Reactive oxygen species generation required for autophagy induction during butyrate- or propionate-induced release of damage-associated molecular patterns from dying gingival epithelial Ca9-22 cells.

PURPOSE: Bacterial cells in mature dental plaque produce a high concentration of short-chain fatty acids (SCFAs) such as butyrate and propionate. SCFA-treatment on human gingival epithelial Ca9-22 cells induced cell death. However, the exact mechanism underlying cell death remains unclear. In this study, the relationship between reactive oxygen species (ROS) and autophagy induction during SCFA-induced cell death was examined. METHODS: Human gingival epithelial Ca9-22 cells were treated with butyrate or propionate to induce cell death and the number of dead cells were measured using SYTOX-green dye. A siRNA for ATG5 and N-acetylcysteine (NAC) were used for autophagy reduction and ROS-scavenging, respectively. Release of damage-associated molecular patterns (DAMPs) such as Sin3A-associated protein 130 (SAP130) and high-mobility group box 1 (HMGB1) were detected using western blot. RESULTS: Reducing autophagy significantly suppressed SCFA-induced Ca9-22 cell death. ROS generation was observed upon SCFA treatment, and scavenging ROS with NAC decreased cell death. NAC also reduced the SCFA-induced increase in microtubule-associated protein 1 light chain 3B (LC3B)-I and LC3B-II, and mitigated the release of DAMPs. CONCLUSION: The findings suggest that ROS generation is necessary for autophagy, which is required for SCFA-induced cell death and accompanying DAMP release.

Possible roles of short-chain fatty acids produced by oral bacteria in the development of alveolar osteitis.

PURPOSE: Alveolar osteitis (dry sockets) is a painful condition characterized by a limited immune response. It is typically caused by the removal of blood clots from extracted tooth sockets, which leads to the fermentation of trapped food remnants by oral bacteria in the cavities, producing high concentrations of short-chain fatty acids (SCFAs). This study examined the effects of SCFAs on immunity and bone metabolism. METHODS: Mouse macrophage Raw264.7 cells were treated with oral bacteria supernatants or SCFA mixtures, and inducible nitric oxide synthase (iNOS) levels were determined by western blot. The same cells were treated with SCFA mixtures in the presence of receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoclast-like cells were counted. MC3T3-E1 cells were treated with SCFA mixtures and stained with alizarin red S. RESULTS: Raw264.7 cells treated with oral bacterial culture supernatants of Porphyromonas gingivalis and Fusobacterium nucleatum inhibited lipopolysaccharide (LPS)-induced iNOS production, likely due to SCFA content. SCFA mixtures mimicking these supernatants inhibited the number of RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive cells and MC3T3-E1 cell mineralization. CONCLUSION: These data suggest that SCFAs produced by P. gingivalis and F. nucleatum may reduce the inflammatory response and mildly induce mineralization of the alveolar walls. These results may contribute to the understanding of alveolar osteitis.

PAR2-dependent phosphorylation of TRPV4 at the trigeminal nerve terminals contributes to tongue cancer pain.

OBJECTIVE: This study aimed to clarify the interactions between the tongue and primary afferent fibers in tongue cancer pain. METHODS: A pharmacological analysis was conducted to evaluate mechanical hypersensitivity of the tongues of rats with squamous cell carcinoma (SCC). Changes in trigeminal ganglion (TG) neurons projecting to the tongue were analyzed using immunohistochemistry and western blotting. RESULTS: SCC inoculation of the tongue caused persistent mechanical sensitization and tumor formation. Trypsin expression was significantly upregulated in cancer lesions. Continuous trypsin inhibition or protease-activated receptor 2 (PAR2) antagonism in the tongue significantly inhibited SCC-induced mechanical sensitization. No changes were observed in PAR2 and transient receptor potential vanilloid 4 (TRPV4) levels in the TG or the number of PAR2-and TRPV4-expressing TG neurons after SCC inoculation. In contrast, the relative amount of phosphorylated TRPV4 in the TG was significantly increased after SCC inoculation and abrogated by PAR2 antagonism in the tongue. TRPV4 antagonism in the tongue significantly ameliorated the mechanical sensitization caused by SCC inoculation. CONCLUSIONS: Our findings indicate that tumor-derived trypsin sensitizes primary afferent fibers by PAR2 stimulation and subsequent TRPV4 phosphorylation, resulting in severe tongue pain.

Radiological and histochemical study of bone regeneration using the costal cartilage in rats.

