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Multiple Changes of Immunologic Parameters

in Prisoners of WarAssessments After Release
From a Camp in...

Article in JAMA The Journal of the American Medical Association · August 1993
DOI: 10.1001/jama.1993.03510050061028

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Clinical Investigation

Multiple Changes of lrnmunologic

Parameters in Prisoners of War
Assessments After Release From a Camp in ManjaCa, Bosnia
Dragan Dekaris, MD, PhD; Ante Sabioncello, PhD; Renata Maiuran, PhD; Sabina Rabatic, PhD;
lvna Svoboda-Beusan, PhD; Nives Ljubic RaEunica, MD; Jelka TomaSic, PhD

0bjective.-To assess immune reactivity in men just released from a war pris- United Nations High Commissioner for
oner camp. Refugees (UNHCR). For this study, 305
Participants.-Random sample of 29 men from a group of 764 liberateddetain- detainees were selected based on the fol-
ees in war prisoner camp in Bosnia, 15 matched healthy control subjects, and pre- lowing criteria: (1) arrival within 48 hours
war historical control subjects. before testing; (2) informed consent (each
person examined signed the declaration
Main Outcome Measures.-Report on immune reactivity parameters, such as requested by theUNHCR to assure that
lymphocyte immunophenotypes, natural killer cell and phagocyte function, serum participating in the testingwas absolutely
cytokines, and hormones. voluntary) (3)aged 17to CXl years (very old
Results.--Compared with control subjects, detainees had significantly lowered and vely young were excluded);and (4)no
red blood cell count, hemoglobin mass concentration, hematocrit, total serum pro- apparent infectious or other diseases de-
teins, and albumin level, while the percentage and count of monocytes and non- tectable by a physician during examina-
segmented neutrophils were increased. Flow cytometry revealed a significant in- tion prior to venipuncture. From this
crease in percentage of activated lymphocytes, activated T lymphocytes, Tcls group, 80 detainees were randomly se-
lymphocytes, B lymphocytes, and total HLA-DR lymphocytes. The absolute counts lected for venipuncture. One detainee was
of activated lymphocytes and activated T lymphocytes were also significantly subsequently excluded because of an ap-
parent infection, leaving 29 detainees for
increased. The percentages of naive Thli lymphocytes and the ratio of CD4:CD8
study.
lymphocytes were decreased. The in vitro natural killer cell cytotoxic activity and The 44 subjects included in this study
phagocytic functions of ingestion and digestion were significantly depressed. Se- consisted of two groups of men. The
rum interferon, serum cortisol, and prolactin were also significantly lowered. Serum first group comprised the 29 detainees
tumor necrosis factor was increased. (age, 17 to 59 years [median, 48 years];
Conclusions.-Alterations in the main parameters of the immune system and weight, 56 to 92 kg [median, 69.5 kg];
depression of important immune effector functions may have resulted from the height, 160 to 186 cm [median, 175 em];
psychological stress, physical deprivation, and malnutrition experienced by these and imprisoned for 4 to 7 months). The
war camp prisoners during their detainment. second group included 15 healthy
matched control subjects from the in-
stitute's laboratory staff from Zagreb,
Croatia, far from the war zone (age, 18
THE INFLUENCE of stress1-"nd tainees in these camps. However, to our to 58 years [median, 48 years]; weight,
physical deprivation, such as malnutri- knowledge, no studies on measurable 62 to 86 kg [median, 70 kg]; and height,
tion:-l1 on immune functions is now well parameters of immune system of such 167 to 187 cm [median, 176 em]). De-
documented. Life in areas submitted to detainees have been published. tainees declared a weight loss of 5 to 37
continuous and prolonged war opera- Therefore, the aim of this study was kg (median, 15.5 kg) during the impris-
tions is certainly stressful. This is es- to identify possible alternations of im- onment in the camp. Ten milliliters of
pecially true for the persons displaced munologic parameters in prisoners re- heparinized blood and 5 mL of blood for
from their homes and kept in prison leased from the Bosnian Serb-run pris- serum separation were collected by veni-
camps. The life conditions in ManjaEa on camp in ManjaEa. puncture between 9 and 10 AM. Blood
prison camp in Bosnia are reported to was processed within 90 minutes. For
have been harshlal"'and have been doc- MATERIALS AND METHODS comparison, we added historical controls
umented by reports on general medi- Study Population from gender- and age-matched groups
call4 and psychicl~roblemsamong de- from before the war.
In November 1992, a group of 764 de-
tainees was released from Manjak and
transported to the Croatian town of Kar- Hematologic Values
From the Institute of Immunology, University of lovac. Transport from ManjaEa to Karlo- A complete blood cell count was per-
Zagreb (Croatia).
Reprmt requests to lnst~tuteof Immunology. Rock- vac lasted 8 to 10 hours. In Karlovac the formed with a Coulter T-660 counter
efellerova 10. 41000 Zagreb, Croat~a(Dr Dekar~s) detainees were received by officers of the (Coulter Electronics Inc, Hialeah, Fla),

