Human Enterocyte-like Cell Line Research Articles

Adhesion Conditions of Bifidobacterium pseudocatenulatum KAKii to Human Enterocyte-like Caco-2 Cell Lines

Attachment ability of bifidobacteria strains to the human intestinal surface is an important criterion as a probiotic candidate. However, attachment activity is influenced by external and internal conditions. This study was conducted to screen cell surface hydrophobicity and adhesion scores of bifidobacteria strains. Attachment conditions (pH and exposure time) of selected strains to human enterocyte-like Caco-2 cell lines were subsequently investigated. Three different solvents (n-hexadecane, Toluene, and Xylene) were used in cell surface hydrophobicity analysis. Based on the results obtained, xylene presented consistent cell hydrophobicity activity in all strains used. Bifidobacterium pseudocatenulatum KAKii (wild type strain) gave promising cell hydrophobicity activity with no significant difference (p > 0.05) when compared to Lactobacillus plantarum NBRC 3070 with xylene as a solvent, and also presented a significantly higher attachment score (p < 0.05) compared to all strains used. The influence of pH and time exposure on adhesion of B. pseudocatenulatum KAKii to Caco-2 cells revealed that this strain was favored to attach to the intestinal cell line at pH 6 and after 120 min of exposure. Further optimization of attachment conditions will be carried out.

Method of preservation and type of protective agent strongly influence probiotic properties of Lactococcus lactis: A complete process of probiotic preparation manufacture and use

In order to assess the essential probiotic properties of a strain dedicated for administration in humans and animals, characteristics of finally formulated products, rather than the cells solely, seems to be of crucial importance. In this study, composition of protective blends for manufacture of L. lactis probiotic powders was optimized using a statistical experimental design. The powders, generated by either spray- or freeze-drying techniques, were subsequently subjected to storage testing, and in vitro digestion in simulated stomach and small intestine. Finally, maintenance of adherence capability to human enterocyte-like cell lines, was evaluated. Our data demonstrated that 10% trehalose ensures the highest viability of L. lactis bacteria upon both drying techniques (viability of 60–68%). Moreover, skimmed milk-protected spray-dried cells exhibit the highest resistance to harsh environmental conditions of stomach (53.9 ± 7.6% survival rate) and higher adhesion ability to HT-29 cell line after digestion (528 ± 29 cells per 100 epithelial cells).

Characterization of Candidate probionts isolated from human breast milk.

This study was designed to isolate and identify the potential probionts present in 32 healthy mothers' breast milk. Microbial culture media and 16SrRNA sequencing were used to isolate and identify the bacteria and all isolates were analyzed for their antagonistic potential, resistance to acidic pH, bile salts and survival under simulated gastric and intestinal conditions. The colonization potential was further assessed based on adherence to human enterocyte-like Caco-2 cell lines. The breast milk samples harbored significant numbers of Gram positive and catalase negative (85%) bacteria. Based on 16SrRNA sequencing, these isolates were identified as Lactobacillus casei, L.gasseri, L.fermentum, L.plantarum, Pediococcus acidilactici, and Enterococcus facieum. Among the isolates, P. acidilactici was the most frequent species (71%) present in these samples. Few Gram and catalase positive isolates, Staphylococcus aureus and S.hominiis were also observed. The isolates were viable and unviable in pH 3 and 1.5, respectively, while all isolates survived in 1.0% bile salt. As putative probionts, P.acidilactici 1C showed a significantly higher percentage of adhesion to Caco-2 cells (p< 0.05)than the other two isolates L.plantarum 7A and E.facieum 2C. Bacterial strains isolated from human breast milk were shown to have probiotic properties including anti-infective protection and may be considered as future therapeutics for infants.

Enterocyte-Associated Microbiome of the Hadza Hunter-Gatherers.

By means of a recently developed non-invasive ex vivo minimal model based on the interaction of the human enterocyte-like HT29 cell line and fecal slurries, we explored the enterocyte-associated microbiome of 21 Hadza hunter-gatherers and nine urban living Italians. Though reductionist, this model allows inferring the microbiota structural and functional arrangement as it interacts with enterocytes. Microbial suspensions obtained from Hadza or Italian stools were first evaluated for structural integrity by high resolution-scanning electron microscopy and co-incubated with HT29 cell monolayers. The enterocyte adherent microbiota fraction was then characterized by 16S rRNA gene sequencing and predictive functional profiling using PICRUSt. Compared to Italians, the Hadza enterocyte-associated microbiome was characterized by a greater amount of adhesive microorganisms with pathogenic potential, such as Proteobacteria, Erysipelotrichaceae, Enterococcus, Clostridium and Sarcina. These compositional characteristics were reflected in a functional enrichment in membrane transport, signal transduction, signaling molecules and interaction. Our results depict a new interesting mutualistic configuration of the enterocyte-associated microbiome in Hadza, stressing the importance of microbe-host interaction at the mucosal surface along the course of human evolution.

