Frederick G. Ferguson

Expansions of peripheral blood CD8 T-lymphocyte subpopulations and an association with cytomegalovirus seropositivity in the elderly: the Swedish NONA immune study

Results from a previous longitudinal study, the Swedish OCTO-Immune study, indicated that the combination of higher CD8 peripheral blood lymphocytes (PBLs), decreased CD4 PBLs, and poor proliferative response to mitogenic stimulation in very old humans were associated with an increased 2 year mortality. In follow up studies this combination of immune parameters was significantly associated with a CD4/CD8 ratio less than one and positive IgG serologic titers to cytomegalovirus (CMV). The present study, the Swedish NONA-Immune study, was an extension of that study, using a new sample of the very old. The results of this study confirmed the results of the previous study and extended the surface marker profile of the PBLs, indicating that the CD4/CD8 ratio change is associated with increased CD8 cells, decreased CD4 cells, and lymphocyte activation. The predominant phenotypes of the CD3+CD8+ cells were CD27-, CD28-, CD56+, and CD57+, CD45RA+, and double marked CD45RA+RO+. As in the OCTO study, the NONA-Immune data indicated that the changes are associated significantly with seropositive responses to CMV.

Age-related change in peripheral blood T-lymphocyte subpopulations and cytomegalovirus infection in the very old: the Swedish longitudinal OCTO immune study

Results from the previous times (Times 1-3) of the Swedish longitudinal OCTO immune study indicated that a combination of high CD8 and low CD4 percentages and poor T-cell proliferation in PBL was associated with a higher 2-year mortality in a sample of very old Swedish individuals. The combination of immune parameters was closely related to an inverted CD4/CD8 ratio. In the present study at Time 4 (T4) results are reported from the final follow-up of this longitudinal study, 8 years after it was initiated in 1989. An additional goal at this time point was to examine the immune system alterations in the very old in relation to evidence of lymphocyte activation and cytomegalovirus antibody status. In the present study immune system changes were identified that suggest a loss of T-cell homeostasis, as reflected by a decrease in the number of CD4 cells and a very significant increase in the number of CD8 cells in individuals with an inverted CD4/CD8 ratio. When considered over the duration of the OCTO study the inversion occurred in a high percentage (32%) of the individuals included in the original sample and was associated with non-survival. At T4 the changes were apparent in a number of the T-cell subsets, but particularly in the CD8+CD28-and CD57+ subsets. T-cell activation was significantly associated with the inversion of the CD4/CD8 ratio. In this very old sample the subset alterations were associated with evidence of cytomegalovirus (CMV) infection.

Changes in CD8 and CD4 lymphocyte subsets, T cell proliferation responses and non-survival in the very old: the Swedish longitudinal OCTO-immune study

Results from a previous longitudinal study indicated that a combination of high CD8 and low CD4 percentages and poor T cell proliferation in peripheral blood lymphocytes was associated with higher mortality in a subgroup of a sample of very old Swedish individuals. The present study examined whether those results could be confirmed at a subsequent 2-year time interval by investigating if additional individuals from the same original sample had developed the immune profile associated with higher mortality. Subgroups were formed by cluster analysis and similar to our previous results, this follow-up study identified a subgroup of subjects (n = 18) with an immune profile which again included high CD8, low CD4 percentages and poor mitogen response and was associated with higher mortality. Over the 2-year period 12 additional individuals: (1) Developed this immune profile; and (2) Could be identified by changes in their CD4:CD8 ratios which progressively decreased over the study period. These results confirm our original study and indicate that in this very old sample, over a subsequent 2 year period, additional individuals moved into the cluster at risk for higher mortality.

Age-related changes in immune parameters in a very old population of Swedish people: a longitudinal study.

This study used a longitudinal design to examine age-related changes in a well-defined sample of Swedish people ranging from 86 to 92 years of age at baseline. The longitudinal design encompassed three measurement occasions with 1 year intermeasurement intervals. The results were analyzed by multivariate analyses of variance (MANOVA), which is useful for comparing individuals over time. Healthy middle-aged subjects (39 years SD +/- 5.8) served as controls. The proliferative responses to Concanavalin A (Con A), a T-cell mitogen, indicated significant lower levels in responses of the old when the two groups were compared. The MANOVA revealed no significant change in mitogen responses over measurement occasions in the old sample as compared with the young. However, when cell types and lymphocyte subpopulations were examined, significant differences were found between the two age groups in many of these parameters and for some (lymphocyte percentages and numbers, CD3 numbers) the MANOVA indicated significant decreases over the measurement occasions in the very old. The results also consistently indicated significant intraindividual correlations in cell types, lymphocyte subpopulations, and mitogen responses over time.

The effects of group housing on the research use of the laboratory rabbit

This project evaluated the influence of group housing on common aspects of research use of female laboratory rabbits (Oryctolagus cuniculus). Eight rabbits housed individually in conventional cages were compared to a second group of 8 housed as a social group in a proportionately larger enclosure. The group housing method provided increased opportunities for exercise, social contact, and a more novel environment. As a function of housing style, the 2 experimental groups were compared on humoral and delayed hypersensitivity response, feed intake, growth rate, and selected physiological parameters that are considered to reflect stress in most species. Single and group housed rabbits did not significantly differ in physiological and immunological measurements, indicating that the practical research performance (immune response, stress level, growth rates etc.) of these rabbits was not significantly affected by group housing compared with the more traditional single housing. Analysis of group social behaviour indicated that the rabbits preferred small social groups, had preferences for microenvironments within the enclosure, and exhibited behaviours that are not possible when housed singly. Group housing appeared to be a successful method for enriching the environment of female rabbits and aspects of it should be considered in the approach to housing rabbits used in research.

