Influence of age on the immunological activity and capacity of the CBA mouse.

The immunological activity and capacity were studied in the CBA mouse as a function of its age. The activity was determined by the number of immunoglobulin containing (C-Ig) cells in different lymphoid organs and the immunoglobulin levels of the serum in non-artificially stimulated animals. It was confirmed that in older age the bone marrow takes over from the spleen the role of the major site of immunoglobulin production. A clear decrease in the number of C-Ig cells was observed in the mesenteric lymph nodes and the Peyer's patches. The Ig serum remained constant after the sixth month of age, with the exception of an increase of IgG1 and IgG,2B. There was a striking increase in variation between the individual animals with advancing age. From these data it can be concluded that the B-cell system of old animals is as active as that of young adult animals. The immunological capacity of CBA mice of various ages was assessed by measuring the levels of specific antibodies after the administration of human serum albumin in complete Freund's adjuvant. A severe decline of the primary and the secondary response was observed on ageing. The reaction of three year old animals was negligible. The discrepancy between the declining immunological capacity and the constant or increasing immunological activity is explained by an age-related deficiency of the T-cell compartment in the spleen.

Autoantibodies in highly aged humans.

The presence of 14 different autoantibodies was determined in 65 persons, aged 95 years and older, without overt disease. The prevalence of positive anti-immunoglobulin latex tests, of autoantibodies against nuclear components and against thyroid microsomes was significantly increased. This selective increase of autoantibodies of low titre and without cluster formation is considered to be the result of a loss of control within the immune system due to ageing, rather than as a sign of latent disease.

Age-related changes of the antigen-specific antibody formation in vitro and PHA-induced T-cell proliferation in individuals who met the health criteria of the Senieur protocol.

The antigen-specific antibody secretion in vitro after immunisation with the primary T-cell dependent antigen Helix pomatia Haemocyanin (HPH) was investigated in both young and elderly individuals, who all met the health admission criteria for immunogerontological studies as detailed in the SENIEUR protocol. In addition, elderly non-Senieur persons were incorporated in this study. Young and elderly Senieur volunteers were fully comparable in terms of the occurrence of anti-HPH antibody secreting cells after in vitro simulation of peripheral blood mononuclear cells with variable doses of the antigen. In contrast, the non-Senieur elderly showed a lower number of anti-HPH antibody secreting cells in vitro. PHA-conditioned medium did enhance this in vitro response, whereas the addition of IL-2 remained ineffective. The PHA-induced T-cell proliferation was found to be somewhat impaired in elderly Senieur individuals and significantly lower in elderly non-Senieur individuals compared to young healthy persons. Using an immunofluorescence double staining technique after BrdU incorporation, the phenotype of the proliferating cells was determined. Again the total number of proliferating cells was impaired in the non-Senieur elderly. No changes in the relative contribution of CD4+ or CD8+ cells to the number of proliferating cells were found in the different age groups. On the other hand, a significantly lower number of proliferating cells with IL-2 receptor expression were detected in the non-Senieur individuals, which could account for the lack of response to IL-2 in this group. Our study clearly shows that so-called age-associated immune deficiency can be the result of disease and not necessarily of the ageing process itself.

Influence of ageing on antibody formation in vivo after immunisation with the primary T-cell dependent antigen Helix pomatia haemocyanin.

