Published Ahead of Print on July 18, 2007 as 10.1097/PSY.

0b013e3180cc2c61
Effects of Chronic Stress and Interleukin-10 Gene Polymorphisms on
Antibody Response to Tetanus Vaccine in Family Caregivers of Patients With
Alzheimer’s Disease
JIAN LI, MD, PHD, LINDA G. COWDEN, RN, JANICE D. KING, HT(ASCP) DAVID A. BRILES, PHD, HARRY W. SCHROEDER, JR., MD, PHD,
ALAN B. STEVENS, PHD, RODNEY T. PERRY, PHD, ZUOMIN CHEN, MD, MICAH S. SIMMONS, MS, HOWARD W. WIENER, PHD,
HEMANT K. TIWARI, PHD, LINDY E. HARRELL, MD, PHD, AND RODNEY C. P. GO, PHD
Objective: To assess the effects of psychological stress on the antibody response to tetanus vaccine adjusting for cytokine gene
polymorphisms and other nongenetic factors in caregivers of patients with Alzheimer’s disease (AD). Methods: A family-based
follow-up study was conducted in 119 spouses and offspring of community-dwelling patients with AD. Psychological stress was
measured by the Perceived Stress Scale (PSS) and the Center for Epidemiologic Studies Depression (CES-D) scale at baseline and
1 month after the vaccination. Nutritional status, health behaviors, comorbidity, and stress-buffering factors were assessed by
self-administered questionnaires, 10 single nucleotide polymorphisms (SNP) from six selected cytokines genotyped, and anti-
tetanus toxoid immunoglobulin G (IgG) concentrations tested using enzyme-linked immunosorbent assays. The effects of stress and
other potential confounders were assessed by mixed models that account for familial correlations. Results: The baseline PSS score,
the baseline CES-D score, the interleukin-10 –1082 A⬎G SNP GG genotype, and the baseline anti-tetanus IgG were inversely
associated with antibody fold increase. Conclusion: Both psychological stress and cytokine gene polymorphisms affected antibody
fold increase. The study provided additional support for the detrimental effects of psychological stress on the antibody response to
tetanus vaccine. Key words: psychological stress, cytokine genes, antibody response, vaccine.

AD ⫽ Alzheimer’s disease; CES-D ⫽ Center for Epidemiologic
Studies Depression; PSS ⫽ Perceived Stress Scale; ELISA ⫽ en-
zyme-linked immunosorbent assays; SNP ⫽ single nucleotide poly-
morphisms.
INTRODUCTION
T he detrimental effects of chronic psychological stress on
vaccine-induced antibody response have been documented
in different populations (1,2), with the most consistent reports
found in spousal caregivers of patients with dementia (3–9).
One of the major pathways through which chronic psycholog-
ical stress affects immune functions is the chronic activation
of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in
the chronic elevation of systemic glucocorticoids (10 –12). At
higher than physiological concentration, glucocorticoids can
lead to a wide range of immune dysregulations, with one of
the notable changes including the inhibition of cytokine syn-
thesis and the imbalance of the Th1/Th2 cytokine network
(10 –12). Several lines of evidence have indicated that the
stress-induced imbalance of the Th1/Th2 cytokine network
From the Department of Epidemiology and International Health (J.L.,
L.G.C., R.T.P., Z.C., M.S.S., H.W.W., R.C.P.G.), School of Public Health,
University of Alabama at Birmingham, Birmingham, Alabama; Department of
Microbiology and Immunology (J.D.K., D.A.B.), University of Alabama at
Birmingham, Birmingham, Alabama; Departments of Clinical Immunology and
Medicine (H.W.S.), University of Alabama at Birmingham, Birmingham, Ala-
bama; Department of Medicine (A.B.S.), Texas A&M University System Health
Science Center, Huston, TX; Department of Biostatistics (H.K.T.), Section on
Statistical Genetics, University of Alabama at Birmingham, Birmingham, Ala-
bama; Department of Neurology (L.E.H.), School of Medicine, University of
Alabama at Birmingham, Birmingham, Alabama.
Address correspondence and reprint requests to Jian Li, Department of
Epidemiology and International Health, School of Public Health, University
of Alabama at Birmingham, Birmingham, Alabama.
Current address: Jian Li, Global Product Safety, Eli Lilly and Company,
DC 2139, Indianapolis, IN 46285. E-mail: lijianx@lilly.com
This research was supported by Grant NS045934-06 from the National
Institute of Neurological Disorders and Stroke and Grant 2RO1 AG09029-15
from the National Institute of Aging.
Received for publication October 9, 2006; revision received March 21,
2007.
DOI: 10.1097/PSY.0b013e3180cc2c61
underlies a wide range of immune changes and diseases
among people subject to chronic stress (13–16).
Stress is commonly measured by subjective assessments
using standard questionnaires, such as Perceived Stress Scale
(PSS) (17) and the Center for Epidemiologic Study Depres-
sion (CES-D) scale (18) or objective stress biomarkers, such
as salivary cortisol (7,19 –21). Studies using those stress mea-
sures have reported inconsistent effects of stress on vaccine-
induced antibody response. Whereas Vedhara et al. (7) found
that the mean salivary cortisol concentrations were inversely
correlated with the immunoglobulin G (IgG) antibody titers to
one viral strain in the influenza vaccine in spousal dementia
caregivers, Burns et al. (22) reported that undergraduate stu-
dents with low titer to hepatitis vaccine had significantly
lower cortisol levels compared with those with higher anti-
body titer. Moreover, many dementia caregiver studies found
no correlation between antibody response to influenza vaccine
and subjective stress measures (4,5,7,8); neither was antibody
response associated with caregiving variables, including years
spent caregiving, the number of hours spent on caregiving
daily, or the extent of the patients’ cognitive impairment (4,8).
However, when caregiving status was used as a surrogate for
stress levels, it was consistently reported that compared with
the age- and gender-matched noncaregivers, the current and
former caregivers had weaker vaccine-induced antibody re-
sponse (4,8).
One of the reasons for those inconsistencies lies in the
complexity of assessing psychological stress and antibody
response. The commonly used questionnaires measure global
stress and depressive symptoms rather than caregiving-spe-
cific stress; thus, the differences in stress levels between
caregivers and noncaregivers can be attributed to life events
other than caregiving. The same is true for objective measures
such as salivary cortisol because the HPA axis can be acti-
vated by stressors other than dementia caregiving. In addition
to psychological stress, antibody response can be influenced
by other factors such as comorbidity and nutrition (23). Fur-