JAMA, August 4, 1993-Vol270, No. 5 lmrnunologic Parameters in Prisoners of War-Dekaris et al 595

Reprinted from JAMA Q The Journal of the American Medical Assoclafion
August 4, 1993 Volume 270
Copyright 1993, American Medical Association
Table 1 .--Hematologic Findings in Detainees and Control Subjects done using a suitable in-house standard
Detainees Control Subiects
(n,atural human leukocyte INF, Insti-
tute of Immunology, Zagreb) cali-
, , brated to first international standard
lnterouartile lnterouartile
Parameter Median FIahge Median FIinge P* recombinant human interferon alfa (&31
Wh~teblood cells x 1 0% 75 6 5-85 67 6 0-76 NS 514) (NIRSC).
Red blood cells, x 1 012/L 4.51 4.1-4.7 4.87 4.6-5.1 c.001 For the determination of tumor ne-
-
Hemwlobin, a/L - 137 126-143 150 145156 ~:.001 crosis factor (TNF) and lymphotoxin,
Hematwrit 0.40 0.37-0.42 0.44 0.42-0.46 -:.OOI the samples of serum were incubated
Mean corpuscular volume, fL 91 87-93 90 87-94 NS
for 24 hours with monolayers of L929
Platelets. x 10% 198 148-219 21 6 166-246 NS
cells (ECACC) ("one day assay""'). The
Lymphocytes, 90 27 22-33 29 23-36 NS
test samples were diluted in a culture
medium containing actinomycin D (Sig-
Monocytes, % 4 3-6 2 1-5 <r.05
ma Chemical Co, St Louis, Mo). Recom-
Segmented neutrophils, % 64 57-70 64 59-72 NS
binant TNFa and TNFp (lymphotoxin)
Nonsegmented neutrophils, % 0 0-1 0 0-0 c.05
(Genzyme Corp, Cambridge, Mass) were
Eosinoohils. % 2 1-4 1 1-2 NS
tested on each plate as a positive con-
Basophils, 96 0 0-1 0 0-1 NS
trol.
Plasma cells, % 0 0-0 0 0-0 NS After the suitable incubation time, for
Myelocytes, % 0 0-0 0 0-0 NS each bioassay, the cell proliferation, cy-
Lymphocytes, x 1@/L 2.11 1.52-2.46 2.07 1.63-2.50 NS topathic effect, or cytotoxicity was es-
Monwytes, x1 09/L 0.31 0.18-0.58 0.15 0.08-0.28 c.05 timated by colorimetric assay," ie, tet-
Segmented neutrophils, x109IL 4.63 3.88-5.68 4.20 3.58-5.47 NS razolium salt MTT was added to all wells
Nonsegrnented neutrophils, x1O9/L 0 0-0.08 0 0-0 .~05 of a 96-well microplate. To dissolve for-
Eosinoohils. x109/L 0.14 0.08-0.25 0.09 0.06-0.15 NS mazan-protein c o m p l e x e ~the
, ~ extrac-
Basophils, x 1O9/L 0 0-0.07 0 0-0.05 NS tion buffer sodium dodecyl sulfate/N,N-
Platelets. x 1 WL 0 0-0 0 0-0 NS dimethylformamide was added at the
Myelocytes, x 109/L 0 0-0 0 0-0 NS end of incubation. Optical densities were
Total serum proteins, gR 73 68-83 88 83-93 c..OOl measured a t 580 nm using a Multiscan
Serum albumin. a/L 45 41-47 57 51 -64 .c.001 MCCIc%Omicroplate reader (Labsystem
Oy, Helsinki, Finland). The readings
*Probability of reject~ngthe null hypothesis. NS indicates not significant.
from the spectrophotometer were trans-
ferred directly to a computer, using Mul-
and leukocyte differential count was cording to the previously described tech- ticalc software (Pharmacia, Wallac Oy,
done on May-Griinwald-Giemsa- nique.ls Turku, Finland) for data evaluation and
stained smears. Total proteins in the quality control.
sera were determined by the biuret test. Polymorphonuclear Phagocytic When appropriate, to demonstrate
