Alterations in Epithelial and Mesenchymal Intestinal Gene Expression During Doxorubicin-Induced Mucositis in Mice In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin -LRB- DOX -RRB- - induced mucositis in mice . Intestinal segments were collected on various days after DOX treatment . DOX-induced damage at day 1 -- 2 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers . Concurrently , T-cell factor-4 -LRB- TCF4 -RRB- levels increased and the epithelial differentiation enhancing factor , bone morphogenic protein-4 -LRB- BMP4 -RRB- , decreased . During severe damage -LRB- day 3 -RRB- , BMP4 levels were significantly increased , which inversely correlated with epithelial proliferation . At the same time , the expression of the epithelial differentiation markers was increasing again . At day 7 , BMP4 levels were down-regulated , while the levels of the epithelial differentiation markers and TCF4 were normalized again . These data suggest that in response to DOX-induced damage , BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved . Introduction The highly proliferating small intestine is very susceptible to chemotherapy-induced damage -LSB- 31 , 47 -RSB- . This side effect , often referred to as mucositis , is very painful and can be life threatening due to the enhanced risk of bacterial translocation caused by loss of epithelial barrier function . Each year , around 500,000 patients worldwide suffer from this side effect -LSB- 7 -RSB- for which there is still no definitive prophylaxis or treatment . Drug-induced damage to the highly proliferating stem cells and progenitors located in the crypts of Lieberkühn showed that different classes of cytostatic drugs affect epithelial crypt cells of different topographical and hierarchical status -LSB- 12 , 13 , 24 -RSB- . Methotrexate -LRB- MTX -RRB- , 5-fluorouracil and vincristine for example , induce damage high in the proliferative compartment at crypt position 9 -- 11 . Damage induced by MTX is well characterized and shows severe morphological changes characterized by epithelial flattening , villus-atrophy , and specific de-differentiation of enterocytes . The last one is indicated by a decreased expression of the enterocyte-specific enzymes but a maintained expression of goblet and Paneth cell-specific genes -LSB- 29 , 38 , 41 , 45 , 46 -RSB- . Doxorubicin -LRB- DOX -RRB- is a cytostatic drug that also frequently causes severe mucositis . However , DOX-induced damage is not well characterized -LSB- 28 -RSB- . DOX preferentially attacks cells at crypt position 4 -- 6 , which is just above or at the same level as the intestinal stem cells -LSB- 12 , 32 -RSB- . DOX is therefore thought to induce more severe damage , especially to the proliferating compartment at the bottom of the crypts , as for example MTX . In clinical practice , DOX is often used in treatment of solid tumors , leukemia , and lymphomas in both adult and childhood cancer patients -LSB- 16 , 18 , 21 , 28 -RSB- . Recent studies revealed a dynamic cascade of events leading to chemotherapy-induced mucositis -LSB- 35 -RSB- . Not only are the specialized epithelial cells affected by cytostatic-drug treatment , but also the underlying submucosal connective tissue . Under normal physiological circumstances , the epithelium maintains a cross-talk with the mesenchyme . Epithelial-mesenchymal interactions are critical for the normal morphogenesis and maintenance of the crypt-villus axis -LSB- 15 , 23 -RSB- . Bone morphogenic protein -LRB- BMP -RRB- - and Wnt-signaling pathways are two of the main pathways involved in embryonic and adult intestinal development -LSB- 5 , 33 -RSB- . BMPs are morphogenes that constitute a large group of structurally and functionally related proteins of the TGF-beta superfamily . BMP signaling pathways have a key role in organogenesis -LSB- 2 , 37 -RSB- , gastrointestinal development and intestinal homeostasis in adults -LSB- 8 -- 11 , 14 , 27 -RSB- . After intestinal development , BMP4 is exclusively expressed in the intravillus and intracrypt mesenchymal cells , including those adjacent to the intestinal stem cells -LSB- 8 , 10 -RSB- . Paracrine BMP signaling occurs specifically in the villus from the mesenchyme to the adjacent epithelium -LSB- 8 -RSB- , suggesting involvement in epithelial-mesenchymal interactions as a mesenchymal signaling molecule . The Wnt-signaling pathway also has been implicated in the regulation of the intestinal epithelial proliferation/differentiation balance in vitro -LSB- 43 -RSB- . In the intestine of mice deficient for transcription factor TCF4 , the main Wnt pathway transcription factor in the intestinal epithelium , loss of proliferative compartments and epithelial cell differentiation are found -LSB- 17 -RSB- . Interestingly , the BMP - and Wnt pathways appear to be linked , as was shown by the fact that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell proliferation and subsequent epithelial differentiation -LSB- 8 , 10 -RSB- . The objective of this study was to develop an experimental mucositis mouse model to characterize DOX-induced intestinal damage and subsequent repair . In addition , we aimed to correlate the alterations in morphology , epithelial homeostasis , and gene expression with changes in BMP4 and TCF4 expression . This , in order to gain insight into possible modulation of the epithelial-mesenchymal cross-talk and progenitor compartment during chemotherapy-induced intestinal damage and regeneration . Materials and methods Animals Animal experiments were performed with permission of the Animal Ethics Committee of the Erasmus MC-Sophia . Upon arrival at our institute , 10-week-old male BALB/c mice -LRB- Harlan , Horst , The Netherlands -RRB- were housed individually during the whole experiment in micro-isolator cages under specific pathogen-free conditions with free access to a standard palletized diet -LRB- Hope farms , Woerden , The Netherlands -RRB- and water . After 1 week of adjustment to the new environment , the mice were divided into three groups and injected intravenously with doxorubicin -LRB- DOX -RRB- -LRB- Doxorubicin , Pharma Chemie , Haarlem , The Netherlands -RRB- on two subsequent days . At day − 1 and 0 , the first group of mice was injected with a low dose of DOX of 6 and 4 mg/kg -LRB- low dose -RRB- respectively , a second group was injected with a medium dose of 8 and 5 mg/kg -LRB- medium dose -RRB- and a third group was injected with a high dose of 10 and 6 mg/kg -LRB- high dose -RRB- . Controls were given equivalent volumes of 0.9 % NaCl . Mice in the low - and high-dose group were sacrificed at day 1 , 2 , 3 and 7 after the final DOX injection ; mice in the medium dose group were only sacrificed at day 3 and 7 . One hour before sacrifice , the mice were injected with 120 μl 10 mg/ml 5 - Bromo-2 ' deoxyUridine -LRB- BrdU -RRB- -LRB- Sigma-Aldrich , Zwijndrecht , The Netherlands -RRB- , an uridine analog , to locate the proliferating cells . Per time point 4 -- 6 DOX-treated animals and 2 -- 4 control animals were sacrificed . Segments of mid-jejunum were collected and either processed immediately for histological analyses or snap-frozen in liquid nitrogen for storage at − 80 °C and subsequent protein isolation . Histochemistry Five-millimeter segments of mid-jejunum were fixed in 4 % paraformaldehyde in phosphate-buffered saline -LRB- PBS -RRB- , dehydrated and embedded in Paraplast Plus -LRB- Sherwood Medical , Den Bosch , The Netherlands -RRB- as previously described -LSB- 45 -RSB- . Four μm sections were routinely stained with hematoxylin -LRB- Vector Laboratories , Burlingame , CA -RRB- and eosin -LRB- Sigma-Aldrich -RRB- to study morphological alterations of the crypts and villi . Immunohistochemistry was performed as described previously -LSB- 45 -RSB- with some minor modifications . The sections for BrdU staining required an extra adjustment to this protocol of HCL incubation , washing with borate buffer , and pepsin treatment as described before -LSB- 34 -RSB- . In short , the sections were blocked as described and incubated overnight with the following antibodies diluted in PBS : to visualize BrdU incorporation , mouse monoclonal anti-BrdU -LRB- 1:250 , Roche Applied Sciences , Indianapolis , IN -RRB- was used , as an enterocyte marker rabbit polyclonal anti-rat Sucrase-Isomaltase -LRB- SI -RRB- -LRB- 1:9000 in PBS , kindly provided by Dr. K.Y. Yeh -LSB- 49 -RSB- -RRB- was used and as a goblet cell-specific marker rabbit polyclonal anti-rat trefoil factor family -LRB- TFF3 : 1:3000 , kindly provided by Prof. Dr. D.K. Podolsky -RRB- was used . Furthermore , BMP expression was visualized with anti-BMP4 -LRB- 1:100 , R&D Systems , Abingdon , UK -RRB- . Immunoreactions were detected using Vectastain ABC Elite Kit -LRB- Vector Laboratories , Burlingame , CA -RRB- and 3,3 ′ - diaminobenzidine tetrahydrochloride -LRB- Sigma-Aldrich , Zwijndrecht , The Netherlands -RRB- . Crypt and - villus length Longitudinal sections of crypts and their corresponding villi were selected so that the base -LRB- marked by Paneth cells -RRB- , middle and top of the crypt were all in the plane of section and thus well orientated . The depth of ten crypts and the length of ten villi were measured on three slides per animal , four animals per time point , with the use of a Nikon Eclipse E800 microscope and IM 500 software . Protein dot blotting The expression of enterocyte markers was detected and quantified as described previously -LSB- 46 -RSB- . Briefly , 5-mm segments of the mid-jejunum were homogenized and protein concentrations were measured using the BCA Protein Assay Reagent -LRB- Pierce , Rockford , IL -RRB- and 50 μg protein of each homogenate was dot-blotted on nitrocellulose -LRB- Protran BA83 , 0.2 μm ; Schleicher & Schuell , Dassel , Germany -RRB- . Hereafter , blots