Involvement of a Rarely Used Splicing SD2b Site in the Regulation of HIV-1 vif mRNA Production as Revealed by a Growth-Adaptive Mutation.

We have previously reported an HIV-1 mutant designated NL-Y226tac that expresses Vif at an ultra-low level, being replication-defective in high-APOBEC3G cells, such as H9. It carries a synonymous mutation within the splicing SA1 site relative to its parental clone. In order to determine whether a certain mutant(s) emerges during multi-infection cycles, we maintained H9 cells infected with a relatively low or high input of NL-Y226tac for extended time periods. Unexpectedly, we reproducibly identified a g5061a mutation in the SD2b site in the two independent long-term culture experiments that partially increases Vif expression and replication ability. Importantly, the adaptive mutation g5061a was demonstrated to enhance vif mRNA production by activation of the SA1 site mediated through increasing usage of a rarely used SD2b site. In the long-term culture initiated by a high virus input, we additionally found a Y226Fttc mutation at the original Y226tac site in SA1 that fully restores Vif expression and replication ability. As expected, the adaptive mutation Y226Fttc enhances vif mRNA production through increasing the splicing site usage of SA1. Our results here revealed the importance of the SD2b nucleotide sequence in producing vif mRNA involved in the HIV-1 adaptation and of mutual antagonism between Vif and APOBEC3 proteins in HIV-1 adaptation/evolution and survival.

Atypical immunohistochemical patterns can complement the histopathological diagnosis of oral premalignant lesions.

BACKGROUND: The histopathological diagnosis of oral premalignant lesions (OPLs) such as oral epithelial dysplasia (ED) and carcinoma in situ (CIS), as well as epithelial hyperplasia (EHP), is important for the early detection and precise treatment of oral squamous cell carcinoma. However, the standardization of detection and treatment by histological criteria alone remains challenging owing to the complicated and varied histology. We evaluated practically useful immunohistochemical (IHC) markers that might complement the histopathological diagnosis of OPLs. METHODS: We re-evaluated the histopathological diagnoses and IHC patterns of Ki-67, TP53, CK13, and CK17 in 200 cases of OPLs and performed multiple logistic regression analyses for their predictive accuracy. RESULTS: We identified and compared atypical IHC patterns in OPLs and in the normal epithelium. Ki-67 expression showed specific patterns in categorized OPLs as EHP, low-grade dysplasia (LED), high-grade dysplasia (HED), and CIS. Multiple logistic regression analyses in the quadrant categories revealed that EHP and CIS had high predictive accuracies of 90.1% and 96.2%, respectively, and in binary categories, combined EHP and LED versus combined HED and CIS showed predictive accuracies of 92.1% and 89.9%, respectively. Binominal logistic regression analysis between each quadrant category revealed satisfactory predictive accuracy of EHP vs. LED, LED vs. HED, and HED vs. CIS (75.2%, 78.9%, and 87.9%, respectively), and Ki-67 showed the highest adjusted odds ratio, followed by TP53. CONCLUSION: The proposed atypical IHC patterns might serve as useful markers to supplement the morphological diagnosis of OPLs, and established IHC methods for Ki-67 and TP53 might provide stable results.

Establishment and characterization of a clear cell odontogenic carcinoma cell line with EWSR1-ATF1 fusion gene.

OBJECTIVE: Clear cell odontogenic carcinoma (CCOC) is a rare malignant odontogenic tumor (MOT) characterized by sheets and lobules of vacuolated and clear cells. To understand the biology of CCOC, we established a new cell line, CCOC-T, with EWSR1-ATF1 fusion gene from a mandible tumor with distant metastasis and characterized this cell line. MATERIALS AND METHODS: To detect the EWSR1-ATF1 fusion gene, we used three CCOC cases, including the present case, by RT-PCR and FISH analysis. We characterized established CCOC-T cells by checking cell growth, invasion and the expression of odontogenic factors and bone-related factors. Moreover, the gene expression profile of CCOC-T cells was examined by microarray analysis. RESULTS: Histologically, the primary tumor was comprised of cords and nests containing clear and squamoid cells separated by fibrous septa. In addition, ameloblastomatous islands with palisaded peripheral cells were observed, indicating probable odontogenic origin. This tumor expressed the fusion gene EWSR1-ATF1, which underlies the etiology of hyalinizing clear cell carcinoma (HCCC) and potentially that of CCOC. We found a breakpoint in the EWSR1-ATF1 fusion to be the same as that reported in HCCC. Established CCOC-T cells grew extremely slowly, but the cells showed highly invasive activity. Moreover, CCOC-T cells expressed bone-related molecules, odontogenic factors, and epithelial mesenchymal transition (EMT)-related molecules. CONCLUSION: To the best of our knowledge, this is the first report on the establishment of a CCOC cell line. CCOC-T cells serve as a useful in vitro model for understanding the pathogenesis and nature of MOT.

Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties.

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of the Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. ERM cells are unique epithelial cells that remain in periodontal tissues throughout adult life. They have a functional role in the repair/regeneration of cement or enamel. Here, we isolated odontogenic epithelial cells from ERM in the periodontal ligament, and the cells were spontaneously immortalized. Immortalized odontogenic epithelial (iOdE) cells had the ability to form spheroids and expressed stem cell-related genes. Interestingly, iOdE cells underwent osteogenic differentiation, as demonstrated by the mineralization activity in vitro in mineralization-inducing media and formation of calcification foci in iOdE cells transplanted into immunocompromised mice. These findings suggest that a cell population with features similar to stem cells exists in ERM and that this cell population has a differentiation capacity for producing calcifications in a particular microenvironment. In summary, iOdE cells will provide a convenient cell source for tissue engineering and experimental models to investigate tooth growth, differentiation, and tumorigenesis.