PURPOSE: The purpose of this study was to conduct basic research on the possibility of using cartilage tissue for hard-tissue reconstruction and to observe morphological changes in the transition of the cartilage to bone. METHODS: A 4-mm diameter bone defect was created in the right mandibular angle of rats. Cartilage, autologous bone, and artificial bone were grafted into the defect. Computed tomography (CT) was performed to measure the increase in bone volume. Further histological evaluation of the grafted site was performed. RESULTS: At 12 weeks, CT show that bone formation in the costal cartilage group was comparable to that in the autogenous bone group. Histologically, in the artificial bone group, a clear boundary was observed between the existing bone and defect, whereas in the costal cartilage and autologous bone groups, laminar plate bone repair of the defect was observed. CONCLUSION: The findings in this study suggest that bone reconstruction achieved with cartilage grafting is almost equivalent to that with autogenous bone grafting and that bone reconstruction using cartilage is clinically feasible. In future, if regenerated cartilage is successfully applied clinically, bone reconstruction using regenerated cartilage may be feasible.

Periodontopathic Bacterium Fusobacterium nucleatum Affects Matrix Metalloproteinase-9 Expression in Human Alveolar Epithelial Cells and Mouse Lung.

BACKGROUND/AIM: Despite evidence of an association between pulmonary diseases and periodontopathic bacteria, the molecular mechanisms remain unknown. Matrix metalloproteinase-9 (MMP9) plays important roles in pneumonia, chronic obstructive pulmonary disease, and asthma; therefore, we assessed the effects of Fusobacterium nucleatum on MMP9 expression in mouse lung and A549 human alveolar epithelial cells. MATERIALS AND METHODS: Heat-killed F. nucleatum was administered to the trachea of mice or added to A549 cell cultures. MMP9 expression was determined using real-time PCR and western blotting. The involvement of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) in MMP9 expression was examined. RESULTS: F. nucleatum induced expression of MMP9 in mouse lung and bronchoalveolar lavage fluid. In A549 cells, F. nucleatum induced production of MMP9 protein and mRNA in a density-dependent manner; this was inhibited by inhibitors of extracellular-regulated kinase 1/2 and NF-κB, but not of p38 and Jun N-terminal protein kinase. CONCLUSION: F. nucleatum may contribute to the onset of pulmonary diseases via MMP9 expression through extracellular-regulated kinase 1/2 and NF-κB activation.

Nuclear localization of propiece IL-1α in HeLa cells.

PURPOSE: The study aimed to examine the nuclear localization of propiece interleukin (IL)-1α (ppIL-1α) and extracellular release rates of ppIL-1α, pIL-1α, and mIL-1α. METHODS: The subcellular localization of IL-1α molecules was observed in HeLa cells transfected with green fluorescent protein (GFP)-tagged IL-1α. Extracellular release efficiency was examined using N-terminal HiBiT-tagged IL-1α. The nuclear localization status of ppIL-1α was examined by incubating ppIL-1α transfectants with 0.1% Triton X-100 solution or with complete medium on ice. RESULTS: The results indicated the diffuse cytoplasmic and nuclear localization for m and p and ppIL-1, respectively. All IL-1α forms were released from the cells even in the steady state, and the release efficiency was 25%, 13%, and 8% for mIL-1α, pIL-1α, and ppIL-1α, respectively. Under oxidative stress condition, GFP-mIL-1α was totally diminished, but weak staining of GFP-pIL-1α and GFP-ppIL-1α was detected; nuclear localization of GFP-ppIL-1α was completely abolished by 0.1% Triton X-100 treatment, however, it remained in the nucleus after culture in complete medium on ice. CONCLUSION: The results of this study showed that ppIL-1α was localized in the nucleus and released extracellularly even in the steady state. Moreover, its cellular localization is not firm, and it is presumed to be floating in the nucleoplasm.

Fusobacterium nucleatum exacerbates chronic obstructive pulmonary disease in elastase-induced emphysematous mice.

Exacerbation of chronic obstructive pulmonary disease (COPD) is associated with disease progression and increased mortality. Periodontal disease is a risk factor for exacerbation of COPD, but little is known about the role of periodontopathic bacteria in this process. Here, we investigated the effects of intratracheal administration of Fusobacterium nucleatum, a periodontopathic bacteria species, on COPD exacerbation in elastase-induced emphysematous mice. The administration of F. nucleatum to elastase-treated mice enhanced inflammatory responses, production of alveolar wall destruction factors, progression of emphysema, and recruitment of mucin, all of which are symptoms observed in patients with COPD exacerbation. Hence, we propose that F. nucleatum may play a role in exacerbation of COPD.

Pannexin 1-Mediated ATP Signaling in the Trigeminal Spinal Subnucleus Caudalis Is Involved in Tongue Cancer Pain.

