Cellular recognition in vitro by mouse lymphocytes. Effects of neonatal thymectomy and thymus graft restoration on alloantigen and PHA stimulation of whole and gradient-separated subpopulations of spleen cells.

The effects of thymectomy and thymus graft restoration upon the in vitro primary responses to alloantigens and PHA have been studied. It has been found that neonatal thymectomy substantially eliminates both PHA reactivity and responsiveness to alloantigens assayed in vitro in host spleen cell populations. Analysis of albumin density gradient-separated subpopulations of the spleen and thymus in such animals was also performed. It was found that the total and proportional representation of the individual density subpopulations was identical in neonatally thymectomized, in normal, and in thymectomized and thymus graft-restored animals. Therefore, thymectomized mice appear to retain a nonfunctioning, small, dense, lymphocyte population. Reconstitution of thymic-dependent in vitro reactivity was nearly complete when syngeneic, but not allogeneic or semisyngeneic thymus was employed. Occasional partial restoration did occur when F(1) thymus was employed, but never when allogeneic thymus was grafted. The grafted thymus contained PHA and alloantigen-reactive cells in a large, less dense B layer subpopulation, whereas the restored animals, as in the case of normals, showed these reactivities to be a property of a small, more dense cell population.

Cellular recognition by mouse lymphocytes in vitro. I. Definition of a new technique and results of stimulation by phytohemagglutinin and specific antigens.

The media and culture conditions required for in vitro stimulation of mouse lymphoid cells are described. The medium was arginine-rich and contained heat-inactivated human serum. A component of the human sera necessary for stimulation of the cells was a natural mouse cell agglutinin, which affected both background stimulation and the degree of induced stimulation with phytohemagglutinin (PHA). Absorption of the agglutinin from the human serum rendered the medium incapable of sustaining DNA synthesis in the presence of PHA. The response to PHA of mouse spleen and thymus cells was age-dependent and, although this response was not present at birth, it rapidly rose to adult levels. Spleen cells from mice immunized with bacillus Calmette-Guérin (BCG) or sheep erythrocytes (SRBC) showed increased in vitro reactivity to added purified protein derivative (PPD) or SRBC stroma, dependent on the time of immunization. The dose response curve for the SRBC stroma stimulated, immune spleen cells is compatible with a theory of cell to cell interaction being necessary for an in vitro reaction to antigen. The possible role of the mouse cell agglutinin (AMLG) is discussed.

Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly donors.

Using cDNA probes to human interleukin 2 (IL2) and interleukin 2 receptor (IL2R), the amount of IL2 and IL2R mRNA produced by PHA stimulated peripheral blood mononuclear cells from young (less than 40 yr) and old (greater than 60 yr) donors was quantitated. Stimulated cell cultures from each individual were also examined for proliferative ability, expression of membrane IL2R, membrane IL2R density, and for the amount of IL2R shed into the culture supernatant. Induction of IL2 and IL2R mRNAs were decreased in cells from elderly individuals, as were the levels of IL2 secretion, the percentage of IL2R+ T cells and the density of membrane IL2R per cell. The results suggest that decreased expression of both IL2 and IL2R mRNA contributes to the low synthesis of IL2 and membrane IL2R, respectively, and is partially responsible for the diminished proliferative activity observed in lymphocytes from the elderly.

Spontaneous or natural killer cytotoxicity of K562 erythroleukemic cells in normal patients.

Peripheral blood mononuclear cells from 200 normal individuals, ages 20 to 95 years, were evaluated for their capability to mediate spontaneous or natural killer (NK) cytotoxicity using the K562 erythroleukemic cells as targets. The results of a 4-hr 51Cr specific release assay demonstrated that the NK activity of normal individuals is independent of age, sex, and smoking habits. Although varying greatly among individuals, the NK activity of 25 persons restudied after a mean 20-month interval remained stable. Deficient NK lytic activity is not characteristic of elderly individuals.

Age differences in phagocytosis by polymorphonuclear leukocytes measured by flow cytometry.

