M. M. GODLEWSKI1, M. S£UPECKA1,2, J. WOLIÑSKI2, T. SKRZYPEK3, H. SKRZYPEK4, T. MOTYL1, R. ZABIELSKI1,2

INTO THE UNKNOWN - THE DEATH PATHWAYS IN THE NEONATAL GUT EPITHELIUM

1Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland

2The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jab³onna, Poland

3Department of Animal Biochemistry and Physiology, Faculty of Veterinary Medicine,Agricultural University of Lublin, Lublin, Poland

4Department of Zoology and Ecology, Catholic University of Lublin, Lublin, Poland.

Apoptosis is a fundamental process in the development of the fast growing intestinalmucosa. Apoptotic cells are present along the whole length of the villi and in thecrypts. The mechanisms involved in the induction of apoptosis in the gut mucosa arestill unknown. Cytokines are believed to play a role in auto- and paracrine modelsbecause the cells are dying in so-called "packets" containing neighboring cells. In therapidly developing gut of neonates, the apoptosis rate is transiently reduced in thefirst days of life, enhancing the growth of mucosa. Afterwards, apoptosis plays a rolein the exchange of the enterocyte population, facilitating maturation of the mucosa.The presence of autophagic cells has been confirmed for the first time in thedeveloping gut. Deprivation of growth factors during feeding artificial milk formulaled to an increased apoptosis rate. Supplementation with leptin reduced cellapoptosis and increased the mitosis-to-apoptosis ratio. Autophagy was alsodiminished. The key to healthy gut mucosa growth in early life, especially in fast-growing animals, is colostrum, which supplies nutritional and defensive componentstogether with supplementary growth factors, cytokines and hormones essential forgrowth and maturation of gut mucosa.

K e y w o r d s : apoptosis, autophagy, enterocyte, gut development, leptin, mitosis

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2005, 56, Supp 3, 7�24

www.jpp.krakow.pl

Review articles

APOPTOSIS PATHWAYS

The term apoptosis originates from the Greek word "falling", as in the fallingof leaves or flower petals. It was proposed in 1972 by Kerr, Wyllie and Curie (1)to distinguish this genetically programmed, physiological and suicidal form ofcell death from necrosis. Apoptosis is a fundamental process in cell biology,which contributes to the survival of every multicellular organism. This so-calledphysiological death facilitates removal of unnecessary (at a given point), old,defective and mutated cells during embryogenesis, development, remodeling andinvolution. It also underlies cancer therapy and prevention. The life of amulticellular organism is sustained by a dynamic equilibrium between mitosisand apoptosis, growth and involution. Maintenance of this equilibrium withincertain boundaries allows physiological remodeling of a tissue. A shift in thisequilibrium towards any extreme is considered pathological. Uncontrolledmitosis or impaired apoptosis results in neoplastic and autoaggressive diseases,whereas excessive apoptosis leads to degenerative disorders such as Alzheimer'sand Parkinson's diseases or AIDS.

Apoptosis is the classical form of programmed cell death that coexists withautophagy, death associated with formation of autophagolysosomes, anoikiswhich results from the detachment from the lamina propria, amorphosisassociated with distortion of the cytoskeleton, and the recently described mitoticcatastrophe (2).

Programmed cell death is induced by a variety of factors that can be dividedinto four major groups (3). 1. Physical factors such as gamma and UV irradiation(4, 5), hyperthermia and hydrostatic pressure. 2. Trophic- and growth factordeficiency resulting from their prolonged deprivation or increased cell number(6). Removal of specific growth factors can also initiate apoptosis i.e. neuronalcells deprived of NGF. 3. Hormones and cytokines such as glucocorticoids,thyroid hormones, TNF-α (7), or TGF-β1 (8, 9). 4. Cytotoxic drugs and factors:granzyme A and B (10, 11), reactive oxygen species (12), inhibitors of enzymeslike camptothecin (13), and antimetabolites (2-chloro-2'deoxyadenozine) (14).Various apoptosis-inducing factors work via different induction mechanisms thatmerge into one common pathway of promotion (reversible, regulated by the Bcl-2 family proteins), and degradation (irreversible, executed by caspases) in a laterstep of apoptosis.

