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An investigation into the role of ghrelin peptides in inflammatory

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An investigation into the role of ghrelin peptides in inflammatory Powered By Docstoc
					 Establishment of a mouse model of
  colitis and its use to evaluate the
  anti-inflammatory effects of two
           ghrelin peptides


                     Samia Taufiq
           Bachelor of Applied Science (Honours)

         School of Life Sciences, Faculty of Science
                Masters of Applied Science
                            2009




                       Supervisors:

  A/Prof Mike McGuckin - Mater Medical Research Institute
                          (MMRI)
Dr. Lisa Chopin - Queensland University of Technology (QUT)
Dr. Penny Jeffery - Mater Medical Research Institute (MMRI)




                              1
           Keywords

Ghrelin, GHSR, Δ4 peptide, DSS, IBD




                 2
                                     Abstract
       Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the
release of growth hormone, and has recently been shown to ameliorate inflammation.
Recent studies have suggested that ghrelin may play a potential role in inflammation-
related diseases such as inflammatory bowel diseases (IBD). A previous study with
ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment
decreased the clinical severity of colitis and inflammation and prevented the
recurrence of disease. Ghrelin may be acting at the immunological and epithelial
level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal
epithelial cells. The current project investigated the effect of ghrelin in a different
mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two
molecular weight forms of DSS were used as they give differing effects (5kDa and
40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in
the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with
ghrelin suppressed colitis in the proximal colon as indicated by reduced
accumulative histopathology scores (p=0.03). Whilst there was a trend toward
reduced scores in the mid and distal colon in these mice this did not reach
significance. Ghrelin did not affect histopathology scores in the 40kDa model. There
was no significant effect on the number of regulatory T cells or TNF-α secretion
from cultured lymph node cells from these mice.
        The discovery of C-terminal ghrelin peptides, for example, obestatin and the
peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have
raised questions regarding their potential role in biological functions. The current
project investigated the effect of Δ4 peptide in the DSS model of colitis however no
significant suppression of colitis was observed. In vitro epithelial wound healing
assays were also undertaken to determine the effect of ghrelin on intestinal epithelial
cell migration. Ghrelin did not significantly improve wound healing in these assays.
In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa
DSS model. The potential mechanisms behind this effect and the disparity between
these results and those published previously will be discussed.




                                          3
                              Table of Contents


1. Chapter One: Introduction............................................11
2. Chapter Two: Literature Review...................................14
   2.1. Inflammatory Bowel Disease
      2.1.1. Genetic factors associated with IBD
      2.1.2. The intestinal barrier in IBD
      2.1.3. Aberrant immune response in IBD
   2.2. Animal models of IBD
      2.2.1. Chemically-induced models
      2.2.2. Genetically engineered models
   2.3. Current treatments for IBD
      2.3.1. New treatments for IBD need to be evaluated
   2.4. Ghrelin
      2.4.1. Ghrelin gene derived peptides
      2.4.2. Ghrelin O-Acyltransferase (GOAT)
      2.4.3. The role of Ghrelin in inflammation and IBD
      2.4.4. Ghrelin in mouse models of colitis
   2.5. Relevance of project
   2.6. Conclusion
3. Chapter Three: Materials and Methods.......................36
   3.1. Mice
   3.2. Induction of colitis and experimental design
   3.3. Assessment of inflammation: symptoms and inflammatory score
   3.4. Haematological analysis
   3.5. Tissue collection
   3.6. Histological assessment of colitis
   3.7. Isolation and culture of Mesenteric lymph node (MLN) cells
   3.8. Enzyme-Linked ImmunoSorbent Assay (ELISA) for TNF-α
   3.9. Mesenteric lymph nodes (MLN) T Regulatory Cells counting using Flow
       Cytometry

                                             4
    3.10. Cell Culture
    3.11. Wound assay
    3.12. Statistical Data analysis
    3.13. Presentation of Results

4. Chapter Four: Results..................................................43
    4.1. Effect of ghrelin peptide treatment on clinical colitis scores in C57BL/6 and
          BALB/c mice administered with dextran sodium sulphate (DSS)
    4.2. Treatment with ghrelin peptides does not affect bodyweight change in mice
          with DSS-induced colitis
    4.3. Effect of ghrelin peptides on colon shortening in C57BL/6 and BALB/c
          mice given DSS
    4.4. Blood analyses of BALB/c and C57BL/6 mice
    4.5. Treatment with ghrelin peptides suppressed histolopathological colitis in
          C57BL/6 mice
    4.6. Measurement of TNF-α from mesenteric lymph node lymphocyte cultures
    4.7. Enumeration of CD4+CD25+Foxp3+ T regulatory cells in mesenteric lymph
          nodes
    4.8. Treatment with ghrelin did not improve wound healing in the HT29 cell line

5. Chapter Five: Discussion..............................................74
Appendix............................................................................80
    1.1      Score sheet for mice undergoing DSS Treatment
    1.2      Assessment of DSS colitis
    1.3      Table A.1
             Figure A.1

References...........................................................................84




                                            5
              List of Illustrations and Diagrams


A. Chapter Two: Literature Review
  1. Figure 1. Mucosal immune responses in IBD.
  2. Figure 2. Alternative splicing of mouse ghrelin gene generates multiple
     ghrelin peptides.
  3. Figure 3. A potential mechanism of ghrelin peptides in inflammation.



B. Chapter Four: Results
1. Figure 1. Clinical evidence of colitis in BALB/c mice treated with DSS and
   ghrelin peptides.
2. Figure 2. Clinical evidence of colitis in C57BL/6 mice treated with DSS and
   ghrelin peptides.
3. Figure 3. Changes in body weight in BALB/c mice treated with DSS and
   ghrelin peptides.
4. Figure 4. Changes in body weight in C57BL/6 mice treated with DSS and
   ghrelin peptides.
5. Figure 5. Colon lengths of mice treated with DSS for 8 days.
6. Figure 6. Two-Way ANOVA comparison of colon lengths of BALB/c and
   C57BL/6 mice treated with ghrelin peptides with 5kDa DSS.
7. Figure 7. Haematological parameters in BALB/c mice treated with DSS and
   ghrelin peptides for 8 days.
8. Figure 8. Haematological parameters in C57BL/6 mice treated with DSS and
   ghrelin peptides for 8 days.
9. Figure 9. Histological colitis scores in BALB/c mice treated with DSS and
   ghrelin peptides.
10. Figure 10. Histological colitis scores in C57BL/6 mice treated with DSS and
   ghrelin peptides.
11. Figure 11. Representative H&E sections of colons dissected from 6 week old
   Naive C57BL/6 mice.
12. Figure 12. Representative histology images of C57BL/6 mice treated with
   5kDa DSS.

                                     6
13. Figure 13. Representative histology images of C57BL/6 mice treated with
   40kDa DSS
14. Figure 14. Histological assessment of inflammation in the Proximal colon of
   BALB/c and C57BL/6 mice.
15. Figure 15. Histological assessment of inflammation in the Distal colon of
   BALB/c and C57BL/6 mice.
16. Figure 16. TNF-α concentration in mesenteric lymph node cultures from
   mice treated with DSS and ghrelin peptides
17. Figure 17. Representative flow cytometry plots of T regulatory cell
   populations in mesenteric lymph nodes in the BALB/c mice.
18. Figure 18. Representative flow cytometry plots of T regulatory cell
   populations in mesenteric lymph nodes in the C57BL/6 mice.
19. Figure 19. T regulatory cell numbers in mesenteric lymph nodes in BALB/c
   (A) and C57BL/6 (B) mice.
20. Figure 20. Analysis of wound assay on HT29 cells to measure the effect of
   various ghrelin concentrations in wound healing.
21. Figure 21. Pre-treatment of HT29 cells with various concentrations of ghrelin
   to measure its effect in wound healing.




                                      7
                               Abbreviations

IBD         Inflammatory Bowel Disease
GHS-R1a     Growth Hormone Secretagogue Receptor 1a
DC          Dendritic Cell
CD          Crohn’s disease
UC          Ulcerative Colitis
Treg        Regulatory T cells
IEC         Intestinal epithelial cells
IL-1β       Interleukin-1β
Il-12       Interleukin-12
IFN-γ       Interferon- γ
TNF-α       Tumour Necrosis Factor- α
IL-10       Interleukin-10
TGFβ        Transforming growth factor beta
NF-κB       Nuclear Factor-κB
DSS         dextran sodium sulphate
IL-10 -/-   Interleukin-10 knock out
TNBS        2,4,6-Trinitrobenzene sulfonic acid
MLNs        Mesenteric lymph nodes
Th          T helper cells
CD4         Cluster of Differentiation 4
CD25        Cluster of Differentiation 25
Foxp3       Forkhead box P3
TLR         Toll-like Receptors
TCR         T-cell receptor
NEMO        NF-κB essential modulator
GOAT        Ghrelin O-Acyltransferase
PPAR-γ      Peroxisome proliferator-activated receptor- γ
LPS         lipopolysaccharides




                                          8
               Statement of Original Authorship



“The work contained in this thesis has not been previously submitted to
meet requirements for an award at this or any other higher education
institution. To the best of my knowledge and belief, the thesis contains
no material previously published or written by another person except
where due reference is made.”


Samia Taufiq




Signature


Date




                                   9
                        Acknowledgement


This study was performed at the Mater Medical Research Institute
(MMRI) – Mucosal Diseases Program, under team leader, Associate
Professor Michael McGuckin. I would specially like to thank my MMRI
supervisor- Dr. Penny Jeffery and my Queensland University of
Technology (QUT) principal supervisor - Dr. Lisa Chopin for their right
guidance and help. I am grateful to Dr. Rajaraman Eri, Deborah Roche,
Sharyn B. Tauro, Patricia Lusby, and the rest of the team from the
Mucin and IBD lab for their valuable contribution. Lastly, I would like
to thank my parents, sister, husband, and friends for supporting me
throughout my degree.




                                  10
                       Chapter One: Introduction

       Inflammatory bowel disease (IBD) is characterized as a chronic
inflammatory condition which predominantly affects the gastrointestinal tract. IBD
can be further classified into two chronic disorders: Crohn’s disease (CD) and
ulcerative colitis (UC). CD is characterised by patchy transmural inflammation
affecting any area of the gastrointestinal tract, whereas UC is limited to the colon,
causing continuous mucosal inflammation also involving the rectum. The etiology of
IBD is not completely known, but it is thought to arise from an abnormal
immunological response to antigens present in the gut lumen (Fiocchi, 1998). IBD is
affected by a combination of environmental, genetic, and immunological
abnormalities which influences the immune response within the intestinal mucosa
leading to inflammation (Hanaeur et al., 1996; Podolsky et al., 2002). An immune
response is generated when immune cells, including monocytes, macrophages, T
cells, and dendritic cells (DC) detect microbes (Guillot et al., 2004). Once an antigen
is detected DCs migrate to the mesenteric lymph nodes and stimulate naive T cells,
which are primarily dominated by mucosal CD4+ lymphocytes (Uhlig et al., 2006).
The CD4+ T cells differentiation is controlled by cytokines which determine the type
of inflammatory response to occur. Pro-inflammatory cytokines such as interleukin
(IL-12), interferon- γ (IFN-γ) and tumour necrosis factor (TNF-α) play a major role
in colonic tissue destruction, and are released upon failure to regulate T cell
responses in the intestinal or colonic mucosa and in response to infection (Weinstein
et al., 1997; Gonzalez-Rey et al., 2006). Animal studies suggest that the presence of
regulatory T cells (Tregs), which produce the anti-inflammatory cytokines, IL-10
and transforming growth factor beta (TGFβ), supports the immune balance in the
normal gut by restoring mucosal tolerance (Mowat, 2003; Kelsen et al., 2005).
       Much of our knowledge of the pathogenesis of IBD comes from numerous
animal models, which are designed to mimic the clinical features seen in human
IBD. Two such types of experimental animal models are discussed in this review, the
chemically induced colitis models and the genetically induced colitis models. Both
types of models can reflect immune cell and epithelial cell dysfunction. Even though
these animal models do not completely exhibit the complexity of the human



                                          11
diseases, they are useful tools to investigate mechanisms of colitis and to evaluate
new therapies for IBD.
       Current therapies used in IBD have significant side effects and many are
ineffective for severe and relapsing IBD. We, therefore, require therapeutic agents
which will suppress the aberrant immune response seen in IBD (to downregulate
pro-inflammatory cytokine release) and also repair the damaged epithelium. A
potential anti-inflammatory agent is ghrelin, a 28 amino acid gut peptide hormone.
Ghrelin inhibits the production of pro-inflammatory cytokines in vitro and exerts
strong protective actions on the gastric mucosa thereby helping to accelerate the
healing of lesions (Dixit et al., 2004; Li et al., 2004b; Konturek et al., 2006). Ghrelin
treatment is also effective in ameliorating experimental colitis in mice, however,
studies by Zhao et al., (2006) have shown that the activation of the ghrelin receptor
can trigger a pro-inflammatory response and can also activate the production of NF-
κB, a pro-inflammatory transcription factor, in human colonocytes in vitro. These
authors suggest that the release of pro-inflammatory cytokines during colonic
inflammation could directly trigger the upregulation of ghrelin during colitis through
the activation of NF-κB. Due to these differing theories, it is important to further
investigate the role of ghrelin in inflammation and IBD, in order to understand the
complete mechanism of its actions.
       It is now known that multiple ghrelin peptides are produced from the
preproghrelin pro-hormone and these include the mature ghrelin hormone and
obestatin, the C terminal peptide of proghrelin which is thought to be active (Zhu et
al., 2006 ). A splice variant of the ghrelin gene results in an exon 4-deleted
proghrelin isoform (Jeffery et al., 2003). As of yet, no study has clearly shown
whether the unique C-terminal peptide of the exon 4-deleted proghrelin isoform is
functionally active or not. Its mRNA expression in the colon or mucosal epithelial
cells has also not been investigated. The human orthologue, exon 3-deleted
proghrelin, is expressed in a wide range of human tissues including human prostate
and breast cancer cell lines, and is upregulated in cancer tissue as compared to
normal tissue (Jeffery et al., 2002; Jeffery et al., 2005a, b). It is important to examine
the functional significance of the exon 4-deleted proghrelin isoform in animal
models of colitis to determine if it has similar anti-inflammatory properties to
ghrelin.


                                           12
       This study will investigate whether the administration of ghrelin and Δ4
proghrelin peptide can ameliorate intestinal inflammation in the DSS model of
colitis. We hypothesise that ghrelin peptides will reduce colitis and rescue epithelial
damage by stimulating proliferation and migration.




                                          13
                     Chapter Two: Literature Review

Inflammatory Bowel Disease
         Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of
the gastrointestinal tract present in two forms: Crohn’s disease (CD) and ulcerative
colitis (UC). IBD affects over 61,000 people in Australia, out of which 28,000
people are affected with CD and 33,000 with UC (ACCA report). There are around
776 new cases of CD and 846 cases of UC diagnosed each year (ACCA). Even
though IBD can be diagnosed at any age it is more prevalent between the ages 15 to
40. Disease morbidity can be significantly higher in younger patients, with the risk
of lifelong problems in relation to emotional well being, physiological growth,
reproductive health issues, education, and employment (Van Limbergen et al.,
2007).
The unique pathophysiological feature of IBD is the close apposition of the intestinal
immune system to high concentrations of intraluminal bacteria. There has been
strong evidence which supports that the dysregulation of the normally controlled
immune response to commensal bacteria in a genetically susceptible individual
causes IBD (Cho, 2008). IBD is thought to be characterised by dysfunction of
mucosal T cells, altered cytokine production, and cellular inflammation which
ultimately leads to damaging the distal small intestine and the colonic mucosa
(Fiocchi, 1998; Hanauer & Present, 2003). Whilst it is difficult to diagnose a patient
with IBD in the early stages, clinical symptoms including abdominal pain, rectal
bleeding, malabsorption and weight loss aid in the diagnosis. (Fiocchi, 1998;
Dieleman & Heizer, 1999). However, in some cases extra-intestinal manifestations
are found to affect skin, joints and eyes in both CD and UC patients (Russel et al.,
2004).
         Although the pathogenesis of IBD is still unclear it is thought to result from a
combination of genetic, environmental, and immunologic abnormalities in which an
uncontrolled immune response within the intestinal lumen leads to inflammation in
genetically predisposed individuals (Yu et al., 2004). Environmental factors such as
drinking, and variations in food intake affect the pathophysiology of IBD (Sakamoto
et al., 2005). The risk of smoking has been found to be increased in patients with
CD, but decreased in patients with UC (Podolsky, 2002). The luminal environment,

                                           14
in particular the luminal flora, play a crucial role in IBD (Korzenik & Podolsky,
2006), for instance, an active balance between the presence of commensal flora and
the dampening of protective host mechanisms is essential to maintain non-inflamed
mucosa. Even though intestinal bacteria play a pivotal role in the development of
IBD, their contribution to inflammation remains unclear (Ott et al., 2004). Studies
about the relationship of microbes-host are limited due to lack of knowledge of the
diversity and complexity of the microbial flora (MacDonald & Monteleone, 2000).


Genetic factors associated with IBD
       Genetic factors also play a significant role in the pathogenesis of IBD in CD
and UC. NOD2 has been identified (also known as CARD15/IBD1) as a
susceptibility gene in CD using positional cloning and other gene approaches (Hugot
et al., 2001; Ogura et al., 2001). Since then several other genes have also been
implicated in CD, such as IBD5, IL23R, and ATG16L1 (Peltekova et al., 2004; Duerr
et al., 2006; Hampe et al., 2007; Silverberg et al., 2007). NOD2 is a pattern-
recognition receptor which functions as an intracellular sensor for bacterial
peptidoglycan and can also be activated by a small bioactive component of
peptidoglycans, muramyl dipeptide (MDP) (Giardin et al., 2003; Inohara et al.,
2003). The activation of NOD2 by the MDPs results in the activation of NF-κB and
mitogen-activated protein (MAP) kinase signalling pathways (Kobayashi et al.,
2005). ATG16L1, an autophagy-related gene, is involved in the degradation of
intracellular pathogens, regulation of cell signalling and of T-cell homeostasis
(Levine & Deretic, 2007). Since autophagy results in restricting the growth of certain
microorganisms, a mutation in the gene would therefore result in the reduction of
clearance of pathogens and allow a more permissive growth to the intracellular
bacterial pathogens (Amano et al., 2006). The association of CD with
polymorphisms in NOD2 and the ATG16L1 suggests that alterations in the
recognition and intracellular processing of bacterial components may have a role in
the immunopathogenesis of the disease (Cho, 2008). However, studies with NOD2-
deficient mice, which showed the absence of intestinal inflammation, have
highlighted that the dysregulation of the NOD2 pathway alone is insufficient to
induce CD (Pauleau & Murray 2003; Kobayashi et al, 2005). Therefore, being a
carrier of any of these genes does not necessarily lead to developing colitis leading to
IBD. The susceptibility of developing IBD results from the change in microbial
                                          15
factors, which is a consequence from the influence of environmental factors. Genetic
factors affecting the barrier function and the innate and adaptive immunity in IBD
are also affected by the alterations between commensal microbes and the mucosa
leading to intestinal inflammation (Van Limbergen et al., 2007).