Albumin was then quantitated by elec- Activity that the particular cytokines caused the
trophoresis in the cellulose acetate fol- Isolated polymorphonuclear neutro- tested effects, we used monoclonal an-
lowed by densitometry. philic leukocytes were washed, and their tibodies in neutralization assay.
ingestion, digestion, and antibody-de-
lmmunophenotypingof Lymphocytes pendent cellular cytotoxic abilities to- Serum Hormone Levels
Immunophenotyping was performed ward opsonized sheep red blood cells All samples were radioimmunoas-
by a lysed whole-blood method using a labeled with chromium 51 were mea- sayed in duplicates on the same day.
panel of Becton Dickinson monoclonal sured as previously described.'" Cortisol, triiodothyronine, and thyrox-
reagents (Immunocytometry Systems, ine were assayed by cortisol (polyeth-
San Jose, Calif) to CD3/CD16+CD56, Bioassay for Cytokines ylene glycol),triiodothyronine (polyeth-
TCRIHLA-DR, CD4/CD45RA, CD81 Interleukin 2 (IL-2) in serum was ylene glycol), and thyroxine (polyethyl-
CD25, CD5dCD20, and CD711CD56, di- quantified by dilution analysis19 using ene glycol) diagnostic kits (Institute of
rectly conjugated to fluorescein isothio- IL-Z-dependent CTLL cells (European Immunology, Zagreb). Prolactin and
cyanate or.phycoerythrin. The samples Collection of Animal Cell Cultures p-endorphin were assayed by Allegro
were analypql on the FACScan flow cy- [ECACC], PHLS Centre for Applied prolactin and Allegro p-endorphin kits
tometer (Becton Dickinson) within 24 Microbiology and Research, Porton (Nichols Institute Diagnostics, San Juan
hours using the SimulSET software as Down, Salisbury, United Kingdom). In- Capistrano, Calif).
described in detail by Reichert et al.lG terim reference reagent for human IL-2
From each sample, 5000 lymphocytes (first international standard, 1987, 861 Statistics
were acquired and the nonspecific stain- ,504,National Institute of BiologicalStan- The descriptive statistics of popula-
ing, assessed by an isotype control, ad- dards and Control [NIBSC], Poters Bar, tion distribution used were median and
justed to less than 1%. The data of lym- Hertfordshire, United Kingdom) was interquartile range because of the asym-
phocyte subsets were expressed as a used to calibrate in-house standards for metric distribution of most of the pa-
percentage of total lymphocytes, and ab- use in each assay. rameters tested. A comparison of the
solute numbers were calculated on the The method for interferon (IFN) as- results obtained in detainees and con-
basis of laboratory differential and white say used Wish cells (ECACC) a s indi- trol subjects was performed by Mann-
blood cell counts. cator cells and vesicular stomatitis vi- Whitney U test and two-tailed proba-
rus as a challenge virus, which is based bility (P value), by which the null hy-
Natural Killer (NK) Cell Activity on the inhibition by I F N of the cyto- pothesis (H,,)can be rejected, was indi-
A standard chromium 51 release as- pathic effert caused by vesicular sto- cated. Multiple comparison of two tested
say of NK activity was performed ac- matitis virus infection. All tests were groups and historical (prewar) control