There are two major pathways of apoptosis: receptor and mitochondrial (Fig.1). Apoptotic signals that activate death receptors (CD95, TNF-R1, DR-5, TGFβ-R) localized on the cellular membrane facilitate their oligomerization,recruitment of an adapter molecule (like Fas-associated death domain - FADD)and procaspase 8. In this way the death-inducing signaling complex (DISC) iscreated. In DISC, procaspase 8 is autoproteolytically cleaved into two subunits.Subsequently, a heterotetramer of two large and two small subunits is formed tocreate an active form of caspase 8 (15, 16). Caspase 8 has the potential for

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autoactivation and direct cleavage of procaspase 3, thereby propagatingapoptosis.

The mitochondrial pathway of apoptosis originates in activation andheterooligomerization of proapoptotic proteins belonging to the Bcl-2 family, suchas BAX and BID. BAX - BID complexes translocate to mitochondria where theyinduce the release of cytochrome c either directly or through complexes withmembrane proteins (17-19). Cytochrome c together with Apaf-1 and procaspase 9forms a 700 kDa complex called the apoptosome where activation of caspase 9occurs (20-23). Together with cytochrome c, ROS, calcium ions (Ca2+) and AIF arereleased from mitochondria. AIF translocates into the nucleus where it facilitatesNUC-18 DNase activation and nuclear protein degradation (24, 25). During theapoptosis process, Ca2+ is also released from the endoplasmic reticulum. It is anecessary component for activation of calpain and DNases (i.e. DNase-I andNUC-1). Caspase 9 participates in activation of other caspases, i.e. caspase 3 and7 responsible for cellular protein degradation (26). Caspase 8 and 9 are thus calledregulator caspases, whereas caspase 3 and 7, executor caspases. The two pathwaysof apoptosis merge, as caspase 9 is capable of either directly or indirectlyactivating caspase 8 (26-29) and caspase 8 mediates the proteolytic activation ofBID (18). One of the first targets of caspase 3 is PARP. PARP is an enzymeactivated in the presence of DNA strand breaks (30, 31). It catalyzes modificationof histones, topoisomerases I and II, DNA polymerase α and other DNA-binding

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Fig. 1. Death receptor (yellow) and mitochondrial (blue) pathways of apoptosis and theirinteractions. Details explained in the "Pathways of apoptosis" section.

proteins engaged in the DNA repair system (32, 33). The complexity of theapoptotic process and interactions between the different proteins involved arepresented in Figure 2.

Apoptosis and mitosis within the gut endothelium were quantified asdescribed by Biernat et al. (34) and Woliñski et al. (35). For confocal analysisthe cells were labeled with secondary Alexa Fluor 488 conjugated antibodies and7-AAD to counterstain the DNA. The following primary antibodies were used:anti-caspase 3, anti-TGF-β1, anti-p53 (FITC-conjugated) and anti-MAP I LC-3.Next, the gut mucosa was visualized using the FV-500 (Olympus Poland)confocal laser scanning microscope system. The combinations of excitation /emission were: Argon 488 nm laser vs. 505-525 nm filter for Alexa Fluor 488and FITC, and HeNe 543 nm laser vs. 610 nm filter for 7-AAD. For scanningelectron microscopy the samples were fixed in 10% buffered formaldehyde,dehydrated in a series of alcohols, chemically dried in HMDS (1,1,1,3,3,3 -hexamethyldisilazane), sputter-coated (Au/Pd 30 nm) and examined in a LEO1430 VP (EHT 15kV).

POSTNATAL DEVELOPMENT OF THE SMALL INTESTINAL EPITHELIUM

Intensive growth of the small intestine starts several weeks before parturitionand is characterized by a faster growth rate than the fetal body as a whole (36).Small intestine growth further accelerates after birth following the first

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Fig. 2. Interactions between proteins involved in apoptosis

