Progenitor identification and SARS-CoV-2 infection in human distal lung organoids.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.

Quantitative analysis of ß1,6GlcNAc-branched N-glycans on ß4 integrin in cutaneous squamous cell carcinoma.

α6ß4 integrin plays pivotal roles in cancer progression in several types of cancers. Our previous study using N-glycan-manipulated cell lines demonstrated that defects in N-glycans or decreased ß1,6GlcNAc-branched N-glycans on ß4 integrin suppress ß4 integrin-mediated cancer cell adhesion, migration, invasion, and tumorigenesis. Furthermore, immunohistochemical analysis has shown that colocalization of ß1,6GlcNAc-branched N-glycans with ß4 integrin was observed in cutaneous squamous cell carcinoma (SCC) tissue. However, until now there has been no direct evidence that ß1,6GlcNAc-branched N-glycans are upregulated on ß4 integrin in cutaneous SCC. In the present study, we performed an ELISA analysis of ß1,6GlcNAc-branched N-glycans on ß4 integrins as well as ß4 integrins in cell lysates from human normal skin and cutaneous SCC tissues. The SCC samples showed a 4.9- to 7.4-fold increase in the ratio of ß1,6GlcNAc-branched N-glycans to ß4 integrin compared with normal skin samples. These findings suggest that the addition of ß1,6GlcNAc-branched N-glycans onto ß4 integrin was markedly elevated in cutaneous SCC tissue compared to normal skin tissue. The value of ß1,6GlcNAc-branched N-glycans on ß4 integrin may be useful as a diagnostic marker associated with cutaneous SCC tumor progression.

Integrin ß4 Is an Effective and Efficient Marker in Synchronously Highlighting Lymphatic and Blood Vascular Invasion, and Perineural Aggression in Malignancy.

Lymphovascular invasion (LVI) and perineural invasion (PNI) are 2 important pathologic parameters and need to be accurately assessed in multiple malignancies. Integrin ß4, a member of the integrin family, has been reported to be positively expressed in vascular endothelia, peripheral nerves, and a collection of epithelia. However, little is known about the effectiveness of ß4 immunostaining on the recognition of LVI and PNI. Herein, we explored the applicability of ß4 immunostaining in stomach, thyroid, and breast cancers. Parallel immunostaining of D2-40, CD34, and S-100 was performed as controls for lymphatic endothelia, vascular endothelia, and neural fibers, respectively. The results demonstrated that ß4 concurrently stained the lymphatic and vascular endothelia, and the peripheral nerves. Both LVI and PNI were clearly and accurately outlined by ß4 immunostaining. ß4 was also expressed in the majority of tumor cells, enabling recognition of LVI and PNI encroached by small tumor clusters. In contrast to D2-40 and CD34, ß4 staining was not observed in stromal cells, and therefore it facilitated differentiation between the shrinkage cleft and LVI. According to our results, ß4 staining strikingly increased the diagnostic accuracy and interobserver concordance for LVI and PNI compared with hematoxylin and eosin staining alone. Finally, the applicability of ß4 was confirmed in 9 other types of malignancies, including cancers of the colon, prostate, esophagus, lung, kidney, uterus, tongue, bladder, and liver. Collectively, ß4 is a reliable marker for synchronous detection and diagnosis of LVI and PNI.

Cytotoxic tumour-infiltrating T lymphocytes influence outcome in resected pancreatic ductal adenocarcinoma.

BACKGROUND: We studied the prognostic effect of CD3-, CD8- and CD103-positive T lymphocytes in a cohort of 165 patients with resected pancreatic ductal adenocarcinomas (PDACs) of the treatment group (adjuvant gemcitabine) and the untreated control group of the CONKO-001 study. METHODS: Immunohistochemical stainings on tissue microarrays (TMAs) against CD3, CD8 and CD103 were performed according to standard procedures. RESULTS: A high number of CD8-positive lymphocytes were significantly and independently associated with longer disease-free survival (DFS) and overall survival (OS) in the overall study population. Median DFS/OS were 7.4/18.1 months for patients with a low number of CD8-positive intratumoural lymphocytes (≤42 per 1 mm tissue core) and 12.7/25.2 months for patients with high numbers (>42 per 1-mm tissue core; p = 0.008/0.020; HR 0.62/0.65). The ratio of intraepithelial to total CD103-positive lymphocytes, but not total numbers of CD103-positive lymphocytes or CD103-positive intraepithelial lymphocytes, was associated with significantly improved DFS and OS in the overall study population (p = 0.022/0.009). Median DFS/OS was 5.9/15.7 for patients with a ratio of intraepithelial to total CD103-positive intratumoural lymphocytes higher than 0.3 and 11.6/24.7 for patients with a lower ratio. CONCLUSION: T-lymphocyte subpopulations might be prognostic in resectable PDAC but need standardization and verification by further studies.