Elderly persons have increased morbidity and mortality due to bacterial infections. Since the polymorphonuclear leukocyte (PMN) is a major defense against bacterial infection, we utilized fluorescent microspheres and flow cytometry to examine phagocytosis by PMNs from 55 young and middle-aged adults (mean age 41.5 yrs) and two groups of elderly subjects: one group of 35 healthy individuals (mean age 74.1 years) living at home, and a second group of 11 residents (mean age 83.1 years) with severe mental and physical disabilities, living in a domiciliary care facility. We determined the percent phagocytic PMNs, the number of microspheres per PMN, and the number of microspheres per 100 PMNs. The mean number of microspheres per phagocytic PMN was similar for all groups. Statistically significant differences were found between the young and middle-aged group and the healthy or ill elderly groups for the percent phagocytic PMNs (75.3% vs 51.5% and 43.8%), and the number of microspheres per 100 phagocytic PMNs (197.3 vs 131.4 and 103.2). There were no significant differences in these parameters between healthy and debilitated elderly subjects. These data document that there is an age-related increase in representation of a population of PMNs which have a defect in phagocytic ability.

Identification of HIV-1 envelope glycoprotein in the serum of AIDS and ARC patients.

Binding of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein (gp120) has been reported to alter the function and surface antigen expression of lymphocytes and monocytes in vitro. To determine whether these in vitro findings could be relevant in vivo, we searched for the presence of this antigen in the serum of patients with AIDS and the AIDS-related complex (ARC). Using an antigen capture enzyme-linked immunosorbent assay (ELISA) with polyclonal anti-gp120 antibody, we detected envelope antigens (gp160/120) in serum of 22 of 32 AIDS patients. In contrast, an ELISA using solid-phase recombinant CD4 to capture gp160/120 failed to detect any positives. A modification of the anti-gp120-based ELISA identified gp160/120-IgG immune complexes in all of 11 AIDS patients tested and in 4 ARC patients who were negative for gp160/120 antigen. We conclude that gp160/120, predominantly in the form of immune complexes, can be identified as circulating antigen in patients with AIDS. The potential pathogenic consequences of this antigenemia, its relation to soluble CD4 therapy, and its application as a clinical marker of disease merit further study.

Influence of age upon the metabolism of zinc in livers of C57BL/6J mice.

The kinetics of accumulation and loss of zinc from the liver following subcutaneous administration of 10 mg of zinc per kg were examined in young adult (6 months old) and old (24 months old) male C57BL/6J mice. After zinc treatment, total liver zinc concentrations rose equally in both groups and returned to basal levels at 96 h post treatment. However, differences were found in the subcellular distribution of zinc in these two age groups. The concentration of zinc in the cytosolic fraction (104,000 g supernate) prepared from the livers of old mice attained its maximum at 24 h post treatment. In contrast, the concentration of zinc in the cytosolic fraction of liver from young adult mice peaked at 48 h post treatment. This difference in accumulation of zinc in the cytosol was reflected by differences in the binding of zinc to metallothionein, a cytosolic transition metal binding protein. In old mice the highest amounts of zinc bound to metallothionein were found at 24 h post treatment: in young adults the maximal zinc binding to this protein occurred at 48 h post treatment. Examination of the relationship between cytosolic zinc contents and the binding of zinc to metallothionein in young adult and old mice suggested similar regulatory processes in the two age groups. Thus, age-dependent differences in accumulation and loss of zinc from the cytosolic fraction of liver probably reflect factors other than differences in regulation of the synthesis of metallothionein by this essential metal.

HIV infection and aging: mechanisms to explain the accelerated rate of progression in the older patient.

Age is an important predictor of progression in HIV infections. Not only do older individuals' develop AIDS more rapidly than younger persons, they die more quickly after developing an AIDS-defining illness. While the elderly have higher morbidity and mortality rates from viral and bacterial infections, the mechanism(s) responsible for the more rapid progression of HIV infection in older individuals has not been described. Our results demonstrate that the destruction of T cells in both young and old HIV infected patients progresses at the same rate. HIV 1-infected cells from older individuals do not appear more susceptible to immune mediated destruction. The more rapid progression appears due to an inability of older persons to replace functional T cells that are being destroyed. These findings suggest that improved survival in older HIV infected individuals will require more aggressive antiretroviral therapies as well as continued research to identify and preserve immune system elements that control the virus.

