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Original article

Effect of zinc on immune functions in patients with

pulmonary tuberculosis
Miguel Guzman-Rivero a,c, , Aleida Verduguez-Orellana a , Marisol Cordova a ,
Luis Maldonado b , Marcos Medina a , Edgar Sejas a , Bjrn kesson c,d
a
Instituto de Investigaciones Bio-Mdicas (IIBISMED), Universidad Mayor de San Simn, Cochabamba, Bolivia
b
Laboratorio de Investigacin Mdica (LABIMED), Universidad Mayor de San Simn, Cochabamba, Bolivia
c
Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, Sweden
d
Department of Clinical Nutrition, Skne University Hospital, Lund, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: The tuberculosis infection triggers in the host a complex immune response and the involvement of CD4+
Received 20 November 2013 lymphocytes and interferon-gamma (INF-) in the control processes has been reported. Since nutritional
Accepted 1st January 2014 status, e.g. regarding zinc may have potent effects on the immune response, we conducted a zinc supple-
mentation study to gain more knowledge on its effects on immune function in pulmonary tuberculosis.
Keywords: Twenty-one patients with pulmonary tuberculosis completed the 3-month study. Ten of them got 45 mg
Zinc zinc daily and 11 of them got placebo in addition to drug therapy. Immunoglobulins in plasma and cell
Interferon-gamma
proliferation, IFN- production and CD markers in peripheral blood mononuclear cells (PBMC) were
CD markers
Immunoglobulins
measured. >The immune system of the patients was activated as reected by the increased concentra-
tion of immunoglobulins in plasma. Still, there was no difference in the ability of the PBMC to proliferate
and produce INF- in response to concanavalin A between patients and controls. Moreover, there were
no signicant differences in these variables between the zinc-supplemented and placebo groups after
3 months. In other experiments, the addition of zinc sulphate or iron sulphate in vitro to PBMC tended to
decrease the number of CD4+ cells.
Conclusions: The immune system of the tuberculosis patients maintained its activity and in response to
pharmacological therapy the immune response seemed to maintain a Th1 orientation. It was not possible
to document a role of zinc supplementation for the immune response.
2014 Elsevier Masson SAS. All rights reserved.

1. Introduction delayed-type hypersensitivity response to tuberculin [3]. There

Active tuberculosis triggers in the host an immune response
with the production of crucial cytokines [1]. This can also favour
the progression of the infection to chronicity, especially when
regulatory T-cells cause the deactivation of cytokines [2,3]. Exper-
imental evidence in murine models showed that lung and spleen
CD4+ lymphocytes were more protective than CD8+ lymphocytes
in tuberculosis but also indicated the pivotal protective role of
interferon-gamma (INF-) [4,5]. Moreover in patients with active
tuberculosis, the immune response is characterized by a depressed
Corresponding author. Biomedical Nutrition, Department of Pure and Applied
Biochemistry, Lund University, PO Box 124, SE-22100 Lund, Sweden.
Tel.: +46 46 222 9607; fax: +46 46 222 4611.
E-mail addresses: Miguel.Guzman@tbiokem.lth.se,
miguelguzmanrivero@yahoo.es (M. Guzman-Rivero), aleidav 15@hotmail.com
(A. Verduguez-Orellana), nmarcordova@yahoo.com (M. Cordova),
luamal@hotmail.com (L. Maldonado), marcosmebu@gmail.com (M. Medina),
edgarsejas@yahoo.com.ar (E. Sejas), bjorn.akesson@tbiokem.lth.se (B. kesson).
is also evidence of a depression of PBMC proliferation in vitro
after stimulation with PPD and a decreased expression of IL-2,
IL-4 and INF- in these cells and an overexpression of IL-10 and
transforming growth factor-beta in monocytes [4,6]. Available data
also indicate that in tuberculosis, the pro-inammatory CD4 Th17
cells are activated, producing IL-17A, IL-17F, tumor necrosis factor,
IL-22 and in some cases also INF- [3]. In another study, a signif-
icantly lower PPD-induced INF- production was found together
with a higher IL-10 production in vitro in patients with tubercu-
losis compared to their controls although the absolute number
of lymphocytes was similar. Moreover, after 6 months of phar-
macological therapy, the in vitro INF- production was increased
non-signicantly and the levels were instead much inuenced by
gene polymorphisms [7].
Zinc is a potent mediator of host resistance to infection because
it can inuence the innate and adaptive immune response in many
ways [810]. It can increase the release of INF- and other cytokines
by PBMC although at high concentrations [11], and induce the pro-
liferation of CD8+ T-cells in combination with an exposure to IL-2.

