Immunological Investigations, 37:858–870, 2008

ISSN: 0882-0139 print / 1532-4311 online

DOI: 10.1080/08820130802447629

Thymulin, A Thymic Peptide,

1532-4311
0882-0139
LIMM
Immunological Investigations,
Investigations Vol. 37, No. 8, September 2008: pp. 1–18

Prevents the Overproduction

of Pro-Inflammatory Cytokines
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and Heat Shock Protein Hsp70

in Inflammation-Bearing Mice
Anti-inflammatory
S. M. Lunin et al. Effect of Thymulin

S. M. Lunin, M. O. Khrenov, T. V. Novoselova,

S. B. Parfenyuk, and E. G. Novoselova
For personal use only.

Institute of Cell Biophysics, Russian Academy of Science, Pushchino Scientific Center,

Moscow Region, Russia
The effects of synthetic analogue of peptide hormone thymulin, which is normally pro-
duced by thymic epithelial cells, on immune cells activity and blood cytokine profile
had been studied in male NMRI mice with acute inflammation induced by injection of
lipopolysaccharide from gram-negative bacteria (LPS, 250 μg/100 g of body weight).
Inflammation induced by LPS resulted in accumulation of several plasma pro-inflam-
matory cytokines, IL-1β, IL-2, IL-6, TNF-α, interferon-γ, and also IL-10, anti-inflam-
matory cytokine. Thymulin previously injected in dose of 15 μg/100 g body weight,
prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also
prevented LPS-induced up-regulation of production of several cytokines by spleen lym-
phocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of
TNF-α production in macrophages cultivated with LPS. In addition, thymulin lowered
the peak of Hsp70 production induced by LPS treatment. The results indicate that
thymulin having significant anti-inflammatory effect may be promising in clinical
application.

Keywords Endotoxin, Inflammation, Thymulin, Cytokines, Lymphocytes, Macrophages.

INTRODUCTION
Chronic and acute inflammations and sepsis as theirs sequelae continue to be
one of main causes of morbidity and mortality. Thus, almost half a million

This article is not subject to United States copyright laws.

Address correspondence to E. G. Novoselova, Institute of Cell Biophysics, Russian
Academy of Science, Pushchino Scientific Center, 142290 Moscow Region, Russia;
E-mail: elenanov_06@mail.ru
 Anti-inflammatory Effect of Thymulin 859

patients developing sepsis are yearly estimated in USA and Europe (Salvo
et al., 1995). Basically, dysregulation of the immuno-inflammatory responses
may occur in inflammation, leading to excessive or inappropriate release of
mediators. There is convincing evidence of oxidative damages in organism
with sepsis (Macdonald et al., 2003). It has been recently shown that bacterial
toxin, LPS, applied in vivo, resulted in NF-κB activation (Blackwell et al.,
2000). The present study was designed to test whether inflammatory response
of mouse immune cells could be improved by thymic hormone, thymulin.
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Hypothalamo-pituitary-adrenal axis as a stress-response system provides

a basis for central regulation of inflammatory reactions and neuro-immune
interactions. It has been shown that a thymus gland is tightly integrated into
the endocrine system (Savino and Dardenne, 2000), and thymus is considered
by some authors as a part of the hypothalamo-pituitary-adrenal axis. Thymic epi-
thelial cells produce many factors controlling immature T-cells to promote their
maturation. Multicomponent preparations, isolated from thymus gland, as well
as synthetic analogs of thymic peptides, have been used for treatment of immuno-
For personal use only.

deficiencies, particularly expressed as T-cells dysfunctions (Arion et al., 1997).