Expression of c-fos, c-jun and jun B in peripheral blood lymphocytes from young and elderly adults.

The expression of c-fos, c-jun and jun B proto-oncogenes was studied in phytohemagglutinin (PHA) activated peripheral blood lymphocytes (PBL) from young and aged humans. Specific mRNAs for c-fos and c-jun were detectable within 30 min after cell activation and reached maximal levels within 2 h. Both c-fos and jun B mRNAs decreased to pre-activation levels within 6 h, while c-jun mRNA remained elevated. In PHA-activated PBL, no age-related differences were observed in c-fos or jun B mRNA expression. However, c-jun mRNA levels decreased significantly (1.73 +/- 0.08 vs. 1.16 +/- 0.09 arbitrary units, P < 0.01, young vs. old) in PBL from elderly individuals activated with PHA. Because previous work has demonstrated that T cells from elderly individuals may display normal proliferative responses when activated via the anti-CD2 pathway, c-jun and jun B mRNA expression was also studied in anti-CD2-activated purified T cells. No age-related differences were found in the expression of either of these two proto-oncogenes by anti-CD2 activated T cells. These results suggest that the decreased IL-2 production and proliferative response displayed by PHA-activated PBL from elderly adults may be related to age-related changes in c-jun mRNA expression and in the ratio of c-fos to c-jun mRNA.

Bcl-2 transfection protects Hut78 cell line from different types of cytotoxic effector cells.

The human T lymphoma cell line Hut78 was transfected with Bel-2 gene or with control G418 Neomycin resistance-conferring plasmid. Bcl-2 transfected and Neomycin selected Hut78 cells expressed about ten-fold greater Bcl-2 protein than the neo-transfected controls. Susceptibility of Bcl-2-transfected Hut78 cells to human NK cells, human mixed lymphocyte reaction-generated cytotoxic effector cells, and mouse cytotoxic spleen cells generated by immunization with Hut78 cells was examined. In all systems tested, Bcl-2-transfected Hut78 target cells were significantly less susceptible to lysis than the neo-transfected control target cells. These results suggest that in Hut78 cells, Bcl-2 gene product may confer partial resistance to different types of cytotoxic effector mechanisms.

Immunoglobulin heavy chain junctional diversity in young and aged humans.

The causes of observed deficiencies to the humoral immune response in aged humans are unknown. Since a major source of antibody diversity is generated at the VH-D-JH junctional regions of the immunoglobulin heavy chain, we determined whether differences in junctional diversity are manifested with aging. We compared the CDR3 regions of IgM heavy chain transcripts isolated from young adult and aged humans. A PCR assay that measures CDR3 length in the majority of mu-heavy chains showed the same average size and normal range of CDR3 length in aged individuals as observed in young adults. To characterize the features of junctional diversity of aged adults in more detail, we determined the CDR3 sequences of a subset of the mu-heavy chain repertoire that utilizes members of the VH 5 family. In general CDR3 length, D family usage, and JH gene usage were similar in aged compared to young adults. Thus, in contrast to dramatic changes in heavy chain junctional diversity associated with fetal to adult development, no major differences were found between young and aged adults. Since the CDR3 repertoire generated in aged individuals appears to be as diverse as that observed in younger adults, the decline in humoral immunocompetence with aging cannot be attributed to a restriction in heavy chain junctional diversification processes.

In vitro erythrocidal activity of activated spleen cells from young and old mice.