Psychosomatic Medicine 69:551–559 (2007) 551

0033-3174/07/6902-0551
Copyright © 2007 by the American Psychosomatic Society

thermore, vaccine-induced antibody response is influenced by
genetic factors (23). Various human leukocyte antigen (HLA)
loci and cytokine gene polymorphisms have been found to be
associated with the responsiveness to several vaccines (24 –
26). Twin studies have further estimated that genetic effects
account for about 60% of the variation in the antibody levels
in response to the hepatitis B vaccines and at least 40% of the
variation in anti-tetanus IgG titers after vaccination with tet-
anus toxoid (TT) (27,28). More importantly, twin studies have
demonstrated that a) non-HLA genes, such as cytokines, have
played major roles in response to vaccines that primarily
induce antibody production and b) ⬎50% of the variations in
antibody levels are determined by the non-HLA loci (27,28).
Taken together, the differences in the antibody response be-
tween the caregivers and the noncaregivers are the result of
the complex interplay between stress due to caregiving and
other causes, other nongenetic factors, and host genetic make-
up. Thus, it is critical to account for other genetic and non-
genetic factors to better delineate the effect of stress on
antibody response.
The current study examined the effects of psychological
stress on the antibody response to tetanus vaccine adjusting
for cytokine gene polymorphisms and other nongenetic factors
in the spouses and offspring of community-dwelling patients
with Alzheimer’s disease (AD). First, given that in many
families both the spouses and the adult offspring are directly
or indirectly involved with the caregiving for the community-
dwelling patients with AD, it is important to study the vari-
ability of stress among family members. The family study
design also provided a unique advantage in controlling for
many unmeasured environmental exposures and genes shared
by family members and in estimating the degrees to which
one’s genetic make-up influence the antibody response. Sec-
ond, because the cross-talk between the neuroendocrine and
immune system is mediated through Th1/Th2 cytokines and
cytokine productions are under genetic control (10 –12), a
number of antagonistic and synergistic Th1 (interleukin (IL)-
1␤, IL-2, IL-12, tumor necrosis factor (TNF)-␣,) and Th2
(IL-10, IL-4) cytokines were chosen. Those cytokines have
been found to play roles in influencing antibody response to a
variety of vaccines (26,29,30). Finally, psychological stress
was assessed by the PSS (17) and depressive symptoms by the
CES-D scale (18). It is important to note that those scales do
not capture caregiving-specific stress bur rather global stress,
which allowed us to study the effect of stress in a broader
context. Moreover, recent intervention research aimed at im-
proving caregiver’s mental health outcome (31,32) pointed to
the importance of examining stress-buffering factors such as
social support (33–35), social activities (36), and positive
aspects of caregiving (37). Other nongenetic confounders in-
cluded age, gender, comorbidity, nutritional status, and base-
line anti-TT IgG. We hypothesized that people with different
stress levels would have differential antibody response to the
tetanus vaccine after adjusting for the cytokine genes, stress-
buffering factors, and other nongenetic factors.
552
J. LI et al.
METHODS
Study Population
The study population consisted of 47 Caucasian families, with a total
sample size of 119 individuals. Each family consisted of the spouse of the
community-dwelling patient with AD and one or two of their biological
offspring. Twenty-five families contained one spouse and two offspring and
22 families contained one spouse and one offspring. Between February 2005
and February 2006, 178 families were initially approached in the Memory
Disorders Clinic at the University of Alabama at Birmingham (UAB) where
the family members accompanied the patients with AD for follow-up visits,
43 of whom agreed to participate after subsequent phone contacts. Four
families were recruited from the previous participants in the MIRAGE study
(Multi-Institutional Research on Alzheimer’s disease Genetic Epidemiology,
UAB site). The study was approved by the Institutional Review Board of
UAB.
To be eligible for the study, the spouses had to be community-dwelling
and take care of the patients with AD at home, be able to give consent, and
live within a 2- to 4-hour driving distance from Birmingham, Alabama. The
offspring had to be at least 19 years of age to give legal consent. Additionally,
all the family caregivers were limited to Caucasians to avoid population
stratification. An individual was excluded from participation for any of the
following conditions: vaccination with TT within the past 3 years, allergy to
vaccine components or a history of a neurological reaction after a previous
vaccine dose, major psychiatric illnesses or degenerative neurological disor-
ders, autoimmune diseases, human immunodeficiency virus infection, sple-
nectomized patients, transplant recipients, serious trauma within the previous
6 months, primary immunodeficiency, chemotherapy or radiation treatment
for cancer within the past 5 years, immunosuppressant medications within the
past 3 months, history of blood coagulation disorder, and pregnancy or
lactation.
Collection of Nongenetic Factors
The nongenetic information was collected using seven well-validated
instruments, including Social Support (33–35), Social Activities (36),
Positive Aspects of Caregiving (37), Caregiver Health and Health Behav-
iors (38,39), Mini Nutritional Assessment (MNA) (40), PSS (17), and
CES-D scale (18). The PSS and CES-D were administered face-to-face
with the study participants at baseline and 1 month after vaccination
during home visits, whereas the rest of the questionnaires were mailed to
the participants after they gave verbal consent over the phone and were
collected at the first home visit. The average time between the dates when
the questionnaires were mailed to the study participants and the dates
when the questionnaires were collected at the first home visit was 3 weeks.
Perceived Stress
The PSS (17) measures the degree to which situations in one’s life are
perceived as stressful and has been commonly used in caregiver studies. There
are ten questions asking how often one felt or thought a certain way in the past
month, with five responses ranging from never, almost never, sometimes,
fairly often, to very often. A total score is obtained by summing across all 10
items. The possible scores range from 0 to 40, with higher scores indicating
higher stress levels.
Depressive Symptoms
The CES-D short form (18,41) measures the current levels of depressive
symptoms. It contains 10 of the original 20 items, including 1) I was bothered
by things that don’t usually bother me, 2) I had trouble keeping my mind on
what I was doing, 3) I felt depressed, 4) I felt that everything I did was an
effort, 5) I felt hopeful about the future, 6) I felt fearful, 7) My sleep was
restless, 8) I was happy, 9) I felt lonely, 10) I could not get “going.” The
questionnaire has a 4-point scale indicating the frequency of their occurrence
in the past week. The summation of all 10 items generates a total score
ranging from 0 to 30, with higher scores indicating greater frequencies or
numbers of depressive symptoms.
Psychosomatic Medicine 69:551–559 (2007)
STRESS AND IMMUNITY
Social Support
The Social Support scale measures four broad categories of social support
construct: received support with separate items for emotional, tangible, in-
