No correlation to collagen synthesis disorders in patients with Perthes' disease: a nationwide Swedish register study of 3488 patients.

BACKGROUND: Mutations of the COL2A1 gene have been identified in patients with Perthes' disease. Several studies have hypothesised a connection between Perthes' disease and collagen synthesis disorders, especially COL2A1-related disorders, but no large studies on the subject have been made. The aim of this study was thus to discover if there is a connection between patients presenting with Perthes' disease, and collagen synthesis disorders. A secondary aim was to see if the children with both disorders had less optimal birth characteristics than the rest. METHODS: Swedish national registers were used to collect data on children diagnosed with Perthes' disease or a collagen synthesis disorder. These registers include all births in Sweden, and data from both outpatient and in-hospital visits. A wide range of data is included besides diagnoses. All children with follow-up data to the age of 15 years were included. Pearson's chi-square was used for analysis. Statistical significance was further analysed with Fisher's Exact Test. RESULTS: In total, 3488 children with either diagnosis were included. 1620 children had only Perthes disease, while 1808 children had only a collagen synthesis disorder. Five children were found to have both the diagnosis Perthes' disease and a collagen synthesis disorder. One child was large for their gestational age and none of the children had a low birthweight. Two of the children were moderately preterm. CONCLUSIONS: The distinct lack of overlap in such a large body of material raises doubt about a connection between the presentation of Perthes' disease and collagen synthesis disorders, either COL2A1-related or not. We could not find an overrepresentation of less optimal birth characteristics either.

Dynamic phenotypic restructuring of the CD4 and CD8 T-cell subsets with age in healthy humans: a compartmental model analysis.

In healthy humans, phenotypic restructuring occurs with age within the CD3+ T-lymphocyte complement. This is characterized by a non-linear decrease of the percentage of 'naive' (CD45RA+) cells and a corresponding non-linear increase of the percentage of 'memory' (CD45R0+) cells among both the CD4+ and CD8+ T-cell subsets. We devised a simple compartmental model to study the age-dependent kinetics of phenotypic restructuring. We also derived differential equations whose parameters determined yearly gains minus losses of the percentage and absolute numbers of circulating naive cells, yearly gains minus losses of the percentage and absolute numbers of circulating memory cells, and the yearly rate of conversion of naive to memory cells. Solutions of these evaluative differential equations demonstrate the following: (1) the memory cell complement 'resides' within its compartment for a longer time than the naive cell complement within its compartment for both CD4 and CD8 cells; (2) the average, annual 'turnover rate' is the same for CD4 and CD8 naive cells. In contrast, the average, annual 'turnover rate' for memory CD8 cells is 1.5 times that of memory CD4 cells; (3) the average, annual conversion rate of CD4 naive cells to memory cells is twice that of the CD8 conversion rate; (4) a transition in dynamic restructuring occurs during the third decade of life that is due to these differences in turnover and conversion rates, between and from naive to memory cells.

The effect of DHEAS on influenza vaccination in aging adults.

OBJECTIVE: To determine whether simultaneous administration of dehydroepiandrosterone sulfate (DHEAS) exhibits adjuvant activity in the immune response of aging humans by supplementing influenza vaccination with the maximum single dose of DHEAS that could be practically injected subcutaneously (approximately 7.5 mg). DESIGN: A randomized, double-blind, placebo-controlled trial of DHEAS injection with 1993-94 and 1994-95 influenza vaccine in older subjects. In addition, initial safety, tolerability, and control testing with 1993-94 influenza vaccine was conducted in young subjects. SETTING: An urban primary care geriatrics clinic. PARTICIPANTS: Seventy-eight older adult volunteers (mean age 78.61 +/- 3.43 years, range 73-90 years) and 20 younger controls (< 40 years, means age 32.76 +/- 5.39 years) were recruited from clinic and community advertising. Subjects were free of disease or medication known to affect immune function. MEASUREMENTS: Immune responses to vaccine at 0, 2, and 4 weeks were measured by vaccine antigen-induced lymphoproliferation in peripheral blood mononuclear cells (PBMC) and serum antibody response by hemagglutination inhibition (HI). RESULTS: The maximum DHEAS dose that could be practically administered subcutaneously was 7.5 mg. Baseline DHEAS levels were significantly lower in older adults (52.1 vs 236.4 micrograms/dL, P < .001). The 1993 old adult DHEAS group HI response tended to be higher for the H3N2 Beijing antigen but not for the H1N1 or B antigen. In subjects with HI titers less then 1:40 for the H3N2 Beijing antigen (n = 29), the post-vaccination titer response tended to be higher among the 16 subjects who received DHEAS (P = .06). The peak response for the H3N2 antigen was associated with the initial DHEAS serum concentration in the DHEAS and placebo groups (R2 = .22, P = .04 and R2 = .21, P = .06, respectively). No significant differences were found for antibody responses to the H1N1 and B antigens or vaccine-antigen induced lymphoproliferation. CONCLUSION: A one-time supplemental dose of DHEAS with influenza vaccination appeared to enhance the specific HI antibody response to the 1993-94 H3N2 antigen in a small group of older adults. These findings were limited to those with lower prevaccination titers and lower DHEAS concentrations. Although clinical implications of these findings for influenza vaccine are uncertain, these results suggest additional detailed immunologic investigations on the role of DHEAS in the aging human immune response are warranted.

