The aim of this study was to assess the distribution and apoptosis of the most prevalent lymphocytes' subpopulations in the blood, and in addition to evaluate *H. pylori*-induced lymphocyte apoptosis, proliferation, and cytokine synthesis pattern (IL-12p40, IFN-gamma, TNF-alpha, IL-6 and IL-10) in *H. pylori*-infected and noninfected children with gastritis with the goal of comparing the results with those obtained from the control group formed by

The study was undertaken according to Helsinki declaration with approval from the ethics committee of Collegium Medicum Nicolaus Copernicus University in Bydgoszcz, Poland. Informed consent was obtained from all the parents of patients and patients older than 16

A total of 136 consecutive subjects older than 8 years of age with dyspeptic symptoms and residing in the Kuivia-Pomeranin district of Poland were included in this study. Exclusion criteria included: 1) previous diagnosis of other inflammatory disease, such as celiac disease, inflammatory bowel disease or allergy; 2) gastric perforation or hemorrhage, history of surgery, bleeding disorders, or evidence of other clinical conditions or intestinal

Each subject underwent an endoscopic examination of the upper part of the gastrointestinal tract as a result of reporting permanent abdominal pain. Three antral biopsies were taken

*H. pylori*-induced lymphocyte apoptosis were also tested.

dyspeptic noninfected patients without gastritis.

**2. Materials and methods**

**2.1 Patients**

years of age.

parasites.

*H. pylori* strain 25A *cagA*-positive (*H. pylori cagA*+) was obtained from the Department of Microbiology and Clinical Immunology, Children's Memorial Hospital, Warsaw, Poland. Bacteria were washed twice with PBS pH 7.2 and adjusted to a density of 21 x 108 cells ml-1 in PBS.

#### **2.3 Isolation of cells**

At the time of endoscopy 10 ml of venous blood was obtained for immunologic testing from each patient. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Isopaque-Ficoll (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) gradient centrifugation according to the procedure laid down by the manufacturer. Isolated PBMCs were resuspended in a PBS or culture medium made up of RPMI 1640 (Gibco, Paisley, UK) supplemented with 5% heat-inactivated human ABRh+ serum and gentamicin (40 μg ml-1).

#### **2.4 Phenotyping of cell surface antigens**

Freshly isolated PBMCs were stained with fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated mouse anti-human monoclonal antibodies (BD Biosceinces,

Lymphocyte Apoptosis, Proliferation and Cytokine

**2.8 Cytokine assay in PBMC culture**

level of significance was set at *p* < 0.05.

Antral mucosa

PMN cells

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; MN: mononuclear cells;

according to the update Sydney system: 0, none; 1, mild; 2, moderate; 3, marked)

0 (0-2) 0 (0-1)

0

MN cells (intensity)

2 (1-3) 2 (0-3)

0

PMN: polymorphonuclear cells.

**3.1 Gastric mucosa histology**

**2.9 Statistical analyses**

**3. Results**

mucosa.

Children

Control group

*Hp*+ *Hp*-

Synthesis Pattern in Children with *Helicobacter pylori* Infection 177

is given as the stimulation index [SI], calculated by dividing the cpm obtained after

Cytokine concentration (IL-12p40, IFN-gamma, TNF-alpha, IL-6 and IL-10) in cell culture supernatants was determined after 72 hours of *H. pylori cagA*+ or Con A stimulation of PBMCs (3 x 106 ml-1) using ELISA (Opt-EIA system, BD Biosciences, San Diego CA, USA), according to the manufacturer's procedure. Dose-response experiments performed for each cytokine indicated that the maximal secretion was obtained with 6.25 x 106 bacteria ml-1, corresponding to a ratio of 2:1 (bacteria to PBMC) and 12.5 μg ml-1 of Con A that also induced the optimal proliferative response. For analysis of spontaneous cytokine secretion

Data was expressed as means with standard deviation [SD] or medians with 95% confidence intervals (CI). The results were compared using the paired Student's *t* test or the Mann-Whitney's *U*-test as appropriate, depending on the normality of the data distribution, with STATISTICA for Windows release 5.0 (StatSoft, Tulusa, OK, USA) For normal distribution, variables were analyzed by the Kolmogorov-Smirnov test with Lilieforse correction. Statistical significance was considered as *p* < 0.05. The gastric inflammation was correlated with the percentage of positive cells using Spearman's coefficient of rank correlation. The

The intensity and activity of antral gastritis were greater in the *H. pylori*-infected children (*Hp*+) as compared to children with gastritis where *H. pylori* infection was excluded (*Hp-*) (*p* < 0.05), (Table 1). Neither atrophy nor intestinal metaplasia were seen in children's gastric

(activity) Atrophy Intestinal

0 0 0

0 0 0

Table 1. Gastric mucosa histology of *Hp*+ and *Hp-* children, and control group (median score

metaplasia *H. pylori*

1 (0-3)

0 0

stimulation by the cpm in corresponding cultures without a stimulator.

the cells were cultured alone in complete medium for 72 hours.

New York, USA), specific for cell-surface markers with the following combinations: CD3/CD4, CD3/CD8, CD4/CD45RA, CD4/CD45RO, CD8/CD45RA, CD8/CD45RO. Simultest LeucoGate, (CD45-FITC/CD14-PE), and isotype control (IgG1-FITC/IgG2-PE) antibodies (BD Biosceinces, San Diego, CA, USA) were included for each staining panel. Fluorescent staining was performed as described (Helmin-Basa et al., 2011). Briefly, 1 x 106 PBMCs in PBS were incubated with the indicated monoclonal antibodies for 20 min at RT in the dark. After one washing in PBS cell samples were submitted to the flow cytometric method for quantification of apoptosis using 7-Amino-Actinomycin D staining.

#### **2.5 7-amino-actinomycin D (7-AAD) staining**

After cell surface labeling, samples (1x106 cells) were incubated for 5-10 min at RT in the dark with 5 μl (0.25 μg) 7-AAD (BD Biosciences, San Diego, CA, USA). Then the samples (20 000 events per sample) were acquired on a FACScan flow cytometry (BD Biosciences, San Diego, CA, USA) equipped with a single 488 nm Argon laser, recorded in list mode and registered on logarithmic scales. 7-AAD emission was detected in the FL-3 channel (>650 nm). Analysis was performed with a Macintosh computer with the BD CellQuest Software (BD Biosciences).

#### **2.6 Annexin V and propidum iodide staining in PBMC culture**

After 72 hours culture of PBMCs (3 x 106 ml-1) with *H. pylori cagA*+ (6.25 x 106 bacteria ml-1), tetanus toxoid (TT, 5 UI ml-1, Sigma-Aldrich) or without the stimulator, the percentage of live and dead lymphocytes were evaluated by flow cytometry (FACScan, BD Biosciences), using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego CA, USA). Briefly, 5 μl of Annexin V-FITC was added to 1 x 105 cells ml-1, incubated for 20 minutes at RT in the dark, washed, and the cells were resuspended in 190 μl of binding buffer. Then 10 μl of propidium iodide was added 5-10 min before analysis by FACScan flow cytometry. The lymphocytes were gated and the live, apoptotic and dead cells were displayed on an FL-1 versus FL-3 fluorescence dot plot. Annexin V-/PI- cells represent live cells, the Annexin V+/PI- cells represent early apoptotic cells, the Annexin V+/PI+ cells are late apoptotic cells, and the Annexin V-/PI+ represent dead cells.

#### **2.7 Proliferation assay in PBMC culture**

The assay was made according to the procedure described previously (Gackowska et al., 2006, Michalkiewicz et al., 2003). Briefly, freshly isolated PBMCs were diluted in culture medium to a final concentration of 3 x 106 ml-1 and transferred in a volume of 100 μl (3 x 105) to flat-bottomed 96–well microplates (Costar, Cambridge, UK). Subsequently, 100 μl of culture medium alone (control) or containing live *H. pylori cells cagA*+ (6.25 x 106 cells ml-1) or 20 μl of Concanavalin A (Con A 12.5 μg ml-1, Sigma-Aldrich) or tetanus toxoid (TT 5 UI ml-1, Sigma-Aldrich) was added. The cells were cultured in triplicates for seven (stimulation with *H. pylori cagA*+ and TT) or three (stimulation with Con A) days. Lymphocyte proliferation was assessed by pulsing the cells with 1 μCi 3H thymidine (Amersham, Little Chalfont, UK) for the last 16 hours of the incubation period. The cultures were then harvested onto glass filter strips using an automated multisample harvester (Skatron, Lier, Norway) and analysed for 3H-thymidine incorporation by liquid scintillation counting. Data is given as the stimulation index [SI], calculated by dividing the cpm obtained after stimulation by the cpm in corresponding cultures without a stimulator.

#### **2.8 Cytokine assay in PBMC culture**

Cytokine concentration (IL-12p40, IFN-gamma, TNF-alpha, IL-6 and IL-10) in cell culture supernatants was determined after 72 hours of *H. pylori cagA*+ or Con A stimulation of PBMCs (3 x 106 ml-1) using ELISA (Opt-EIA system, BD Biosciences, San Diego CA, USA), according to the manufacturer's procedure. Dose-response experiments performed for each cytokine indicated that the maximal secretion was obtained with 6.25 x 106 bacteria ml-1, corresponding to a ratio of 2:1 (bacteria to PBMC) and 12.5 μg ml-1 of Con A that also induced the optimal proliferative response. For analysis of spontaneous cytokine secretion the cells were cultured alone in complete medium for 72 hours.

#### **2.9 Statistical analyses**

Data was expressed as means with standard deviation [SD] or medians with 95% confidence intervals (CI). The results were compared using the paired Student's *t* test or the Mann-Whitney's *U*-test as appropriate, depending on the normality of the data distribution, with STATISTICA for Windows release 5.0 (StatSoft, Tulusa, OK, USA) For normal distribution, variables were analyzed by the Kolmogorov-Smirnov test with Lilieforse correction. Statistical significance was considered as *p* < 0.05. The gastric inflammation was correlated with the percentage of positive cells using Spearman's coefficient of rank correlation. The level of significance was set at *p* < 0.05.

#### **3. Results**

176 Clinical Flow Cytometry – Emerging Applications

New York, USA), specific for cell-surface markers with the following combinations: CD3/CD4, CD3/CD8, CD4/CD45RA, CD4/CD45RO, CD8/CD45RA, CD8/CD45RO. Simultest LeucoGate, (CD45-FITC/CD14-PE), and isotype control (IgG1-FITC/IgG2-PE) antibodies (BD Biosceinces, San Diego, CA, USA) were included for each staining panel. Fluorescent staining was performed as described (Helmin-Basa et al., 2011). Briefly, 1 x 106 PBMCs in PBS were incubated with the indicated monoclonal antibodies for 20 min at RT in the dark. After one washing in PBS cell samples were submitted to the flow cytometric

After cell surface labeling, samples (1x106 cells) were incubated for 5-10 min at RT in the dark with 5 μl (0.25 μg) 7-AAD (BD Biosciences, San Diego, CA, USA). Then the samples (20 000 events per sample) were acquired on a FACScan flow cytometry (BD Biosciences, San Diego, CA, USA) equipped with a single 488 nm Argon laser, recorded in list mode and registered on logarithmic scales. 7-AAD emission was detected in the FL-3 channel (>650 nm). Analysis was performed with a Macintosh computer with the BD CellQuest Software

After 72 hours culture of PBMCs (3 x 106 ml-1) with *H. pylori cagA*+ (6.25 x 106 bacteria ml-1), tetanus toxoid (TT, 5 UI ml-1, Sigma-Aldrich) or without the stimulator, the percentage of live and dead lymphocytes were evaluated by flow cytometry (FACScan, BD Biosciences), using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego CA, USA). Briefly, 5 μl of Annexin V-FITC was added to 1 x 105 cells ml-1, incubated for 20 minutes at RT in the dark, washed, and the cells were resuspended in 190 μl of binding buffer. Then 10 μl of propidium iodide was added 5-10 min before analysis by FACScan flow cytometry. The lymphocytes were gated and the live, apoptotic and dead cells were displayed on an FL-1 versus FL-3 fluorescence dot plot. Annexin V-/PI- cells represent live cells, the Annexin V+/PI- cells represent early apoptotic cells, the Annexin V+/PI+ cells are late apoptotic cells,

The assay was made according to the procedure described previously (Gackowska et al., 2006, Michalkiewicz et al., 2003). Briefly, freshly isolated PBMCs were diluted in culture medium to a final concentration of 3 x 106 ml-1 and transferred in a volume of 100 μl (3 x 105) to flat-bottomed 96–well microplates (Costar, Cambridge, UK). Subsequently, 100 μl of culture medium alone (control) or containing live *H. pylori cells cagA*+ (6.25 x 106 cells ml-1) or 20 μl of Concanavalin A (Con A 12.5 μg ml-1, Sigma-Aldrich) or tetanus toxoid (TT 5 UI ml-1, Sigma-Aldrich) was added. The cells were cultured in triplicates for seven (stimulation with *H. pylori cagA*+ and TT) or three (stimulation with Con A) days. Lymphocyte proliferation was assessed by pulsing the cells with 1 μCi 3H thymidine (Amersham, Little Chalfont, UK) for the last 16 hours of the incubation period. The cultures were then harvested onto glass filter strips using an automated multisample harvester (Skatron, Lier, Norway) and analysed for 3H-thymidine incorporation by liquid scintillation counting. Data

method for quantification of apoptosis using 7-Amino-Actinomycin D staining.

**2.6 Annexin V and propidum iodide staining in PBMC culture**

/PI+ represent dead cells.

**2.7 Proliferation assay in PBMC culture**

**2.5 7-amino-actinomycin D (7-AAD) staining**

(BD Biosciences).

and the Annexin V-

#### **3.1 Gastric mucosa histology**

The intensity and activity of antral gastritis were greater in the *H. pylori*-infected children (*Hp*+) as compared to children with gastritis where *H. pylori* infection was excluded (*Hp-*) (*p* < 0.05), (Table 1). Neither atrophy nor intestinal metaplasia were seen in children's gastric mucosa.

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; MN: mononuclear cells; PMN: polymorphonuclear cells.

Table 1. Gastric mucosa histology of *Hp*+ and *Hp-* children, and control group (median score according to the update Sydney system: 0, none; 1, mild; 2, moderate; 3, marked)

Lymphocyte Apoptosis, Proliferation and Cytokine

significant differences between the groups.

and controls).