2210-5239/$ see front matter 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.bionut.2014.01.004

Please cite this article in press as: Guzman-Rivero M, et al. Effect of zinc on immune functions in patients with pulmonary tuberculosis.
Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.01.004
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In such studies, the addition of zinc can also affect the prolifera-
tion of different cell types in response to various mitogenic stimuli
although an excessive supplementation by zinc could also have a
deleterious effect on immune functions [810].
In the present study, we tried to gain more knowledge on the
effects of zinc supplementation on the immune functions of pul-
monary tuberculosis patients as well as on the effects of in vitro
addition of zinc and iron on the activity of PBMCs from these
patients in response to mitogen stimulus.
2. Material and methods
2.1. Patients
The patients were recruited in the districts of Southern
Cochabamba city in Bolivia as described elsewhere (Guzman-
Rivero et al. submitted). Forty patients having active pulmonary
tuberculosis based on the demonstration of acid- and alcohol-
resistant Bacilli in stained smears of sputum samples were
contacted, and 29 of them were selected since they met the
additional inclusion criteria of age 1550 years and no previous
tuberculosis episodes. The exclusion criteria were extra-pulmonary
tuberculosis, HIV infection, pregnancy, lactation, use of nutritional
supplements, and presence of diabetes mellitus, chronic renal fail-
ure or liver disease. All patients completed a health questionnaire
prior to entering the study and gave their verbal consent for inclu-
sion into the study. Twenty-one patients completed the study and
8 left the study for different reasons (Guzman-Rivero et al. submit-
ted).
2.2. Control subjects
The controls were age- and gender-matched subjects without
any clinical signs of previous tuberculosis infection and also resi-
dents in the same area as the corresponding patients. Exclusion
criteria were pregnancy, lactation, use of nutritional supplements
or regular medication, presence of diabetes mellitus, chronic renal
failure or liver disease. All controls completed a health question-
naire prior to entering the study and gave their verbal consent for
inclusion into the study.
2.3. Study design
Patients were randomly allocated to receive zinc or placebo
coded capsules for 3 months as described elsewhere (Guzman-
Rivero et al. submitted). Each zinc capsule contained 315 mg of zinc
gluconate (corresponding to 45 mg zinc) and each placebo capsule
contained 315 mg of cornstarch, both specially prepared for our
study by a company (Farmacia Artesanal, Cochabamba, Bolivia).
All patients received simultaneously the standardized treatment
regime for active tuberculosis of adults proposed by PHAO/WHO
[12,13] (two months of daily doses of isoniazid, rifampicin, pyraz-
inamide and ethambutol followed by 4 months of daily doses of
isoniazid and rifampicin, dosed in mg/kg body weight) together
with one capsule per day (zinc or placebo) taken in the fasting state
early in the morning. Controls were not given any zinc or placebo
capsules. No dietary advice was given to the subjects in the study.
2.4. Collection of blood samples
For patients blood was sampled twice, at time zero (T0) before
the start of the treatment and after 3 months (end of zinc supple-
mentation, T1), and for controls, blood was sampled at time zero
only. Blood was collected by venipuncture (after 12 h of fasting and
30 min of relaxing) into heparinized tubes. Plasma was obtained
by centrifugation, aliquoted and stored at 80 C until processing.
Please cite this article in press as: Guzman-Rivero M, et al. Effect of zinc on immune functions in patients with pulmonary tuberculosis.
Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.01.004
The cell pellet was used for isolation of mononuclear cells in the
IIBISMED laboratory, Cochabamba, Bolivia.
2.5. Immunoglobulin and C-reactive protein measurements
The measurement of plasma immunoglobulins (IgA, IgG and
IgM) and C-reactive protein (CRP) concentration was performed
in the laboratory of Skne University Hospital, Lund, Sweden. The
samples were transported on dry ice from Bolivia to the laboratory.
The analytical methods involved the addition of antibodies directed
to the different Igs and the resulting agglutinates were measured
by turbidimetry on a Cobas instrument using accredited methods.
2.6. Proliferation of peripheral blood mononuclear cells (PBMC)
This was performed as described [14]. Briey, blood samples
were collected into heparinized tubes and plasma was removed
after centrifugation (5000 g for 10 min) and replaced by twice the
volume of phosphate-buffered saline (PBS). The diluted blood was
layered onto Histopaque (density 1.077) and centrifuged for 30 min
at 3000 g. The PBMCs were washed twice with RPMI-1640 con-
taining penicillin/streptomycin (1:100) and nally re-suspended
in the same medium. The suspension was adjusted to a nal
concentration of 1 106 cells/mL after performing a manual cell
counting (dilution 1:100 with Trk solution). The viability was
determined in a Neubauer hemocytometer after dilution 1 + 1 with
Trypan blue solution and the mean (SD) cell viability was 84 (9)
%. PBMC were cultured in RPMI-1640 medium, fetal bovine serum
(FBS) and antibiotics. For stimulation of cells, concanavalin A (Con
A) was added at a nal concentration of 5 g/mL, and in other
wells mixtures of Con A plus zinc (1 mol/L) or iron (1 mol/L)
(both as sulphate) were added. The nal volume of the culture
was 300 L/well and the cultures were performed in triplicate for
6 days. Proliferation was measured at the end of culture period as
total DNA content by propidium iodide staining [15]. Briey, the
cells were permeabilized for 30 min with 500 L of 100% ethanol
and then incubated in the dark for 30 min with 500 L of propidium
iodide at 10 g/mL in PBS. Fluorescence (excitation, 535 nm; emis-
sion, 612 nm) was measured in a uorometer (Fluoroskan Ascent ,
cat. no 5210450, Labsystems Thermo Scientic, Helsinki, Finland).
The uorescence values for the triplicate cultures were averaged
and expressed as relative uorescence units (RFU).
2.7. Measurement of production of interferon-gamma (INF)
and of CD4+/CD8+ lymphocyte subpopulations in cultured
peripheral blood mononuclear cells
Cells were stimulated by the addition of Con A and trace ele-
ments during culture as described above but with an incubation
time of 3 days. For measurement of INF-, the cultured samples
were centrifuged, and the supernatant was stored at 80 C. The
cell pellet was re-suspended in one volume (500 L) of FBS and one
volume of 20% DMSO in RPMI and stored in liquid nitrogen until cell
phenotype analysis. Supernatants of cell culture were later thawed
and an ELISA Quantikine test kit was used for measurement of
the concentration of INF-. The detection limit was 8.0 pg/mL (data
supplied by the manufacturer) and the coefcient of variation was
6% for our assays.
Cells preserved in liquid nitrogen were thawed by keeping the
vials at 37 C for 2 min and then suspended in PBS solution, cen-
trifuged at 3000 g for 10 min and re-suspended in 1% FBS in PBS. The
CD cell markers BD FACSCountTM kit was used for quantication of
CD4+ and CD8+ following the manufacturers instructions. Briey,
the cells were xed in formaldehyde solution, and monoclonal anti-
bodies uorescence-labelled against CD4+/8+ markers were added
and the samples were measured in a FACSCountTM instrument
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system. The results were expressed in number of cells/L for CD4+