Acceleration of tumor growth due to dysfunction in M1 macrophages and enhanced angiogenesis in an animal model of autoimmune disease.

Both autoimmunity and tumor immunity are immune responses against self-tissues or cells. However, the precise similarity or difference between them remains unclear. In this study, to understand a novel mechanism of tumor immunity, we performed transplantation experiments with a murine autoimmune model, C57BL/6J (B6)/lpr mice. A melanoma cell line, B16F10 cells, or granulocyte macrophage colony-stimulating factor- overexpressing B16F10 (B16F10/mGM) cells were transplanted into B6 or B6/lpr mice. Tumor growth by transplanted B16F10/mGM cells was significantly accelerated in B6/lpr mice compared with that in B6 mice. The accumulation of M1 macrophages in the tumor tissues of B6/lpr recipient mice was significantly lower compared with that in the control mice. In vitro co-culture experiment showed that impaired differentiation into M1 macrophages was observed in B6/lpr mice. The number of tumor vessels and vascular endothelial growth factor (VEGF) expression were also significantly enhanced in the tumor tissues of B6/lpr mice compared with those in the B6 mice. Moreover, VEGF expression was correlated with the increased expression of hypoxia-inducible factor-1α in the tumor tissues of B6/lpr mice. These results suggest that dysfunctional tumor immunity and enhanced angiogenesis in autoimmunity influence tumor growth.

Aromatase controls Sjögren syndrome-like lesions through monocyte chemotactic protein-1 in target organ and adipose tissue-associated macrophages.

Several autoimmune diseases are known to develop in postmenopausal women. However, the mechanism by which estrogen deficiency influences autoimmunity is unknown. Aromatase is an enzyme that converts androgens to estrogens. Herein, we used female aromatase gene knockout (ArKO) mice as a model of estrogen deficiency to investigate the molecular mechanism that underlies the onset and development of autoimmunity. Histological analyses showed that inflammatory lesions in the lacrimal and salivary glands of ArKO mice increased with age. Adoptive transfer of spleen cells or bone marrow cells from ArKO mice into recombination activating gene 2 knockout mice failed to induce the autoimmune lesions. Expression of mRNA encoding proinflammatory cytokines and monocyte chemotactic protein-1 increased in white adipose tissue of ArKO mice and was significantly higher than that in wild-type mice. Moreover, an increased number of inflammatory M1 macrophages was observed in white adipose tissue of ArKO mice. A significantly increased monocyte chemotactic protein-1 mRNA expression of the salivary gland tissue in ArKO was found together with adiposity. Furthermore, the autoimmune lesions in a murine model of Sjögren syndrome were exacerbated by administration of an aromatase inhibitor. These results suggest that aromatase may play a key role in the pathogenesis of Sjögren syndrome-like lesions by controlling the target organ and adipose tissue-associated macrophage.

A critical role for thymic stromal lymphopoietin in nickel-induced allergy in mice.

Ni is the most frequent cause of contact allergy induced by metals. However, the underlying mechanism of this induction is unknown. Our previous research demonstrates that activation of dendritic cells (DCs) through p38MAPK/MKK6 is required for Ni-induced allergy in mice. In the current study, we investigated the cellular and molecular mechanisms underlying Ni-induced allergy using a mouse model that involves injecting Ni into the ear, with or without Freund's incomplete or complete adjuvants. Nickel had greater potential to cause allergic reactions compared with palladium and gold. Among the proteins expressed at higher levels in mice with Ni-induced allergy, we focused on thymic stromal lymphopoietin (TSLP), which is produced in abundance by keratinocytes. We detected increased expression of the TSLP receptor (TSLPR) in DCs from cervical lymph nodes of mice with Ni-induced allergy, suggesting that DCs in ear tissues were activated through TSLPR signaling induced by keratinocyte-derived TSLP. Furthermore, delayed-type hypersensitivity reactions in mice with Ni-induced allergy were decreased significantly by injection of a Tslp-short interfering RNA along with atelocollagen in the ear skin. These results suggest that Ni allergy may be triggered by a TSLP/TSLPR-mediated interaction between epithelial and immune cells.

Matrix metalloproteinase-13 (MMP-13) directly and indirectly promotes tumor angiogenesis.

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.

Fas-independent T-cell apoptosis by dendritic cells controls autoimmune arthritis in MRL/lpr mice.

BACKGROUND: Although autoimmunity in MRL/lpr mice occurs due to a defect in Fas-mediated cell death of T cells, the role of Fas-independent apoptosis in pathogenesis has rarely been investigated. We have recently reported that receptor activator of nuclear factor (NF)-κB ligand (RANKL)-activated dendritic cells (DCs) play a key role in the pathogenesis of rheumatoid arthritis (RA) in MRL/lpr mice. We here attempted to establish a new therapeutic strategy with RANKL-activated DCs in RA by controlling apoptosis of peripheral T cells. Repeated transfer of RANKL-activated DCs into MRL/lpr mice was tested to determine whether this had a therapeutic effect on autoimmunity. METHODS AND FINDING: Cellular and molecular mechanisms of Fas-independent apoptosis of T cells induced by the DCs were investigated by in vitro and in vivo analyses. We demonstrated that repeated transfers of RANKL-activated DCs into MRL/lpr mice resulted in therapeutic effects on RA lesions and lymphoproliferation due to declines of CD4(+) T, B, and CD4(-)CD8(-) double negative (DN) T cells. We also found that the Fas-independent T-cell apoptosis was induced by a direct interaction between tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) on T cells and TRAIL on Fas-deficient DCs in MRL/lpr mice. CONCLUSION: These results strongly suggest that a novel Fas-independent apoptosis pathway in T cells maintains peripheral tolerance and thus controls autoimmunity in MRL/lpr mice.