The in vivo antibody response to the primary T-cell dependent antigen Helix pomatia Haemocyanin (HPH) was studied, in order to detect the possible presence of a humoral immune deficiency in ageing. The IgG subclass distribution of the specific antibodies was also determined. In order to define a dose of HPH which could be used to discriminate between the responsiveness of healthy and immunocompromised individuals, we first established a dose-response curve for this antigen in 60 healthy young volunteers. Their responses were compared with the responses of a group of patients suffering from end stage renal failure. The patients who were treated with haemodialysis showed a significantly lower IgM, IgG and IgA anti-HPH antibody response after immunisation with a dose of 30 micrograms HPH, which could be restored by increasing the antigen dose. Patients treated with continuous ambulant peritoneal dialysis and a group of elderly persons, selected according to the Senieur protocol, showed no impairment of antibody formation after immunisation with 30 micrograms HPH, but in the non-Senieur elderly the anti-HPH antibody response was significantly lower. Furthermore, Senieur and non-Senieur elderly persons showed a diminished IgG2 anti-HPH antibody formation, whereas in the elderly non-Senieur individuals and in the patients with renal insufficiency, IgG1 and IgG3 anti-HPH antibodies were also diminished. This study clearly shows that the so-called age-associated immune deficiency can be the result of disease and is not necessarily due to the ageing process itself.

Serum immunoglobulin class and IgG subclass levels and the occurrence of homogeneous immunoglobulins during the course of ageing in humans.

Serum levels of IgM and IgA classes and of IgG subclasses were determined and related to the presence of homogeneous immunoglobulin components (H-Ig) in volunteers equally distributed in age groups from 25 to 98 years, who all met the Senieur admission criteria for immunogerontological studies. In addition, sera of non-Senieur volunteers aged 75 years and older were included. Furthermore, the amount of IgD was determined in sera of Senieur individuals equally distributed in age groups from 15 to 98 years. In the Senieur persons, the contribution of the IgG subclasses and the IgM and IgA classes to the pool of serum immunoglobulins remained relatively unchanged during the course of ageing. In comparison with Senieur individuals aged 25-34 years, a slight increase in IgM and IgA levels was observed from the age 35 to 44 onwards and in IgG1 from the age 55 to 64 onwards. The variability of the immunoglobulin concentrations increased during ageing. The most prominent observation was the continuous decline of serum IgD starting in young adults. The non-Senieur persons differed from their Senieur age-matched counterparts mainly by the elevated IgG2 and IgA levels. During the course of ageing, H-Ig mainly of low concentration were detected at an increasing frequency in the Senieur persons and even more frequently in the elderly non-Senieur volunteers. Although in some individuals the elevation of immunoglobulin levels correlated with the appearance of H-Ig within the corresponding isotype, this relationship was not conclusive for all sera investigated. These results suggest that the rise of serum levels of individual immunoglobulin isotypes associated with ageing is usually the consequence of a polyclonal B cell activation. The occurrence of H-Ig and the decline of serum IgD in aged Senieur persons indicate that these are, at least partly, true phenomena of ageing and not always the consequence of disease.

Monoclonal gammopathies in human aging: increased occurrence with age and correlation with health status.

To determine the incidence of monoclonal gammopathies (MG) in relation to the aging process as such, and to evaluate the influence of disease on the occurrence of MG, we studied 439 elderly subjects aged 75-84 years. These individuals were categorized into 4 groups on the basis of their health status. There was a group of "optimally healthy" elderly, a group of "apparently healthy" residents of homes for the aged, a group of geriatric outpatients and a group of randomly chosen inpatients from a general hospital. Whereas no MG were detected in a control group of healthy young subjects aged 25-34 years, the frequency of MG in the aged groups ranged from 11% in the "optimally healthy" aged group to 38% in the inpatients group. In a tentative classification according to possible cause, most of the MG belonged to the pathogenetic category of immunodeficiency. There was a clear association of the occurrence of monoclonal gammopathies of this category with the health status.

Necessity of the assessment of health status in human immunogerontological studies: evaluation of the SENIEUR protocol.