and CD8+ and as the ratio CD4+/CD8+.

2.8. Statistical analysis

The SPSS software was used. The MannWhitney U-test was per-
formed for two non-normally-distributed continuous variables to
make the comparisons between zinc-supplemented and placebo
patient groups and between controls and patient data at T0 or T1.
The Wilcoxon signed-rank test for non-normally-distributed con-
tinuous variables was performed for paired comparisons between
different incubation conditions. The Spearman correlation test was
used to assess correlations. No correction for multiple testing was
made.

2.9. Ethical considerations

Ethics permission for all procedures involving human volun-

teers was obtained from the Ethics Committee of the Medical
Faculty, Universidad Mayor de San Simn, Cochabamba.

3. Results
3.1. Immunoglobulins in plasma
Twenty-one patients supplemented with zinc or placebo for
3 months (T0 to T1) completed the study. During the supplemen-
tation period, the concentration of zinc in plasma increased from
11 (0.6) mol/L to 14 (1.5) mol/L in the supplemented group and
the mean increment was 22% compared to 6% in the placebo group
(Guzman-Rivero et al, submitted). The plasma concentrations of
IgA, IgG and IgM, at T0 were all signicantly increased compared
with the corresponding controls except for IgM in the placebo group
(Table 1). In both supplementation groups, the concentrations of
all three immunoglobulins decreased from T0 to T1 and this was
signicant for IgA in both groups (P = 0.005) but the extent of the
decrease was not signicantly different between the two supple-
mentation groups (P > 0.05). IgG in the zinc-supplemented group
also decreased signicantly with time. Nevertheless, the concen-
trations at T1 were still higher compared to the data from controls.
For IgG signicant differences were found compared to controls in
the zinc-supplemented group (P = 0.017) and for IgA (P = 0.017) and
IgG (P = 0.05) in the placebo group at T1.
Fig. 1. Effects of in vitro addition of concanavalin A, zinc sulphate and iron sulphate
on the proliferation of PBMC isolated from patients with pulmonary tuberculosis
before and after zinc supplementation and drug treatment and from controls. PBMC
were cultured for 6 days and then harvested. Data were expressed as median (IQR).
Wilcoxons signed-rank test, P values for ConA-Zn ConA T0 = 0.51 (zinc-
supplemented), 0.21 (placebo), T1 = 0.28, 0.95, Control = 0.51, 0.21; for ConA-
Fe ConA T0 = 0.20, 0.91, T1 = 0.51, 0.64, Control = 0.17, 0.026. PBMC = peripheral
blood mononuclear cells, Con A = concanavalin A.
3.4. Effects of in vitro addition of zinc or iron on PBMC
The possible effects of in vitro addition of zinc sulphate and iron
sulphate on PBMC proliferation and INF- production were studied
(Figs. 1 and 2). The addition of Con A markedly stimulated INF-
production. The further addition of zinc or iron did not result in
any additional effect in any of the two groups (Figs. 1 and 2).
In the same experiments, the number of cells expressing CD4+
and CD8+ surface markers were studied after the addition of zinc
or iron (Table 3). There were signicant decreases in the number of
CD4+ cells in the zinc-supplemented group in the comparison Con
A-zinc versus Con A at T1 (p = 0.015) and in the placebo group in
the comparison Con A-iron versus Con A at T0 (p = 0.018) (Table 3).

3.5. Correlations among immunity parameters

3.2. PBMC proliferation and INF- production
In the zinc-supplemented group, there were small non-
signicant increases in PBMC proliferation (P = 0.24) and INF-
production (P = 0.44) after 3 months of anti-tuberculosis therapy
and zinc supplementation (Table 2), but the values were close to
those obtained in the placebo group and the controls. There were
no statistical differences between these data of patients from both
groups at T0 or T1 compared to their controls (Table 2). This was
partly due to the large variation in the data.
3.3. CD4+/CD8+ cell markers
The addition of Con A to PBMC cultures did not consistently
affect the number of CD4+ and CD8+ cells (Table 3). There were
no statistical differences for the CD4+/CD8+ ratio between the zinc-
supplemented and placebo groups (P = 0.96 and 0.42) (Table 2). The
number of CD4+ and CD8+ cells was signicantly higher at T1 than
at T0 in the zinc-supplemented group but not in the placebo group
(Table 3).
Correlations were calculated for some immunity variables in
both patients and controls (Table 4). The strongly signicant corre-
lations between cell proliferation and INF- production probably
reect variations in cell number and activity to a large extent. Also,
the correlations between CD4+/CD8+ ratios in incubations with and
without Con A probably mainly show methodological consistency.
Most other correlation coefcients did not show statistical signif-
icance. It can be speculated that the positive correlation between
plasma IgA and CD4+/CD8+ ratio might be related to the recovery
from tuberculosis.
4. Discussion
The humoral immune response measured through plasma
immunoglobulin concentrations showed increased concentrations
of all three immunoglobulins in patients compared to controls
(Table 1). These results were similar to other studies that reported
signicant increase of plasma concentrations of specic IgG, IgM
and IgA against different antigens of Mycobacterium tuberculosis

Please cite this article in press as: Guzman-Rivero M, et al. Effect of zinc on immune functions in patients with pulmonary tuberculosis.
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Table 1
Plasma concentration of immunoglobulins in patients with pulmonary tuberculosis patients at baseline (T0) and after supplementation for 3 months (T1) and in controls.