formational subscales; satisfaction with the support, with separate items for
the overall satisfaction with tangible, emotional, and informational support
received (34); social network of family, friends, and confidants (35); and
negative interactions with others (33). An overall total social support score is
calculated by summing 15 questions. The score ranges between 0 and 53, with
higher scores indicating higher levels of social support.
Social Activities
The Social Activities scale measures the impact of caregiving on one’s
ability to engage in desirable social/leisure activities (36). The form consists
of seven questions asking the caregivers how often they have been able to
participate in various social/leisure activities (e.g., quiet time, attending
church). The response to these items should be summed to form a total score
ranging from 0 to 14, with higher scores indicating greater amounts of time
for social/leisure activities.
Positive Aspects of Caregiving
The Positive Aspects of Caregiving scale contains 11 items phrased as
statements about the caregivers’ mental state in relationship to the caregiving
experience and their ability to cope with the caregiving-related stress by
emphasizing the positivity of the experiences (37). The response options are
on a 5-point agree/disagree scale. The total scores range from 0 to 36, with
higher scores indicating more positive feelings toward caregiving.
Health Behaviors and Comorbidity
The Caregiver Health and Health Behavior instrument assesses caregivers
general and perceived health, comorbidities, and preventive health behavior
(38,39). The section that measures comorbidity included 12 disease condi-
tions; the total score ranges from 0 to 12, with higher scores indicating higher
numbers of comorbidity.
Nutritional Status
The MNA (40) includes anthropometric measurements, a dietary ques-
tionnaire, global assessment, and self-assessment. The possible scores range
between 0 and 30. The nutritional assessment consists of three categories:
malnourished ⫽⬍17; at risk of malnutrition ⫽ 17 to 23.5; and well nourished
⫽ⱖ24.
Home Visits for Blood Collection
and Stress Assessment
There were two home visits. On the first visit, the research staff conducted
informed consent. Then, 30 ml of venous blood was drawn followed by the
injection of 0.5 ml TT adsorbed (Aventis Pasteur, Swiftwater, PA, USA)
administered intramuscularly into the deltoid muscle on one arm. Weight,
height, midarm, and calf circumferences were also measured. On the second
visit scheduled 4 weeks after the vaccination, the same amount of blood (30
ml) was drawn. On both visits, the research staff conducted face-to-face
interviews using the PSS and CES-D scale.
Antibody Testing
Sera samples were analyzed for their content of antibodies reactive with the
TT antigen using an enzyme-linked immunosorbent assays (ELISA) (42). Paired
pre- and postvaccination serum samples were tested for anti-TT IgG in duplicates
for the total sample of 119 vaccinees. Microtiter 96-well plates (NUNC Roskilde,
Denmark) were coated overnight at 4°C in phosphate-buffered saline (PBS) at
pH ⫽ 7.2 with 1 ␮g/ml TT (Aventis Pasteur, Swiftwater PA, USA). All assays
included a control plate coated with bovine serum albumin (BSA) to verify the
specificity of the assays for the coating antigen. The low levels of reactivity with
the BSA plates were subtracted from the values on the antigen-coated plates.
Plates were washed with PBS containing 0.05% Tween 20 (ELISA wash buffer).
The plates were blocked with PBS containing 1% BSA for 1 hour at room
temperature followed by incubation with the subjects’ prediluted sera (1:500 for
Psychosomatic Medicine 69:551–559 (2007)
the prevaccination samples and 1:4000 for the postvaccination samples; a few
samples were diluted at 1:90 and 1:250 for the pre- and postvaccination samples,
respectively) overnight at 4°C, then washed with ELISA wash buffer. The plates
were incubated with a secondary antibody biotin-conjugated goat antihuman
immunoglobulin antiserum IgG (H ⫹ L) (Southern Biotechnology Associates,
Birmingham, AL) for 1 hour at room temperature. After another rewash with
ELISA wash buffer, plates were incubated with streptavidin-alkaline phosphatase
(Southern Biotechnology Associates, Birmingham, AL) for 1 hour at room
temperature. After the final wash with ELISA wash buffer, the plates were
developed with p-nitrophenyl phosphate (Sigma, St. Louis, MO, USA) and
absorbance was read at 405 nm in a microplate reader. All antibodies against TT
were standardized to human antibodies with a known concentration.
Single Nucleotide Polymorphisms Genotyping
Ten single nucleotide polymorphisms (SNP) on six cytokine genes (IL-
1␤, IL-2, IL-4, IL-10, IL-12␤, TNF-␣) were genotyped with the ABI 7500
Fast Real-Time PCR System using the Taqman 5⬘ nuclease assay for allelic
discrimination. The Applied Biosystems (ABI) assay-on-demand service was
used to order probes and primers for the 10 cytokine SNP available publicly
at www.appliedbiosystems.com. The total volume for PCR reactions was 5
␮l, including 1 ␮l genomic DNA (33 ng/␮l), 1.25 ␮l sterile water, 2.5 ␮l of
TaqMan Universal PCR Master Mix, and 0.25 ␮l TaqMan SNP Genotyping
Assay Mix containing 18 ␮M of a given primer and 4 ␮M of a given probe.
Plates were preread, run, and postread on the 7500 Fast real-time PCR
System. Standard mode conditions were used for all the assays. Sequence
Detection software version 1.3 (Applied Biosystems) was applied for the
allelic discrimination analysis.
Statistical Analysis
Outcome Variable
The pre- and postvaccination anti-TT IgG (pre-IgG and post-IgG) con-
centrations were log10 transformed to achieve normality for the residuals of
the models before analyses. The outcome variable is the TT-induced antibody
fold increase, defined as log10 (post-IgG/pre-IgG).
Nongenetic Factors
The baseline CES-D and PSS scores were the main exposures of interest.
Nine other covariates were also adjusted for, including gender, age, baseline
anti-TT IgG, nutrition score, comorbidity score, positive aspects of caregiving
score, social support score, social activities score, and the days between the
two home visits.
Genetic Analysis
Mendelian transmission of the 10 SNP markers was checked using the
MARKERINFO procedure in S.A. G. E; any errors were corrected or set to
missing values before the analysis. Hardy-Weinberg equilibrium was con-
firmed for all loci using the Mantel-Haenszel exact ␹2 goodness-of-fit tests in
47 spouses. The additive and dominant effects of each of the 10 SNP markers
on the antibody fold increase were assessed one at a time in the presence of
all of the above nongenetic covariates. SNPs that were significant in the
multivariable setting were entered together with all the other nongenetic
covariates to form the full models.
Full Models
There were two full models: the CES-D model and the PSS model. The
CES-D and the PSS scores, although highly correlated, measure two inde-
pendently predictive constructs (17): PSS for perceived stress and CES-D for
depressive symptoms. Therefore, the effects of PSS and CES-D were assessed
in different models. Except for the stress measures, the two models share the
same genetic and nongenetic covariates described above.
The association between antibody response and the genetic and non-
genetic factors were performed using the Proc Mixed procedure in SAS
(SAS Institute, Cary, NC), with the unstructured covariance matrix spec-
ified to account for the familial correlations. The significance levels used
an ␣ of 0.05.
553
 J. LI et al.