Efficiency and Specificity of Gene Deletion in Lung Epithelial Doxycycline-Inducible Cre Mice.

The transgenic mouse strains surfactant protein C-reverse tetracycline transactivator (SP-C-rtTA), club cell secretory protein (CCSP)-rtTA, and tetracycline operator (TetO)-Cre have been invaluable for spatiotemporally regulating gene deletion in the pulmonary epithelium. In this study, we measured the efficiency and specificity of gene deletion that can be achieved in these mice using the Rosa26-eYFP reporter. Triple-transgenic mice (tTg or rtTA/TetO-Cre/Rosa-eYFP) were bred and treated with various doxycycline (dox) regimens to induce gene deletion, which was then quantified in various cell populations by flow cytometry. In these crosses, we found that the TetO-Cre transgene must be transmitted through the female parent to avoid germline gene deletion. With dox exposure during lung development, SP-C-tTg mice deleted in ∼65-75% of alveolar epithelial type II (ATII) cells, but in only ∼45-50% of the integrin ß4+ population, which consisted of club cells and distal lung progenitor cells. In contrast, CCSP-tTg mice deleted in ∼50% of ATII cells and ∼80% of integrin ß4+ cells. Upon dox treatment of adults, deletion in ATII cells and integrin ß4+ cells in SP-C-tTg mice dropped significantly to ∼20% and ∼6%, respectively, whereas CCSP-tTg mice deleted in ∼57% of ATII and ∼40% of integrin ß4+ cells. Interestingly, untreated CCSP-tTg mice also deleted in ∼40% of integrin ß4+ cells, indicating significant leakiness of CCSP-tTg in ß4+ cells. In all mouse groups, minimal deletion occurred in mouse tracheal epithelial cells or in mesenchymal or hematopoietic cells. These data provide the first quantitative, side-by-side comparison of the deletion efficiency for these widely used transgenic mouse strains.

Elevated integrin α6ß4 expression is associated with venous invasion and decreased overall survival in non-small cell lung cancer.

Lung cancer carries a poor prognosis and is the most common cause of cancer-related death worldwide. The integrin α6ß4, a laminin receptor, promotes carcinoma progression in part by cooperating with various growth factor receptors to facilitate invasion and metastasis. In carcinoma cells with mutant TP53, the integrin α6ß4 promotes cell survival. TP53 mutations and integrin α6ß4 overexpression co-occur in many aggressive malignancies. Because of the high frequency of TP53 mutations in lung squamous cell carcinoma (SCC), we sought to investigate the association of integrin ß4 expression with clinicopathologic features and survival in non-small cell lung cancer (NSCLC). We constructed a lung cancer tissue microarray and stained sections for integrin ß4 subunit expression using immunohistochemistry. We found that integrin ß4 expression is elevated in SCC compared with adenocarcinoma (P<.0001), which was confirmed in external gene expression data sets (P<.0001). We also determined that integrin ß4 overexpression associates with the presence of venous invasion (P=.0048) and with reduced overall patient survival (hazard ratio, 1.46; 95% confidence interval, 1.01-2.09; P=.0422). Elevated integrin ß4 expression was also shown to associate with reduced overall survival in lung cancer gene expression data sets (hazard ratio, 1.49; 95% confidence interval, 1.31-1.69; P<.0001). Using cBioPortal, we generated a network map demonstrating the 50 most highly altered genes neighboring ITGB4 in SCC, which included laminins, collagens, CD151, genes in the EGFR and PI3K pathways, and other known signaling partners. In conclusion, we demonstrate that integrin ß4 is overexpressed in NSCLC where it is an adverse prognostic marker.

MUC5AC interactions with integrin ß4 enhances the migration of lung cancer cells through FAK signaling.

