Administration of Anti-Reg

Domenico Viterbo
1
, Gordon E Callender
1
, Theresa DiMaio
2
,Cathy M Mueller
1
,
Tamar Smith-Norowitz
3
, Michael E Zenilman
1
, Martin H Bluth
1,4
Departments of
1
Surgery,
2
Pathology, and
3
Pediatrics, SUNY Downstate Medical Center.
Brooklyn, NY, USA.
4
Department of Pathology, Wayne State School of Medicine. Detroit MI, USA
ABSTRACT
Context The regeneration protein family (Reg), which includes Reg I and PAPII, is expressed in pancreas acinar cells, and increases
in acute pancreatitis. We have demonstrated that Reg gene knockdown worsens severity of acute pancreatitis in the rat and
hypothesize that the proteins offer a protective effect in this disease. Objective We investigated the ability of anti-Reg and anti-PAP
antibody to neutralize pancreatic Reg protein and affect pancreatitis severity. Intervention Pancreatitis was induced in rats by
retrograde ductal injection of 4% sodium taurocholate. Animals Eighty-four rats: 48 with induced pancreatitis, 30 sham operated,
and 6 normal animals. Setting Intraductal anti-Reg I and/or anti-PAPII antibody was administered at induced pancreatitis and sham
operated subgroups of 6 rats each. Main outcome measure Serum and pancreata were harvested 24 and/or 48 hours later and
assessed for pancreatitis severity by pancreatic wet weight, serum C-reactive protein (CRP), amylase, PAPII levels, and
histopathology. Results Animals induced with pancreatitis with administration of anti-Reg/PAP antibodies had significantly higher
wet weights compared with taurocholate and histopathological analysis revealed that anti-Reg/PAP treated animals had worse tissue
inflammation and necrosis compared with controls. Serum CRP, amylase, and Reg levels did not significantly differ between
experimental and sham control groups. Conclusions Administration of anti-Reg/PAP antibody worsened taurocholate-induced organ
specific pancreatitis. These data suggest that the Reg family of proteins is protective in acute pancreatitis.
INTRODUCTION
Acute pancreatitis has a spectrum of severity ranging
from a mild, self-limiting course treated with
conservative methods, to a more aggressive variety
characterized by sepsis, pancreatic necrosis and
hemorrhage. It is estimated that 25% of patients with
acute pancreatitis will progress in severity and require
operative management or die [1]. Pancreatic
regenerating protein may play a role in the
pathophysiology of acute pancreatitis. The regeneration
family of proteins (Reg), which include Reg I
(pancreatic stone protein) and Reg III (pancreatitis-
associated protein: PAP), are a family of proteins
minimally expressed in normal pancreas but strongly
induced in acute pancreatitis [2, 3, 4]. We have
previously demonstrated that antisense mediated gene
knockdown of Reg/PAP in vivo worsens pancreatitis
[5]. In those studies, inhibition of Reg/PAP expression
significantly worsened pancreatitis in that serum
amylase activity, pancreas wet weight, reflecting
edema, and serum C-reactive protein levels all
increased in antisense-treated animals compared with
controls. Furthermore, histopathologic evaluation of
pancreas revealed worsened edema, elevated leukocyte
infiltration, and fat necrosis after antisense-treatment
compared with controls [5]. Here we examined the
ability of anti-Reg/anti-PAP antibodies to neutralize
Reg/PAP proteins and their affect on pancreatitis
severity.
MATERIALS AND METHODS
Experimental Design
Seventy-eight 225 g Sprague-Dawley male rats
(Charles River, Wilmington, MA, USA) were utilized
for this model in the experimental sodium taurocholate
induced pancreatitis (n=48) and in the control (sham
operated; n=30) groups. In addition, 6 normal rats were
also studied.
Received April 24th, 2008 - Accepted May 13th, 2008
Key words Pancreatitis; pancreatitis-associated protein
Abbreviations NaT: 4% sodium taurocholate; NaT-N: 4% sodium
taurocholate plus non specific antibody; NaT-P: 4% sodium
taurocholate plus anti-PAPII; NaT-R: 4% sodium taurocholate
plus anti-Reg I; NaT-RP: 4% sodium taurocholate plus anti-Reg I
plus anti-PAPII; Reg: regeneration protein family; PAP:
pancreatitis-associated protein; S: sham operated control rats; S-N
sham operated plus non specific antibody; S-P: sham operated plus
anti-PAPII; S-R: sham operated plus anti-Reg I
Correspondence Martin H Bluth
Wayne State University School of Medicine, Department of
Pathology, 8203 Scott Hall, 540 E. Canfield, Detroit, Michigan
48201, USA
Phone: +1-313.577.1102; Fax: +1-313.577.0057
E-mail: mbluth@med.wayne.edu
Document URL http://www.joplink.net/prev/200901/03.html

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16
Induction of Pancreatitis
Retrograde intra-ductal infusion of 4% sodium
taurocholate (NaT) in saline was performed using a
polyethylene catheter (0.011” and 0.024”, internal and
outer diameter, respectively; Adams, Parsipanny, NJ,
USA) as previously described [5]. All rats were first
anesthetized using sodium pentobarbital (Abbott
Laboratories, Abbott Park, IL, USA) using a loading
dose of 40 mg/kg administered intraperitoneally. A
midline incision was then performed. The common bile
duct was identified and cannulated in an antegrade
direction with PE-10 tubing (Fisher Scientific,
Pittsburgh, PA, USA) such that the proximal end of the
tube was beyond the ampulla of Vater in the
duodenum. The bile duct was then ligated to prevent
the flow of bile, and 4% NaT in sterile saline was
consistently infused into the pancreatic duct at a rate of
1 mL/kg over 10 min [5].