Thymic peptides can be regarded as hormones since the scope of its
actions is not limited to thymus tissue. Thymic hormones are known to pro-
mote the complete maturation of peripheral thymus-originated lymphocytes
(Inagaki-Ohara, 1996). T-lymphocytes act in coordination with macrophages
and natural killer cells, thus the thymic hormones indirectly control these
cells. Moreover, the direct effect of thymic peptide on natural killer cells has
been shown (Merlino and Marsh, 2001). Nevertheless, a role of thymic pep-
tides in mature immune cell regulation yet has not been studied.
One of the best known thymic peptides is thymulin, a nonapeptide with
zinc-dependent biological activity. Actually thymulin consists of zinc and a pep-
tide, named serum thymic factor (FTS). This hormone induces the thymocytes
proliferation and promotes the occurrence of several biomarkers inherent in
these cells, e.g., CD90, CD3, CD4, and CD8 (Savino and Dardenne, 2000).
The production of thymic hormones is controlled via the neuroendocrine
system. For example, thymulin was shown to induce the production of pitu-
itary thyreotropin, which stimulates the release of thyreoid hormones (Goya
et al., 1994), and thyreoid hormones in turn increase the production of thymu-
lin by thymic epithelial cells (Fabris and Mocchegiani, 1985). Similar correla-
tions were demonstrated between thymulin and sex hormones (Zaidi et al.,
1988), growth hormone, prolactine (Goya et al., 1994), and stress-related hor-
mones. In fact, fluctuations of these plasma hormones concentrations are
accompanied by corresponding fluctuations of thymulin concentrations (Hadley
et al, 1997; Savino et al., 1988).
Thymus gland hormones interact with other hormonal systems that con-
trol metabolism, energy balance and stress-response, and, on the other hand,
thymus is considered as central organ of immune system. So, it can be
 860 S. M. Lunin et al.

assumed that thymic hormones may be direct regulators of immune cells

responses to stress signals. Now there are few reports concerned the role of
thymus gland in inflammation. The objective of present study was to evaluate
the anti-inflammatory activity of thymulin in mouse inflammation model.
Taking into account an important role of cytokines and heat shock proteins in
the primary responses to toxic signals, the aim of the present study was to
examine the protective activity of thymulin by measuring its effect on cytok-
ine production, cytokine secretion, as well as the production of heat shock pro-
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tein Hsp70, important indicators of cellular stress.

MATERIALS AND METHODS

Animals and Substances. Male NMRI mice with body weight 25–30 g and
age of 3 months were kept in standard laboratory conditions (20–22°C, 10–14 h
light-dark cycle, 65% humidity) supplied with food and water ad libitum. The
standard mouse food pellets contained a balanced diet with proteins, vitamins,
For personal use only.

minerals, etc. The procedures followed were in accordance with institutional

guidelines and Guidelines for Ethical Conduct in the Care and Use of Animals.
Inflammation was induced by a single intraperitoneal injection of
lipopolysaccharide (LPS) from Escherichia coli (Serotype 026.B6, “Sigma”,
USA), 250 μg/100 g body weight. Thymulin solution was prepared from serum
thymic factor (American Peptides, USA) with added the equimolar concentra-
tion of ZnCl2 (Dardenne et al., 1982). Saline with ZnCl2 added in the same
concentration was used as a control solution.
Four groups of animals were used: 1, mice injected with thymulin (15 μg/100 g
of body weight) followed with saline injection in half an hour afterwards; 2, mice
injected with saline followed by LPS injection in half an hour afterwards; 3,
mice injected with thymulin followed by LPS injection half an hour afterwards,
and 4, mice injected with ZnCl2 + saline followed by saline injection half an hour
afterwards. Animals of the latter group served as controls. Mice were decapi-
tated 6 hours after LPS injection in parallel with corresponding control groups.
Plasma, peritoneal macrophages, and spleen lymphocytes were obtained as
described next. A total of 30 animals were used for experiments, all measures
were carried out individually for each mice.
In Vitro Study. The production of TNF-α by peritoneal macrophages was
also studied in vitro. Macrophages were cultivated for 24 h in 24-well plates
with medium containing thymulin (5 ng/ml), or LPS (1 μg/ml), or thymulin
(5 ng/ml) applied half an hour before LPS (1 μg/ml). Cells that cultivated without
thymulin and LPS additions served as controls.
Isolation of Blood Plasma. Blood was collected after decapitation, kept
for 3–5 h at 4°C, centrifuged at 2000 rpm, and supernatants were collected.
Isolation of mouse peritoneal macrophages and spleen lymphocytes were pro-
vided as described earlier (Novoselova et al., 2006). The peritoneal cells were
 Anti-inflammatory Effect of Thymulin 861