Inhibition of enteropathogens adhesion to human enterocyte-like HT-29 cells by a dairy strain of Propionibacterium acidipropionici

Adhesion to the host intestinal mucosa is considered relevant for orally delivered probiotics as it prolongs their persistence in the gut and their health promoting effects. Classical propionibacteria are microorganisms of interest due to their role as dairy starters as well as for their functions as probiotics. Propionibacterium acidipropionici Q4, is a dairy strain isolated from a Swiss-type cheese made in Argentina that displays probiotic potential. In the present work we assessed the ability of this strain to adhere to the human enterocyte-like HT-29 cell line and to counteract the adhesion of two common human enteropathogens, such as Escherichia coli C3 and Salmonella Enteritidis 90/390. The results were compared with those obtained with the well-known probiotic Lactobacillus rhamnosus GG. P. acidipropionici Q4 showed a high adhesion capacity, even higher than the reference strain L. rhamnosus GG (42.3±4.4% and 36.2±2.3%, respectively), whereas adhesion of enteropathogens was significantly lower (25.2±2.2% for E. coli and 21.0±3.4% for S. Enteritidis). Propionibacteria as well as lactobacilli were able to inhibit by exclusion and competition the adherence of E. coli C3 and S. Enteritidis 90/390 whereas only L. rhamnosus GG displaced S. Enteritidis from HT-29 intestinal cells. Inhibition of pathogens by propionibacteria was not exerted by antimicrobials or coaggregation but was mainly due to exclusion by cell surface components, such as proteins and carbohydrates. The relevance of cell surface proteins (CSP) for preventing pathogens infection was confirmed by their concentration dependent effect observed for both pathogens: 100 µg/ml of CSP inhibited E. coli attachment almost as untreated propionibacteria, whereas it partially inhibited the attachment of S. Enteritidis. Results suggest that P. acidipropionici Q4 could be considered for the development of propionibacteria containing functional foods helpful in counteracting enteropathogen infection.

Intracellular Retention and Subsequent Release of Bovine Milk Lactoferrin Taken Up by Human Enterocyte-Like Cell Lines, Caco-2, C2BBe1 and HT-29

Lactoferrin (LF) is an iron-binding glycoprotein contained in milk and other exocrine fluids, and is believed to have multiple biological functions. We investigated the intracellular dynamics of LF taken up by three lines of human enterocytes and the subsequent release of internalized LF by using two-site ELISA and confocal microscopy. LF taken up by Caco-2 cells was kept partially intact within the cells and subsequently released to the medium as degraded fragments of 30-50 kDa. The retention and subsequent release of LF by Caco-2 cells were much more abundant than those of ovalbumin, ovomucoid and lysozyme. Such results characteristic of LF were also similarly observed in C2BBe1 and HT29 cells more markedly. LF was detected as punctate signals and partially colocalized with the lactoferrin receptor, intelectin-1, in the respective cytoplasm and nuclei of Caco-2 and C2BBe1 cells. In contrast, LF within the HT-29 cells was detected as much smaller punctate signals scattered in the cytoplasm.

The effects of proinflammatory cytokines on expression of serotonin transporters in human enterocyte-like cell line

Objective To determine the effect of proinflammatory cytokines including interleukin-lbeta (IL-1β) and tumor necrosis factor-Alpha (TNF-α) on human serotonin transporter (SERT) in human enterocyte-like cell line Caco-2.Methods Caco2 cells were cultured for 5 days,then were divided into control group.IL-1β treated group (50 ng/ml) and TNF-α treated group (50ng/ml) for 2.24,48 and 72 hours.The expression of SERT mRNA was detected by RT-PCR at 2,24 and 48 hours and its protein expression was measured by Western blotting at 24,48 and 72 hours.Results The expression of SERT mRNA at 2,24 and 48 hours was lower both in IL-1β treated group (1.393±1.184,1.064±0.625 and 1.013±0.415,respectively) and TNF-α treated group (1.000±0.000,0.829±0.162and 0.945±0.147,respectively) in comparison with control group (2.282±1.367,1.586±0.421 and 1.86 ±O.496,respectively).There was significant difference among three groups (P<0.01).The expression of SERT protein at 24,48 and 72 hours was lower in IL-1β treated group and TNF-α treated group than in control group.Conclusions The IL-1β and TNF-α can inhibit the expressions of SERT mRNA and protein in Caco-2 cells,which indicates that IL-1β and TNF-α may change visceral sensitivity by influencing peripheral 5-hydroxytryptamine levels. Key words: Colonic neoplasms Interleukins; Tumor necrosis factor-Alpha; Serotonin transporter; Cell line,tumor