0%

medium

(a*p* < 0.05), *versus* controls (b*p* < 0.05).

control group

Hp cagA+

TT

b b b b b

a

b b

a

medium

Hp cagA+

b

b b b

Hp+ children Hp- children Control group

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; *Hp*, *H. pylori*; TT, tetanus toxoid. Results represent median values obtain from n independent experiments: *Hp*+ children and controls: n = 6; *Hp*- children: n = 5. Statistically significant differences (Mann-Whitney's *U*-test): *versus Hp*- children

Fig. 1. The apoptosis of lymphocytes in PBMC culture in *Hp*+ and *Hp*- children and the

TT

medium

Hp cagA+

TT

dead cells

late apoptotic cells early apoptotic cells live cells

20%

40%

Percentage of cells [%]

60%

80%

a

100%

**3.4 The apoptosis of lymphocytes in** *H. pylori***-induced PBMCs**

Synthesis Pattern in Children with *Helicobacter pylori* Infection 179

*Hp*- (*p* = 0.01 and *p* < 0.01) children as compared with controls, but the percentages of apoptotic CD4+ and CD8+ T cells were higher in the former group (*p* < 0.01 and *p* = 0.02) (Table 3). Moreover, we found that the naive CD4+ and CD8+ T-cell subsets (CD45RA+) and memory CD4+ T-cell subset (CD45RO+) showed an increased apoptosis in both *Hp*+ (*p* = 0.02, *p*  < 0.01 and *p* = 0.01) and *Hp*- (*p* < 0.01, *p* = 0.02 and *p* < 0.01) children, but with no statistically

The level of lymphocyte early apoptosis was unchanged in *H. pylori*- and TT-stimulated culture of PBMCs as compared with medium in both the *Hp*+ and *Hp*- children. However, *Hp*+ children had a higher level of spontaneous and TT-induced early apoptosis of lymphocytes than the *Hp*- ones (*p* = 0.02 and *p* < 0.01) (Fig. 1). Lymphocyte late apoptosis in *H. pylori*- and TT-induced PBMCs was equally high in both *Hp*+ and *Hp*- children (*Hp*+: *p* = 0.01 and *p* = 0.09; *Hp*-: *p* = 0.01 and *p* = 0.01) when compared to the controls. Moreover, both *Hp*+ and *Hp*- children had a low proportion of live lymphocytes in PBMC culture with *H. pylori* and TT (*Hp*+: *p* = 0.05 and *p* < 0.01; *Hp*-: *p* = 0.01 and *p =* 0.02), with no significant differences between the groups. However, only *Hp*- children have high percentage of dead (necrotic) cells in culture PBMC especially after TT stimulation (*p* < 0.01 *versus Hp*+ children

#### **3.2 The distribution of T-cell subsets in peripheral blood**

There was an equal percentage of CD4+ and CD8+ T cell subsets in peripheral blood in the *Hp*+ and *Hp*- children and the control group (Table 2). However, the naive CD8+ T-cell subset (CD45RA+) was higher in the *Hp*+ children when compared to the control group *(p* < 0.01). In *Hp*- children, the proportion of memory CD8+ T-cell subset (CD45RO+) was increased (*p* = 0.01, *versus* control group). However, there was no correlation between the percentage of naive and memory T-cell subsets and the gastric inflammation scores in *Hp*+ and *Hp*- children.

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; SD, standard deviation. Statistically significant differences (Student's *t* test): *versus* controls (a*p* < 0.05).

Table 2. The distribution of T-cell subsets in peripheral blood of *Hp*+ and *Hp*- children and the control group

#### **3.3 Apoptosis of T-cell subsets in peripheral blood**

Flow cytometric quantification of apoptotic T cell subsets in freshly isolated PBMCs showed significantly more apoptotic CD4+ and CD8+ T cells in both *Hp*+ (*p* < 0.01 and *p* < 0.01) and

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; SD, standard deviation. Statistically significant differences (Student's *t* test): *versus* controls (a*p* < 0.05), *versus Hp*- children (b*p* < 0.05).

Table 3. Apoptosis in T-cell subsets in peripheral blood of *Hp*+ and *Hp*- children and the control group

There was an equal percentage of CD4+ and CD8+ T cell subsets in peripheral blood in the *Hp*+ and *Hp*- children and the control group (Table 2). However, the naive CD8+ T-cell subset (CD45RA+) was higher in the *Hp*+ children when compared to the control group *(p* < 0.01). In *Hp*- children, the proportion of memory CD8+ T-cell subset (CD45RO+) was increased (*p* = 0.01, *versus* control group). However, there was no correlation between the percentage of naive and memory T-cell subsets and the gastric inflammation scores in *Hp*+

> *Hp*- children (n = 15)

36.96±6.76 27.57±6.61 26.31±6.15 16.37±0.58 29.72±7.17 9.91±0.52a

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; SD, standard deviation. Statistically

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; SD, standard deviation. Statistically significant differences (Student's *t* test): *versus* controls (a*p* < 0.05), *versus Hp*- children (b*p* < 0.05). Table 3. Apoptosis in T-cell subsets in peripheral blood of *Hp*+ and *Hp*- children and the

Table 2. The distribution of T-cell subsets in peripheral blood of *Hp*+ and *Hp*- children and

Flow cytometric quantification of apoptotic T cell subsets in freshly isolated PBMCs showed significantly more apoptotic CD4+ and CD8+ T cells in both *Hp*+ (*p* < 0.01 and *p* < 0.01) and

> *Hp*- children (n = 15)

4.50 ± 2.12a 5.01 ± 1.92a 4.62 ± 1.58a 7.37 ± 1.42a 5.45 ± 1.85a 5.76 ± 1.51

Control group (n = 12)

Control group (n = 12)

2.13 ± 2.00 1.48 ± 1.27 0.36 ± 0.07 0.55 ± 0.34 0.68 ± 0.21 4.17 ± 1.55

38.98±5.92 27.04±3.91 22.95±0.65 15.21±4.10 25.24±1.34 6.76±1.79

**3.2 The distribution of T-cell subsets in peripheral blood**

Means ± SD

(n = 13)

significant differences (Student's *t* test): *versus* controls (a*p* < 0.05).

**3.3 Apoptosis of T-cell subsets in peripheral blood**

T lymphocyte subsets *Hp*+ children

Means ± SD

10.48 ± 5.80a,b 8.46 ± 4.34a,b 5.24 ± 2.16a 5.11 ± 1.94a 4.69 ± 0.79a 6.12 ± 2.44

(n = 13)

39.95±4.78 28.89±5.72 29.51±8.62 16.74±6.50 34.95±1.72a 10.44±2.78

T lymphocyte subsets *Hp*+ children

and *Hp*- children.

CD3+CD4+ % CD3+CD8+ % CD4+CD45RA+ % CD4+CD45RO+ % CD8+CD45RA+ % CD8+CD45RO+ %

the control group

CD4+ % CD8+ % CD4+CD45RA+ % CD4+CD45RO+ % CD8+CD45RA+ % CD8+CD45RO+ %

control group

*Hp*- (*p* = 0.01 and *p* < 0.01) children as compared with controls, but the percentages of apoptotic CD4+ and CD8+ T cells were higher in the former group (*p* < 0.01 and *p* = 0.02) (Table 3). Moreover, we found that the naive CD4+ and CD8+ T-cell subsets (CD45RA+) and memory CD4+ T-cell subset (CD45RO+) showed an increased apoptosis in both *Hp*+ (*p* = 0.02, *p*  < 0.01 and *p* = 0.01) and *Hp*- (*p* < 0.01, *p* = 0.02 and *p* < 0.01) children, but with no statistically significant differences between the groups.

#### **3.4 The apoptosis of lymphocytes in** *H. pylori***-induced PBMCs**

The level of lymphocyte early apoptosis was unchanged in *H. pylori*- and TT-stimulated culture of PBMCs as compared with medium in both the *Hp*+ and *Hp*- children. However, *Hp*+ children had a higher level of spontaneous and TT-induced early apoptosis of lymphocytes than the *Hp*- ones (*p* = 0.02 and *p* < 0.01) (Fig. 1). Lymphocyte late apoptosis in *H. pylori*- and TT-induced PBMCs was equally high in both *Hp*+ and *Hp*- children (*Hp*+: *p* = 0.01 and *p* = 0.09; *Hp*-: *p* = 0.01 and *p* = 0.01) when compared to the controls. Moreover, both *Hp*+ and *Hp*- children had a low proportion of live lymphocytes in PBMC culture with *H. pylori* and TT (*Hp*+: *p* = 0.05 and *p* < 0.01; *Hp*-: *p* = 0.01 and *p =* 0.02), with no significant differences between the groups. However, only *Hp*- children have high percentage of dead (necrotic) cells in culture PBMC especially after TT stimulation (*p* < 0.01 *versus Hp*+ children and controls).

*Hp+*, *H. pylori*-infected children; *Hp-*, noninfected children; *Hp*, *H. pylori*; TT, tetanus toxoid. Results represent median values obtain from n independent experiments: *Hp*+ children and controls: n = 6; *Hp*- children: n = 5. Statistically significant differences (Mann-Whitney's *U*-test): *versus Hp*- children (a*p* < 0.05), *versus* controls (b*p* < 0.05).

Fig. 1. The apoptosis of lymphocytes in PBMC culture in *Hp*+ and *Hp*- children and the control group

Lymphocyte Apoptosis, Proliferation and Cytokine

and the control group

and the control group

Concentration of IFN-gamma [pg ml-1

]

Concentration of IL-12p40 [pg ml-1]

Synthesis Pattern in Children with *Helicobacter pylori* Infection 181

Hp cagA+ Con A

Hp+ children Hp- children Control group

Hp cagA+ Con A

Hp+ children Hp- children Control group

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp+, H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ children: n = 27; *Hp*- children: n = 27; controls: n = 9. Statistically significant differences (Mann-Whitney's *U*-test):

Fig. 3. Concentration of IFN-gamma in culture supernatants of PBMC in *Hp*+ and *Hp*- children

*versus* Con A stimulation. (b*p* < 0.05), *versus Hp-* children (c*p* < 0.05), *versus* controls (d*p* < 0.05).

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp+, H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent medians obtained from n independent experiments: *Hp*+ children: n = 29; *Hp*- children: n = 23; controls: n = 9. Statistically significant differences (Mann-Whitney's *U*-test): *versus*

Fig. 2. Concentration of IL-12p40 in culture supernatants of PBMC in *Hp*+ and *Hp*- children

b

b,d c,d

Con A stimulation. (b*p* < 0.05), *versus Hp-* children (c*p* < 0.05), *versus* controls (d*p* < 0.05).

<sup>b</sup> b,d

#### **3.5 The proliferative response in** *H. pylori***-induced PBMCs**

The changes in the mode of lymphocyte proliferation involved: a) lower lymphocyte response to *H. pylori* in the *Hp*+ children than in the *Hp*- ones (*p* = 0.03) and control group (*p* < 0.01); b) increased response to Con A in the *Hp*+ as compared to the *Hp*- children (*p* = 0.02) and control group (*p* = 0.04). However, lymphocytes of *Hp*+ and *Hp*- children and control group did not differ in their response to TT (Table 4).

*Hp*+, *H. pylori*-infected children; *Hp*-, noninfected children; CI, confidence intervals. Statistically significant differences (Mann-Whitney's *U*-test): *versus* controls (*ap* < 0.05), *versus Hp*- children (*bp* < 0.05).

Table 4. PBMC proliferative response in *Hp*+ and *Hp*- children and the control group

#### **3.6 The pattern of cytokine expression in** *H. pylori***-induced PBMCs**

The levels of *H. pylori*- or Con A-induced cytokines did not exceed the control values in both *Hp*+ and *Hp*- children. However, the profile of cytokine synthesis showed a different pattern: 1) IL-12p40 synthesis (Fig. 2): *H. pylori* was a poor IL-12p40 inducer both in the *Hp*+ and *Hp*- children (*p* < 0.01). However, in the *Hp*+ children production of IL-12p40 was higher than in the *Hp*- ones (*p* = 0.02). In contrast, Con A stimulation gave rise to a similar IL-12p40 level in both groups of children. Finally, the IL-12p40 synthesis profile in the *Hp*children was similar to that of the control group with a better response to *H. pylori* than to Con A (*p* = 0.04 and *p* < 0.01). 2) IFN-gamma synthesis (Fig. 3): *H. pylori-*induced IFNgamma synthesis remained unchanged in the *Hp*+ children, but it was slightly lower in the *Hp*- ones (*p* = 0.01) as compared with the control group. Con A was a significantly better IFN-gamma inducer than *H. pylori* in both *Hp*+ and *Hp*- children with no significant differences between the two groups. 3) TNF-alpha synthesis (Fig. 4): In the *Hp*- children, *H. pylori* and Con A-induced TNF-alpha levels were lower than in the control group (*p* < 0.01 and *p* = 0.01). However, Con A was a stronger inducer of TNF-alpha in the *Hp*+ children than in the *Hp*- ones (*p* = 0.02). 4) IL-6 synthesis (Fig. 5): *H. pylori* was a poorer IL-6 inducer in the *Hp*+ children than in the *Hp*- ones (*p* = 0.03) and control group (*p* = 0.03); this tendency was maintained with Con A being a worse stimulator of IL-6 in the *Hp*+ than in the *Hp*- children (*p* < 0.01). 5) IL-10 synthesis (Fig. 6): *H. pylori* was a better IL-10 inducer in the *Hp*+ than in *Hp*- children (*p* = 0.03). However, in both the *Hp*- children and control group Con A was a better IL-10 inducer than *H. pylori* (*p* = 0.05).

The changes in the mode of lymphocyte proliferation involved: a) lower lymphocyte response to *H. pylori* in the *Hp*+ children than in the *Hp*- ones (*p* = 0.03) and control group (*p* < 0.01); b) increased response to Con A in the *Hp*+ as compared to the *Hp*- children (*p* = 0.02) and control group (*p* = 0.04). However, lymphocytes of *Hp*+ and *Hp*- children and

> *Hp*- children (n = 59, 21 or 13)

[3.50/45.68]

[3.06/25.68]

[111.44/199.25]

6.29

169.12

4.27

*Hp*+, *H. pylori*-infected children; *Hp*-, noninfected children; CI, confidence intervals. Statistically significant differences (Mann-Whitney's *U*-test): *versus* controls (*ap* < 0.05), *versus Hp*- children (*bp* < 0.05).