Age- and strain-related differences in murine spleen cell responses to different activation signals

The effect of age on the response of splenocytes to activation with anti-CD3 mAb and a combination of anti-CD3 mAb and TPA, as evidenced by interleukin-2 (IL-2) and interleukin-4 (IL-4) production and cell proliferation, was examined in the C57BL/6 and DBA/2 murine strains. Depending on the mode of activation, there were age and strain differences in IL-2 and IL-4 production. With all modes of activation, cells from the old C57BL/6 mice produced less IL-2 than their young counterparts. In DBA/2 mice there was no age-related difference in IL-2 production with anti-CD3 mAb activation alone, whereas when the same cell population was activated with anti-CD3 mAb and TPA an age-associated decrease in IL-2 production occurred. In both strains, there was an age-related increase in IL-4 production with anti-CD3 mAb activation. After addition of TPA, however, there was an age-related decrease in IL-4 production. An age-related decline in the proliferation occurred with all modes of activation in both mouse strains. There were also strain-related differences in proliferation after the addition of forskolin, an inhibitor of Th1-cell function. While forskolin inhibited the proliferation of cells from the young C57BL/6 mice only, in the DBA/2 mice proliferation of cells was inhibited in both age groups. There were no strain-related differences in inhibition by anti-transferrin receptor (TrfR) mAb, although cells from the old mice were slightly more sensitive to this inhibition.

The effect of selected arachidonic acid metabolites on natural killer cell activity

The effect of arachidonic acid (AA) metabolites of lipoxygenase(s) was evaluated on natural killer (NK) cell activity in Fischer F344 rat splenic lymphocytes and compared with prostaglandin E2 (PGE2), a known inhibitor of NK cell lytic activity. It was observed that 5(S),12(S)-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-diHETE, EZEZ) inhibited NK cell activity to a degree comparable to the inhibitory effects of PGE2. This compound maximally inhibited NK cell activity at concentrations of 10(-6) and 10(-8) M. PGE2 and 5(S),12(S)-diHETE (EZEZ) inhibited NK activity to an identical degree at all concentrations and effector:target (E:T) cell ratios tested. Of the other lipoxygenase pathway metabolites screened, 8(S),15(S)-all trans-diHETE and 8(S),15(S)-diHETE (EZEZ) also inhibited NK activity, but only at 10(-6) M and a 50:1 E:T cell ratio. These findings provide further evidence that the lipoxygenase and cyclooxygenase pathways produce metabolites which can modulate NK cell function, and that 5(S),12(S)-diHETE (EZEZ), which has not been previously tested for effects on NK cells, may have a significant immunoregulatory role.

An in vitro and in vivo analysis of murine immunocompetence during pregnancy and lactation

Spleen cells from pregnant and lactating BALB/c mice were depressed in their cytolytic capabilities after in vivo immunization with the allogeneic EL4 lymphoma. However, in vitro spleen cells from both syngeneic (BALB/c X BALB/c) and allogeneic (BALB/c X C57BL/6J) matings responded with proliferative and cytolytic responses which were comparable to virgin controls. Upon secondary in vitro stimulation, in vivo primed maternal cells had responses which were similar to virgin controls. In addition, the in vivo sensitized maternal spleen cells adoptively immunized in irradiated allogeneic recipients responded like the virgin controls. In these studies, suppressor cells could not be detected in either nonimmune or immune maternal spleen cell populations.

Immune Risk Phenotypes and Associated Parameters in Very Old Humans: A Review of Findings in the Swedish NONA Immune Longitudinal Study

In the previous OCTO immune longitudinal study of free-living subjects >85 yr. selected for good health, we identified an Immune Risk Phenotype (IRP) associated with increased mortality. The IRP was characterised by high CD8+, low CD4+ T-cell counts and a poor T-cell proliferative response, inversion of the CD4/CD8 ratio and evidence of persistent cytomegalovirus infection. In the NONA immune longitudinal study the aim was to examine whether the same IRP parameters applied to a population-based sample aged 86–94 years who were not selected for very good health. More sophisticated analytical parameters were studied, as well as the role of inflammatory processes in relation to longevity. The immune panel included the analysis of T-cell subsets inflammatory markers, virus serology, cytokines, TCR clonotype mapping and functional and phenotypic analysis of virus-specific CD8+ cells by HLA/peptide multimers, in collaborations between participants of the EU funded T-CIA project.

REDUCTION IN ANIMAL NUMBERS REQUIRED FOR ANTISERA PRODUCTION USING THE SUBCUTANEOUS CHAMBER TECHNIQUE IN RABBITS

The purpose of this study was to determine if the subcutaneous chamber technique would reduce the number of rabbits required for antisera production by enabling serial use of individual animals for multiple antigens. Rabbits were assigned to immunization protocols against two antigens in series that involved combinations of chamber implantation, Freunds adjuvant and injection of antigen either by the subcutaneous route or by direct inoculation into the chamber. Results indicate the systemic immune response to both antigens achieved similar magnitude and duration by use of either Freunds adjuvant or direct inoculation of antigen into the chamber, suggesting that chamber use may be able to replace use of Complete Freunds adjuvant for many antigens. Rabbits re-used for a second antigen were equally successful in production of significant titres in both serum and chamber fluid without evidence of either a significant inflammatory response due to the chronic presence of the implant or a decrease in the yield of antisera harvested from the chamber. These results support the advantages of chamber use as reported by others and demonstrate that the chamber technique can significantly extend the productive life of an individual animal that would otherwise be euthanized following a single use in antisera production.