Immunoglobulins in plasma Time 0 Time 1 Control P value

mean (SD) mean (SD) mean (SD)

T0 vs T1a T0 vs ctrla T1 vs ctrla T1T0Zn vs placebob

Zinc-supplemented group (n = 10)

IgA (g/L) 4.1 (1.3) 3.3 (1.1) 2.6 (0.9) 0.005 0.047 0.22 0.32
IgG (g/L) 17.9 (4.2) 16.3 (3.9) 11.9 (1.7) 0.047 0.005 0.017 0.97
IgM (g/L) 1.7 (0.3) 1.5 (0.3) 1.3 (0.4) 0.13 0.013 0.093 0.63
Placebo group (n = 10)
IgA (g/L) 4.7 (1.2) 3.6 (0.9) 2.6 (0.9) 0.005 0.005 0.017
IgG (g/L) 18.4 (3.6) 16.8 (3.3) 12.2 (2.0) 0.20 0.005 0.005
IgM (g/L) 1.5 (0.9) 1.4 (0.8) 1.3 (0.6) 0.10 0.87 0.79
a
Wilcoxons signed-rank test.
b
MannWhitney test of changes from T0 to T1.

Table 2
Immunological assays in PBMC isolated from patients with pulmonary tuberculosis before and after zinc supplementation.

Type of test T0 T1 Control P values

SP Con A SP Con A SP Con A Con A treatment

T0-T1a T0-Ctrla T1-Ctrla T0b T1b

(Zn vs Placebo)

Zinc-supplemented group (n = 10)

Cell prolif (RFU) 1.2 1.4 1.3 2.7 1.4 2.7 0.24 0.72 0.39 0.86 0.11
(0.5) (2.4) (1.1) (5.5) (0.5) (2.2)
INF- (pg/mL) 4 329 5 483 4 496 0.44 0.59 0.96 0.29 0.44
(1) (551) (0.3) (673) (0.3) (808)
CD4+/CD+ ratio 0.9 1.1 1.4 1.2 1.1 1.0 0.89 0.36 0.78 0.96 0.42
(0.9) (2.2) (0.8) (0.6) (0.8) (0.8)
Placebo group (n = 10)
Cell prolif (RFU) 0.9 1.6 1.2 1.6 1.0 1.3 0.72 0.93 0.53
(0.4) (2.1) (0.5) (1.9) (0.8) (2.3)
INF- (pg/mL) 4 110 5 65 5 516 0.89 0.16 0.36
(0.8) (441) (0.5) (631) (1) (591)
CD4+/CD+ ratio 1.5 1.3 1.6 1.8 1.4 1.5 0.25 0.95 0.87
(1.7) (2.6) (1.3) (1.3) (1.5) (0.7)

The supplementation with zinc and placebo administration proceeded for 3 months (from T0 to T1) in combination with drug treatment. Controls were only studied at
T0. PBMC were isolated and cultured for 6 days (cell proliferation) and for 3 days (INF- and CD markers). Data were expressed as median (IQR). PBMC: peripheral blood
mononuclear cells; SP: spontaneous proliferation; Con A: concanavalin A; RFU: relative uorescence units; Cell prolif: cell proliferation; INF-: interferon-gamma.
a
Wilcoxons signed-rank test, for T1 vs T0 and T0 or T1 vs control for each group.
b
MannWhitney test for zinc-supplemented vs placebo groups.

Table 3
Effects of in vitro addition of concanavalin A, zinc sulphate and ferrous sulphate on the number of CD4+ and CD8+ cells (cells/L) of PBMC.

