Odontogenesis-Associated Phosphoprotein (ODAPH) Overexpression in Ameloblasts Disrupts Enamel Formation via Inducing Abnormal Mineralization of Enamel in Secretory Stage.

Odontogenesis-associated phosphoprotein (ODAPH) is a recently discovered enamel matrix protein. Our previous study demonstrated that knockouting out Odaph in mice resulted in enamel hypomineralization. To further investigate the effect of Odaph on enamel mineralization, we constructed an Odaph overexpression mouse model, controlled by an amelogenin promoter. Our histological analysis of OdaphTg mice revealed that the enamel layer was thinner than in WT mice. An uneven, thinner enamel layer was confirmed using micro-computed tomography (uCT). It was subsequently found that the Tomes' processes lost their normal morphology, resulting in the loss of the enamel prism structure. These results indicate that Odaph overexpression in ameloblasts led to enamel dysplasia. In conjunction with this, Odaph overexpression hindered Amelx secretion, and may result in endoplasmic reticulum stress. Interestingly, uCT revealed that enamel had higher mineral density at the secretory stage; due to this, we did the histological staining for the mineralization-related proteins Alkaline phosphatase (ALPL) and Runt-related transcription factor 2 (RUNX2). It was observed that these proteins were up-regulated in OdaphTg mice versus WT mice, indicating that Odaph overexpression led to abnormal enamel mineralization. To confirm this, we transfected ameloblast-like cell line (ALC) with Odaph overexpression lentivirus in vitro and identified that both Alpl and Runx2 were strikingly upregulated in OE-mus-Odaph versus OE-NC cells. We concluded that the ectopic overexpression of Odaph in ameloblasts led to abnormal enamel mineralization. In summary, Odaph profoundly influences amelogenesis by participating in enamel mineralization.

The synergistic effects of TGF-ß1 and RUNX2 on enamel mineralization through regulating ODAPH expression during the maturation stage.

Transforming growth factor ß1 (TGF-ß1) and Runt-related transcription factor 2 (RUNX2) are critical factors promoting enamel development and maturation. Our previous studies reported that absence of TGF-ß1 or RUNX2 resulted in abnormal secretion and absorption of enamel matrix proteins. However, the mechanism remained enigmatic. In this study, TGF-ß1-/-Runx2-/- and TGF-ß1+/-Runx2+/- mice were successfully generated to clarify the relationship between TGF-ß1 and RUNX2 during amelogenesis. Lower mineralization was observed in TGF-ß1-/-Runx2-/- and TGF-ß1+/-Runx2+/- mice than single gene deficient mice. Micro-computed tomography (µCT) revealed a lower ratio of enamel to dentin density in TGF-ß1-/-Runx2-/- mice. Although µCT elucidated a relatively constant enamel thickness, variation was identified by scanning electron microscopy, which revealed that TGF-ß1-/-Runx2-/- mice were more vulnerable to acid etching with lower degree of enamel mineralization. Furthermore, the double gene knock-out mice exhibited more serious enamel dysplasia than the single gene deficient mice. Hematoxylin-eosin staining revealed abnormalities in ameloblast morphology and arrangement in TGF-ß1-/-Runx2-/- mice, which was accompanied by the absence of atypical basal lamina (BL) and the ectopic of enamel matrix. Odontogenesis-associated phosphoprotein (ODAPH) has been identified as a component of an atypical BL. The protein and mRNA expression of ODAPH were down-regulated. In summary, TGF-ß1 and RUNX2 might synergistically regulate enamel mineralization through the downstream target gene Odaph. However, the specific mechanism by which TGF-ß1 and RUNX2 promote mineralization remains to be further studied.

Expression and localization of amelotin, laminin γ2 and odontogenesis-associated phosphoprotein (ODAPH) on the basal lamina and junctional epithelium.

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic.

Runx2 deficiency in junctional epithelium of mouse molars decreases the expressions of E-cadherin and junctional adhesion molecule 1.