Diminished cell-cell binding by lymphocytes from healthy, elderly humans: evidence for altered activation of LFA-1 function with age.

We studied the kinetics of cell-cell adhesion by monocyte-depleted peripheral blood lymphocytes with a stirred-cuvette aggregometer using samples from healthy young (mean age = 25 years) and healthy elderly (mean age = 75 years) human donors. This was a very reproducible assay, as there was virtually no intraindividual variability and very little interindividual variability among donors of the same age group. In all cases, aggregation of cells began immediately upon addition of the protein kinase C activator phorbol myristate acetate (PMA) or the calcium ionophore ionomycin, and continued until reaching an asymptotic limit by 20 minutes or less. Unstimulated cells did not aggregate during this time. The rate of aggregate formation and total amount of aggregation (using young donors' cells) varied between PMA- and ionomycin-stimulated cells, suggesting different mechanisms for initiating cellular aggregation, or possibly, different peripheral blood lymphocyte subsets affected by these activating agents. However, for both stimuli, the rate of aggregation and the total amount of cellular aggregation were significantly lower with the elderly donors' cells. Further, aggregation was Ca2+/Mg2+ dependent, and the reaction required metabolically active cells, as the reaction was inhibited by the addition of sodium azide and 2-deoxy-D-glucose in both donor groups. Pretreating cells with the actin polymerization inhibitor cytochalasin B resulted in 35-40% inhibition of aggregation among young donors' cells, although, interestingly, there was no apparent effect upon the cells capable of forming aggregates in the old donor group. In both donor groups, aggregation by activated cells could be partially blocked by pretreating cells with monoclonal antibodies directed against the alpha and beta chains of the integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18). These results show that there is diminished cell-cell binding among lymphocytes from healthy, elderly humans, and this is partly due to altered activation of LFA-1 function with age.

Aging and lymphocyte function: a model for testing gerontologic hypotheses of aging in man.

Advanced age in humans and experimental animal models has been consistently associated with declines in the in vivo and in vitro responsiveness of T lymphocytes. The declines in vitro responses cannot be explained by decrease in numbers of differentiated T cells or by age-associated changes in the proportion of CD4+ 'helper' to CD8+ 'cytotoxic/suppressor' T cells. However, recent studies have demonstrated a decline with age in numbers of what appear to be antigenically 'naive' or 'virgin' T cells, accompanied by a proportionate increase in 'memory' T cells which mediate anemnestic or recall responses to previously encountered antigens. The findings lead to the hypothesis that thymic involution leads to decline in input of newly differentiated 'naive' T cells, while T cell number in peripheral tissues is maintained by accumulation of longer lived 'memory' T cells due to lifetime antigen exposure. So far, limited data suggest that increasing average biological age of the memory T cells which accumulate with age is largely responsible for declines in vitro activation responses of human peripheral blood T cells and that a subset of these memory T cells becomes refractory to activation stimuli as a consequence of in vivo cellular aging. Approaches to testing this hypothesis are presented and the implications of these findings for use of memory T cells as a model for investigating the mechanisms of in vivo cellular aging, are discussed.

Effects of preliminary culture on the membrane microviscosity of lymphocytes from young and old donors. Microviscosity correlates with mitogenic response.