Comparison of cytokine modulation by natural peroxisome proliferator–activated receptor γ ligands with synthetic ligands in intestinal-like Caco-2 cells and human dendritic cells—potential for dietary modulation of peroxisome proliferator–activated receptor γ in intestinal inflammation

BackgroundPeroxisome proliferator–activated receptor γ (PPARγ) plays a role in the regulation of intestinal inflammation and is activated by both natural (polyunsaturated fatty acid; PUFAs) and synthetic (troglitazone) ligands. The fatty acid content of defined formula diets may play a role in mediating the antiinflammatory effect, but the mechanism is unclear. ObjectiveWe evaluated to what extent the effect of PUFAs on intestinal inflammation is mediated via PPARγ. DesignThe human enterocyte-like cell line Caco-2 and human dendritic cells were stimulated by interleukin (IL) 1β and lipoprotein polysaccharide, respectively, in the presence of PPARγ agonists (troglitazone or PUFAs) or antagonist (GW9662). Five PUFAs were tested: α-linolenic acid (ALA), conjugated linoleic acid (CLA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and γ-linolenic acid (GLA). Cytokine production was measured by enzyme-linked immunosorbent assay and PPARγ, I-κB, and inducible nitric oxide synthase (iNOS) expression by Western blot. ResultsIn Caco-2 cells, IL-6 secretion was significantly decreased by troglitazone, DHA, EPA, and GLA. IL-8 production was significantly decreased by troglitazone, ALA, DHA, EPA, and GLA. PPARγ expression was significantly increased by troglitazone, DHA, and EPA. iNOS expression was significantly decreased by troglitazone, DHA, and EPA. Troglitazone and PUFAs at 0.1 μmol/L tended to increase the expression of I-κB. Addition of GW9662 reversed the effect of troglitazone and PUFAs at 0.1 μmol/L on IL-8 production and decreased the expression of PPARγ. EPA and DHA also modulated the dendritic cell response to lipoprotein polysaccharide. ConclusionsThe tested PUFAs exerted an antiinflammatory effect in vitro in both models. This effect of PUFAs in Caco-2 cells is similar to that of troglitazone on intestinal inflammation mediated by PPARγ, and the potency of the antiinflammatory effect is linked to the number of double bonds.

Effect of long-term fluoxetine treatment on the human serotonin transporter in Caco-2 cells

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) broadly used in the treatment of human mood disorders and gastrointestinal diseases involving the serotoninergic system. The effectiveness of this therapy depends on repeated long-term treatment. Most of the long-term studies in vivo of SSRI effects on serotoninergic activity have focused on their effects on autoreceptors or postsynaptic receptors. The chronic effect of SSRIs on the activity of the serotonin transporter (SERT) has been less studied and the results have been contradictory. The aim of this study was to determine the specific effect of long-term fluoxetine treatment on human serotonin transporter (hSERT) in vitro, by using the human enterocyte-like cell line Caco-2. Results show that fluoxetine diminished the 5-HT uptake in a concentration-dependent way and that this effect was reversible. Fluoxetine affected mainly the hSERT transport rate by reducing the availability of the transporter in the membrane with no significant alteration of either the total hSERT protein content or the hSERT mRNA level. These results suggest that the effect of fluoxetine on the expression of hSERT is post-translational and has shown itself to be independent of PKC and PKA activity. This study may be useful to clarify the effect of the long-term fluoxetine therapy in both gastrointestinal and central nervous system disorders.

Role ofBifidobacterium longum in the induction of apoptotic deletion in the human enterocyte-like Caco-2 cell line

Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced byB. longum. This has been valuedin vitro, performing the incubation of threeB. longum strains with enterocyte-like Caco-2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells withB. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherentB. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues.