precipitated, washed twice in DMEM medium, 1.5 × 106 cells were resus-
pended in RPMI 1640 medium containing gentamycin, HEPES (Sigma, USA),
and 10% FCS, placed into 24-well plates (1 ml per well), and kept for 2 h at
37°C in the presence of 5% CO2. The supernatant fluid was carefully removed,
the adhered cells were washed in RPMI 1640 medium, and the monolayer of mac-
rophages was incubated in 1 ml of the medium for additional 24 h at 37°C in
humidified atmosphere containing 5% CO2. After incubation, the cell suspension
was subjected to three cycles of freeze-thawing and stored at −20°C until assay.
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Spleen lymphocytes were suspended in RPMI 1640 medium containing 1%

L-glutamine, HEPES, 0.5% gentamycin, 5 × 10–5 M β-mercaptoethanol (Sigma,
USA), and 10% FCS, and 1.5 × 106 cell/ml were incubated in 24-well plates for
72 h at 37°C in the atmosphere with 5% CO2. After the incubation, the superna-
tants were stored at −20°C.
Cytokines Assay. Concentration of cytokines in the supernatants of lym-
phocytes, in macrophages lyzates, and in the mouse blood serum was deter-
mined by ELISA. ELISA Development Kits for mouse TNF-α, mouse IL-1β,
For personal use only.

mouse IL-2, mouse IL-6, mouse IL-10, and mouse IFN-γ (Peprotech, USA)
were used. In order to visualize the binding, 100 μl of an ABTS green dye
(Sigma, USA) dissolved in 0.05 M citrate buffer (pH 4.0) with 0.01% H2O2 was
applied. The optical density was measured at 405 nm with the plate spectro-
photometer (Multiscan EX, Thermo Electron Corporation).
Western Blot Procedure. Spleen lymphocytes were suspended in phosphate-
buffered saline (PBS, pH 7.4) containing 0.01% PMSF and disrupted by sonication,
and the insoluble debris was removed by centrifugation at 18000 g for 40 min.
After measuring the protein concentration by the Bradford method (Bradford,
1976), 0.01 mg of total protein was subjected to gel electrophoresis, which was
performed according to Laemmli in 12% PAGE (Laemmli, 1970). After electro-
phoresis, proteins were transferred onto nitrocellulose membranes and filters were
blocked for 1 h with Tris-buffered saline (TBS, pH 7.4) containing 5% of dry milk.
Nitrocellulose membranes were washed in PBS with 0.05% Tween-20 and placed
for 2 hours in a solution containing antibody (anti-Hsp72 antibody, an inducible
form of Hsp70 protein (clone SPA-812, StressGen Biotechnologies, Canada).
After washing, the nitrocellulose membranes were incubated for 1 hour
with the anti-rabbit IgG biotinylated antibody (Jackson ImmunoResearch,
USA), and peroxidase-conjugated streptavidin (IMTEK, Russia) was finally
added for 1 hour. Loading control was performed using mouse monoclonal
Anti-Human Tubulin beta (US Biological, USA, Swampscott, Massachusetts).
ECL-plus chemiluminescent cocktail (Amersham/GE Healthcare, UK) was
used to develop the immunostaining of blots following the manufacturer’s
instructions by exposing the blot to Kodak film.
NO Production Assay. Macrophages, 106 cells/ml, were cultivated in 24-well
plates (Costar, USA) in DMEM without pH indicator at 37°C in humidified
atmosphere containing 5% CO2. The medium was supplemented with 1 mM
 862 S. M. Lunin et al.

sodium pyruvate, 25 mM HEPES, 2 mM l-glutamine, 3% fetal bovine serum.

Supernatants were collected at 21 h and assayed for NO2. The Griess reagent
containing a mixture of 1% sulphanilamide/0.1% naphthylethylene diamine
dihydrochloride and 2.5% H3PO4 (1:1) was used to estimate NO2 that served as
indicator of NO production. After cultivation, 100 μl of each sample was placed
into a 96-well plate with 100 μl of Griess reagent. Ten minutes later the optical
density was measured on the plate reader at 590 nm.
Statistical Analysis. Differences between all treatment groups were com-
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pared by analysis of variance. Tukey's test for unequal N comparisons was

performed when the overall F-value was statistically significant (p < 0.05).