Table 4. PBMC proliferative response in *Hp*+ and *Hp*- children and the control group

The levels of *H. pylori*- or Con A-induced cytokines did not exceed the control values in both *Hp*+ and *Hp*- children. However, the profile of cytokine synthesis showed a different pattern: 1) IL-12p40 synthesis (Fig. 2): *H. pylori* was a poor IL-12p40 inducer both in the *Hp*+ and *Hp*- children (*p* < 0.01). However, in the *Hp*+ children production of IL-12p40 was higher than in the *Hp*- ones (*p* = 0.02). In contrast, Con A stimulation gave rise to a similar IL-12p40 level in both groups of children. Finally, the IL-12p40 synthesis profile in the *Hp*children was similar to that of the control group with a better response to *H. pylori* than to Con A (*p* = 0.04 and *p* < 0.01). 2) IFN-gamma synthesis (Fig. 3): *H. pylori-*induced IFNgamma synthesis remained unchanged in the *Hp*+ children, but it was slightly lower in the *Hp*- ones (*p* = 0.01) as compared with the control group. Con A was a significantly better IFN-gamma inducer than *H. pylori* in both *Hp*+ and *Hp*- children with no significant differences between the two groups. 3) TNF-alpha synthesis (Fig. 4): In the *Hp*- children, *H. pylori* and Con A-induced TNF-alpha levels were lower than in the control group (*p* < 0.01 and *p* = 0.01). However, Con A was a stronger inducer of TNF-alpha in the *Hp*+ children than in the *Hp*- ones (*p* = 0.02). 4) IL-6 synthesis (Fig. 5): *H. pylori* was a poorer IL-6 inducer in the *Hp*+ children than in the *Hp*- ones (*p* = 0.03) and control group (*p* = 0.03); this tendency was maintained with Con A being a worse stimulator of IL-6 in the *Hp*+ than in the *Hp*- children (*p* < 0.01). 5) IL-10 synthesis (Fig. 6): *H. pylori* was a better IL-10 inducer in the *Hp*+ than in *Hp*- children (*p* = 0.03). However, in both the *Hp*- children and control group

**3.6 The pattern of cytokine expression in** *H. pylori***-induced PBMCs**

Control group (n = 13)

[90.60/181.82]

10.48 [8.26/24.16]

121.52

2.44 [0.71/5.01]

**3.5 The proliferative response in** *H. pylori***-induced PBMCs**

control group did not differ in their response to TT (Table 4).

Medians [CI]

*Hp*+ children (n = 46, 14 or 8)

[190.63/513.91]

[0.45/18.02]

Con A was a better IL-10 inducer than *H. pylori* (*p* = 0.05).

3.28a,b [1.55/26.09] 312.12a,b

7.77

Type of stimulator

*H. pylori cagA*+

Concanavalin A

Tetanus toxoid

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp+, H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent medians obtained from n independent experiments: *Hp*+ children: n = 29; *Hp*- children: n = 23; controls: n = 9.

Fig. 2. Concentration of IL-12p40 in culture supernatants of PBMC in *Hp*+ and *Hp*- children and the control group

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp+, H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ children: n = 27; *Hp*- children: n = 27; controls: n = 9.

Fig. 3. Concentration of IFN-gamma in culture supernatants of PBMC in *Hp*+ and *Hp*- children and the control group

Lymphocyte Apoptosis, Proliferation and Cytokine

0

200

the control group

**4.  Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ and *Hp*children: n = 23; controls: n = 8. Statistically significant differences (Mann-Whitney's *U*-test): *versus* Con

Fig. 6. Concentration of IL-10 in culture supernatants of PBMC in *Hp*+ and *Hp*- children and

In the present study *H. pylori*-infected and noninfected children with gastritis and the control group were compared with respect to 1) the distribution and apoptosis of T lymphocyte subsets in the blood, and 2) lymphocyte apoptosis, proliferation and cytokine synthesis pattern in the culture PBMCs. Additionally, the lymphocyte phenotypes of

We report that the group of *H. pylori*-infected children have: 1) an elevated proportion of naive CD8*+* (CD45RA+) T-cell population along with an increase in the percentage of apoptotic CD4+ and CD8+ T cells in the blood, 2) high spontaneous early lymphocyte apoptosis, low lymphocyte proliferation to *H. pylori* but a high response to Con A, and 3) slightly increased IL-10 expression along with decreased IL-6 expression in *H. pylori*induced PBMC culture. Children with gastritis but without infection had a high percentage of memory CD8+ T-cell subset (CD45RO+) in the blood and a low INF-gamma and TNFalpha in *H. pylori*-induced PBMC culture. Both *H. pylori*-infected and noninfected children with gastritis were characterized by 1) an increased percentage of apoptotic naive and memory CD4+ and CD8+ T-cell subsets in blood, 2) a high percentage of late apoptotic lymphocytes, along with a low proportion of live lymphocytes, and a low IL-12p40

children with gastritis were correlated with gastric inflammation scores.

b

b

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp*+, *H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ children: n = 19; *Hp*-children: n = 22; controls: n = 10. Statistically significant differences (Mann-Whitney's *U*-test): *versus Hp-* children (c*p* < 0.05), *versus* controls (d*p* < 0.05).

Fig. 4. Concentration of TNF-alfa in culture supernatants of PBMC in *Hp*+ and *Hp*- children and the control group

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp*+, *H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ children: n = 21; *Hp* children: n = 12; controls: n = 11. Statistically significant differences (Mann-Whitney's *U*-test): *versus Hp-* children (c*p* < 0.05), *versus* controls (d*p* < 0.05).

Fig. 5. Concentration of IL-6 in culture supernatants of PBMC in *Hp*+ and *Hp*- children and the control group

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp*+, *H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ and *Hp*children: n = 23; controls: n = 8.

Fig. 6. Concentration of IL-10 in culture supernatants of PBMC in *Hp*+ and *Hp*- children and the control group

#### **4.  Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ children: n = 21; *Hp* children: n = 12; controls: n = 11. Statistically significant differences (Mann-Whitney's *U*-test):

Fig. 5. Concentration of IL-6 in culture supernatants of PBMC in *Hp*+ and *Hp*- children and

*Hp*, *H. pylori*; *Hp+*, Con A, Concanavalin A, *Hp*+, *H. pylori*-infected children; *Hp-*, noninfected children. Blue horizontal bars represent median values obtained from n independent experiments: *Hp*+ children: n = 19; *Hp*-children: n = 22; controls: n = 10. Statistically significant differences (Mann-Whitney's *U*-test):

Fig. 4. Concentration of TNF-alfa in culture supernatants of PBMC in *Hp*+ and *Hp*- children

d

d

1600 1400

1200

1000 800 600

Concentration TNF-alfa [pg ml ]-1

c

*versus Hp-* children (c*p* < 0.05), *versus* controls (d*p* < 0.05).

c,d c

*versus Hp-* children (c*p* < 0.05), *versus* controls (d*p* < 0.05).

400

200

0

and the control group

0

200 400

the control group

600 800

1000

Concentration of IL-6 [pg ml

 -1]

1200 1400

1600

In the present study *H. pylori*-infected and noninfected children with gastritis and the control group were compared with respect to 1) the distribution and apoptosis of T lymphocyte subsets in the blood, and 2) lymphocyte apoptosis, proliferation and cytokine synthesis pattern in the culture PBMCs. Additionally, the lymphocyte phenotypes of children with gastritis were correlated with gastric inflammation scores.

We report that the group of *H. pylori*-infected children have: 1) an elevated proportion of naive CD8*+* (CD45RA+) T-cell population along with an increase in the percentage of apoptotic CD4+ and CD8+ T cells in the blood, 2) high spontaneous early lymphocyte apoptosis, low lymphocyte proliferation to *H. pylori* but a high response to Con A, and 3) slightly increased IL-10 expression along with decreased IL-6 expression in *H. pylori*induced PBMC culture. Children with gastritis but without infection had a high percentage of memory CD8+ T-cell subset (CD45RO+) in the blood and a low INF-gamma and TNFalpha in *H. pylori*-induced PBMC culture. Both *H. pylori*-infected and noninfected children with gastritis were characterized by 1) an increased percentage of apoptotic naive and memory CD4+ and CD8+ T-cell subsets in blood, 2) a high percentage of late apoptotic lymphocytes, along with a low proportion of live lymphocytes, and a low IL-12p40 expression in culture PBMCs.

Lymphocyte Apoptosis, Proliferation and Cytokine

of circulating CD4+ and CD8+ T-cell subsets.

(Bontems at al. 2003, Lopes et al. 2005).

apoptosis levels.

Synthesis Pattern in Children with *Helicobacter pylori* Infection 185

We report here that lymphocytes of both *H. pylori-*infected and noninfected children with gastritis proved to be much more sensitive to *H. pylori* bacteria and tetanus toxoid-induced apoptosis than lymphocytes of the controls. These observations indicate that gastritis itself (independent of ongoing *H. pylori*-infection) makes peripheral lymphocytes sensitive to apoptosis. However, the higher early spontaneous lymphocyte apoptosis in *H. pylori*infected children may suggest that ongoing *H. pylori* infection can make the lymphocyte apoptosis rate faster, possibly due to their prolonged contact with *H. pylori in vivo*. Our study of apoptotic lymphocytes in blood confirm that *H. pylori* infection increases apoptosis

In general, our results concerning lymphocyte apoptosis are partly in agreement with other reports suggesting that *H. pylori-*induced apoptosis could be the reason for modest *H. pylori*induced proliferation of T cells in *H. pylori*-infected subjects (Schmees et al., 2007). Some authors suggest that the apoptotic mechanism possibly involves Fas-bearing T cells through induction of FasL expression (Wang et al., 2000, 2001). Also, gastric T cells have been reported to express FasL and undergo apoptosis *in situ* following *H. pylori* infection as has also been shown previously (Galgani et al., 2004). The role of *cag* PAI in induction of T cell death through Fas/FasL interaction has also been strongly suggested on the basis that *cag*  PAI-deficient strains of *H. pylori* were not able to induce apoptosis in T cells (Wang et al., 2001). In line with these results, Galgani e*t al*. (Galgani et al., 2007) have shown that *cagA+* strains of *H. pylori* were highly effective inducers of apoptosis in human monocytes, but not in monocyte-derived dendritic cells. Our results also suggest that at least in the model of *H. pylori*-induced PBMCs *cagA* expression in the stimulating bacteria affected lymphocyte

Low *H. pylori*-induced IL-12p40 production found in the PBMCs of *H. pylori*-infected children may result either from limited number of IL-12 producing cells or from the defects in IL-12 regulation. Gastritis itself also limits the PBMC abilities in terms of IL-12p40 synthesis since *H. pylori*-activated PBMCs of noninfected children with gastritis also showed lower IL-12p40 production than controls; however, the values were higher than those of infected subjects. This observation may indicate that children gastritis itself, regardless of *H. pylori* infection, down-regulates IL-12p40 expression in *H. pylori*-induced PBMCs, and that *H. pylori*-infection makes this process more profound. Gram-negative bacteria have been found to induce rather low IL-12 synthesis in PBMCs but high IL-10 levels (Hessle et al., 1999, 2000). In this model system IL-12 is produced mainly by monocytes and is up-regulated by T cells, since their removal from PBMCs decreases the bacteria-induced IL-12 production (Hessle et al., 1999). Low IL-12p40 levels found in the *H. pylori cagA+*-induced PBMCs of the *H. pylori*infected children and in a lesser degree also in the noninfected children with gastritis may result from two processes. Firstly, a high apoptosis of CD4+ T cells participating in the upregulation of IL-12 production by monocytes, and secondly, hypo-reactivity and/or apoptosis of monocytes directly induced either by *H. pylori* action (Galgani et al. 2004), or indirectly mediated by a relative excess of IL-10 (D'Andrea et al., 1993). Low IL-12 production may play a role in a complex and still poorly understood mechanism of rather limited *H. pylori*-mediated pro-inflammatory responses usually observed in children

We have found that the production of INF-gamma decreased in noninfected children with gastritis but not in *H. pylori*-infected ones. This is in contrast with the IL-12p40 findings, and

An increase in the percentage of CD45RA-bearing CD8+ T-cell subsets in the *H. pylori*infected children may indicate that the infection with *H. pylori* induces the thymic T-cell renewal leading to the generation of T cells with high expression of CD45RA isoform. These results differ from those obtained in another study, also examining blood, which showed an unchanged proportion of circulating naive T cell subset (CD45RA+) in a group of *H. pylori*infected children [Helmin-Basa et al., 2011]. This discrepancy might result from a different number of examined children and the age of the children evaluated in previous study. However, similar changes have been observed in the gastric antral mucosa using quantitative immunohistochemistry [Maciorkowska et al., 2004].

Other differences that we have observed in *H. pylori*-infected children were the increase in the percentage of apoptotic CD4+ and CD8+ T lymphocytes in the blood and spontaneous early apoptosis of lymphocyte in PBMC culture. Similar changes have been observed in previous studies that evaluated the apoptosis of gastric mucosa inflammatory cells showing an increased apoptosis of CD4+ T-cell subset using immunohistochemistry methods [Guarner et al., 2003, Kotłowska-Kmieć et al., 2009].

The noninfected children with gastritis had an elevated proportion of CD45RO-positive CD8+ T-cell subset in the blood. These results are analogous to our previous study that also showed an increase in the percentage of circulating memory T cells with very high expression of CD45RO isoform in a group of noninfected children with gastritis [Helmin-Basa et al., 2011]. This suggests the activation of thymic–generated CD45RA+ cells leading to the transition of the CD45RA isoform into CD45RO.

Additionally, our results have shown that circulating naive (CD45RA+) and memory (CD45RO+) T-cell subsets display high apoptosis in the *H. pylori*-infected and noninfected children with gastritis. CD45RO+ cells may provide pro-inflammatory signals that contribute to the gastric inflammation. Hence, high apoptosis, especially of the memory T cells, can make the gastritis self-limited regardless of *H. pylori* infection.