Disease is frequent in ageing, and the many conflicting results in studies of the ageing process can be due to the presence of factors such as underlying disease or the use of medication. For immunogerontology, a solution to this problem was initiated in 1984 by a working party of EURAGE, the European Community's Concerted Action Programme on Ageing and Diseases. A protocol defining strict admission criteria to studies of ageing, the SENIEUR protocol, was elaborated. This protocol intends to limit the influence of disease and/or medication and to standardize admission criteria to immunogerontological studies. In subjects fulfilling the SENIEUR criteria, we found less immunological defects with ageing than generally stated. This could mean that many studies performed in not-optimally healthy subjects describe defects that are not a consequence of the ageing process, but could be a result of underlying disease or of the influence of medication. For lymphocyte subsets, certain changes are only found in the comparison of SENIEUR groups of young and aged, while other changes are only found when non-healthy groups are compared. The occurrence of monoclonal gammopathies and autoantibodies was increased in ageing, but was also influenced by health status. Experience of other groups, and the objections against the protocol are discussed.

Admission criteria for immunogerontological studies in man: the SENIEUR protocol.

Immunogerontological studies in man have often led to conflicting results. One of the main reasons is the selection of the subjects to be studied. Admission criteria such as "apparently healthy" or "without overt disease" seem insufficient to exclude underlying disease which might influence the immune system and thereby the results. In an attempt to solve this problem, the SENIEUR protocol described in this paper was developed by a working party in the framework of the EURAGE Concerted Action Programme on Ageing of the European Community. This protocol establishes strict admission criteria for immunogerontological studies in man based on clinical information and laboratory data, and it sets limits to pharmacological interference. The use of this protocol will lead to standardization between centers and also to a closer study of the influence of age as such on the immune system. These findings in the immunologically "optimally aged" can also serve as reference values for immunogerontological studies in subjects who do not meet the SENIEUR criteria. In this way the use of this protocol can contribute to the dissection of the influence of disease versus ageing on the immune system.

The specificity of commercial conjugates to human immunoglobulins: results of performance tests on monoclonal and polyclonal plasma cells and lymphocytes.

The activity and the specificity of 29 fluorochrome conjugated antisera against human immunoglobulin heavy and light chains were evaluated by performance testing with the direct technique of immunofluorescence using plasma cells and lymphocytes as biological substrates. Fifteen conjugates gave satisfactory results in the detection and classification of cytoplasmic and surface bound immunoglobulins and were therefore considered specific. Fourteen conjugates did not meet the required standards. The usefulness of the biological substrates for quality control tests is discussed.

Hairy cell leukemia: its place among the chronic B cell leukemias.

Hairy cell leukemia is a chronic B cell leukemia. The presence of surface Ig (SIg) of gamma or multiple isotypes on the cells locates HCL at a rather mature stage of B cell differentiation. The reactivity of HC with McAb is in accordance with this concept (T65-, OKM1+, FMC7+, BA-1-). To relate HCL to other chronic B cell leukemias, a morphologic classification was developed that distinguishes, besides HCL, five subtypes of chronic B cell leukemia. CLL showed weak staining for SIg of mu +/- delta class. The phenotype with McAb was T65+, OKM1-, FMC7-, BA-1+. Prolymphocytic transformation of CLL had essentially the same membrane phenotype. LPL often had brighter SIg of mu +/- delta class with gamma or multiple isotypes in about half of the cases. McAb gave a T65-or+, OKM1-or+, FMC7-or+, BA-1+ phenotype. The same surface-marker profile was found in CL. Finally, PLL showed bright SIg of gamma or multiple isotypes in the majority of cases and reactivity with McAb according to a T65-or+, OKM1+, FMC7+, BA-1+ pattern. The various immunologic phenotypes of the morphologic subtypes showed a considerable overlap. The various chronic B cell leukemias should be located in the scheme of B cell differentiation in the sequence CLL-LPL/CL-PLL-HCL.

Microfluorometric evaluation of the specificity of fluorescent antisera against mouse immunoglobulins with the defined antigen substrate spheres (DASS) system.