The experimental animals received a total volume of 1
mL/kg of 4% sodium taurocholate into the pancreatic
duct and were subdivided into subgroups of 6 rats each
which were simultaneously administered with non
specific antibody (NS IgG: 1.6-2 mg; Sigma, St Louis,
MO, USA) (NaT-N), anti-Reg I alone (NaT-R), anti-
PAPII alone (NaT-P), anti-Reg I together with anti-
PAPII (NaT-RP), while no antibody was administered
to 6 rats (NaT).
Controls
Controls consisted of sham-operated rats which
underwent open laparotomy with infusion of saline
alone (S), saline with non-specific antibodies (S-N),
saline with PAPII alone (S-P), and saline with anti-Reg
I alone (S-R).
Anti-Reg/PAP Antibody for in vivo Administration
Monoconal anti-Reg I antibody was purified from
mouse ascites fluid after immunization with a Reg I
producing hybridoma cell line [6]. Polyclonal anti-
PAPII antibody was similarly obtained after injection
of a 31 aminoacid PAP oligopeptide protein sequence
(TMGQQPNGGGWEWSNSDVLNYLNWDGDPSST)
into rabbit (Cocalico Biologicals Inc, Reamstown, PA,
USA). This sequence represents a hydrophilic region of
PAPII and is distinct from Reg I. The gene sequence
coding for this protein was directionally cloned into
PGEX-5X-1 plasmid (Amersham Biosciences/GE
Healthcare, Piscataway, NJ, USA) using primer EcoRI
(F-agcagaattcgaagactcccagaaggc agtgccctctacacg) and
XhoI (R-ctcactcgag gtc tac tgc ttg aac ttg cag aca aaa
ggt aat tcc aca tc) linked sequences generating a PAPII-
GST fusion protein. Recombinant plasmids were
transformed into BL21 competent cells (Stratagene, La
Jolla, CA, USA) and purified protein was obtained by
glutathione column affinity chromatography [7].
PAPII peptide used to generate anti-PAPII antibody
was generated by the Alignment Program within the
ExPASy software (Expert Protein Analysis System;
http://www.expasy.org). Reg isoforms were compared
and sequence homology obtained. Subsequent analysis
demonstrated a unique amino acid sequence which had
minimal overlap with other Reg isoforms (Figure 1).
Anti-Reg I did not cross react with PAPII protein and
anti-PAP antibody did not cross react with Reg I
protein.
Concentration of Anti-Reg/PAP Antibody
Reg I
We developed a direct enzyme linked immunosorbent
assay (ELISA) to determine the amount of antibody
required to completely saturate the amount of
endogenous Reg I found within the pancreatic ductal
system. To this end 8 µg/mL of Reg I antigen were
loaded on a microplate, after which varying
concentrations of monoclonal Reg I antibody was
added and a saturation curve was generated; the slope
of OD absorbance plateaued at 40 µg/mL Reg I
antibody. We were able to obtain 200 µL of pancreatic
juice per rat. Pancreatic juice contains about 7 µg/µL
total protein (Bradford assay, St Louis, MO, USA) and
Reg I protein comprises about 15% of pancreatic juice
(1 µg/µL) [8]; an average rat would contain about 200
µg of Reg I protein. We therefore postulated that
injection of 100 µL of 20 mg/mL (i.e., 2 mg total) of
monoclonal Reg I antibody would effectively saturate
endogenous Reg I protein at a 10:1 antibody:protein
ratio.
PAPII
Unlike Reg I, the concentrations of PAPII in both the
pancreatic juice and serum of healthy animals is
minimal [3, 9]. In contrast, and consistent with earlier
studies in our laboratory [8, 10], serum concentrations
increase over 200 fold in pancreatitic rats compared
with healthy controls (about 220±50 µg/mL vs. less
than 2 µg/mL, P<0.05) [10], but are still a fraction of
Reg I levels in serum and pancreatic juice. We injected
1.6 mg (total) of polyclonal anti-PAPII antibody into
the pancreatic duct based on pilot studies which
demonstrated that this amount of antibody would
maximize pancreatitis (histology) presumably by
inhibiting endogenous PAPII protein in the pancreatic
parenchyma in acute pancreatitis.
Western Analysis
Purified anti-Reg/PAP antibodies (Reg I, PAPII) were
tested for Reg specificity in vivo. Rats were induced
with NaT pancreatitis and pancreatic juice or pancreas
Figure 1. Peptide fragment used for anti-PAPII antibody generation:
sequence comparison and homology between Reg I and PAPII.

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17
was harvested after 24 hours. Pancreatic juice or
homogenized pancreas tissue lysate was run on 15%
acrylamide gel and subsequently blotted with
appropriately diluted antibody (anti-Reg I antibody,
1:1,000; anti-PAP antibody, 1:400) [7].