Junctional epithelium (JE) attaching to the enamel surface seals gaps around the teeth, functioning as the first line of gingival defense. Runt-related transcription factor 2 (Runx2) plays a role in epithelial cell fate, and the deficiency of Runx2 in JE causes periodontal destruction, while its effect on the barrier function of JE remains largely unexplored. In the present study, hematoxylin-eosin (H&E) staining revealed the morphological differences of JE between wild-type (WT) and Runx2 conditional knockout (cKO) mice. We speculated that these changes were related to the down-regulation of E-cadherin (E-cad), junctional adhesion molecule 1 (JAM1), and integrin ß6 (ITGB6) in JE. Moreover, immunohistochemistry (IHC) was conducted to assess the expressions of these proteins. To verify the relationship between Runx2 and the three above-mentioned proteins, human gingival epithelial cells (HGEs) were cultured for in vitro experiment. The expression of Runx2 in HEGs was depleted by lentivirus. Quantitative real-time PCR (qRT-PCR) and Western blotting analysis were adopted to analyze the differences in mRNA and protein expressions. Taken together, Runx2 played a crucial role in maintaining the structure and function integrality of JE via regulating the expressions of E-cad and JAM1.

Hesperetin as an adjuvant augments protective anti-tumour immunity responses in B16F10 melanoma by stimulating cytotoxic CD8+ T cells.

Hesperetin (HES) is a dihydroflavone with the molecular formula of C16H14O6. It has been reported that Hesperetin has antioxidant and anticancer effects. Recent studies showed that it can also regulate immune responses. To assess its potential function as a vaccine adjuvant, we formulated HES with inactivated B16F10 melanoma cells and determined whether it would enhance the activation of antigen-presenting cells by experiments in vivo and in vitro. We found that HES activated the PI3K-Akt signalling pathway in antigen-presenting cells (APCs), enhanced cytotoxic T lymphocyte (CTL) responses and deactivated tolerogenic T cells. We also observed that inactivated B16F10 cells in combination with HES vaccine inhibited the growth of mice tumours, resulting in improved overall survival compared to the effects of inactivated B16F10 cell vaccine. To verify that CD8+ T cells play a key role in inhibiting the development of melanoma, we transferred the sorted CD8+ T cells from immunized mice to B16F10 challenged models and found that the survival rate of tumour-bearing mice was significantly prolonged. Taken together, these results suggest that hesperetin can be used as a potential adjuvant to improve tumour immune responses and antigen immunogenicity.

HCMV-encoded IE2 induces anxiety-depression and cognitive impairment in UL122 genetically-modified mice.

Although there is a high risk of mood disorders and cognitive impairment in congenital human cytomegalovirus (HCMV) infections, the molecular pathogenetic mechanisms of HCMV have not yet been fully determined. In this study, we show that immediate-early 2 (IE2) protein modulates affective and cognitive behaviors. We used a UL122 genetically-modified mice model that can continuously express IE2 protein. We used a series of animal behavior tests to determine the relationship between HCMV-encoded IE2 and psychiatric disorders. In open-field, elevated plus-maze test and tail suspension tests, we found that UL122 genetically-modified mice displayed more anxiety-depression behavior than did wild-type (WT) mice. The Morris water maze test and novel object recognition test showed that spatial learning and memory were lower in UL122 genetically-modified mice model than in WT mice. Level of fibroblast growth factor 2 (FGF2) protein in the hippocampus cornu ammonia areas (CA1, CA3) and dentate gyrus (DG) of the experimental group was significantly lower, consistent with immunohistochemical staining and western blot for neuron-specific nuclear protein (NeuN) and glial fibrillary acidic protein (GFAP). Levels of SYP and PSD-95 proteins were lower in the hippocampus UL122 genetically-modified mice. These data suggest the importance of HCMV-encoded IE2 for studying anxiety and depression behaviors and for the spatial learning and memory. This would help to further explain the molecular pathological mechanism of psychiatric disorders caused by HCMV infection.

Intrahepatic T-Cell Receptor ß Immune Repertoire Is Essential for Liver Regeneration.