Membrane microviscosity was assessed by a fluorescence polarization technique in fresh and precultured human peripheral blood lymphocytes of young and old subjects. Membrane microviscosity was significantly higher in fresh, non-treated cells of old donors as compared to young adults. Preincubation of cells in culture medium supplemented with pooled human serum diminishes the original microviscosity difference between the age groups. The observed increase in membrane fluidity correlates with the improvement of the mitogen-induced proliferative response due to preculturing cells from aged subjects. The results support the suggestion that membrane microviscosity can affect the proliferative response of lymphocytes, and it may play a role in the decline of the immune responsiveness in the elderly.

Diminished heat-shock protein synthesis following mitogen stimulation of lymphocytes from aged donors.

Studies of the diminished mitogen-induced proliferative response of T lymphocytes from older subjects show that aging must result in some defect(s) in the intracellular events required for transition from the G0 or quiescent state through the prereplicative interval and into the first S phase of the cell cycle. This conclusion is supported by observations of diminished inducibility of the lymphokine IL-2 and its receptor during aging. The current study demonstrates that decreased proliferative response to phytohemagglutinin (PHA) is also paralleled by decreased induction during the prereplicative interval of two of the most strongly enhanced proteins in mitogen-activated T cells: HSP90 and P73, which are also members of the heat-shock protein family. Diminished induction of HSP90 and P73 is observed in lymphocytes from older subjects (mean age 75), regardless of differences in health status of the subject populations. These results suggest that in vivo aging of human T cells results in a general defect in the induction of gene products required for transition from quiescence into the S phase of the cell cycle and that diminished T cell proliferation in advanced age is not due to a specific, "intrinsically immunologic" defect in induction of IL-2 and the IL-2 receptor.

Expression of the human T cell antigen receptor complex in advanced age.

Several studies of T cell-associated surface marker expression in older subjects "with no evidence of diseases known to affect immune function" have suggested that diminished expression of the constant portion (CD3) of the human T cell antigen-receptor complex (CD3/Ti) may be associated with aging. To determine if there is a decrease with age in expression of this complex, we determined the percent of cells positive for WT31, an antibody that recognizes a constant epitope on Ti alpha-beta heterodimers, CD3, CD4 and CD8 by immunofluorescence on peripheral blood lymphocytes from young controls (19-31 years), and 2 well characterized populations of older donors (greater than 69 years). The first group of older donors had no history of chronic or recent acute illness and saw a physician only for routine medical care. The second group was selected from patients seen in a geriatrics clinic for diagnoses that included osteoarthritis and cardiopulmonary disorders. There were no significant differences in the percent of cells positive with WT31, or with antibodies to CD4 and CD8 between young adults and the 2 groups of older donors. The clinic subjects showed a consistent decrease in percent of CD3+ lymphocytes in peripheral blood mononuclear cells compared to young adults (5-10% reduction) with less difference between the healthy older subjects and young adults. In the clinic subjects this reduction appears to be due to a decrease in CD3+, WT31-lymphocytes. These cells represent about 5-6% of the total T cell population in the young and healthy older group and are known to express CD3 in combination with Ti gamma-delta dimers. We show here that these cells are about equally distributed in high and low density lymphocytes after Percoll fractionation. We conclude that significant quantitative reduction in expression of the CD3/Ti antigen-receptor complex on human T cells is not a feature of the primary aging process and cannot account for diminished in vitro proliferative response of healthy subjects with age. However, diminished abundance of T cells expressing products of the Ti gamma-chain in less healthy older subjects may predispose to or result from diseases not usually associated with altered immune function.

Lymphocyte proliferative response to PHA and anti-CD3/Ti monoclonal antibodies, T cell surface marker expression, and serum IL-2 receptor levels as biomarkers of age and health.