SP ConA ConA-Zn ConA-Fe P value

ConA-SPa ConA-Zn ConA-Fe SPb ConAb ConA ConA

ConAa ConAa Znb Feb

Zinc-supplemented group (n = 9)
CD4+ cell/L
Time (0) 27 (36) 46 (50) 33 (50) 19 (38) 1.00 0.78 0.17 0.11 0.66 0.55 0.82
Time (1) 104 (90) 121 (123) 77 (104) 105 (102) 0.31 0.015 0.95 0.67 0.74 0.96 0.41
Ctrl 51 (71) 44 (67) 33 (78) 64 (61) 0.48 0.35 0.77 0.55 0.30 0.24 0.26
CD8+ cell/L
Time (0) 28 (19) 22 (18) 29 (28) 22 (28) 0.89 0.77 0.93 0.40 0.28 0.71 0.50
Time (1) 66 (96) 90 (112) 81 (124) 86 (113) 0.14 0.34 0.52 0.67 0.14 0.54 0.25
Ctrl 45 (36) 56 (35) 47 (31) 51 (39) 0.77 0.94 0.95 0.26 1.00 0.44 0.55
Placebo group (n = 10)
CD4+ cell/L
Time (0) 45 (63) 54 (88) 35 (63) 32 (39) 0.81 0.21 0.018
Time (1) 83 (102) 80 (97) 77 (82) 103 (110) 0.33 0.93 0.12
Ctrl 48 (146) 51 (180) 49 (190) 81 (138) 0.53 0.65 0.76
CD8+ cell/L
Time (0) 34 (48) 36 (127) 25 (61) 29 (51) 0.77 0.96 0.88
Time (1) 59 (71) 47 (59) 70 (60) 63 (53) 0.07 0.94 0.49
Ctrl 53 (70) 40 (97) 50 (77) 59 (51) 0.51 0.64 0.39

Ctrl: control; PBMC were isolated from patients with pulmonary tuberculosis before and after zinc supplementation and drug treatment and from controls. PBMC were
cultured for 3 days and then harvested. Data were expressed as median (IQR). Abbreviations as in Table 2.
a
Wilcoxons signed-rank test.
b
MannWhitney test for zinc-supplemented vs placebo groups.

Please cite this article in press as: Guzman-Rivero M, et al. Effect of zinc on immune functions in patients with pulmonary tuberculosis.
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Table 4
Correlations among indices of immune function in patients with pulmonary tuberculosis and their controls.

Variable Cell prolif Cell prolif IFN- Con A CD4+/CD8+ CD4+/CD8+ CRP IgA IgG IgM
SP Con A SP Con A

Cell prolif 0.67*** 0.68*** 0.11 0.54* 0.05 0.12 0.32 0.26
SP 0.78*** 0.65*** 0.03 0.04 0.04 0.22 0.30 0.18
Cell 0.76*** 0.60** 0.10 0.45 0.14 0.14 0.07 0.18
prolif 0.61** 0.05 0.07 0.09 0.30 0.24 0.04
Con A
IFN- 0.05 0.11 0.09 0.44 0.03 0.21 0.08 0.21
Con A 0.15 0.16 0.10 0.38 0.12 0.33
CD4+/CD8+ 0.16 0.23 0.39 0.74*** 0.13 0.45 0.28 0.13
SP 0.91*** 0.06 0.60* 0.29 0.11
CD4+/CD8+ 0.07 0.16 0.49* 0.79*** 0.32 0.28 0.34 0.15
Con A 0.11 0.45 0.37 0.08
CRP 0.29 0.49* 0.15 0.36 0.23 0.32 0.18 0.08
0.15 0.32 0.03
IgA 0.31 0.15 0.24 0.22 0.10 0.19 0.16 0.01
0.15 0.20
IgG 0.11 0.04 0.20 0.10 0.13 0.05 0.14 0.38
0.53*
IgM 0.26 0.09 0.17 0.06 0.16 0.14 0.09 0.13

Spearman correlation coefcients were calculated for controls (lower left) and patients (upper right), upper values at T0 and lower values at T1. Abbreviations: SP/Con A,
PBMC cultured without or with the addition of Con A; Cell Prolif, IFN-, CD4/CD8, measurement of cell proliferation, IFN- production and CD4+/CD8+ cell ratio. Measurement
in plasma of C-reactive protein (CRP), immunoglobulins A (IgA), G (Ig G) and (IgM).
*
P < 0.05.
**
P < 0.01.
***
P < 0.002.