596 JAMA. August 4, 1 9 9 S V o l 2 7 0 , No. 5 immunologic Parameters in Prisoners of War-aekaris et al

subjects, if available, was performed by Table 2.-Lymphocyte Subpopulations in Detainees and Control Subjects
means of computing the Conover's in- Percentaaes Absolute Numbers. x109/L
equality if the H , was rejected by 8 8

Kruskal-Wallis test corrected for ties." Detainees Control Subjects Detainees Control Subjects
(n=29) (n=15) (n=29) (n=15)

lnterquartile lnterquartile lnterquartile lnterquartile

RESULTS Median Ranae Median Ranae Median Ranae Median Ranae
Hematologic Findings
In peripheral blood some significant dif-
ferences between detainees and healthy
control subjects were found (Table 1). In
detainees, the red blood cell count, hemo-
globin mass concentration, hematocrit, to- TCR 73 69-81 80 70-83 1.41 1.18-1.80 1.48 1.22-2.00
tal serum protein, and albumin level were TCR'IDR 61 54-65 68 60-74* 1.12 0.90-1.56 1.24 0.99-1.87
decreased, while the percentage and TCR'IDR' 13 10-19 7 7-13* 0.26 0.18-0.40 0.17 0.12-0.20*
count of monocytes and nonsegmented TCR IDR' 15 13-18 13 7-14* 0.30 0.23-0.42 0.22 0.15-0.33
neutrophils were increased. HLA-DR 28 26-35 20 17-25t 0.62 0.46-0.70 0.35 0.34-0.52*

Lymphocyte Subsets
Lymphocyte subsets and activation
A "

markers quantified as a percentage of

total lymphocytes and an absolute count
are presented in Table 2. Two-color anal-
ysis allowed the simultaneous assess-
ment of two populations and three sub-
populations defined by two markers for
each of the combinations studied.
Concerning the three populations of
lymphocytes (T, B, and NK cells), the
percentage of R lymphocytes (CD20 and
TCR-/DR ')was increased in detainees,
as well as the percentage and count of
1ymphoc.ytes expressing HLA-DR. To-
tal T lymphocytes (characterized by
TCR, CD8, or CD5) were not altered,
but the percentage of naive Thli cells
(CD4+/CD45RAi) was decreased and
that of Tc/s cells (CD8) was increased.
As a result, the CD4:CDX ratio in de-
tainees at 1.15 was lower than in control
subjects a t 1.59 (Pc.05). Regarding the
expression of activation markers (CD25
and CD71, and HLA-DR for T lympho- subjects compared with detainees and tainees, TNF concentration was signif-
cytes), detainees demonstrated higher historical control subjects (median, 4; icantly increased, and I F N concentra-
percentages and absolute numbers of interquartile range, 4 to 7). tion was decreased. Interleukin 2 con-
activated T lymphocytes (TCR+/DR+) centration was similar in detainees and
and lymphocytes with CD71. NK Cell Function control subjects.
Results obtained in detainees and The in vitro NK cell cytotoxic activity
control subjects were compared with was significantly depressed in detain- Serum Hormone Levels
the prewar historical control subjects ees at all tested effector to target cell Serum cortisol and prolactin levels
for CD3 (n=9), TCR (n=13), CD5 (n= ratios (Table 3), despite the unchanged were simficantly lower in detainees
16), CD20 (n=40), HLA-DR (n=7), CD4 percentages and counts of any NK phe- compared with control subjects. Serum
(n=39), CDX (n=36), CD56 (n=24), notype characteristics as determined by cortisol levels were lower in detainees
CD16,56 (n=9), CD25 (n=27), and CD71 flow cytometry (Table 2). compared with historical control sub-
(n=9). Multiple comparison by Kruskal- jects (Table 6).
Wallis test revealed no difference be- Phagocytic Function
tween the three groups for CD3, TCR, The polymorphonuclear phagocyte's COMMENT
CD5, CD4, CD56, and CD16,56 mark- functions-ingestion and digestion but This study simultaneously assessed
ers. Increased percentages of CD20, not extracellular cytotoxicity (antibody- multiple immunologic parameters in a
HLA-DR, and CD8 lymphocytes were dependent cellular cytotoxicity)-were group of prisoners of war. Some of the
found in detainees compared with both significantly depressed in detainees (Ta- main immune response effector func-
groups of control subjects. The only ex- ble 4). The decrease of ingestion capac- tions, for example, NK and peripheral
ceptions were increased percentages of ity was particularly pronounced. blood phagocyte activity, were signifi-
CD25 (Pc.01) found in both detainees cantly depressed in these detainees.
and control subjects compared with his- Serum Cytokine Concentrations Stress14 and protein-energy malnutri-
torical control subjects (median, 4; in- Serum concentrations of TNF, IFN, tionx-l1could have caused these effects,
terquartile range, 3 to 7) and decreased and IL-2 were determined in detainees although we cannot examine these two
percentages of CD71 (P<.01) in control and control subjects (Table 5). In de- possible causes independently.