The intestinal barrier in IBD
       The intestinal barrier is primarily made up of biofilm with commensal
bacteria, the mucous layer and the epithelium and innate immune defences, including
macrophages, dendritic cells, and neutrophils. Any damage caused to this barrier can
subsequently result in the persistent activation of the immune system (Figure 1).
Immune cells including monocytes, macrophages, T cells and DCs participate in the
detection of microbes, as there is constant communication between the luminal flora
and the underlying dense network of innate and adaptive immune cells in the
epithelium (Guillot et al., 2004). The intestinal epithelium consists of intestinal
epithelial cells (IEC) which are important for the absorption and transportation of
nutrients and the formation of a protective mucosal barrier in the gut which is
normally rapidly regenerated after damage (Han et al., 2003; Okamoto & Watanabe,
2004; Cario & Podolsky, 2005). IECs express Toll-like receptors (TLRs), which are
a family of innate immune recognition receptors that control adaptive immune
responses and induce antimicrobial effector pathways, which, therefore, lead to the
elimination of host-threatening pathogens (Guillot et al., 2004; Cario, 2005).


Aberrant immune response in IBD
       The mucosal immune response is initiated by the sensing of microbes, which
later activates the adaptive immune response (Xavier & Podolsky, 2007). The
activated immune response, the characteristic of IBD, is mostly dominated by the
mucosal CD4+ T lymphocytes (Uhlig et al., 2006). The differentiation of CD4+ T
cells is tightly controlled by cytokines which determine the type of inflammatory
response that occurs in the host. Failure to regulate T cell responses in the intestinal
or colonic mucosa causes the release of pro-inflammatory cytokines which leads to
death of enterocytes and inflammation of the tissue (Weinstein et al., 1997).
Recruited macrophages cause damage to epithelial barrier by producing reactive
oxygen radicals, nitric oxide radicals, and pro-inflammatory cytokines such as
interleukin-12 (IL-12), interleukin 1β (IL-1β), interferon-γ (IFN-γ) and tumour
                                          16
necrosis factor (TNF-α) which play a major role in colonic tissue destruction
(Podolsky, 2002).
       Regulatory T cells (Tregs), are characterised as being CD4+,CD25+ Foxp3+
and produce IL-10 and transforming growth factor beta (TGFβ) which have anti-
inflammatory effects in the gut (Mowat, 2003; Uhlig et al., 2006). Without the
presence of any inflammatory mediators, TGFβ results in the development of Foxp3+
Treg cells, which then suppress inflammatory responses (Kim & Rudensky 2006).
Interestingly, if TGFβ cooperates with IL-6 (a pro-inflammatory cytokine) it results
in the generation of Th17 cells, which are involved in numerous inflammatory
diseases, including colitis (Fantini et al., 2007). This suggests that TGFβ can be both
anti-inflammatory and pro-inflammatory depending on the expression of other
cytokines. Thus the combination of TGFβ and IL-6 results in the inhibition of Treg
cell development and the differentiation of naive CD4+ T cells into Th17 T cells
(Bettelli et al., 2006). Theoretically, the potential role for Tregs to suppress IBD in
humans via the production of TGFβ is also limited. Monteleone et al., (2004) have
shown high levels of Smad7, which prevents TGFβ signalling and also down-
regulates the immune response, in inflammatory cells in IBD lesions. In contrast, Yu
et al., (2006) have claimed that Treg cells in the lamina propria have evolved to
suppress immune responses to the resident commensal bacteria, and play a pivotal
role in modulating the clinical range of UC. The true role of Treg cells in the IBD
immune responses in both humans and mouse models of colitis requires further
examination.
       Dendritic cells are the dominant antigen presenting cell (APC) type in the
lamina propria. They form an extensive network beneath the intestinal epithelium
and can sample luminal antigens via long processes which project through the
interstices of epithelial cells (Chieppa et al., 2006). The sampling of bacteria by
resident DCs is enabled by direct dendritic cell-microbial contact (Niess et al., 2005;
Chieppa et al., 2006). Once stimulated, DCs then migrate to mesenteric lymph nodes
(MLN) where they promote the differentiation of naive T cells into effector and
regulatory T cells. The cytokines secreted by DCs, then stimulates the differentiation
of naive CD4+ T cells into a Th1, Th2, Th17 or regulatory T cells subsets. Aberrant
activity of lamina propria DCs has been suggested to be a vital component of the
immune response in IBD patients (Niess, 2008).


                                          17
                                                                            Lumen




           IEC

                                            Inflammation
                      DC                                      DC             Lamina
                                                                             propria
                                     DC


                                                        IL-12
                                                        TNF-α
                                                        IFN-γ

                                              CD4+ cells            Macrophages
                       Regulatory T cells

 Figure 1. Mucosal immune responses in IBD. An immune response is generated when
 foreign pathogens gain entry through the intestinal epithelial cells (IECs) which act as a
 protective barrier. These foreign antigens then activate dendritic cells (DCs), and
 macrophages in the lamina propria. CD4+ cells release an over production of pro-
 inflammatory cytokines, such as- IL-12, TNF-α, IFN-γ which results in severe damage of
 the colonic tissue. Regulatory T cells, which normally suppress inflammation, maybe
 impaired in IBD.


       The recruitment of activated neutrophils, DCs, and macrophages in the
lamina propria initiates the local immune response, which leads to inflammation
(Fort et al., 1998). The master regulator of cytokine expression is the transcription
factor NF-κB, which resides in the cytoplasm and translocates to the nucleus upon
activation. NF-κB is a pro-inflammatory transcription factor that plays an important
role in immune, inflammatory, and stress responses where it induces the expression
of target genes to promote cell cycle progression, cell survival, adhesion, invasion,
and angiogenesis. However, the inappropriate activation of NF-κB promotes
uncontrolled inflammation (Finco et al., 1997; Bharti & Aggarwal, 2002).


Animal models of IBD
       A significant portion of our understanding of IBD comes from studies in
animal models of intestinal inflammation (Blumberg et al., 1999; Strober et al.,
2002; Pizarro et al., 2003). These animal models have been designed to imitate
different forms of IBD, but they still fail to represent the exact mechanisms of

                                            18
human diseases. However, they are still useful for investigating important aspects
such as the pathogenic mechanisms during the initiation of colitis, and can also act as
important tools in studying the therapeutic effects of drugs in IBD.
       There are numerous animal models used to study the inflammatory
mechanisms in IBD, but the two most commonly used are chemically-induced colitis
and genetically-engineered colitis (Table 1). This review will primarily focus on
these two models used in IBD.


Intestinal inflammation- epithelial integrity
Chemically-induced models
       The intestinal epithelium not only acts as a physical but also as an
immunological barrier which prevents the direct contact of the intestinal mucosa
with the luminal bacteria. Therefore, any damage to the intestinal barrier can be
crucial for the development of IBD, as luminal antigens and microorganisms can
easily gain entry into the mucosa resulting in the initiation of inflammatory
responses (Wirtz & Neurath, 2000). Two models which highlight the intestinal
epithelial cell function in IBD are the dextran sodium sulphate (DSS)-induced model
and 2,4,6-Trinitrobenzene sulfonic acid (TNBS) induced colitis model.
       The TNBS model is very useful in studying aspects of gut inflammation,
including cytokine secretion patterns, mechanisms of oral tolerance and
immunotherapy (Wirtz et al., 2007). In this model the TNBS solution has to remain
in the colon lumen to create reproducible results. Therefore, this model requires
constant observation when inducing colitis. Higher doses of these compounds can
lead to substantial death rates resulting from colon damage (Wirtz et al., 2007).
There is a high variability within experimental groups, and with the reproducibility
of the injury and inflammation, which are either strain-specific or specific to the lot
of TNBS (Fedorak & Madsen, 2000). For example, C57BL/6 mice are relatively
resistant to TNBS, whereas BALB/c mice demonstrate higher severity of colitis.
Significant care needs to be executed during the intrarectal step to make sure that the
gut is not damaged or punctured.
       The DSS model in comparison to the TNBS model is a simple oral
administration of these polymers. DSS is a heparin-like polysaccharide which
contains three sulphate groups per molecule. DSS in drinking water can induce both
acute and chronic colitis in mice, depending on the time course of the oral
                                          19
administration of DSS (Vowinkel et al., 2004). DSS-induced colitis exhibits
morphological and pathophysiological features that resemble human UC (Hamilton
et al., 1983; Okayasu et al., 1990; Cooper et al., 1993). Features such as superficial
ulcerations, mucosal damage, production of cytokines and other inflammatory
mediators, and leukocyte infiltration are similar to the human UC (Okayasu et al.,
1990; Cooper et al., 1993; Elson et al., 1995). The direct toxic effect of these
polymers on the colonic epithelium affect on the integrity of the mucosal barrier
(Fedorak & Madsen, 2000), stimulate macrophage activation, and the alteration of
colon microflora have all been implicated in the pathogenesis of DSS-induced colitis
(Okayasu et al., 1990; Cooper et al., 1993). Kitajima et al., (2000) have shown that
colitis in mice treated with 40kDa DSS was more severe when compared to mice
treated with 5kDa DSS, and there was also a difference in location of colitis in the
colon.
         The severity of DSS-induced colitis depends on the molecular weight,
sulphate percentage (of DSS), and the dosage and duration of the DSS administration
(Okayasu et al., 1990; Yamada, et al., 1992; Cooper et al., 1993; Axelsson et al.,
1996). Therefore, these factors have to be considered when the DSS-colitis is used in
mice. As is the case with TNBS model, DSS induced colitis is also affected by the
mouse strain, the age of the mice, individual differences in the intestinal microflora
between the animal groups, the dosage applied to these animals and the duration of
DSS induction (Wirtz et al., 2007). BALB/c mice develop mild to severe colitis with
5% DSS, whereas C57BL/6 mice develop mild to severe colitis with 2% DSS.
However, in comparison to other colitis models, this model can produce acute,
chronic or relapsing forms of colitis by changing the concentration and cycle of
administration of DSS (Okayasu et al., 1990). This model is widely used in the
exploration of immunological mediated aspects of chronic mucosal inflammation,
and also for evaluating new potential therapeutic agents.


Intestinal inflammation- cells of the adaptive immune system
Genetically engineered models
         The genetically engineered models of IBD have allowed the exploration of
the interaction between genetic and environmental factors (Fedorak & Madsen,
2000). Although, these models do not necessarily duplicate all the histologic and
clinical characteristics associated with IBD, they do exhibit features which are
                                         20
similar to human disease, such as the development of colitis and acute inflammation
(Fedorak & Madsen, 2000; Wirtz et al., 2000).           There are several genetically
engineered models, but one of the most commonly used is the IL-10 knock-out,
mouse which causes a disease with similar features to CD in humans (Van Deventer
et al., 1997; Schreiber et al., 2000). Other transgenic models such as T-cell receptor
(TCR) mutant mice and NEMO (NF-κB essential modulator) are also briefly
discussed in this review.
       IL-10 is a potent anti-inflammatory cytokine which down-regulates activation
of Th1 cells, and also inhibits macrophage inflammatory cytokine production
including TNF-α, IL-1, and IL-2, and other T-cell associated macrophage activity
(Schreiber et al., 2000). In this model, the IL-10 gene is inactivated by targeted
mutation (Fedorak et al., 2000). Furthermore, IL-10-/- mice experience a
“spontaneous” enterocolitis which is dependent on the presence of luminal flora
(Kuhn et al., 1993). IL-10-/- mouse studies have shown that a Th1 cell response is
generated, which in normal mice is suppressed by Th2 cell production of IL-10
(Berg et al., 1996). These increased levels of pro-inflammatory cytokines in IL-10 -/-
mice imply that the uncontrolled cytokine production by the macrophages
contributes to the pathogenic response (Rennick et al., 1997; Fedorak & Madsen,
2000). However, the disease onset of this model is prolonged, and the expression of
colitis can take several months (Kuhn et al., 1993). The severity of the disease in this
spontaneous gene targeted model can be inconsistent and depends on environmental
factors such as commensal flora (Wirtz et al., 2007). However, there are variations in
the IL-10-/- mice such as the differences in the strain of mice, and also in the
phenotype of colitis. Nevertheless, this model of IBD has similar features as CD in
humans, which helps to develop our understanding of the pathogenesis of the
disease.
       Models such as TCR deficient mouse colitis and NEMO -/- have similar
features to UC (Mombaerts et al., 1993; Schmidt-Supprian et al., 2000). The TCR
deficient model and NEMO -/-, like IL-10 -/- mice, also take a long time to develop
colitis, but the colitis develops differently. TCR mice develop chronic colitis
spontaneously with an increase in T and B cell production. In comparison, NEMO -/-
mice develop colitis by extensive epithelial destruction, suggesting that the epithelial
integrity is damaged by the invasion of bacteria from the lumen into the mucosa
(Nenci et al., 2007; Zaph et al., 2007). Zaph et al., (2007) showed that NEMO -/-
                                          21
mice are unable to eliminate infection by pathogens, suggesting that NF-κB plays an
important role in IECs against pathogens through protective immune responses.


      Table 1. Different mouse models used to induce colitis and their
                               mechanisms.
  Mouse models         Mode of            Mechanisms                  References
                     administration     underlying colitis
Chemical induction
TNBS                 Intrarectal       Breakage of mucosal       Morris et al., 1989;
                                       barrier resulting in      Neurath et al, 1995;
                                       Th1 response (similar     Elson et al., 1996;
                                       to CD) with increased     Strober et al., 2002;
                                       production of             Wirtz et al., 2007
                                       inflammatory
                                       cytokines (IL-12,
                                       IFN-γ, and TNF-α)
                                       and chemokines from
                                       infiltrating cells and
                                       isolated macrophages
DSS                  Oral              Acute colitis is          Fedorak & Madsen,
                                       stimulated through        2000
                                       the direct toxic effect
                                       of DSS on colonic
                                       epithelial cells
                                       affecting the integrity
                                       of the mucosal barrier
Genetically engineered
IL-10 -/-            Genetically       Lack of anti-             Kuhn et al., 1993
                     transferred       inflammatory
                                       cytokine IL-10,
                                       causes spontaneous
                                       colitis due to
                                       suppressed immune
                                       tolerance
TCR mutant mice      Genetically       Spontaneous colitis;      Mombaerts et al., 1993
                     transferred       Lack majority of
                                       CD4+ and CD8+ T
                                       cells. Suggested to be
                                       associated with an
                                       increase in Th2
                                       response similar to
                                       UC
NEMO -/-             Genetically       Blocks NF-κB by           Schmidt-Supprian et
                     transferred       pro-inflammatory          al., 2000
                                       cytokines and
                                       interferes with the
                                       generation of
                                       lymphocytes. Similar
                                       to UC




                                        22
          Each mouse model has its advantages and disadvantages, and this highlights
the importance of utilising a number of different models to trial drugs or helps
understand specific features of IBD.


Current treatments for IBD
          Over the years, there has been intense study regarding the treatment of IBD.
However, new treatments which suppress inflammation and restore damaged mucosa
are required. Table 2 summarises the commonly used drugs and their mechanisms of
action.
          5-ASA or mesalamine used to treat patients with IBD in its active disease
state is known to maintain patients in remission (Sutherland & Shaffer, 1993).
Although it is considered to be a safe drug, its efficacy rates are not very high.
Nevertheless, past studies have shown that there seems to be a dose–response
relationship in the efficacy for the treatment of both UC and CD (Khan et al., 1977;
Khan et al., 1980; Singleton et al., 1993). However, long-term treatment with
mesalamine in CD is not as effective as it is for UC.
          In comparison, corticosteroids reduce inflammation in the intestine of
patients with UC and CD. They have anti-inflammatory actions by suppressing the
immune system, however, this results in patients being more susceptible to
infections. Corticosteroids are unable to keep UC or CD disease in remission and
also cannot prevent the re-occurrence of the disease after surgery. The side-effects
related to corticosteroids are dependent on the dose and the duration of the therapy
(Hanauer et al., 1991; Lindmark, 1993). Therefore, long-term uses of corticosteroids
are discouraged, due to the risk of long-lasting side effects. Patients who take these
drugs are at a risk of developing osteoporosis and long-term use in children can
result in delayed growth. Immunomodulators such as AZA and 6-MP have a slow
onset of action compared to corticosteroids (Dieleman et al., 1991). Since these
drugs suppress the immune system there are greater chances of infection. 6-MPs also
cause severe hepatotoxicity in a significant number of patients (Dieleman et al.,
1991).




                                           23
         Table 2. Therapeutic treatments and their mechanisms in IBD.
Therapeutic drugs      Disease    Mechanisms of         Side effects           References
                                     Action
5-ASA/Mesalamine      CD &       Inhibits IL-1, NF-   Kidney related         Jarnerot, 1994;
                      mild-      κB, TNF-α            problems, when         Egan et al., 1999;
                      moderate   activation, and B    administered in        Kaiser et al.,
                      UC         cells and oxygen     high doses             1999; Nikolaus et
                                 radical production                          al., 2000; Weber
                                                                             et al., 2000;
                                                                             Sutherland et al.,
                                                                             2002 & 2003
Corticosteroids       Moderate- Down-regulates the    Dyspepsia,             Lindmark, 1993;
                      severe    activation of NF-     hyperglycemia,         Auphan et al.,
                      CD & UC κB, and also            alteration of fat      1995; Brattsand &
                                decreases cytokine    distribution, high     Linden, 1996;
                                production by         risk of infection,     Hanauer & Kane,
                                inducing cytokine     and adrenal            2000
                                inhibition.           suppression
                                Decreases IL-1 and
                                IL-2 production
Immunomodulators      CD        Inhibits cell         Nausea, fever,         Sandborn, 1996;
- AZA & 6-MP                    proliferation in      rash, joint pain,      Hanauer & Kane,
                                DNA synthesis         diarrhea, renal        2000
                                phase of the cell     dysfunction, and
                                cycle                 high risk of
                                                      infection
Infliximab            CD         Blocks the action    Allergic reaction      Knight et al.,
                                 of TNF-α by          or a delayed           1993; D’Haens et
                                 binding to it and    hypersensitivity       al., 1999;
                                 prevents its         reaction, high         Hanauer et al.,
                                 signalling to the    blood pressure,        2002
                                 TNF-α receptors on   chest pain,
                                 the surface of       difficulty in
                                 leukocytes and       breathing, and
                                 epithelial cells     high risk of
                                                      infection (i.e.
                                                      respiratory)
Probiotics            CD & UC    Unknown, but has     Not many known, Hart et al., 2003;
                                 been suggested to    but may cause anMach, 2006;
                                 suppress growth or                   Ebtissam, 2007
                                                      allergic reaction if
                                 the epithelial       taken with
                                 binding by           another
                                 pathogenic           medication or
                                 bacteria. Increases  supplement
                                 production of
                                 protective
                                 cytokines, IL-10,
                                 and suppress
                                 production of pro-
                                 inflammatory
                                 cytokines, TNF-α
       *5-ASA (5-Aminosalicylates); AZA (Azathioprine); 6-MP (6-Mercaptopurine)



                                           24
        The monoclonal antibody infliximab is a mouse-human chimeric antibody to
TNF-α (Knight et al., 1993). TNF-α is one of the key cytokines that triggers and
sustains the inflammatory response (D’Haens et al., 1999; Hanauer et al., 2002). Due
to immunogenity, patients being treated with infliximab can form human anti-
chimeric antibodies (HACA), but co-treatment with immunomodulators can reduce
the risk of HACA formation (Kuhbachler & Foisch, 2007). There is also a risk of
decreased white and red blood cells, as well as decreased platelet count. Even though
they are an efficacious method for treating CD the disadvantage with this therapy is
that it is relatively expensive and infection is a serious side-effect. There are other
antibodies in the pipeline for treatment of CD. IL-23R gene is associated with CD,
but has also been reported in patients with UC (Duerr et al., 2006). IL-23 is
expressed at high levels by activated macrophages and DCs and the functional IL-23
cytokine is comprised of p19 and p40. Blocking antibodies specific for p40 inhibits
IL-23 and IL-12 induced signaling. This administration of the p40-specific
antibodies has proved to be a promising approach for the treatment of CD (Mannon
et al., 2004).
        A contentious class of therapeutic agents for treatment of IBD are probiotics.
Probiotics are living microorganisms which are present in the intestine as normal
flora and are important to the health and well-being of the host (Campieri et al.,
2001). Increasing evidence suggests that gastrointestinal microflora are involved in
the pathogenesis of IBD in genetically susceptible subjects with immunological
dysregulation (Dotan & Rachmilewitz, 2005). Supporting this hypothesis is the
observation that there is an increase in the number of microorganisms and a change
in various populations of normal flora in IBD patients (Ebtissam, 2007). The
interactions between the commensal microflora and the intestinal mucosa stimulate
inflammatory activity (Dotan & Rachmilewitz, 2005; Bai & Quyang, 2006). A recent
study by Fedorak (2008) has identified the mechanism of action of probiotics to have
direct effect on epithelial cell function and intestinal health, including enhancing
epithelial barrier function, modulating epithelial cytokine secretion into an anti-
inflammatory dominant profile, altering mucus production, changing bacterial
luminal flora, modifying the innate and systemic immune system, and inducing
regulatory T-cell effects. However, for probiotics to have a therapeutic effect on
IBD, their therapeutic mechanism of action has to be associated with the pathogenic
mechanism of action of the disease (Fedorak 2008). Furthermore, recent studies have
                                          25
shown that the administration of probiotics ameliorates inflammation by exerting
positive effects on epithelial cell and mucosal immune system dysfunction which
form the basis of the inflammation (Boirivant & Strober, 2007). Nevertheless, the
use of probiotics for therapy requires both in vitro and in vivo study to prove their
effectiveness in IBD.
       The use of antibiotics such as metronidazole and ciprofloxacin seem to have
a scientific rationale in treating IBD especially in the long-term treatment of CD and
in the management of pouchitis (Guslandi, 2005). However, the use of antibiotics in
IBD is still debated as they are administered for long periods of time resulting in
tolerability problems as well as adverse side effects (Guslandi, 2005; Perencevich &
Burakoff, 2006).