similar with those found in patients with mild or moderate tuber-

Fig. 2. Effects of in vitro addition of concanavalin A, zinc sulphate and iron sulphate
on the INF- concentrations (pg/mL) in supernatants of PBMC isolated from patients
with pulmonary tuberculosis before and after zinc supplementation and drug treat-
ment and from controls. PBMC were cultured for 3 days and then harvested. Data
were expressed as median (IQR).
Wilcoxons signed-rank test, P values for ConA-Zn ConA T0 = 0.61 (zinc-
supplemented), 0.57 (placebo), T1 = 0.88, 0.73, Control = 0.59, 0.31; for ConA-
Fe ConA T0 = 0.88, 0.40, T1 = 0.51, 0.87, Control = 0.57, 0.015. PBMC = peripheral
blood mononuclear cells, Con A = concanavalin A.
among tuberculosis patients compared to their controls [1618],
including high levels of the subclasses IgG1 and IgG3 [19]. The
supplementation with zinc for 3 months during conventional anti-
tuberculosis therapy did not affect these variables signicantly
compared to placebo. Still, a previous review indicated slight
changes in the immunoglobulin concentration after zinc supple-
mentation [20].
The PBMC proliferation at T0 and T1 was not signicantly
changed by zinc supplementation (Table 2). These results are
culosis [14] and in healthy vaccinated subjects with BCG [21].
Regarding INF- production, a high production of INF- in both
zinc-supplemented and placebo groups was found at T0 and T1
(Fig. 2) although without signicant changes from T0 to T1 after
zinc supplementation (Table 2). The maintained production of IFN-
found by us indicates that the cellular immune response in these
patients was activated.
Regarding the number of CD4+ and CD8+ cells, we found that
these were signicantly higher at T1 than at T0 in the zinc-
supplemented group (Wilcoxon test P < 0.05) but not in the placebo
group. In another study of tuberculosis patients who did not receive
any type of supplementation, a lower number of CD4+ and CD8+
cells was found [22]. In our results also the CD4+/CD8+ ratio did
not show signicant changes from T0 to T1, similar to the results
of another study in tuberculosis [23]. Still another study reported a
lowering of the CD4+/CD8+ ratio in multidrug resistant tuberculosis
[24].
The in vitro addition of zinc or iron sulphate to the Con A-
incubated PBMC gave no signicant effects on cell proliferation and
the production of INF- (Figs. 1 and 2). Still the addition of zinc
produced at T1 a signicant decrease of the number of CD4+ cells
in the zinc-supplemented group whereas in the placebo group a
decrease was found at T0 after addition of iron (Table 3). Although
this may be chance ndings, it can also be explained by zinc addition
not affecting the INF- production or cell proliferation but instead
inhibiting the allogeneic reaction in the mixed lymphocyte culture
[25,26].
Because the immune system operates simultaneously through
its humoral and cellular responses, we investigated the possi-
ble correlations between some of the measurements performed
(Table 4). Others authors reported a negative correlation between
the level of IgG antibodies against PPD with cell proliferation [27]
and also between IgG against lipoarabinomannan (a component of
cell wall from M. tuberculosis) and in vitro production of INF- [27].
In our study, we did not nd correlations between the levels of
immunoglobulins and the cell proliferation or production of INF-
. This may be due to the fact that we did not measure the level
of immunoglobulins against specic antigens from M. tuberculosis
and the limited number of patients.

Please cite this article in press as: Guzman-Rivero M, et al. Effect of zinc on immune functions in patients with pulmonary tuberculosis.
Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.01.004
G Model
BIONUT-209; No. of Pages 6 ARTICLE IN PRESS
6 M. Guzman-Rivero et al. / Biomedicine & Preventive Nutrition xxx (2014) xxxxxx
The results of the present study showed that both the humoral
and cellular immune response of the tuberculosis patients were
activated as reected by the increased concentration of plasma
immunoglobulins and by the maintained ability of the PBMC to pro-
liferate and produce INF- in response to stimuli. No effects of oral
zinc supplementation on PBMC proliferation, production of INF-
and the CD4+/CD8+ ratio were observed. At the in vitro level, the
addition of zinc sulphate or iron sulphate to PBMC cultures tended
to produce a reduction of the number of CD4+ cells. Still the num-
ber of patients in the present study was rather small and it must be
regarded as a pilot study.
Disclosure of interest
The authors declare that they have no conicts of interest con-
cerning this article.
Acknowledgements
The study was part of a collaborative program between Uni-
versidad Mayor de San Simn and Lund University on Health
and Nutrition supported by SIDA (Swedish International Develop-
ment Agency). Further support was obtained from the EU project
ECNIS2.
We thank Ms. Fabiola Omonte-Mendoza for help in the blood
sampling and other laboratory procedures and Dr. Daniel Illanes,
Dr. Lieselotte Cloetens and Prof Leif Blow for helpful discus-
sions.
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