were blocked for 1 h with blocking buffer containing 50 mM Tris , pH 7.8 , 5 % -LRB- wt/vol -RRB- nonfat dry milk powder -LRB- Campina Melkunie , Eindhoven , The Netherlands -RRB- , 2 mM CaCl2 , 0.05 % -LRB- vol/vol -RRB- Nonidet P40 -LRB- BDH , Brunschwig Chemie , Amsterdam , The Netherlands -RRB- and 0.01 % Antifoam B -LRB- Sigma-Aldrich -RRB- . Blots were incubated overnight at 4 °C with rabbit polyclonal anti-rat SI -LRB- 1:1000 -LSB- 49 -RSB- -RRB- , and rabbit polyclonal anti-rat trefoil factor family -LRB- TFF3 : 1:1500 -RRB- diluted in blocking buffer . After washing with blocking , the buffer blots were incubated with 125I-labeled protein A -LRB- specific activity 30 mCi/mg , Amersham Biosciences , Roosendaal , The Netherlands -RRB- for 2 h at room temperature . Specific binding of 125I-labeled protein A to the enterocyte marker antibodies was measured using PhosphorImager detection . The elicited signal was quantified by ImageQuant software -LRB- Molecular Dynamics , B&L systems , Zoetermeer , The Netherlands -RRB- and the expression of TFF3 and SI was expressed per 50 μg protein of tissue . Average expression levels of TFF3 and SI in the mid-jejunum were calculated per mouse , followed by calculation of the mean expression of TFF3 and SI per time point studied . Subsequently , the average expression of TFF3 and SI of control mice was set at 100 % . The specificity of the above-described antibodies was previously confirmed by Western-blot analysis -LSB- 46 -RSB- . Western-blot analysis The same protein homogenate was used as described for protein dot blot analysis . Twenty μg of protein was loaded per lane and run on a 12.5 % SDS-PAGE . The separated proteins were transferred to nitrocellulose membranes -LRB- Protran BA83 , 0.2 μm -RRB- and blocked for 1 h at room temperature in blocking buffer as described above . The blots were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer : mouse monoclonal anti-human PCNA , clone PC10 -LRB- 1:250 , Novo Castra Laboratories , Newcastle upon Tyne , UK -RRB- and mouse monoclonal anti-human BMP4 , clone 3H2 -LRB- 1:100 -RRB- , -LRB- Novocastra Laboratories , Newcastle upon Tyne , UK -RRB- , rabbit polyclonal anti-human cleaved Caspase-3 antibody -LRB- 1:1000 , Cell Signaling , Beverly , MA -RRB- , and mouse monoclonal anti-human TCF-4 , clone 6H5-3 -LRB- 1:250 , Upstate , Waltham , MA -RRB- . After washing with PBS , 0.2 % Tween-20 blots bound antibodies were revealed using HRP conjugated goat anti-mouse or rabbit anti-goat secondary antibodies -LRB- 1:1000 -RRB- and SuperSignal West Femto Luminol Enhancer kit -LRB- Pierce , Rockford , IL -RRB- . The signal was detected and quantified by the ChemiGenius gel documentation system -LRB- Syngene , Cambridge , UK -RRB- and the expression of the specific proteins analyzed was expressed per 20 μg protein of tissue . Average expression levels of PCNA , BMP4 , TCF4 , and caspase-3 in the mid-jejunum were calculated per mouse , followed by calculation of the mean expression of these specific proteins per time point studied . Subsequently , the average expression of PCNA , BMP4 , TCF4 , and caspase-3 in control mice was set at 100 % . Statistical analysis Changes in protein expression levels during damage and regeneration were statistically analyzed using the Kruskal-Wallis H-test and the Mann-Whitney U-test . A p < 0.05 was considered statistically significant . Data are presented as the mean ± standard error of the mean -LRB- SEM -RRB- . Results Dose-response analysis of DOX-induced mucositis in an experimental mouse model To optimize the dose of DOX necessary to induce severe intestinal damage -LRB- i.e. , villus atrophy , crypt loss and flattening of the epithelial cells -RRB- a dose-response curve was performed . Thereto , mice were divided in three treatment groups : low dose , medium dose , and high dose -LRB- see Materials and methods , section Animals for details -RRB- . In the high-dose treatment group , two of the six mice died at day 4 . Necropsy showed an excess of fluid in the abdominal cavity of unknown source . Because of the elapsed time after death , morphological evaluation could not be performed . Morphological analysis of the low-dose treatment group at days 1 and 2 -LRB- data not shown -RRB- , revealed only a slight increase in crypt length , which became more pronounced at day 3 -LRB- Fig. 1B -RRB- . An increase in crypt length was also seen in the medium dose group at day 3 . -LRB- Note the medium-dose treatment group was not studied at days 1 and 2 -RRB- . No other morphological changes were seen in the low dose - -LRB- days 1 -- 3 -RRB- and medium-dose group -LRB- day 3 -RRB- . In contrast , in the high-dose treatment group we did see severe morphological damage -LRB- Fig. 1D -- F -RRB- . Specifically , severe villus atrophy , crypt lengthening , crypt loss , perturbation of crypt arrangement , flattening of crypt and villus epithelium and inflammation were observed at day 3 -LRB- Fig. 1F -RRB- . Moreover , the latter morphological damage was already seen , although in a milder degree , at days 1 and 2 after the high-dose DOX treatment -LRB- Fig. 1D , day 1 and Fig. 1E , day 2 -RRB- . At day 7 , the intestinal morphology was completely restored in the low-dose and medium-dose group -LRB- data not shown -RRB- , but was still affected in the high-dose group . As in the latter group , the crypt epithelium still showed signs of regeneration , like new crypt formation and less epithelial flattening -LRB- Fig. 1G -RRB- . Quantitative analysis of crypt and villus length To quantify the amount of morphological damage induced by the different doses of DOX , the length of the crypts and villi were measured -LRB- Fig. 1H -- I -RRB- . In the high-dose group , but not in the low-dose or medium-dose group , a significant increase in crypt length was already observed at day 2 -LRB- data not shown -RRB- . At day 3 the crypt length was significantly increased in each treatment group compared to the control group -LRB- Fig. 1H -RRB- . By day 7 , the length of the crypts had returned to control levels in the low-dose and medium-dose group , but were still increased in the high-dose group . This increase in crypt length , however , showed a trend towards normal levels because the crypt length at day 3 was 41 % above control level but at day 7 this was still only 13 % above control level . The length of the villi did not change in the low-dose or the medium-dose treatment group on each day investigated . However , in the high-dose treatment group the length of the villi were significantly decreased on days 1 -- 3 and 7 -LRB- Fig. 1H , day 3 and 1I , day 7 -RRB- . Because severe intestinal mucositis -LRB- day 3 -RRB- and morphological regeneration -LRB- day 7 -RRB- were seen only in the high-dose treatment group , we continued our studies with the high-dose DOX treatment model . Effects of high-dose DOX treatment on enterocyte-specific gene expression To gain insight into the functional capacity of the intestinal epithelium after DOX treatment , a sucrase-isomaltase -LRB- SI -RRB- immunohistochemical staining was performed . SI is an enterocyte-specific disaccharidase responsible for sucrose degradation . SI is expressed in the brush border of differentiated villus enterocytes directly after weaning -LSB- 42 -RSB- and is considered an intestinal epithelial differentiation marker . At day 1 and 2 after DOX treatment SI was expressed in the brush border of enterocytes along the entire villi , comparable to the control epithelium -LRB- Fig. 2A , control ; B , day 1 ; C , day 2 -RRB- . At day 3 , SI staining showed a patchy pattern with weak staining on less affected parts of the epithelium and even absence of staining on severely damaged epithelial parts of the sections -LRB- Fig. 2D -RRB- . At day 7 , during the regenerative phase , the SI staining pattern was comparable to control again -LRB- Fig. 2E -RRB- . To quantify SI protein expression , protein dot-blot analyses were performed using SI-specific antibodies -LRB- Fig. 2F -RRB- . The SI protein expression level at day 1 was similar to the control situation . At day 2 , SI expression levels were significantly decreased and remained significantly decreased at day 3 . At day 7 , the SI expression levels regained to control levels . Effects of DOX treatment on goblet cell-specific gene expression Goblet cell-specific gene expression after high-dose DOX treatment was analyzed by the expression of trefoil factor family 3 -LRB- TFF3 -RRB- . TFF3 is a bioactive peptide , produced and secreted by goblet cells , that is involved in epithelial protection and repair -LSB- 6 , 22 -RSB- . Immunohistochemical staining of TFF3 showed TFF3 expression by goblet cells in crypts and villi of the jejunum -LRB- Fig. 3 -RRB- . At day 1 , the TFF3 staining was not visibly altered compared to the control situation -LRB- Fig. 3B -RRB- . At day 2 , the immunohistochemical detection of TFF3 decreased visibly , especially in the goblet cells localized in the crypts and lower part of the villi where TFF3 protein could hardly be detected/was absent -LRB- Fig. 3C -RRB- . At day 3 , during most severe morphological damage , TFF3 staining by goblet cells in the crypt was reconstituted -LRB- Fig. 3D -RRB- . Day 7 showed complete regeneration of TFF3 -LRB- Fig. 3E -RRB- . Subsequently , a protein dot-blot analysis was performed -LRB- Fig. 3F -RRB- using TFF3-specific antibodies to quantify TFF3 levels . At day 1 , the TFF3 expression was already significantly decreased to 50 % of control level and maintained at this low level at day 2 . At day 3 , concomitant with the most severe morphological damage , TFF3 expression returned to control level . TFF3 protein expression was again comparable to control situation at day 7 . Effect of high-dose DOX treatment on epithelial proliferation Localization of proliferation was studied by immunohistochemical detection of incorporated BrdU -LRB- Fig. 4 -RRB- . In controls -LRB- Fig. 4A -RRB- BrdU was mainly localized in cells from the bottom of the crypt up to three quarter of the crypt length . At day 1 and 2 the proliferative zone broadened and progressively moved upwards to the crypt-villus junction . At day 3 , during severe morphological damage , BrdU-positive cells formed a scattered pattern throughout the remaining crypt structures of the small intestine . In the regenerative phase , at day 7 BrdU-positive cells were located in the lower part of the crypts again.Fig . 