Accepted Manuscript

Title: Altered expression of hsp27 and hsp70 in distal oesophageal mucosa in patienswith gastro-oesophageal reflux disease subjected to fundoplication

Authors: T. Rantanen, T. Honkanen, T. Paavonen, L. Rantanen, N. Oksala

PII: S0748-7983(10)00555-X

DOI: 10.1016/j.ejso.2010.10.013

Reference: YEJSO 3070

To appear in: European Journal of Surgical Oncology

Received Date: 4 April 2010

Revised Date: 9 September 2010

Accepted Date: 26 October 2010

Please cite this article as: Rantanen T, Honkanen T, Paavonen T, Rantanen L, Oksala N. Alteredexpression of hsp27 and hsp70 in distal oesophageal mucosa in patiens with gastro-oesophagealreflux disease subjected to fundoplication, European Journal of Surgical Oncology (2010), doi: 10.1016/j.ejso.2010.10.013

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2Author manuscript, published in "European Journal of Surgical Oncology (EJSO) 37, 2 (2011) 168"

DOI : 10.1016/j.ejso.2010.10.013

1

ALTERED EXPRESSION OF HSP27 AND HSP70 IN DISTAL OESOPHAGEAL MUCOSA IN PATIENS WITH GASTRO-OESOPHAGEAL REFLUX DISEASE SUBJECTED TO FUNDOPLICATION T. Rantanen1,2, T. Honkanen3, T. Paavonen3, L. Rantanen3, N. Oksala4 1 Department of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland 2 Department of Surgery, Kanta-Häme Central Hospital, Hämeenlinna, Finland 3 Department of Pathology, Tampere University Hospital, Tampere, Finland 4Division of Vascular Surgery, Department of Surgery, Tampere University Hospital, Tampere, Finland Corresponding Author: Tuomo K. Rantanen, MD, PhD Department of Gastroenterology and Alimentary Tract Surgery Tampere University Hospital P.O. Box 2000 FI-33521 Tampere, Finland Email:tuomo.rantanen@uta.fi Fax:358-3-31164339 Tel.:358-3-31164617

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Abstract

Background Gastro-oesophageal reflux disease (GERD) is a risk factor for oesophageal

adenocarcinoma. Although fundoplication cures reflux symptoms and oesophagitis, it remains

controversial whether it is capable of preventing the development of oesophageal adenocarcinoma.

Hsp27 and Hsp70 are associated with the development of cancer, whereas the effect of

fundoplication on them is not known.

Methods The expression of Hsp27 and Hsp70 was assessed semiquantitatively from biopsies of

oesophageal mucosa for a prospective cohort of 19 patients with GERD treated with fundoplication

and 7 controls without GERD. Upper gastrointestinal endoscopy with biopsies from the

oesophagogastric junction (EGJ) and the distal and proximal oesophagus were performed

preoperatively (19 patients) and after recovery from GERD at 6 (19 patients) and 48 months (16

patients) postoperatively.

Results The expressions of both Hsp27 (p=0.001) and Hsp70 (p=0.002) in the distal oesophagus

were lower in patients preoperatively and at 48 months postoperatively (p<0.001 for both) than in

controls. The patients’ Hsp27 and Hsp70 levels were lower preoperatively in the proximal

oesophagus (p=0.048 for both) than in controls. Both Hsp27 (p=0.002) and Hsp70 (p=0.003) were

lower in the distal oesophagus preoperatively and at 48 months postoperatively (p=0.003 for Hsp27,

p=0.004 for Hsp70) than in the proximal oesophagus.

Conclusions Our results indicate that there may be some factor interfering with the mucosal

defence system of the distal oesophagus in GERD that is uninfluenced by fundoplication and not

associated with the acid-reflux-normalizing effect.

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Key words: Heat shock proteins, GERD, Fundoplication, Follow-up, Oesophageal adenocarcinoma

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Introduction

Gastro-oesophageal reflux disease (GERD) is a common disorder in Western countries, with a

prevalence of roughly 10% in an adult population (1). Not only the disturbing symptoms but also

the fact that GERD is a risk factor for oesophageal adenocarcinoma (2) increase its importance.