MUC5AC is a secretory mucin aberrantly expressed in various cancers. In lung cancer, MUC5AC is overexpressed in both primary and metastatic lesions; however, its functional role is not well understood. The present study was aimed at evaluating mechanistic role of MUC5AC on metastasis of lung cancer cells. Clinically, the overexpression of MUC5AC was observed in lung cancer patient tissues and was associated with poor survival. In addition, the overexpression of Muc5ac was also observed in genetically engineered mouse lung adenocarcinoma tissues (Kras(G12D); Trp53(R172H/+); AdCre) in comparison with normal lung tissues. Our functional studies showed that MUC5AC knockdown resulted in significantly decreased migration in two lung cancer cell lines (A549 and H1437) as compared with scramble cells. Expression of integrins (α5, ß1, ß3, ß4 and ß5) was decreased in MUC5AC knockdown cells. As both integrins and MUC5AC have a von Willebrand factor domain, we assessed for possible interaction of MUC5AC and integrins in lung cancer cells. MUC5AC strongly interacted only with integrin ß4. The co-localization of MUC5AC and integrin ß4 was observed both in A549 lung cancer cells as well as genetically engineered mouse adenocarcinoma tissues. Activated integrins recruit focal adhesion kinase (FAK) that mediates metastatic downstream signaling pathways. Phosphorylation of FAK (Y397) was decreased in MUC5AC knockdown cells. MUC5AC/integrin ß4/FAK-mediated lung cancer cell migration was confirmed through experiments utilizing a phosphorylation (Y397)-specific FAK inhibitor. In conclusion, overexpression of MUC5AC is a poor prognostic marker in lung cancer. MUC5AC interacts with integrin ß4 that mediates phosphorylation of FAK at Y397 leading to lung cancer cell migration.

Splicing abnormality of integrin ß4 gene (ITGB4) due to nucleotide substitutions far from splice site underlies pyloric atresia-junctional epidermolysis bullosa syndrome.

BACKGROUND: Pyloric atresia-junctional epidermolysis bullosa syndrome (PA-JEB) is a rare subgroup of epidermolysis bullosa, which is inherited disorder characterized by skin fragile. PA-JEB is caused by mutation of ITGB4 or ITGA6, which encodes integrin ß4 or α6, respectively. OBJECTIVE: To clarify the molecular basis of PA-JEB and to expand the mutational database, we carried out the mutational analysis of a 29-year-old Japanese PA-JEB patient. METHODS: Standard methods were used to prepare, PCR-amplify, and sequence DNA or mRNA in peripheral blood or skin samples, respectively. RESULTS: Sequence analysis revealed two novel mutations in ITGB4, c.264+2TtoA and c.1762-25TtoA. The paternal c.264+2TtoA resided within a splice site consensus region and generated two splice variants resulting in a premature termination codon (PTC). The maternal c.1762-25TtoA was a unique mutation because of its location, 25 bp away from the splice site, and resided in branch-point consensus sequence. This c.1762-25TtoA substitution resulted in generation of two abnormal transcripts each with a PTC. Genotype-phenotype correlation in this case was also unique because the proband showed a non-lethal phenotype regardless of both mutations resulted in only abnormal transcripts with a PTC. CONCLUSION: The present case expands the mutational database and further elucidates the genotype-phenotype correlation for this rare disease, PA-JEB.

Clonal culture of adult mouse lung epithelial stem/progenitor cells.

Clonal culture of stem cells is crucial for their identification, and the characterization of the cellular and molecular mechanisms that regulate their proliferation and differentiation. In the adult mouse lung, epithelial stem/progenitor cells are defined by the phenotype CD45(neg) CD31(neg) EpCAM(pos) CD104(pos) CD24(low). Here we describe a tissue dissociation and flow cytometry strategy for the detection and isolation of adult mouse lung epithelial stem/progenitor cells, and a three-dimensional colony-forming assay for their clonal culture in vitro.

The role of phosphoinositide 3-kinase in adhesion of oral epithelial cells to titanium.