RESULTS
Baseline Caregiver Characteristics
Table 1 describes the baseline characteristics of the family
caregivers. Overall, the mean ⫾ standard deviation age was
57.1 ⫾ 16.0 years; 60% of the participants were females; only
two people reported using alcohol and smoking cigarettes in
the past month at the time of sample ascertainment; the most
common self-reported disease conditions were arthritis
(32.8%), hypertension (27.7%), heart disease (14.3%), and
psychiatric disorders (12.6%). According to the MNA, an
overwhelming majority of the participants were well nour-
ished (84%) and none of them was malnourished. Medication
usage indicated that 24.4% of the caregivers took ⱖ3 prescrip-
tion drugs per day, but the specific drug names were not
obtained. The CES-D and PSS scores were 7.6 ⫾ 4.9 and
14.8 ⫾ 6.1, respectively. The scores for the stress-buffering
factors were 19.7 ⫾ 4.8 for social support, 8.1 ⫾ 3.1 for social
activities, and 25.4 ⫾6.7 for positive aspects of caregiving.
The geometric mean for the baseline antitetanus IgG concen-
trations was 7.0 ␮g/ml (95% Confidence Interval range: 0.1–
104.3). The average days between the two home visits were
27 ⫾ 5 days.
Cytokine SNP Association
Univariate analyses conducted in both additive and domi-
nant models showed that none of the cytokine genes was
associated with the baseline antitetanus IgG (data not shown).
In the multivariable setting, the dominant effect of IL-10 –
1082 A⬎G SNP (GG versus GA ⫹ AA) was associated with
the antibody fold increase in the presence of all other nonge-
netic covariates in both the PSS model (p ⫽ .01) and the
CES-D model (p ⫽ .0004) (Table 2). Thus, the model that
contained the dominant effects of the IL-10 –1082 A⬎G SNP
and all other nongenetic covariates formed the final models.
Predictors of Antibody Response
The log-transformed geometric means of the pre- and post-
vaccination antitetanus IgG concentrations declined by age
(p ⬍ .0001 for pre-IgG; p ⫽ .0008 for post-IgG), whereas the
crude antibody fold increase increased by age (p ⫽ .03).