Heat shock response is a characteristic and conserved protective response of a tissue to a stimulus,

such as heat, by a complex cascade, the end-product of which is the synthesis of heat shock proteins

(3). Heat shock proteins (Hsps) function as molecular chaperons in normal and stressful conditions.

The significance of Hsps in gastroduodenal protection has been demonstrated (4). Their expression

has been reported in response to diverse chemical or physical stresses, including oxidative stress,

which has been shown to play a role in the pathogenesis of GERD and also in malignant

transformation in Barrett`s oesophagus (3, 5, 6). In addition, heat shock proteins (Hsps) promote

tumourigenesis by inhibiting apoptosis in multiple ways (7). Hsps can be classified according to

their molecular weight and function into distinct families. Hsp70 minimises the aggregation of

newly synthesised proteins (8) and protects cells not only from heat (9) but also from most

apoptotic stimuli. It renders cells highly resistant to cell death induced by TNF, monocytes,

oxidative stress, ceramide, terminal differentiation, caspase-3 over-expression and several

chemotherapeutic drugs (10, 11). Hsp27 is capable of blocking apoptosis induced by death

receptors, monocytes and hydrogen peroxidase (10, 12). It protects cells from oxidative-stress-

induced death by increasing the cellular gluthathione levels and by neutralising the toxic effects of

oxidised proteins through its chaperone-like activity (11)

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There are few studies dealing with Hsps in the oesophageal epithelium with animal or ex vivo

models, and the data is conflicting (13-18).

Nissen fundoplication, the most common procedure, is quite effective in curing reflux symptoms

and reflux oesophagitis. However, the results are contradictory regarding the value of antireflux

surgery in cancer prevention (19, 20). In addition, the effect of fundoplication on the expression of

Hsp27 and Hsp70 in oesophageal mucosa is unknown.

Therefore, we examined the effect of fundoplication on the expression of Hsp27 and Hsp70 in the

mucosa of the EGJ, the distal oesophagus and the proximal oesophagus over a 48-month follow-up

for a prospective cohort of 20 patients with GERD treated by fundoplication, comparing the results

with those of seven control patients without GERD.

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Patients and Methods

Patients

The study group comprised 20 consecutive GERD patients (median age 57, range 27–66;

male/female ratio 14/6) who underwent fundoplication at the Kanta-Häme Central Hospital over a

three-month period in 1998. Preoperatively, all patients had objectively verified symptomatic

GERD, including abnormal 24-hour pH monitoring values in the distal oesophagus (median pH<4

15.0%, range 5.2%–55.4%) (21). In addition, upper gastrointestinal endoscopy with biopsies was

performed on every patient preoperatively. Using the Savary-Miller grading system (22), the

preoperative endoscopy revealed non-erosive reflux disease (NERD) in 8 (40%) and erosive reflux

disease (ERD) in 12 (60%) patients with either Barrett’s epithelium or granulation tissue due to

inflammation – of these 12, 1 (5%) patient suffered from grade I, 4 (20%) patients from grade II and

7 (35%) from grade IV oesophagitis (6 patients with Barrett's epithelium and 1 with an oesophageal

ulcer). For one patient (grade II oesophagitis without Barret’s epithelium), the preoperative and

postoperative biopsy material at 6 months were insufficient for analysis. This asymptomatic patient

(normal pH monitoring at 6 months) also refused endoscopy at 48 months. Therefore, the final

numbers of analysed patients were 19 (preoperatively), 19 (6 months postoperatively) and 16 (48

months postoperatively; NERD in 7, grade II in 3, grade IV without Barrett’s epithelium in 1 and

with Barrett’s epithelium in 5 patients).

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In all cases, medical treatment with proton pump inhibitors (PPIs; 18 patients) or H2 blockers (2

patients) had failed, or the patients were dependent on these medications and requested surgical

treatment. All medications used for GERD were withdrawn for two weeks before the operation.

Controls

The control group included seven Caucasians with neither reflux symptoms nor endoscopic or

histological evidence of reflux disease (median age 60, range 32–69; male/female ratio 4/3). The

controls had undergone endoscopy either for non-reflux dyspepsia (5 cases) or asymptomatic

anaemia (2 cases).

Surgical procedure

Laparoscopic (19) or open (1 patient with associated ventral hernia repair) Nissen-Rossetti

fundoplication was used. The surgical technique has been described earlier (23).