New treatments for IBD need to be evaluated
       Peptides isolated from the gut have shown promising effects in animal
models of IBD. Gut peptides such as ghrelin, vasoactive intestinal peptide (VIP),
cortistatin, and somatostatin have been shown to exhibit anti-inflammatory effects
(van Bergeijk & Wilson, 1997; Delgado et al., 1999; Dixit et al., 2004; Li et al.,
2004; Gonzalez-Rey et al., 2006; Szliter et al., 2007). Ghrelin and VIP suppress
inflammation through both the innate and adaptive immune systems (Delgado et al.,
1999; Dixit et al., 2004; Szliter et al., 2007). In a toxin-induced colitis study, it was
found that ghrelin had anti-inflammatory effects and decreased the severity of colitis
and inflammation and prevented the recurrence of the disease (Gonzalez-Rey et al.,
2006). It significantly reduced the clinical symptoms and pathology by down-
regulating both pro-inflammatory and Th-1 mediated immune responses (Gonzalez-
Rey et al., 2006). Ghrelin has been shown to affect cell proliferation in colon cancer
cell lines (Taufiq et al., in preparation). Our work demonstrates that nanomolar
concentration of ghrelin significantly increases the proliferation of human
colonocytes. Novel treatments are required which will not only suppress
inflammation by acting directly on the immune system, but also repair and restore
the injured epithelium to prevent further damage. Therefore, ghrelin peptides are
promising therapeutic candidates for treating IBD and other inflammatory diseases.




                                           26
Ghrelin
         Ghrelin is a 28-amino acid peptide which is octanoylated at its third amino
acid and is the endogenous ligand for the ghrelin receptor, the growth hormone
secretagogue receptor (GHS-R1a) (Kojima et al., 1999). Ghrelin is mainly produced
by stomach tissue and it has an unusual acyl modification on its critical serine-3
residue (Kojima et al., 1999). The acyl modification is important for the activation of
the GHS-R1a (Kojima et al., 1999). As well as stimulating growth hormone (GH)
release (Kojima et al., 1999), ghrelin also influences appetite (Nakazato et al., 2001),
energy balance (Inui, 2001), and gastric motility (Sibilia et al.,2002). Ghrelin
circulates in the plasma in two forms: acylated (octanoylated) and non-acylated (des-
octanoyl or des-acyl) ghrelin. Ghrelin is predominantly expressed in the entero-
endocrine cells of the stomach although co-expression of ghrelin and the GHS-R1a
have been observed widely throughout human and mouse tissues at the mRNA or
protein levels, albeit at low levels (Date et al., 2000). Ghrelin expressing cells, also
known as X/A-like cells, are found in the stomach, where they are not in contact
with the lumen but are close to the capillaries (Korbontis et al., 2004). Ghrelin is
found in the fundus of the stomach and also in a smaller number of immunopositive
cells in the small and large intestine (Korbontis et al., 2004). Lee et al., (2002) have
shown the presence of ghrelin within the mucosa of both rat stomach and colon, with
the highest levels being in the stomach. It has also been demonstrated in RT-PCR
analyses that ghrelin receptor mRNA expression is present in many peripheral
organs, such as heart, lung, liver, kidney, pancreas, stomach, small and large
intestines, adipose tissues, and immune cells which indicate that ghrelin has multiple
functions in these tissues (Guan et al., 1997; Hattori et al., 2001; Gnanapavan et al.,
2002).
         In our study we also observed mRNA and protein expression of ghrelin and
its receptor, GHS-R1a, in colon epithelial cell lines (Taufiq et al., in preparation). In
particular ghrelin and its receptor are expressed in the Caco-2 cell line, which is
similar in biochemical and morphological aspects to normal intestinal enterocytes.
As summarised in Table 3, ghrelin has been shown to affect various factors including
cell proliferation and inflammation. The expression of ghrelin and its receptor have
been identified in T cells, which suggest that ghrelin levels found in IBD patients can




                                           27
clarify the possible involvement of ghrelin in intestinal inflammation (Hattori et al.,
2001; Dixit et al., 2003; Dixit et al., 2004).
       Ghrelin also affects epithelial cell proliferation in several different cell types
(Pettersson et al., 2002; Jeffery et al., 2003; Kim et al., 2004; Mazzocchi et al.,
2004; Zhang et al., 2004; Jeffery et al., 2005 a, b; Maccarinelli et al., 2005;
DeVriese et al., 2005). We have observed in our previous study that ghrelin
increased cell proliferation in Caco-2 (colon epithelial) cell lines by nearly 50% with
the administration of nanomolar concentration of ghrelin (Taufiq et al., in
preparation). However some studies have shown that ghrelin inhibits proliferation
(Xia et al., 2004). The effect of ghrelin on cell proliferation has been shown to have
conflicting results (Nanzer et al., 2004), however, these apparent discrepancies may
be due to different cells types and receptor expression.


                 Table 3. Multiple Effects of Ghrelin function.
        Parameter                 Effects                       References
GH release                                       Kojima et al., 1999

Appetite                                         Nakazato et al., 2001

Obesity                                          Tschop et al., 2000

Inflammation                        ?            Dixit et al., 2004; Dixit et al., 2009; Li et
                                                 al., 2004; De Smet et al., 2009
Cardiovascular functions

       Cardiac output                            Nagaya et al., 2001;

        Blood pressure                           Nagaya & Kangawa, 2003
Gastric functions

       Gastric motility                          Sibilia et al.,2002

Cell Proliferation                  ?            Jeffery et al., 2002; 2003; 2005a, b;
                                                 DeVriese et al., 2006

Ghrelin gene derived peptides
       The 28 amino acid peptide hormone, ghrelin is derived proteolytically from a
precursor of 117 amino acids (Kojima et al., 1999). Zhu et al (2006) have shown that
cleavage of the 23 amino acid signal sequence yields pro-ghrelin, which has a
glycine as its N terminal residue. However, the C terminus of the mature ghrelin is

                                            28
generated via the prohormone convertase 1/3 (PC1/3), which cleaves after the
arginine-28 of pro-ghrelin, creating the mature 28 amino acid peptide (Zhu et al.,
2006). Furthermore, several potentially functional peptide hormones can be derived
from alternative splicing of the ghrelin gene, as can be seen in Figure 2. These
include the mature ghrelin peptide, obestatin, and the novel Δ4 C-terminal proghrelin
peptide (Jeffery et al., 2003; Jeffery et al., 2005a,b). It has been predicted that the
exon 4-deleted variant is produced potentially by an alternative splicing mechanism
such as exon skipping (Jeffery et al., 2005a,b). The exclusion of exon 4 from mouse
preproghrelin mRNA transcript results in a cDNA frameshift that, when translated,
encodes a unique 16 amino acid C-terminal peptide sequence (Δ4 proghrelin
peptide) which is highly conserved (Jeffery et al., 2003; Jeffery et al., 2005a,b).
However, whether this unique C-terminal peptide of the Δ4 proghrelin peptide is
functionally active or not has not been determined.


                                                Exon
          5’                                                                           3’
                         1       2         3             4             5
                        Transcription, translation, and alternative splicing



      2             3                4           5           2             3       5
                             Cleaved and post-translationally modified



               2             3              4                2             3        5
                   ghrelin               obestatin               ghrelin       Δ4 peptide

  Figure 2. Alternative splicing of mouse ghrelin gene generates multiple ghrelin
  peptides.



Ghrelin O-Acyltransferase (GOAT)
       Ghrelin is the only protein in animals that is known to be modified by O-
acylation with octanoate, an eight-carbon fatty acid, which is necessary for the
central action of ghrelin (Kojima & Kangawa, 2005), however, the enzyme which
catalyses this modification has only recently been discovered (Yang et al., 2008).


                                                29
The polytopic membrane-bound enzyme which attaches octanoate to serine-3 of
ghrelin has been named Ghrelin O-Acyltransferase (GOAT). GOAT belongs to the
family of Membrane-Bound O-Acyltransferases (MBOATs), which attach fatty acids
to lipids and proteins (Hofmann, 2000). GOAT is a unique enzyme since it is the
first family member which transfers a medium-chain fatty acid such as octanoate,
whereas previously studied enzymes transferred long-chain fatty acids of at least 16
carbons (Yang et al., 2008). Yang et al, (2008) further discuss that since ghrelin is
the only known octanoylated protein in animals, it is perhaps the sole substrate for
GOAT. The relative distribution of GOAT mRNA in different mouse tissue reveals
that like ghrelin, GOAT is highly expressed in the stomach (Yang et al., 2008).
Gutierrez et al., (2008) have identified and characterised human GOAT, and have
also demonstrated the occurrence of ghrelin and GOAT in stomach and pancreas
tissue implicating a crucial role of GOAT in the acylation of ghrelin in pancreatic
function. Since ghrelin is primarily localised to a minor population of X/A cells it
will be interesting to determine whether GOAT is also restricted to these cells (Yang
et al., 2008). There was a moderate level of expression of GOAT mRNA in mouse
tissue of the colon suggesting that is does play some role in the acylation of ghrelin
in the colon. Since we have demonstrated the expression of ghrelin in some colonic
epithelial cells, it would therefore be interesting to identify the role of GOAT and its
acylation of ghrelin in both human and mouse colon. GOAT is now being studied as
a critical molecular target in developing novel therapy for obesity, type-2 diabetes,
and perhaps in inflammatory-mediated diseases (Gutierrez et al., 2008).


The role of Ghrelin in inflammation and IBD
       Ghrelin has been described as a potent anti-inflammatory factor which
inhibits the production of pro-inflammatory cytokines through activated monocytes
and endothelial cells, and results in protection from endotoxic shock (Dixit et al.,
2004; Li et al., 2004). An inhibitory effect of ghrelin on cytokine mRNA expression
might be responsible for the inhibition of splenic T cell proliferation by ghrelin in
vitro (Xia et al., 2004). Ghrelin also modulates an immune response in several
disease processes such as: arthritis (Granado et al., 2005) pancreatitis (Dembinski et
al., 2003), stomach inflammation (Ariyasu et al., 2004; Konturek et al., 2006) and
intestinal colitis (Gonzalez-Rey et al., 2006). In a study by Konturek et al., (2006) it
was found that due to its anti-inflammatory properties, ghrelin exerts strong
                                          30
protective actions on the gastric mucosa and also accelerates healing of lesions.
Ghrelin and its receptor have been identified in T cells, and can inhibit the activation
of cytokines including, IL-1β, IL-6, TNF-α, and also leptin (Dixit et al., 2003).
Ghrelin also reduces Th1 (IL-2 and IFN-γ) cytokine mRNA expression in activated
lymphocytes, and almost completely inhibits Th2 cytokine mRNA expression (Xia et
al., 2004). What is also interesting is that serum levels of ghrelin are increased in
patients with IBD (Karmiris et al., 2006). Therefore, elevated serum levels are a
feature of active IBD, suggesting a role for endogenous ghrelin in active human IBD
(Peracchi et al., 2006).
       NF-κB is considered to play an important role in inflammation and IBD
pathogenesis (Finco et al., 1997; Bharti & Aggarwal, 2002). Ghrelin has strong anti-
inflammatory effects in human endothelial cells, potentially mediated by the
inhibition of NF-κB activation. Li et al., (2004) have also shown that ghrelin inhibits
TNF-α-induced NF-κB activation, whereas Zhao et al., (2006) claim that the up-
regulation of ghrelin during colitis is through NF-κB activation. Nevertheless, Zhao
et al., (2006) have not addressed the functional importance of the overexpression of
the ghrelin receptor in pathogenesis of colitis. Due to the differences in results,
further research into the role of ghrelin in inflammation and IBD is required to
understand the complete mechanism of its actions.


Ghrelin in mouse models of colitis
       A study by Gonzalez-Rey et al., (2006) used a TNBS-induced colitis model
which, like CD, causes an archetypal CD4+ Th1 cell-mediated colitis, to investigate
the effect of ghrelin treatment on IBD. In this study the anti-inflammatory effect of
ghrelin decreased the severity of colitis and inflammation and prevented the
recurrence of the disease (Gonzalez-Rey et al., 2006). The therapeutic effect was
linked with the down-regulation of both pro-inflammatory cytokines (IL-12, IFN-γ,
and TNF-α) and the Th1-driven autoimmune response and increased levels of the
anti-inflammatory cytokine IL-10 (Gonzalez-Rey et al., 2006). Ghrelin treatment
reduced colonic infiltration of neutrophils, and macrophages due to the down-
regulation of multiple chemokines and cytokines in the lamina propria. Interestingly,
lamina propria mononuclear cells and mesenteric lymph node (MLN) T cells, which
were isolated from ghrelin treated mice, secreted lower levels of pro-inflammatory
cytokines in vitro upon activation (Dixit et al., 2004; Gonzalez-Rey et al., 2006).
                                          31
The study did not investigate the effects of ghrelin on epithelial barrier damage and
did not look at the expression of ghrelin and its receptor, GHSR, in the colonic
specimens from the mice. The histological sections did not specify from what region
of the colon the histopathology scores were derived. This is important especially in
the 5kDa DSS model, where the location of damage induced by DSS is dependent
upon the molecular weight of DSS and the mouse strain. Also the cytokine and
chemokine contents from the protein extracts were measured from only the TNBS
model and not the DSS model. Since these two models induce different kinds and
degree of colitis, it is important to measure the levels of cytokine/chemokines from
each model.


Relevance of Project
       The advancement in the understanding of mucosal immunology in IBD in
recent years has generated a robust variety of potential therapeutics. These potential
therapeutic agents may be useful in treating CD and UC however; thorough pre-
clinical studies need to be undertaken. The recent discovery of the anti-inflammatory
actions of ghrelin in colitis, have raised many questions related to the role of ghrelin
in the colonic epithelial barrier and the mucosal immune system. According to
current studies it is evident that ghrelin does influence production of inflammatory
cytokines and transcription factors by both antigen presenting cells and immune
effector cells, and is an element of co-regulation between the endocrine and the
immune system. The recent discovery of exon 4-deletd preproghrelin and its high
expression in the stomach creates a new aspect in the field of ghrelin research.
        A significant knowledge gap is whether ghrelin acts as a pro- or anti-
inflammatory mediator during immune responses. This research project will
investigate the mechanism of ghrelin peptides by treating mice with colitis with
different concentrations to determine whether the ghrelin peptides will ameliorate
colitis. This study will also compare the disease severity of acute colitis induced by
DSS in two different mouse strains. It has been identified that C57BL/6 mice are
more susceptible than BALB/c mice to DSS-induced colitis (Sasaki et al., 2008). In
a study by Kitajima et al., (2000) the molecular weight of the DSS polymers used to
induce colitis made a huge impact on the severity of colitis and epithelial damage of
the mucosa. They showed that the severity and primary location of colitis differ with


                                          32
administration of DSS at different molecular weights for 7 days in mice (Kitajima et
al., 2000). Their study concluded that colitis induced by 40kDa DSS was more
severe than that induced by 5kDa DSS in BALB/c mice. The differences in genetic
background are also a major determinant of the inflammatory response. While the
inflammatory response in C57BL/6 is driven by Th1, the response in BALB/c mice
is Th2-driven. This reflects the fact that BALB/c mice have a defective Th1 response
and are more susceptible to bacterial infections such as Leishmania major. C57BL/6
infected mice, in comparison, effectively mount a Th1 response, leading to the
clearance of the infection followed by healing (Sacks & Noben-Trauth, 2002). This
leads us to predict that the induction of colitis in these two strains of mice will be
relatively different both histopathologically and immunologically. Therefore, our
project will use 5kDa and 40kDa of DSS to develop colitis in BALB/c and C57BL/6
mice, predicting that mice treated with 40kDa will develop more severe colitis
compared to mice treated with 5kDa.
       The first aim of this project is: To determine whether the administration of
ghrelin and Δ4 proghrelin peptide will ameliorate intestinal inflammation in two
different mouse strains with DSS-induced colitis. We hypothesise that ghrelin
peptides will reduce colitis and rescue epithelial damage through proliferative
mechanisms. Since IBD is a multifactorial disease which is caused by both
environmental and immunological factors, we will examine the influence of the
ghrelin peptides in two different animal strains which will be treated with different
molecular weights of DSS to induce colitis. This will be the first study to investigate
the functional significance, if any, of Δ4 proghrelin peptide in DSS-induced colitis
mouse models. Mice in the DSS model will develop acute colitis, and will be treated
with the ghrelin peptides. The inflammatory activity will be measured by scoring
histopathology in the colon and measuring the expression of pro-inflammatory
cytokines and the relative abundance of Treg cells from the mesenteric lymph nodes.
       Another knowledge gap is whether ghrelin is working at the epithelial level.
It has been previously demonstrated by our group that ghrelin and its receptor are
expressed in some colon cancer cells, but whether the protective mechanism in
colitis occurs at an epithelial level is still not clear. Previous studies have revealed
that both exogenous and endogenously produced ghrelin increases proliferation in
some in vitro cell systems (Waseem et al., 2008). Therefore the second aim of this
project is: To investigate whether exogenous ghrelin modulates intestinal
                                          33
proliferation/migration using colon cancer cell lines, as in vitro models of
intestinal epithelium. We hypothesise that ghrelin peptides improve epithelial would
healing in colon cancer cell line- HT29. In this model, the cells are grown to
confluence and then insulted by creating a “wound”. The cells are then treated with
different concentrations of ghrelin peptides over 48 hours, photographed at regular
intervals. This study would act as a model for intestinal epithelial growth and/or
recovery after treatment with ghrelin.
        In a study by Konturek et al., (2006) it was found that due to its anti-
inflammatory properties, ghrelin exerts strong protective actions on the gastric
mucosa and also accelerates healing of lesions. Therefore, it would be interesting to
identify whether ghrelin will cause wound healing in the colonic epithelium. The
exact mechanism through which ghrelin may mediate proliferation and migration are
not clear.