TABLE 1. Baseline Characteristics of Family Caregivers

Total Sample Spouses Offspring

n ⫽ 119 n ⫽ 47 n ⫽ 72

Demographic characteristics
Age (in years), mean ⫾ SD 57.1 ⫾ 16.0 72.4 ⫾ 9.2 45.9 ⫾ 9.3
Gender (n, %)
Male 47 (39.5) 19 (40.4) 28 (38.9)
Female 72 (60.5) 28 (59.6) 44 (61.1)
Health behaviors, yes, n (%)
Alcohol drinking in the past month 2 (1.7) 0 2 (2.8)
Cigarette smoking in the past month 2 (1.7) 0 2 (2.8)
Comorbidity
Overall comorbidity score, mean ⫾ SD 1.8 ⫾ 1.7 2.6 ⫾ 1.8 1.2 ⫾ 1.3
Disease conditions, yes, n (%)
Arthritis 39 (32.8) 22 (46.8) 17 (23.6)
Hypertension 33 (27.7) 22 (46.8) 11 (15.3)
Heart disease 17 (14.3) 10 (21.3) 7 (9.8)
Kidney disease 8 (6.7) 4 (8.5) 4 (5.6)
Chronic lung disease 5 (4.2) 4 (8.5) 1 (1.4)
Liver disease 1 (0.8) 0 1 (1.4)
Psychiatric disease 15 (12.6) 7 (14.9) 8 (11.1)
Nutritional status, yes, n (%)
Malnourished 0 0 0
At risk for malnutrition 19 (15.9) 7 (14.9) 12 (16.7)
Well-nourished 100 (84.0) 40 (85.1) 60 (83.3)
Taking ⬎3 prescription drugs per day, yes, n (%) 29 (24.4) 20 (42.6) 9 (12.5)
Stress measures, mean ⫾ SD
Baseline CES-D score 7.6 ⫾ 4.9 9.0 ⫾ 4.9 6.6 ⫾ 4.6
Baseline PSS 14.8 ⫾ 6.1 15.9 ⫾ 6.1 14.1 ⫾ 5.8
Stress-buffering factors, mean ⫾ SD
Social support 19.7 ⫾ 4.8 19.1 ⫾ 4.8 20.4 ⫾ 4.8
Social activities 8.1 ⫾ 3.1 7.6 ⫾ 2.8 8.6 ⫾ 3.3
Positive attitude of caregiving 25.4 ⫾ 6.7 25.3 ⫾ 6.1 25.4 ⫾ 7.2
Baseline anti-tetanus IgG concentration in ␮g/ml 7.0 (0.1–104.3) 2.3 (0.1–53.3) 14.5 (1.4–104.3)
(95% CI)
Days between two home visits, mean ⫾ SD 27 ⫾ 5 27 ⫾ 5 27 ⫾ 5

SD ⫽ standard deviation; CES-D ⫽ Center for Epidemiologic Studies Depression; PSS ⫽ Perceived Stress Score; IgG ⫽ immunoglobulin G; CI ⫽ confidence
interval.

554 Psychosomatic Medicine 69:551–559 (2007)

STRESS AND IMMUNITY

TABLE 2. Genotype Frequencies of Selected Cytokines and Their Association With the Baseline Antitetanus IgG and the Antibody Fold Increase
in Family Caregivers

Genotype Frequencies, n (%) p

Cytokine Genes SNP ID Nucleotide Change
Total Spouses HW CES-D PSS
n ⫽ 119 n ⫽ 47 p Modela Modelb