RESULTS

Blood Plasma Cytokines

To characterize immune status of mice during inflammation, with or with-
For personal use only.

out thymulin pretreatment, the level of interleukins, IL-1β, IL-2, IL-6, IL-10,
tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ) has been measured in
blood plasma from four animal groups, as indicated in Figure 1.
Thymulin injected alone did not significantly affect plasma cytokine levels
except for TNF-α and IL-10, but tendency to lowering the concentrations of

Figure 1: The effect of thymulin injection on cytokine concentrations in blood plasma from
healthy and LPS-treated mice. Each value is average mean ± SD from four mice; the mea-
surements were made for each individual mouse in quadruplicate. Data are expressed in
pg/ml of plasma. *Significantly different from control, p < 0.05. ^Significantly different from
LPS-group, p < 0.05.
 Anti-inflammatory Effect of Thymulin 863

almost all pro-inflammatory cytokines was observed. It is necessary to emphasize

that thymulin induced a statistically significant decrease in the TNF-α concentra-
tion of plasma from healthy mice (p < 0.05). On the contrary, the concentration of
anti-inflammatory cytokine, IL-10, was significantly increased in thymulin-
injected healthy animals (p < 0.05).
Treatment with bacterial toxin provided an expected increase in plasma
concentrations of all cytokines studied, namely IL-1β, IL-2, IL-6, IL-10, TNFα,
and IFNγ. Pretreatment of LPS-injected animals with thymulin restored the
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level of pro-inflammatory cytokines to control values. Interestingly, concentra-

tion of anti-inflammatory cytokine IL-10 remained significantly higher when
compared with controls, but it was significantly lower than plasma IL-10 con-
centration in LPS-injected mice.

Production of Cytokines and Nitric Oxide

It is obvious that accumulation of plasma cytokines during inflammation
denotes the possible changes in cytokine production by immune cells. There-
For personal use only.

fore the production of several cytokines by macrophages and lymphocytes had

been measured in experimental animals. In addition, the production of nitric
oxide (NO) as one of the important inflammation mediator produced by mac-
rophages was determined.
Peritoneal Macrophages. Figure 2 shows that the thymulin injection did
not induce a significant effect on the NO production by peritoneal macrophages.
On the contrary, LPS application considerably increased the production of NO
as compared to control. It should be noted that thymulin injected before LPS did
not modulate the NO production by peritoneal macrophages.
As to cytokines, thymulin injection stimulated production of TNF-α and IL-10
in macrophages from healthy mice. Endotoxin injection provided about 6-fold
increase in TNF-α, and 2-fold increase in IL-10 production by peritoneal mac-
rophages. Interestingly, application of both the thymic hormone and the toxin did
not demonstrate an additive effect. Conversely, when mice received both hormone
and LPS, TNF-α and IL-10 production in the cells reduced to the control levels.
Notably, thymulin added to macrophages in vitro demonstrated similar
changes in TNF-α production. The results of in vitro effect of thymulin on TNF-
α production by isolated peritoneal macrophages measured with or without
endotoxin supplement are shown in Figure 3. Thymulin or low dose of LPS
added separately to cultivated macrophages induced stimulation of TNF-α pro-
duction by the cells in vitro. But thymulin added to LPS-treated macrophages
prevented the increase of the TNF-α production, and moreover, thymulin addi-
tion brought down pro-inflammatory cytokine production up to control values.
Spleen Lymphocytes. Figure 4 shows the production of IL-2, the activator
of T-cells proliferation and differentiation, and interferon-γ, T-lymphocyte-
specific pro-inflammatory cytokine in spleen lymphocytes. Thymulin injection
 864 S. M. Lunin et al.
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For personal use only.