Alteration of T cell surface marker expression with a decrease of CD3 positive cells relative to the number of CD4 and CD8 positive cells, diminished in vitro proliferative response to mitogenic stimuli like PHA and antibodies to the CD3/Ti complex, and increase in serum IL-2 receptor levels, are among the changes in immunologic parameters that have been associated with advanced age. To distinguish between effects of the primary aging process and diseases of aging not known to be directly related to immune function, we investigated these variables in two well characterized populations of elderly donors (greater than 70 years) and a young adult control group (less than 35 years). The first group of older donors reported no evidence of significant chronic or recent acute illness and saw a physician only for routine medical care. The second group was randomly selected from individuals seen in a geriatric medicine clinic for diagnoses that included osteoarthritis and cardiopulmonary disorders. Altered surface marker expression and increased serum IL-2 receptor levels were seen only in the second group. On the other hand, lymphocyte proliferative responses to PHA, Leu 4 (anti-CD3) and a monoclonal antibody to the beta-chain of the T cell antigen receptor (WT31) were significantly decreased in both populations. Because we would expect primary aging to affect even extremely fit individuals of advanced age, we conclude that decrease in T cell proliferative response may represent a biomarker of primary aging in man. The alteration in surface marker expression and increased IL-2R levels in serum appear to be effects secondary to non-immunologic disease rather than aging.

Do immature T cells accumulate in advanced age?

The hypothesis that decreased T cell function in the elderly involves an increased number of less differentiated T cells was examined. Three markers known to change during thymocyte development were analyzed; ratio of adenosine deaminase (ADA) to purine nucleoside phosphorylase (PNP), lactate dehydrogenase (LD) H/M subunit ratios and the T cell associated antigens, T3, T4, T8 and T10. Cells tested were from 10 old (greater than 75 years) and 10 young (less than 35 years) persons with equal numbers of males and females in each group. Before analysis, cells were purified into three groups; unfractionated, and monocyte depleted T cell and B cell enriched populations. Results for ADA/PNP ratios showed no significant differences between old and young in any of the fractions analyzed. H/M ratios however, were significantly reduced in all three fractions from old donors when compared with young. Surface marker distribution pattern as illustrated by the T3 - (T4 + T8) difference was lower in samples from old donors but not significantly so. There was a very significant reduction in percent cells positive for T3 in all three fractions from old persons. Although some of the changes seen in these markers could be due to a failure of normal differentiation, they could also be caused by the general phenomenon of altered gene expression known to occur with advanced age in a variety of non-lymphoid cells. The absence of any difference in the ADA/PNP ratio suggests that T cell dysfunction in the elderly may not be due to increased numbers of less differentiated cells as a result of thymic involution.

Evidence for expansion of a population of lymphocytes with reduced or absent T3 expression in aged human donors.

In a previous report, we described an unusual pattern of T cell associated surface marker expression in unfractionated mononuclear cells from aged donors; an excess of T4 and T8 positive cells relative to T3 positive cells. This study further characterizes these cells on the basis of density, adherence to nylon wool and quantitative expression of cell surface markers. We find that the population of lymphocytes responsible for the unusual surface marker expression is of low density, adheres to nylon wool, and is present in small numbers in young donors. The adherent cells have a reduced quantitative expression of the T3 antigen, no change in the antigen density of T4 and T8, and have increased expression of the T10 antigen. These cells do not have the characteristics of large granular lymphocytes, monocytes, B cells with unusual marker expression, or thymocytes poised for export to peripheral blood. We suggest that these cells, found in increased numbers in aged humans, may represent an expansion of a population of T lymphocytes with absent or reduced T3 antigen expression found normally in smaller numbers in young adults. T lymphocyte antigen receptor density has been quantitatively linked to expression of the T3 antigen. Thus, our results imply that aging may lead to decreased T cell surface antigen density, which may account in part for decline in T cell function with age.

Enhancement of mitogen response and surface marker analysis of lymphocytes from young and old donors after preliminary incubation in vitro.

We have examined the detailed kinetics of PHA induced proliferation of freshly isolated mononuclear cells from young and old human donors. Our studies confirm that donors over the age of 60 years may have a decreased number of PHA responsive cells and that these cells have a significantly diminished rate of entry into the first cell cycle. Age of the donor does not affect the time at which significant numbers of cells appear in first S-phase, the duration of first S-phase, the doubling time in exponential growth, and thymidine uptake per cell. Cells from young and old donors were also cultured in medium supplemented with pooled human serum for 1 or 2 days prior to the PHA response assay. After 2 days of preliminary culture, the PHA response in both young and old is significantly enhanced, with a greater enhancement in the old. The basis of this enhancement appears to be a significant increase in rate of entry into the first cell cycle. None of the other kinetic parameters were significantly altered. The magnitude of the rate enhancement in old donors' cells eliminated the difference in entry rate between young and old after 2 days of preliminary culture. The same rate enhancing effects were seen in the presence of serum pooled from young or old donors. There is no statistically significant change in the number of responding cells after preliminary culture. We had previously suggested, based on lactate dehydrogenase (LD) subunit ratio and patterns of T cell associated surface markers, that less differentiated subpopulations of T cells exist in elderly donors. After preliminary culture, no significant change was seen in the proportions of cells positive for T3, T4, T8, T10 and Ia surface antigens and the unusual pattern of surface marker distribution was still present on the old donors' cells. It appears that the greater rate enhancement in old donors' cells after preliminary culture may not be due to induced maturation of the possibly less differentiated T cell populations described previously. The results do suggest that these subpopulations may be non-responsive to PHA.