Highly purified MOPC-21 IgG1, MOPC-173 IgG2a, MOPC-195 IgG2b, MOPC-104 E IgM, and MOPC-315 IgA paraproteins, heterogeneous mouse IgG, Fab and Fc fragments of heterogeneous IgG were prepared and coupled to Sepharose beads. These beads were then used as artificial substrates to test the specificity of fluorescent antisera against mouse immunoglobulins by microflurometry. By comparing the visual evaluation of strained plasma cells and measurements on beads, the highest permissible percentage (FITC) and 6% for a tetramethyl rhodamine iso thiocyanate (TRITC) conjugate. By application of these criteria, 1 out of 7 tested commercial antisera and 6 out of 8 conjugates prepared in this laboratory proved to be satisfactory. The most common impurities were anti-light chain antibodies, as revealed by their reaction with Fab. With the bead system, a good impression of the specificity of an antiserum can be obtained. It gives, however, only approximate information on whether the conjugate will cause a high background staining in the biological specimen.

Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.

Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.

Prevalence of dementia in the 'oldest old' of a Dutch community.

OBJECTIVE: To estimate the prevalence rate of dementia in subjects 85 years of age and over. DESIGN: A two-phase design with the Mini-Mental State Examination (MMSE) in the screening phase and the Geriatric Mental State Schedule (GMS) in the diagnostic phase. SETTING: Community survey including subjects in residential care. SUBJECTS: All (n = 1,259) inhabitants of Leiden, The Netherlands, aged 85 years and over on December 1, 1986. First phase participation rate was 71% (17% dropout due to death); second phase participation rate was 82%. MAIN OUTCOME MEASURE: DSM-III diagnosis of dementia without further specification of the etiology of the dementia. RESULTS: An overall prevalence rate of 23% (95% C.I.: 19%-26%) was found. This included 12% mild dementia, 7% moderate and 4% severe dementia. The prevalence rate was higher among women (24%) than among men (18%). It increased with age from 19% (95% C.I.: 16%-22%) in the group of 85-89 years to 32% (95% C.I.: 26%-39%) in the group of 90-94 years to 41% (95% C.I.: 25%-58%) in the 95+ group. CONCLUSION: A fifth of the 85+ and a third of the 90+ population suffer from dementia with an indication that half of the 95+ population is affected. With the expected steep rise in the number of the oldest old, dementia will stay a major health problem in the near future.

Reference values for the Mini-Mental State Examination (MMSE) in octo- and nonagenarians.

The Mini-Mental State Examination (MMSE) was used in a population survey of all inhabitants of Leiden, the Netherlands, over 85 years (n = 1258). In this paper we report on 532 subjects without neurological or psychiatric disease. Results show that the median score and lowest quartile cut-off score remain high until the tenth decade (median score = 28, lowest quartile cut-off score = 26). Thus age, in itself, is not a major limitation in using the MMSE. In this study a comparatively low level of education (the majority had 6 to 7 years of education) did not affect the results on the MMSE in a negative way, nor did we find an association with the use of psychoactive drugs.

Intracellular inclusion bodies in 14 patients with B cell lymphoproliferative disorders.

Two types of intracytoplasmic inclusion were detected by immunofluorescence microscopy in 12 patients with chronic lymphocytic leukaemia and two patients with a leukaemic phase of well differentiated lymphocytic lymphoma. Further analysis with light- and electron microscopy, showed that most inclusion bodies were rod-like crystalline structures. However, in three patients they consisted of amorphous vesicular precipitates. Immunological studies revealed the presence of immunoglobulins of the same class and type at the cell surface as well as in the inclusion bodies. The monoclonal immunoglobulins were all of lambda type except in two cases. The origin of immunoglobulin inclusion bodies in B cell malignancies is discussed in relation to published data and our own observation in one patient followed during treatment.

Longevity and heredity in humans. Association with the human leucocyte antigen phenotype.