JAMA, August 4, 1993--Vol 270, No. 5 Immunologic Parameters in Prisoners of War-Dekaris et al 597
Table 3.-Natural Killer Cell Function (Percentages of Cytotoxicity) in Detainees, Control Subjects, and It seems that, at the moment of test-
Historical Control Subjects ing, the immune depression was not a
Effector: Taraet Cell Ratio result of the immunosur)pressive action
I I of glucocorticoick,"1since ~lucocorticoids
25:l 5O:l 100:l were lower in the serum s a m ~ l eof
s cam11
Control subjects detainees. If a weight loss of more than
No. 15 15 15
9 kg, decreased food intake for more
Med~an(~nterquart~le
range) 29 (21-30) 45 (42-53) 60 (55-66)
than 2 weeks, and a decreased serum
P* 001 01 01
' <
albumin level (<X2 g-/I,) are used as a
Detainees definition for protein-energy malnutri-
NO.
Med~an(interquart~leranqe) 7 (2-18) 17 (8-28) 15 (8-34)
tion,I0the detainees met two of the three
required criteria (weight loss and de-
creased food intake). In comparison with
Historical control subjects
No. control subjects, they also had a signif-
Median iinterauartile ranoe) ... 61 (35-721 71 159-80)
icantly decreased serum albumin levels,
but none of the detainees had albumin
'Probab~l~tyof rejectmg null hypothesis for detainees vs control subjects. levels less than 22 g/L.
tDetainees vs historical control subjects. Ellipses mdicate data not available By flow cytometry immunophenotyp-
ing we sought to analyze the basic lym-
phocyte antigens. Each population was
c,xpressed as a percentage of total lgm-
Table 4.-Phagocytic Functions (Percentages): Ingestion (ING), Digestion (DIG), and Antibody-Dependent
Cellular Cvtotoxicitv (ADCC) in Detainees. Control Subiects, and Historical Control Subiects
phocytes and an absolute count. When
significant, the trends of these two mere
ING DIG ADCCS always parallel; however, fewer differ-
Control subjects ences were detected by the analysis of
No.* 13 13 13 absolute counts. The possible explana-
Median (interquartilerange) 32 (23-41) 27 (23-40) 69 (65-73) tion for this discrepancy may lay in the
Pt .r.01 ,:.05 NS greater variability of results expressed
Detainees as absolute counts, owing to cumulat'Ion
No. 29 29 29 of errors in white blood cell counts and
Median (interquartilerange) 18 (12-25) 20 (15-24) 58 (47-70) differential determinations.lh
pt c.01 <..05 NS In detainees, the percentage of naive
Historical control subjects Thli cells (CD4-/CI)45RA+) u-as lowered
No. 23 18 23
while the percentage of the T d s cell pop-
Median iinterauart~leranae) 41 127-531 31 118-401 63 137-76)
ulation was increased, resulting in lower
"Two subjects' samples did not contain a complete blood cell count to perform all tests. CDCCDX ratios. This ratio has been re-
tprobability of rejecting null hypothesis for detainees vs control subjects. ported to be significantly decreased in
*Detainees vs historical control Subjects.
5NS indicates not significant. malnourished subjects.'~"n increased
percentage and count of lymphocytes
with late ( 0 7 1 and HLA-DR) but not
with early (CD25) activation markers was
also found. The increased percentage and
Table 5.-Serum Cytokine Levels: Tumor Necrosis Factor (TNF), Interferon (IFN), and Interleukin 2 (IL-2) absolute number of HLA-DR ' lymphw
Expressed as Units per Milliliter in Detainees and Control Subjects
cytes in detainees waq the consequence of
Detainees Controls increased percentage of total R lympho-
I I I I cytes and activated T 1;vmphoq;tes (both
lnterquartile lnterquartile
Cytokine No. Median Range No. Median Range P* expressing HLA-DR).
TNF 29 500 125-500 15 0 0 .: ,001 Natural killer cells are considered to
IFN 29 0 0 15 11 7-13 <:..001 be important in host immunosurveil-
lan~e.~'The percentage and count of cells
with NK cell markers were unchanged
"Probability of rejecting null hypothesis. NS indicates not significant. in detainees. In contrast, in vitro func-
tion of NK cells was sipificantly tle-
pressed among detainees. A recently de-
scribed clinical syndrome, referred to as
Table 6.-Serum Hormone Level in Detainees. Control Subiects. and Historical Control Subiects "low NK syndrome," is characterized
by a low NK cell activity with the num-
Controls Detainees Historical Controls ber of cells with NK phenotype in the
I I I -I
lnterquartile lnterquartile lnterquartile normal range."' A decreased NK cell
Hormone Median Ranae P* Median Ranae P t t Median* Ranaef activity in protein-energy malnutrition
Triiodothyronine,nrnol/L 2.2 2.1-2.3 NS 2.3 2.2-2.5 NS 2.1 1.8-2.3 has also been reported." The mechanism
Thvroxine. nrnollL 125 113-141 NS 132 112-143 NS 123 96-140 underlying the evident decline of NK
Cortisol, nrnoVL 521 447-705 <:.01 364 259-452 c.01 469 386-579 cytotoxicity in detainees is not clear.
Prolactin, ngImL 6.7 4.7-11.6 ,..05 5 3.5-6.4 ... ... ... However, since pelsons with chronic low
B-Endomhin. ~ d m L 24.5 16.4-38.8 NS 24.6 15.3-44.3 ... .. . ... NK activity are a t risk for infections,17
this finding could be of practical impor-
"Probability of rejectmg null hypothesis for detainees vs control subjects. NS ind~catesnot significant
tDeta~neesvs h~stor~cal control subjects tance.
*Ell~psesindicate data not available. Impaired phagocytic functions have