IL-1␤ rs16944 ⫺511 T⬎C TT 8 (6.8) 2 (4.3) .42 .1 0.1

TC 59 (50.0) 22 (46.8)
CC 51 (43.2) 23 (48.9)
rs1143627 ⫺31 C⬎T CC 47 (47.9) 22 (56.4) 1.00 .3 .4
CT 48 (48.9) 15 (38.4)
TT 3 (3.0) 2 (5.1)
IL-2 rs2069762 ⫺384 T⬎G GG 66 (56.4) 28 (59.6) .75 .9 .9
GT 45 (38.5) 15 (31.9)
TT 6 (5.1) 4 (8.5)
IL-4 rs2070874 ⫺33 C⬎T CC 85 (72.6) 35 (74.5) 1.00 .5 .6
CT 28 (23.9) 11 (23.4)
TT 4 (3.4) 1 (2.1)
IL-10 rs1800872 ⫺592 A⬎C AA 54 (45.8) 22 (46.8) .81 .7 .8
AC 50 (42.4) 18 (38.3)
CC 14 (11.9) 7 (14.9)
rs1800896 ⫺1082 A⬎G AA 30 (25.6) 12 (25.5) 1.00 .01 .01
AG 54 (46.2) 22 (46.8)
GG 33 (28.2) 13 (27.7)
IL-12␤ rs2546890 ⫹8275 A⬎G AA 28 (23.9) 14 (30.4) 1.00 .5 .3
AG 60 (51.3) 22 (47.8)
GG 29 (24.8) 10 (21.7)
rs3212227 ⫹159 A⬎C CC 3 (2.7) 1 (2.2) .80 .7 .9
AC 28 (24.8) 8 (17.8)
AA 82 (72.6) 36 (80.0)
TNF-␣ rs361525 ⫺238 A⬎G AA 1 (0.9) 0 .65 .2 .1
AG 20 (16.9) 10 (21.3)
GG 97 (82.2) 37 (78.7)
rs1800629 ⫺308 A⬎G AA 4 (3.4) 1 (2.1) .74 .8 .4
AG 39 (33.3) 16 (34.0)
GG 74 (63.3) 30 (63.8)

SNP ⫽ single nucleotide polymorphisms; HW ⫽ Hardy-Weinberg equilibrium; CES-D ⫽ Center for Epidemiologic Studies Depression; PSS ⫽ Perceived Stress
Score; IL ⫽ interleukin.
a
Full model with CES-D score as the main exposure.
b
Full model with perceived stress scores as the main exposure.

Overall, 70.6% of the people had protective immunity at

baseline and 96.6% reached protective immunity after vacci-
nation (data not shown). TABLE 3. Univariate Analysis on Nongenetic Factors and Antibody
Univariate analyses examining the association between Fold Increase
baseline antitetanus IgG levels and the baseline nongenetic
factors (CES-D score, PSS score, age, gender, nutrition, co- Regression
Predictors p
Coefficient
morbidity, social support, social activities, and positive as-
pects of caregiving) showed that only age (p ⬍ .0001) and Age .009 .03
comorbidity score (p ⫽ .02) were significantly associated Gender .064 .63
(data not shown). When further univariate analyses were per- Baseline antitetanus IgG ⫺.604 ⬍.0001
formed between those same nongenetic factors and the anti- Baseline CES-D ⫺.029 .03
Baseline PSS scores ⫺.015 .15
body fold increase (Table 3), age became a positive predictor Social support .024 .07
(p ⫽ .03), whereas the baseline antitetanus IgG (p ⬍ .0001) Social activities .003 .89
and the baseline CES-D scores (p ⫽ .03) were negative Positive aspects of caregiving .004 .70
predictors; the baseline PSS score was not a significant pre- Days between two visits ⫺.005 .67
dictor for antibody fold increase in the univariate analysis Nutrition .009 .76
Comorbidity .060 .14
(p ⫽ .15).
In the full models, the baseline antitetanus IgG levels, the CES-D ⫽ Center for Epidemiologic Studies-Depression scale; PSS ⫽ Per-
GG genotype of the IL-10 –1082 A⬎G SNP, the baseline PSS ceived Stress Score.

Psychosomatic Medicine 69:551–559 (2007) 555

 J. LI et al.

TABLE 4. Predictors for Antibody Fold Increase in Family Caregivers

PSS Model CES-D Model

Predictors
Regression Regression
p p
Coefficient Coefficient

Age ⫺.0078 .05 ⫺.0064 .12

Gender .049 .62 .031 .75
Baseline antitetanus IgG ⫺.62 ⬍.0001 ⫺.61 ⬍.0001
Baseline PSS scores ⫺.019 .01 — —
Baseline CES-D scores — — ⫺.032 .004
Social support .0091 .46 .011 .36
Social activities ⫺.011 .55 ⫺.012 .48
Positive aspects of caregiving ⫺.0023 .78 ⫺.0044 .59
Days between 2 visits ⫺.00038 .97 ⫺.0015 .88
Nutrition .013 .54 .0055 .79
Comorbidity .036 .32 .034 .35
IL-10–1082 A⬎G SNP (GG versus AA ⫹ AG) ⫺.35 .01 ⫺.34 .01

PSS ⫽ Perceived Stress Score; CES-D ⫽ Center for Epidemiologic Studies-Depression scale; IgG ⫽ immunoglobulin G; IL ⫽ interleukin.