We also found that the lymphocytes of the *H. pylori*-infected children had reduced proliferation in *H. pylori cagA+*-induced PBMC culture.  Several mechanisms have been proposed as an explanation for this phenomenon, for example the active suppression of T and B lymphocyte proliferation potential, suppression of monocytes activation, inhibitory action of several virulent *H. pylori* products including CagA protein and VacA cytotoxin, and others (Boncristino et al., 2003; Wang et al., 2001). Profound low lymphocyte response to *H. pylori cagA*+ in infected children with high proliferation to antigen specific inducer (tetanus toxoid) and T -cell mitogen (Con A) in both the *H. pylori-*infected and noninfected children with gastritis, as well as in the controls leads to a number of conclusions. First-ofall, a low lymphocyte proliferation in response to *H. pylori* induction selectively found in the *H. pylori-*infected children is highly specific only for *H. pylori*. Secondly, the ongoing *H. pylori* infection creates conditions for *H. pylori* specific low T lymphocyte proliferation. This profile of response is also typical of gastric mucosa T cells in *H. pylori*-infected adults showing unresponsiveness to *H. pylori* antigens (Lungren et al., 2005; Ren et al., 2000) unless costimulatory cytokines are present (Ren et al., 2000).

An increase in the percentage of CD45RA-bearing CD8+ T-cell subsets in the *H. pylori*infected children may indicate that the infection with *H. pylori* induces the thymic T-cell renewal leading to the generation of T cells with high expression of CD45RA isoform. These results differ from those obtained in another study, also examining blood, which showed an unchanged proportion of circulating naive T cell subset (CD45RA+) in a group of *H. pylori*infected children [Helmin-Basa et al., 2011]. This discrepancy might result from a different number of examined children and the age of the children evaluated in previous study. However, similar changes have been observed in the gastric antral mucosa using

Other differences that we have observed in *H. pylori*-infected children were the increase in the percentage of apoptotic CD4+ and CD8+ T lymphocytes in the blood and spontaneous early apoptosis of lymphocyte in PBMC culture. Similar changes have been observed in previous studies that evaluated the apoptosis of gastric mucosa inflammatory cells showing an increased apoptosis of CD4+ T-cell subset using immunohistochemistry methods

The noninfected children with gastritis had an elevated proportion of CD45RO-positive CD8+ T-cell subset in the blood. These results are analogous to our previous study that also showed an increase in the percentage of circulating memory T cells with very high expression of CD45RO isoform in a group of noninfected children with gastritis [Helmin-Basa et al., 2011]. This suggests the activation of thymic–generated CD45RA+ cells leading to

Additionally, our results have shown that circulating naive (CD45RA+) and memory (CD45RO+) T-cell subsets display high apoptosis in the *H. pylori*-infected and noninfected children with gastritis. CD45RO+ cells may provide pro-inflammatory signals that contribute to the gastric inflammation. Hence, high apoptosis, especially of the memory T

We also found that the lymphocytes of the *H. pylori*-infected children had reduced proliferation in *H. pylori cagA+*-induced PBMC culture.  Several mechanisms have been proposed as an explanation for this phenomenon, for example the active suppression of T and B lymphocyte proliferation potential, suppression of monocytes activation, inhibitory action of several virulent *H. pylori* products including CagA protein and VacA cytotoxin, and others (Boncristino et al., 2003; Wang et al., 2001). Profound low lymphocyte response to *H. pylori cagA*+ in infected children with high proliferation to antigen specific inducer (tetanus toxoid) and T -cell mitogen (Con A) in both the *H. pylori-*infected and noninfected children with gastritis, as well as in the controls leads to a number of conclusions. First-ofall, a low lymphocyte proliferation in response to *H. pylori* induction selectively found in the *H. pylori-*infected children is highly specific only for *H. pylori*. Secondly, the ongoing *H. pylori* infection creates conditions for *H. pylori* specific low T lymphocyte proliferation. This profile of response is also typical of gastric mucosa T cells in *H. pylori*-infected adults showing unresponsiveness to *H. pylori* antigens (Lungren et al., 2005; Ren et al., 2000) unless

cells, can make the gastritis self-limited regardless of *H. pylori* infection.

quantitative immunohistochemistry [Maciorkowska et al., 2004].

[Guarner et al., 2003, Kotłowska-Kmieć et al., 2009].

the transition of the CD45RA isoform into CD45RO.

costimulatory cytokines are present (Ren et al., 2000).

We report here that lymphocytes of both *H. pylori-*infected and noninfected children with gastritis proved to be much more sensitive to *H. pylori* bacteria and tetanus toxoid-induced apoptosis than lymphocytes of the controls. These observations indicate that gastritis itself (independent of ongoing *H. pylori*-infection) makes peripheral lymphocytes sensitive to apoptosis. However, the higher early spontaneous lymphocyte apoptosis in *H. pylori*infected children may suggest that ongoing *H. pylori* infection can make the lymphocyte apoptosis rate faster, possibly due to their prolonged contact with *H. pylori in vivo*. Our study of apoptotic lymphocytes in blood confirm that *H. pylori* infection increases apoptosis of circulating CD4+ and CD8+ T-cell subsets.

In general, our results concerning lymphocyte apoptosis are partly in agreement with other reports suggesting that *H. pylori-*induced apoptosis could be the reason for modest *H. pylori*induced proliferation of T cells in *H. pylori*-infected subjects (Schmees et al., 2007). Some authors suggest that the apoptotic mechanism possibly involves Fas-bearing T cells through induction of FasL expression (Wang et al., 2000, 2001). Also, gastric T cells have been reported to express FasL and undergo apoptosis *in situ* following *H. pylori* infection as has also been shown previously (Galgani et al., 2004). The role of *cag* PAI in induction of T cell death through Fas/FasL interaction has also been strongly suggested on the basis that *cag*  PAI-deficient strains of *H. pylori* were not able to induce apoptosis in T cells (Wang et al., 2001). In line with these results, Galgani e*t al*. (Galgani et al., 2007) have shown that *cagA+* strains of *H. pylori* were highly effective inducers of apoptosis in human monocytes, but not in monocyte-derived dendritic cells. Our results also suggest that at least in the model of *H. pylori*-induced PBMCs *cagA* expression in the stimulating bacteria affected lymphocyte apoptosis levels.

Low *H. pylori*-induced IL-12p40 production found in the PBMCs of *H. pylori*-infected children may result either from limited number of IL-12 producing cells or from the defects in IL-12 regulation. Gastritis itself also limits the PBMC abilities in terms of IL-12p40 synthesis since *H. pylori*-activated PBMCs of noninfected children with gastritis also showed lower IL-12p40 production than controls; however, the values were higher than those of infected subjects. This observation may indicate that children gastritis itself, regardless of *H. pylori* infection, down-regulates IL-12p40 expression in *H. pylori*-induced PBMCs, and that *H. pylori*-infection makes this process more profound. Gram-negative bacteria have been found to induce rather low IL-12 synthesis in PBMCs but high IL-10 levels (Hessle et al., 1999, 2000). In this model system IL-12 is produced mainly by monocytes and is up-regulated by T cells, since their removal from PBMCs decreases the bacteria-induced IL-12 production (Hessle et al., 1999). Low IL-12p40 levels found in the *H. pylori cagA+*-induced PBMCs of the *H. pylori*infected children and in a lesser degree also in the noninfected children with gastritis may result from two processes. Firstly, a high apoptosis of CD4+ T cells participating in the upregulation of IL-12 production by monocytes, and secondly, hypo-reactivity and/or apoptosis of monocytes directly induced either by *H. pylori* action (Galgani et al. 2004), or indirectly mediated by a relative excess of IL-10 (D'Andrea et al., 1993). Low IL-12 production may play a role in a complex and still poorly understood mechanism of rather limited *H. pylori*-mediated pro-inflammatory responses usually observed in children (Bontems at al. 2003, Lopes et al. 2005).

We have found that the production of INF-gamma decreased in noninfected children with gastritis but not in *H. pylori*-infected ones. This is in contrast with the IL-12p40 findings, and

Lymphocyte Apoptosis, Proliferation and Cytokine

The study was supported by grant UMK 20/2010.

**6. Acknowledgment**

2004), pp, 387-404

285-292

226

21

**7. References**

Synthesis Pattern in Children with *Helicobacter pylori* Infection 187

protect from more aggressive forms of *H. pylori*-induced inflammation, but on the other

Acheson, D. W.& Luccioli S. (2004). Microbial-gut interactions in health and disease.

Bodger, K., Bromelow, K., Wyatt, J. I. & Heatley, R. V. (2001). Interleukin 10 in

Boncristiano, M., Paccani, S. R., Barone, S., Ulivieri, C., Patrussi, L., Ilver, D., Amedei, A.,

Bontems, P.; Robert F.; Van Gossum A.; Cadranel S. &; Mascart F. (2003). *Helicobacter*

D'Andrea, A., Aste-Amezaga, M., Valiante, N. M., Ma, X., Kubin, M. & Trinchieri, G. (1993).

Fan, X. J., Chua, A., Shahi, C.N., McDevitt. J, Keeling, P. W. & Kelleher, D. (1994). Gastric T

Gackowska, L., Michalkiewicz, J., Krotkiewski, M., Helmin-Basa, A., Kubiszewska, I.,

Galgani, M., Busiello, I., Censini, S., Zappacosta, S., Racioppi, L. & Zarrilli, R. (2004).

Guarner, J., Bartlett, J., Whistler, T., Pierce-Smith, D., Owens, M., Kreh, R., Czinn, S. & Gold,

Mucosal immune responses. *Best Pract Res Clin Gastroenterol*, Vol.18, No.2, (April

*Helicobacter pylori* associated gastritis: immunohistochemical localisation and in vitro effects on cytokine secretion. *J Clin Pathol,* Vol.54, No.4, (April 2001), pp,

D'Elios, M. M., Telford, J. L. & Baldari, C. T. (2003). The *Helicobacter pylori* vacuolating toxin inhibits T cell activation by two independent mechanisms. *J Exp*

*pylori* modulation of gastric and duodenal mucosal T cell cytokine secretions in children compared with adults. *Helicobacter*, Vol.8, No.3, (June 2003), pp, 216-

Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells.

lymphocyte responses to *Helicobacter pylori* in patients with *H pylori* colonisation.

Dzierzanowska, D. (2006). Combined effect of different lactic acid bacteria strains on the mode of cytokines pattern expression in human peripheral blood mononuclear cells. *J Physiol Pharmacol.* Vol.57, No.9, (November 2006), pp, 13-

*Helicobacter pylori* induces apoptosis of human monocytes but not monocytederived dendritic cells: role of the cag pathogenicity island. *Infect Immun,* Vol.72,

B. D. (2003). Can pre-neoplastic lesions be detected in gastric biopsies of children with *Helicobacter pylori* infection? *Pediatr Gastroenterol Nutr*, Vol. 37, No.3,

hand may also participate in the failure of eliminating the infection.

*Med,* Vol.198, No.12, (June 2003), pp, 1887-1897

*J Exp Med*, Vol.178, No.3, (September 1993), pp, 1041-1048

*Gut*, Vol.35, No.10, (October 1994), pp, 1379-1384

No.8, (August 2004), pp, 4480-4485

(September 2003), pp, 309-314

indirectly suggests that in the PBMC model system, *H. pylori cagA+*-induced IFN-gamma production essentially does not depend on IL-12p40. This observation confirms the results of other researchers showing that PBMC stimulation with Gram-negative bacteria results in a rather low and separately-independent IFN-gamma and IL-12 expression (Hessle et al., 2000).

In the *H. pylori*-infected children, but not in the noninfected ones, a relatively high *H. pylori cagA+*-mediated IFN-gamma synthesis was connected with relatively elevated IL-10 production (both responses were on the level of the controls). This observation may indicate a possible role of IL-10 in the down-regulation of IFN-gamma in the course of *H. pylori*infection in children. This issue has been studied extensively by others (Holck et al., 2003). *H. pylori* bacteria (live or killed) and their antigens have been found to induce a higher IL-10 production in PBMCs of *H. pylori-*infected than noninfected adults (Haeberle et al., 1997; Jakob et al., 2001; Windle et al., 2005).

To the best of our knowledge, our report is the first showing a relative increase in IL-10 production in culture PBMCs of *H. pylori*-infected children. These results are consistent with previous findings, indicating that *H. pylori* infection is related to a high expression of IL-10 in the gastric mucosa both on mRNA and protein level (Bodger et al., 2001; Hida et al., 1999). IL-10 has long been considered a potent anti-inflammatory cytokine strongly implicated in *H. pylori* infection (Bodger et al., 2001). On the one hand it may protect from the harmful effects of potentially pro-inflammatory responses induced by *H. pylori,* but on the other, it may inhibit the protective mechanisms of immune responses directed against *H. pylori* antigens.

We also found that *H. pylori cagA+* was a much lower IL-6 inducer in the culture PBMCs of *H*. *pylori*-infected than noninfected children with gastritis. Diminished IL-6 induction may play a role in the hypo-responsiveness of PBMCs to *H. pylori* stimulation in infected children. In our model system IL-6 is produced both by T cells and monocytes. IL-6 is a known strong co-stimulatory cytokine that plays a significant role in providing second signal to antigen-induced T cells (Shi et al., 1989).

In this study, *H. pylori* evoked a similar activity in stimulating TNF secretion in PBMCs of both *H. pylori*-infected and noninfected children. However, in the former group there was a tendency to a slightly lower TNF-alpha level. This partially confirms the findings of earlier studies in the stomach in which *H. pylori*-infected children and adults showed a similar concentration of this cytokine (Bontems et al., 2003). Unchanged or slightly decreased production of this cytokine in *H. pylori cagA+*-mediated PBMCs may also be contributing to the limited pro-inflammatory responses in children with gastritis.

### **5. Conclusion**

In conclusion, *H. pylori* infection in children results in: a) increased percentage of peripheral blood memory CD8+ T cells, b) high apoptosis of circulating CD4+ and CD8+ T-cell subsets, c) *H. pylori*-specific-peripheral hypo-responsiveness (low lymphocyte proliferation and IL-12p40 expression), and d) unchanged *H. pylori* dependent pro- inflammatory responses (IFN-gamma, TNF-alfa, respectively), associated with a high expression of IL-10 but low expression of IL-6. This response pattern, together with a high T-cell subsets apoptosis, may protect from more aggressive forms of *H. pylori*-induced inflammation, but on the other hand may also participate in the failure of eliminating the infection.

#### **6. Acknowledgment**

The study was supported by grant UMK 20/2010.