1Morphology of the murine small intestine after low , medium - or high-dose DOX treatment . Morphology of the jejunum of a control mouse -LRB- A -RRB- , at day 3 after low-dose DOX -LRB- B -RRB- and at day 3 after medium-dose DOX -LRB- C -RRB- , both were mildly affected by DOX treatment . Morphology of the jejunum at day 1 -LRB- D -RRB- and day 2 -LRB- E -RRB- after high-dose DOX treatment , which appeared progressively affected and severely deteriorated at day 3 -LRB- E -RRB- . At day 7 , the regenerative phase , the intestinal morphology of the mice in the low - and medium-dose group was completely restored to control situation -LRB- data not shown -RRB- . The morphology at day 7 of the mice treated in the high-dose group was regenerating , showing new crypt formation but still villus atrophy . These photographs are representative examples of a group of four animals per time-point . To quantify the effect of these different doses of DOX treatment , crypt and villus lengths were measured . Crypt and villus lengths of control mice were set at 100 % . At day 3 -LRB- A -RRB- , the length of the crypts in all three DOX treatment groups was significantly increased in comparison to the control group . Villus length in the low - and medium-dose group remained at control level . The length of the villi of the high-dose group decreased significantly , indicating significant villus atrophy after high-dose DOX treatment . At day 7 -LRB- B -RRB- crypt and villus lengths of the low - and medium-dose DOX treatment groups showed complete restoration . The crypts of the high-dose treatment group were still significantly longer , but this increase in crypt length was declining compared to day 3 -LRB- day 3 41 % , day 7 13 % -RRB- . The length of the villi was still significantly decreased at day 7 , showing no signs of regeneration . * P < 0.05 versus control . The bars are expressed in mean + SEMFig . 2Effect of high-dose DOX treatment on the enterocyte-specific SI expression in the small intestinal epithelium . SI expression of the jejunum of a control mouse -LRB- A -RRB- , at day 1 -LRB- B -RRB- and at day 2 -LRB- C -RRB- . The brush border staining at day 1 and 2 is comparable to control situation . At day 3 -LRB- D -RRB- , SI staining showed a patchy pattern ; some parts of the brush border showed SI expression , on some other parts of the brush border SI expression was absent -LRB- → -RRB- . SI expression at day 7 -LRB- E -RRB- showed complete regeneration , and was equal to the control situation . SI protein expression levels were analyzed by dot-blot technique and the SI expression of control mice was set at 100 % -LRB- F -RRB- . The expression of SI remained stable at day 1 , but decreased significantly at day 2 and 3 . During the regenerative phase , at day 7 , the SI expression reached control level again . * P < 0.05 day 2 and 3 versus control . The bars are expressed in mean + SEMFig . 3Effect of high-dose DOX treatment on the goblet-specific TFF3 expression in the small intestine . Immunohistochemical staining of the goblet cell-specific protein TFF3 -LRB- A -- E -RRB- . TFF3 expression of the jejunum of a control mouse -LRB- A -RRB- , at day 1 -LRB- B -RRB- , at day 2 -LRB- C -RRB- , at day 3 -LRB- D -RRB- and at day 7 -LRB- E -RRB- . In the control situation , TFF3 was expressed in all goblet cells in the crypts and villi , at day 1 -LRB- B -RRB- , the amount of goblet cells expressing TFF3 maintained at control level . At day 2 -LRB- C -RRB- , TFF3 expression decreased visibly , the goblet cells localized in the crypts showed no staining . At day 3 -LRB- D -RRB- , during severe morphological damage , TFF3 expression of goblet cells in the crypt was reconstituted . TFF3 expression at day 7 -LRB- E -RRB- was regenerated and comparable to control situation . TFF3 protein expression levels were analyzed by dot-blot technique , the TFF3 expression of control mice was set at 100 % -LRB- F -RRB- . TFF3 expression decreased significantly at day 1 and day 2 , the expression was decreased to 50 % of control level . During severe morphological damage at day 3 , the TFF3 expression increased almost to control level . Day 7 showed TFF3 expression even above control level . * P < 0.05 day 1 and 2 versus control . The bars are expressed in mean + SEMFig . 4Effect of high-dose DOX treatment on epithelial proliferation . The localization of epithelial proliferation was visualized by detection of BrdU incorporation . BrdU incorporation of the jejunum of a control mouse -LRB- A -RRB- and mice at day 1 -LRB- B -RRB- , at day 2 -LRB- C -RRB- , at day 3 -LRB- D -RRB- and at day 7 -LRB- E -RRB- . At day 1 , the BrdU-positive cells were slightly migrated upwards in the crypt , at day 2 the BrdU-positive cells were seen along the entire length of the crypts . At day 3 the BrdU expression decreased and showed a scattered pattern on some crypts and villi . The location of BrdU-positive cells at day 7 was confined again to the lower part of the crypts . PCNA , Caspase-3 , TCF4 and BMP4 protein expression levels were analyzed by Western-blot technique -LRB- F -RRB- , the PCNA expression of control mice was set at 100 % -LRB- G -RRB- . The expression of PCNA protein showed a non-significant trend of increased expression at day 1 , and a significant increase at day 2 . PCNA expression at day 3 and 7 showed a decreasing trend to around 20 % of control level . * P < 0.05 day 1 and 2 versus control . The bars are expressed in mean + SEM The changes in proliferation induced by DOX were quantified by Western-blot analysis of PCNA protein expression -LSB- 4 , 40 -RSB- using a specific monoclonal PCNA antibody . PCNA protein expression after MTX treatment -LRB- Fig. 4F , G -RRB- showed a trend of increased expression at days 1 . At day 2 , this increased expression was significant compared to control levels , indicating that high DOX treatment did not induce an arrest in proliferation at day 1 and 2 . At day 3 , PCNA expression was decreased to around 20 % of the normal level and was still 50 % of control level at day 7 . The latter data indicate that the inhibition of epithelial proliferation sustained during the regenerative phase . Overall , the PCNA levels correlated with the immunohistochemical data -LRB- Figs. 4A -- E vs. 4G -RRB- Effect of high-dose DOX treatment on enterocyte apoptosis Besides proliferation , the influence of DOX treatment on apoptosis was quantified with a polyclonal antibody against cleaved Caspase-3 -LRB- Fig. 4F , 5A -RRB- . Caspase-3 is one of the key executioners of apoptosis -LSB- 26 -RSB- . The expression of cleaved Caspase-3 protein was significantly increased at day 1 and 2 , at the beginning of DOX-induced morphological damage . At day 3 , during severe morphological damage , and day 7 , during regeneration , expression of cleaved caspase-3 was comparable to control level . Increased apoptosis at day 1 and 2 was primarily located along the crypt axis -LRB- Fig. 5B showing day 1 after MTX treatment -RRB- -RRB- Fig. 5Effect of high-dose DOX treatment on epithelial apoptosis . Caspase-3 protein expression of the control mice , analyzed by Western-blot technique -LRB- Fig 4F -RRB- , was set at 100 % -LRB- A -RRB- . At day 1 and 2 , the Caspase-3 levels increased significantly in comparison to the control levels . At day 3 , during severe morphological damage the caspase-3 expression maintained at control level , the same was seen at day 7 , during the regenerative phase . The increase in apoptosis at day 1 is primarily located in the crypt region -LRB- B -RRB- . * P < 0.05 day 1 and 2 versus control . The bars are expressed in mean + SEM . → indicates apoptotic cells Effect of high-dose DOX treatment on TCF-4 protein expression To gain insight into the effects of DOX on the epithelial stem cell compartment , the expression of TCF-4 was quantified . TCF-4 is a transcription factor of the Wnt-signaling pathway and is expressed in the gut epithelium in a gradient fashion that is highest at the base of the crypts -LSB- 1 , 19 -RSB- . TCF-4 − / − mice lose the intestinal epithelial progenitor and stem cell population before crypt formation can be established -LSB- 17 -RSB- . Therefore , TCF-4 has a role in intestinal epithelial stem cell maintenance . The TCF-4 expression -LRB- Figs. 4F and 6 -RRB- remained very stable despite DOX treatment . Only at day 1 the TCF-4 expression increased significantly , although little in absolute sense . But at day 2 , day 3 and day 7 , the TCF4 protein expression remained at control level.Fig . 