598 JAMA, August 4, 1993--Vol270. No. 5 lrnmunologic Parameters In Prisoners of War D e k a r i s et al

been previously described in malnour-
ished micei1 and men.xr%o of three
phagocytic functions tested in the de-
tainees were depressed. As neutrophils
have a critical role in the host defense
against various microbial antigens," this
finding is of clinical importance.
In our study, TNF was significantly
elevated, and IFN was decreased in de-
tainees. We measured the bioactive
molecules of these cytokines. Patholog-
ical conditions may result from an ex-
cessive production and activity of
TNFa,"' which is the principal mediator
of systemic responses to sepsis and in-
jury.a Recent work suggests that pro-
duction of several cytokines (interleu-
kin 1and IL-2 and IFN-y) is decreased
in protein-energy malnutrition.@
Various hormones exert a significant
impact on the immune re~ponse.~;"Im-
munosuppressive and anti-inflammato-
ry actions of glucocorticoids are well
known." Acute stress results in an in-
crease of circulating corticosterone lev-
els, but in chronic stress, corticosterone
levels return to control range.3LBelow
normal circulating cortisol levels in the
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View publication stats
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declared brief combat experience (6
weeks) before imprisonment. All others
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the day of imprisonment. Since the de-
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This study was supported in part by Croatian Min-
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"Immunoreactivity in War Refugees in Croatia!'
The authors wish to thank Becton Dickinson, Heidel-
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knowledge the help provided by Lars Lynge Niel-
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