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.

Characterization of the murine cyclin-dependent kinase inhibitor gene p27Kip1.

The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in regulating cell-cycle progression. p27Kip1 directly inhibits the catalytic activity of cyclin/cdks (cyclin-dependent kinase) complexes and/or interferes physically with cyclin/cdks activation by CAK. Interestingly, the expression level of p27Kip1 mRNA was maximal in resting Go T-cells and rapidly declined following anti-CD3 activation. We report here the cloning of p27Kip1 gene from murine genomic DNA and the functional analysis of the promoter of the p27Kip1 gene. The gene consists of at least three exons and spans more than 5.6 kb of DNA. Primer extension and nuclease S1 protection analysis revealed two major transcription initiation sites. The promoter region lacked a TATA box but contained potential binding sites for the transcriptional factors including two Sp1, CRE, Myb and NFkB located at positions -153, -178, -286, -875, and -1011, respectively. To analyze the regulatory mechanisms controlling p27Kip1 gene expression, we characterized the 5'-flanking region from nt -1609 to +178. The -326 to -615 region contained positive regulatory elements.

Identification of genes differentially expressed in T cells following stimulation with the chemokines CXCL12 and CXCL10.

BACKGROUND: Chemokines are involved in many biological activities ranging from leukocyte differentiation to neuronal morphogenesis. Despite numerous reports describing chemokine function, little is known about the molecular changes induced by cytokines. METHODS: We have isolated and identified by differential display analysis 182 differentially expressed cDNAs from CXCR3-transfected Jurkat T cells following treatment with CXCL12 or CXCL10. These chemokine-modulated genes were further verified using quantitative RT-PCR and Western blot analysis. RESULTS: One hundred and forty-six of the cDNAs were successfully cloned, sequenced, and identified by BLAST. Following removal of redundant and non-informative clones, seventeen mRNAs were found to be differentially expressed post treatment with either chemokine ligand with several representing known genes with established functions. Twenty-one genes were upregulated in these transfected Jurkat cells following both CXCL12 and CXCL10, four genes displayed a discordant response and seven genes were downregulated upon treatment with either chemokine. Identified genes include geminin (GEM), thioredoxin (TXN), DEAD/H box polypeptide 1 (DDX1), growth hormone inducible transmembrane protein (GHITM), and transcription elongation regulator 1 (TCERG1). Subsequent analysis of several of these genes using semi-quantitative PCR and western blot analysis confirmed their differential expression post ligand treatment. CONCLUSIONS: Together, these results provide insight into chemokine-induced gene activation and identify potentially novel functions for known genes in chemokine biology.

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.

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.

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.

Characterization of the murine cdc2 gene.

A 1204 bp full-length cDNA encoding murine cdc2 was isolated and used as a probe to obtain four overlapping cdc2 genomic clones which span the entire cdc2 sequence. Characterization of these clones revealed that the cdc2 mRNA is distributed over 28 kb and in 8 exons ranging in size from 63 to approximately 303 bp. Since the 5'-untranslated sequence is interrupted by intron 1, the initiation codon is located in exon 2. The PSTAIRE region is in exon 3, and the stop codon is in exon 8. Primer extension analysis of cdc2 mRNA isolated from immobilized anti-CD3 activated T-cells demonstrated that the murine cdc2 gene utilizes multiple transcriptional start sites. No consensus sequence for a TATA box exists at an appropriate position within the promoter region. Instead, several putative transcription factor binding sites for PEA3, CREB, C/EBP, E box factor, YY1, ATF-like, Spl, and E2F were detected. Analysis of the promoter activity of the 5'-flanking region suggests that the sequence between -188 to -38, which possesses two Spl and one E2F motif, contains a major positive regulatory activity for the expression of murine cdc2.

Ibogaine acts at the nicotinic acetylcholine receptor to inhibit catecholamine release.

In an effort to determine mechanisms of action of the putative anti-addictive agent ibogaine, we have measured its effects on catecholamine release in a model neuronal system, cultured bovine chromaffin cells. Various modes of stimulating catecholamine release were used including nicotinic ACh receptor activation, membrane depolarization with elevated K+ and Na+ channel activation with veratridine. In addition, because ibogaine has been reported to interact with kappa opioid receptors, we tested whether kappa receptor antagonists could reverse ibogaine's effects on catecholamine release. Ibogaine, at low concentration (<10 microM) was found to selectively inhibit nicotinic receptor-mediated catecholamine release, while having no significant effect on release evoked by either veratridine or membrane depolarization with elevated K+. The inhibitory actions of ibogaine and the kappa agonists were not reversed by preincubation with the opioid antagonists nor-binaltorphimine or naltrexone, suggesting that these inhibitory effects are not mediated by the kappa opioid receptor. The effects of low dose (10 microM) ibogaine were rapidly reversible, while the inhibitory effects of higher ibogaine doses persisted for at least 19 h following ibogaine washout. The results provide evidence for a mechanism of action ibogaine at the nicotinic ACh receptor. The results are consistent with a model in which the initial high transient brain concentrations (100 microM) of ibogaine act at multiple cellular sites and then have a selective action at the nicotinic ACh receptor cation channel following its metabolism to lower brain concentrations. The present findings are relevant to potential anti-addictive actions of ibogaine and to the development of drugs to combat nicotine addiction.