Follow-up

Endoscopy was performed at the beginning of the operation (20 patients) and 6 months (20 patients)

and 48 months (16 patients) after fundoplication. Oesophageal 24-hour pH recording was

performed on 19 patients after 6 months. The one patient (biopsy-confirmed Barrett’s epithelium)

who refused 24-hour pH monitoring at 6 months had a normal fundic wrap at endoscopy along with

a normal finding in a barium swallow. This patient also refused endoscopy at 48 months. All 4

patients who refused endoscopy at 48 months were asymptomatic. All interviews and pre- and

postoperative endoscopies with biopsies were performed by one of the authors (TKR).

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Biopsy protocol

At the beginning of the operation, endoscopy was performed and biopsies were obtained from the

proximal and distal oesophagus and from the EGJ using Olympus FB-53U-1 biopsy forceps. Two

biopsies were taken from each biopsy site. Biopsies of the proximal oesophagus were taken 20 cm

above the EGJ. Biopsies of the distal oesophagus were taken from macroscopic lesions and, if there

was no lesion, 5 cm above the EGJ. For patients with Barrett’s metaplasia, biopsies were obtained

from proximal to the squamocolumnar junction (SCJ). Specimens of the EGJ were obtained 1 cm

under the EGJ. The biopsies were fixed in 10% buffered formalin solution. With regard to the

postoperative endoscopies as well as the control subjects, biopsies were taken and handled using the

same criteria as preoperatively.

Immunohistochemistry

The endoscopic samples were submitted in 10% formalin, routinely processed and embedded in

paraffin, and 3–5 µm paraffin sections were cut onto Superfrost Plus microscope slides (Menzel-

Gläser, Braunschweig, Germany). The following monoclonal antibodies were used for

immunohistochemical stainings: anti-heat shock protein (HSP) 27 (clone G3.1, 1:400, Labvision

corporation Neomarkers, Fremont California, USA) and anti-heat shock protein (HSP) 70 (clone

C92F3A-5, 1:50, Stressgen Bioreagents, Michigan, USA). Immunostaining was performed using

the Ventana BenchMark LT Automated IHC Stainer (Ventana Medical System, Arizona, USA)

with the Ultraview Universal DAB detection kit (catalogue No. 760-500, Ventana Medical System,

Arizona, USA). The fully automated processing of bar code labelled slides included the baking of

the slides as well as solvent-free deparaffinisation and antigen retrieval. Each step of the Ventana

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Ultraview Universal DAB detection kit procedure was optimized with the BenchMark LT

instrument and preset. The Ventana High Temperature Liquid Coverslip (LCS, catalogue No:650-

010, Ventana) was applied throughout the automated protocol as appropriate. Tissue slides were

rinsed between steps with the Ventana Tris-based Reaction buffer (catalogue No. 950-300,

Ventana). Deparaffinization was performed using Ventana EZ Prep solution (catalogue No 950-

100, Ventana), and Epitope retrieval was performed using CC1: Tris-EDTA buffer pH 8.0

(catalogue No 950-124,Ventana) at 95–100°C for 30 minutes with anti-HSP27 and for 60 minutes

with anti-HSP70. Endogenous peroxidase was blocked with UV-Inhibitor 3% H202 (Ventana) for 4

minutes at 37°C. After rinsing, the slides were incubated at 37°C for 32 minutes with specific

antibodies, followed by the application of Ventana Ultraview HRP Universal Multimer (8 minutes

37°C). Antibodies were detected using diaminobenzidine as chromogen, and slides were

counterstained with haematoxylin before mounting. The specificity of the immunohistochemistry

was controlled by omitting the primary antibodies or replacing them with irrelevant antisera. Known

positive tissue samples were also used to confirm the staining reliability of all separate staining

patches.