6Effect of high-dose DOX treatment on TCF-4 protein expression . TCF-4 protein expression of control mice , analyzed by Western-blot technique -LRB- Fig. 4F -RRB- , was set at 100 % . At day 1 , TCF-4 protein expression increased significantly . At day 2 , day 3 , and day 7 , the TCF-4 protein expression remained stable at control level . * P < 0.05 day 1 versus control . The bars are expressed in mean + SEM Effect of high-dose DOX treatment on expression of Bone morphogene 4 -LRB- BMP4 -RRB- To examine the role of BMP4 signaling in intestinal homeostasis the expression of BMP4 was quantified . BMP4 is crucial for epithelial-mesenchymal cross-talk and intestinal homeostasis . BMP4 is expressed in the intravillus and intracrypt mesenchymal cells -LSB- 8 , 10 -RSB- , inhibition of BMP signaling causes intestinal architectural abnormalities -LSB- 8 -RSB- and decreased differentiation as seen in tumorigenesis -LSB- 27 -RSB- . BMP4 protein expression showed a mild decrease in expression level at day 1 after DOX treatment -LRB- Figs. 4F , 7A -RRB- . At day 2 , this decrease in BMP4 expression level progressed and was almost significant -LRB- P = 0.05 -RRB- in comparison to the control level . Thereafter , BMP4 day 2 but not to control . Day 7 showed a non-significant decrease in expression compared to control mice . BMP4 was predominantly expressed in the intravillus mesenchym as shown by immunohistochemistry -LRB- Fig. 7B , C -RRB- , both in control as in MTX-treated mice.Fig . 7Effect of high-dose DOX treatment on expression of the morphogen BMP4 . The BMP4 protein expression of the control mice , analyzed by Western-blot technique -LRB- Fig. 4F -RRB- , was set at 100 % -LRB- A -RRB- . Expression of the morphogen BMP4 protein showed a decreasing trend at day 1 , which was at the brink of significance -LRB- P = 0.05 -RRB- at day 2 in comparison to control expression . At day 3 , the BMP4 expression showed an increasing trend . This increase in expression was significant in comparison to day 2 . Day 7 showed a decrease in expression again beneath the level of control expression . The localization of BMP4 expression did not change at day three -LRB- C -RRB- in comparison to control situation -LRB- B -RRB- . * P < 0.05 day 2 versus control , ** P < 0.05 day 2 versus day 3 . The bars are expressed in mean + SEM Discussion This study revealed that DOX , in a dose of 10 and 6 mg/kg induced severe morphological damage to the small intestine of mice within 3 days , which was almost completely regenerated by day 7 . Moreover , it revealed that the intestine was virtually not or much less affected by lower doses of DOX . Mucositis induced by the chosen dose of DOX was characterized by an increasing degree of intestinal morphological damage at day 1 and 2 , which correlated with a significant increase in both apoptosis and proliferation . During this phase of epithelial hyper-proliferation , the epithelial cells lost their highly differentiated status as measured by a significant down-regulation of epithelial-specific SI at days 2 -- 3 . The decreased expression of TFF3 at days 1 -- 2 could be caused by a decrease in goblet cell differentiation , but on the other hand , could also be the result of increased TFF3 secretion . At day 3 , the time-point when intestinal damage was most severe , the morphology was characterized by severe villus atrophy , a significant rise in crypt length , epithelial flattening , crypt loss , inhibition of proliferation and impaired epithelial differentiation . During morphological regeneration , at day 7 , proliferation started to return to control level , and SI and TFF3 expression levels were normalized again . In order to be able to prevent or treat chemotherapy-induced mucositis , it is essential to know if different cytostatic drugs induce the same or different kinds of intestinal damage . Potten et al. demonstrated that there is a general tendency for antibiotics , like DOX , and radiation to damage the lower cell positions in the crypt near or at the position of the stem cells -LRB- position 4 -- 6 -RRB- -LSB- 12 -RSB- . Alkylating agents on the other hand mainly damage cells at position 6 -- 8 . Anti-metabolites like MTX and a microtubule dissociating agents act on higher cell positions -LSB- 9 -- 11 -RSB- . It is , however , unclear if cytostatic drugs attacking at the lowest positions in the crypts cause a different kind of damage than drugs damaging cells at higher positions . If we compare the DOX-induced mucositis as studied presently with the well-characterized MTX-induced mucositis -LSB- 34 , 38 , 44 -- 46 , 48 -RSB- , then it is clear that there are many similarities and few discrepancies between the two . Although the two drugs affected cells of different hierarchical height -LSB- 12 -RSB- , they both caused apoptosis , villus atrophy , epithelial flattening , crypt loss and a temporary loss of SI expression and TFF3 expression -LSB- 45 , 46 , 48 -RSB- . Since SI is involved in carbohydrate metabolism and TFF3 is involved in mucosal repair -LSB- 6 , 22 , 30 , 42 -RSB- , these data suggest impaired absorption and mucosal repair after DOX as well as after MTX treatment . In contrast , both MTX -LSB- 45 -RSB- and DOX hardly affect the expression of lysozyme by Paneth cells in the crypts -LRB- data not shown -RRB- . Decreased levels of TFF3 after both DOX and MTX was at the same time as epithelial hyper-proliferation -LSB- 45 , 48 -RSB- , but changes in proliferation , induced by MTX treatment followed a different time-line compared to DOX treatment . MTX causes proliferation inhibition within 1 day , followed by a period of hyper-proliferation during severe intestinal damage -LSB- 34 , 45 -RSB- . DOX treatment leads to immediate hyper-proliferation -LRB- day 1 and 2 -RRB- with subsequent inhibition of proliferation during severe morphological damage -LRB- day 3 -RRB- . Moreover , the cell-fate specific affect of MTX on goblet cells causing goblet cells to accumulate in the crypt and at the top of the villus -LSB- 45 , 46 -RSB- was not seen after DOX treatment . The reason for these discrepancies remains to be further investigated , but might be directly related to the difference in topographical height -LRB- and thus status -RRB- of the cells vulnerable to the two different drugs . Overall , however , the similarities in intestinal responses after DOX or MTX treatment are striking . This suggests that there may be common pathways involved in intestinal damage and repair . Historically , chemotherapy - or radiation-induced mucositis was believed to be solely due to damage to dividing epithelial cells at the bottom of the crypts -LSB- 20 -RSB- . However , recently it has become clear that other parts of the intestinal mucosa and submucosa might also be involved -LSB- 35 , 36 -RSB- . Here we provide evidence for a mesenchymal contribution to the damage by showing that BMP4 , a very important lamina propria derived-morphogen in the small intestine -LSB- 8 , 10 -RSB- , is affected by DOX treatment . BMP4 was modulated by DOX during the onset of damage at days 1 and 2 . BMP4 expression decreased almost significant at day 2 , which correlated well with an increasing degree of morphological damage , increased proliferation , and loss of epithelial differentiation as measured by the decreased SI and likely TFF3 expression . Very recently , a link between the BMP and the Wnt pathways has been demonstrated . It was shown that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell proliferation and subsequent epithelial differentiation -LSB- 8 , 10 -RSB- . Here we show that there indeed might be a close relationship between BMP and Wnt pathways , because at day 1 when BMP4 expression is decreased , expression of TCF4 , a Wnt effector , increased significantly , which correlated well with increased proliferation and inhibited epithelial differentiation . At day 2 , BMP4 remained low , whereas TCF4 returned to a normal level and remained at control level during the following days . At day 3 , when damage was most severe , BMP4 was increased , which inversely correlated with proliferation , and correlated with epithelial differentiation , as suggested by the recovery of TFF3 expression level . BMP is involved in epithelial-mesenchymal signaling -LSB- 8 -RSB- and therefore we conclude that the data presented in this study indicate that epithelial-mesenchymal cross-talk is modulated during onset of DOX-induced damage in order to stimulate proliferation instead of differentiation and during severe intestinal damage to induce differentiation and inhibit proliferation . During the regenerative phase , at day 7 , BMP4 expression level was down-regulated again , which could be a response to the shortage in number of crypts , since a blockage of BMP4 has been shown to cause stem cells to divide , leading to newly formed crypts -LSB- 8 , 10 -RSB- . Therefore , our findings are in line with the roles of the BMP and Wnt/TCF pathway in epithelial homeostasis/morphogenesis . Furthermore , the decrease in BMP4 might also indirectly cause the observed decrease in SI expression , because inhibition of BMP4 stimulates Wnt signaling , and Wnt signaling itself induces SOX9 , a negative regulator of Cdx2 , which is a SI transcriptional activator -LSB- 3 , 10 , 39 -RSB- . In addition , BMP4 has been shown to be directly involved in HNF-1α expression , also a well-known activator of SI transcription -LSB- 25 -RSB- . Thus , the decrease in BMP4 might result in a decrease in Cdx2 and HNF-1α expression , two of the most important activators of SI transcription -LSB- 39 -RSB- . Currently , it is not known whether the Wnt or BMP4 signaling pathways regulate TFF3 gene expression . In conclusion , high-dose DOX induces severe damage to the epithelium , which closely resembles damage induced by MTX , indicating that general mechanisms of damage and repair are involved . We show that signaling pathways involving BMP4 and TCF4 and thus epithelial-mesenchymal cross-talk are modulated by DOX-induced damage in such a way that homeostasis of the progenitor compartment is restored by initially inducing cell proliferation and inhibiting differentiation and subsequently inducing differentiation , inhibiting proliferation and promoting crypt fission . Understanding these mechanisms is essential to develop clinical strategies to prevent chemotherapy-induced mucositis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