Figure 2: The effect of thymulin on production of TNF-α, IL-10, and nitric oxide by peritoneal
macrophages from healthy and LPS-injected mice. Each value is average mean ± SD from
for mice; the measurements were made for each individual mouse in quadruplicate. Data
are expressed in pg/106 cells for cytokines (A) and mmol/106 cells for NO2 concentration (B).
*Significantly different from control, p < 0.05.
 Anti-inflammatory Effect of Thymulin 865

150 *

125

100
pg/10^6 cells

control
*
75 thymulin
LPS
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50 thymulin+LPS

^
25 *

0
TNF α

Figure 3: The in vitro effects of thymulin on TNF-α production by isolated peritoneal mac-
For personal use only.

rophages cultivated with or without LPS. Cells were pooled from six mice. Each sample in
triplicate was an aliquot from pooled suspension. Measurements were made in nonuple
for each cultivated sample. Data are expressed in pg/106 cells. Each value is average
mean ± SD. *Significantly different from control, p < 0.05. ^Significantly different from LPS-
group, p < 0.05.

250 *

225

200

175 * * *

150 control
pg/10^6 cells

thymulin
125
^ LPS
100 thymulin+LPS
75

50

25

0
IL-2 IFN γ

Figure 4: The effect of thymulin on IFN-γ and IL-2 production by spleen lymphocytes from
healthy and LPS-injected mice. Each value is average mean ± SD from four mice; the measure-
ments were made for each individual mouse in quadruplicate. Data are expressed in pg/106
cells. *Significantly different from control, p < 0.05. ^Significantly different from LPS-group,
p < 0.05.
 866 S. M. Lunin et al.

resulted in increase of IL-2 production (p < 0.05) by splenocytes, and hormone

had no effect on IFN-γ production. LPS treatment raised both cytokines
production. Pretreatment of LPS-treated animals with thymulin resulted in
decrease of IFN-γ production up to control level, and did not affect IL-2
production as compared to LPS-treated group.

Heat-Shock Protein Hsp72 Production

To analyze the defense system in immune cells during inflammation, with
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or without thymulin application, the production of Hsp72, an inducible form of

heat-shock protein of Hsp70 family in splenic lymphocytes was measured in
above four groups of animals.
Figure 5 shows that acute inflammation increased Hsp72 production in
spleen lymphocytes. The thymulin injection alone did not significantly
For personal use only.

Figure 5: The effect of thymulin on Hsp72 production by spleen lymphocytes from healthy and
LPS-injected mice. Western blot analysis of extracts from isolated mice lymphocytes, using
anti-Hsp72 antibody (top) or anti-tubulin antibody (bottom). Marker is recombinant Hsp72
protein (StressGen, Canada). Results are representative for four independent experiments.
The order of stains corresponds to the columns of histograms. Histograms calculated as rela-
tive units correspondingly to internal control are the results of Hsp72 blots densitometry by pro-
gram QAPA from four independent experiments. *Significantly different from control, p < 0.05.
^Significantly different from LPS-group, p < 0.05.
 Anti-inflammatory Effect of Thymulin 867

change the production of Hsp72 in healthy mice, but pretreatment with

thymulin of LPS-injected mice resulted in significant decrease of Hsp72
production by spleen lymphocytes when compared to mice treated with
LPS only.

DISCUSSION
The present study was aimed to characterize the immune system regulation
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by thymic hormone thymulin using animal inflammation model. Our findings

indicate that thymulin itself has no considerable effect on the cytokine profile
of healthy animals. This is agreed with the fact that the hormone is non-toxic
in very large dose range (Reggiani et al., 2006), and it has no marked effect on
animal physiological status.
To study the protective effect of thymulin during inflammation, we used a
well-known animal model with inflammation induced by lipopolysaccharide
from gram-negative bacteria. LPS induces a potent immune cell response
For personal use only.

resulting in release of cytokines and a number of other bioactive molecules.