Conclusion
        The current therapies for treating patients with severe IBD are often
ineffective and have been associated with numerous side effects. We thus require
ideal treatments which will not only suppress inflammation by acting directly on the
immune system but will also repair the injured colonic mucosa to prevent any further
damage. The recent discoveries of the anti-inflammatory actions of the ghrelin
peptides in the immune system are promising. With the help of experimental animal
models, which have clinical manifestations similar to those observed in IBD, we can
investigate the immunological, pathological, and physiological features of intestinal
inflammation in IBD. These models can help us to elucidate the influence of ghrelin
on the immune system including on T cells, dendritic cells, and regulatory T cells.
Figure 3 summarises a hypothesised role of ghrelin peptides in the immune system,
where they potentially target DCs which may affect T cells directly and cause the
production of anti-inflammatory cytokines to suppress inflammation caused in
colitis. Therefore, future studies in the role and underlying mechanisms of novel
ghrelin peptides in IBD will contribute to the development of new targets for
medical therapies.




                                         34
                                                                  Lumen




    IEC
                                                                  Lamina
                DC       Ghrelin                     DC           propria
                         peptides

                     IL-10                   IL-12
                                             TNF-α
             IL-10                           IFN-γ
             TGFβ
             Foxp3                                         Macrophages
                                    CD4+ cells
               Regulatory T cells

Figure 3. A potential mechanism of ghrelin peptides in inflammation. We
hypothesise that ghrelin peptides can alter the functions of DC and cause them to
tolerise and induce the differentiation of Foxp3 expressing Treg cells. Treg cells
will then secrete the production of anti-inflammatory cytokines, such as IL-10 and
TGFβ, and suppress the production of pro-inflammatory cytokines such as IL-12,
TNF-α, IFN-γ in response to IEC barrier injury. Ghrelin could also work directly
on mucosal epithelial cells and T cells to repress inflammation.




                                        35
              Chapter Three: Materials and Methods

3.1 Mice
       Female BALB/c and C57BL/6 mice, six weeks old, were purchased from
Animal Resources Centre (WA, Australia) and housed in individual filtered air cages
within the MMRI animal handling facility under 12:12-h light-dark cycles. The mice
were fed a standard rodent pellet diet and tap water and were acclimatized for a
minimum of one week before the start of the study. All animal experiments were
approved by the University of Queensland animal ethics committee (AEC No.
MMRI/397/08).


3.2 Induction of colitis and experimental design
       Experimental colitis was induced by the administration of dextran sodium
sulphate (DSS) dissolved in drinking water. Two molecular weights of DSS were
used: 40kDa (ICN Biomedicals, Costa Mesa, CA) and 5kDa (Wako Pure Chemicals,
Tokyo, Japan). BALB/c mice were given five percent DSS and C57BL/6 mice
received two percent DSS as they are more susceptible to DSS-induced colitis (Wirtz
et al., 2007). Mice were divided into groups of six per cage/treatment and the
experiment was performed twice (N=10-12 per treatment; four animals died
spontaneously during two separate experiments). DSS was administered for eight
days   and   mice    were   sacrificed   on   day   eight.   Octanoylated    ghrelin
(1nmol/mouse/day), ghrelin Δ4 peptide (1nmol/mouse/day) or sterile phosphate-
buffered saline (PBS,vehicle control) were injected intraperitoneally on days four
and six. The time period for DSS treatment and timing of injections were optimised
by the lab previously, and is also a published protocol (Gonzalez-Rey et al., 2006).
Control mice (naives) received tap water only. Ghrelin (Auspep, Parkville, Victoria)
and ghrelin Δ4 peptide (Mimotopes, Clayton, Victoria) were diluted in sterile PBS
immediately before injection.


3.3 Assessment of inflammation: symptoms and inflammatory score
       The development of colitis was assessed daily by measurement of body
weight and evaluation of stool consistency, faecal bleeding, and diarrhoea. Clinical
symptoms, including shivering, hunching and ruffling of the coat were evaluated


                                         36
daily using clinical score indices as described more fully in the score sheet
(Appendix 1.1). Body weight on day zero was taken as 100% weight and change in
weight each day was measured as a deviation from day zero weight. The
presence/severity of diarrhea was scored on a scale of 0 to 3 (0= normal; 1= slightly
soft but well formed; 2=soft and not well formed; 3=watery faeces with mucus).
Similarly, rectal bleeding was defined as faeces containing visible blood or gross
rectal bleeding scored on a scale of 0 to 3 (0= no traces of blood; 1= specks of blood
in the faeces; 2= blood present in faeces; 3= prominent blood in the faeces and/or
sticking to fur around the rectum). The cumulative score for diarrhea and rectal
bleeding was calculated by adding the scores for each day.


3.4 Haematological analysis
       Blood samples were collected on the final day of the experiment (day eight).
Blood was collected from the mice either by tail vein bleed, cardiac puncture (in
extreme cases where mice were too sick to bleed) or submandibular puncture,
following which the mice were sacrificed by either asphyxiation or cervical
dislocation. For tail vein bleeds the animals were placed in containers and heated for
5-10 minutes with an infrared heat lamp to promote vasodilation. Each animal was
then placed in a plastic restrainer which only exposed the tail. A sterile scalpel blade
was used to cut one of the lateral veins in the tail to collect blood into eppendorf
tubes containing 2µl heparin (BD Biosciences). Haematocrit, total leukocyte counts
and differential white cell counts were measured in the blood samples (50µl) using
the Sysmex SF3000 Haematology Analyser (Sysmex Corp, Kobe, Japan).


3.5 Tissue collection
       Mice were euthanized by either asphyxiation or cervical dislocation on day 8.
Post-mortem, the colons were excised, opened longitudinally and washed gently in
PBS to remove faecal debris and their lengths were measured. The colons were then
dissected in half with one half rolled in the “swiss roll” formation and fixed in
neutral buffered 10% formalin solution. From the remaining colon, 5mm sections
from distal and proximal colon were dissected for RNA and protein extraction. For
RNA extraction, the colon tissues were crushed immediately in 0.5ml Trizol
(Invitrogen, Carlsbad, CA) and snap frozen on dry ice. For protein extraction, the
colon samples were snap frozen in liquid nitrogen and stored at -80°C until the full
                                          37
protein extraction protocol could be performed. The mesenteric lymph nodes (MLN)
were also dissected from each mouse and placed into complete RPMI (RPMI-1640
supplemented with 100U/mL penicillin/streptomycin, 2nmol/L L-glutamine,
50µmol/L β-mercaptoethanol (Invitrogen), and 10% heat-inactivated foetal calf
serum (Invitrogen) and kept on ice until processing.


3.6 Histological assessment of colitis
       Colon swiss rolls collected in 10% formalin were transferred to 70% ethanol
24h later. These were then sent to the histology service at the Queensland Institute of
Medical Research (QIMR) and were dehydrated, embedded in paraffin and 5 micron
sections were cut for immunohistochemical analysis or for haematoxylin and eosin
(H&E) staining. H&E stained sections were examined to assess the degree of colitis
in each mouse. Sections were examined at various magnifications using an Olympus
BX50 microscope and representative digital photographs were taken. Histological
colitis severity was evaluated based on microscopic features including crypt length,
ulceration, goblet cell loss and inflammatory cell infiltration (full details in scoring
sheet, Appendix 1.2). The grading system used for this project is well established in
our laboratory (Heazlewood et al., 2008) and the scoring was done by two blinded
investigators.


3.7 Isolation and culture of Mesenteric lymph node (MLN) cells
       Whole mesenteric lymph nodes, adjacent to the proximal colon were
dissected from the mice. The lymph nodes were then gently crushed between sterile
glass slides to disaggregate cells and then passed through a 40µm cell strainer
(Falcon, BD Biosciences, Franklin Lakes, NJ) to remove fat and connective tissue.
Lymph node cells were then washed in fresh RPMI, counted using a
haemocytometer, obtained in a single-cell suspension and plated into 24 well plates
at a concentration of 2 X 106 cells/mL, in the presence of 50ng/ml phorbol 12-
myristyl 13-acetate (PMA, Sigma) and 750ng/ml ionomycin (Sigma) to stimulate
immune cells. The culture supernatants were collected at 48h time point and
centrifuged at 400g for 5min to remove cells and debris and were then aliquoted and
stored at -80°C. TNF-α production in culture supernatants was determined after 48h
culture as described below.


                                          38
3.8 Enzyme-Linked ImmunoSorbent Assay (ELISA) for TNF-α
       ELISA was performed to measure the level of mouse TNF-α in stimulated
mesenteric lymph node cultures from mice treated with DSS and mice co-treated
with ghrelin peptides using BD ELISA kits (BD Biosciences). Capture antibody for
TNF-α was diluted 1:250 in coating buffer (1M sodium carbonate) and was plated
100μl/well in a 96 well microplate (Nunc, Roskilde, Denmark). The plate was
sealed and left overnight at 4°C. The following day the capture antibody solutions
were aspirated and washed 3 times with 300μl/well wash buffer (0.05% Tween in
PBS) in an automatic microplate washer (BioTek ELx405, Winooski, Vermont).
After the plate was washed it was inverted and blotted on an absorbent paper to
remove any residual debris. The plates were then blocked with assay diluent (3%
Bovine Serum Albumin in PBS, 200μl/well) and incubated at room temperature (RT)
for 1h. At this point the lymph node culture supernatants were thawed on ice. The
plate was washed in the plate washer 3 times as described above. The standard for
the cytokine, TNF-α, and the supernatants were diluted 1:10 – 1:20 in the assay
diluent. The supernatants and the standard (100μl/well) were pipetted into the
appropriate wells in the plates were sealed and incubated at RT for 2h. The plates
were then washed 5 times. The detection antibody (1:250) was diluted in the assay
diluent and then 100μl diluted detection antibody was added to each well. The plate
was once again sealed and incubated at RT for 1h. This was followed by another 5
washes in the plate washer. Next, the enzyme reagent (Sav-HRP) 1:250 was diluted
in assay diluent and 100μl was added to each well. The plate was sealed and
incubated for 30 mins at RT. The plate was then washed 7 times, which was
followed by adding 100μl/well of tetramethylbenzidine (TMB) Substrate Solution
(BD Bioscience) and incubation at RT for 30 mins in the dark. This was followed by
the addition of 50μl of stop solution (1M H2SO4) to each well. The plate was then
analysed in a Sunrise microplate reader (Tecan, Zurich, Switzerland) at an
absorbance of 450nm.


3.9 Mesenteric lymph nodes (MLN) T Regulatory Cells counting using Flow
Cytometry
       Mesenteric lymph nodes were crushed, using sterile glass slides, after
dissection and filtered to make a single cell suspension (as described in section 3.7).
Lymph node cells (106 cells) were then washed in ice-cold RPMI complete medium
                                          39
and then centrifuged at 400g for 5 mins (at 4°C). Cell pellets were resuspended in
flow cytometry staining buffer (0.05% BSA, 0.09% NaN3 in PBS), vortexed and
centrifuged again. All antibodies were purchased from eBioscience. Supernatant was
aspirated, and the cells were then co-stained for the T regulatory cell surface
molecules CD4 and CD25 (0.125µg/106 cells of CD4-FITC and 0.06µg/test of
CD25-PE antibodies in 100µl flow staining buffer). Samples were incubated at 4°C
for 30 min in the dark, and then washed with cold PBS (1ml). Cells were centrifuged
at   400g   for     5   min,     and   cell    pellets   were   resuspended   in   1ml
Fixation/Permeabilization solution (eBioscience) and vortexed. The samples were
incubated at 4°C for 18h in the dark. The cells were then washed twice with
permeabilization buffer (1ml, eBioscience), centrifuged, and supernatant aspirated.
The samples were then blocked with 1-2µg/test Fc Block (anti-mouse CD16/32
clone, eBioscience) in permeabilization buffer (100µl) at 4°C for 15 min to minimise
non-specific binding of the Foxp3 antibody to macrophage and lymphocyte Fc
receptor. After the blocking step, 0.5µg/test anti-mouse/rat Foxp3 (clone FJK-16s)
antibody or isotype control in permeabilization buffer was added to the samples and
incubated for a minimum of 30 mins in dark at 4°C. Control samples included auto-
fluorescent controls (staining buffer only), CD4-FITC single stained control, CD25-
PE single stain control, Foxp3-APC single stain control and APC isotype control.
The cells were then washed in permeabilization buffer (1ml), centrifuged and
supernatant was aspirated. Cells were resuspended in cold PBS (400µl) and analysed
on a LSRII flow cytometer (BD). Gates were drawn to include the lymphocyte
population and exclude dead cells, erythrocytes and debris and were based on
forward and side scatter profiles. Gating strategy and compensation data, including
PMT voltages (included in Appendix 1.3).            Data were analysed using FlowJo
software (Tree Star, Harvard).


3.10 Cell Culture
       The HT29 colon cancer cell line obtained from ATCC was cultured in
complete RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-
inactivated foetal calf serum (FCS), 50 units/ml penicillin G and 50µl/ml
streptomycin sulphate and 5mmol/ml L-glutamine (Invitrogen). The cells were
cultured in 80 cm2 cell culture flasks (Nunc) at 37°C in a Sony incubator with 5%


                                              40
CO2. The cells were tested routinely by the Scientific Support Staff at the MMRI and
were found to be free from Mycoplasma.


3.11 Wound assay
        HT29 cells (1x106cells/well) were plated into 6 well plates (Nunc) and
cultured until they reached 100% confluence. Once confluent the cells were then
washed twice with 37ºC PBS and then serum starved overnight in 0.1% FCS in
RPMI. Prior to creating the wounds, the cells were treated with Mitomycin C
(10µg/ml, Sigma, St Louis, MO) for two hours to inhibit cell proliferation. This
concentration of Mitomycin C was determined empirically and does not induce
apoptosis in colon cells. A sterile p200 pipette tip was used to create a “wound” (3
scratches per well). The cells were washed again once with warm PBS (1ml/well) to
remove cell debris and to smooth the edge of the scratch and then replaced with 2ml
of media with different concentrations (0-10nM) of ghrelin (Auspep) and EGF
(100ng/ml, Sigma) as a positive control. The cells were photographed (Canon EOS
40D, Australia) under phase-contrast microscopy (Olympus CKX41, USA), at 0, 12,
24, and 48h time points. Wound closure was determined quantitatively by using
ImageJ (National Institutes of Health). The percentage of wound closure was
calculated as (initial area-final area)/initial x 100. Experiments were performed once,
with two wells per treatment, where each well had three scratches.


3.12 Statistical Data analysis
    All data were expressed as mean + S.D. Data were analysed using GraphPad
Prism (v5.01), statistics analysis software program (San Diego, CA). Parametric tests
such as One-way ANOVA followed with Tukey’s post hoc comparison was used to
determine statistically significant differences in the mean values between the groups.
Where necessary, Two-way ANOVA was used to analyse two independent factors.
Non-parametric analysis using Mann-Whitney U-test was used when comparing two
groups. The statistical test used and the sample sizes for each analyses are provided
within the figure legend. Probability values p<0.05 were considered statistically
significant.




                                          41
3.13 Presentation of Results
   The data presented in the results section examines two different variables (mouse
strains and DSS molecular weight) as well the efficacy of ghrelin in colitis.
Therefore, the results are presented in a comparative format. The total clinical scores
and body weight change are shown over a period of time to indicate the start of
colitis and the effect of ghrelin. These data were further split into 5kDa DSS +/-
ghrelin peptides treated mice vs. 40kDa DSS +/- ghrelin peptide treated mice.
However, the rest of the data presented evaluates the efficacy of the ghrelin peptides
in comparison to PBS in the different molecular weights of DSS treated mice in each
mouse strain.




                                          42
                            Chapter Four: Results

4.1 Effect of ghrelin peptide treatment on clinical colitis scores in C57BL/6 and
BALB/c mice administered with dextran sodium sulphate (DSS)
Eight weeks old female BALB/c and C57BL/6 mice were induced with colitis by
treating them with 5% and 2% DSS, respectively over 8 days. The DSS treatment
was conducted with two molecular weights- 5kDa and 40kDa which differentially
affect the proximal and distal large intestine. The mice were injected with ghrelin
peptides on day 4 and day 6 via i.p. injections. A pilot study was conducted with the
administration of ghrelin peptides prior to day 4, however no significant benefit was
observed over the two day injection protocol. The total clinical scores were
evaluated daily on clinical symptoms such as diarrhoea, rectal bleeding, shivering,
hunching and ruffling (described in more detail in the score sheet, Appendix 1.1).
Changes in bodyweight change were not included in the clinical scores because
ghrelin is a known appetite stimulant. In BALB/c mice clinical scores were higher
when treated with the larger molecular weight of DSS (40kDa) (Figure 1B) than the
5kDa DSS group (Mann-Whitney U-test, p=0.0001; Figure 1A) on day 7. In
contrast, in the C57BL/6 mice total clinical scores were higher when treated with the
5kDa DSS (Figure 2A) as compared to the 40kDa DSS (Figure 2B), but this did not
reach statistical significance (Mann-Whitney U-test, p=0.309). Treatment with
ghrelin or ∆4 peptide did not suppress clinical colitis in BALB/c mice whether they
were given 5kDa or 40kDa DSS (Figures 1A & B). However, C57BL/6 mice
administered 5kDa DSS (Figure 2A) did display significantly less clinical signs of
colitis when they were treated with ghrelin (p= 0.012) by day 8. There were no
clinical scores recorded for the BALB/c mice on day 8 due to an oversight.
Therefore, a comparison between the two strains of mice on that day could not be
measured.