Evaluation of HSP27 and HSP70 Expression

Epithelial expression of Hsp27 and Hsp70 was determined in stratified epithelia of the proximal and

distal oesophagus and in simple cuboidal epithelia of the EGJ. In clear cases evaluation was

performed semiquantitatively by one highly experienced observer who had no knowledge of patient

history. In problematic cases the analysis was checked by another observer, after which consensus

was reached. The Hematoxylin&Eosin staining intensities of Hsp27 and Hsp70 were scored from 0

to 3 according to the following scale: 0 = no cells were stained, 1 = faint immunoreactivity, 2=

moderate and 3= strong positive staining (24). Cell positivities (the percentage of positive epithelial

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cells from the whole epithelium) were defined as follows: 0 = no cells were stained, 1=1%–25%

positive cells, 2 =26%–75% positive cells, 3 = >75% positive cells (25). The final score was based

on the results of both the analysis of intensity and cell positivity.

All patients gave a written informed consent. This study was approved by the Ethics Committee of

Kanta-Häme Central Hospital, Hämeenlinna, Finland.

Statistical methods

Values are expressed as median and range, unless otherwise stated. Differences at different time

points between controls and patients were compared using the Mann-Whitney U-test, while changes

between time points were analysed with the Friedman or Wilcoxon test. In addition, the Jonckheere-

Terpstra test also was used. Non-parametric methods were applied because of the shape of the

distributions of variables. Statistical calculations were carried out with SPSS software version 15.0

for Windows (SPSS, Inc., Chicago, Il, USA). A p value of <0.05 was considered significant.

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Results

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Clinical assessment

At 6 months postoperatively, the oesophageal acid exposure in pH monitoring was normalised in all

patients (21). At endoscopy, erosive oesophagitis had healed in all cases (22).

After 4 years all 16 patients had a normal fundic wrap, and none of the 16 patients who underwent

endoscopy at that point presented with erosive oesophagitis. Of the 6 patients with Barrett’s

epithelium at baseline, 4 demonstrated no change in the length of the Barrett’s epithelium over the

follow-up period, and in 1 patient the length was reduced from 10 to 5cm. One of the 6 patients for

whom the length of the Barrett’s epithelium remained unchanged at 6 months refused to undergo

endoscopy at 4 years.

The effect of fundoplication on expressions of HSP27 and HSP70

No difference between pre- and postoperative values on the expression of Hsp27 were found at any

location. When compared to controls, the patients’ expression of Hsp27 was lower in the distal

(p=0.001) and proximal oesophagus (p=0.048) preoperatively, in the distal oesophagus (p<0.001) at

6 months and in the distal oesophagus (p<0.001) at 48 months (Table 1, Fig. 1).

As regards the different time points, Hsp70 was lower in the EGJ at 6 months than at 48 months

(p=0.038). When compared to controls, the patients’ preoperative Hsp70 values were lower in the

distal (p=0.002) and proximal oesophagus (p=0.048), as were their postoperative values in the distal

oesophagus at 6 months (p=0.001) and 48 months (p<0.001) (Table 2, Fig. 2).

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No differences were found between Hs27 and Hsp70 at any location preoperatively or during

follow-up.

The grade of preoperative oesophagitis (23) had no correlation with the Hsp27 and Hsp70

expressions at any location. However, we did find a trend at 48 months suggesting that the levels of

Hsp27 in distal oesophageal mucosa decrease with increasing severity of GERD (p=0.041) (Table

3).

Hsp27 was lower in the distal oesophagus than the proximal oesophagus preoperatively (p=0.002)

as well as at 6 (p=0.006) and at 48 months (p=0.003) postoperatively. Furthermore, Hsp27 was

lower in the distal oesophagus than the EGJ at 6 months (p=0.025).

Hsp70 was lower in the distal oesophagus than the proximal oesophagus preoperatively (p=0.003),

at 6 (p=0.010) and at 48 months (p=0.004).

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Discussion

The most important finding of the present study was that in the distal oesophagus, both Hsp27 and

Hsp70 were lower in patients with GERD than in controls preoperatively as well as over a 48-

month follow-up, while no difference was found between patients and controls as regards the

proximal oesophagus postoperatively. Furthermore, no pre- or postoperative differences were

observed between patients and controls in the expression of either Hsp27 or Hsp70 in the EGJ. Our

results indicate the presence of some unknown factor diminishing the mucosal defence capacity in

the distal oesophagus, which is unaffected by fundoplication.