scores, and the baseline CES-D scores were significant pre-
dictors for the antibody fold increase (Table 4). In both the
CES-D and the PSS models, the baseline IgG levels were
inversely associated with the antibody fold increase (p ⬍
.0001); people with the GG genotype of the IL-10 –1082 A⬎G
SNP had lower antibody fold increase compared with those
with the AA ⫹ AG genotype (p ⫽ .01). The PSS and the
CES-D scores had a negative impact on the antibody fold
increase, with every point increase in the PSS score associated
with 6.2% less antibody fold increase (p ⫽ .01) and every
point increase in the CES-D score associated with 7.0% less
antibody fold increase (p ⫽ .004).
DISCUSSION
The current study found that the baseline CES-D score, the
baseline PSS score, the GG genotype of the IL-10 –1082 A⬎G
SNP, and the baseline antitetanus IgG levels were inversely
associated with antibody fold increase in response to tetanus
vaccine adjusting for other potential confounders. Because the
CES-D and PSS scores reflect general stress and not caregiving-
specific stress, these findings have broader implication for the
detrimental effect of global stress on vaccine-induced antibody
response. Moreover, these findings demonstrate that antibody
response is under the influences of key cytokine genes and
other nongenetic factors.
It is important to note that, in this study, stress was assessed
differently from previous ones (4,8). First, instead of using
caregiving status as a proxy for stress, the current study
analyzed CES-D scores and the PSS scores as independent
predictors and was thus able to directly quantify the separate
effects of the PSS and the CES-D scores on the antibody
response in the family caregivers. The results showed that
every point increase in the CES-D score translated into about
7% less antibody fold increase and every point increase in the
PSS score about 6% less antibody fold increase. Second, the
association between stress and the antibody response was
adjusted for a number of immunoregulatory cytokine genes
and some nongenetic factors that have influence on immune
response, including age, gender, nutritional status, comorbid-
ity, baseline antitetanus IgG levels, and stress-buffering fac-
tors. A good case in point is comorbidity. Some previous
reports on the difference in antibody response between care-
givers and noncaregivers may be partially explained by the
fact that the noncaregivers are typically healthier than care-
givers. In this study, adjusting for comorbidity provides better
control for the confounding by health status than using care-
giver status itself. Further, the differences in the univariate
analyses and multivariate analyses pointed to the importance
of covariate adjustment. For instance, the PSS score was not
significant in univariate analysis but became significant in the
presence of other genetic and nongenetic factors, suggesting
the presence of genetic or nongenetic confounders. In addition
to covariate adjustment in statistical models, the current study
adopted a family study design that included both the spouse
and the offspring of the patients with AD. In this study,
whereas all the spouses lived with patients with AD at home
and were the primary caregivers, the majority of the offspring
lived in close proximity to their parents and helped with the
caregiving on a regular basis. The inclusion of the offspring
who live close to their parents provided the unique advantage
in accounting for many unmeasured environmental exposures
and genes shared by the family members, thus strengthening
the control for confounding. Finally, most previous studies
examined vaccine-induced antibody response as a dichotomous
variable, such as beyond certain thresholds (2–9). The current
study, however, assesses the antibody fold increase as a contin-
uous variable, which provides greater power in detecting factors
with moderate effects.
It is important to adjust for the baseline antitetanus IgG
levels because everyone in the current study had the tetanus
vaccination previously, e.g., 71% of the study participants had
protective antitetanus IgG levels at baseline. Furthermore, the
postvaccination antibody titer is known to be determined by
the prevaccination antibody titer: the lower the prevaccination