Tissue Analysis for Edema
Pancreata were harvested at 24 and 48-hour after
pancreatitis induction plus/minus anti-Reg/PAP
treatment and severity was assessed by pancreatic wet
weight (edema) and histopathology. To determine the
degree of edema secondary to pancreatitis, the weight
of the whole pancreas following harvesting was
compared to the weight of the rat at the time of the
pancreatectomy [5]. The pancreatic wet weight
(milligrams of pancreas weight/grams of total body
weight of rat; mg/g) was thus obtained from each rat
belonging to each group (based on condition and time)
and averaged together.
Histopathological Analysis
The head of the pancreas from each rat was fixed in
10% buffered formaldehyde solution (Fisher Scientific,
Pittsburgh, PA, USA). Slides were generated for each
pancreas collected using four to six micron sections,
and stained with hematoxylin and eosin (H&E). The
histological severity of pancreatitis was examined by
pathologists who performed the evaluation in a blinded
fashion by using previously described criteria [11, 12].
The degree of inflammation, hemorrhage, leukocytic
infiltration, and tissue necrosis were determined for
each specimen by using a scale ranging from zero
(representing the least severity) to four (representing
the greatest severity).
Serum Analysis for Amylase and CRP
Whole blood from each of the rats was obtained at the
time of the pancreatectomy. Serum was separated and
assayed for amylase (U/mL) and CRP levels (mg/dL)
by the Clinical Laboratory at SUNY Downstate
Medical Center.
Serum Analysis for Reg/PAP
Serum Reg levels obtained from experimental and
control rats were determined by enzyme linked
immunosorbent assay (ELISA) [3, 13].
Reg I
After measuring the amount of total protein within rat
pancreatic juice (collected from normal rats), we
estimated that 15% of the protein content was Reg I
[8]. For each well, 8 µg/mL of purified rat Reg I
protein was plated on a 96-well plate and left to
incubate overnight. After serial washing with
phosphate-buffered solution (PBS) and blocking with
3% BSA in PBS, a standard curve using our Reg I
monoclonal antibody (ranging from 2.7 to 85 µg/mL)
as our primary antibody was generated and a saturation
curve produced as previously described [3].
PAPII
Goat anti-rat PAPII polyclonal antibody (R&D
Biosystems, Minneapolis, MN, USA) (100 µg/µL
diluted in coating buffer (0.05 M carbonate-
bicarbonate, pH 9.6; Bethyl Labs Inc., Montgomery
TX, USA), was plated in 96 well plates (100 µL/well)
and incubated overnight at 4°C. Plates were washed
three times with wash solution (50 mM Tris-buffered
saline, pH 8.0, 0.05% Tween 20; Bethyl Labs,
Montgomery, TX, USA) after which blocking postcoat
solution (50 mM Tris-buffered saline, pH 8.0, 1.0%
BSA; Bethyl Labs, Montgomery, TX, USA) was added
and plates were incubated for 30 min at room
temperature. Plates were washed three times with wash
solution and experimental and control rat sera,
appropriately diluted in dilution buffer (50 mM Tris-
buffered saline, pH 8.0, 1.0% BSA, 10% Tween 20;
Bethyl Labs, Montgomery, TX, USA), were added and
incubated for 60 min at room temperature. Plates were
washed 5 times with wash solution and biotinylated
anti-rat PAPII polyclonal antibody (R&D Systems,
Minneapolis, MN, USA) in sample diluent (1:200; 100
µL/well) was added and plates were incubated for 1
hour at room temperature. Plates were washed 5 times
with wash buffer and streptavidin-HRP (R&D Systems,
Minneapolis, MN, USA), (1:200 in sample diluent; 100
µL/well) was added and plates were incubated for 1
hour at room temperature. Plates were washed 5 times
in wash solution, were developed for 15 min in the
dark using 3,3,5,5, tetramethylbenzidine (TMB)
substrate (100 µL/well; Bethyl Labs, Montgomery, TX,
USA), according to manufacturer’s recommendations,
and the reaction stopped with H2SO(2 mol/L). Plates
were read at 450 nm using a microplate reader (Bio-
Rad, Hercules, CA, USA). Standard curves were
generated using recombinant PAPII protein (used to
generate anti-PAPII antibody above: Bethyl Labs,
Montgomery, TX, USA) appropriately diluted in
normal rat serum/PBS (1:3). Range of assay was 0.075-
7.5 ng/µL with coefficient of variation (CV) no greater
than 9.1% [13].
ETHICS
Institutional
guidelines
regarding
animal
experimentation were followed according to the criteria
outlined in the "Guide for the Care and Use of
Laboratory Animals (1996)" prepared by the National
Academy of Sciences.
STATISTICS
All data were expressed as mean±standard error of the
mean (SEM) representing a minimum of six animals
per group. Median values of the histopathology scores
were also evaluated. During initial studies the
statistical analysis was performed by using the
unpaired Student’s independent-sample t-test for
between group comparisons, which consisted of control
vs. insulted (NaT) animals, and the paired Student’s t-
test within groups observed over two different time

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points. Upon compilation of all data sets representing
all groups (controls and NaT insulted) analysis of
variance (ANOVA) with Tukey post-hoc multiple
comparison procedure was applied when three or more
groups were pairwise analyzed. Initially ANOVA was
employed among all control groups (normal and sham
plus/minus antibody treatment). If no significance was
obtained then normal animal data was compared with
all insulted groups (NaT plus/minus antibody
treatment). If there were differences among controls
groups then all controls would be compared with all
insulted groups. The Statistical Package for Social
Sciences (Version 11.0; SPSS, Inc., Chicago, IL, USA)
was used. Statistical significance was defined as two-
tailed P value less than 0.05.