The 35S CaMV plant virus promoter is active in human enterocyte-like cells

The 35S cauliflower mosaic virus (CaMV) promoter is commonly used to drive transgene expression in the genetically engineered (GE) crop plants that have been commercialized so far. Whether, and how far, the 35S promoter might be active in mammalian cells has been scientifically unsettled and controversial. Very recently it was established that the 35S promoter is transcriptionally active following transient reporter gene transfections in continuous cell lines of human [J Biotechnol 103:197–202, 2003] and hamster ovary [Environ Biosafety Res 3:41–47, 2004] fibroblasts. The initial exposure of a human organism to DNA from GE food takes place in the gastrointestinal tract (GIT). Hence, we have now investigated the promoter capacity of 35S in human enterocyte-like cells. We constructed expression vectors with 35S promoter inserted in front of two reporter genes encoding firefly luciferase and green fluorescent protein (GFP), respectively, and performed transient transfection experiments in the human enterocyte-like cell line Caco-2. It was demonstrated that the 35S CaMV promoter was able to drive the expression of both reporter genes to significant levels, although the protein expression levels might seem modest compared to those obtained with the strong promoters derived from human cytomegalo virus (hCMV) and simian virus 40 (SV40). Furthermore, computer-based searches of the 35S CaMV DNA sequence for putative mammalian transcription factor binding motifs gave a high number of hits. Some of the identified motifs indicate that transcriptional activation by the 35S CaMV promoter may be stronger in other human and animal cell types than in those investigated so far.

Influence of polarisation and differentiation on interaction of 43-kDa outer-membrane protein of Aeromonas caviae with human enterocyte-like Caco-2 cell line

It has been recognised that adherence and invasion to host cells are important steps in the pathogenesis of entero-pathogenic bacteria, including Aeromonas caviae. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interaction of A. caviae isolates to Caco-2 cells in different polarisation and differentiation conditions. The adherence of A. caviae may be related to accessibility of host cell basolateral receptors. Aggregative A. caviae isolates, grown at 22 degrees C, were more adherent in both non-polarised and undifferentiated Caco-2 cells and EGTA-treated polarised and differentiated Caco-2 cells. Furthermore, monolayers pre-incubated with 43-kDa outer-membrane protein (OMP) or A. caviae strains pre-incubated with rabbit IgG anti-43-kDa OMP decreased adherence of some A. caviae strains to EGTA-treated polarised and differentiated Caco-2 cells, suggesting an interaction of 43-kDa OMP with basolateral cell receptors. Bacterial cells were observed adhering to microvilli and to plasma membrane on both the apical and basal surfaces of the monolayer. Pedestal-like formation with cytoskeletal rearrangement was also observed. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. Furthermore, A. caviae were observed free in the cytosol of Caco-2 cells, suggesting escape form cytoplasmatic vacuoles.

Actin mediates Encephalitozoon intestinalis entry into the human enterocyte-like cell line, Caco-2

Microsporidia are spore-forming obligate intracellular eucaryotes that parasitize eukaryotic cells.Encephalitozoon intestinalis (formerly Septata intestinalis) is a microsporidian species of emerging medical importance, responsible for chronic diarrhoea in immunocompetent patients and enteritis and systemic infections in HIV-1 infected patients. Infection of host enterocytes has been demonstrated in HIV-1-infected patients. However, the mechanisms of entry ofE. intestinalis into host enterocytes have not been studied and remain hypothetically based on diacytosis, a model involving the injection of microsporidian sporoplasm through the polar tubule into the host cell. An electron microscopy based study recently challenged this hypothesis. We studied the entry of E. intestinalis into intestinal epithelial cells by infecting the human enterocyte-like cell line Caco-2. Entry was mediated by directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on E. intestinalis uptake, colocalization of E. intestinalis and F-actin and engulfment of E. intestinalis into Caco-2 cell protrusions. Confocal- and electron microscopy observations also suggested that after initial contacts through the posterior pole of the microsporidian spore, the basolateral surface of Caco-2 cells may be the portal of entry for E. intestinalis sporoplasm. Our observations allowed us to propose a new, actin-based model to describe the entry of microsporidia into enterocytes.

Identification of a Bile Acid-responsive Element in the Human Ileal Bile Acid-binding Protein Gene: INVOLVEMENT OF THE FARNESOID X RECEPTOR/9-cis-RETINOIC ACID RECEPTOR HETERODIMER