administration of colostrum. Thus, in the neonatal pig the small intestine doublesits weight and increases its length by 30% within the first 3 days of postnatal life(37). Accordingly, the intestinal crypt depth and villi length increase by 40 and 35percent, respectively (34, 38). Growth is, on the one hand, associated withmucosa (in particular, enterocyte) incrustation by colostrum proteins (39). Thesize of a single enterocyte changes from 169 ± 27 µm2 in unsuckling neonates to229 ± 26 and 242 ± 34 µm2 (P<0.001) in 24-hour-old and 7-day-old piglets,respectively. There was a 3 - 4-fold difference in intestinal protein synthesisdescribed between piglets fed colostrums or milk formula and those fed water.The differences in intestinal protein mass seemed to be a result of IgGaccumulation and retention as it was markedly higher in piglets fed colostrumwhile no significant difference was observed between water and milk formula fedones (40). On the other hand, mucosa growth is tied to an increased cellproliferation rate, which can be deduced from the enhanced mucosa DNA content(41). In pig neonates we observed a 40 to 50% increase in the mitotic index in thecrypt region of the jejunum within the first 1-2 days after birth (34). Cellproliferation increases, resulting in shortening the cell renewal cycle from 20 daysin the fetal intestinal epithelium to 2-3 days in the neonatal tissue. Besidesintensive growth, in the early postnatal period the small intestinal epitheliumundergoes an essential maturation process. The enhanced proliferation in thecrypt area is linked to major structural and functional remodeling (42).Morphologically, the so-called "fetal type" enterocytes containing largelysosomal vacuoles are replaced with "adult type" enterocytes that do not containthe vacuoles. In the "fetal type" enterocytes, two categories of lysosomalvacuoles, transport and digestive, have been described (43). The former, presenton the entire length of the small intestine but only within the first two days afterbirth, are involved in transferring macromolecules in their intact form from thegut lumen into the circulation through the enterocyte. The transport vacuolesallow the biologically active colostral molecules, including immunoglobulins, topass the gut epithelium without affecting their activity. Lysosomal vacuoles of thelatter category are present in the lower half of the small intestine and seem to beresponsible for intracellular digestion of nutrients and for controlling the pH ofthe gut lumen (44). The digestive vacuoles are observed for about 3-4 weeks inthe pig ileum (42). Functionally, the activity of brush border lactase,aminopeptidases A and N, and dipeptidase IV is markedly reduced within a fewdays after birth (45). The high absorption rate of large molecules associated withthe open "gut barrier" is abolished about two days after birth (43, 46, 47).

Stem cells, which constitute a replication center for the gut epithelium,occupy the lower third of the intestinal crypts (Fig. 3). The mitosis rate is highin the small intestine crypts. The highest percentage of dividing cells wasobserved in the mid-jejunum on the second postnatal day (Table 1). The fate ofthe newly constituted epithelial cells depends on the direction they "choose".Some cells migrate down to the crypt bottom and transform into Paneth cells.

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Fig. 3. Scheme of intestinalvillus and crypts, with stem-cell region marked in orangeand Panneth-cell region ingreen. The fate of the gutepithelial cell depends onthe direction of their move-ment. Cells that migrate tothe villi differentiate intoenterocytes, goblet cells(~5%) or endocrine cells(~1%) and are characterizedby quick turnover, with alife-span seldom exceeding48 hours. Cells directeddownwards into the depth ofthe crypts differentiate intoPaneth cells. These cellswith life-span reaching 21days produce lysozyme anddefensins and are involvedin protection of the gut.

Table1. Mitotic and apoptotic indexes, and mitosis-to-apoptosis ratio in whole gut cross sections ofnewborn (unsuckling - 0 days), suckling (24 hours, 7 days) and weaned (12 weeks) piglets.$According to Woliñski et al. (35). $$According to Biernat et al. (34).

Piglets Newborn Suckling Suckling Weaned 0 days 24 hours 7 days 12 weeks

Mitotic index$ 4.50% 6.10% 4.45% 4.80%

Apototic index# $$ 21.8% 15.9%** 21.8% 25.1%

Mitosis/Apoptosis 0.20 0.38 0.20 0.19

# whole gut crossections; ** P<0.01

With a life span exceeding 20 days, these cells produce lysozyme and defensinsand are responsible for the protection of gut mucosa (48). The majority ofepithelial cells migrate upward to the top of the villi and differentiate into gobletand endocrine cells and enterocytes. Enterocytes responsible for the secretoryand absorptive functions of the gut epithelium are the most abundant. Thepopulation of mucus-producing goblet cells and endocrine cells make upapproximately 5% and 1% of the epithelial cells covering the villi, respectively(Fig. 3). Their life span seldom exceeds 48 hours and the quick turnover is basedon the dynamic equilibrium between cell mitosis and apoptosis. In neonatalpiglets, enhanced mitosis was observed parallel with a significant decrease inapoptosis during the first two days after birth, resulting in transient elevation ofthe mitosis/apoptosis ratio (34). This phenomenon substantially contributes tothe enlargement of the gut mucosa. The mitosis rate then stabilizes until theweaning period when another increase is observed, but this time it is associatedwith enhanced apoptosis (Tab. 1) (34). The mitosis-to-apoptosis ratio is a goodmarker of gut growth intensity. In particular, it clearly demonstrates that the firstrapid phase of postnatal growth is not only due to passive incrustation of themucosa with colostrum proteins but also due to an increased number of epithelialcells. This parameter allows differentiating between intensive growth of the gutmucosa occurring during the first 24 h of postnatal life from remodeling of thegut that occurs after the weaning of piglets, when the mitosis-to-apoptosis ratioremains unchanged while mitosis increases.