T lymphocytes synergize with the cellular immune system to promote hepatocyte regeneration. The T-cell receptor (TCR) immune repertoire is closely associated with the host immune response and regenerative proliferation. High-throughput sequencing of TCR provides deep insight into monitoring the immune microenvironment. Here, we aimed to determine the role of the TCRß immune repertoire in liver regeneration (LR). We investigated hepatic regeneration in TCRß chain-deficient (tcrb-/- ) mice by two-thirds partial hepatectomy (PHx) method. Our results demonstrated that tcrb-/- mice revealed a reduced capacity for LR, which was characterized by impaired hepatocyte proliferation and enhanced hepatocyte apoptosis. Dysregulation of inflammatory signaling activation and inflammatory factors was observed in regenerated tcrb-/- livers. Simultaneously, significantly altered immunocyte levels and aberrant cytokine levels were observed during hepatic regeneration. In addition, we first determined the profile of the TCRß immune repertoire during LR, indicating that PHx resulted in remarkably lower TCRß diversity in intrahepatic T lymphocytes. Conclusion: Taken together, our data suggest that TCRß deficiency gives a rise to aberrant intrahepatic immune microenvironment that impairs LR, and the TCRß reconstitution is required for hepatic immunocyte recruitment and activation during LR.

Extracranial bone metastases from recurrent anaplastic astrocytoma on FDG PET/CT: A case report a care-compliant article.

OBJECTIVE: Extracranial bone metastases from astrocytoma are rare and frequently detected as part of multiorgan metastases. It is extremely rare for astrocytoma to have extracranial bone metastases alone. The importance of whole-body fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) imaging in evaluating extracranial metastasis (ECMs) has not been described effectively due to the rarity of this event. The purpose of our case report is to emphasize the role of FDG PET/CT in the assessment of tumor recurrence and extracranial bone metastases from anaplastic astrocytoma. METHODS AND MATERIALS: A 25-year-old woman was firstly admitted with a 4-month history of progressive blurred vision, and 2-month history of intermittent headache. Presurgical MRI imaging revealed a large mass in the left trigone of lateral ventricle. Subsequently, she underwent tumor resection, radiotherapy and chemotherapy. A final pathological diagnosis of anaplastic astrocytoma (WHO III) was made. Nearly 12 months after the surgery, the follow-up brain MR imaging revealed a contrast-enhanced lesion in the site of operative region. Whole-body FDG PET/CT imaging was performed to evaluate the situation. RESULTS: Postoperative brain FDG PET/CT showed an abnormal focal FDG uptake corresponding to the contrast-enhanced lesion in the operative area, suggesting a tumor recurrence. Whole-body FDG PET/CT also showed multiple FDG-avid osteosclerotic lesions in the body. It was highly suggestive of extracranial bone metastases. A subsequent open bone biopsy of FDG-avid lesion in right iliac crest was performed. Histopathological and immunohistochemical findings indicated characteristic of glioma. The patient died 1 month later, nearly 13 months after the initial diagnosis. CONCLUSIONS: ECMs from anaplastic astrocytoma are extremely rare but they do occur. Whole-body FDG PET/CT imaging with inclusion of brain was valuable in differentiating tumor recurrence from radiation necrosis and in detecting uncommon extracranial bone metastases from anaplastic astrocytoma, which were closely related to prognosis of this disease.

Blastic Plasmacytoid Dendritic Cell Neoplasm Evaluated by FDG PET/CT.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy. A 34-year-old man with a biopsy-proven BPDCN underwent FDG PET/CT for staging. FDG PET/CT revealed multiple mild FDG-avid cutaneous lesions on the chest and back, involvement of left inguinal lymph node, and a markedly increased FDG-avid subcutaneous mass in the left lower leg.

Polysplenia Syndrome With Splenic and Skeletal Muscle Metastases From Thyroid Carcinoma Evaluated by FDG PET/CT: Case Report and Literature Review: A Care-Compliant Article.