The effect of fundoplication on the expression of Hsp27

Our hypothesis was that fundoplication could normalise the expression of Hsp27 in the EGJ as well

as the distal and proximal oesophagus in parallel with the termination of acid reflux. In contrast,

however, fundoplication had no effect on Hsp27 expression in the distal oesophagus. Six months

postoperatively, pH recording was normalised in every patient, and at 48 months all patients were

free of GERD symptoms and their fundic wrap was normal at endoscopy. Therefore, it is unlikely

that insufficient fundoplication could explain the present results. It is possible that some irreversible

changes have occurred in the distal oesophagus that cannot be restored by the antireflux procedure.

The expression of Hsp27 in GERD

Controversy prevails concerning the expression of Hsp 27 in the oesophagus after gastro-

oesophageal reflux (16- 18, 26). Oesophageal microvascular endothelial cells (HEMEC) have been

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found to respond to acidic pH stress by the induction of Hsp27 in an ex vivo human study (16). In

addition, Hsp27 has been demonstrated to be increased in oesophageal tissue after 12 weeks’

gastroduodenal reflux in rats (17). This is in contrast to the present study, as we found a lower level

of Hsp27 in the distal oesophagus among patients with GERD than among controls without GERD.

In another study, Hsp27 expression was found abundant in a normal human oesophagus (19), but it

then decreased in Barrett’s epithelium (18, 26), which is thought to be a consequence of chronic

gastro-oesophageal reflux. The latter finding is in accordance with the results found in the present

study. Wide variation in the degree of expression of Hsps has been observed between different cell

types and tissues (27, 28). Our results therefore cannot be compared to those of the study by Soldes

et al. (1999) as such, because we took all biopsies from squamous epithelium, also in the cases of

patients with Barrett’s epithelium, and Soldes et al. (1999) took the biopsies from Barrett’s

epithelium.

The reasons behind the differing expressions of Hsp27 after reflux found in ex/in vivo models and

human material can be several, the most important being the chronic exposure to reflux resulting in

adaptive responses. Accordingly, long-lasting GERD in studies dealing with human material has led

to decreased levels of Hsp27, as was found in an ex vivo model when acidic reflux had lasted at

least 24 hours (16).

The effect of fundoplication on the expression of Hsp70

In the current study, fundoplication could not normalise the expression of Hsp70 in the distal

oesophagus, although it was effective in the proximal oesophagus. This is in line with the effect of

fundoplication on the expression of Hsp27 in the distal oesophagus. Our findings are in conflict

with the results of the study by Johnston et al. (29) who found high levels of Hsp70 expression in

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oesophageal and laryngeal specimens taken from controls and patients with laryngopharyngeal

reflux. The reasons for the differences between our study and the report by Johnston and colleagues

(29) are unknown. Conflicting data concerning the expression of Hsp70 after reflux has been

presented in animal and ex vivo models (13, 16), some demonstrating increased (14) and others

reporting decreased levels after reflux (13). However, Hsp70 levels have been shown to disappear

after recovery (13), which is in line with the result presented in our study only as regards the

proximal oesophagus.

The effect of fundoplication on different biomarkers

We have demonstrated previously that oxidative stress of the oesophageal mucosa remains

increased even 4 years after successful fundoplication and the healing of macroscopic oesophagitis

(23), although others have found substantially lower expressions of cyclooxygenase-2 (COX-2) and

interleukin 8 (IL-8) after fundoplication (30, 31). Yet, in the study reporting the latter, the IL-8 level

in Barrett’s mucosa, although significantly reduced, failed to normalise fully (31). Previously, we

have demonstrated with the present cohort that the glutathione content (GSH) was lower in patients

both pre- and postoperatively when compared with controls (23). It has been shown that part of the

mechanism of Hsp27 in protecting cells from death caused by reactive oxygen species is its

capacity to increase cellular GSH levels (11). In the present study, Hsp27 was lower among patients

both pre- and postoperatively. Therefore, parallel ameliorations of Hsp and GSH strongly suggest

the presence of some unknown factor unaffected by fundoplication.