#### **7. References**

186 Clinical Flow Cytometry – Emerging Applications

indirectly suggests that in the PBMC model system, *H. pylori cagA+*-induced IFN-gamma production essentially does not depend on IL-12p40. This observation confirms the results of other researchers showing that PBMC stimulation with Gram-negative bacteria results in a rather low and separately-independent IFN-gamma and IL-12 expression (Hessle et al.,

In the *H. pylori*-infected children, but not in the noninfected ones, a relatively high *H. pylori cagA+*-mediated IFN-gamma synthesis was connected with relatively elevated IL-10 production (both responses were on the level of the controls). This observation may indicate a possible role of IL-10 in the down-regulation of IFN-gamma in the course of *H. pylori*infection in children. This issue has been studied extensively by others (Holck et al., 2003). *H. pylori* bacteria (live or killed) and their antigens have been found to induce a higher IL-10 production in PBMCs of *H. pylori-*infected than noninfected adults (Haeberle et al., 1997;

To the best of our knowledge, our report is the first showing a relative increase in IL-10 production in culture PBMCs of *H. pylori*-infected children. These results are consistent with previous findings, indicating that *H. pylori* infection is related to a high expression of IL-10 in the gastric mucosa both on mRNA and protein level (Bodger et al., 2001; Hida et al., 1999). IL-10 has long been considered a potent anti-inflammatory cytokine strongly implicated in *H. pylori* infection (Bodger et al., 2001). On the one hand it may protect from the harmful effects of potentially pro-inflammatory responses induced by *H. pylori,* but on the other, it may inhibit the protective mechanisms of immune responses directed against *H. pylori*

We also found that *H. pylori cagA+* was a much lower IL-6 inducer in the culture PBMCs of *H*. *pylori*-infected than noninfected children with gastritis. Diminished IL-6 induction may play a role in the hypo-responsiveness of PBMCs to *H. pylori* stimulation in infected children. In our model system IL-6 is produced both by T cells and monocytes. IL-6 is a known strong co-stimulatory cytokine that plays a significant role in providing second

In this study, *H. pylori* evoked a similar activity in stimulating TNF secretion in PBMCs of both *H. pylori*-infected and noninfected children. However, in the former group there was a tendency to a slightly lower TNF-alpha level. This partially confirms the findings of earlier studies in the stomach in which *H. pylori*-infected children and adults showed a similar concentration of this cytokine (Bontems et al., 2003). Unchanged or slightly decreased production of this cytokine in *H. pylori cagA+*-mediated PBMCs may also be contributing to

In conclusion, *H. pylori* infection in children results in: a) increased percentage of peripheral blood memory CD8+ T cells, b) high apoptosis of circulating CD4+ and CD8+ T-cell subsets, c) *H. pylori*-specific-peripheral hypo-responsiveness (low lymphocyte proliferation and IL-12p40 expression), and d) unchanged *H. pylori* dependent pro- inflammatory responses (IFN-gamma, TNF-alfa, respectively), associated with a high expression of IL-10 but low expression of IL-6. This response pattern, together with a high T-cell subsets apoptosis, may

2000).

antigens.

**5. Conclusion**

Jakob et al., 2001; Windle et al., 2005).

signal to antigen-induced T cells (Shi et al., 1989).

the limited pro-inflammatory responses in children with gastritis.

Lymphocyte Apoptosis, Proliferation and Cytokine

2003), pp, 1755-1762

2005), pp, 5612-5619

ISSN

pp, 760-770

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Lopes, A. I.; Quiding-Jarbrink, M.; Palha, A.; Ruivo, J.; Monteiro, L.; Oleastro, M.; Santos, A.

infection. *Clin Diagn Lab Immunol*, Vol.12, No.8, (August 2005), pp, 994-1002 Lungren, A., Suri Payer, E., Enarsson, K., Svennerholm, A. M. & Lundin, B .S. (2003).

Lundgren, A., Trollmo, C., Edebo, A., Svennerholm, A. M. & Lundin, B. S. (2005).

Maciorkowska, E., Kondej-Muszyńska, K., Kasacka, I., Kaczmarski, M., Kemona, A. (2004).

Malfitano, A. M. , Cahill, R., Mitchell, P., Frankel, G., Dougan, G., Bifulco, M., Lombardi, G.,

Michalkiewicz, J., Krotkiewski, M., Gackowska, L., Wyszomirska-Gołda, M., Helmin, A.,

Peek, R. M. Jr., Miller, G. G., Tham, K. T., Perez-Perez, G. I., Zhao, X., Atherton, J. C. &

Queiroz, D. M., Rocha, G. A., Mendes, E. N., Carvalho, A. S., Barbosa, A. J., Oliveira, C. A. &

Ren, Z., Pang, G., Lee, R., Batey, R., Dunkley, M., Borody, T. & Clancy, R. (2000). Circulating

Schmees, C., Prinz, C., Treptau, T., Rad, R., Hengst, L., Voland, P., Bauer, S., Brenner, L.,

Shi, T., Liu, W. Z., Shi, G. Y.& Xiao, S. D. (1989). Synergistic interactions of IL-1 and IL-6 in T

Soares, T. F.; Rocha, G. A.; Rocha, A. M.; Correa-Oliveira, R.; Martins-Filho, O. A.; Carvalho,

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*Helicobacter pylori*-specific CD4+ T cells home to and accumulate in the human *Helicobacter pylori*-infected gastric mucosa. *Infect Immun*, Vol.73, No.9, (September

Memory cells in the antral mucosa of children with Helicobacter pylori infection.

Lechler, R. I. & Bamford K. B. (2006). *Helicobacter pylori* has stimulatory effects on

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Harris, P.R., Wright, S. W., Serrano, C., Riera, F., Duarte, I., Torres, J., Peña, A., Rollán, A.,

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Helmin-Basa, A., Michalkiewicz, J., Gackowska, L., Kubiszewska, I., Eljaszewicz, A.,

Hessle, C., Hanson, L. A., Wold & A. E. (1999). Lactobacilli from human gastrointestinal

Hessle, C., Andersson, B. & Wold, A. E. (2000). Gram-positive bacteria are potent

Hida, N., Shimoyama, T. Jr., Neville, P., Dixon, M. F., Axon, A. T., Shimoyama, T. Sr. &

Jakob, B., Birkholz, S., Schneider, T., Duchmann, R., Zeitz, M. & Stallmach, A. (2001).

Karlsson, H., Hessle, Ch. & Rudin, A. (2002). Innate immune response to human neonatal

Karttunen, R., Crowe, Haeberle, H. A., Kubin, M., Bamford, K. B., Garofalo, R., Graham, D.

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No.10, (October 1997), pp, 4229-4235

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Characterization of lymphocytic infiltrates in *Helicobacter pylori*-associated gastritis.

Mierzwa, G., Bala, G., Czerwionka-Szaflarska, M., Prokurat, A. & Marszalek, A. (2011). Pediatric *Helicobacter pylori* infection and circulating T-lymphocyte activation and differentiation. *Helicobacter*, Vol. 16, No.1, (February 2011), pp, 27-35,

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Gastric mucosal cytokine responses in *Helicobacter pylori*-infected patients with gastritis and peptic ulcers. Association with inflammatory parameters and bacteria load. *FEMS Immunol Med Microbiol*, Vol.36, No.3, (May 2003), pp, 175-

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Periapoptotic markers in children with *Helicobacter pylori* infection. *Med Wieku*

**11**

*Shanghai, China*

**The Effect of Epigallocatechin Gallate (EGCG)**

**and Metal Ions Corroded from Dental**

*School of Stomatology, Tongji University,*

**Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues**

Jiansheng Su, Zhizen Quan, Wenfei Han, Lili Chen and Jiamei Gu

Metallic materials are the basis of dental materials science, which is an important part of dentistry. As a result of their good mechanical properties and some biological properties, metals are widely used in oral rehabilitation of dentition and mandibular defects, orthodontic and dental implants, as well as in the equipment for dental operations. Commonly used metals include nickel chromium, cobalt chromium, titanium, and silver palladium alloy; of these, nickel-chromium and cobalt-chromium alloys are in widespread clinical use in China because of their stable biological properties and moderate prices.

The oral environment is a very complex electrolyte environment with a large number of microorganisms, in which the metal prosthesis corrodes and releases metal ions. Microbial corrosion is a major contributor to this process. The products of bacterial metabolism (including organic and inorganic acids) can directly affect the pH on the surface or interface of a metal prosthesis, and thus affect the electrochemical reactivity, eventually promoting corrosion. Metal ions are increased in the saliva and oral soft tissue (e.g. gingiva & tongue smear) of patients who wear alloy prostheses. The metal ions released from dental casting alloys can potentially reduce the metabolism of cells, inhibit cell proliferation and have other toxic effects in vitro. Ions released from commonly used Ni-Cr alloys may induce adverse reactions, such as gingival inflammation and discoloration, oral cell mutation or cancer. Cobalt ions from Co-Cr alloy have been reported to stimulate allergic reaction. Another ion that may be released, molybdenum, though is not a main component of Co-Cr alloy, can potentially cause mutagenesis, inactive some important enzymes, such as alkaline

Microbial corrosion refers to electrochemical reactions aroused or catalyzed by microorganisms, rather than the metal specifically corroded by microorganisms. Microbial

**1. Introduction**

**1.1 The application of dental metallic materials**

phosphatase, and inhibit chromium absorption via leukocytes.

**1.2 The biological effects of dental casting alloys**

in *Helicobacter pylori* infection according to age. *Scand J. Immunol,* Vol.62, No.1, (July 2005), pp, 63-70

## **The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues**

Jiansheng Su, Zhizen Quan, Wenfei Han, Lili Chen and Jiamei Gu *School of Stomatology, Tongji University, Shanghai, China*

#### **1. Introduction**

190 Clinical Flow Cytometry – Emerging Applications

Wang, J., Fan, X., Lindholm, C., Bennett, M., O'Connoll, J., Shanahan, F., Brooks, E. G.,

Wang, J., Brooks, E. G., Bamford, K. B., Denning, T. L., Pappo, J.& Ernst, P. B. (2001).

Windle, H. J., Ang, Y. S., Athie-Morales, V., McManus, R. & Kelleher, D. (2005). Human

*Infect Immun,* Vol.68, No.7, *(*July 2000), pp, 4303-4311

2005), pp, 63-70

926-934

25-32.

in *Helicobacter pylori* infection according to age. *Scand J. Immunol,* Vol.62, No.1, (July

Reyes, V. E & Ernst, P. B. (2000). *Helicobacter pylori* modulates lymphoepithelial cell interactions leading to epithelial cell damage through Fas/Fas ligand interactions.

Negative selection of T cells by *Helicobacter pylori* as a model for bacterial strain selection by immune evasion. *J Immunol*, Vol.167, No.2, (July 2001), pp,

peripheral and gastric lymphocyte responses to *Helicobacter pylori* NapA and AphC differ in infected and uninfected individuals. Gut, Vol.54, No.1, (January 2005), pp,

#### **1.1 The application of dental metallic materials**

Metallic materials are the basis of dental materials science, which is an important part of dentistry. As a result of their good mechanical properties and some biological properties, metals are widely used in oral rehabilitation of dentition and mandibular defects, orthodontic and dental implants, as well as in the equipment for dental operations. Commonly used metals include nickel chromium, cobalt chromium, titanium, and silver palladium alloy; of these, nickel-chromium and cobalt-chromium alloys are in widespread clinical use in China because of their stable biological properties and moderate prices.

The oral environment is a very complex electrolyte environment with a large number of microorganisms, in which the metal prosthesis corrodes and releases metal ions. Microbial corrosion is a major contributor to this process. The products of bacterial metabolism (including organic and inorganic acids) can directly affect the pH on the surface or interface of a metal prosthesis, and thus affect the electrochemical reactivity, eventually promoting corrosion. Metal ions are increased in the saliva and oral soft tissue (e.g. gingiva & tongue smear) of patients who wear alloy prostheses. The metal ions released from dental casting alloys can potentially reduce the metabolism of cells, inhibit cell proliferation and have other toxic effects in vitro. Ions released from commonly used Ni-Cr alloys may induce adverse reactions, such as gingival inflammation and discoloration, oral cell mutation or cancer. Cobalt ions from Co-Cr alloy have been reported to stimulate allergic reaction. Another ion that may be released, molybdenum, though is not a main component of Co-Cr alloy, can potentially cause mutagenesis, inactive some important enzymes, such as alkaline phosphatase, and inhibit chromium absorption via leukocytes.

#### **1.2 The biological effects of dental casting alloys**

Microbial corrosion refers to electrochemical reactions aroused or catalyzed by microorganisms, rather than the metal specifically corroded by microorganisms. Microbial

The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental

inhibiting telomerase activity.

via inhibition of JNK.