4.2 Treatment with ghrelin peptides does not affect bodyweight change in mice
with DSS-induced colitis
There was no significant effect on body weight in mice with DSS-induced colitis
when treated with ghrelin peptides. A decrease in body weight was observed from
day 5, in both the BALB/c (Figure 3) and C57BL/6 mice DSS-treated mice (Figure


                                         43
4). However, the main difference observed was that each strain of mice responded
differently towards the different molecular weights of DSS. There was a significant
difference in bodyweight in the BALB/c mice between the 5kDa DSS + PBS group
(Figure 3A) when compared to the 40kDa DSS + PBS group (Figure 3B). There was
a significant drop in bodyweight in from day 6, p=0.002 in the 40kDa DSS + PBS
group mice in comparison to the naive mice, and also on day 7, p<0.0001 and day 8,
p<0.0001, using Mann-Whitney U-test. In the BALB/c mice when the 5kDa DSS +
PBS group was compared to the 5kDa DSS + ghrelin group on days 6, p=0.77; day
7, p=0.71 and day 8, p=0.98, there was no significant difference observed. No
significance was observed with the Δ4 peptide treatment in the BALB/c mice, on day
6, p=0.51, day 7, p=0.97 and day 8, p=0.79, using a Mann-Whitney U-test. In
contrast, the reverse was found in the C57BL/6 group of mice where the 5kDa DSS
+ PBS group (Figure 4A) progressively lost weight from day 6 when compared to
the 40kDa DSS + PBS group (Figure 4B). The BALB/c mice did not lose weight
when treated with the smaller molecular weight of DSS (5kDa) whereas when
treated with 40kDa DSS there was a significant drop in weight. Conversely, the
C57BL/6 mice were resistant to the 40kDa DSS treatment with no significant weight
change. However, in comparison to the BALB/c, we did notice a significant decrease
of initial body weight loss in the C57BL/6 5kDa DSS treated mice. The C57BL/6
5kDa DSS + PBS group lost significant weight from day 6, p=0.0002 and on day 7,
p=0.0001 and day 8, p=0.002, in comparison to the naive mice; Mann-Whitney U-
test. There was no significant difference with ghrelin treatment in the 5kDa DSS
group, on day 6, p=0.69, day 7, p=0.31 or day 8, p=1.00, using a Mann-Whitney U
test. Similarly, there was no significant effect of ghrelin and its peptide, Δ4, on body
weight in C57Bl/6 mice treated with DSS (5kDa or 40kDa).


4.3 Effect of ghrelin peptides on colon shortening in C57BL/6 and BALB/c mice
given DSS
Colon lengths were measured in all the groups to examine whether ghrelin peptides
were able to rescue colitis. Mice treated with DSS had shrunken and swollen colons
as compared to the naive/healthy group of mice due to apoptosis (Maki et al., 2005).
The colon lengths in the 40kDa DSS groups of BALB/c mice (Figure 5A) were
much shorter in length as compared to the 5kDa DSS group, Mann-Whitney U-test:
p<0.0005. This observation is in agreement with the greater loss of body weight, in
                                          44
the 40kDa DSS. In C57BL/6 mice (Figure 5B) there was no significant difference in
colon lengths between the two molecular weights of DSS, in contrast to the BALB/c
mice. Colitis was induced because the colons of the 5kDa DSS + PBS group were
significantly shorter in length when compared to the naive C57BL/6 mice, p=0.0001,
Mann-Whitney U-test. There was also a significant difference in colon length
between the naive C57BL/6 mice and the 40kDa DSS + PBS group, p=0.002, using a
Mann-Whitney U-test. However, a Two-Way ANOVA (Figure 6A and B) test did
not show any significant difference between the colon lengths of the ghrelin peptides
treated groups when compared to the DSS + PBS groups in either strain of mice in
both the 5 and 40kDa DSS groups.




                                         45
   A                                      Total Clinical scores BALB/c
                              8

                              7



    Total Clinical Scores
                              6

                              5

                              4

                              3

                              2

                              1

                              0
                                  0   1      2       3           4      5       6   7
                                                          Days


   B                                      Total Clinical scores BALB/c
                              8

                              7
      Total Clinical Scores




                              6

                              5

                              4

                              3

                              2

                              1

                              0
                                  0   1      2        3          4       5      6   7
                                                          Days
                                                 Naives
                                                 5kDa DSS + PBS
                                                 5kDa DSS + 1nmol Ghrelin
                                                 5kDa DSS + 1nmol ∆4 peptide
                                                 40kDa DSS + PBS
                                                 40kDa DSS + 1nmol Ghrelin
                                                 40kDa DSS + 1nmol ∆4 peptide

Figure 1. Clinical evidence of colitis in BALB/c mice treated with DSS and
ghrelin peptides. BALB/c mice were assessed on the severity of colitis by
calculating total clinical scores daily with evaluation of stool consistency, faecal
bleeding and diarrhoea. Clinical symptoms such as shivering, hunching and ruffling
were also evaluated daily using clinical score indices as described in complete detail
in the score sheet (Appendix 1.1). Black arrows indicate administration of ghrelin
peptides or PBS on Days 4 and 6. Clinical scores of BALB/c mice treated with 40kDa
DSS (B) were higher when compared to 5kDa DSS (A) on day 7, (Mann-Whitney U-
test, p=0.0001). (N=11-12 mice/group, Mean ± S.D.) The data is representative of 2
individual studies.
                                                          46
   A                                                Total Clinical scores C57BL/6
                                 8


                                 7


                                 6
                                                                                               *p=0.012
     Total Clinical Scores
                                 5


                                 4


                                 3


                                 2


                                 1


                                 0
                                        0       1    2     3          4       5       6          7        8
                                                                    Days


   B                                                Total Clinical scores C57BL/6
                                        8


                                        7


                                        6
                Total Clinical Scores




                                        5


                                        4


                                        3


                                        2


                                        1


                                        0
                                            0   1     2     3          4      5       6          7        8
                                                                     Days

                                                                Naives
                                                                5kDa DSS + PBS
                                                                5kDa DSS + 1nmol Ghrelin
                                                                5kDa DSS + 1nmol ∆4 peptide
                                                                40kDa DSS + PBS
                                                                40kDa DSS + 1nmol Ghrelin
                                                                40kDa DSS + 1nmol ∆4 peptide
Figure 2. Clinical evidence of colitis in C57BL/6 mice treated with DSS and
ghrelin peptides. C57BL/6 mice were assessed on severity of colitis by calculating
total clinical scores daily with evaluation of stool consistency, faecal bleeding and
diarrhoea. Clinical symptoms such as shivering, hunching and ruffling were also
evaluated daily using clinical score indices as described in complete detail in the score
sheet (Appendix 1.1). Black arrows indicate administration of ghrelin peptides or PBS
on Days 4 and 6. There was a significant improvement in colitis in mice treated with
5kDa DSS with ghrelin by day 8 (A). Mann-Whitney, U-test, *p=0.012. However,
there was no such improvement in the 40kDa DSS group (B). (N=11-12 mice/group,
Mean ± S.D). The data is representative of 2 individual studies.
                                                                     47
   A                                                         Body weight change BALB/c
                                               120




     Percentage of initial weight change
                                               110




                                               100




                                                90
                                                     0   1    2       3       4       5       6        7       8
                                                                            Days

   B                                                         Body weight change BALB/c
                                               120


                                                                                                  ** p=0.002
         Percentage of initial weight change




                                               110
                                                                                                      *** p<0.0001

                                               100



                                                90



                                                80



                                                70
                                                     0   1     2      3       4       5       6        7       8
                                                                            Days

                                                                   Naives
                                                                   5kDa DSS + PBS
                                                                   5kDa DSS + 1nmol Ghrelin
                                                                   5kDa DSS + 1nmol ∆4 peptide
                                                                   40kDa DSS + PBS
                                                                   40kDa DSS + 1nmol Ghrelin
                                                                   40kDa DSS + 1nmol ∆4 peptide
Figure 3. Changes in body weight in BALB/c mice treated with DSS and ghrelin
peptides. Body weight of mice was expressed as percentage of initial weight.
BALB/c mice were given 5% (5kDa DSS/40kDaDSS) with or without the presence
of ghrelin peptides. Black arrows indicate administration of ghrelin peptides or PBS
on Days 4 and 6. There was a significant difference in body weight in the BALB/c
group, ***p=0.0009, between the 5kDa DSS + PBS (A) and 40kDa DSS + PBS (B)
using Mann-Whitney, U-test on day 6, day 7, ***p<0.0001, and day 8,
***p<0.0001. Colitis was induced from day 6, **p=0.002, to day 7, ***p<0.0001,
and on day 8, ***p<0.0001 in BALB/c mice treated with 40kDa DSS using Mann-
Whitney, U-test, when compared to the Naïve mice. (N=11-12 mice/group, Mean ±
SD).
                                                                            48
    A                                                        Body weight change C57BL/6

                                               120




        Percentage of initial weight change
                                               110



                                               100



                                                90



                                                80



                                                70
                                                     0   1      2     3      4      5      6       7       8
                                                                           Days


    B                                                        Body weight change C57BL/6

                                               120
         Percentage of initial weight change




                                               110



                                               100



                                                90



                                                80



                                                70
                                                     0   1      2     3      4       5      6          7       8
                                                                            Days
                                                                    Naives
                                                                    5kDa DSS + PBS
                                                                    5kDa DSS + 1nmol Ghrelin
                                                                    5kDa DSS + 1nmol ∆4 peptide
                                                                    40kDa DSS + PBS
                                                                    40kDa DSS + 1nmol Ghrelin
                                                                    40kDa DSS + 1nmol ∆4 peptide
Figure 4. Changes in body weight in C57BL/6 mice treated with DSS and ghrelin
peptides. Body weight of mice was expressed as percentage of initial weight.
C57BL/6 mice were given 2% DSS (5kDa DSS / 40kDa DSS) with or without the
presence of ghrelin peptides. Black arrows indicate administration of ghrelin peptides
or PBS on days 4 and 6. There was a significant difference in body weight in the
C57Bl/6 group, **p=0.0046, between the 5kDa DSS + PBS (A) and 40kDa DSS +
PBS(B) using Mann-Whitney, U-test on day 6, ***p=0.0003, day 7, ***p<0.0001, day
8, ***p<0.0001. Colitis was induced with 5kDa DSS from day 6, p=0.0002; day 7,
p=0.0001 and till day 8, p=0.002, Mann-Whitney, U-test when compared to the naïve
mice. (N=11-12 per group, Mean ± SD). 49
                     A                          Colon length BALB/c

                                                     ***p<0.0005
                                      120
                                      110
                                      100
                                          90



                 Colon length (mm)
                                          80
                                          70
                                          60
                                          50
                                          40
                                          30
                                          20
                                          10
                                           0
                                             SS es




                                                        in

                                                          e

                                                G S



                                             ol tide




                                                          e
                                       m lG S




                                                pe n
                                                pe n
                                            D ptid




                                                      id
                                                        i
                                            ∆4 reli
                                                      B
                                                      B




                                            ∆4 rel
                                                     el
                                  SS a D aiv

                                         nm + P




                                         nm + P




                                                   pt
                                                  hr




                                                   p

                                                  h
                                                  h
                      k D a D Da p e
                                                 N




                                                G
                              SS + 1 SS
                                             ol
                    40 kD 40k ∆4
                                             o




                                          m
                                         ol
                                         ol
                                         ol




                                        1n
                                       D

                              SS + 1




                                       m



                                       m
                                     5k




                                    1n
                                    1n
                                    1n



                                  SS

                                  +
                                  +
                                D
                              a




                            D
                            D
                            D
                         5k




                          a
                          a



                        40
                       D
                     5k




                                                     Treatment Groups



                     B
                                                Colon length C57BL/6

                                          120                       **p=0.002
                                          110    ***p=0.0001
                                          100
                                           90
                      Colon length (mm)




                                           80
                                           70
                                           60
                                           50
                                           40
                                           30
                                           20
                                           10
                                            0
                                               SS ves




                                          1n S + e

                                        m lG S



                                        m lG e



                                                         e
                                        m lG S

                                                 pe in




                                          1n pe n



                                                 pe in
                                                       d




                                                      id



                                                      id
                                                       i
                                                     PB
                                     1n mo PB




                                              ∆4 rel



                                              ∆4 el
                                              ∆4 rel

                                              D pti




                                                    pt



                                                    pt
                                   SS a D ai




                                                   hr
                                                   h




                                                   h
                                                  +
                                                  N




                                               S

                                               o



                                               o
                                             m



                                             m
                                           ol
                                           ol
                                           ol

                                           a
                                          1n



                                         D
                                         D
                                       5k




                               40 0k

                               SS +
                                SS +




                                     1n
                                     1n
                                     a
                                     4
                                  kD

                                   +
                                   +
                                 D
                       5 k Da




                              D
                              D
                           5k




                            a
                            a




                        kD
                          D




                      40




                                                     Treatment Groups


Figure 5. Colon lengths of mice treated with DSS for 8 days. There was a
significant difference (Mann-Whitney U-test: ***p<0.0005) in colon lengths between
the 5kDa DSS + PBS group and the 40kda DSS + PBS group in the BALB/c mice
(A). In the C57BL/6 group (B) there was no significant difference in the DSS groups.
However, the naïve C57BL/6 mice had a significant difference in colon length when
compared to the 5kDa DSS + PBS mice, ***p=0.0001 and with the 40kDa DSS +
PBS mice, **p=0.002 using a Mann-Whitney, U-test. (N=11-12 mice/group, Mean ±
S.D).
                                                               50
                 A                Colon length
                                         ***
                         120
                                                                   5kDa DSS + PBS
                         100
     Colon length (mm)




                                                                   5kDa DSS + 1nmol Ghrelin
                          80

                          60

                          40

                          20

                           0
                                                        6
                                    /c




                                                     L/
                                 LB




                                                  7B
                               BA




                                               C5




           B
                                  Colon length
                                         ***
                         120
                                                                 5kDa DSS + PBS
                                                                 5kDa DSS + 1nmol ∆4 peptide
  Colon length (mm)




                         100

                          80

                          60

                          40

                          20

                           0
                                                     6
                                  /c




                                                  L/
                                 LB




                                                 7B
                               BA




                                               C5




Figure 6. Two-Way ANOVA comparison of colon lengths of BALB/c and
C57BL/6 mice treated with ghrelin peptides with 5kDa DSS. There was a
significant difference between the strain of mice (Two-way ANOVA,
***p<0.0005) in colon lengths between the 5kDa DSS + PBS group in the
BALB/c mice and C57BL/6 group. However, there was no significant difference
in the DSS + PBS group compared to the DSS + Ghrelin group (A) nor the DSS
+ Δ4 peptide (B) in either strain of mice (N=11-12 mice/ group, Mean ± S.D).




                                                            51
4.4 Blood analyses of BALB/c and C57BL/6 mice
The mice were bled before they were culled on Day 8. Around 50µl of blood was
collected from each mouse; however due to severity of colitis this was not always
possible. When mice were dehydrated, the method of blood collection was cardiac-
puncture. The blood was then run through a Sysmex SF3000 Haematology Analyser
(Sysmex Corp, Kobe, Japan). Figures 7 and 8, show a summary of the blood analysis on
BALB/c and C57BL/6 mice. There was no significant difference in the haematocrit
(HCT) count between the different DSS-treated groups in the BALB/c strain (Figure
7A). The HCT of the 40kDa DSS + PBS, mean 48%, were lower when compared to the
40kDa DSS + Ghrelin group, 55% but this was not found to be statistically significant
(p=0.5 Mann-Whitney, U-test). When comparing between the strains of mice it was
found that the C57BL/6 mice HCT (Figure 8A) were much lower following DSS when
compared to the BALB/c strain of mice. In contrast, the leukocyte levels in the BALB/c
mice treated with 40kDa DSS (Figure 7B) were relatively higher than the C57BL/6
group (Figure 8B). The 40kDa DSS treated BALB/c mice had a slightly increased
leukocyte count when compared to the 5kDa DSS treated group. There was no
significant difference observed between the various DSS groups of the C57BL/6 mice
treated with or without the ghrelin peptides. Similar results observed in the leukocyte
count were found with the lymphocyte count in the BALB/c (Figure 7C) and C57BL/6
mice (Figure 8C). The levels of leukocyte and lymphocytes in the 40kDa DSS group in
the BALB/c mice were elevated but this may have been due to technical error. In some
cases, when the mice were too severely dehydrated, it was difficult to obtain enough
blood, therefore the collected blood samples were then diluted in PBS to obtain enough
sample to run through the Sysmex machine. This reason may have accounted for the
elevated levels of the leukocyte and lymphocyte count.




                                          52
A                   Haematocrit BALB/c                                                B                                   Leukocyte BALB/c
                                                                                                                     80

                    80                                                                                               70
                    70




                                                                                               Leukocyte (x10^9/L)
                                                                                                                     60
                    60                                                                                               50
    Haematocrit %




                    50
                                                                                                                     40
                    40
                                                                                                                     30
                    30
                                                                                                                     20
                    20
                                                                                                                     10
                    10
                                                                                                                      0
                     0




                                                                                                                 1n pe i n
                                                                                                                                   s



                                                                                                               kD 4 p lin

                                                                                                                                   e
                                                                                                                                BS



                                                                                                                                   e



                                                                                                                                   e
                                                                                                                                   S




                                                                                                                                  in
                                                                                                     DS S + DS aive




                                                                                                                       SS id




                                                                                                             1n mo ptid



                                                                                                                               id
                                                                                                             1n mo PB




                                                                                                                      ∆4 rel
                                                                                                                                l
                                                                                                             40 l ∆ hre




                                                                                                                    d ∆ hre
                                                                                                           1n nm + P
                                                                                                   DS SS a D ept




                                                                                                                             pt
                   S + 1 SS ves




                                            e
                                          BS

                            1n 4 p lin
                           m ol G BS

                                    p in




                                            e
                             o l Gh e
                                    pe lin




                                                                                                                  o l Gh
                                                                                                                           +




                                                                                                                          pe
                                                                                                                           N
                                         id
                                 SS tid




                                         id




                                                                                                                          G
                                                                                                                        lG
         kD a 40k ∆4 rel




                                                                                                                         S
                                ∆4 re
                                ∆ hre
                                        P




                       1n nm + P




                                       pt
                        1n mo ept
                 DS SS a D ai




                                                                                                                        l
                                                                                                                       ol

                                                                                                                       4
               DS SS a D ep
                                      h
                              nm +
                                     N




                                    G




                                                                                                Da DS Da

                                                                                                                 1n

                                                                                                                  o
                                  l
                          m ol




                                                                                                                 ol
                                                                                                                 1
                                                                                                                m




                                                                                                                m
                                                                                                              5k




                                                                                                              m
                                                                                                               +
                             ol




                            ol
                            D
            Da D kD




                            1




                           m




                                                                                                           +
                          +
                        1n




                                                                                                         +
                                                                                                        D
                         5




                                                                                                        S




                                                                                                      S
                                                                                                      a
                                                                                             5 k Da
                      +




                     +




                                                                                           40 0kD
                    D




                                                                                                  5k
                   S
         5 k Da




                                                                                                a
       40 0kD




                                                                                                4
                                                                                             kD
              5k




             a
            4




                                                                                                                             Treatment Groups
                         Treatment Groups



                                   C
                                                                        Lymphocyte BALB/c

                                                                   60

                                                                   50
                                            Lymphocyte (x10^9/L)




                                                                   40

                                                                   30

                                                                   20

                                                                   10

                                                                    0
                                                                1n pe n
                                                                1n S + s



                                                                              in

                                                                1n S + e
                                                                               S



                                                                                e



                                                                                e
                                                                        G S




                                                                        pe in
                                                                             ve




                                                                    DS ptid




                                                                             id



                                                                             id
                                                                              i
                                                           1n mo PB
                                                                            PB




                                                                    ∆4 rel
                                                                    ∆4 rel
                                                           40 l ∆4 rel




                                                                           pt



                                                                           pt
                                                         SS a D ai



                                                                          h




                                                                 o l Gh
                                                                 ol Gh
                                                       DS Da pe
                                                                         N




                                                                      l
                                                                      l
                                                                     ol
                                                                     S




                                                           1 n mo
                                                                   m
                                                                 o
                                                                D




                                                              m
                                                              m




                                                              m
                                                            5k




                                                              k

                                                             +
                                                             +
                                                           1n



                                                           S
                                                         +
                                                         +
                                                       D




                                                       S
                                                      SS




                                                    DS
                                                     a
                                             5 k Da
                                                    D



                                                  kD
                                                 5k




                                                  a
                                                  a



                                                40

                                              kD
                                                D




                                            40




                                                                            Treatment Groups



Figure 7. Haematological parameters in BALB/c mice treated with DSS and ghrelin
peptides for 8 days. Mice were bled on day 8 (final day of experiment) and 50µl of blood
samples were collected for measuring haematocrit (A), leukocyte (B) and lymphocyte (C)
counts using the Sysmex SF3000 Haematology Analyser. There was no significant
difference observed between the DSS and ghrelin treated groups or in comparison to the
naive mice. The levels of leukocyte and lymphocyte in the 40kDa DSS group were
elevated but this may have been due to technical error. (N= 11-12 mice/group, Mean ±
S.D).