BACKGROUND: Oral epithelial cells (OECs) adhesion to titanium may improve the success rate of implant restoration. PURPOSE: We investigated the mechanism by which OECs adhere to titanium dental implants. MATERIALS AND METHODS: (1) After culturing rat OECs on titanium plates (Ti) or culture dishes in the presence or absence of a phosphoinositide 3-kinase (PI3K) activator or inhibitors and/or growth factors, and OEC morphology under these conditions were analyzed. (2) Right maxillary first molars were extracted and replaced with experimental implants. The rats were treated with or without growth factors. RESULTS: (1) Cell adherence was lower of OECs on Ti than in those on culture dishes, as were the levels of integrin ß4 and the continuity of F-actin structures. After PI3K inhibition, markedly reducing adherence to both substrates. In contrast, PI3K activation with activator or insulin-like growth factor restored the OEC adherence and the expression of adhesion molecules on Ti to the levels seen in OECs cultured on dishes. Cell migration was inhibited by PI3K activation. (2) High expression of integrin ß4 was observed in the peri-implant epithelia of PI3K-activated rats. CONCLUSION: These findings suggest that PI3K plays an important role in the adhesion of OECs to Ti.

A rare population of CD24(+)ITGB4(+)Notch(hi) cells drives tumor propagation in NSCLC and requires Notch3 for self-renewal.

Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small-cell lung cancer (NSCLC). CD24(+)ITGB4(+)Notch(hi) cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a nonredundant role in tumor cell propagation in two mouse models and in human NSCLC. The TPC population is enriched after chemotherapy, and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC.

Tissue-engineered mucosa is a suitable model to quantify the acute biological effects of ionizing radiation.

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.

Initial attachment, subsequent cell proliferation/viability and gene expression of epithelial cells related to attachment and wound healing in response to different titanium surfaces.

OBJECTIVES: A tight seal between the epithelium and the dental implant surface is required to prevent bacterial inflammation and soft tissue recession and therefore to demonstrate a long-term success. Surface hydrophilicity was recently shown to promote osseointegration. The aim of this study was to investigate the influence of surface hydrophilicity in combination with surface topography of Ti implant surfaces on the behavior and activation/differentiation of epithelial cells using a set of in vitro experiments mimicking the implant-soft tissue contact. METHODS: Hydrophobic acid-etched (A) and coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and modSLA surfaces were produced. The behavior of an oral squamous cell carcinoma cell line (HSC-2) grown on all surfaces was compared through determination of cell attachment and proliferation/viability (CCK-8 and MTT assay), time-lapse microscopy of fluorescence labeled cells and determination of gene expression by real time polymerase chain reaction. RESULTS: Within the surfaces with similar wettability cell spreading and cell movements observed by time-lapse microscopy after one day of incubation were most pronounced on smoother (A and modA) surfaces compared to rougher (SLA and modSLA) surfaces. Within the surfaces with similar roughness the hydrophilic surfaces (modA and modSLA) showed more cell spreading and cell activity compared to the hydrophobic surfaces (A and SLA). The relative gene expressions of cytokeratin14, integrin α6, integrin ß4, vinculin, transforming growth factor (TGF)-ß, TGF-ß1, and TGF-ß3 were decreased in HSC-2 on all four types of Ti surfaces compared to control surfaces (tissue culture polystyrene; p<0.01) and there was no significant difference of gene expression on the four different implant-surfaces. SIGNIFICANCE: We have demonstrated that for proliferation and spreading of HSC-2 cells the smoother and hydrophilic surface is optimal (modA). These results suggest that surface hydrophilicity might positively influence the epithelial seal around dental implants. All tested titanium surfaces downregulate cell attachment, cell proliferation, expression of adhesion promoters, and cytokines involved in wound healing in HSC-2 cells compared to control surfaces.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip.

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, ß1 subunits of α3ß1 heterodimer and ß4 subunit of integrin α6ß4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of ß1, ß4 and α3 integrins, and SISCCs lacked ß1, ß4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin ß1, ß4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype.

Is CD10 a reliable marker of invasive colorectal cancer?