morphogens
intestinal differentiation
bone morphogenetic proteins


Recurrent respiratory papillomatosis : an overview of current thinking and treatment Human papillomaviruses -LRB- HPV -RRB- infection in benign laryngeal papillomas is well established . The vast majority of recurrent respiratory papillomatosis lesions are due to HPV types 6 and 11 . Human papillomaviruses are small non-enveloped viruses -LRB- > 8 kb -RRB- , that replicate within the nuclei of infected host cells . Infected host basal cell keratinocytes and papillomas arise from the disordered proliferation of these differentiating keratinocytes . Surgical debulking of papillomas is currently the treatment of choice ; newer surgical approaches utilizing microdebriders are replacing laser ablation . Surgery aims to secure an adequate airway and improve and maintain an acceptable quality of voice . Adjuvant treatments currently used include cidofovir , indole-3-carbinol , ribavirin , mumps vaccine , and photodynamic therapy . The recent licensing of prophylactic HPV vaccines is a most interesting development . The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance . Large multi-centre collaborative clinical trials are therefore required . Even so , sufficient clinical follow-up data would take several years . Introduction Sir Morrell Mackenzie -LRB- 1837 -- 1892 -RRB- was the first to recognize papillomas as a lesion of the laryngo-pharyngeal system in children in the late 1800s . It is now apparent that these benign tumours may occur at other parts of the upper gastrointestinal and respiratory tracts , and in all age groups . It was not until the 1940s that Chevalier Jackson -LRB- 1865 -- 1958 -RRB- first coined the term `` juvenile laryngeal papillomatosis '' . The prevalence of laryngeal papillomatosis has been estimated at between four to seven cases per million person-years in the Western World -LSB- 4 , 5 , 25 , 43 -RSB- . Furthermore , the incidence of recurrent respiratory papillomatosis -LRB- RRP -RRB- has been estimated at about 2 per 100,000 in adults and 4 per 100,000 in children -LSB- 9 -RSB- . The disease can be categorized into adult onset and juvenile onset forms . Age of first presentation of disease is usually in the teens -LRB- 50 % -RRB- for the juvenile onset form but can be as early as the first year of life . Initial presentation in the adult form tends to peak in the third and fourth decades . It is now well established that human papillomaviruses -LRB- HPVs -RRB- are the aetiological agent of many benign and malignant tumours arising from epidermal tissues . They are a necessary cause of the second most common female cancer worldwide , cancer of the cervix -LSB- 7 , 45 -RSB- , and strongly associated with several other ano-genital cancers such anal , penile , vulval and vaginal carcinomas -LSB- 17 -RSB- . Furthermore , there is mounting evidence of at least some head and neck cancers associated with HPV infection -LSB- 15 , 17 , 23 -RSB- . These malignancies are associated with ∼ 15 high risk -LRB- HR -RRB- types , in particular HPV 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 68 , 73 and 82 . Benign tumours such as common warts , flat warts and genital warts are caused by low risk types such as HPV 1 , 2 , 3 , 4 , 6 , 10 , 11 and others . HPV 6 and 11 have been described as the dominant types found in RRP -LSB- 10 -RSB- . Despite the benign nature of these lesions , there is significant morbidity and occasional mortality due to multiple recurrences which necessitate hospital admission for surgical removal . Dissemination or extension of the growths into the lower airways indicates a poorer prognosis . The clinical behaviour is variable and lesions can regress , persist and in rare instances , progress to carcinoma if other environmental factors such as smoking or irradiation are involved . Epidemiology Human papillomaviruses infection in benign laryngeal papillomas is well established . One Danish study showed that 95 % of solitary laryngeal papillomas were positive for HPV DNA by in situ hybridization -LSB- 26 -RSB- . Another study from Hong Kong found that 59 % of laryngeal papillomas showed the presence of HPV 6 , 11 , 16 , 18 subtypes , with 6 and 11 the dominant types -LSB- 10 -RSB- . Malignant transformation of some lesions has been described in association with HPV 11 integration into the genome and mutation of p53 -LSB- 32 -RSB- . Infection with HPV 11 is more likely to be associated with the development of distal airways disease than HPV 6 . Clinical manifestation of disease prior to three years of age is a further risk factor associated with distal spread of disease -LSB- 1 , 47 -RSB- . The vast majority of RRP lesions are due to HPV types 6 and 11 , and the reservoir for these types is the human ano-genital tract . Ano-genital HPV is the commonest sexually transmitted viral infection and the prevalence of clinically apparent genital warts is thought to be the `` tip of the iceberg '' of HPV infection . Koutsky et al. estimates that ∼ 10 -- 20 % of the US population between 15 and 49 years of age have molecular evidence of infection and that another 60 % have had prior infection -LSB- 22 -RSB- . In the UK , there were well over 81,000 incident genital wart infections reported from genitourinary medicine clinics in 2005 -LSB- 16 -RSB- and this has been increasing every year . Such high background prevalence suggests that there is a risk of mother to child transmission at the time of delivery , especially if there are noticeable genital warts . Data supporting this hypothesis showed that a history of maternal condylomata during pregnancy was associated with a 200-fold risk of RRP in the child -LSB- 37 -RSB- . An uncomplicated vaginal delivery in a mother with HPV infection has been estimated to carry a risk of transmission of 1:80 -- 1:1,500 -LRB- median of 1:400 -RRB- -LSB- 34 -RSB- . In one group of children with juvenile laryngeal papillomatosis , 54 % had a maternal history of vulval condylomata at the time of delivery -LSB- 13 -RSB- . Another study found that in 77 mothers with condylomata at delivery , 9 children -LRB- 11.6 % -RRB- were later diagnosed with juvenile laryngeal papillomatosis -LSB- 21 -RSB- . The route of transmission is likely to be different in the juvenile onset and adult onset forms of RRP . Evidence for this has been suggested by a case control study , in which the risk factors for both forms were compared -LSB- 20 -RSB- . The authors found that adult onset patients were more likely to have had more sexual partners and oral sex than their controls . Patients with the juvenile form were more likely to have been born to teenage mothers and first-born children compared to their controls . Aetiological and histopathological features Papillomatous lesions preferentially occur anatomically at the sites of `` transformation zones '' , where squamous epithelia abut ciliated columnar epithelia but can infect anywhere in the respiratory tract -LSB- 28 -RSB- . The classical sites for recurrent disease in the upper aero-digestive tract would be the nasopharyngeal area of the soft palate , limen vestibuli , midzone of laryngeal area of the epiglottis , upper and lower margins of the ventricle , vocal fold undersurface , carina and bronchial spurs . It is interesting to note that papillomata have been observed at tracheotomy sites and tracts where the iatrogenic induction of change of epithelialization also occurs -LSB- 19 -RSB- . Human papillomaviruses are small nonenveloped viruses -LRB- ∼ 8 kb -RRB- , with a double stranded circular DNA genome encapsulated within an icosahedral capsid that replicate within the nuclei of infected host cells . The genome codes for 8 -- 10 