RESULTS
Generation of PAPII Antibodies
The Reg protein family which contains Reg I and
PAPII, possesses considerable homology to one
another [3]. In light of this, the sequences of Reg I and
PAPII were compared and a unique hydrophilic region
for PAPII was identified: T Met G Q Q P N G G G W
E W S N S D V L N Y L N W D G D P S S T. As
shown in Figure 1 this oligopeptide contains about
20% homology to Reg I. This distinction allows for
analysis of antibody-mediated neutralization of
individual Reg proteins with minimal overlap; cross
reactivity was not observed (data not shown).
Specificity of Anti-Reg/PAP Antibody
Prior to injection of antibody, the specificity to the Reg
I and PAPII proteins were confirmed by sodium
dodecyl sulphate - polyacrylamide gel electrophoresis
(SDS-PAGE) and Western blot. Protein isolated from
pancreatic juice or pancreas obtained from animals
induced with 4% NaT demonstrated a band at
approximately 17 kDa representing Reg protein [3]
when blotted with monoclonal Reg I antibody against
human and rat Reg I, (Figure 2; a1 and a2,
respectively). Similarly, a 17 kDa band was observed
with anti PAPII antibody blotted against pancreata
obtained from rats induced with NaT pancreatitis
(Figure 2, c1) when compared with pancreata obtained
from normal rats (Figure 2, c2). In contrast, non-
specific control IgG did not demonstrate any bands
representative of Reg I or PAPII (Figure 2, b1 and b2).
Wet Weight
Animals induced with NaT pancreatitis had higher
pancreatic wet weights at 24 hours when compared
Figure 2. Validation of specificity of monoclonal anti-human Reg I
antibody to human and rat Reg I protein using SDS-PAGE and
Western blot. Lanes a1 and a2 demonstrate reactivity of monoclonal
antibody against human and rat Reg I protein respectively, revealing
a band at approximately 17 kDa; Reg I protein was obtained from
pancreatic juice from human or rat with pancreatitis (ammonium
sulfate precipitation). Lane contained normal mouse IgG against
human Reg I and rat Reg I (b1 and b2 respectively), which did not
reveal any bands. Lane c: anti-PAPII antibody blotted against
pancreas obtained from NaT induced pancreatitis (c1) or normal
pancreas (c2).
Table 1a. Pancreatic wet weights (mean±SEM; mg/g).
Groups (6 rats in each group)
0 h
24 h
48 h
P values a
Normal
4.07±0.06
N/A
N/A
-
S: Sham
N/A
4.54±0.21
3.93±0.16
0.056
S-N: Sham + non specific IgG
N/A
3.68±0.18
NT
-
S-P: Sham + anti-PAPII
N/A
4.72±0.16
NT
-
NaT: 4% sodium taurocholate
N/A
5.61±0.25
4.64±0.14
0.007
NaT-N: 4% NaT + NS IgG
N/A
5.71±0.20
6.04±0.21
0.286
NaT-P: 4% NaT + anti-PAPII
N/A
5.53±0.19
NT
-
NaT-R: 4% NaT + anti-Reg I
N/A
5.90±0.16
6.60±0.21
0.024
NaT-RP: 4% NaT + anti-Reg I+ anti-PAPII
N/A
6.57±0.25
NT
-
paired Student’s t-test
N/A: not applicable; NT: not tested
Table 1b. Results of the pairwise comparison of the pancreatic wet weight after 24 hours among groups (P values are reported in the table).
S
S-N
S-P
NaT
NaT-N NaT-P NaT-R NaT-RP
S: Sham
-
0.004
0.997
0.002
0.001
0.005
<0.001
<0.001
S-N: Sham + non specific IgG
0.004
-
0.005
<0.001
<0.001
<0.001
<0.001
<0.001
S-P: Sham + anti-PAPII
0.997
0.005
-
0.017
0.005
0.040
0.001
<0.001
NaT: 4% sodium taurocholate
0.002
<0.001
0.017
-
1.000
1.000
0.959
0.010
NaT-N: 4% NaT + NS IgG
0.001
<0.001
0.005
1.000
-
0.998
0.997
0.029
NaT-P: 4% NaT + anti-PAPII
0.005
<0.001
0.040
1.000
0.998
-
0.854
0.004
NaT-R: 4% NaT + anti-Reg I
<0.001
<0.001
0.001
0.959
0.997
0.854
-
0.177
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
<0.001
<0.001
<0.001
0.010
0.029
0.004
0.177
-
ANOVA with Tukey post-hoc multiple comparison.
Table 1c. Results of the pairwise comparison of the pancreatic wet
weight after 48 hours among groups (P values are reported in the
table).