We have recently reported that activated mouse spleen mononuclear cells (MNCs) efficiently lyse autologous erythrocytes in vitro (Saxena and Chandrasekhar, 2000). In the present study, we have investigated erythrocyte-depleting ability (EDA) of spleen MNCs from young and old mice. Time kinetics of survival of erythrocytes in mitogen-activated spleen cell cultures indicated that the erythrocyte depletion was significantly faster in young spleen cell cultures than in the old. Poorer EDA of old MNCs was in spite of the fact that the susceptibility to lysis actually increased in erythrocytes from old mice. Erythrocytes opsonized by a hamster anti mouse Fas monoclonal antibody, were destroyed with a much greater efficiency by young MNCs, whereas the corresponding effect of opsonization was only moderate for old MNCs. Depletion of macrophages from MNC preparations by plastic adherence as well as carbonyl-iron and magnet treatment had a marginal if any effect on EDA of young and old mouse MNCs, indicating that a lower lymphocyte-associated erythrocidal activity as one of the factors responsible for overall lower EDA associated with spleen derived MNCs of old mice.

Age-related effects in T cell activation and proliferation.

Age-associated thymic involution manifests its effects in a variety of ways that are related to a loss of T cell function. These include the appearance of a non-functional subset of T cells that increase in representation with age. Moreover there is a loss of T cell proliferative ability, a decline in the synthesis and release of interleukin-2 (IL-2), a decline in the ability of the T cell to express the IL-2 receptor, and a loss of control activity. This loss of control is demonstrated by the age-related appearance of autoantibodies and an increase in the elaboration of inflammatory cytokines such as TNF, IFN, IL-6, and TGF. A major part of the basis for the loss of T cell function is an inability of the T cell to respond to activation signals that are transmitted through the membrane binding of specific stimulatory signals. Transduction events, differentiation signals, and a loss of control mechanisms are all parts of a complicated picture of age-related immune deficiencies.

Cytolytic activity of mitogen activated old and young mouse spleen cells against tumor target cells expressing high or low levels of Fas antigen.

Sensitivity of Fas expressing tumor cells (high levels in Hut78 & Jurkat; low levels in P815) toward the cytotoxic Con-A (5 microg/ml) activated spleen cells from young (12 to 16 week old males) and old (2 year old males) mice were studied. The spleen cells from young mice activated for a day showed high levels of cytotoxic activity against Hut78 and Jurkat cell lines but not against P815 cells. The cytotoxic activity against P815 cells were detected in the spleen cells from old but not young mice following a longer period of Con-A activation (three days). Comparable levels of cytotoxic activity against Hut78 and Jurkat cells were observed in the spleen cells from both young and old mice following three days of activation. Treatment of Hut78 cells with anti-Fas antibody affected the tumor cells become resistant against the cytotoxic activity of the spleen cells from young mice in a dose dependent manner however P815 cells were not affect by the anti-Fas antibody treatment. These results show that there are differences in the sensitivity of target tumor cells toward Con-A induced cytotoxic spleen cells from young and old mouse. Mitogen-induced cytotoxic lymphocytes from young mouse spleen appear to kill targets through mechanisms involving Fas antigen, specially, in early stage (1 day) of activation. Old mouse spleen cells generated high levels of cytotoxic cells in later phase (3 days), which appear to kill through Fas-unrelated mechanisms.

Absolute peripheral blood lymphocyte count and subsequent mortality of elderly men. The Baltimore Longitudinal Study of Aging.

In this 16-year longitudinal study of 105 healthy elderly men, we analyzed one aspect of immunosenescence--a decline in the absolute number of peripheral blood lymphocytes--with particular reference to its relationship with subsequent mortality. It was found that there was a significantly (P less than .01) lower absolute lymphocyte count (1432 +/- 55/mm3; mean +/- SEM) within three years of death when compared with five years (1719 +/- 89/mm3) or 10 years (1715 +/- 98/mm3) before death. There was no relationship between this decrease in lymphocytes and age at death, smoking status, or prior cardiac illness. Previous cross-sectional studies have yielded conflicting data on age-related decreases in lymphocytes which may have been the result of an unrecognized selection process that either eliminated or included subjects who were close to death.

Reduced infectivity of cold-adapted influenza A H1N1 viruses in the elderly: correlation with serum and local antibodies.