genes -LRB- median of eight -RRB- . The late L1 and L2 genes code for the viral capsid proteins , the early proteins E1 and E2 are responsible for viral replication and transcription , and E4 appears to aid virus release from infected cells . The early genes E6 and E7 have transforming ability in in vitro assays for HR types but LR types have little to no ability for this feature -LSB- 2 , 33 , 42 -RSB- . Electron microscopic analysis reveals the virion to be ∼ 55 nm in diameter and the capsid to be comprised of 72 pentameric capsomers . The predominant protein in the capsid consists of the L1 protein , with a smaller proportion of L2 embedded deep within the protein shell . It is this L1 protein which provides the dominant antigenic epitopes recognized by neutralizing antibodies and forms the basis for the bivalent -LRB- GlaxoSmithKline -RRB- and quadrivalent -LRB- Merck , Hohenbrumn , Germany -RRB- vaccines currently available . The virus is thought to bind to and gain entry to its host cell , the basal keratinocyte , by microtrauma or abrasions to the surface epithelium . The receptor has not been definitively identified but α6-integrin and heparin sulphate may play important roles in viral entry -LSB- 11 , 18 , 30 -RSB- . Following infection and uncoating , the virus is thought to maintain its genome as a low copy number episome in the basal cells . It has been suggested that expression of E1 and possibly E2 , may be sufficient for basal maintenance of viral episomes -LSB- 50 -RSB- . Viral early proteins E6 , E7 , E1 and E2 are expressed at low level in early passage cell lines derived from naturally occurring low-grade cervical lesions and the viral genome is maintained at around 10 -- 200 copies per cell -LSB- 8 , 41 -RSB- . The viral genome is amplified in differentiating keratinocytes via rolling-circle amplification that will synthesize sufficient viral genome for packaging -LSB- 3 , 12 -RSB- . This requirement for differentiating epithelial cells is a key part of the virus life cycle but the normal restraint on cell cycle progression appears to be abolished by the E6 and E7 proteins and normal terminal differentiation is retarded -LSB- 36 -RSB- . These E6 and E7 effects on key apoptotic proteins such as Rb and p53 have been demonstrated in HPV 16 and other high-risk subtypes in in vitro assays -LSB- 24 , 27 , 28 -RSB- . HPV 6 and 11 E6 and E7 proteins do not readily bind to or degrade the p53 or Rb proteins -LSB- 14 , 31 , 46 -RSB- . This suggests that alternative mechanisms of altered cellular growth and proliferation may exist for the low-risk subtypes of HPV . Furthermore , there are little data on HPV 6 and 11 life cycles , replication , maintenance and viral production in respiratory cells . It is not known if these are similar to or different to disease in the ano-genital region . Treatment Surgical Surgical debulking is currently the treatment of choice ; newer surgical approaches utilizing microdebriders are replacing laser ablation . Surgical excision aims to secure an adequate airway and improve and maintain an acceptable quality of voice -LSB- 49 -RSB- . HPV is present in the normal macroscopically unaffected mucosa and it is currently not possible to distinguish infected cells with a normal appearance from uninfected epithelia . Repeated recurrences are frequent , however , repeated attempts to treat the papillomas may cause serious complications -LSB- 40 -RSB- . Current practice in the treatment of RRP was recently evaluated by a questionnaire in the UK -LSB- 44 -RSB- . Various lasers such as CO2 , KTP , and pulsed dye were found to be the preferred method of surgical removal of RRP in children -LSB- 49 -RSB- . Spontaneous ventilation -LRB- 65.3 % -RRB- is the preferred method of anaesthesia . The frequent recurrence of papillomas has resulted in the use of different adjuvant treatments alongside surgical removal of macroscopically obvious in the attempt to improve outcomes . In the future , advances in the understanding of the immune response to HPV may improve our treatment modalities and prevention strategies . Adjuvant treatment Adjuvant treatments currently used include cidofovir , indole-3-carbinol , ribavirin , mumps vaccine , and photodynamic therapy . As with surgical management , viral persistence occurs following treatment with these adjuvant modalities . Intralesional cidofovir may help control papilloma regrowth and reduce disease severity in many children with RRP -LSB- 39 -RSB- . In most cases , cidofovir would appear to be less efficacious in producing disease eradication . There appears to be little evidence to support prolonged treatment regimes -LRB- i.e. more than eight treatments -RRB- -LSB- 35 -RSB- . Subcutaneously injected cidofovir has been tested on cartilage in a rabbit model -LSB- 39 -RSB- . There was a positive dose-response relationship which existed for gross skin changes ; however , there was no dose-response relationship for severity of change in the epithelium . Higher doses of cidofovir than commonly are used in the treatment of RRP may be safe , although the effects of repeat application and long-term complications are not yet known . In animals , cidofovir is carcinogenic -LRB- mammary adenocarcinoma in rats -RRB- , embryotoxic and teratogenic -LSB- 48 -RSB- . Care must be taken in humans that the possibility of pregnancy is excluded when usage is considered . In view of the severe nephrotoxicity shown when intravenous cidofovir is administered to animals and humans , caution would be advised for repeated intralesional or subcutaneous applications for RRP treatment . The less common complications of bone marrow toxicity , iritis and uveitis may also arise and vigilance is required from clinicians . Controlled trials failed to provide sufficient evidence to draw reliable conclusions about the effectiveness of antiviral agents as adjuvant therapy in the management of RRP . Further research is required before any specific antiviral adjuvant therapy can be recommended . Factors leading to virus activation in RRP have not been recognized , however , extra-oesophageal acid reflux disease -LRB- EERD -RRB- has been suggested as a possible factor , initially by Borkowski et al. -LSB- 6 -RSB- , and then by a group from Harvard in 2005 -LSB- 29 -RSB- . There is clinical evidence suggesting a link between the presence of EERD and RRP . Inflammation induced by acid exposure may result in the expression of HPV in susceptible tissues . Therefore , treatment of EERD should be considered in all patients with difficult to control RRP with EERD . Despite currently available surgical and adjuvant management options , tracheotomy may become necessary in selected patients with extensive disease . Decannulation should be performed as early as possible to avoid further spread of viral infection and improve the quality of life . The primary cause of papilloma extension to the lower airways appears to be iatrogenic , i.e. the tracheotomies performed in children with laryngeal papillomatosis -LRB- 92.5 % of cases -RRB- . This was reported in a case group of 448 children with RRP treated in St. Vladimir Moscow Children 's Hospital between 1988 and 2003 -LSB- 38 -RSB- . The recent licensing of prophylactic HPV vaccines is a most interesting development . In particular , the quadrivalent vaccine from Merck & Sanofi-Pasteur -LRB- Gardasil ® -RRB- which shows efficacy against HPV 6 , 11 , 16 , 18 subtypes , may be anticipated to impact upon the incidence of RRP . The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance . Large multi-centre collaborative clinical trials are therefore required . Even so , sufficient clinical follow-up data would take several years .