APOPTOSIS IN THE GUT EPITHELIUM

Until recently, apoptosis in the gut was the subject of only a few reports. Theapoptotic cells were localized in the top of the villi of adult human subjects androdents, and in the upper one third of the villi in monkeys (49, 50). Theseobservations came from the gut of adult animals and supported the view ofapoptosis as a common way of eliminating old, used epithelial cells from the villi.Species-specific differences are observed in the fate of eliminated epithelial cells.In adult mice and rats, the apoptotic cells are detached and desquamated into theintestinal lumen (49). In the guinea pig and monkey, apoptotic cells are not onlydesquamated but some of them are phagocytosed by macrophages present in thegut mucosa. In the end of last millennium, the first observations confirmed thepresence of apoptotic cells in the intestinal crypt region as well (51). Our studiesin newborn animals confirmed the localization of apoptotic cells in intestinalcrypts (34, 52). In contrast to the observations made in adult animals, in neonatalpiglets apoptotic cells were present along the entire length of the villi, includingthe lower half (Figs. 4 and 6). Similar pictures have also been found recently inchicken broilers (52). In the neonatal pig gut mucosa, dying cells are eliminatedinto the lumen as single cells or groups of cells (Fig. 4b), or shed with the entire

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top of the villus (Fig. 4c and 6a). They can also be brought under the epitheliumlayer where they undergo phagocytosis (Fig. 4d). Interestingly, unlike the reportsdescribing apoptosis in the gut of adults, the cells in neonatal piglets seem to diein packets (Fig. 5a and 6c-f), suggesting involvement of auto- or paracrinefactor(s) in the induction phase of apoptosis (34, 52). Furthermore, the closeproximity of the observed apoptotic cells in neighboring villi imply transmissionof the apoptogenic signal via the luminal space (Fig. 5b). This peculiar pattern ofapoptosis was observed in mucosa slides using conofocal microscopy and, evenmore convincingly, with scanning electron microscopy (Fig. 6a) as groups ofapoptotic cells observed along the entire length of the villi. In three-dimensionalSEM images, the surface of the villi is flat with honeycomb-shaped outlines of

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Fig. 4. Localization of apoptotic cells in the gut mucosa of neonatal piglets. The late apoptotic cells,characterized by high expression of active caspase-3 (green fluorescence), were localized in thecrypts and at the basis of the villi (a), in the middle of the villi (b) and on the top of the villi (c andd) Cells from the top of the villi were either shed to the gut lumen (c) or internalized under theenterocyte layer where they underwent phagocytosis (d). Gut cross sections were stained with AlexaFluor 488-conjugated secondary antibodies against caspase-3 (green fluorescence) and 7-AADagainst the DNA (red fluorescence).

the top of the enterocytes. The groups of apoptotic cells are either sunken orprotrude by 1-2 µm over the epithelial cell surface (Fig. 6 c-f).

The mechanisms of apoptosis induction in the gut epithelium remain elusive.A decrease in anti-apoptotic members of the Bcl-2 family as a result of totalparenteral nutrition, so-called death from unuse, was suggested by Wildhaber etal. (53), increased free radical formation associated with passive smoking in ratswas observed by Wang et al. (54), and infections of the gastrointestinal tract wereassociated with increased apoptosis (55). All of the above-mentionedmechanisms may apply to enterocyte apoptosis in the neonatal gut as well, but themost significant apoptosis-inducing mechanism during the physiologicaldevelopment of the gut epithelium seems to be local deprivation of growth factorsand tissue hormones like EGF, IGF, IGF-BP, and leptin (35, 56). This inductionpathway presumably is especially important during the first days of life as themucosa of newborn animals grows extremely rapidly and, simultaneously, the guthas limited ability to produce its own growth factors. Thus the neonatal gutheavily depends on external regulators coming with colostrum and milk (57). Ourrecent data showing that the apoptosis rate in gut mucosa is higher in formula-fednewborns as compared with sow-reared newborns strongly support thishypothesis (Table 2).