                                                                                 53
A                        Haematocrit C57BL/6                                       B                               Leukocyte C57BL/6
                                                                                                              80

                    80                                                                                        70




                                                                                        Leukocyte (x10^9/L)
                    70                                                                                        60
                    60                                                                                        50
    Haematocrit %




                    50                                                                                        40
                    40                                                                                        30
                    30                                                                                        20
                    20
                                                                                                              10
                    10
                                                                                                               0
                     0




                                                                                                                          s



                                                                                         kD 40 0kD 4 p lin

                                                                                                         1n S + e

                                                                                                                         S




                                                                                                           o l Gh e



                                                                                                                          e
                                                                                                                         S




                                                                                                                        in



                                                                                                                  pe in
                                                                                                               SS e




                                                                                                               S tid




                                                                                                                        d



                                                                                                                       id
                                                                                                     1 n mo P B
                                                                                                     1n mo PB




                                                                                                              ∆4 rel



                                                                                                              ∆4 rel
                                                                                                              D aiv



                                                                                                                     re




                                                                                                     1n mo pti



                                                                                                                     pt
                                                                                                                   ep
                                                                                                           ol Gh




                                                                                                          o l Gh
                                                 lin
                                  + SS es




                                        SS tide

                                                   S



                                                    e



                                                    e
                                                   S




                                   1n pe n



                                                  in




                                                                                                                   +




                                                                                                         1n pe
                                                                                                                   N
                               1n mo ptid



                                                id
                                                  i
                               1 n mo P B
                                               PB




                                       ∆4 rel



                                       ∆4 rel
                                       D aiv



                                              he




                                              pt




                                                                                                                l



                                                                                                                l
                                                                                                                l
                                              p




                                                                                                              ∆
                                    o l Gh



                                    o l Gh




                                                                                                              D
                                            G



                                            +
                                            +



                                           pe




                                           pe
                                            N




                                                                                                           a
                                                                                                  D kDa

                                                                                                         1n
                                  m ol




                                         l



                                         l
                                       ∆4




                                                                                                         m
                                                                                                         m




                                                                                                         m
                               1n m



                                       D




                                                                                                SS +
                                                                                                 SS +
                                    1n




                                                                                                       5
                                    ol

                                    a
                            D Da




                                   1n




                                                                                                     a
                                                                                                   SS



                                                                                                     4
                  kD 40k kD



                                  m



                                  m




                                                                                             a kD

                                                                                                   +
                                                                                                   +
                 5 k Da 5 k




                         SS +
                               40
                        D SS




                               a




                                                                                        5 k Da
                             D

                             +
                             +




                                                                                               D
                                                                                               D
                                                                                            5k
                          SS




                                                                                             a
                                                                                           D
                        D
                      5k




                                                                                       40
                      a
                      a
                    D




                                                                                                                      Treatment Groups
                40




                             Treatment Groups



                                   C                                 Lymphocyte C57BL/6

                                                                60

                                                                50
                                         Lymphocyte (x10^9/L)




                                                                40

                                                                30

                                                                20

                                                                10

                                                                 0
                                                  SS + 1 SS es



                                                                          in

                                                                            e

                                                           m lG S




                                                           m lG e



                                                                            e
                                                           m lG S




                                                                   pe n




                                                                   pe n
                                                                D ptid




                                                                        id




                                                                        id
                                                                          i




                                                                          i
                                                                       PB
                                                                       PB




                                                                ∆4 rel




                                                                ∆4 el
                                                                ∆4 rel
                                                                        v




                                                                      pt




                                                                      pt
                                                      SS a D ai




                                                                     hr
                                                                     h




                                                                     h
                                                             nm +
                                                             nm +



                                           kD 40k kDa pe
                                                                    N




                                                  SS + 1 SS
                                                                 o




                                                                 o




                                                                 o
                                                              m

                                                             ol
                                                             ol
                                                             ol




                                                            1n
                                                           D
                                                         5k




                                                        1n
                                                        1n
                                                        1n

                                                        40

                                                        a
                                                      D

                                                      +
                                                      +
                                                    D
                                                  a




                                                D
                                                D
                                                D
                                             5k




                                              a
                                              a
                                            D
                                          5k




                                         40




                                                                         Treatment Groups




    Figure 8. Haematological parameters in C57BL/6 mice treated with DSS and
    ghrelin peptides for 8 days. Mice were bled on day 8 (final day of experiment)
    and 50µl of blood samples were collected for measuring haematocrit (A),
    leukocyte (B) and lymphocyte (C) counts using the Sysmex SF3000 Haematology
    Analyser. There was no significant difference observed between the DSS and
    ghrelin treated groups or in comparison to the naive mice. (N= 11-12 mice/group,
    Mean ± S.D).




                                                                                54
4.5 Treatment with ghrelin peptides suppressed histolopathological colitis in C57BL/6
mice
Figures 9 and 10 show the summary of total histology scores, which were scored by two
blinded individuals. The colons were fixed in formalin in a “swiss roll” manner, which
were then sent for paraffin embedding followed by sectioning and finally H&E stained
(Figure 11). The scores were based on inflammation severity, epithelial damage (i.e.
crypt loss, goblet cell loss, crypt abscesses, and ulceration), infiltration extent; whether
there was cell infiltration around the base of the crypt, or more in the muscularis
mucosa resulting in oedema, and finally on a overall percentage involving crypt
abscesses, loss of crypt and ulcers (more details of scoring system in Appendix 1.2).
Figures 9 and 10 show a comparison in the colon sections between all of the groups in
the BALB/c and C57BL/6 mice. The colon sections observed for scoring were:
proximal colon, mid colon, and distal colon. The sections were observed and
photographed at various magnifications.
       Ghrelin treatment in the 5kDa DSS induced colitis group in the C57BL/6 mice
was more effective as compared to the 40kDa DSS. Ghrelin was able to suppress
inflammation to some extent in the proximal colon of the C57BL/6 mice (Figure 10A)
as compared to BALB/c mice in Figure 9A. There was less goblet cell loss, fewer crypt
abscesses and less cell infiltration in the proximal colon when the C57BL/6 mice were
treated with ghrelin (Figure 12D-F). The crypt architecture was intact with no
formations of ulcers in the distal colon when compared to the PBS treated group (Figure
12A-C). Two-way ANOVA analysis showed that the total histological scores of the
5kDa DSS treated group in the proximal colon were decreased when treated with
ghrelin. A statistical significance (p=0.03) was found between the 5kDa DSS + PBS
group and the 5kDa DSS + Ghrelin group in the C57BL/6 mice (Figure 14A). There
was also a significant difference (p<0.0001) in the total histology scores between the
two strains of mice, when treated with the same molecular weight of DSS (5kDa). This
is consistent with the previous results where treatment with 5kDa DSS was more severe
in the C57BL/6 mice as compared to the BALB/c strain of mice. Interestingly, there
also seemed to be an effect with the Δ4 peptide which caused a slight decrease in the
histology scores in the proximal colon (Figure 14B) in the C57BL/6 mice. Less goblet
cell loss was observed however there was some infiltration present in the mid and distal
colon (Figure 12H and I). Even though, this result was not statistically significant
(p=0.06) it indicates that there was a slight difference in the total histology score
                                            55
between the 5kDa DSS + PBS and the 5kDa DSS + Δ4 peptide groups in the C57BL/6
mice.
        There was not any significant difference between the PBS and ghrelin peptides
treated groups in the mid colon both in the BALB/c (Figure 9B) and the C57BL/6
(Figure 10B) strain of mice. However, there was a very slight improvement with ghrelin
treatment in the 5kDa DSS group in the C57BL/6 mice in the total histology score. In
Figure 12E, presence of goblet cells can be seen when compared to the mid colon in the
5kDa DSS + PBS group (Figure 12B). Figure 10A, and B indicate that the C57BL/6
group of mice had a higher total histology score when compared to the respective
treatment groups in the BALB/c mice.
        Figure 9C and 10C shows the summary of histological scores in the distal colon.
Interestingly, there was a slight improvement discovered in the C57BL/6 mice (Figure
10C) with the treatment of ghrelin in the 5kDa DSS treated group. However, a Two-
way ANOVA indicate that these trends were not significant p=0.28 (Figure 15A). In
Figure 12F, the crypt architecture and goblet cells are present; however some
infiltration can also be noticed. There was less histological damage in the C57BL/6
mice treated with 40kDa DSS when compared to the 5kDa DSS. There was less damage
in the proximal colon with mice treated with 40kDa DSS + PBS (Figure 13A). Ghrelin
treatment did not have any effect in the proximal and mid colon in the C57BL/6 mice
treated with 40kDa DSS. There was small improvement noticed in the distal colon of
the mice treated with ghrelin (Figure 13F), as there were less crypt abscesses and less
crypt loss seen in comparison with mice treated with PBS (Figure 13C). In contrast, the
Δ4 peptide did not have any effect in the C57BL/6 mice in the 40kDa DSS group
(Figure 13G-I).
        Unlike its effect in C57BL/6 mice, ghrelin peptides were unable to repress
colitis in the 5 and 40kDa DSS treated mice in the BALB/c group. It was also observed
that the naive mice in the BALB/c group were unexpectedly exhibiting mild colitis in
the proximal, mid and distal colons (Figures 9). Furthermore, some mice treated with
ghrelin and Δ4 peptide only, without DSS also exhibited mild inflammation. However,
it was interesting to observe that BALB/c mice treated with Δ4 peptide only, showed no
sign of inflammation in the distal colon when compared to the naive mice. Regardless
of the naive mice scoring, there was no difference in histological colitis scores between
the 5kDa DSS and the 40kDa DSS. Even though, the 40kDa DSS group seemed to have
worse colitis as compared to the 5kDa DSS group in terms of body weight loss and
                                           56
clinical scores, no such difference was observed at the histology level. Since the
BALB/c mice unexpectedly showed low grade inflammation in the naive group, this
potentially skewed the results for the other treatment groups making clear
interpretations difficult.




                                       57
 A Proximal colon BALB/c                                                              B                             Mid colon BALB/c

                     20                                                                                       20


                     15                                                                                       15
   Histology score




                                                                                            Histology score
                     10                                                                                       10


                      5                                                                                         5


                      0                                                                                         0
                   1n 4 p lin
                   1n S + es


                                 lin

                         S tide




                   m l G ide



                                   e
                     o Gh S




                   m ol G BS




                           pe n




                                                                                                             1n S + es




                                                                                                             1n pe lin
                                                                                                                               n

                                                                                                                               e




                                                                                                               o l Gh e



                                                                                                                               e
                                                                                                               ol Gh S




                                                                                                                       pe in
                                                                                                              ol Gh S
                                id
                1n mo PB




                                 i




                                                                                                    SS + DS tid




                                                                                                                             d



                                                                                                                            id
                                                                                                    a 40k ∆4 reli



                                                                                                                   o l PB
                                                                                                          1n mo PB
                       D aiv




                       ∆4 rel
                4 l ∆4 re




                               e




                                                                                                                             l
                               P



                               t


                              pt




                                                                                                                  D aiv




                                                                                                          1n mo pti

                                                                                                                  ∆4 re
                                                                                                                  ∆4 re
                             hr
              SS Da pep




                      m ep




                                                                                                                          pt
              + 1nm S +




                                                                                                       SS Da pep
                            h




                                                                                                        + 1nm S +
                            N




                                                                                                                        N
                         l
                        S




                                                                                                                     l
                                                                                                                    l
                       D




                                                                                                                   S
                       o
                       ∆


                    ol
                    ol
              SS a




                                                                                                       SS a
             D kD



                   m




                                                                                                      D kD
    kD a D 0k




                                                                                                             m
                                                                                                             m




                                                                                                             m
           SS +
            SS +




                1n
                1n
                 5




                                                                                                     SS +




                                                                                                          1n
                                                                                                           5
              +




                                                                                                        +


                                                                                                      D
   5 k Da




                                                                                            5 k Da
          D
          D



        kD




                                                                                                   D
                                                                                                   D



                                                                                           40 kD
       5k




        a
        a




                                                                                                5k
      40




                                                                                                 a
                                                                                                 a
      D




                                                                                               40
                                                                                             kD
                                                                                               D
  40




                          Treatment Groups                                                                                 Treatment Groups



                                   C                      Distal colon BALB/c


                                                         20


                                                         15
                                       Histology score




                                                         10


                                                          5


                                                          0
                                                                                                                           in ide
                                                             pe i n
                                                                     s



                                                             pe n

                                                                     e




                                                                     e
                                                     m ol G BS




                                                             G S




                                                                                                                         el
                                                          SS e




                                                    + DS tid




                                                                  id
                                             a 40k ∆4 eli



                                                                   B




                                                                                                                              t
                                                                   l




                                                                                                                       hr ep
                                                         D aiv




                                                         ∆4 re
                                                                 P




                                                        m +P



                                                                pt
                                                               hr

                                                                p



                                                               h
                                                 + 1nm +




                                                                                                                     G
                                                              N




                                                                                                                            p
                                                     1n S




                                                                                                                   ol ∆ 4
                                                          ol




                                                                                           ol                    m     l
                                                                                                              1 n mo
                                                SS a
                                                      ol
                                                SS a




                                                      D




                                                                                          m
                                               D kD




                                                                                     1n                          1n
                                              SS +
                                                  1n
                                                   5




                                                                                 +
                                                                             S
                                               D




                                                                          DS
                                     5 k Da
                                            D



                                          kD




                                                                      a
                                         5k

                                          a




                                                                 kD
                                        40
                                        D




                                                               40
                                                              Treatment Groups


Figure 9. Histological colitis scores in BALB/c mice treated with DSS and ghrelin
peptides. There was no significant difference between the naive and 5kDa DSS or
40kDa DSS group, or with treatment of ghrelin and Δ4 peptide in the proximal colon
(A). In the Mid colon (B) there was no significant difference between the 5kDa DSS +
PBS group and the ghrelin peptide treated groups. There was also no significant
improvement in colitis with ghrelin and Δ4 peptide treatment in the distal colon (C).
Histological colitis severity was evaluated based on microscopic features including crypt
length, ulceration, goblet cell loss and inflammatory cell infiltration (full details in the
scoring sheet in Appendix 1.2). (N= 11-12 mice/group, Mean ± S.D).
                                                                          58
  A                                                                        B
           Proximal colon C57BL/6                                                                  Mid colon C57BL/6

                                                                                              20
                        20
                             *p=0.03

                        15                                                                    15




                                                                            Histology score
      Histology score




                        10                                                                    10



                         5                                                                     5


                         0
                      1n pe lin                                                                0
                                       s


      kD a D 0kD 4 elin

                                       e
                             lG S




                        o l Gh e



                                       e
                      m ol G BS




                               pe in




                                                                                            1n pe lin
                                                                                                              s



                                                                             kD a D 0kD 4 elin

                                                                                                              e

                                                                                             ol Gh S



                                                                                              o l Gh e



                                                                                                              e
                                                                                            m ol G BS




                                                                                                      pe in
                            SS e




             SS + DSS tid




                                     d



                                    id
                  1n mo PB




                                     l




                                                                                                  SS e




                                                                                                  SS d




                                                                                                            d



                                                                                                           id
                                                                                                  o l PB
                           D aiv




                  1n mo pti

                           ∆4 re
                           ∆4 re




                                                                                                            l
                                   P




                                  pt




                                                                                                 D aiv




                                                                                                 D pti




                                                                                        1n mo pti

                                                                                                 ∆4 re
                                                                                                 ∆4 re
                                 hr




                                                                                                          P
                                  p




                                                                                                         pt
                                                                                                        hr
                                 h
                                +
                + 1nm +


                               pe
                                N




                                                                                       + 1nm +
                                                                                       + 1nm +



                                                                                                      pe
                                                                                                       N
                             l




                                                                                                    l
                           ∆




                                                                                                 ∆
                       ol
                        ol

                SS a
                SS a




                      1n




                                                                                              ol

                                                                                       SS a
                                                                                       SS a
               D kD




                      m



                      m




                                                                                      D kD




                                                                                            m



                                                                                            m
              SS +
                  1n
                    5




                                                                                    SS +
                                                                                     SS +




                                                                                        1n
                                                                                        1n
                                                                                          5
                  4




                                                                                        4
                +
     5 k Da




                                                                            5 k Da
            D
            D



    40 kD




                                                                                   D
                                                                                   D



                                                                           40 kD
         5k




          a
          a


        40




                                                                                5k
        D




                                                                                 a
                                                                                 a


                                                                               40
                                                                               D
                             Treatment Groups                                                         Treatment Groups



                                       C
                                                          Distal colon C57BL/6
                                                          20



                                                          15
                                        Histology score




                                                          10



                                                           5



                                                           0
                                                         1n pe lin
                                                                         s



                                         kD a D 0kD 4 elin

                                                         1n S de
                                                         m l G BS



                                                         m l G de



                                                                         e
                                                           ol Gh S




                                                                pe in
                                                              SS e




                                                                      id
                                                              ol PB




                                                                       l
                                                             D aiv




                                                              S ti




                                                     1n mo pti

                                                             ∆4 re
                                                             ∆4 re
                                                     1n mo P




                                                                   pt
                                                                    r
                                                   SS a pep




                                                                  h
                                                                  h
                                                                 +
                                                   + 1nm +
                                                                 N




                                                             ∆

                                                             D




                                                          ol
                                                          ol
                                                   SS a
                                                  D kD



                                                         m



                                                SS +
                                                 SS +
                                                     1n
                                                       5




                                                     4



                                                   +
                                        5 k Da




                                               D
                                               D



                                       40 kD
                                            5k




                                             a
                                             a


                                           40
                                           D




                                                               Treatment Groups


Figure 10. Histological colitis scores in C57BL/6 mice treated with DSS and ghrelin
peptides. There was a statistical difference in the proximal colon (A) of mice treated with
5kDa DSS + PBS in comparison to 5kDa DSS + Ghrelin, *p=0.03, using a Two-Way
ANOVA. There was a slight improvement in the proximal colon with 5kDa DSS + Δ4
peptide, however this was not significant, p=0.06, using a Two-Way ANOVA. There was
no difference observed in the mid colon (B) within the treatment groups. There again was a
slight improvement observed in the 5kDa DSS + Ghrelin group when compared to the
5kDa DSS + PBS group in the distal colon (C), p=0.28, but this was not significant. No
significant difference was noticed in the 40kDa DSS group. Histological colitis severity
was evaluated based on microscopic features including crypt length, ulceration, goblet cell
loss and inflammatory cell infiltration (full details in the scoring sheet in Appendix 1.2).
(N= 11-12 mice/group, Mean ± S.D).
                                                                     59
                 A




B                                                 C




                      D




Figure 11. Representative H&E sections of colons dissected from 6 week old Naive
C57BL/6 mice. Histology pictures (A) show an overall “swiss roll” formation of the colon
(X40 magnification). Histological severity was assessed as per the scoring system attached
in the Appendix. The circles in histology pictures represent goblet cells in the (B) proximal
region of the colon, (C) mid colon and (D) distal colon (all X200 magnification).
Representative digital photographs were taken using an Olympus BX50 microscope. The
arrows from picture A (X40 magnification) indicate which section of the colon was observed
for histology in X200 magnification.