S
NaT
NaT-N
NaT-R
S
-
0.003
0.001
<0.001
NaT
0.003
-
0.999
0.846
NaT-N
0.001
0.999
-
0.890
NaT-R
<0.001
0.846
0.890
-
ANOVA with Tukey post-hoc comparison.

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19
with sham controls (5.61±0.25 mg/g vs. 4.54±0.21
mg/g, P=0.002) (Table 1). Pancreatitic animals treated
with anti-Reg I or anti-PAPII antibodies did not
significantly differ from NaT treated animals alone
(P=0.959 and P=1.000). In contrast, pancreatitis
animals treated with both anti-Reg I and anti-PAPII at
24 hours demonstrated significantly increased
pancreatic wet weight (NaT-RP vs. NaT: 6.57±0.25
mg/g vs. 5.61±0.25; P=0.010). Furthermore, at 48-hour
post pancreatitis induction, the NaT treated animals
demonstrated improvement of edema compared with
24-hour time point (NaT 48 h vs. NaT 24 h: 4.64±0.14
mg/g vs. 5.61±0.25 mg/g, P=0.007) whereas anti-Reg I
treated animals continued to demonstrate worsening
evidence of edema (NaT-R 48 h vs. NaT-R 24 h:
6.60±0.21 mg/g vs. 5.90±0.16 mg/g, P=0.024). These
data suggest that the degree of edema was worse when
an animal received anti-Reg I and anti-PAPII antibody
treatment and may continued to worsen over time.
Amylase
Animals induced with NaT pancreatitis had higher
serum amylase levels at 24 hours when compared with
sham controls (NaT vs. S: 4,250±471 U/L vs.
2,489±617 U/L, P=0.047) (Table 2). However, similar
to pancreatic wet weight results, pancreatitic animals
treated with anti-Reg I or anti-PAPII antibodies did not
differ from NaT treated animals alone (P=0.996 and
P=0.512, respectively). Although, pancreatitis animals
treated with both anti-Reg I and anti-PAPII antibodies
(4,502±756 U/L, P=1.000) did not significantly differ
from NaT treated animals, they also trended toward
increased amylase levels. Furthermore, at 48-hour post
pancreatitis induction, the NaT pancreatitis animals
treated with non-specific IgG demonstrated reduction
of serum amylase (NaT-N 48 h vs. NaT-N 24 h:
2,610±281 U/L vs. 4,155±356 U/L, P=0.007) whereas
anti-Reg I treated animals continued to maintain
elevated amylase levels (NaT-R 48 h vs. NaT-R 24 h:
4,879±1,281 U/L, vs. 4,789±563U/L, P=0.950). These
data suggest that serum amylase remained elevated
over time when an animal received anti-Reg I antibody
treatment in the setting of taurocholate-induced
pancreatitis.
Serum C Reactive Protein and PAP Levels
CRP
Animals induced with NaT pancreatitis had non-
significantly higher serum CRP levels at 24 hours
when compared with sham controls (NaT vs. S:
1.73±0.68 mg/dL vs. 1.43±0.46 mg/dL, P=0.999).
Furthermore, pancreatitic animals treated with anti-Reg
I or anti-PAPII antibodies either individually or in
combination did not differ from NaT treated animals
alone (Table 3).
PAP
Animals induced with NaT pancreatitis had
significantly higher serum PAP levels at 24 hours when
compared with sham controls (298±33 µg/mL vs. 20±6
µg/mL, P<0.001). However, pancreatitic animals
treated with anti-Reg I or anti-PAPII antibodies either
individually or in combination did not differ from NaT
treated animals alone (Table 3).
Histopathology
Animals induced with chemical pancreatitis
demonstrated worsening pancreatitis by histopathology
Table 2a. Serum amylase levels (mean±SEM; U/L).
Groups (6 rats in each group)
0 h
24 h
48 h
P values a
Normal
1,551±31
N/A
N/A
-
S: Sham
N/A
2,489±617
1,543±75
0.159
S-N: Sham + non specific IgG
N/A
2,923±453
NT
-
S-P: Sham + anti-PAPII
N/A
3,187±306
NT
-
NaT: 4% sodium taurocholate
N/A
4,250±471
3,119±1,381
0.456
NaT-N: 4% NaT + NS IgG
N/A
4,155±356
2,610±281
0.007
NaT-P: 4% NaT + anti-PAPII
N/A
5,606±650
NT
-
NaT-R: 4% NaT + anti-Reg I
N/A
4,789±563
4,879±1,281
0.950
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
N/A
4,502±756
NT
-
paired Student’s t-test
N/A: not applicable; NT: not tested
Table 2b. Results of the pairwise comparison of the serum amylase levels after 24 hours among groups (P values are reported in the table).
S
S-N
S-P
NaT
NaT-N NaT-P NaT-R NaT-RP
S: Sham
-
0.999
0.977
0.047
0.245
0.001
0.027
0.081
S-N: Sham + non specific IgG
0.999
-
1.000
0.541
0.636
0.005
0.134
0.310
S-P: Sham + anti-PAPII
0.977
1.000
-
0.792
0.863
0.017
0.292
0.553
NaT: 4% sodium taurocholate
0.047
0.541
0.792
-
1.000
0.512
0.996
1.000
NaT-N: 4% NaT + NS IgG
0.245
0.636
0.863
1.000
-
0.421
0.987
1.000
NaT-P: 4% NaT + anti-PAPII
0.001
0.005
0.017
0.512
0.421
-
0.943
0.757
NaT-R: 4% NaT + anti-Reg I
0.027
0.134
0.292
0.996
0.987
0.943
-
1.000
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
0.081
0.310
0.553
1.000
1.000
0.757
1.000
-
ANOVA with Tukey post-hoc comparison.
