Hala Ghattas

In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection

Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium‐enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24‐hr intravenous infusion of 6,6‐D2‐glucose, CD3– CD16+ NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence‐activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n = 5), deuterium enrichment was maximal ∼ 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (± standard deviation) proliferation rate was 4·3 ± 2·4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 ± 7·6 × 106 cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6·9 ± 4·0%/day; half‐life (T½) < 10 days]. Healthy elderly subjects (n = 8) had lower proliferation and production rates (P = 2·5 ± 1·0%/day and 7·3 ± 3·7 × 106 cells/l/day, respectively; P = 0·04). Similar rates were seen in patients chronically infected with human T‐cell lymphotropic virus type I (HTLV‐I) (P = 3·2 ± 1·9%/day). In acute infectious mononucleosis (n = 5), NK cell numbers were increased but kinetics were unaffected (P = 2·8 ± 1·0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV‐I infection and normalize rapidly following acute Epstein–Barr virus infection.

Measurement and modeling of human T cell kinetics

The ability to measure, describe and interpret T cell kinetics is pivotal in understanding normal lymphocyte homeostasis and diseases that affect T cell numbers. Following in vivo labeling of dividing cells with 6,6‐D2‐glucose in eight healthy volunteers, peripheral blood T cells were sorted by CD4, CD8 and CD45 phenotype. Enrichment of deuterium in DNA was measured by gas chromatography‐mass spectrometry. A novel model of T cell kinetics, allowing for heterogeneity within T cell pools, was used to analyze data on acquisition and loss of label and calculate proliferation and disappearance rates for each subpopulation. Proliferation rates for CD45RO+CD8+ cells and CD45RO+CD4+ cells were 5.1% and 2.7% /day, respectively (equivalent doubling times: 14 and 26 days). CD45RA+CD8+ lymphocytes and CD45RA+CD4+ lymphocytes had slower proliferation rates, 0.5% and 0.6% / day, respectively (doubling time about 4 months). Disappearance rates of labeled cells were similar for all cell types (7%–12% / day) and exceeded corresponding proliferation rates. This disparity may be understood conceptually in terms of either phenotypic heterogeneity (rapid versus slow turnover pools), or history (recently divided cells are more likely to die). The new kinetic model fits the data closely and avoids the need to postulate a large external source of lymphocytes to maintain equilibrium.

Direct Measurement of T Cell Subset Kinetics In Vivo in Elderly Men and Women

The age-associated decline in immunocompetence is paralleled by changes in the proportions of PBL subpopulations. In turn, the size and composition of the peripheral lymphocyte pool is determined by input from the thymus and bone marrow and by the balance of proliferation and death in each lymphocyte subpopulation. We compared the kinetics of lymphocyte subtypes in young (seven of eight CMV seronegative) and healthy elderly human subjects (six of seven CMV seropositive), using deuterated glucose DNA labeling in vivo to measure rates of T cell proliferation and disappearance. For CD45RO+ cells of both CD4+ and CD8+ subtypes and for CD4+CD45RA+ cells the kinetics of proliferation and disappearance were remarkably similar between elderly and young subjects. In the young, the kinetics of CD8+CD45RA+ cells with a naive phenotype resembled those of CD4+CD45RA+ cells. However, CD8+CD45RA+ T cells from the elderly exhibited a predominantly primed phenotype, and for this subset, although the proliferation rate was similar to that of other CD45RA+ cells, the disappearance rate of labeled cells was greatly decreased compared with that of all other T cell subsets. Our data provide a direct demonstration that there are no substantial changes in in vivo kinetics for most T cell populations in healthy elderly compared with young subjects. However, primed CD8+CD45RA+ cells show unusual kinetic properties, indicating the persistence of these cells in the blood and dissociation of proliferation from disappearance.

Rapid turnover of T cells in acute infectious mononucleosis

During acute infectious mononucleosis (AIM), large clones of Epstein‐Barr virus‐specific T lymphocytes are produced. To investigate the dynamics of clonal expansion, we measured cell proliferation during AIM using deuterated glucose to label DNA of dividing cells in vivo, analyzing cells according to CD4, CD8 and CD45 phenotype. The proportion of labeled CD8+CD45R0+ T lymphocytes was dramatically increased in AIM subjects compared to controls (mean 17.5 versus 2.8%/day; p<0.005), indicating very rapid proliferation. Labeling was also increased in CD4+CD45R0+ cells (7.1 versus 2.1%/day; p<0.01), but less so in CD45RA+ cells. Mathematical modeling, accounting for death of labeled cells and changing pool sizes, gave estimated proliferation rates in CD8+CD45R0+ cells of 11–130% of cells proliferating per day (mean 47%/day), equivalent to a doubling time of 1.5 days and an appearance rate in blood of about 5×109 cells/day (versus 7×107 cells/day in controls). Very rapid death rates were also observed amongst labeled cells (range 28–124, mean 57%/day),indicating very short survival times in the circulation. Thus, we have shown direct evidence for massive proliferation of CD8+CD45R0+ T lymphocytes in AIM and demonstrated that rapid cell division continues concurrently with greatly accelerated rates of cell disappearance.

Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose

Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water (2H2O). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-2H2]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10–24 h; sampling, ∼3 weeks; DNA extraction/derivatization, 2–3 d; and GC/MS analysis, ∼2 d.

Effect of moderate anaemia on later mortality in rural African children

Severe anaemia in childhood is associated with increased mortality, although evidence relating moderate anaemia to child survival is scarce. We aimed to investigate this association. We did a case-control study of children with moderate anaemia, and compared haemoglobin concentrations measured up to a year before death for 403 children (age range 28 days to 15 years) with those from children who survived (matched for age and sex). Data were obtained from long-term health records (1950-97) of a rural Gambian research centre. Excluding an acute effect in this last week of life, no evidence was recorded of lower haemoglobin concentrations in the children who died than in survivors, or of any general or disease-specific effects of non-severe anaemia (70-110 g/L) on mortality.