Enhanced proliferation of crypt stem cells results in increased susceptibilityand frequency of replication errors and mutations, which trigger the p53-

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Fig. 5. Packet pattern of late apoptotic cells in the gut mucosa characterized by a high level of activecaspase-3 (green fluorescence). In contrast to the adult gut, the apoptotic cells in the gut of newbornpiglets are grouped in packets, suggesting involvement of auto- / paracrine factor(s) in regulation ofthe process (a). Furthermore, the signal seemed to be transmitted to the neighboring villi throughthe lumen of the gut (b). Gut cross-sections were stained with Alexa Fluor 488-conjugatedsecondary antibodies against caspase-3 (green fluorescence) and 7-AAD against the DNA (redfluorescence).

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Fig. 6. Three-dimensionalscanning electron micro-photographs of gut mucosaof neonatal piglets. Apopto-tic cells are manifestedeither as sunken or protru-ding by 1-2 mm over theepithelial cell surface: a) anoverview of the intestinalmucosa showing theapoptotic cells along theentire length of the villi(magnification 300x); b)shedding of the entire top ofthe villi (2500x); c - esequence of microphoto-graphs showing packets ofapoptotic cells localized inthe middle section of thevillus (magnificationranged from 3000x to10000x); f) packet of theapoptotic cells localized atthe base of the villus(15000x).

Table 2. Mitotic and apoptotic indexes, and mitosis-to-apoptosis ratio in isolated gut mucosa in 7-day-old piglets reared with sows, fed milk formula alone or milk formula supplemented with leptin(10 µg/kg bw every 8 h).

Piglets Kept with sows Milk formula Milk formula + leptin*7 days 7 days 7 days

Mitotic index 4.45% 3.45% 5.12%

Apototic index# 11.0% 14.5% 12.9%

Mitosis/Apoptosis 0.41 0.23 0.39

* +10 µg leptin/kg bw every 8 h; # isolated mucosa

dependent cell cycle arrest and, in case the error cannot be repaired, the p53- andBcl-2 family-dependent death pathways (58). The epithelial cells expressing p53were restricted solely to the crypt region and base of the villi.

TGF-β1-induced apoptosis is an another pathway involved in cell deathpromotion that cannot be excluded. TGF-β1 has been recently proved to be apotent apoptosis-promoting factor in the epithelial cells of the mammary gland (9,59, 60). This cytokine was observed to be secreted from macrophages upon theuptake of apoptotic bodies (49) and since it acts as paracrine factor, it mayinfluence the neighboring gut epithelial cells forcing them into the receptor(extrinsic) pathway of apoptosis (Fig. 7). The involvement of TGF-β1 in inducingapoptosis in the gut mucosa could explain why the epithelial cells die in packetsrather than alone, as shown in Figs. 5 and 6.

AUTOPHAGY IN THE GUT EPITHELIUM

Autophagy is a newly described form of programmed cell death set apart fromapoptosis because of differences in molecular and morphological patterns. In cellsthat undergo autophagic death the prominent observed feature is the formation ofautophagolysosomes where cell organelles are degraded (61) resulting in totaldestruction of the cytosol compartment of the cell with no changes observed

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Fig. 7. Expression of TGF-β1 in the intestinal mucosaof neonatal piglets. TheTGF-β1-positive cellswere observed in the entiremucosa layer, but the mostwere localized at the lowerthird of the villi. Gut crosssections were stained withAlexa Fluor 488-conjuga-ted secondary antibodiesagainst TGF-β1 (greenfluorescence) and 7-AADagainst the DNA (redfluorescence).

within the nucleus (62-65). Cathepsins are enzymes catalyzing this pathway of celldeath with cathepsin-B and -D suggested to be the most potent. There is a crosslinkbetween the apoptosis and autophagic pathways (66) since cathepsin-B is capableof activating BID via N-terminal cleavage (67) and cathepsin-D, of activatingBAX (68). Both cathepsins are capable of activating executor caspases 3 and 7(69) (Fig. 8). The recent studies of Lamparska-Przybysz et al. on MCF-7, amammary epithelial cell line, (70) suggest that autophagy is a complementaryform of cell death to apoptosis, and is switched on in cells that are more resistantto typical apoptosis-inducing signals in the death-promoting environment. Inmammalian cells, autophagy is regulated by homologues of the ATG and AUTfamily of yeast genes. Beclin 1, a homolog of the yeast autophagy protein Atg6 isrequired for vacuolar transport and can induce autophagy in human cells (71). Themammalian homologue of the yeast Atg8 gene codes microtubule-associatedprotein I (MAP I) light chain 3 (LC-3), which binds with autophagic membranesand is currently the only reliable marker of autophagosomes (72-75). Our studiesconfirmed the presence of MAP I LC-3-positive, autophagic cells within the gut in