                                            60
      Prox 5kDa DSS + PBS                  Mid 5kDa DSS + PBS                   Distal 5kDa DSS + PBS

  A                                    B                                  C




Prox 5kDa DSS + 1nmol Ghrelin Mid 5kDa DSS + 1nmol Ghrelin Distal 5kDa DSS + 1nmol Ghrelin

 D                                   E                                    F




 Prox 5kDa DSS + 1nmol Δ4                Mid 5kDa DSS + 1nmol Δ4              Distal 5kDa DSS + 1nmol Δ
         peptide                                 peptide                                peptide

 G                                   H                                    I




     Figure 12. Representative histology images of C57BL/6 mice treated with 5kDa DSS.
     Histological severity was assessed as per the scoring system attached in the Appendix 1.2. In mice
     treated with 5kDa DSS + PBS (A-C) goblet cell loss (black circle), loss of crypt architecture,
     crypt abscesses (black arrow), infiltration (red arrow), and ulceration were observed. In
     comparison, mice treated with 1nmol ghrelin (D-F) and 1nmol delta 4 peptide (G-I) showed less
     goblet cell loss and infiltration.


                                                   61
    Prox 40kDa DSS + PBS              Mid 40kDa DSS + PBS                Distal 40kDa DSS + PBS

A                                 B                                  C




Prox 40kDa DSS + 1nmol                                                   Distal 40kDa DSS + 1nmol
                                      Mid 40kDa DSS + 1nmol
        Ghrelin                                                                   Ghrelin
                                             Ghrelin

D                                 E                                  F




Prox 40kDa DSS + 1nmol Δ4         Mid 40kDa DSS + 1nmol Δ4               Distal 40kDa DSS + 1nmol Δ4
         peptide                          peptide                                   peptide

G                                H                                   I




    Figure 13. Representative histology images of C57BL/6 mice treated with 40kDa DSS.
    Histological severity was assessed as per the scoring system attached in the Appendix 1.2. In
    mice treated with 40kDa DSS + PBS (A-C) goblet cell loss, loss of crypt architecture, crypt
    abscesses and infiltration (red arrow), were starting to be observed. Mice treated with 1nmol
    ghrelin (D-F) and 1nmol delta 4 peptide (G-I) also showed goblet cell loss and infiltration.



                                                62
                 A
                                            Proximal colon
                                                     ***
                                      20

        Total Histology scores
                                                                        *     5kDa DSS + PBS
                                                                              5kDa DSS + 1nmol Ghrelin
                                      15


                                      10


                                       5


                                       0



                                                                    6
                                                /c




                                                                 L/
                                             LB




                                                              7B
                                           BA




                                                           C5




              B
                                            Proximal colon

                                      20
             Total Histology scores




                                                                             5kDa DSS + PBS
                                                                             5kDa DSS + 1nmol ∆4 peptide
                                      15


                                      10


                                       5


                                       0
                                                                    6
                                                /c




                                                                 L/
                                             LB




                                                              7B
                                           BA




                                                           C5




Figure 14. Histological assessment of inflammation in the Proximal colon of
BALB/c and C57BL/6 mice. There was a significant difference (A) between the
5kDa DSS + PBS vs. 5kDa DSS + Ghrelin (Two-way ANOVA, *p=0.03), where
treatment with ghrelin reduced colitis. A significant difference between the severity of
colitis was also found between the two strains of mice (***p<0.0001). Treatment with
Δ4 peptide (B) was unable to reach statistical significance in suppressing colitis in the
C57BL/6 mice (Two-way ANOVA, p=0.06). (N=11-12 mice/group, ± Mean S.D).
The data is representative of 2 individual studies.




                                                                        63
            A
                                        Distal colon

                               20
      Total Histology scores                                         5kDa DSS + PBS
                                                                     5kDa DSS + 1nmol Ghrelin
                               15


                               10


                                5


                                0


                                                           6
                                         /c




                                                        L/
                                      LB




                                                     7B
                                    BA




                                                  C5




           B
                                        Distal colon

                               20
      Total Histology scores




                                                                    5kDa DSS + PBS
                                                                    5kDa DSS + 1nmol ∆4 peptide
                               15


                               10


                                5


                                0
                                                          6
                                         /c




                                                       L/
                                      LB




                                                    7B
                                    BA




                                                 C5




Figure 15. Histological assessment of inflammation in the Distal colon of
BALB/c and C57BL/6 mice. There was no significant difference between the 5kDa
DSS + PBS vs. 5kDa DSS + Ghrelin (A) and 5kDa DSS + PBS vs. 5kDa DSS + Δ4
peptide (B). There was a slight, but not significant, improvement in treatment with
ghrelin and Δ4 peptide in the C57BL/6 mice. No difference was seen in the BALB/c
mice treated with PBS compared to Ghrelin or the Δ4 peptide. Colitis severity was
assessed on the scoring system of histology scores attached in the Appendix 1.2.
(N=11-12 mice group, Mean ± S.D). The data is representative of 2 individual
studies.




                                                               64
4.6 Measurement of TNF-α from mesenteric lymph node lymphocyte cultures
The levels of Th1 pro-inflammatory cytokine, TNF-α, in stimulated mesenteric lymph
node (MLN) cultures from mice treated with DSS and mice co-treated with ghrelin
peptides was measured using an ELISA. MLN leukocytes were treated with 50ng/ml
PMA and 750ng/ml ionomycin to stimulate immune cells for 48h. Even though there
was no effect of treatment of ghrelin in either strain of mice for both the 5kDa DSS or
the 40kDa DSS, the naïve mice in both the strains had unexpectedly high levels of
TNF-α in comparison to what had been established by our lab (TNF-α levels in cultures
from naive C57BL/6 mice: 250pg/ml; Heazlewood et al., 2008).              Figure 16A
demonstrates that the naïve BALB/c mice had similar levels of TNF- α, when compared
to the other groups. Ghrelin did slightly decrease the levels of TNF- α in the 5kDa DSS
group in the BALB/c mice (Figure 16A), this result is not significant and a conclusion
cannot be made since there is not any difference in TNF-α levels between the naives
and the 5kDa DSS + Ghrelin. These elevated levels of TNF-α are in conjunction with
the histology results where the naïve BALB/c mice were scoring for low grade
inflammation. However, similar TNF-α levels were also seen in the C57BL/6 naïve
mice (Figure 16B). These mice were healthy and had no inflammation in the
histological assessment. Similarly to the BALB/c mice, there was no difference in the
TNF-α levels in the C57BL/6 mice following DSS treatment. C57BL/6 mice treated
with 40kDa DSS + Ghrelin did slightly decrease the levels of TNF-α when compared to
40kDa DSS + PBS, however, this did not reach significance (Mann-Whitney, p=0.25).




                                          65
                 A            BALB/c TNF-α levels 48h
                          5000


                          4000


                          3000




                  pg/ml
                          2000


                          1000


                               0




                                                                              in
                                       s




                                                                               S




                                                                                e
                                               S


                                                            in
                                    ve




                                                                             id
                                                                  PB
                                              PB


                                                          el




                                                                            el

                                                                           pt
                                 ai




                                                        hr




                                                                          hr
                                                                 +
                                           +




                                                                         pe
                               N




                                                                         G
                                                    G

                                                            SS
                                         SS


                                                   ol




                                                                       ol


                                                                      ∆4
                                                           D
                                     D




                                                                      m
                                                m




                                                                             ol
                                                          a
                                     a

                                              1n




                                                                  1n
                                                      kD
                                 D




                                                                            m
                               5k




                                                                 +

                                                                        1n
                                           +

                                                    40

                                                            SS
                                        SS




                                                                       +
                                                           D

                                                                   SS
                                     D




                                                          a
                                    a




                                                                  D
                                                      kD
                                D




                                                                 a
                              5k




                                                    40


                                                             kD
                                                           40




                                                   Treatment Groups




                B             C57BL/6 TNF-α levels 48h

                             5000


                             4000


                             3000
                     pg/mL




                             2000


                             1000


                                 0
                                                            SS es




                                                          ∆4 elin


                                                           SS d e

                                                                      S

                                                          ∆4 elin


                                                                      e
                                                                     S




                                                                   id
                                                                    B
                                                                  PB
                                                                   v




                                                                   i

                                                       nm + P
                                                                 pt




                                                                 pt
                                                                ai




                                                                hr




                                                                hr
                                                               +




                                                              pe




                                                              pe
                               N




                                                              G




                                                              G
                                                           ol




                                                           ol
                                                          D
                                                          D

                                                         m




                                                       ol
                                                       ol

                                                       a
                                   a

                                                      1n




                                                    kD
                                D




                                                     m
                                                     m




                                                     1
                              5k


                                                    +




                                                  1n
                                                  1n




                                                   +
                                                  40
                                                SS




                                                 S

                                                +
                                                +




                                     kD DS
                                    D




                                            SS
                                             SS
                               a




                                            a

                                           D
                                           D
                               D




                                        kD
                             5k




                                         a
                                         a
                                     D




                                      40
                                   5k




                                   40




                                                Treatment Groups


Figure 16. TNF-α concentration in mesenteric lymph node cultures from mice
treated with DSS and ghrelin peptides. To determine the cytokine profile between
each treatment group, mesenteric lymph nodes, adjacent to proximal colons were
dissected from the mice, on day 8. The lymph nodes were then obtained in a single-
cell suspension, plated, and then stimulated with 50ng/ml PMA and 750ng/ml
ionomycin. The culture supernatants were collected after 48h time points. Cytokine
levels of TNF-α in BALB/c (A) and C57BL/6 (B) mice were determined using an
ELISA. There was so statistical significance observed between the different
treatment groups and the naive mice (N=3-6 mice/group, Mean ± S.D).

                                                                 66
4.7 Enumeration of CD4+CD25+Foxp3+ T regulatory cells in mesenteric lymph
nodes
Figures 17 and 18 demonstrate flow cytometric analysis of T regulatory (Treg) cells
from BALB/c and C57BL/6 mice respectively. The x-axis represents CD25 staining in
cells CD4+ gated against, y-axis staining for the Foxp3 transcription factor. Figure 19A
shows that there was not any significant difference in the Treg cells between the PBS
treated and the ghrelin treated group in both the 5kDa and 40kDa DSS in the BALB/c
mice. There was a slight increase in the 40kDa DSS + Δ 4 peptide as compared to the
40kDa DSS + PBS; however this did not reach significance, Mann-Whitney U-test,
p=0.06. There was a significant increase in Treg cells in C57BL/6 mice treated with
5kDa DSS + Ghrelin when compared to the naïve mice, Mann-Whitney, U-test
p=0.0009. There was also a slight increase in the percentage of Treg cells in the
C57BL/6 mice (Figure 19B) in the 5kDa DSS + Ghrelin group as compared to the 5kDa
DSS + PBS, however using a Mann-Whitney U-test failed to show significance, p=0.2.
There was no statistical difference observed between each treatment group in both the
strains of mice (Figure 19). Ghrelin did not seem to have a substantial effect in
significantly increasing the proportion of CD4+CD25+Foxp3+ cells in MLNs from
BALB/c and C57BL/6 mice.




                                          67
           5kDa DSS + PBS             5kDa DSS + 1nmol Ghrelin         5kDa DSS + 1nmol Δ4 peptide




          40kDa DSS + PBS             40kDa DSS + 1nmol Ghrelin 40kDa DSS + 1nmol Δ4 peptide
Foxp3




                                       CD25 – gated on CD4+
              Isotype control




        Figure 17. Representative flow cytometry plots of T regulatory cell populations in
        mesenteric lymph nodes in BALB/c mice. Cells were stained with CD4-FTIC, CD25-
        PE, Foxp3-APC. The percentage of CD4+ CD25+Foxp3+ cells was determined using
        LSRII Flow Cytometer and shown within the gate on each graph. Data were analysed
        using FlowJo software (Tree Star, Harvard). N=11-12 mice/group.


                                               68
                    Naives                       5kDa DSS + PBS                5kDa DSS + 1nmol Ghrelin




        5kDa DSS + 1nmol Δ4 peptide             40kDa DSS + PBS               40kDa DSS + 1nmol Ghrelin
Foxp3




        40kDa DSS + 1nmol Δ4 peptide




                                           CD25 – gated on CD4+
        Figure 18. Representative flow cytometry plots of T regulatory cell populations in
        mesenteric lymph nodes in C57BL/6 mice. Cells were stained with CD4-FTIC, CD25-PE,
        Foxp3-APC. The percentage of CD4+ CD25+Foxp3+ cells was determined using LSRII
        Flow Cytometer and shown within the gate on each graph. Data were analysed using FlowJo
        software (Tree Star, Harvard). N=11-12 mice/group.


                                                     69
                  A
                    BALB/c MLN regulatory T cells

                                          30




                      CD4+CD25+Foxp3+
                                          20



                                          10



                                                      0
                                                                                                        in          e
                                                            s




                                                                                        in


                                                                               SS d e

                                                                                         S
                                                                                         S
                                                                                                      el        tid
                                                         ve




                                                                                    PB
                                                                   PB


                                                                                     el
                                                                                                    hr




                                                                                      i
                                                                                                             ep



                                                                                   pt
                                                      ai




                                                                                  hr




                                                                                  +
                                                                  +




                                                                                 pe
                                                                                                   G
                                              N




                                                                          G                                p
                                                                                                ol
                                                              SS



                                                                                                        ∆4
                                                                       ol

                                                                              ∆4

                                                                              D
                                                          D




                                                                                              nm     ol
                                                                     m

                                                                           ol

                                                                            a
                                                      a

                                                                  1n



                                                                                            1
                                                                         kD
                                          D




                                                                                                    m
                                                                          m

                                                                                          +      1n
                                        5k


                                                              +

                                                                       1n

                                                                       40
                                                                                      SS
                                                          SS




                                                                                               +
                                                                      +




                                                                                     D
                                                                                            SS
                                                          D

                                                                  SS




                                                                                   a
                                                      a




                                                                                kD        D
                                                               D
                                  D




                                                                                        a
                                5k




                                                                              40
                                                              a




                                                                                     kD
                                                        D
                                                      5k




                                                                                   40
                                                                          Treatment Groups




                  B
                    C57BL/6 MLN regulatory T cells

                                                      30
                                    CD4+CD25+Foxp3+




                                                                       **
                                                      20




                                                      10




                                                          0
                                           1n pe lin
                                                             s



                                                            in

                                                   S de

                                            ol Gh S



                                            o l Gh e


                                                             e
                                                 lG S




                                                    pe in
                                 SS + DSS ive




                                                           d



                                                          id
                                                o l PB
                                      1n mo PB




                                                           l
                                                         el
                                               DS ti




                                      1n mo pti

                                               ∆4 re
                                               ∆4 re




                                                       pt
                                                      hr
                                    SS a pep
                                                       a




                                    + 1nm +
                                                     +
                                                     N




                                                  l
                         kD a D 0kD 4
                                               ∆
                                            ol
                             a DSS Da
                                           1n




                                          m
                                          m




                                          m
                                        5k




                                         +
                                      1n
                                      4
                                    +




                                  S
                               DS
                        5 k Da
                               D



                       40 kD
                            5k




                             a
                           40
                           D




                                                                          Treatment Groups


Figure 19. Regulatory T cell numbers in mesenteric lymph nodes in BALB/c
(A) and C57BL/6 (B) mice. The percentage of CD4+ CD25+Foxp3+ cells was
determined using LSRII Flow Cytometer. Data were analysed using FlowJo
software. There was no significant difference observed between the different
DSS groups and with ghrelin treatment. However, there was significant increase
in Treg cells in C57BL/6 (B) mice when comparing the naive mice to the 5kDa
DSS + Ghrelin treated group, **p=0.0009, using a Mann-Whitney U-test.
(N=11-12 mice/group, Mean ± S.D).


                                                                                  70
4.8 Treatment with ghrelin did not improve wound healing in the HT29 cell line
Wound healing assay was used as a model for intestinal epithelial growth and recovery.
To measure the effect of ghrelin in cell migration/wound healing, the colon cancer cell
line HT29 was obtained. HT29 cells were grown to confluence and were treated with
Mitomycin C (to inhibit proliferation) for two hours prior to creating the “wound”.
Once a scratch/wound was created the cells were immediately treated with various
concentrations of ghrelin (0.01, 0.1, 1, 10nM), media only as negative control and EGF
(100ng/ml) as positive control and were photographed at different time points (0, 10,
24, 48h). Images taken were analysed using the ImageJ program to quantify wound
closure in the cells. As can be seen from Figure 20A there was not any significant effect
in wound closure after 10 h of treatment with ghrelin. There was no difference between
the different concentrations of ghrelin in the wound closure of HT29 cells. Treatment
with EGF caused a significant increase in the cells after 10h (One-way ANOVA,
Tukey’s post hoc: p<0.001) and 24h (p<0.05) treatment. However, there was not any
difference observed between the media only (negative control) and different
concentrations of ghrelin treated cells. Figure 21, shows HT29 cells were pre-treated
with ghrelin for one hour before the “wound” was created. After 10hr (Figure 21A) of
treatment with ghrelin there was not any difference observed between any group. Even
the EGF did not cause any significant increase in wound closure after 10 and 24 h.
There was a slight increase in wound closure after 24h in the HT29 cells treated with
0.1nM ghrelin. However, results from One-way ANOVA, with Tukey’s post hoc tests
revealed that this was not statistically significant.




                                              71
            A
                                                                    HT29 10h wound
                                                        40




            Percent of wound closure
                                                        30

                                                                         **
                                                        20



                                                        10



                                                         0
                                                                     F


                                                                               G




                                                                                                           G
                                                                                          G
                                                               l




                                                                                                G
                                                             ro


                                                                    EG


                                                                             nM




                                                                                                      nM
                                                                                          M


                                                                                                M
                                                           nt




                                                                                        1n


                                                                                              1n
                                                         co




                                                                           01




                                                                                                    10
                                                                                      0.
                                                                         0.
                                                    eg
                                                    N




                                                                     Treatment Groups


             B
                                                                    HT29 24h wound
                                                        40

                                                                     *
                         Percent of wound closure




                                                        30



                                                        20



                                                        10



                                                          0
                                                                     F


                                                                                  G




                                                                                                       G
                                                                                          G
                                                                l




                                                                                                G
                                                              ro


                                                                    EG


                                                                              nM




                                                                                                      nM
                                                                                          M


                                                                                                M
                                                            nt




                                                                                        1n


                                                                                              1n
                                                          co




                                                                            01




                                                                                                    10
                                                                                      0.
                                                                          0.
                                                        eg
                                                    N




                                                                         Treatment Groups


Figure 20. Analysis of wound assay on HT29 cells to measure the effect of
various ghrelin concentrations in wound healing. Cells were photographed at
different time points (0, 10, 24, 48h). Graph represents the percentage of wound
closure of HT29 cells over a period of 48 h. * indicates p<0.05; **p<0.001
(one-way ANOVA with Tukey’s post hoc comparisons). Data represent 1
experiment. (N=6 per treatment group, Mean ± S.D).