Intestinal bile acid-binding protein (I-BABP) is a cytosolic protein that binds bile acids (BAs) with a high affinity. In the small intestine, its expression is restricted to the ileum where it is involved in the enterohepatic circulation of BAs. Using the human enterocyte-like Caco-2 cell line, we have recently shown that BAs increased I-BABP gene expression. To determine whether this regulation occurs in vivo, the effect of BA depletion or supplementation was studied in mice. A dramatic drop in I-BABP mRNA levels was observed in mice treated with the BA-binding resin cholestyramine, whereas an increase was found in animals fed with taurocholic acid. BAs are physiological ligands for the nuclear farnesoid X receptor (FXR). Both FXR and I-BABP are co-expressed along the small intestine and in Caco-2 cells. To determine the role of FXR in the regulation of I-BABP expression, the promoter of the human I-BABP gene was cloned. In Caco-2 cells, cotransfection of FXR and RXRalpha is required to obtain the full transactivation of the I-BABP promoter by BAs. Deletion and mutation analyses demonstrate that the FXR/RXRalpha heterodimer activates transcription through an inverted repeat bile acid responsive element located in position -160/-148 of the human I-BABP promoter. In conclusion, we show that FXR is a physiological BA sensor that is likely to play an essential role in BA homeostasis through the regulation of genes involved in their enterohepatic circulation.

Poliovirus infection induces apoptosis in CaCo-2 cells

The effects of poliovirus infection in CaCo-2 cells, a human enterocyte-like cell line are described. Infected cells were examined by a combination of techniques, including optical and electron microscopy, cytofluorimetric analysis of DNA content, and DNA agarose gel electrophoresis. Results obtained by the different experimental approaches demonstrate that poliovirus infection in enterocyte-like cells can result in an apoptotic process.

Influence of temperature and growth phase on expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes.

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117-124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42 degrees C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42 degrees C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25 degrees C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37 degrees C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.

Glucose and amino acid metabolism in Caco-2 cells maintained in a serum- and hormone-free medium.

Glucose and amino acid metabolism in a human enterocyte-like cell line (Caco-2) cultured in a serum- and hormone-free medium was examined and effects of epidermal growth factor (EGF) on these cells determined. These cells took up glutamine and converted it to alanine and proline. EGF stimulated the metabolism significantly.

Lectin reactivity and virulence among strains of Listeria monocytogenes determined in vitro using the enterocyte-like cell line Caco-2.

Forty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested for lectin agglutination using a four-lectin set. Lectin reactivity (i.e. agglutination by one or more of the four lectins) was observed in all 12 clinical isolates, 16 of the 23 food isolates and eight of the 11 collection strains. Virulence was evaluated in vitro based on strains' ability to invade the human enterocyte-like cell line Caco-2. In gentamicin survival experiments, recovery of viable intracellular bacteria among lectin-unreactive strains was usually 100-1000-fold lower than among lectin-reactive haemolytic strains, and lower than among nonhaemolytic strains. Considerable cytopathogenic effects were produced by lectin-reactive haemolytic strains in trypan-blue-stained cell monolayers, whereas lectin-unreactive and nonhaemolytic strains produced no detectable cytopathogenic effect. Among lectin-reactive strains, the number of listerial cells associated with Caco-2 monolayers was more than tenfold greater than among lectin-unreactive strains. Cell invasion was inhibited by pretreatment of Caco-2 cells with sugars recognized by the lectins or of listeriae with enzymes which removed the same sugars from the bacterial surface. The results suggest that the study of lectin interactions could be helpful in understanding the pathogenicity potential of isolates of food and environmental origin.

Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29.

Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were alpha-lactalbumin, beta-lactoglobulin, apo-lactoferrin, and Fe(3+)-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that beta-lactoglobulin, apo- and Fe(3+)-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.

Induction of apoptosis in HT-29 cells infected with SA-11 rotavirus.

Rotavirus infection is associated both in vivo and in vitro with a series of subcellular pathological alterations leading to cell lysis. It has been suggested that these modifications can play a key role in the pathogenesis of rotavirus-associated diarrheal disease. We describe the effects of SA-11 rotavirus infection in HT-29 cells, a human enterocyte-like cell line. Cytological analyses suggested that the viral-induced cytopathic process, including chromatin clumping, can be referred to as apoptosis, the cell death pathway alternative to necrosis. A time course of the process was performed to investigate whether rotavirus-associated cell death showed specific injury signs. HT-29-infected cells were analyzed by scanning and transmission electron microscopy and features of apoptosis such as blebbing of the plasma membrane, peripheral condensation of chromatin, and fragmentation of the nucleus were observed. Specific changes occurring in cell-substrate adhesion and in some organelles relevant for viral maturation, i.e., rough endoplasmic reticulum, were detected. These findings indicate a role for apoptosis in the rotavirus infection process and its related cytopathology, and also suggested that specific histological alterations such as derangement of enterocytes are associated with the pathogenesis of rotavirus-induced diarrheal disease and could be a direct consequence of viral-triggered apoptosis.