Ibogaine selectively inhibits nicotinic receptor-mediated catecholamine release.

The effects of ibogaine, a putative anti-addictive drug, on stimulated catecholamine release were examined in cultured chromaffin cells to clarify its mechanism(s) of action. Low concentrations of ibogaine (1-10 microM) had a selective inhibitory action on nicotinic receptor-mediated catecholamine release, while higher concentrations (100 microM) inhibited additional modes of stimulated catecholamine release. These results suggest a selective inhibitory action of ibogaine at the nicotinic acetylcholine receptor, possibly at the receptor ion channel site.

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.

Collagenase production by cloned populations of rabbit synovial fibroblasts.

Monolayers of rabbit synovial fibroblasts treated experimentally with phorbol myristate acetate produce large amounts of collagenase and prostaglandin E2 and have been a suitable experimental model for the proliferative/destructive lesion of rheumatoid arthritis. We used X-irradiation to prolong the in vitro lifespan of these cells so that cloned populations could be studied. By a number of criteria, X-irradiation did not alter the cells to make them unrepresentative of synovial fibroblasts. With limiting dilution techniques, we simultaneously isolated three clones. These clones were shown to have different growth rates and to produce different amounts of collagenase and prostaglandin E2. Rates of protein synthesis, measured by incorporation of 3H-leucine, were similar for all three clones. Our data support the concept that particular populations of synovial cells may contribute selectively to the joint destruction seen in rheumatoid disease.

Enumeration of T lymphocyte subsets by monoclonal antibodies in young and aged humans.

The percentage and absolute number of peripheral blood mononuclear cells reactive with monoclonal antibodies identifying mature thymocytes and T cells (T3+), helper/inducer cells (T4+), and suppressor/cytotoxic cells (T8+) was determined in 19 young (mean age 35 yr) and 31 elderly (mean age 72 yr) individuals. The percent representation but not the absolute number of T cells (T3+) declined significantly (p less than 0.001) in the elderly, and the decline was attributable to both an absolute and relative decrease in the representation of the subpopulation of cytotoxic/suppressor (T8+) cells. The percentage and number of helper/inducer (T4+) T cells was comparable in both age groups.

Human B cell function in normal individuals of various ages. 1. In vitro enumeration of pokeweed-induced peripheral blood lymphocyte immunoglobulin-synthesizing cells and the comparison of the results with numbers of peripheral B and T cells, mitogen responses, and levels of serum immunoglobulins.

The effect of age on the in vitro generation of immunoglobulin-secreting cells in pokeweed mitogen-stimulated cultures was examined using a staphylococcal protein A plaque assay. Although there was no statistically significant decrease with age in the numbers of plaque-forming cells, subjects whose cells failed to produce immunoglobulin were four times more common amongst individuals over 55 years of age. Simultaneously-measured T and B lymphocyte numbers. 3H-thymidine incorporation by mitogen-stimulated cultures, and serum immunoglobulins were comparable in both the young and the aged.

Mitogenic activity of 12-O-tetradecanoyl phorbol-13-acetate on peripheral blood lymphocytes from young and aged adults.

The effect of age on the proliferative response to 12-O-tetradecanoyl phorbol-13-acetate (TPA) was examined using peripheral blood lymphocytes from 185 adults. TPA-induced DNA synthesis measured by cellular 3H-thymidine incorporation was found, like the responses of cells activated by PHA and Con A, to markedly diminish with advancing age. The presence of indomethacin (1 microgram/ml) or Ro 20-5720 (10 micrograms/ml) in TPA activated cell cultures, unlike PHA stimulated cultures, did not result in augmentation of 3H-thymidine incorporation by cells from elderly individuals. These results demonstrate that prostaglandin synthesizing suppressor cells are not responsible for the age-related depression of cellular immune function observed in TPA activated cells and confirm the observation that decreased production and/or utilization of soluble mediators, such as IL-2, may account for the diminished mitogen responsiveness of lymphocytes from elderly individuals.

Paraben allergy.

A hydrocortisone preparation containing methylparaben and propylparaben provoked bronchospasm and pruritus when given intravenously to an asthmatic patient, whereas another hydrocortisone preparation without paraben preservative did not. Direct and passive transfer (Prausnitz-Küstner) skin tests for immediate hypersensitivity to parabens were positive. Parabens, frequently employed as bacteriostatic agents, are capable of producing immunologically mediated, immmediate systemic hypersensitivity reactions.

A transient granulocyte killing defect secondary to a varicella infection.

A varicella infection in a previously healthy young girl was complicated by bacterial sepsis, arthritis, and osteomyelitis in multiple locations. This secondary complication caused by Staphylococcus aureus was associated with a transient defect in granulocyte function and an alteration in the representation of CD4 and CD8 positive lymphocyte subpopulation. The mechanism responsible for secondary bacterial infections following varicella may be due to transient defects in granulocyte function.