Pain is one of the most severe concerns in tongue cancer patients. However, the underlying mechanisms of tongue cancer pain are not fully understood. We investigated the molecular mechanisms of tongue cancer-induced mechanical allodynia in the tongue by squamous cell carcinoma (SCC) inoculation in rats. The head-withdrawal threshold of mechanical stimulation (MHWT) to the tongue was reduced following SCC inoculation, which was inhibited by intracisternal administration of 10Panx, an inhibitory peptide for pannexin 1 (PANX1) channels. Immunohistochemical analyses revealed that the expression of PANX1 was upregulated in the trigeminal spinal subnucleus caudalis (Vc) following SCC inoculation. The majority of PANX1 immunofluorescence was merged with ionized calcium-binding adapter molecule 1 (Iba1) fluorescence and a part of it was merged with glial fibrillary acidic protein (GFAP) fluorescence. Spike frequencies of Vc nociceptive neurons to noxious mechanical stimulation were significantly enhanced in SCC-inoculated rats, which was suppressed by intracisternal 10Panx administration. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive (IR) neurons increased significantly in the Vc after SCC inoculation, which was inhibited by intracisternal 10Panx administration. SCC inoculation-induced MHWT reduction and increased pERK-IR Vc neuron numbers were inhibited by P2X7 purinoceptor (P2X7R) antagonism. Conversely, these effects were observed in the presence of P2X7R agonist in SCC-inoculated rats with PANX1 inhibition. SCC inoculation-induced MHWT reduction was significantly recovered by intracisternal interleukin-1 receptor antagonist administration. These observations suggest that SCC inoculation causes PANX1 upregulation in Vc microglia and adenosine triphosphate released through PANX1 sensitizes nociceptive neurons in the Vc, resulting in tongue cancer pain.

Porphyromonas gingivalis gingipains potentially affect MUC5AC gene expression and protein levels in respiratory epithelial cells.

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.

Oxytocin-Dependent Regulation of TRPs Expression in Trigeminal Ganglion Neurons Attenuates Orofacial Neuropathic Pain Following Infraorbital Nerve Injury in Rats.

We evaluated the mechanisms underlying the oxytocin (OXT)-induced analgesic effect on orofacial neuropathic pain following infraorbital nerve injury (IONI). IONI was established through tight ligation of one-third of the infraorbital nerve thickness. Subsequently, the head withdrawal threshold for mechanical stimulation (MHWT) of the whisker pad skin was measured using a von Frey filament. Trigeminal ganglion (TG) neurons innervating the whisker pad skin were identified using a retrograde labeling technique. OXT receptor-immunoreactive (IR), transient receptor potential vanilloid 1 (TRPV1)-IR, and TRPV4-IR TG neurons innervating the whisker pad skin were examined on post-IONI day 5. The MHWT remarkably decreased from post-IONI day 1 onward. OXT application to the nerve-injured site attenuated the decrease in MHWT from day 5 onward. TRPV1 or TRPV4 antagonism significantly suppressed the decrement of MHWT following IONI. OXT receptors were expressed in the uninjured and Fluoro-Gold (FG)-labeled TG neurons. Furthermore, there was an increase in the number of FG-labeled TRPV1-IR and TRPV4-IR TG neurons, which was inhibited by administering OXT. This inhibition was suppressed by co-administration with an OXT receptor antagonist. These findings suggest that OXT application inhibits the increase in TRPV1-IR and TRPV4-IR TG neurons innervating the whisker pad skin, which attenuates post-IONI orofacial mechanical allodynia.

Cost of establishing a general dentistry department in a small or medium-sized hospital.

To contribute to future dental healthcare policies, this study compiled data on hospital expenses and follow-ups conducted after a hospital dentistry department was established. In addition, the management status and reports on the utility and challenges of establishing a dentistry department were analyzed. The dentistry department was established through fund raising and inaugurated in May 2009. The depreciation period was set at 7 years, and income and expenditure during the 7 years 8 months after opening were compiled. In total, 17.22 million yen was needed for the dentistry department. The average income from dental care was 21.59 million yen per year, and expenditure amounted to 21.54 million yen per year. The findings indicated that a general dentist able to systemically manage patients was essential in a chronic-care hospital. Moreover, the present findings indicate that if general dentistry consultations were performed without excessive investments, after adjusting for personnel expenses, such an initiative would neither yield considerable income nor produce a substantial deficit. Finally, it is imperative to develop staff who are familiar with the costs and management of hospital dentistry and to increase medical fees for consultations with elderly patients.

Bone marrow-derived mesenchymal stem cells enhance bone marrow regeneration in dental extraction sockets.