Familial alopecia areata. HLA antigens and autoantibody formation in an American family.

Ten members of a white American family, spanning three generations, were studied. Three family members from two different generations were affected with hair loss. Two had alopecia universalis; one had alopecia areata. All subjects were HLA-typed using 131 antiserum samples obtained from multiparous female donors defining 41 HLA-A and HLA-B antigen specificities. Six haplotypes were identified. The affected persons and four other family members shared a common haplotype, HLA-A2,B40. The OKT4 (helper), OKT8 (suppressor-cytotoxic) cells, OKT4-OKT8 (helper-suppressor-cytotoxic) ratios and the percentage of B cells found were comparable for both the 12 control subjects and the family members studied. However, family members showed increased autoantibody formation, decreased T-cell percentages, and concanavalin A-induced suppression of the normal lymphocyte response to mitogens.

Suppressor cell number and function in alopecia areata.

Several values of immunologic function were studied and correlated with disease activity and extent in 14 patients with alopecia areata, alopecia totalis, or alopecia universalis and in a concurrently studied age- and sex-matched control group. As compared with the control group, the patients showed a significantly increased incidence of autoantibody formation, increased concanavalin A-induced suppression of the normal lymphocyte response to mitogens, an increased proportion of suppressor-cytotoxic cells in the peripheral blood, and a decrease in the absolute B-cell count. Absolute total T-cell counts, quantitative serum immunoglobulin determinations, and lymphocyte proliferation after exposure to the mitogens--concanavalin A, phytohemagglutinin, and pokeweed--and to tetanus antigen were comparable for both groups. Neither the percentage of concanavalin A-induced suppression of the normal lymphocyte response to mitogens nor the helper-suppressor ratio correlated significantly with the extent of hair loss. However, patients, particularly those who demonstrated spontaneous regrowth of hair, had increased concanavalin A-induced suppression in conjunction with an increase in the proportion of peripheral suppressor cells.

Unusual pattern of surface marker expression on peripheral lymphocytes from aged humans suggestive of a population of less differentiated cells.

Monoclonal antibodies were used to detect the surface antigens T3, T4, T8, and T10 on the peripheral blood lymphocytes of 26 aged (14 female and 12 male, mean age 89) and 28 young (14 female and 14 male, mean age 29) subjects. In the aged subjects, independent of the sex of the donor, the sum of the percent or absolute number of T4- and T8-positive cells was significantly greater than the number of T3-positive cells (p less than 0.001). There was also a significant increase in the percent and absolute number of cells positive for the T10 antigen with age (p less than 0.01). Analysis of individual cell surface markers revealed that the percent and absolute number of T3-positive cells was decreased only in old females, with no difference between old males and the young donors. The expression of T4 was not affected by age or sex, but both the percent and absolute number of T8-positive cells were decreased in females relative to males, with no effect due to age. These findings are consistent with the presence of a population of peripheral T cells in advanced age with a thymocyte-like pattern of surface marker expression. This conclusion is supported by previous work showing a less differentiated pattern of LDH isoenzyme distribution in the T cells of persons of advanced age.

Evidence for a less differentiated subpopulation of lymphocytes in people of advanced age.

The ratio of the lactate dehydrogenase subunits, H/M, is shifted toward lower values in thymocytes, and in high-density T cells in normal peripheral blood. The results presented here indicate that the T cells, but not B cells or monocytes, of donors greater than 75 years' old have a significantly reduced H/M ratio relative to young adults. This reduced H/M ratio appears to be correlated with a diminished percentage of OKT3 positive cells in the old donors. However, the density distribution of cells from young and old on Percoll gradients indicates that the T cells with lower H/M ratio in the elderly may represent a distinct, and possibly unique, less differentiated T cell type.