Table 2c. Results of the pairwise comparison of the serum amylase
levels after 48 hours among groups (P values are reported in the
table).
S
NaT
NaT-N
NaT-R
S
-
0.065
0.087
0.012
NaT
0.065
-
0.999
0.846
NaT-N
0.087
0.999
-
0.773
NaT-R
0.012
0.846
0.773
-
ANOVA with Tukey post-hoc comparison.

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20
as evidenced by increased leukocytic infiltration,
hemorrhage and necrosis (Table 4). When NaT animals
were injected with anti-Reg I and anti-PAPII
antibodies, pancreatitis severity worsened as evidenced
by increased leukocytic infiltration and tissue necrosis
when compared with non specific antibody treatment
(Table 4, Figure 3). This suggests that anti-Reg
treatment directly worsened local pancreatitis severity
as demonstrated by histopathology.
DISCUSSION
In our present study we have shown in vivo evidence
that Reg proteins provide protection from severity of
pancreatitis in that antibody neutralization of Reg
proteins correlated with worsening pancreatitis on the
local pancreas tissue. Reg proteins are well-known
components in pancreatic juice. In 1979, DeCaro et al.
described a novel protein found in patients with
chronic pancreatitis; initially it was named human
pancreatic stone protein (PSP) [14]. Subsequently, after
further analysis, it was later renamed to pancreatic
thread protein (PTP) by Gross et al. [15] and finally the
name was changed to pancreatic Reg I, for it was
shown to be expressed in regenerating islet cells
following a near total pancreatectomy [16, 17]. We
have shown that Reg I is mitogenic to both pancreatic
beta- and ductal cells and co-precipitates with trypsin
although the mechanism of action is yet to be
elucidated [18]. Reg III, an isoform of the Reg protein
family, is also known as pancreatitis associated protein
(PAP) is comprised of three additional isoforms of
PAPI, PAPII and PAPIII. The Reg proteins are coded
for by genes present on chromosome 9 in the rat, span
about 100 kb and possess over 60% homology with one
another [3]. The exact function of Reg is largely
unknown. Knockout studies of Reg I by Unno et al. did
not demonstrate any significant physiological changes
in a mouse animal model [19]. This could be due to
redundancies in the Reg gene family which are able to
compensate for one another in the event of individual
Reg gene damage. To this end, Bodeker et al. have
shown that PAPI interacts with PAPII, PAPIII and
lithostatin (Reg I alpha) as well as itself to form
homo/heterodimers, [20] which suggests that
individual Reg proteins may provide overlapping
function for other members of the Reg protein family.
The large span of the entire Reg gene family renders
classical gene knockout strategies, including Cre-Lox,
an ineffective means to study Reg function in vivo. We
have previously employed gene knockdown strategies
to study Reg III. In those studies antisence mediated
gene knockdown using a consensus sequence coding
for a bioactive protein component common to all three
PAP isoforms (GGWEWSN) [21], was able to
decrease gene expression of PAPI, PAPII, and PAPIII
and worsened pancreatitis severity in an animal model
of acute pancreatitis [5]. Our studies are consistent with
recent studies by Gironella et al. who have shown that
PAP has anti-inflammatory effects in PAP knockout
mice induced with pancreatitis [22]. However, in
Table 3a. Serum CRP and PAP levels evaluated after 24 hours (means±SEM).
Groups (6 rats in each group)
CRP levels (mg/dL)
PAP levels (µg/mL)
Normal
NT
<5
S: Sham
1.43±0.46
20±6
S-N: Sham + non specific IgG
1.42±0.41
25±9
S-P: Sham + anti-PAPII
1.44±0.66
29±8
NaT: 4% sodium taurocholate
1.73±0.68
298±33
NaT-N: 4% NaT + NS IgG
1.65±0.60
300±26
NaT-P: 4% NaT + anti-PAPII
1.77±0.61
330±20
NaT-R: 4% NaT + anti-Reg I
1.70±0.60
316±33
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
1.79±0.65
345±28
NT: not tested
Table 3b. Results of the pairwise comparison of the serum CRP levels after 24 hours among groups (P values are reported in the table).
Groups
S
S-N
S-P
NaT
NaT-N NaT-P NaT-R NaT-RP
S: Sham
-
1.000
1.000
0.999
1.000
0.997
0.998
0.997
S-N: Sham + non specific IgG
1.000
-
0.999
1.000
1.000
1.000
1.000
1.000
S-P: Sham + anti-PAPII
1.000
0.999
-
1.000
1.000
1.000
1.000
1.000
NaT: 4% sodium taurocholate
0.999
1.000
1.000
-
1.000
1.000
1.000
1.000
NaT-N: 4% NaT + NS IgG
1.000
1.000
1.000
1.000
-
1.000
1.000
1.000
NaT-P: 4% NaT + anti-PAPII
0.997
1.000
1.000
1.000
1.000
-
1.000
1.000
NaT-R: 4% NaT + anti-Reg I
0.998
1.000
1.000
1.000
1.000
1.000
-
1.000
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
0.997
1.000
1.000
1.000
1.000
1.000
1.000
-
ANOVA with Tukey post-hoc comparison.