Bone marrow-derived mesenchymal stem cells (BMMSCs) remain the most widely used source of osteogenic cells in bone tissue engineering research. A cell-based treatment for alveolar ridge augmentation has received attention as an alternative to bone grafting. In the present study, BMMSC transplantation into tooth extraction sockets of C57BL/6J mice was evaluated for alveolar ridge regeneration. The first right maxillary molars were extracted, and then BMMSCs (PDGFRα+ Sca-1+ CD45- TER119- cells) isolated from femoral and tibial bone marrow were immediately transplanted into the extraction sockets. A control group underwent the same procedure except for BMMSC transplantation. Bone formation in the sockets was evaluated using micro-computed tomography and histological and immunohistochemical analyses. At 3 weeks, bone formation in the sockets was more advanced in the experimental group than in the control group. Histological analysis at 6 weeks after transplantation showed that the sockets in the experimental group also contained a greater quantity of bone marrow. Interestingly, socket bone mineral density was lower in the experimental group than in the control group at 6 weeks. These findings suggest that BMMSC transplantation accelerates bone healing and augments bone marrow formation in tooth extraction sockets.

Distribution of aquaporin-5, transforming growth factor-ß1 and laminin during regeneration of atrophic rat submandibular glands after duct ligation.

The aim of this study was to determine the localization of aquaporin-5 (AQP5), transforming growth factor-ß1 (TGF-ß1) and laminin during regeneration of the rat submandibular gland. After duct ligation for 7 days, the regenerating glands were collected on days 0, 1, 3, 7, and 14 after ligation release to study the process of regeneration. Immunohistochemical staining revealed apical expression of AQP5 in many acinar cells, strong expression in intercalated ducts (ICDs) of the normal submandibular gland at Day 14, and strong expression in duct-like structures (DLSs) during regeneration from Day 0 to 7. However, a few AQP5-negative acinar cells were detected during regeneration. At Day 0, immunopositivity for TGF-ß1 was detected in connective tissue. At Days 3 and 7 during regeneration, TGF-ß1 immunostaining was observed in DLSs, which were surrounded by α-smooth muscle actin-positive thickened myoepithelial cells. Laminin staining was strong in the thickened basement membrane of DLSs at Day 3 during regeneration, but weak around acinar cells at Day 14. These findings suggest that TGF-ß1 is involved in the environment around DLSs, myoepithelial cells and laminin, that DLSs have the same functional properties as ICDs, and that AQP5-negative acinar cells may be mucous cells.

Endothelin Signaling Contributes to Modulation of Nociception in Early-stage Tongue Cancer in Rats.

BACKGROUND: Patients with early stage tongue cancer do not frequently complain of tongue pain. Endothelin-1 signaling is upregulated in the cancerous tongue at the early stage. We tested the hypothesis that endothelin-1 signaling contributes to the modulation of tongue nociception. METHODS: Squamous cell carcinoma cells were inoculated into the tongue under general anesthesia. Lingual mechanical sensitivity under light anesthesia using forceps from days 1 to 21 (n = 8) and the amounts of endothelin-1 and ß-endorphin in the tongue on days 6, 14, and 21 (n = 5 to 7) were examined after the inoculation. The effect of endothelin-A or µ-opioid receptor antagonism on the mechanical sensitivity was examined (n = 5 to 7). RESULTS: Lingual mechanical sensitivity did not change at the early stage (days 5 to 6) but increased at the late stage (days 13 to 14). The amount of endothelin-1 increased (25.4 ± 4.8 pg/ml vs. 15.0 ± 5.2 pg/ml; P = 0.008), and endothelin-A receptor antagonism in the tongue induced mechanical hypersensitivity at the early stage (51 ± 9 g vs. 81 ± 6 g; P = 0.0001). The µ-opioid receptor antagonism enhanced mechanical hypersensitivity (39 ± 7 g vs. 81 ± 6 g; P < 0.0001), and the amount of ß-endorphin increased at the early stage. CONCLUSIONS: ß-Endorphin released from the cancer cells via endothelin-1 signaling is involved in analgesic action in mechanical hypersensitivity at the early stage.

Diverse calcification patterns of ameloblastic fibro-odontoma on radiographic examination.