**2.2 Biological effects of EGCG**

Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues 193

EGCG is also the most active component of green tea leaves. It has been shown to have biological effects, including antimutagenicity, antitumorigenesis, free radical scavenging, etc. Epidemiologic studies demonstrated that a low rate of tumorigenesis may be associated with the habit of drinking tea. Experiments in animal models and cells also suggested that green tea extract may contribute to the inhibition of the generation and development of tumors. In addition to the above-mentioned effect, EGCG, as reported, plays a critical role in inducing apoptosis of malignant cells, regulating key elements of signal pathway and

EGCG is a potent antioxidant component that has demonstrated great antioxidant protection in experimental studies. It is also involved in protecting against free-radical DNA damage, and interferes with the binding of cancer-causing agents to cellular DNA. Besides, EGCG plays a role in inhibition of lipid peroxidation and inducement of apoptosis of malignant cells by regulating various signal pathways. For instance, EGCG may induce

EGCG can negatively regulate protein serine/threonine phosphatase-2A (PP-2A) to positively regulate p53-dependent apoptosis. In addition: EGCG induces apoptosis of JB6 cells, which is associated with hyperphosphorylation of p53 and up-regulation of the proapoptotic gene, Bak (Qin et al., 2008); the interruption of the VEGF (vascular endothelial growth factor) signaling pathway by EGCG results in caspase activation and subsequent malignant cell death; EGCG induces stress signals by damaging mitochondria and ROSmediated JNK activation in MIA PaCa-2 pancreatic carcinoma cells; EGCG-mediated caspase activation induces proteolytic cleavage of the NF-kappa B/p65 subunit, leading to the loss of transactivation domains, and driving the cells towards apoptosis; EGCG show its cytotoxicity to PC-3 cells by up-regulating the 67LR and the mitochondria-mediated apoptosis pathway (Zhang et al., 2008); and finally, EGCG treatment resulted in dosedependent inhibition of TNF-α-induced production of MMP-1 and MMP-3 at the protein

The mechanism of the biological effects of EGCG may differ for different cell categories. Treatment of PC-3 cells with EGCG resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. In contrast, EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells (Albrecht et al., 2008). In HLE cells (hepatoma cell line), EGCG induced apoptosis, but not cell-cycle arrest, and appears to have down-regulated Bcl-2α and Bcl-xl by inactivation of NF-kappaB. Oral administration of EGCG showed similar effects in HLE xenograft tumors; in normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. It is c-Jun N-terminal kinase (JNK) signaling that mediates EGCGinduced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis

Research has indicated that the 67-kDa laminin receptor (67LR) mediates epigallocatechin gallate (EGCG)-induced cell growth inhibition and reduction of myosin regulatory light chain (MRLC) phosphorylation at Thr-18/Ser-19, which is important for cytokinesis through

apoptosis of cells through binding to the antiapoptotic proteins Bcl-2 and Bcl-xL;

and mRNA levels in RA synovial fibroblasts (Yun et al., 2008).

corrosion requires appropriate conditions for microbial reproduction, and is often the result of multi-microbial symbiosis and interaction. In the oral environment, dental casting alloys are sensitive to micro-organisms and tarnish, e.g. nickel, chromium, copper, aluminum, iron, palladium and zinc. It is reported that Actinomyces viscosus has a significant influence on the electrochemical corrosion of Ni-Cr alloy and Gold alloy; the existence of streptococcus mutans increases the corrosion of Ni-Cr alloy. On the other hand, oral bacteria can produce lactic acid and acetic acid. When fluoride ions, hydrogen peroxide and lactic acid exist simultaneously in the mouth, the corrosion of titanium would greatly increase. The biggest corrosion threat to iron is glucose, followed by acetic acid (Park et al., 2007). As mentioned above, the oral bacteria play an important role in material deterioration. Oral microorganisms should be considered when discussing biocompatibility of prostheses.

In conjunction with the corrosion process of dental casting alloys, many metal ions are absorbed within the digestive system. Per unit ion concentration, K+, Cd2+, V2+, Ag+, Hg2+, Sb3+, Be2+, and In3+ have higher toxicity, while Sn4+, Zr4+, Nb5+, and Mo6+ have lower toxicity. Cr6+ and Be2+ have the highest toxicity towards the cell membrane integrity and protein synthesis of human gingival fibroblasts; Ni2+ is moderately toxic to these cells, and Cr3+ and Mo6+ show the lowest toxicity. Ni2+, Co2+, Ti4+, and V3+ could affect DNA synthesis, ALP (alkaline phosphatase) activity and calcification processes of osteoblast-like cells in vitro. Bone marrow cells can be significantly damaged when exposed to Cr6+ for 48h; Co2+, Mo6+, and Ni2+ also showed moderate toxicity; V5+ showed significant toxicity after 4 weeks.

The commonly used Ni-Cr alloy (which also contains Be) could cause gingival fibroblast morphology, viability and proliferation changes, increase the production of PCNA (Proliferating cell nuclear antigen) and Bcl-2 (B-cell lymphoma gene 2) proteins, and boost the production of IL-6, IL-8 and other cytokines involved in inflammation. These effects show positive correlations with concentration of Co, Cr and Cu ions. After wearing Ni-Cr alloy (with Be), animal experiments demonstrated apoptosis lymphocytes and DNA damage of buccal epithelial cells, which deteriorated further with increased wearing time; the gold alloy in contrast, produced no side effects. (Su et al., 2006a, 2006b, 2006c, 2008a, 2008b; Yu et al., 2007)

#### **2. The tea application**

Tea, which originated in China, and has spread over the world, has become a popular worldwide drink. Nowadays, over 34 countries produce tea, and there are more than 4 billion tea consumers in over 100 countries.

Tea is not only a refreshing drink, but also a healthy drink for the prevention of radiation sickness, cardiovascular diseases and cancer, with certain pharmacological effects. The main active ingredients in tea are tea polyphenols, which are strong antioxidants. These polyphenolic compounds mainly consist of catechins. They have a health benefits, including lowering blood cholesterol and blood pressure, and anti-cancer, anti-bacterial and anti-viral effects.

#### **2.1 Biological effects of catechin**

The green tea catechins make up approximately 60–80 wt.% of tea polyphenols. (-)-epigallocatechin gallate (EGCG) is the most abundant of the four major catechins which also include (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC).

EGCG is also the most active component of green tea leaves. It has been shown to have biological effects, including antimutagenicity, antitumorigenesis, free radical scavenging, etc. Epidemiologic studies demonstrated that a low rate of tumorigenesis may be associated with the habit of drinking tea. Experiments in animal models and cells also suggested that green tea extract may contribute to the inhibition of the generation and development of tumors. In addition to the above-mentioned effect, EGCG, as reported, plays a critical role in inducing apoptosis of malignant cells, regulating key elements of signal pathway and inhibiting telomerase activity.

#### **2.2 Biological effects of EGCG**

192 Clinical Flow Cytometry – Emerging Applications

corrosion requires appropriate conditions for microbial reproduction, and is often the result of multi-microbial symbiosis and interaction. In the oral environment, dental casting alloys are sensitive to micro-organisms and tarnish, e.g. nickel, chromium, copper, aluminum, iron, palladium and zinc. It is reported that Actinomyces viscosus has a significant influence on the electrochemical corrosion of Ni-Cr alloy and Gold alloy; the existence of streptococcus mutans increases the corrosion of Ni-Cr alloy. On the other hand, oral bacteria can produce lactic acid and acetic acid. When fluoride ions, hydrogen peroxide and lactic acid exist simultaneously in the mouth, the corrosion of titanium would greatly increase. The biggest corrosion threat to iron is glucose, followed by acetic acid (Park et al., 2007). As mentioned above, the oral bacteria play an important role in material deterioration. Oral microorganisms should be considered when discussing biocompatibility of prostheses.

In conjunction with the corrosion process of dental casting alloys, many metal ions are absorbed within the digestive system. Per unit ion concentration, K+, Cd2+, V2+, Ag+, Hg2+, Sb3+, Be2+, and In3+ have higher toxicity, while Sn4+, Zr4+, Nb5+, and Mo6+ have lower toxicity. Cr6+ and Be2+ have the highest toxicity towards the cell membrane integrity and protein synthesis of human gingival fibroblasts; Ni2+ is moderately toxic to these cells, and Cr3+ and Mo6+ show the lowest toxicity. Ni2+, Co2+, Ti4+, and V3+ could affect DNA synthesis, ALP (alkaline phosphatase) activity and calcification processes of osteoblast-like cells in vitro. Bone marrow cells can be significantly damaged when exposed to Cr6+ for 48h; Co2+, Mo6+, and Ni2+ also showed moderate toxicity; V5+ showed significant toxicity after 4 weeks.

The commonly used Ni-Cr alloy (which also contains Be) could cause gingival fibroblast morphology, viability and proliferation changes, increase the production of PCNA (Proliferating cell nuclear antigen) and Bcl-2 (B-cell lymphoma gene 2) proteins, and boost the production of IL-6, IL-8 and other cytokines involved in inflammation. These effects show positive correlations with concentration of Co, Cr and Cu ions. After wearing Ni-Cr alloy (with Be), animal experiments demonstrated apoptosis lymphocytes and DNA damage of buccal epithelial cells, which deteriorated further with increased wearing time; the gold alloy in contrast, produced no side effects. (Su et al., 2006a, 2006b, 2006c, 2008a, 2008b; Yu et al., 2007)

Tea, which originated in China, and has spread over the world, has become a popular worldwide drink. Nowadays, over 34 countries produce tea, and there are more than 4

Tea is not only a refreshing drink, but also a healthy drink for the prevention of radiation sickness, cardiovascular diseases and cancer, with certain pharmacological effects. The main active ingredients in tea are tea polyphenols, which are strong antioxidants. These polyphenolic compounds mainly consist of catechins. They have a health benefits, including lowering blood cholesterol and blood pressure, and anti-cancer, anti-bacterial and anti-viral

The green tea catechins make up approximately 60–80 wt.% of tea polyphenols. (-)-epigallocatechin gallate (EGCG) is the most abundant of the four major catechins which also include (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC).

**2. The tea application**

effects.

billion tea consumers in over 100 countries.

**2.1 Biological effects of catechin**

EGCG is a potent antioxidant component that has demonstrated great antioxidant protection in experimental studies. It is also involved in protecting against free-radical DNA damage, and interferes with the binding of cancer-causing agents to cellular DNA. Besides, EGCG plays a role in inhibition of lipid peroxidation and inducement of apoptosis of malignant cells by regulating various signal pathways. For instance, EGCG may induce apoptosis of cells through binding to the antiapoptotic proteins Bcl-2 and Bcl-xL;

EGCG can negatively regulate protein serine/threonine phosphatase-2A (PP-2A) to positively regulate p53-dependent apoptosis. In addition: EGCG induces apoptosis of JB6 cells, which is associated with hyperphosphorylation of p53 and up-regulation of the proapoptotic gene, Bak (Qin et al., 2008); the interruption of the VEGF (vascular endothelial growth factor) signaling pathway by EGCG results in caspase activation and subsequent malignant cell death; EGCG induces stress signals by damaging mitochondria and ROSmediated JNK activation in MIA PaCa-2 pancreatic carcinoma cells; EGCG-mediated caspase activation induces proteolytic cleavage of the NF-kappa B/p65 subunit, leading to the loss of transactivation domains, and driving the cells towards apoptosis; EGCG show its cytotoxicity to PC-3 cells by up-regulating the 67LR and the mitochondria-mediated apoptosis pathway (Zhang et al., 2008); and finally, EGCG treatment resulted in dosedependent inhibition of TNF-α-induced production of MMP-1 and MMP-3 at the protein and mRNA levels in RA synovial fibroblasts (Yun et al., 2008).

The mechanism of the biological effects of EGCG may differ for different cell categories. Treatment of PC-3 cells with EGCG resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. In contrast, EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells (Albrecht et al., 2008). In HLE cells (hepatoma cell line), EGCG induced apoptosis, but not cell-cycle arrest, and appears to have down-regulated Bcl-2α and Bcl-xl by inactivation of NF-kappaB. Oral administration of EGCG showed similar effects in HLE xenograft tumors; in normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. It is c-Jun N-terminal kinase (JNK) signaling that mediates EGCGinduced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of JNK.

Research has indicated that the 67-kDa laminin receptor (67LR) mediates epigallocatechin gallate (EGCG)-induced cell growth inhibition and reduction of myosin regulatory light chain (MRLC) phosphorylation at Thr-18/Ser-19, which is important for cytokinesis through

The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental

intake of tea polyphenol. This will give the prosthesis a long service life.

wearing titanium dentures should not drink concentrated tea solution.

**3.2 Complexation reactions of tea polyphenols and metal ions**

bands of some functional groups of epicatechin disappeared

nickel-chromium alloy.

Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues 195

passive film on the surface. Treatment with a high concentration of tea polyphenol could further reduce the corrosion tendency of the Co-Cr alloy. Compared with in artificial saliva,

Drinking tea at the recommended daily intake is helpful to enhance the corrosion resistance of Co-Cr alloy. Thus, it is advised that people with Co-Cr alloy prostheses increase their

As titanium has good corrosion resistance, it is more and more widely used in clinical stomatology. In tea polyphenol solutions, with increasing concentration of polyphenols, the corrosion of titanium accelerates as its corrosion resistance decreases. Therefore, while lower concentrations of tea help to increase the corrosion resistance of titanium, if the concentration is much higher than the recommended amount for daily drinking the stability of titanium will be lower, as described above, with potentially harmful effects. So patients

We have studied both artificial saliva, and three different concentrations of polyphenol solutions. Among the three alloys studied, the corrosion rate of the titanium is slowest (i.e. its corrosion resistance is best); the corrosion rate of the nickel-chromium alloy is fastest (so its corrosion resistance is worst, and showed the most serious corrosion in the experiments) and the corrosion resistance of cobalt-chromium alloy is between that of titanium and

Polyphenols that are abundant in green tea contain multiple hydroxyl groups. As a result, green tea has a strong acid-base buffering capacity, and the polyphenols can complex with central ions such as Ni2+, Bi3+, Cr6+, Fe3+, Al3+, Fe2+, Mo6+, Cu2+, and Mn2+, and form chelate rings. A number of studies show that flavonoids are strong chelating agents with Fe3+. Catechins (flavonols) contain epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC). All of them can form complexes with Fe3+. A "B ring" catechol group is required for combination of polyphenols and Fe3+; when the B ring of 3,4 o-hydroxy changes into a 3,4,5-trihydroxy gallic acyl group, the efficiency of the combination of polyphenol and Fe3+ is low. Although gallic acid (another phenolic acid) can form complexes with Fe3+, the binding capacity of the polyphenols is weak because of the galloyl functional group. The functional groups and chemical characterization of polyphenols and metal ions have been studied for a long time. Chemical analysis and X-ray diffraction showed that catechin and Al3+ form a non-crystalline precipitate complex with a ratio of 1:1. Nuclear magnetic resonance spectroscopy shows the chemical shifts of catechins change upon complex formation; infrared absorption spectra show that the absorption

Some experiments show that catechins folded into a cavity-like structure around Zn2+ and Cu2+, capturing Zn2+ and Cu2+ through the ring π-electron clouds of the aromatic compounds and forming complexes with 1:1 ratios. However, many studies suggest tea polyphenols and metal ions can coordinate in a wide variety of ways. The coordination modes of tea polyphenols and metal ions change with changing pH. Al different pH values, catechin and Fe3+ form complexes in different proportions: when pH < 3, Fe3+ and catechin combined at a molar ratio of 1:1; when 3 < pH < 7, the molar ratio was 2:1; and when pH > 7,

the Co-Cr alloy in tea polyphenol has a lower corrosion tendency and corrosion rate.