                                                                               72
            A                                                   HT29 10h wound
                                                             pre-treated with Ghrelin
                                                        40




                            Percent of wound closure
                                                        30



                                                        20



                                                        10



                                                            0
                                                                              F


                                                                             G




                                                                                                 G
                                                                             G
                                                                  l




                                                                                          G
                                                                ro

                                                                           EG

                                                                          nM




                                                                                                nM
                                                                           M


                                                                                       M
                                                              nt




                                                                         1n


                                                                                     1n
                                                            co




                                                                        01




                                                                                              10
                                                                       0.
                                                                      0.
                                                         g
                                                       Ne




                                                                       Treatment Groups



            B                                                  HT29 24h wound
                                                            pre-treated with Ghrelin
                                                       40
             Percent of wound closure




                                                       30



                                                       20



                                                       10



                                                        0
                                                                              F


                                                                             G




                                                                                                 G
                                                                             G
                                                              l




                                                                                          G
                                                            ro

                                                                           EG


                                                                          nM




                                                                                                nM
                                                                           M


                                                                                      M
                                                          nt




                                                                         1n


                                                                                    1n
                                                        co




                                                                        01




                                                                                              10
                                                                       0.
                                                                      0.
                                                       eg
                                                N




                                                                      Treatment Groups

Figure 21. Pre-treatment of HT29 cells with various concentrations of
ghrelin to measure its effect in wound healing. The cells were pre-treated
with various ghrelin concentrations before creating the “wound”. Cells were
then photographed at different time points (0, 10, 24, 48h). Graph represents
the percentage of wound closure of HT29 cells over a period 48h. Data
represent 1 experiment. (N=3 per treatment group, Mean ± S.D).




                                                                            73
                            Chapter Five: Discussion

       This study demonstrates that ghrelin treatment suppresses clinical colitis in
C57BL/6 mice treated with DSS (5kDa) and this is reflected in the reduction in
accumulative histopathological scores in the colon. Significant reduction of
inflammation was found in the proximal colon of these mice, although there also was a
trend towards less inflammation in the mid and distal colons of ghrelin treated mice.
This may have been due to expression of the ghrelin receptor or other receptors which
mediate ghrelin’s actions, being most highly expressed in the proximal colon region.
Peroxisome proliferator-activated receptors (PPAR-γ) are a group of nuclear receptor
proteins, which have been recently shown to interact with ghrelin receptor signalling
(Demers et al., 2008). A study by Avallone et al., (2006) has shown that scavenger
receptor, CD36, and/or GHS-R1a receptor may signal to enhance PPAR-γ activity in
macrophages. PPAR-γ has a major role in mediating anti-inflammatory processes and
the ability from its ligands to inhibit the expression of inflammatory genes in
macrophages and other vascular cells (Lee et al., 2003; Castrillo & Tontonoz, 2004).
Thus, Avallone et al, (2006) speculate that the activation of CD36 and/or GHS-R1a
may impact the inflammatory response in macrophages through PPAR-γ. Therefore it
can be potentially hypothesized that PPAR-γ could play a major role in inflammatory
responses mediated via ghrelin. PPAR-γ is expressed throughout the colon, mostly
occurring in the luminal epithelial cells of the proximal colon (Su et al., 2007).
Therefore this may explain why there was an effect of ghrelin primarily in the proximal
colon and this hypothesis is currently being investigated in our laboratory. Su et al.,
(2007) further showed that PPAR-γ was lowly expressed in the distal colon in the
C57BL/6 mice, which supports our findings that ghrelin showing only slight
suppression of colitis in the distal colon.
       Our results showed mild action of ghrelin in the DSS model in comparison to
the TNBS model used by the Gonzalez-Rey group. The difference in colitis models
could be one of the reasons why a difference in ghrelin’s effect was noticed. Different
mouse strains and animal housing conditions could also result in the variance of results.
The previous study also used the BALB/c mouse strain with 5kDa DSS, however, their
study focused on the TNBS model, and they only showed difference in colon lengths,
clinical scores, and neutrophil activation activity results from their DSS model. Since


                                              74
ghrelin is des-acylated very easily due to instability of the esterification, we made every
effort to preserve the activity of ghrelin however, this could be responsible for the
difference in results. Perhaps in the future the use of more stable analogues of ghrelin
such as the growth hormone secretagogues (Merck Research Laboratories) would be
efficacious.
       A previous study by Melgar et al., (2005) demonstrated that the severity of
acute colitis induced by DSS was strain specific, where the colitis was more chronic in
C57BL/6 but not in the BALB/c mice. Our results showed that colitis in C57BL/6 mice
was more severe with 5kDa DSS treatment and colitis in BALB/c mice was more
severe with 40kDa DSS. Moreover, many studies have shown that the severity of colitis
induced by DSS is dependent on inbred mouse strain (Maehler et al., 1998), the
concentration of DSS (Egger et al., 2000), the molecular weight of DSS (Axelsson et
al., 1996; Kitajima et al., 2000), and the duration of DSS (Cooper et al., 1993). To
further investigate the pathophysiological effect of ghrelin in intestinal inflammation,
we studied the anti-inflammatory effect of ghrelin peptides using different molecular
weights of DSS in two different mouse strains. Our results indicate that treatment with
ghrelin was more effective in the C57BL/6 mouse strain treated with 5kDa DSS as
compared to the 40kDa DSS. C57BL/6 mice treated with 40kDa DSS showed no
beneficial effects of ghrelin, possibly because the severity of colitis in the 40kDa DSS
is more prominently seen in the lower colon (Axelsson et al., 1996). Kitajima et al.,
(2000) examined the effect of different molecular weights of DSS and found that the
severity of disease in the 5kDa DSS was mostly in the caecum and the upper colon. Our
results show that the C57BL/6 mice had severe inflammation in their proximal and
distal colons after being treated with 5kDa DSS in comparison to 40kDa DSS. This
inflammation was improved significantly after treatment with ghrelin peptides in the
proximal colon. BALB/c mice however, had more inflammation in the distal colon with
40kDa DSS treatment. Kitajima et al., (2000) also showed that the severity of colitis in
the 40kDa DSS was more prominently seen in the lower colon of BALB/c Cr Slc mice.
Axelsson et al., (1996), discuss that perhaps the difference in the severity of colitis
caused by the 5kDa and 40kDa DSS may be due to the sulphur content per molecule,
which may play an important role in the induction of colitis. It is suggested that the
larger molecular weight of DSS may be more prone to induce severe colitis in the
region of the colon which is more permeable where large amount of DSS can pass
through (Axelsson et al., 1996). Some in vitro studies of the large intestine with test
                                            75
markers have shown that the proximal colon is more permeable than the distal colon
(Hosoya et al., 1993; Lange et al., 1994; van Meeteren et al., 1998). This therefore
suggests that there is a strong relationship between the colitis induced by different
molecular size forms of DSS and the region of permeability in the mucosal barrier
(Kitajima et al., 2000).
       It is still unknown whether the Δ4 proghrelin peptide is functionally active.
Other prohormone fragments of ghrelin, such as obestatin, have been shown to be
bioactive. Obestatin has been reported to have inhibitory effects on feeding and
digestive motility and therefore has been postulated to antagonize ghrelin actions on
energy homeostasis and gastrointestinal functions (Zhang et al., 2005). However, these
findings have been questioned and further studies are required to determine the
physiological function of obestatin (Gourcerol et al., 2006; Lauwers et al., 2006; Bassil
et al., 2007). We therefore investigated whether the Δ4 peptide had similar or opposing
effects in our colitis models as ghrelin. Our results indicate that whilst there appears to
be mild benefit this is not significant or as potent as ghrelin, but it is mostly working in
the proximal colon. This effect was only seen in the C57BL/6 mice and not in the
BALB/c mice. Therefore, we may need to optimise the delivery of this peptide in mice
and further study of its administration and dosage is being investigated in our
laboratory. We would also need to determine the expression of Δ4 proghrelin peptide in
the both normal and inflamed colon and also in the epithelial cells.
       Regulatory T cells (Treg) play a central role in IBD. They are characterised by
CD4+CD25+Foxp3+ which produce anti-inflammatory cytokines, IL-10 and TGFβ
(Mowat, 2003; Uhlig et al., 2006). In our study we did not find any significant
difference in Treg cells isolated from mesenteric lymph nodes (MLNs) with ghrelin
treatment. There seemed to be a slight increase in the Treg cells with ghrelin treatment
in the C57B/6 mice treated with 5kDa DSS. This may have been because these mice
had severe colitis in comparison to 40kDa DSS treatment. This study however did not
look at the function of Treg cells in these mice. Gonzalez-Rey et al., (2006) found
increased levels of IL-10 and TGFβ in MLNs and the lamina propria mesenteric cells of
their BALB/c mice. Our study failed to show any such difference in the number of Treg
cells in our BALB/c mice treated with ghrelin.
       We also investigated the levels of the Th-1 pro-inflammatory cytokine, TNF-α
in this study. However, we could not determine a conclusion because cultures MLNs
from our naive mice had unexpectedly high levels of TNF-α than normally identified in
                                            76
our lab. This perhaps could be a technical issue, since we had elevated levels of TNF-α
isolated from the MLNs. Other studies have measured pro-inflammatory cytokine levels
using protein extracts from the colon or colon implants to determine the effect of
ghrelin in colitis (Gonzalez-Rey et al., 2006; Heazlewood et al., 2008). There can be
numerous reasons explaining the increased cytokine levels. The MLN cells may have
been over-stimulated with PMA/ionomycin after 48 h. Gonzalez-Rey et al. (2006) used
a low concentration of PMA (10ng/ml) whereas we used a fairly higher concentration
(50ng/ml). Since naïve mice are healthy, at times it is very difficult to distinguish the
lymph nodes from fat. Therefore, there could be some possibility that fat cells were also
incubated along with the MLN cells. If this were the case, then fat cells could have
caused an increase in various cytokine production causing the MLN cells from the
naïve mice to have elevated TNF-α levels. Leptin, a nonglycosylated protein is
produced by adipocytes, it induces activation of cytokine towards a Th1 response and
has been found to enhance mesenteric TNF-α expression (Zhang et al., 1994; Barbier et
al., 2003; Fain et al., 2004). Explants from the lamina propria could also be beneficial
in investigating the effect of ghrelin in colitis in these DSS-induced mice. The
recruitment of immune cells, dendritic cells and macrophages into the lamina propria
could help in determining the mechanism of ghrelin’s action in inflammation. Since it
has been found that ghrelin receptors are expressed in macrophages, B cells, and
dendritic cells, it would be important to investigate the role of ghrelin in these cells
(Dixit et al., 2004). It would be interesting to perform in vitro studies with mouse
intestinal epithelial cells and by insulting the cells with TNF-α or lipopolysaccharides
(LPS) first and then treating the cells with ghrelin to determine the anti-inflammatory
effects of ghrelin.
        We also found that the naïve BALB/c mice had a consistent presence of perhaps
a parasitic infection primarily occurring in the caecum and the proximal colon in the
histology images. It was present in either the luminal region or in the surface
epithelium, but was very rarely seen in the crypt or in the crypt base. It is suspected that
there may be an underlying parasitic infection and a senior parasite expert, Dr. Damien
Stark (Division of Microbiology, SydPath St. Vincent's Hospital) has suggested it to be
Dientamoeba fragilis, after observing histological images taken from the colons of the
BALB/c mice. Perhaps this underlying parasite could be the cause of the low-grade
inflammation occurring in the naïve and ghrelin and delta 4 only mice. D. fragilis is a
trichomonad parasite which has been implicated in causing gastrointestinal disease in
                                            77
humans (Stark et al., 2007). It has been found that D. fragilis infection may be
symptomatic with both acute and chronic infections being reported in children and
adults causing symptoms such as diarrhoea and abdominal pain (Stark et al., 2005).
However, no such infections have been observed in rodents so far. Therefore, it would
be too early to predict the reason of low grade infection in the BALB/c mice. Currently,
there are further investigations taking place to determine if D. fragilis is indeed present
or whether there is another cause of the background inflammation.
       It is not known if ghrelin affects wound healing in the intestinal epithelial cells
of mice so we investigated an in vitro wound assay to determine if it could help repair
the epithelial barrier. Previous study with ghrelin has shown it can increase migration in
astrocytoma cells (Dixit et al., 2006). However, we did not see an improvement in
wound healing in intestinal epithelial cells- HT29. This could have been because we
needed to insult the cells with TNF-α first, so it would interfere with the inherent
restitutive potential of mucosal epithelial cells through inhibition of proliferation, cell
migration and induce cellular apoptosis (Waseem et al., 2008). A study by Waseem et
al., (2008) showed that in the presence of TNF-α, ghrelin promoted the migration of
intestinal epithelial Caco-2 cells. We are confident our assay worked because treatment
with the positive control, EGF, did cause some significant increase in wound closure
after 10 and 24h. However there was not any effect on wound healing in the HT29 cells
with various concentrations of ghrelin. A complete closure with EGF after 24h
treatment could have been a better control in this assay. In the future, it would be useful
to study would closure in HT29 cells using different concentrations of EGF. It would
also be beneficial to study the effects of EGF + ghrelin in HT29 cells and other colon
cancer cells line. This would address whether there are potential synergistic or additive
effects of ghrelin on these cell lines. For future studies it would be useful to examine
the effect of ghrelin in wound healing in mouse epithelial cell lines to confirm the effect
of ghrelin in suppressing colitis in C57BL/6 mice in the proximal colon. Further
investigation would be required to study the role of ghrelin in other intestinal epithelial
cell lines using an siRNA approach and to determine if the down-regulation of ghrelin
and its receptor mRNA decreases the rate of proliferation and migration in these cell
lines. In order to establish the clinical relevance of ghrelin expression, future studies are
required in human colon (histopathological) tissue and this should be performed using
RT-PCR, Western analysis and immunohistochemistry.


                                             78
       In conclusion, we demonstrate that ghrelin was able to partially suppress colitis
in the C57BL/6 mice in the proximal colon and very mildly in the mid and distal colon.
However, these effects were only in the 5kDa DSS model and not in the 40kDa DSS
model. Although these results confirm the anti-inflammatory actions of ghrelin, they are
not in complete agreement with the findings of the Gonzalez-Rey et al., (2006). Our
study only showed improvement with ghrelin in the proximal colon of C57BL/6 mice
treated with a smaller molecular weight of DSS. This study also demonstrates that the
severity of colitis depends very highly on the molecular weight of DSS along with the
strain of mice. Our study showed that ghrelin was able to improve colitis in the
proximal colon, therefore it may be applicable to use ghrelin treatment in IBD in the
future. Patients with Crohn’s Disease of the terminal ileum could benefit with ghrelin
treatment as it is more effective in that region. Future studies examining the expression
of ghrelin and its receptor in human colons would be able to guide us in treatment with
ghrelin.




                                           79
                                       Appendix


 1.1
 SCORE SHEET FOR MICE UNDERGOING DSS TREATMENT

 DAILY OBSERVATIONS
         Experiment number:                                                          ♀/♂
         Mouse number:

         Date and time of challenge:
                                       Dose:
         Pre-challenge weight (g):

DATE
Time post-challenge
Days post-DSS              Day   Day    Day     Day   Day   Day   Day   Day   Day
                            1     2      3       4     5     6     7     8     9
Observations from a
distance
Inactive
Hunched posture
Ruffled fur
Rate of breathing
Crusty Eyes
Shivering
Diarrhoea
Rectal bleeding
On handling
Not inquisitive or alert
Bodyweight (%
change from
start/score)*
Any other abnormal
behaviour or signs
noted
ACTION TAKEN^

NOTES


TOTAL SCORE

 Scoring Details:
 0 = normal, 1 = equivocal symptoms, 2 = mild symptoms, 4 = severe symptoms. Total
 of 4 or over = mouse culled.
 0= no weight loss, 2 = 5-15% weight loss, 4 = >15% weight loss


                                           80
1.2
Assessment of DSS colitis – scoring system adapted from Obermeier Clin Exp
Immunol. 1999; 116(2): 238–245 and Tamaki et al. Gastroenterology 2006; 131:1110-
1121.

Inflammation Severity (IS)                              Infiltration Extent (IE)
0 = none                                                0 = no infiltrate
1 = mild                                                1 = infiltrate around crypt base
2 = moderate                                            2 = infiltrate reaching to muscularis mucosae
3 = severe                                              3 = extensive infiltration reaching the muscularis mucosae and
                                                        thickening of the mucosa with abundant oedema
                                                        4 = infiltration of the submucosa.
Epithelial Damage (ED)                                  Percentage Involvement of Epithelial Damage (PD) (crypt
0 = normal morphology                                   abscessed, crypt loss or ulceration)
1 = some loss of goblet cells /some crypt               0 = 0%
abscesses or damage                                     1 = 1-25%
2 = loss of goblet cells in large areas                 2 = 26-50%
/extensive crypt abscesses or damage                    3 = 51-75%
3 = loss of crypts <5 crypt widths                      4 = 76-100%
4 = loss of crypts > 5 crypt widths, < 20%
ulceration
5 = > 20% ulceration

Experiment …………………………………….. Read by…………………… Blind
Date    / /

No.                 CA                            PC                            MC                    DC/REC
       IS      IE        ED   PD    IS       IE        ED    PD     IS     IE    ED     PD      IS    IE     ED     PD




                                                             81
1.3

TABLE A.1. Photomultiplier (PMT) voltages used for flow cytometric enumeration
of T regulatory cells in mesenteric lymph nodes.


Parameter    PMT Voltages    PMT Voltages      PMT Voltages    PMT Voltages
             C57BL/6 5kDa    C57BL/6 40kDa     BALB/c 5kDa     BALB/c 40kDa
             Figure 18       Figure 18         Figure 19       Figure 19
FSC-A        549             599               599             599
FSC-W        549             599               599             599
FSC-H        549             599               599             599
SSC-A        308             374               368             324
SSC-W        308             374               368             324
SSC-H        308             374               368             324
FITC         484             500               484             451
PE           533             516               522             495
APC          632             604               622             593




                                     82
Foxp3




                         CD25                                               CD4


        Figure A.1. A representative example of the gating strategies employed to
        enumerate T regulatory cells in mesenteric lymph nodes. Erythrocytes,
        adipocytes, granulocytes and cellular debris were eliminated from analysis by
        the use of forward and side scatter profiles. Lymphocytes were further gated on
        size and side-scatter profile. CD4+ T lymphocytes were determined by CD4-
        FITC staining and only these cells were included in the T regulatory cell
        analysis. T regulatory cells were characterised as CD4+CD25+Foxp3+
        (all antibodies from eBioscience).




                                               83
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