Table 3c. Results of the pairwise comparison of the serum PAP levels after 24 hours among groups (P values are reported in the table).
Groups
S
S-N
S-P
NaT
NaT-N NaT-P NaT-R NaT-RP
S: Sham
-
1.000
1.000
<0.001
<0.001
<0.001
<0.001
<0.001
S-N: Sham + non specific IgG
1.000
-
1.000
<0.001
<0.001
<0.001
<0.001
<0.001
S-P: Sham + anti-PAPII
1.000
1.000
-
<0.001
<0.001
<0.001
<0.001
<0.001
NaT: 4% sodium taurocholate
<0.001
<0.001
<0.001
-
1.000
0.964
0.999
0.754
NaT-N: 4% NaT + NS IgG
<0.001
<0.001
<0.001
1.000
-
0.975
1.000
0.794
NaT-P: 4% NaT + anti-PAPII
<0.001
<0.001
<0.001
0.964
0.975
-
1.000
1.000
NaT-R: 4% NaT + anti-Reg I
<0.001
<0.001
<0.001
0.999
1.000
1.000
-
0.980
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
<0.001
<0.001
<0.001
0.754
0.794
1.000
0.980
-
ANOVA with Tukey post-hoc comparison.

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21
contrast their studies used cerulean as the inducing
agent which induce pancreatitis by mechanisms distinct
from NaT induction [23] and pancreatic necrosis was
less severe with gene knockout compared with wild
type mice. Furthermore, their studies investigated
knockout effects of Reg IIIb/Reg II which is
phylogenetically most consistent with the PAPI
isoform of rat and PAPI is only 58% homologous to
PAPII. Our studies investigated the role of PAPII with
anti-PAPII antibodies raised to a oligopeptide sequence
in the loop region of PAPII which has minimal overlap
with any of the other PAP isoforms and its effect either
alone or in conjunction with anti-Reg I antibody on
pancreatitis severity. It is likely that the relationship of
Reg and PAP isoforms to one another directly affect
the disease course. Since gene knockdown technologies
do not always translate into actual protein function [24]
our current studies therefore investigated a protein
knockdown approach by neutralizing Reg proteins with
anti-Reg I and anti-PAPII specific antibodies.
The monoclonal anti-human Reg I antibody used was
specific to Reg I found in the pancreas of rats. Despite
having a homology of approximately 68% between rat
and human Reg I [25], our anti-human antibody
recognized the same epitope on rat Reg I. In addition,
based on the ELISA data observed, we have shown we
were able to saturate endogenous Reg I within the
ductal juice of the rat pancreas. Based on previous
studies [8] that have shown that approximately 15% of
total protein content within rat pancreatic juice contains
Reg, we were able to calculate that a total of 2 mg of
antibody was needed to competitively bind endogenous
Reg within the pancreatic juice. Reg III/PAPII is found
in trace amounts in serum in healthy rats and increases
in acute pancreatitis [3]. To this end since PAPII in
serum and tissue appears to comprise a fraction of Reg
I concentration. We injected 1.6 mg (total) of
polyclonal anti-PAPII antibody into the pancreatic duct
based on pilot studies which demonstrated that this
amount of antibody would maximize pancreatitis
(histology) presumably by inhibiting endogenous
PAPII protein in the pancreatic parenchyma in acute
pancreatitis (data not shown). It is possible that anti-
Reg antibody administration could precipitate
pancreatitis by forming antibody-antigen aggregates,
protein plugs in the pancreatic ducts, or precipitates. In
such a case one would likely expect to find dilated
ducts, distinct areas of periductal leukocytic infiltrate
or evidence of complex deposition in these ducts all of
which were not evident in our studies. This suggests
that the worsening pancreatitis severity observed in
pancreas tissue with anti-Reg or anti-PAP
administration were likely due to antibody
neutralization, although ultrastructural changes cannot
be excluded [26]. Since PAP has been found to co-
localize with intracellular zymogens in acinar cells
[26], studies investigating the effect of anti-zymogen
antibody neutralization in conjunction with anti-Reg
and/or anti-PAP antibody administration with
determination of antibody presence in duct and tissue
are warranted to further elucidate the relationship of
these proteins in the context of acute pancreatitis. It is
also possible that the antibodies may be degraded by
activated proteases when retroperfused into the duct,
thereby underscoring the observed effects. Addition of
protease inhibitor during antibody administration may
have potentiated the observed responses.
The wet weights were used to determine the degree of
edema following induction of pancreatitis. As was
expected, NaT administration demonstrated increased
wet weight compared with shams at 24 or 48 hours. It
is interesting to note that only groups receiving
combined anti Reg I and PAPII antibody treatment had
worsening edema compared with NaT alone, whereas
Figure 3. Photomicrographs of pancreatitis severity. Representative
images of experiments described in Table 4. a. Normal rat pancreas.
b. Pancreas from rats induced with pancreatitis (NaT) alone
demonstrating leukocytic infiltration. c. After anti-Reg I or PAPII
antibody treatment showing increased leukocytic infiltration. d. and
e. After administration of both anti Reg I and PAPII demonstrating
increased necrosis (d.), and leukocytic infiltration and hemorrhage
(e.). (Magnification. a, c, d: 100x; b, e: 200x).