Several arguments support the idea of a link between longevity and heredity, both in experimental animals and in the human species. In mice, genes in the major histocompatibility complex (MHC) are associated with a significant effect on life span. Results of analogous studies in man are confusing and contradictory. We have therefore investigated the question of an association of the human leucocyte antigen (HLA) and longevity in a large and ethnically homogeneous population. Our study population consisted of all 964 available inhabitants aged 85 years and over in the Dutch community of Leiden (pop. 104,000). Our control group comprised 2444 young inhabitants, aged 20-35 years, with an identical ethnic and demographic background. In addition, control groups of different age-brackets from the same region were used. Two antigens differed in frequency: HLA-B40 was lower and HLA-DR5 was higher in the group of 85 years and over, as compared to the control group, aged 20-35 years. Both differences were more evident in females. No major disease associations with HLA-B40 or HLA-DR5 have been reported. It is unlikely that these results are a chance observation: the overall similarity of the HLA pattern of the old and young age groups is a confirmation of their identical ethnic and demographic background and the changes as observed in the different age-groups were gradual. The biological meaning of these results is still unclear.

Microfluorometric evaluation of conjugate-specificity with the defined antigen substrate spheres (DASS) system.

Six fluorescent antihuman Ig preparations were tested for their Ig class specificity by reacting them with highly purified IgG, IgM, IgA, and OVA coupled covalently to Sepharose beads. OVA was used as a measure for nonimmunologic binding. Bead fluorescence was determined by microfluorometry. The amounts of USS and NSS were expressed quantitatively. These data were compared with the performance of these particular conjugates in a biologic system, namely, monoclonal bone marrow cells. Five of the six conjugates satisfied the requirement of monospecific activity; one did not. At a dilution of 1 : 8, the five monospecific conjugates reacted between five and 50 times stronger with their appropriate antigens than with OVA-coupled beads. Cross reactivity with other Ig classes, after correction for OVA staining was maximally 6%. The conjugate that was nonspecific in the bone marrow system gave very high cross reactivity with the Ig-coupled beads. A good correlation was found between OVA bead staining and nonimmunologic binding of conjugates in bone marrow slides. In this respect, conjugates prepared from antibody preparations isolated by solid immunoadsorbents proved to be superior to globulin or whole IgG fractions. Ig coupled to Sepharose beads seems to represent a very promising substrate for conjugate specificity testing.

The prevalence of morbidity in the oldest old, aged 85 and over: a population-based survey in Leiden, The Netherlands.

The Leiden 85-plus study has investigated the prevalence of morbidity in the total population of the Dutch community of Leiden (population 105 000) aged 85 and over, including both independently living and institutionalized elderly. The participation rate of 94% of all living elderly (n = 1037) and 78% of the initial cohort (n = 1259) was exceptionally high. Information was obtained on past and present diseases by taking a medical history. The life-time prevalence for arteriosclerosis and malignancies was 31.9% and 9.7% respectively. High prevalences were found for the non-lethal disabling disorders of hearing and visual impairment (44.4% and 49.9%, respectively) and urinary incontinence (25.6%). The authors conclude that establishing a classical medical diagnosis in the oldest old, which was the goal of this study, is not complete without assessing its effect on the functional ability and the quality of life of the individual.

[Problems of aging in China]. / Ouderdomsproblematiek in China.

A WHO consultancy to the 'National Seminar on Geriatrics' in Beijing provided an opportunity to get some ideas about the problems of ageing in China. Since 1949 life expectancy has doubled and it has been estimated that between now and the end of the century the 65+ group will double to a total of approximately 100 million people. This will be the fastest growing ageing population in history. With more than 1000 million inhabitants China is the largest country in the world and also because of epidemiological facilities it offers unique opportunities to study for instance maximum life-span. Results sofar support the view that this will not exceed the generally accepted figure of 120 years. Institutional care is limited, but this is at least partly offset by the structure of society, having the family as the central unit. Preventive medicine scores high and one should hope that it will contribute to geriatrics, as it has contributed to other parts of medicine.