B I I O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

recurrent respiratory papillomatosis
hpv 6 and 11


Association

analysis

of

the

gene

with

childhood

growth

concentrations

and

type

diabetes

Aims/hypothesis

Insulin-like

growth

is

a

major

childhood

growth

factor

and

promotes

pancreatic

islet

cell

survival

and

growth

in

vitro

We

hypothesised

that

genetic

variation

in

might

be

associated

with

childhood

growth

glucose

metabolism

and

type

diabetes

risk

We

therefore

examined

the

association

between

common

genetic

variation

in

and

predisposition

to

type

diabetes

childhood

growth

and

metabolism

Introduction

Type

diabetes

is

a

common

autoimmune

disorder

that

arises

by

an

interaction

between

genes

and

the

environment

To

date

ten

loci

have

been

identified

including

the

HLA

class

II

genes

the

cytotoxic

T-lymphocyte

associated

protein

-LRB-

-RRB-

locus

protein

tyrosine

phosphatase

non-receptor

type

-LRB-

lymphoid

-RRB-

-LRB-

-RRB-

receptor

alpha

-LRB-

-RRB-

interferon

induced

with

helicase

C

domain

-LRB-

-RRB-

and

four

novel

loci

identified

by

a

genome-wide

association

study

-LSB-

-RSB-

The

protein

products

of

these

genes

play

important

roles

in

antigen

presentation

and

the

cellular

immune

response

highlighting

the

importance

of

the

immune

system

in

the

pathogenesis

of

type

diabetes

However

not

all

patients

with

evidence

of

islet

autoimmunity

develop

complete

beta

cell

failure

-LSB-

-RSB-

Given

the

evidence

to

suggest

that

early

weight

gain

and

growth

in

childhood

are

associated

with

type

diabetes

-LSB-

-RSB-

it

is

possible

that

factors

influencing

insulin

action

-LRB-

body

weight

and

fat

mass

-RRB-

and

islet

function

may

predispose

to

or

influence

the

presentation

of

type

diabetes

Lower

concentrations

predict

the

development

of

glucose

intolerance

in

adults

-LSB-

-RSB-

George

et

al.