67LR. EGCG at a physiological concentration can activate myosin phosphatase by reducing myosin phosphatase 1 (MYPT1) phosphorylation and that may be involved in EGCGinduced cell growth inhibition (Umeda et al., 2008). Matrix metalloproteinase (MMP-9) expression is linked with myeloid cell differentiation, as well as inflammation and angiogenesis processes related to cancer progression. EGCG inhibited MMP-9 secretion in a time- and dose-dependent manner. The gene and protein expression of MMP-9 and of the mRNA stabilizing factor HuR were also inhibited. In an invasion assay, EGCG repressed the invasion of lung carcinoma, and it down-regulated the expression of MMP-9. NF-kappa B localized in the nucleus of the 95-D cells was diminished in a dose-dependent manner in EGCG-treated cells. Thus, the inhibition of tumor invasion by EGCG was shown to be attributed to decreases in the expression of MMP-9 and NF-kappa B, which may result from decrease of intracellular oxidants (Annabi et al. 2007).

EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form; EGCG also enhanced production of intracellular reactive oxygen species (ROS) (Noda et al., 2007).

#### **3. Tea and metal ions**

#### **3.1 The effect of tea polyphenols on the corrosion behavior of dental casting alloys**

Ni-Cr alloy has become prevalent in oral prosthetic applications; although many instances of side effects caused by fixed nichrome dentures have been reported. Gingival inflammation and the appearance of marginal gingival blackening are both ascribed to release of metal ions from the denture. Thus, we wondered if the habit of drinking tea affected the corrosion behavior of dental casting alloys.

As reported, the recommended daily intake of tea is 6-16 g per person, and it differs with age. The major component of green tea, tea polyphenol, accounts for 20–30 wt.% of the dry weight of green leaves. The concentration of weak tea (4–6 g/L green tea) is equivalent to 1.25 g/L tea polyphenol solution, while strong tea (17–25 g/L green tea) is about 5 g/L. An average consumption of green tea (8-13g/L green tea) equates to 2.5g/L tea polyphenol solution.

Experiments showed that the effect of tea polyphenol on Ni-Cr alloy was the most obvious; it enhanced the corrosion rate of Ni-Cr alloy at low concentrations. Tea polyphenol at a concentration of 1.25 g/L had the lowest corrosion resistance compare to the other two groups (the alloy treated with 2.5g/L and 5g/L tea polyphenol ).

With an increase in the concentration of tea polyphenol, the corrosion resistance of Ni-Cr alloy improved. A surface view of Ni-Cr alloy with a scanning electron microscope demonstrated that the surface of the alloy treated with 1.25 g/L tea polyphenol was the roughest, compared with the other samples. It was a very interesting result. Although the exact principle is unclear, the EGCG could formulate an insoluble chelate with Ni and/or Cr, thus precluding further corrosion.

Co-Cr alloy possesses good hardness and high strength, and more importantly does not contain adverse elements as Be and Ni. A great deal of scientific research has demonstrated that the corrosion of the alloy with Cr (>16%) is reduced because of the existence of a

67LR. EGCG at a physiological concentration can activate myosin phosphatase by reducing myosin phosphatase 1 (MYPT1) phosphorylation and that may be involved in EGCGinduced cell growth inhibition (Umeda et al., 2008). Matrix metalloproteinase (MMP-9) expression is linked with myeloid cell differentiation, as well as inflammation and angiogenesis processes related to cancer progression. EGCG inhibited MMP-9 secretion in a time- and dose-dependent manner. The gene and protein expression of MMP-9 and of the mRNA stabilizing factor HuR were also inhibited. In an invasion assay, EGCG repressed the invasion of lung carcinoma, and it down-regulated the expression of MMP-9. NF-kappa B localized in the nucleus of the 95-D cells was diminished in a dose-dependent manner in EGCG-treated cells. Thus, the inhibition of tumor invasion by EGCG was shown to be attributed to decreases in the expression of MMP-9 and NF-kappa B, which may result from

EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form; EGCG also enhanced production of intracellular

**3.1 The effect of tea polyphenols on the corrosion behavior of dental casting alloys**  Ni-Cr alloy has become prevalent in oral prosthetic applications; although many instances of side effects caused by fixed nichrome dentures have been reported. Gingival inflammation and the appearance of marginal gingival blackening are both ascribed to release of metal ions from the denture. Thus, we wondered if the habit of drinking tea

As reported, the recommended daily intake of tea is 6-16 g per person, and it differs with age. The major component of green tea, tea polyphenol, accounts for 20–30 wt.% of the dry weight of green leaves. The concentration of weak tea (4–6 g/L green tea) is equivalent to 1.25 g/L tea polyphenol solution, while strong tea (17–25 g/L green tea) is about 5 g/L. An average consumption of green tea (8-13g/L green tea) equates to 2.5g/L tea polyphenol

Experiments showed that the effect of tea polyphenol on Ni-Cr alloy was the most obvious; it enhanced the corrosion rate of Ni-Cr alloy at low concentrations. Tea polyphenol at a concentration of 1.25 g/L had the lowest corrosion resistance compare to the other two

With an increase in the concentration of tea polyphenol, the corrosion resistance of Ni-Cr alloy improved. A surface view of Ni-Cr alloy with a scanning electron microscope demonstrated that the surface of the alloy treated with 1.25 g/L tea polyphenol was the roughest, compared with the other samples. It was a very interesting result. Although the exact principle is unclear, the EGCG could formulate an insoluble chelate with Ni and/or

Co-Cr alloy possesses good hardness and high strength, and more importantly does not contain adverse elements as Be and Ni. A great deal of scientific research has demonstrated that the corrosion of the alloy with Cr (>16%) is reduced because of the existence of a

decrease of intracellular oxidants (Annabi et al. 2007).

reactive oxygen species (ROS) (Noda et al., 2007).

affected the corrosion behavior of dental casting alloys.

groups (the alloy treated with 2.5g/L and 5g/L tea polyphenol ).

Cr, thus precluding further corrosion.

**3. Tea and metal ions**

solution.

passive film on the surface. Treatment with a high concentration of tea polyphenol could further reduce the corrosion tendency of the Co-Cr alloy. Compared with in artificial saliva, the Co-Cr alloy in tea polyphenol has a lower corrosion tendency and corrosion rate.

Drinking tea at the recommended daily intake is helpful to enhance the corrosion resistance of Co-Cr alloy. Thus, it is advised that people with Co-Cr alloy prostheses increase their intake of tea polyphenol. This will give the prosthesis a long service life.

As titanium has good corrosion resistance, it is more and more widely used in clinical stomatology. In tea polyphenol solutions, with increasing concentration of polyphenols, the corrosion of titanium accelerates as its corrosion resistance decreases. Therefore, while lower concentrations of tea help to increase the corrosion resistance of titanium, if the concentration is much higher than the recommended amount for daily drinking the stability of titanium will be lower, as described above, with potentially harmful effects. So patients wearing titanium dentures should not drink concentrated tea solution.

We have studied both artificial saliva, and three different concentrations of polyphenol solutions. Among the three alloys studied, the corrosion rate of the titanium is slowest (i.e. its corrosion resistance is best); the corrosion rate of the nickel-chromium alloy is fastest (so its corrosion resistance is worst, and showed the most serious corrosion in the experiments) and the corrosion resistance of cobalt-chromium alloy is between that of titanium and nickel-chromium alloy.

#### **3.2 Complexation reactions of tea polyphenols and metal ions**

Polyphenols that are abundant in green tea contain multiple hydroxyl groups. As a result, green tea has a strong acid-base buffering capacity, and the polyphenols can complex with central ions such as Ni2+, Bi3+, Cr6+, Fe3+, Al3+, Fe2+, Mo6+, Cu2+, and Mn2+, and form chelate rings. A number of studies show that flavonoids are strong chelating agents with Fe3+. Catechins (flavonols) contain epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC). All of them can form complexes with Fe3+. A "B ring" catechol group is required for combination of polyphenols and Fe3+; when the B ring of 3,4 o-hydroxy changes into a 3,4,5-trihydroxy gallic acyl group, the efficiency of the combination of polyphenol and Fe3+ is low. Although gallic acid (another phenolic acid) can form complexes with Fe3+, the binding capacity of the polyphenols is weak because of the galloyl functional group. The functional groups and chemical characterization of polyphenols and metal ions have been studied for a long time. Chemical analysis and X-ray diffraction showed that catechin and Al3+ form a non-crystalline precipitate complex with a ratio of 1:1. Nuclear magnetic resonance spectroscopy shows the chemical shifts of catechins change upon complex formation; infrared absorption spectra show that the absorption bands of some functional groups of epicatechin disappeared

Some experiments show that catechins folded into a cavity-like structure around Zn2+ and Cu2+, capturing Zn2+ and Cu2+ through the ring π-electron clouds of the aromatic compounds and forming complexes with 1:1 ratios. However, many studies suggest tea polyphenols and metal ions can coordinate in a wide variety of ways. The coordination modes of tea polyphenols and metal ions change with changing pH. Al different pH values, catechin and Fe3+ form complexes in different proportions: when pH < 3, Fe3+ and catechin combined at a molar ratio of 1:1; when 3 < pH < 7, the molar ratio was 2:1; and when pH > 7,

The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental

mean ± SD for each group

± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues 197

Fig. 1. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with EGCG. The cell density was detected at 24, 48 and 72 h after cell seeding. Cell survival rate was calculated as the number of cells present at these time points compared with the initial cell seeding density. Data points represent the

Fig. 2. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Ni2+ for 48 hours compared with the control group (treatment with Ni2+ only). Data points represent the mean

Fig. 3. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Cr3+ for 48 hours compared with the control group (treatment with Cr3+ only). Data points represent the mean

the molar ratio was 4:1. The stability constants of these complexes are in the range 105–1017 (Sungur & Uzar, 2007) . Electrospray mass spectrometry shows that catechins and iron ions form three different compounds (L1Fe, L12Fe and L1 3Fe with one to three ligands) at different pH values (Elhabiri et al., 2007). Different kinds of polyphenols also have different coordination modes with metal ions. Studies show that EGC and EC combined with Mn2+ through the o-hydroxy of the B ring, while EGCG mainly combines with Mn2+ through the D ring (gallic ring), and then through the B ring (gallate catechin ring), as the coordination bond formed at the D-loop was stronger than that at the B ring. The same tea polyphenols and metal ions can form different complexes in different coordination modes. To form [Al (egcgH-2 )]+, two hydroxyl groups on the D-ring of epigallocatechin gallate become deprotonated, while to form [Al (egcgH-3)]0, first one hydroxyl group on the B-ring and two on the D-ring are deprotonated, then Al3+ combines with two oxygen atoms on the D-ring and one on the B-ring to form a polymeric structure.

In addition, oxidation-reduction reaction can also take place between tea polyphenols and metal ions. The reduction potential of tea polyphenols is high, so it is easy for them to autooxidize under certain conditions. High valence metal ions can oxidize the polyphenols to their quinone or other derivatives, with the metal ions being reduced to their lower valent states. Catechins, the main components of tea polyphenols, have been shown experimentally to reduce Cu2+ and Fe3+ into Cu+ and Fe2+. Because the Cu2+/Cu+ redox potential is low, the Cu2+/Cu+ reaction is easier than the Fe3+/Fe2+ reaction. When excess Fe3+ was added into gallic acid (GA), Fe3+ rapidly combined with the o-hydroxyl on the B-ring of gallic acid, and formed complexes at the ratio of 1:1, as indicated by the formation of a dark blue solution. Subsequently, other complexes can formed electron transfer reactions - Fe3+ was reduced to Fe2+, and GA was oxidized to semiquinone, which then rapidly combined with the remaining Fe3+ to form benzoquinone.

#### **4. Effects of interaction between EGCG and metal ions on cells**

#### **4.1 Effects of interaction between EGCG and metal ions on cell proliferation rate**

We found that EGCG has a strong inhibitory effect on the growth of tongue squamous carcinoma cells; with significant inhibition beginning at 100 µM. In contrast, for gingival fibroblasts, a small inhibition of cell growth was noted at over 150 µM EGCG (Fig. 1). Some studies have shown that Ni2+ has carcinogenic effects, and some have shown that its toxicity and carcinogenic effects are related to its absorption, transport, distribution and retention in cells. Our studies show that when the concentration of EGCG is less than 150 μM, the growth of gingival fibroblasts remains almost unaffected (cell survival rate is more than 95%); when EGCG concentration ranges from 150 μM to 300 μM, a concentration- and time-dependent inhibition of growth of gingival fibroblasts is seen. These results indicated that EGCG may have little promotion on the growth of gingival fibroblasts at low concentrations, and strong inhibition at high concentrations (Fig. 1). Interestingly, we also found that the interactions between EGCG and Ni2+/Co2+ enhanced the inhibition effect of EGCG on cell growth (Fig. 2, Fig. 4), as well as cell morphological changes. Cr3+/Mo6+ have little effect on the growth of tongue squamous carcinoma cells and gingival fibroblast cells, and the interaction of EGCG and Cr3+/Mo6+ produces no significant inhibition effects on tongue squamous carcinoma cells and gingival fibroblast cells (Fig. 3, Fig. 5).

the molar ratio was 4:1. The stability constants of these complexes are in the range 105–1017 (Sungur & Uzar, 2007) . Electrospray mass spectrometry shows that catechins and iron ions form three different compounds (L1Fe, L12Fe and L13Fe with one to three ligands) at different pH values (Elhabiri et al., 2007). Different kinds of polyphenols also have different coordination modes with metal ions. Studies show that EGC and EC combined with Mn2+ through the o-hydroxy of the B ring, while EGCG mainly combines with Mn2+ through the D ring (gallic ring), and then through the B ring (gallate catechin ring), as the coordination bond formed at the D-loop was stronger than that at the B ring. The same tea polyphenols and metal ions can form different complexes in different coordination modes. To form [Al (egcgH-2 )]+, two hydroxyl groups on the D-ring of epigallocatechin gallate become deprotonated, while to form [Al (egcgH-3)]0, first one hydroxyl group on the B-ring and two on the D-ring are deprotonated, then Al3+ combines with two oxygen atoms on the D-ring

In addition, oxidation-reduction reaction can also take place between tea polyphenols and metal ions. The reduction potential of tea polyphenols is high, so it is easy for them to autooxidize under certain conditions. High valence metal ions can oxidize the polyphenols to their quinone or other derivatives, with the metal ions being reduced to their lower valent states. Catechins, the main components of tea polyphenols, have been shown experimentally to reduce Cu2+ and Fe3+ into Cu+ and Fe2+. Because the Cu2+/Cu+ redox potential is low, the Cu2+/Cu+ reaction is easier than the Fe3+/Fe2+ reaction. When excess Fe3+ was added into gallic acid (GA), Fe3+ rapidly combined with the o-hydroxyl on the B-ring of gallic acid, and formed complexes at the ratio of 1:1, as indicated by the formation of a dark blue solution. Subsequently, other complexes can formed electron transfer reactions - Fe3+ was reduced to Fe2+, and GA was oxidized to semiquinone, which then rapidly combined with the

**4. Effects of interaction between EGCG and metal ions on cells**

**4.1 Effects of interaction between EGCG and metal ions on cell proliferation rate**

We found that EGCG has a strong inhibitory effect on the growth of tongue squamous carcinoma cells; with significant inhibition beginning at 100 µM. In contrast, for gingival fibroblasts, a small inhibition of cell growth was noted at over 150 µM EGCG (Fig. 1). Some studies have shown that Ni2+ has carcinogenic effects, and some have shown that its toxicity and carcinogenic effects are related to its absorption, transport, distribution and retention in cells. Our studies show that when the concentration of EGCG is less than 150 μM, the growth of gingival fibroblasts remains almost unaffected (cell survival rate is more than 95%); when EGCG concentration ranges from 150 μM to 300 μM, a concentration- and time-dependent inhibition of growth of gingival fibroblasts is seen. These results indicated that EGCG may have little promotion on the growth of gingival fibroblasts at low concentrations, and strong inhibition at high concentrations (Fig. 1). Interestingly, we also found that the interactions between EGCG and Ni2+/Co2+ enhanced the inhibition effect of EGCG on cell growth (Fig. 2, Fig. 4), as well as cell morphological changes. Cr3+/Mo6+ have little effect on the growth of tongue squamous carcinoma cells and gingival fibroblast cells, and the interaction of EGCG and Cr3+/Mo6+ produces no significant inhibition effects on tongue squamous carcinoma cells and gingival fibroblast

and one on the B-ring to form a polymeric structure.

remaining Fe3+ to form benzoquinone.

cells (Fig. 3, Fig. 5).