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Fig. 8. Autophagic pathway andits crosslink with the apoptosispathways.

piglets fed milk formula and milk formula supplemented with leptin (Fig. 9). Inthe piglets fed milk formula alone, the MAP I LC-3-positive cells were grouped inpackets, similar to cells dying via the apoptotic pathway (compare Fig. 9a withFig. 5). When milk formula was supplemented with leptin, the amount ofautophagic cells decreased and the packet pattern was no longer observed (Fig.9b). In comparison with apoptosis, autophagy seems to play a less significant rolein gut development as the number of MAP I LC-3-positive cells is small ascompared with the number of apoptotic cells. It is possible that autophagy isswitched on in cells with impaired apoptotic machinery, but this theory needsfurther evaluation (70). Several attempts were undertaken to enhance the mitosis-to-apoptosis ratio in the developing gut. Blum & Baumrucker (56) proved thatsupplementation of single growth factors and tissue hormones alone to milkformula was not enough to increase the mitotic ratio in calves. Positive effectswere observed only when growth factors were supplemented to colostrum or milk.Mimicking the effect of feeding milk and colostrum, in particular, to neonates isvery difficult, as they contain a species-specific composition of regulatorypeptides, hormones, cytokines and growth factors as well as albumins capable ofprotecting these bioactive substances from enzymatic digestion (76). So far onlyleptin (Table 2) and IGF (77) were proved to be potent in increasing the mitoticratio to some extent and in reducing the apoptosis of enterocytes in neonates when

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Fig. 9. Autophagy in the gut of neonatal piglets. The autophagic MAP I LC-3-positive(microtubule-associated protein I light chain 3) cells were observed in packets in the gut mucosa ofpiglets fed milk formula (a). The number of autophagic cells was reduced and packet forming wasno longer visible in piglets fed milk formula supplemented with leptin (10 µg/kg bw every 8 h) (b).Gut cross sections were stained with Alexa Fluor 488-conjugated secondary antibodies againstMAP I LC-3 (green fluorescence) and 7-AAD against the DNA (red fluorescence).

supplemented to milk formula (35, 77). Furthermore, preventing enterocyteapoptosis does not mean enhancement of adaptation and development of theintestinal mucosa, as shown by Juno et al. (78). Investigation of gut epitheliumapoptosis may be of help in constructing new artificial milk formulas (79).

In conclusion, there are two major stages of gut development during postnatallife, easily distinguishable by the mitosis-to-apoptosis ratio. The first stage ofintensive development is characterized by a two-fold increase in this ratio. Thisstage occurs immediately after the first meal and is associated with a significantreduction in apoptosis with a parallel increase in the mitotic index. The secondstage, remodeling, is associated with a corresponding increase in the mitosis andapoptosis indexes with no change in the mitosis-to-apoptosis ratio. The packetpattern of cell death via apoptosis pathways suggests involvement of some auto-or paracrine factor(s) controlling the process. The ubiquitous expression of TGF-β1 in the gut of neonatal piglets suggests its role as one of the apoptosis-inducingauto/paracrine factors in the intestinal mucosa. The intensity of apoptosis in gutmucosa of piglets fed milk formula suggests a powerful influence of bioactivefactors from colostrums and milk on gut development, especially during the firstfew days of neonatal life. Finally, although the presence of autophagy wasconfirmed during intestinal mucosa development, it seems to play only acomplementary role to apoptosis.

Acknowledgments: This work was supported by grant nr PBZ-KBN-093/P06/2003 from theNational Committee for Scientific Research, Poland.

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R e c e i v e d : January 28, 2005A c c e p t e d : April 4, 2005

Author's address: Micha³ M. Godlewski, Ph.D., Department of Physiological Sciences, Faculty ofVeterinary Medicine, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland.Tel/Fax: +48-22-8472452.E-mail: mickgodl@hotmail.com

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