We observed an inflammatory response resulted in a release of pro-inflammatory
cytokines IL-1β, IL-2, IL-6, and TNF-α. Besides, the LPS treatment increased
the production of anti-inflammatory cytokine IL-10. In fact, the plasma con-
centrations of these cytokines are important characteristic of inflammation
process (Jin et al., 1997; Victor et al., 2002).
We had shown that pretreatment with thymulin inhibited the release of
inflammatory mediators and prevented the overexpression of heat shock pro-
tein Hsp70. These findings indicate that thymulin decreases overall inflam-
mation response in this animal inflammation model. These results are
coincided with the recently received data, which proved that thymulin
reduced the level of TNFα, IL-1β and IL-6 in the skin and liver of endotoxine-
treated rats, and that thymulin decreased hyperalgesia induced by endotoxine
(Safieh-Garabedian et al., 2002). In addition, we tried to elucidate possible
targets of thymulin activity in inflammation. We found that thymulin
decreased the production of interferon-γ, which was increased during inflam-
mation. This effect indicates that T-lymphocytes may be one of target cell pop-
ulations for the thymulin.
This assumption is not contrary to well known data, which demonstrates
that thymus gland regulates the activity of peripheral T-lymphocytes by stim-
ulation, for example, cells proliferation. In addition, T-cells produce interleu-
kin-2, which stimulates T-cells proliferation. According to our findings, IL-2
production was increased in thymulin-treated, LPS-treated, and LPS/thymu-
lin-treated mice. Despite of increased production of this cytokine, we had not
observed the accumulation of IL-2 in the blood from thymulin injected mice,
but IL-2 concentration increased in LPS-treated mice. These findings suggest
that thymulin controls the IL-2 metabolism in lymphocytes. Indeed, previous
 868 S. M. Lunin et al.

data showed that thymulin changed the number of IL-2 receptors suggesting
the multicomponent relationships between thymulin and interleukin-2 metab-
olism (Chandratilleke and Marsh, 2000).
When studying the effects of thymulin on peritoneal macrophages activity,
one should take into account theirs close interaction with lymphocytes. So, the
effect of hormone on macrophages may be direct and/or lymphocyte-mediated.
Macrophages are major producers of nitric oxide, which is one of the most impor-
tant inflammation mediators. The NO level is markedly increased during stress.
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In our study, the lack of thymulin influence on NO production by macrophages

both in normal animals and in LPS-treated animals could be due to the fact that
macrophages are not primary target for the thymulin. Nevertheless, thymulin
appeared to modulate the production of TNF-α by macrophages. We examined
the in vitro effect of thymulin on isolated macrophages, and our results showed
the close agreement between in vivo and in vitro effect of hormone.
Regardless of the increase in macrophages TNF-α production by LPS
applied in vivo, we have not observed its accumulation in blood plasma. These
For personal use only.

results suggest rapid catabolism of TNF-α in inflammation- bearing animals.

The thymulin also modulated the production of macrophage anti-inflamma-
tory cytokine, IL-10. The IL-10 production was increased in thymulin-treated
and decreased in thymulin/LPS-treated mice. This result also suggests that
thymulin prevents inflammatory response in macrophages.
Another member of cellular defense system, heat-shock protein Hsp70, acts
as molecular chaperone in protein folding, transport and degradation. Members
of this protein family, including Hsp72, prevent and/or repair massive protein
damages. The findings of present study, as well as our previous report
(Novoselova et al., 2006) demonstrate that the hyperactivation of immune cells by
bacterial toxin is accompanied by increase of Hsp70 production. Being non-toxic
hormone, thymulin had no significant effects on the production of Hsp70 in lym-
phocytes from healthy mice. But applied to inflammation-bearing animals,
thymulin induced significant decrease in Hsp70 expression. The decrease in
Hsp70 production indicates that thymulin reduces stress response to endotoxin,
and this finding is confirmed by lowering the pro-inflammatory cytokine concen-
tration in plasma from thymulin/LPS-treated mice.
The present results have demonstrated that thymulin is a potent anti-
inflammatory mediator, and further investigations in this area can bring clin-
ically important results, particularly, in view of non-toxicity of this modulator.

ACKNOWLEDGMENTS
This work was supported by the President Foundation, projects NSH-
2741.2008.4 and by Russian Academy of Sciences Program “Fundamental
Sciences–for Medicine”.
 Anti-inflammatory Effect of Thymulin 869

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