Fig. 1. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with EGCG. The cell density was detected at 24, 48 and 72 h after cell seeding. Cell survival rate was calculated as the number of cells present at these time points compared with the initial cell seeding density. Data points represent the mean ± SD for each group

Fig. 2. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Ni2+ for 48 hours compared with the control group (treatment with Ni2+ only). Data points represent the mean ± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

Fig. 3. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Cr3+ for 48 hours compared with the control group (treatment with Cr3+ only). Data points represent the mean ± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental

alone (Figs. 6–7).

Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues 199

cancer cells could also be increased by the combination of Cr3+ and EGCG. Furthermore, the damage to human gingival fibroblast cells and tongue squamous cancer cells caused by Cr3+ in combination with EGCG was significantly less than that produced by 100 μM EGCG

Fig. 6. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Ni2+ and EGCG combined with

Fig. 7. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Cr3+ and EGCG combined with

Fig. 8. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Co2+ and EGCG combined with

Ni2+. Cells were treated with 100 μM EGCG and/or 200 μM Ni2+ for 48 hours

Cr3+. Cells were treated with 100 μM EGCG and/or 200 μM Cr3+ for 48 hours

Co2+. Cells were treated with 150 μM EGCG and/or 300 μM Co2+ for 48 hours

Fig. 4. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Co2+ for 48 hours. The control group was treated with Co2+ only. Data points represent the mean ± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

Fig. 5. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Mo6+ for 48 hours compared with the control group (treatment with Mo6+ only). Data points represent the mean ± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

#### **4.2 The influence of the combination of metal ions and EGCG on cellular DNA**

DNA is a biological macromolecule, which occupies a central and critical role in the cell as its genetic information. A variety of factors can cause DNA damage in cells, such as ionizing radiation, peroxides, thiols and certain metal ions. It has been reported that Ni2+ can decrease DNA synthesis, change the structure of DNA, reduce protein synthesis and inhibit DNA replication and transcription. It can also cause decrease of succinate dehydrogenase activity and total cellular protein. In addition, Ni2+ complexes with nitrogen compounds may damage DNA in breast cancer cells (Lü et al., 2009). However, Cr3+ is difficult to get into cells, and hence has no significant effect on the human gingival fibroblast cells (Elshahawy et al., 2009).

Single cell gel electrophoresis (SCGE), also called comet assay could detect the DNA damage on single cell level. The parameter of Olive Tail Moment reflects both intensity and extent of DNA damage. Ni2+ in combination with EGCG significantly increased the damage to human gingival fibroblast cells and tongue squamous cancer cells. The damage to tongue squamous

Fig. 4. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Co2+ for 48 hours. The control group was treated with Co2+ only. Data points represent the mean ± SD. n=6

Fig. 5. Cell survival rates in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) upon treatment with combinations of EGCG and Mo6+ for 48 hours compared with the control group (treatment with Mo6+ only). Data points represent the

DNA is a biological macromolecule, which occupies a central and critical role in the cell as its genetic information. A variety of factors can cause DNA damage in cells, such as ionizing radiation, peroxides, thiols and certain metal ions. It has been reported that Ni2+ can decrease DNA synthesis, change the structure of DNA, reduce protein synthesis and inhibit DNA replication and transcription. It can also cause decrease of succinate dehydrogenase activity and total cellular protein. In addition, Ni2+ complexes with nitrogen compounds may damage DNA in breast cancer cells (Lü et al., 2009). However, Cr3+ is difficult to get into cells, and hence has no significant effect on the human gingival fibroblast cells

Single cell gel electrophoresis (SCGE), also called comet assay could detect the DNA damage on single cell level. The parameter of Olive Tail Moment reflects both intensity and extent of DNA damage. Ni2+ in combination with EGCG significantly increased the damage to human gingival fibroblast cells and tongue squamous cancer cells. The damage to tongue squamous

**4.2 The influence of the combination of metal ions and EGCG on cellular DNA**

(\*, p<0.05; \*\*, p<0.01)

mean ± SD. n=6 (\*, p<0.05; \*\*, p<0.01)

(Elshahawy et al., 2009).

cancer cells could also be increased by the combination of Cr3+ and EGCG. Furthermore, the damage to human gingival fibroblast cells and tongue squamous cancer cells caused by Cr3+ in combination with EGCG was significantly less than that produced by 100 μM EGCG alone (Figs. 6–7).

Fig. 6. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Ni2+ and EGCG combined with Ni2+. Cells were treated with 100 μM EGCG and/or 200 μM Ni2+ for 48 hours

Fig. 7. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Cr3+ and EGCG combined with Cr3+. Cells were treated with 100 μM EGCG and/or 200 μM Cr3+ for 48 hours

Fig. 8. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Co2+ and EGCG combined with Co2+. Cells were treated with 150 μM EGCG and/or 300 μM Co2+ for 48 hours

The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental

and EGCG. (Figs. 10–11)

12–13)

Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues 201

showed that: EGCG induces G0/G1 cell cycle arrest and partial apoptosis in the tongue squamous cancer cells. Ni2+ arrests the cell cycle at S in the human gingival fibroblast cells and tongue squamous cancer cells. After exposure to the combination of EGCG and Ni2+, the cell cycle is arrested at G0/G1 and apoptosis is increased. Compared with the Ni2+ induced effect, the effect of the combination of Ni2+ and EGCG is enhancement of the apoptosis of both human gingival fibroblast cells and tongue squamous cancer cells. This indicates that the cytotoxicity of Ni2+ enhanced after co-treatment with Ni2+ and EGCG. In contrast, Cr3+ has no obvious influence on the cell cycle of the human gingival fibroblast cells and tongue squamous cancer cells. The effect on the cell cycle of human gingival fibroblast cells and tongue squamous cancer cells induced by EGCG could be reduced by combination of Cr3+

Fig. 11. Cell cycles for human gingival fibroblast cells during treatment with EGCG, Ni2+,

Both EGCG and Co2+ inhibited the cell cycle of tongue squamous cancer cells, block the DNA synthesis and arrest the cell cycle at G0/G1. However, the combination of EGCG and Co2+ significantly reduced the inhibition effect caused by EGCG or Co2+ individually, and increased the percentage of cells in the G2/M phase. There was no significant change in the cell cycle between the group of Cal-27 cells treated with Mo6+ and the control group, and the cell cycle arrest of Cal-27 cells induced by EGCG could be reduced combination with Mo6+ to the extent that no significant difference was seen compared with the control group. (Figs.

Cr3+, EGCG combined with Ni2+ and EGCG combined with Cr3+

The combination of Co2+ and EGCG produced less DNA damage than either individual component on tongue squamous cancer cells, but increased the damage to human gingival fibroblast cell DNA. Mo6+ in combination with EGCG produced significantly less damage than EGCG alone to both human gingival fibroblast cells and tongue squamous cancer cells (Figs. 8–9).

Fig. 9. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Mo6+ and EGCG combined with Mo6+. Cells were treated with 150 μM EGCG and/or 300 μM Mo6+ for 48 hours

#### **4.3 The influence of interaction of metal ions and EGCG on cell cycle**

Flow cytometry (FCM) is an efficient experimental method for cell cycle detection and estimation of apoptotic cells. Hence, we use FCM to measure the apoptosis. The result

Fig. 10. Cell cycles for tongue squamous cancer Cal-27 cells during treatment with EGCG, Ni2+, Cr3+, EGCG combined with Ni2+ and EGCG combined with Cr3+

The combination of Co2+ and EGCG produced less DNA damage than either individual component on tongue squamous cancer cells, but increased the damage to human gingival fibroblast cell DNA. Mo6+ in combination with EGCG produced significantly less damage than EGCG alone to both human gingival fibroblast cells and tongue squamous cancer cells

Fig. 9. Comet Assay: DNA damage in tongue squamous cancer Cal-27 cells (left) and human gingival fibroblast cells (right) after treatments with EGCG, Mo6+ and EGCG combined with

Flow cytometry (FCM) is an efficient experimental method for cell cycle detection and estimation of apoptotic cells. Hence, we use FCM to measure the apoptosis. The result

Fig. 10. Cell cycles for tongue squamous cancer Cal-27 cells during treatment with EGCG,

Ni2+, Cr3+, EGCG combined with Ni2+ and EGCG combined with Cr3+

Mo6+. Cells were treated with 150 μM EGCG and/or 300 μM Mo6+ for 48 hours

**4.3 The influence of interaction of metal ions and EGCG on cell cycle**

(Figs. 8–9).

showed that: EGCG induces G0/G1 cell cycle arrest and partial apoptosis in the tongue squamous cancer cells. Ni2+ arrests the cell cycle at S in the human gingival fibroblast cells and tongue squamous cancer cells. After exposure to the combination of EGCG and Ni2+, the cell cycle is arrested at G0/G1 and apoptosis is increased. Compared with the Ni2+ induced effect, the effect of the combination of Ni2+ and EGCG is enhancement of the apoptosis of both human gingival fibroblast cells and tongue squamous cancer cells. This indicates that the cytotoxicity of Ni2+ enhanced after co-treatment with Ni2+ and EGCG. In contrast, Cr3+ has no obvious influence on the cell cycle of the human gingival fibroblast cells and tongue squamous cancer cells. The effect on the cell cycle of human gingival fibroblast cells and tongue squamous cancer cells induced by EGCG could be reduced by combination of Cr3+ and EGCG. (Figs. 10–11)

Fig. 11. Cell cycles for human gingival fibroblast cells during treatment with EGCG, Ni2+, Cr3+, EGCG combined with Ni2+ and EGCG combined with Cr3+

Both EGCG and Co2+ inhibited the cell cycle of tongue squamous cancer cells, block the DNA synthesis and arrest the cell cycle at G0/G1. However, the combination of EGCG and Co2+ significantly reduced the inhibition effect caused by EGCG or Co2+ individually, and increased the percentage of cells in the G2/M phase. There was no significant change in the cell cycle between the group of Cal-27 cells treated with Mo6+ and the control group, and the cell cycle arrest of Cal-27 cells induced by EGCG could be reduced combination with Mo6+ to the extent that no significant difference was seen compared with the control group. (Figs. 12–13)

The Effect of Epigallocatechin Gallate (EGCG) and Metal Ions Corroded from Dental

no significant synergistic effect was seen with EGCG+Cr3+/Mo6+.

Co2+ (although this latter effect is seen only in normal fibroblasts).

from Doctoral Fund of Ministry of Education of China<PHONE_NUMBER>0041).

**5. Conclusion**

cancerous cells.

**7. References**

**6. Acknowledgments**

0145-2126

0009-2797

ISSN 0020-1693

0142-9612

Casting Alloys on Cell Cycle Progression and Apoptosis in Cells from Oral Tissues 203

Our results show that Ni2+ in combination with EGCG significantly increases damage to normal gingival fibroblasts and tongue squamous cancer cells, whereas the combination of EGCG and Co2+ increased damage only to gingival fibroblasts. In contrast, EGCG together with either Cr3+ or Mo6+ resulted in significantly less damage than produced by EGCG alone. Moreover, the cell cycle is arrested at G0/G1 and apoptosis after exposure to EGCG+Ni2+ is increased relative to that occurring after treatment with EGCG only. Combining EGCG+Co2+ significantly reduced the growth inhibitory effect caused by EGCG or Co2+ individually, and increased the percentage of cells in the G2/M phase. In contrast,

These results indicate that the toxicity of EGCG may be i) enhanced in the presence of Ni2+, ii) diminished when given in combination with Cr3+ or Mo6+ or iii) partially inhibited by

Our studies have demonstrated interactions between EGCG and metal ions that affect cell cycle progression and apoptosis. However, more research is required to reveal the binding mode (s) of these substances and the potential mechanisms by which they affect normal and

This work was supported by grants from National Natural Science Foundation of China (30840031, 30970726) and from Shanghai (0852nm03600, 11nm0504300, 114119a3700) and

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Fig. 12. Cell cycles for tongue squamous cancer Cal-27 cells during treatment with EGCG, Co2+, Mo6+, EGCG combined with Co2+ and EGCG combined with Mo6+

Fig. 13. Cell cycles for human gingival fibroblast cells (right) during treatment with EGCG, Co2+, Mo6+, EGCG combined with Co2+ and EGCG combined with Mo6+

#### **5. Conclusion**

202 Clinical Flow Cytometry – Emerging Applications