AIM: Previous studies reported that CD10 positive Colorectal Cancer Cells (CRC) characterized by deeply invasive neoplasia. MATERIALS AND METHODS: We have examined 50 pts surgically treated for colorectal cancer on at least 5 years follow up. TNM, grading score and survival have been compared to CD10 expression. RESULTS: Thirty-four out of fifty cases have been analyzed (18 males and 16 female) of whom nineteen were CD10 positive and fifteen were CD10 negative. The remaining 16 cases were droping out. No difference in survival rate between CD10 positive and negative in N0, N1, N2. No difference on survival rate and grading 1, 2, 3. We have then analyzed CD10 positive and CD10 negative cases, according to neoplasia grading, in patients with positive linphonodes N1 and N2. We showed a statistical difference between the CD10 positive/N2 (grading 1.66 +/- 0.5) and the CD10 negative/N2 (grading 3) (p < 0.005). CONCLUSIONS: We can hypothesize that CD10 positive neoplasia display a more invasive behaviour, independently from the N score and the G score, compared to CD10 negative neoplasia.

Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium.

BACKGROUND AND OBJECTIVE: It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. MATERIAL AND METHODS: Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. RESULTS: Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). CONCLUSION: The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.

Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin.

The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that beta4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of beta4 integrin. Suppression of beta4 integrin expression by shRNA and disruption of beta4 integrin function by transfection of dominant-negative beta4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for beta4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between beta4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The beta4 integrin-ezrin interaction appears to be critical for maintenance of beta4 integrin expression. These data begin to integrate ezrin and beta4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.

IgG from patients with bullous pemphigoid depletes cultured keratinocytes of the 180-kDa bullous pemphigoid antigen (type XVII collagen) and weakens cell attachment.

We have shown that binding of bullous pemphigoid (BP)-patient IgG (BP-IgG) causes the internalization of BP180 from the cell membrane. This study examined whether BP-IgG treatment can deplete cultured keratinocytes of BP180, how it affects cellular levels of alpha6 and beta4 integrins (by western blot analysis using monoclonal antibodies to these antigens), and whether it reduces adhesion of cells to the culture dish (by a vibration detachment assay). All BP-IgG or BP sera with high values of BP180-ELISA from 18 BP patients before and after oral corticosteroid treatment showed dramatically decreased BP180 in cells after 6 hours of BP-IgG stimulation, whereas alpha6 and beta4 integrin levels were not decreased. Even IgG from patients in whom oral corticosteroid had suppressed active blistering could deplete cells of BP180, as long as sera retained a high value of BP180-ELISA. On the other hand, reduction of cell BP180 content increased detachment of cells from the dish. These results suggest that BP-IgG reduces hemidesmosomal BP180 content, weakening the adhesion of hemidesmosomes to the lamina densa. In the presence of BP180 deficiency, inflammation generated by BP180 immune-complex formation might then tear the weakened lamina lucida, and this could lead to generation of the BP-specific split at the lamina lucida.

Immunolocalization of laminin and integrin in regenerating junctional epithelium of mice after gingivectomy.

BACKGROUND AND OBJECTIVE: The expression patterns of adhesive proteins and extracellular matrix proteins in regenerating gingival epithelium after gingivectomy are unknown. The aim of this study was to examine the expression of laminin 1, laminin gamma(2) (a specific component of laminin 5), integrin beta(4) and integrin alpha(3) in the regenerating gingival epithelium in order to understand the mechanism of wound healing during reconstitution of the sulcular environment. MATERIAL AND METHODS: The palatal gingivae of the maxillary molars of Institute of Cancer Research mice were excised, and the regenerating tissues were examined 1, 3, 5, 7 and 14 days later. Fresh, non-fixed and non-decalcified frozen sections were prepared and stained using immunofluorescence. RESULTS: At 1 day post-surgery, intense expression of laminin gamma(2), integrin beta(4) and integrin alpha(3) was distinct in the frontal margin of the regenerating oral epithelium. Laminin gamma(2) was diffusely detected on the root surface and in connective tissues beneath the regenerating oral epithelium at 3 and 5 days. At 7 days, laminin gamma(2) was intermittently recognizable in the internal basal lamina (IBL) close to tooth-facing cells, while laminin gamma(2), integrin beta(4) and integrin alpha(3) were observed in the IBL and in the external basal lamina (EBL) of the regenerating junctional epithelium at 14 days. CONCLUSION: These results suggest that secretion of laminin 5 in the connective tissue may induce epithelial cell migration, and that binding of laminin 5 to integrin alpha(6)beta(4) and integrin alpha(3)beta(1) in the IBL may provoke cell adhesion and migration of cells facing the tooth on the enamel surface of the regenerating junctional epithelium.

Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. MATERIAL AND METHODS: We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. RESULTS: Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. CONCLUSION: These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.