The occurrence of ameloblastic fibro-odontoma (AFO) in the oral region is unusual and accounts for 1-3% of all odontogenic tumors. AFO presents mixed radiopaque patterns within the lesion with diverse findings; therefore, it is important to compare this tumor with other odontogenic tumors that radiographically present with calcified bodies. Herein, we observed the calcification patterns within the lesion in seven AFO cases (five males, two females; mean age, 8.3 years; age range, 4-13 years). Periapical and panoramic radiographs were obtained from all seven cases. Five cases underwent conventional computed tomography (CT) scanning, and one underwent cone beam CT. Classification of the calcifications primarily involved the following two characteristics on the X-rays: appearance and location of the lesions. All seven cases were located in the molar regions of the mandible in association with impacted teeth. The calcification patterns of these AFOs were mixed or inhomogeneous within the lesion with various findings, including complex odontoma-like calcifications. However, the patterns differed between panoramic radiography and CT in some cases. The radiolucent lesions in AFO demonstrated varying calcification patterns and were associated with impacted teeth on the CT images.(J Oral Sci 58, 533-537, 2016).

Immunolocalization of FGF-2, -7, -8, -10 and FGFR-1-4 during regeneration of the rat submandibular gland.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play important roles in the development of the submandibular gland. Although regeneration of submandibular glands follows a similar process to their development, it is unknown how FGFs and FGFRs are distributed during regeneration of submandibular gland. The aim of this study was to determine the localization of FGFs and FGFRs during such regenerative processes. After 7 days' obstruction, the submandibular glands were collected at days 0, 1, 3, 7, 11 and 14 after duct release to study regeneration. The regenerative processes of the submandibular gland were investigated by immunohistochemistry for FGF-2, 7, 8, 10 and FGFR-1-4. Immunohistochemical staining revealed that FGF-2 was moderately expressed in the epithelial cells of duct-like structures (DLS) and newly formed acinar cells (NFAC) at days 0-7, and strongly in intercalated duct (ICD) at control gland and Day 7-14. FGF-7 was localized moderately in NFAC and DLS. FGF-8 was localized moderately in the epithelial cells of DLS during regeneration. Strong positive immunoreactions for FGF-10 were found in NFAC and the epithelial cells of DLS during regeneration, as well as the ICD and lateral surfaces of the maturing acinar cells (MAC). FGFR-1 was expressed moderately in the ICD, and weakly in the NFAC and MAC. Positive immunoreactions for FGFR-2 were not observed during regeneration. Additionally, FGFR-4 was detected strongly in the ICD and slightly in NFAC. These findings suggest that FGF-2, -7, -8 and -10 play important roles in NFAC, MAC, and DLS through FGFR-1 and -4 during regeneration of submandibular gland.

Runx1 and Runx3 Are Downstream Effectors of Nanog in Promoting Osteogenic Differentiation of the Mouse Mesenchymal Cell Line C3H10T1/2.

Previously, we reported that the transcription factor Nanog, which maintains the self-renewal of embryonic stem cells (ESCs), promotes the osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 through a genome reprogramming process. In the present study, to clarify the mechanism underlying the multipotency of mesenchymal stem cells (MSCs) and to develop a novel approach to bone regenerative medicine, we attempted to identify the downstream effectors of Nanog in promoting osteogenic differentiation of mouse mesenchymal cells. We demonstrated that Runx1 and Runx3 are the downstream effectors of Nanog, especially in the early and intermediate osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2.

Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control green fluorescent protein (GFP) gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process.

Immunohistological study of small Rho GTPases and ß-catenin during regeneration of the rat submandibular gland.

Our study immunohistochemically evaluated the localization patterns of small Rho GTPases and ß-catenin during regeneration of the rat submandibular gland. After 7 days of obstruction, regenerating glands were collected at days 0, 3, 7, 11 and 14 after duct release to study regeneration. RhoA was detected strongly and RhoC was detected weakly in the cytoplasm of newly formed acinar cells from day 3 to 7, and both RhoA and RhoC at the basal site and cytoplasm were detected moderately from day 11 to 14. RhoB was detected strongly and moderately in the cytoplasm of newly formed and matured acinar cells, respectively, and detected strongly in duct-like structures (DLSs) and intercalated ducts (ICDs). Rac1 was detected at the cell-cell and subcellular region, but ß-catenin was not observed in newly formed acinar cells. Rac1 immunolabeling gradually reduced, and the ß-catenin staining pattern became stronger. p-Rac1, a phosphorylated form of Rac1, was observed in the cytoplasm of newly formed acinar cells. At apical and subcellular region of DLSs and ICDs, Rac1 and ß-catenin were detected. These findings suggest that RhoA and RhoC might be involved in the actin cytoskeleton at the basolateral site of regenerating acinar cells, and RhoB might play a unique role in regenerating acinar cells and in DLSs and ICDs. Rac1 and ß-catenin at the cell-cell region might play important roles in cell-cell adhesion and the differentiation of regenerating acinar cells, as well as actin reconstruction at apical and subcellular regions of DLSs and ICDs.