CSF-1) or 2) CSF-1 plus synergistic factor from conditioned medium of the WEHI-3 myelomonocytic cell line (HPP-CFCW+CSF-1). We have shown that HPP-CFC of both types express relatively more Qa-m7 antigen than CFCCSF-1 and can be separated and enriched on this basis by discontinuous buoyant density centrifugation and fluorescence-activated cell sorting of normal bone marrow. Significant enrichments of HPP-CFCHPlac+CSF-1 (43.5-fold) and HPP-CFCW+CSF-1 (28.8-fold) have been achieved with cloning efficiencies of HPP-CFC in the most enriched fractions reaching 4 to 5%. These results clearly illustrate the fact that there are populations of progenitor cells from normal, unperturbed bone marrow that strictly require a combination of two hemopoietic growth factors (CSF-1 plus synergistic factor) in order to be detected.

DNA sequences showing a delay in cytosine methylation after replication. Time course of methylation in synchronized mammalian cell populations and relationship to DNAase I sensitive domains.

We have shown that in several mammalian cell lines a minor fraction of cytosine methylation is delayed for up to several hours after strand synthesis and that different methylases performed the immediate and the delayed classes of DNA methylation. To investigate the time course of this delayed methylation we have used three different cell lines, two of human and one of hamster origin. These were synchronized by two different methods: mitotic detachment and double hydroxyurea blocks. A uniform picture was obtained with all three cell lines. Delayed methylation of early replicating sequences occurred while cells were still in mid-S-phase, with the maximum rate of delayed methylation occurring in cells in the second half of S and in G2. Delayed methylation seems to be complete before cells entered the next G1-phase. Limited DNAase I hydrolysis of cell nuclei was used to test whether the delay in methylation in some DNA sequences was due to high levels of transcriptional activity. However, DNA sequences exhibiting delayed methylation showed no preferential concentration in or exclusion from DNAase I hypersensitive regions.