556 Psychosomatic Medicine 69:551–559 (2007)

STRESS AND IMMUNITY
titer, the higher the postvaccination levels (43,44). Danilova et
al. (44) found that in healthy Russian young men, those with
prevaccination anti-TT IgG concentrations ⱖ17 ␮g/ml were
unable to mount a three-fold increase after the vaccination.
The negative association between the baseline antitetanus IgG
and the antibody fold increase is consistent with those find-
ings. More importantly, in this study, elderly people (⬎65
years) had much lower baseline antitetanus IgG levels com-
pared with younger people. Thus, adjusting for the baseline
levels also eliminates the confounding by age with the anti-
body fold increase, allowing for better assessment for the
effects of the baseline PSS scores, the baseline CES-D scores,
and the IL-10 –1082 A⬎G SNP on the antibody fold increase
without the confounding by age and the baseline antitetanus
IgG levels.
Furthermore, the findings suggested that individuals carry-
ing the GG genotype of the IL-10 –1082 A⬎G SNP has lower
antibody fold increase compared with those carrying the AG ⫹
AA genotypes. The IL-10 –1082 A⬎G SNP has also been
found to influence the antibody response to hepatitis A and
hepatitis B vaccines (29) and the high IL-10 production was
associated with low antibody response to influenza vaccine in
the elderly (45). IL-10 is a critical anti-inflammatory cytokine
that activates B cells, promotes B cell survival, proliferation,
and differentiation, induces immunoglobulin class switching
to the IgG isotype, and enhances antibody response (46). In
addition to its direct actions on B cells, IL-10 exerts its
immunoregulatory influence by inhibiting key proinflamma-
tory cytokines such as TNF-␣, IL-1␤ (46). More importantly,
the expression of IL-10 is regulated at the transcriptional level
and polymorphisms at the promoter regions of both cytokines
are associated with the variability in their production. For the
IL-10 –1082 A⬎G SNP compared with the ⫺1082 G allele,
the ⫺1082A alleles confer higher binding affinity for the
transcription factor PU.1, which inhibits gene expression and
leads to decreased IL-10 transcription activity in individuals
carrying this allele (47). Turner et al. (48) reported a decreased
IL-10 production in positive individuals with ⫺1082A, but
these results were not confirmed by other studies (49,50).
Taken together, the individual differences in the production of
IL-10 and other cytokines produced during antibody response
combined with the synergistic and antagonistic actions in the
Th1/Th2 cytokine network may determine the magnitude of
the antibody fold increase.
The effects of the IL-10 –1082 A⬎G SNP were consistent
in both the CES-D and the PSS models. However, there are
some unresolved issues. First, this study did not measure
cytokine levels. Therefore, we cannot determine the associa-
tions among the IL-10 polymorphisms, the cytokine levels,
and the antibody response. Second, due to its anti-inflamma-
tory effects, IL-10 has been studied as a potential treatment for
chronic inflammatory diseases for which steroid is the stan-
dard therapy. Clinical studies found that a single dose of IL-10
resulted in a 20% increase in plasma cortisol levels within 24
hours (46). It remains to be seen how the cytokines and the
Psychosomatic Medicine 69:551–559 (2007)
stress hormone cortisol interact with each other in vivo during
stress reactions to influence antibody response.
Consistent with the literature, gender is not a significant
predictor for antibody response. Nor were any of the stress-
buffering factors, including social support, social activities,
and positive aspects of caregiving. Vedhara et al. (9) found
that an 8-week stress management intervention helped im-
prove the caregiver’s immune response to the influenza
vaccine. However, the current study did not provide stress
intervention but assessed the association between the
stress-buffering factors measured at baseline and the anti-
body fold increase. This may explain why we did not
observe the expected beneficial effects from those factors.
Another possibility is that the current study lacks the power
to adequately assess the effects of the stress-buffering
factors because testing stress-buffering effects requires
testing interactions. Other factors, including nutritional sta-
tus, comorbidity, and time between obtaining the two blood
samples, were not associated with antibody fold increase in
univariate or multivariate analyses. There were only two
smokers and two alcohol drinkers in this sample. The lack
of variability in those two variables did not allow us to
adjust for them in the full models.
This study had several limitations. First, to prevent population
stratification, the study participants were restricted to Cauca-
sians—a restriction that limits the generalizability of the study to
other ethnic populations. Population stratification is a spurious
genetic association due to the differences in the allele frequencies
present in different ethnic populations. A good case in point is the
IL-10 –1082 A⬎G SNP. The A and G alleles are almost equally
distributed in Caucasians, but unevenly distributed in other ethnic
populations. Had other ethnic populations being included, the
association between the IL-10 –1082 A⬎G SNP may not have
been detected due to the much lower frequency of the G allele, or
a spurious association may have been detected due to the differ-
ences in the allele frequencies in different ethnic population but
not because of any real genetic impact on antibody response. A
further restriction is that, because the caregivers are a highly
stressed sample, the results may not be generalizable to more
normative samples. Furthermore, one of the most common rea-
sons for declining participation in the study was “already too
stressed out to handle a study.” It was very likely that those with
the highest stress levels were self-selected out of the study,
resulting in an inadequate representation of individuals with the
highest stress levels. Had the highly stressed caregivers been
included in the study, we would expect to see much stronger
effects of stress on the antibody response than what was observed
in the current study. Only 5.3% of the caregivers in this study had
clinical depression based on the CES-D score of ⱖ15 (41,51),
compared with the REACH (Resources for Enhancing Alzhei-
mer’s Caregiver Health) Caucasian cohort where 24.5% in the
control group and 10.5% in the experimental group after inter-
vention were classified as having clinical depression using the
same CES-D score cut-off (32). The mean PSS score in the
current study also seemed to be lower compared with another
caregiver study by Glaser et al. (8): 14.8 ⫾ 6.1 versus 16.7 ⫾
557

2.82. Third, we did not measure caregiving variables, such as
including years spent caregiving, the number of hours spent on
caregiving daily, or the extent of the patients’ cognitive impair-
ment. However, the majority of the caregiving literature has
found no association between antibody response and those vari-
ables (4,8). Moreover, we are more interested in examining the
effects of global stress in a context broader than caregiving-
specific stress. Finally, we did not do analyses on a dichotomous
outcome indicating whether or not an individual has reached
protective tetanus immunity because the tetanus vaccine is highly
immunogenetic and only four people in the study failed to reach
that level after the vaccination.
The original power calculation suggested 50 families with
one spouse and two offspring were needed to test the genetic
association. In reality, 25 families with one spouse and two
offspring and 22 families with one spouse and one offspring
were recruited. This may increase the chance of random error.
However, the strength of the associations, covariate adjust-
ment, the consistency of the results from both the CES-D and
the PSS models, and the internal validity of the associations
suggest our results are valid, but replication of these results in
an independent larger study would lead to greater support for
our hypotheses regarding psychological stress and IL-10 –
1082 A⬎G SNP on the antibody response to TT.
In conclusion, the major findings from the current family-
based association study were that the baseline CES-D score,
the baseline PSS score, and the GG genotype of the IL-10 –
1082 A⬎G SNP were inversely associated with the antibody
response to the tetanus vaccine adjusting for other potential
confounders. The results provided additional evidence on the
detrimental effects of psychological stress on the antibody
response to the tetanus vaccine. It also demonstrated that
antibody response is under the influences of both genetic and
nongenetic factors. From the public health perspective, it is
imperative to raise the awareness of the harmful effects of
chronic stress among healthcare professionals and caregivers
alike, so that the caregivers can seek proper counseling and
support that will help them maintain a strong and healthy
immune system as they care for their loved ones.
We are grateful for the support and trust of the caregivers who
participated in this study. We also thank the staff in UAB Memory
Disorder Clinic and UAB Alzheimer’s Disease Center for their
support and cooperation with the recruitment.
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