Table 4. Histopathology scoring of pancreatitis severity (median values). Animals were sacrificed after 24 hours and pancreata were analyzed for
pancreatitis severity. Severity was scored, by a pathologist blinded to specimens, (key: - no pancreatitis; + minimal; ++ mild; +++ moderate; ++++
severe) [8, 9].
Groups
Edema
Hemorrhage
Leukocyte
infiltration
Tissue
necrosis
Fat
Necrosis
S: Sham
-
-
-
-
-
S-N: Sham + non specific IgG
+
-
-
-
-
S-P: Sham + anti-PAPII
-
-
+
-
-
S-R: Sham + anti-Reg I
-
-
-
-
-
NaT: 4% sodium taurocholate
-
+
++
++
+
NaT-N: 4% NaT + NS IgG
-
++
++
++
+
NaT-P: 4% NaT + anti-PAPII
-
+
+++
++++
++++
NaT-R: 4% NaT + anti-Reg I
-
++
++
+++
++
NaT-RP: 4% NaT + anti-Reg I + anti-PAPII
-
+
++++
++++
++++

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22
individual antibody treatment did not differ from NaT
induced animals. It is likely that with regard to wet
weight, which is an index of pancreatic edema, there
may be redundant protective mechanisms provided by
both Reg isoforms. Only when both isoforms were
neutralized was there evidence of increased edema.
Furthermore, Reg I antibody treatment maintained the
increased state of edema compared with NaT treatment
alone. It could be that although anti-Reg I alone was
unable to worsen pancreatitis compared with NaT
treatment, it was able to maintain the severity induced
by NaT.
Interestingly, although there was increased evidence of
pancreatitis with NaT treatment, compared with sham
controls, as evidenced by increased serum amylase and
PAP levels, there were no differences with
administration of either individual or dual anti
Reg/PAP treatment compared with NaT treatment
alone. Furthermore, there were no observed differences
in CRP levels. This could be due to the fact that these
represent systemic parameters of inflammation [27]
and are not sensitive enough to be affected by antibody
treatment. Additionally, the antibodies utilized in the
ELISA were different from the experimental anti-
PAPII that was administered to rats. Thus, the epitopes
recognized by these antibodies differ, and the ELISA
utilized may not be able to distinguish free PAPII,
bound PAPII or PAPII-anti-PAPII complexes.
Therefore, although the levels of PAPII in sera did not
differ, it could be that the levels of active protein
differed. However, the degree of pancreatic
inflammation and necrosis with anti Reg treatment did
increase with anti-Reg/PAP treatment. Taken together
with the lack of differences in blood based markers of
disease (amylase, CRP, PAP) this suggests that only
the “local” pancreatic parenchyma is affected by
Reg/PAP protein neutralization. We have demonstrated
similar worsening of pancreatitis in the pancreatic
organ with antisense gene knockdown of PAP [5]. In
those studies knockdown of all three PAP isoforms
correlated with increased inflammatory infiltrates and
pancreatic edema. Taken together our data suggest that
Reg proteins have a protective effect on pancreatitis. It
is possible that the family of Reg proteins is critical in
protecting the pancreas during inflammation. Another
member of the Reg family may actually be more
important in diminishing the inflammatory response
following the induction of pancreatitis than Reg I.
Other researchers [2] have also demonstrated that in
acute pancreatitis, the expression of Reg I within the
pancreas increased 100 fold, while the expression of
Reg III/PAP increased on the order of 500-1,000 fold.
In addition, serum levels of Reg III/PAP are markedly
higher in the setting of acute pancreatitis in rats when
compared to Reg I [18, 28]. Reg III/PAP is expressed
in the same gene family, which is localized to 2p12 in
humans [29, 30]. It is therefore likely that Reg I and
Reg III/PAP act in concert to protect and stabilize the
pancreas.
It has been suggested that Reg proteins have mitogenic
and may have anti-apoptotic characteristics [31] and
that Reg and its receptor are upregulated in acute
pancreatitis [32]. Our collective data suggest that by
interfering with the normal physiological functioning
of Reg proteins, the balance between pro- and anti-
apoptotic mediators in pancreatitis is shifted towards
inducing cell death and subsequent necrosis. More in-
depth analysis needs to be conducted to examine this
interplay that Reg proteins may have in preventing cell
death. Finally, since Reg proteins have been reported to
have immunomodulatory effects, they may exert a
direct role on the pathogenesis or progression of
pancreatitis.
Acknowledgements The authors would like to thank
Dr. Sameer Patel, Dr. Gabriel Levi, Gary Papierman,
Marshall Weisberg, Hazel Drew, and Victor Ocasio for
their efforts. In addition, the authors would also like to
acknowledge Hans Von Gizycki for using SPSS to
perform various statistical analyses for this project and
Michael Kruger, MS for his statistical review of the
manuscript
Research grant support This project was funded by
the National Institute of Digestive and Kidney Disease
(R01DK54511, MEZ)
Conflict of interest The authors have no potential
conflicts of interest
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