Criticism concerning the present study

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The present prospective study consisted of objectively verified GERD patients on whom

fundoplication was performed using a standard technique in all cases. We should emphasise that

one of the authors (TKR) performed all the endoscopies, strictly following the same biopsy

protocol. Furthermore, all Hsp27 and Hsp70 evaluations were made by an independent observer

who had no knowledge of patient history. However, we did not perform 24-hour pH monitoring at 4

years, nor did we measure alkaline reflux at any time point. Therefore, we have no exact data on the

degree of acid exposure at the 4-year control point or on the exposure of the mucosa to alkaline

reflux at any time point. In addition, the study group consisted of a rather heterogenic group of

GERD patients, and the number of patients in each group was quite small and no statistically

significant effect of the grade of oesophagitis on the expression of Hsp27 and Hsp70 could be

found. In order to avoid variability related to different mucosal types in the distal oesophagus, we

evaluated the expression of both Hsps among patients with Barrett’s epithelium only from the

squamous epithelium adjacent to the Barrett’s epithelium.

Conclusions

In conclusion, Hsp27 and Hsp70 levels were lower among patients with GERD also after

fundoplication in the distal oesophagus, while in the proximal oesophagus both Hsps reached a

level equal to controls at 48 months’ follow-up. This indicates that there may be some unknown

stress factor present in the distal oesophagus that is rather uninfluenced by the antireflux procedure.

Longer follow-up and larger cohorts are needed to validate our results and to examine whether they

remain similar during long-term follow-up.

Conflict of interest

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There is no conflict of interest.

Acknowledgements

This study was supported by the Competitive Research Funding of the Pirkanmaa Hospital District

and by the Pirkanmaa Cultural Foundation.

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Captions for figures Figure 1. The expression of Hsp27 in the distal esophagus among controls and patients over 48-month follow-up Figure 2. The expression of Hsp70 in the distal oesophagus among controls and patients over 48-months follow-up

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Table 1 The expression of Hsp27 in the EGJ, in distal oesophagus and proximal oesophagus among controls (n=7) and GERD patients preoperatively (n=19) as well as at 6 (n=19) and 48 (n=16) months postoperatively Location controls 0 months 6 months 48 months EGJ 3(0-3) 2(1-3) 3(1-3) 2(0-3) Distal oesophagus 3(3-3) 2(0-3)* 2(0-3)** 1(0-2)*** Proximal oesophagus 3(3-3) 3(2-3)# 3(2-3) 3(2-3) _______________________________________________________________________________

*Significantly lower than controls, p=0.001, **Significantly lower than controls, p<0.001, *** Significantly lower than control, p<0.001 #Significantly lower than controls, p=0.048

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Table 2 The expression of Hsp70 in the EGJ, distal oesophagus and proximal oesophagus among controls (n=7) and GERD patients preoperatively (n=19) as well as at 6 (n=19) and 48 (n=16) months postoperatively ________________________________________________________________________ Location Controls 0 months 6 months 48 months EGJ 3(0-3) 2(1-3) 2(0-3)* 3(0-3) Distal oesophagus 3(3-3) 2(0-3)¤ 2(0-3)¤¤ 2(1-3)¤¤¤ Proximal oesophagus 3(3-3) 3(2-3)# 3(2-3) 3(2-3) ________________________________________________________________________________* Significantly lower than at 48 months, p=0.038 ¤ Significantly lower than controls, p=0.002, ¤¤ significantly lower than controls, p=0.001, ¤¤¤ significantly lower than controls, p<0.001 # Significantly lower than controls, p=0.048

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Table 3 The expression of Hsp27 in the distal oesophagus among patients with NERD, oesophagitis (ESO), Barrett’s epithelium (BE), or oesophagitis or Barrett’s epithelium (ESO/BE) preoperatively (NERD in 8 patients, ESO in 5 patients, BE in 6 patients), at 6 months postoperatively (NERD in 8 patients, ESO in 5 patients, BE in 6 patients) and at 48 months postoperatively (NERD in 7 patients, ESO in 4 patients, BE in 5 patients). _______________________________________________________________________________ Patients 0 months 6 months 48 months NERD 2(0-3) 2(0-3) 2(1-3)* ESO 2(1-3) 1(0-2) 1(1-2)* BE 1(1-2) 2(0-3) 1(0-2)* ESO/BE 2(1-3) 2(0-3) 1(0-2)*

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_______________________________________________________________________________ * trend towards decreasing level of Hsp27 among patients with severe GERD, p=0.041 Figure 1

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Figure 2

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