reported

that

islet

overexpression

of

in

diabetic

mice

enabled

islet

regeneration

and

gradual

correction

of

hyperglycaemia

and

hypoinsulinaemia

-LSB-

-RSB-

A

microsatellite

in

the

region

to

has

been

associated

with

adult

height

cardiovascular

risk

osteoporosis

and

type

diabetes

in

some

-LSB-

-RSB-

but

not

all

-LSB-

-RSB-

studies

Since

common

variation

in

could

alter

circulating

concentrations

and

therefore

body

habitus

insulin

action

and

beta

cell

secretion

we

sought

to

determine

the

associations

if

any

between

and

circulating

concentrations

size

at

birth

both

fasting

and

postprandial

insulin

concentrations

and

type

diabetes

Materials

and

methods

Polymorphism

identification

DNA

samples

from

randomly

selected

type

diabetic

probands

were

amplified

using

specifically

designed

forward

and

reverse

primers

This

provided

probability

of

detecting

single

nucleotide

polymorphisms

-LRB-

SNPs

-RRB-

with

minor

allele

frequencies

-LRB-

MAF

-RRB-

of

probability

for

MAF

and

for

MAF

-LSB-

-RSB-

Resequencing

included

the

kb

region

to

the

gene

all

exons

and

exon

intron

boundaries

and

untranslated

region

Four

kb

segments

in

the

second

intron

kb

intervals

apart

were

also

resequenced

Tag-SNP

selection

The

resequencing

genotype

data

were

used

to

select

SNP

subsets

that

predicted

the

genotypes

of

the

remainder

using

a

coefficient

of

determination

which

measures

the

ability

to

predict

each

known

SNP

genotype

by

linear

regression

on

the

tag-SNP

genotypes

-LSB-

-RSB-

We

considered

only

SNPs

with

a

MAF

in

the

type

diabetes

collection

and

in

the

Avon

Longitudinal

Study

of

Parents

And

Children

-LRB-

ALSPAC

-RRB-

cohort

-LRB-

for

details

see

below

and

Electronic

supplementary

material

-LSB-

ESM

-RSB-

-RRB-

using

a

minimum

of

Genotyping

All

genotyping

data

were

double-scored

to

minimise

error

All

genotypes

were

in

Hardy

Weinberg

equilibrium

-LRB-

p

-RRB-

Populations

studied

All

DNA

samples

were

collected

after

ethics

approval

and

informed

consent

had

been

obtained

Type

diabetes

family

collection

Type

diabetes

families

were

of

white

European

descent

with

two

parents

and

at

least

one

affected

child

The

populations

studied

have

been

described

previously

-LSB-

-RSB-

ALSPAC

Details

of

this

birth

cohort

are

available

on

the

ALSPAC

website

-LRB-

-RRB-

The

children

in

this

study

are

from

a

Children

in

Focus

sub-cohort

-LRB-

full-term

singleton

infants

-RRB-

randomly

selected

from

the

last

months

of

recruitment

for

more

detailed

measurements

of

growth

Birthweight

was

noted

from

hospital

records

and

length

and

head

circumference

were

measured

after

birth

At

age

years

-LRB-

mean

age

years

-RRB-

body

weight

and

height

were

measured

Body

composition

was

assessed

at

age

years

by

whole-body

dual-energy

X-ray

absorptiometry

Internal

SD

scores

were

calculated

for

all

parameters

of

growth

to

adjust

for

age

and

sex

concentrations

were

measured

in

cord

blood

samples

-LRB-

birth

-RRB-

and

in

venous

blood

-LRB-

or

years

of

age

-RRB-

Fasting

insulin

sensitivity

was

assessed

by

the

homeostatic

model

assessment

index

-LRB-

-RRB-

and

insulin

secretion

at

min

post

oral

glucose

by

the

insulinogenic

index

at

age

years

Statistical

analysis

All

statistical

analyses

to

test

the

association

between

and

type

diabetes

were

performed

in

either

Stata

-LRB-

-RRB-

or

R

-LRB-

-RRB-

statistical

systems

Additional

routines

may

be

downloaded

-LRB-

www-gene

cimr.cam.ac.uk

clayton/software

-RRB-

Missing

tag-SNP

genotypes

were

imputed

under

the

hypothesis

and

were

analysed

using

a

multilocus

test

-LSB-

-RSB-

The

microsatellite

CA

repeat

was

analysed

using

TRANSMIT

-LSB-

-RSB-

The

global

effect

of

the

tag-SNP

set

on

each

outcome

variable

was

entered

into

a

multi-locus

regression

model

change

was

taken

as

the

contribution

to

the

total

variation

in

each

outcome

Associations

of

SNPs

with

growth

and

metabolic

phenotypes

were

analysed

using

univariate

ANOVA

-LRB-

general

linear

models

-RRB-

The

association

of

the

CA

repeat

was

analysed

by

comparing

the

wild-type

allele

-LRB-

bp

-RRB-

to

all

other

alleles

-LSB-

-RSB-

For

further

details

on

Materials

and

methods

section

see

ESM

Results

Genetic

variation

in

Resequencing

identified

novel

polymorphisms

consisting

of

SNPs

one

rare

non-synonymous

SNP

in

exon

-LRB-

Ala

to

Thr

-RRB-

and

two

deletion/insertion

polymorphisms

Eleven

SNPs

had

a

MAF

Four

tag-SNPs

-LRB-

Table

-RRB-

were

selected

and

genotyped

in

the

type

diabetes

families

studied

In

ALSPAC

seven

tag-SNPs

were

selected

-LRB-

ESM

Table

-RRB-

Table

summary

of

tag-SNPs

genotyped

in

-LSB-

AT

-RSB-

CTCAAATAGACTTAAC

-LSB-

A/G

-RSB-

TGTTTTCTCAGGTTGG

-LSB-

A/T

-RSB-

CTCAAACTAACCTCAA

-LSB-

A/G

-RSB-

CTGTCTACAllele

coding

-LRB-

minor

-RRB-

AA/GMAF

in

Minor

allele

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-LRB-

-RRB-

Genotype

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Genotype

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

OR

for

minor

allele

-LRB-

CI

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aDeletion/insertion

polymorphismbUntransmitted

-LRB-

pseudocontrol

-RRB-

genotypes

are

estimated

as

previously

described

-LSB-

-RSB-

Multilocus

test

p

and

type

diabetes

The

selected

tag-SNPs

were

genotyped

in

families

-LRB-

parent

child

trios

-RRB-

The

multilocus

test

of

the

tag-SNPs

provided

no

evidence

of

an

association

with

type

diabetes

-LRB-

p

Table

-RRB-

The

microsatellite

CA

repeat

was

genotyped

in

families

and

analysed

using

TRANSMIT

-LSB-

-RSB-

There

was

no

evidence

of

association

-LRB-

p

-RRB-

-LRB-

ESM

Table

-RRB-

Wellcome

Trust

Case

Control

Consortium

data

We

used

data

from

the

Wellcome

Trust

Case

Control

Consortium

-LRB-

WTCCC

-RRB-

genome-wide

association

study

-LSB-

-RSB-

to

test

association

of

the

extended

region

with

type

diabetes

The

region

contained

SNPs

When

the

WTCCC

data

were

analysed

using

a

logistic

regression

model

adjusted

for

variation

in

allele

frequencies

across

Great

Britain

no

evidence

of

association

of

these

SNPs

within

the

region

was

found

-LRB-

ESM

Table

-RRB-

Association

with

concentrations

In

the

ALSPAC

children

multilocus

regression

models

showed

that

the

tag-SNP

set

was

associated

with

protein

levels

at

birth

-LRB-

p

-RRB-

and

weakly

associated

at

age

to

years

-LRB-

p

ESM

Table

-RRB-

Results

of

protein

level

associations

with

individual

SNPs

are

shown

in

Table

However

the

multilocus

tag-SNP

set

did

not

associate

with

any

other

childhood

growth

or

metabolic

phenotype

-LRB-

ESM

Table

-RRB-

No

associations

were

observed

between

the

CA

repeat

and

concentrations

or

any

growth

or

metabolic

phenotype

-LRB-

ESM

Table

-RRB-

Table

protein

levels

-LRB-

ng/ml

-RRB-

at

birth

and

at

age

or

years

by

genotypes

of

seven

tag-SNPs

in

a

representative

birth

cohortParameternGenotype

-LRB-

-RRB-

p

I/D

-LRB-

-RRB-

I/I

-LRB-

-RRB-

D/D

-LRB-

-RRB-

At

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

G/G

-LRB-

-RRB-

G/A

-LRB-

-RRB-

A/A

-LRB-

-RRB-

At

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

A/A

-LRB-

-RRB-

A/T

-LRB-

-RRB-

T/T

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

G/G

-LRB-

-RRB-

A

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

T/T

-LRB-

-RRB-

G

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

T/T

-LRB-

-RRB-

C

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

C/C

-LRB-

-RRB-

A

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

-LRB-

-RRB-

Values

are

mean

-LRB-

SE

-RRB-

for

concentration

and

per

cent

for

genotype

Additive

models

adjusted

for

age

and

sex

Discussion

We

used

a

UK

birth

cohort

ALSPAC

and

a

large

family

collection

of

type

diabetes

to

explore

association

with

a

subset

of

SNPs

generated

by

in-depth

resequencing

of

the

locus

A

subset

of

SNPs

that

effectively

predicted

the

genotype

of

the

remainder

was

subsequently

genotyped

in

these

cohorts

-LSB-

-RSB-

We

report

no

evidence

to

support

the

hypothesis

that

genetic

variation

in

is

associated

with

major

susceptibility

to

type

diabetes

However

genetic

variation

in

this

locus

may

modestly

influence

circulating

concentrations

at

least

at

birth

and

during

childhood

Prior

studies

examining

the

role

of

genetic

variation

in

in

predisposition

to

common

human

disease

have

focused

on

a

microsatellite

in

the

region

to

This

variant

has

been

inconsistently

associated

with

adult

height

and

type

diabetes

-LSB-

-RSB-

Perplexingly

the

same

allele

has

been

associated

with

high

-LSB-

-RSB-

and

low

-LSB-

-RSB-

concentrations

It

is

possible

that

other

common

genetic

variants

in

different

degrees

of

linkage

disequilibrium

with

this

variant

alter

concentrations

and

explain

the

discrepancy

in

the

published

studies

We

found

no

association

between

this

microsatellite

and

circulating

concentrations

growth

or

type

diabetes

To

exclude

the

possibility

that

other

polymorphisms

in

the

gene

predispose

to

disease

we

undertook

a

systematic

analysis

of

gene

variation

using

tagging

SNPs

-LSB-

-RSB-

Our

relatively

large

white

type

diabetes

family

collection

of

European

ancestry

provided

power

to

detect

a

causal

allele

for

type

diabetes

predisposition

with

MAF

and

OR

of

at

the

significance

level

The

study

was

therefore

adequately

powered

to

detect

weak

predisposition

to

type

diabetes

conferred

by

We

did

not

directly

examine

whether

these

variants

alter

the

age

at

diagnosis

of

type

diabetes

since

there

was

no

primary

evidence

of

association

The

possibility

remains

that

variation

in

could

alter

insulin

secretion

or

action

as

well

as

childhood

growth

Therefore

to

examine

these

associations

we

adopted

the

same

strategy

in

an

established

birth

cohort

While

we

demonstrate

that

common

genetic

variation

in

modestly

influences

circulating

concentrations

at

birth

and

during

childhood

we

did

not

find

a

major

contribution

of

to

birthweight

growth

or

response

to

oral

glucose

It

remains

possible

that

maternal

genotypes

that

influence

maternal

metabolism

might

alter

birth

size

but

they

are

unlikely

to

significantly

impact

on

childhood

growth

In

summary

this

large

systematic

study

shows

that

common

variants

modestly

influence

circulating

concentrations

at

birth

and

during

childhood

However

these

variants

are

not

associated

with

birthweight

childhood

growth

and

insulin

secretion

or

action

Moreover

they

do

not

alter

type

diabetes

risk

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

ESM

Table

SNPs

genotyped

in

ALSPAC

children

-LRB-

PDF

KB

-RRB-

ESM

Table

TRANSMIT

analysis

for

association

of

the

microsatellite

in

the

region

of

with

type

diabetes

-LRB-

PDF

KB

-RRB-

ESM

Table

Summary

of

WTCCC

SNPs

in

the

extended

region

-LRB-

PDF

KB

-RRB-

ESM

Table

Multilocus

test

for

associations

with

protein

levels

childhood

growth

and

glucose

metabolism

in

a

representative

birth

cohort

-LRB-

PDF

KB

-RRB-

ESM

Table

Association

testing

for

the

CA

repeat

with

childhood

growth

and

glucose

metabolism

in

a

representative

birth

cohort

-LRB-

PDF

KB

-RRB-

ESM

Table

ESM

Text

-LRB-

PDF

KB

-RRB-

O O O O O O O O B O O O O B I I O O B O O O O O B O O O O O O O O B O O O O O O B I O O O O O O O B O O O O B I I O O O O O O O O O B I O O O O O B I I O O B O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O

children
genetic variation
growth
microsatellite
snp
type 1 diabetes

igf-1
insulin-like growth factor-1
single nucleotide polymorphism
tag-snps