OBJECTIVE: To compare young and elderly adults in terms of their immune responses and rates of infection following intranasal vaccination with a live attenuated influenza virus. DESIGN: Time series, comparing outcomes in young and elderly convenience sample. METHOD: Retrospective laboratory analysis of serum and nasal wash specimens collected during prior studies in which young or elderly volunteers had been inoculated with cold-adapted influenza A/Kawasaki/86 (H1N1) reassortant virus. SETTING: Johns Hopkins Center for Immunization Research. PARTICIPANTS: Healthy young and elderly adults with pre-vaccination serum hemagglutination inhibition (HAI) antibody titers less than or equal to 1:8. OUTCOME MEASUREMENTS: Antibody responses in serum and nasal washes. MAIN RESULTS: The proportion of vaccinees who developed any serum or local antibody response was higher in young compared with elderly subjects (20/20 vs 5/14, P less than 0.0005). Resistance to infection with cold-adapted virus correlated with pre-vaccination levels of serum immunoglobulin G (IgG), serum IgA, and nasal wash IgA antibody to whole virus antigen. Age was highly correlated with a lack of response to vaccine by simple regression, but not when data were adjusted for pre-existing antibody levels. CONCLUSIONS: Cold-adapted reassortant influenza A H1N1 viruses achieve lower rates of infection in elderly than young adults, primarily due to age-related differences in preexisting levels of immunity which may not be reflected by HAI titer.

Laminin concentrations in serum and cerebrospinal fluid in aging and Alzheimer's disease.

Laminin, a basement membrane protein, and a potent promoter of neurite outgrowth, surrounds all peripheral nerves. It appears in the central nervous system during development and reappears in response to injury. In Alzheimer's disease (AD), there is a progressive loss of neurons in specific areas of the brain along with the presence of an increased number of senile plaques and neurofibrillary tangles. Laminin levels have been shown to be increased in injury, so we undertook to examine levels of laminin by radioimmunoassay (RIA), in cerebrospinal fluid and serum of patients with AD and age-matched controls. No difference in the CSF and serum laminin concentrations was found between Alzheimer's disease and age-matched controls. We found a lack of correlation between severity of clinical dementia and laminin concentrations. Finally, we show that the CSF and serum laminin concentrations increase with age.

Acquired immunodeficiency syndrome in the elderly.

Recent data from the US show that since 1990 the number of paediatric patients with AIDS is decreasing while the number of patients with AIDS over age 50 years is increasing. To date, little attention has been given to understanding AIDS risk-taking behaviours, clinical presentations, and therapeutic needs of middle-aged and older HIV-infected individuals. Older HIV-infected individuals deteriorate more rapidly than younger patients due to an accelerated loss of CD4+ helper T cells. Despite recognised age-related physiological differences between young and elderly individuals, scant information about drug optimisation for the treatment of AIDS in older individuals is available. More data need to be collected about this group of AIDS patients, and appropriate treatment strategies designed for their special needs.

Followup study of possible HIV seropositivity among abusers of parenteral drugs in 1971-72.

Serum specimens obtained from a nationwide sample of parenteral drug abusers (PDAs) during the period 1971-72 had previously been screened for human immunodeficiency virus (HIV) antibodies. Some specimens were considered to be positive to both ELISA and Western blot (WB) analysis. These findings have been a topic of controversy, since HIV was not thought to have penetrated at-risk populations at such an early date. This study was a followup of those PDAs with apparent seropositivity to WB analysis. Of 10 persons followed, only one death (in 1985) was documented, and postmortem findings were inconsistent with HIV infection. Eight of the remaining PDAs were traced and found to be alive and well in 1989. Fresh specimens were obtained from the two persons with the strongest 1971-72 WB staining and were found to be both ELISA and WB negative on retesting. Their T-cell parameters were within normal limits. We concluded that the earlier WB results were most likely false positives and that definitive evidence of HIV infection in the U.S. addict population as early as 1971-72 is still lacking.

Immunological aspects of aging and malnutrition: consequences and intervention with nutritional immunomodulators.

As knowledge of molecular and cellular mechanisms of development and aging is elucidated, the need to understand the relationships among tissues, organ systems, health habits, nutrition, physical activity, and environmental factors on successful aging becomes more explicit. Progress in our understanding of how the immune system functions and responds with other factors, such as aging and nutrition, is spawning significant inroads to achieving a successful old age.