Polysplenia syndrome (PSS) is a rare congenital abnormality. Metastases to spleen and skeletal muscle from differentiated thyroid cancer (DTC) are also extremely rare. Our case report aims to present an interesting case of PSS associated with splenic metastasis (SM) and skeletal muscle metastasis (SMM) from advanced papillary thyroid carcinoma which was evaluated on fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT). An 84-year-old Chinese man was admitted with the history of multiple enlarged masses in bilateral neck, right axillary, and inguinal areas for >2 months. The results of ultrasonography examination were highly suggestive of malignancy. The histological results of the following biopsy were consistent with papillary thyroid carcinoma with involvement of multiple regional lymph nodes. He was referred for an FDG PET/CT imaging to evaluate the situation. FDG PET/CT showed that an intense FDG-avid thyroid mass with widespread regional lymph node involvement and distant metastases in the body. Unexpected sites of metastases were detected in the spleens and skeletal muscles. Most interestingly, FDG PET/CT imaging also described the typical imaging findings of PSS including the 2 right-sided spleens, azygos and hemiazygos continuation of inferior vena cava (IVC), right-sided stomach, middle line liver, a short pancreas, preduodenal portal vein (PPV), and malrotation of gut. Whole body FDG PET/CT imaging can accurately evaluate the situation of DTC by detecting regional lymph node involvement, common and rare sites of distant metastases which are closely related to staging, management, and prognosis of this disease. Whole-body FDG PET/CT is also valuable in demonstrating the typical imaging features of PSS.

[Clinical observation on the target therapy of rh-endostin, combined with chemotherapy in advanced non-small cell lung cancer.].

BACKGROUND: The chemotherapy of advanced non-small cell lung cancer is in a Bottleneck. The target therapy of Anti-angiogenesis gradually shows an advantage in the therapy of patients with advanced NSCLC. To investigate the short-term efficacy, safety and the quality of life of the target therapy of rh-Endostin combined with chemotherapy in patients with advanced NSCLC. METHODS: Sixty-two advanced NSCLC patients were randomly divided into either the trial group with chemotherapy plus rh-Endostin or control group with chemotherapy alone. The efficacy and toxicity were evaluated after 2 cycles according to RECIST criteria. RESULTS: The trial groups efficiency rate was 46.87%, while the control group was 26.66%, there was no significant differences of two groups (Chi-square=1.912, P=0.166). The clinical benefit rate was 81.25% in the trial group and 53.33% in control group. There was significant difference of the clinical benefit rate between the trial group and the control group (Chi-square=4.3185, P=0.0377). The score of quality of life in the trial group was significantly higher than that the control group after the treatment (Chi-square=11.233, P=0.0008). There was no significant difference of incidence of toxicities between the trial group and the control group (P>0.05). CONCLUSIONS: Rh-Endostin combined with chemotherapy was effective, reasonable, safe and well tolerated for advanced NSCLC.

Changes of activated circulating endothelial cells and survivin in patients with non-small cell lung cancer after antiangiogenesis therapy.

BACKGROUND: Although antiangiogenesis therapy plays an important role in anti-neoplastic treatment with its recognized efficacy and slight adverse effect, there is no prospective clinical trial to define ideal markers for predicting efficacy of antiangiogenic therapy. This study was undertaken to investigate the changes of activated circulating endothelial cells (aCECs) and survivin after anti-angiogenesis therapy and their significance in predicting the efficacy of the therapy. METHODS: Patients of non-small cell lung cancer (NSCLC) treated with chemotherapy with or without Endostar were observed. The amount of activated CECs was detected by flow cytometry, and the expression of survivin mRNA was determined by real-time polymerase chain reaction (PCR). RESULTS: After treatment, the amount of activated CECs decreased significantly in clinical benefit cases (P = 0.021 in chemotherapy alone, P = 0.001 in chemotherapy plus Endostar), increased in disease progressive cases (P = 0.015 in chemotherapy alone, but P = 0.293 in chemotherapy with Endotatar). After therapy, the expression of survivin mRNA decreased in clinical benefit cases (P = 0.001) and increased in disease progressive cases (P = 0.018). A positive correlation was found between activated CECs and survivin in the chemotherapy group pre- and post-therapy (P = 0.001 and 0.021, respectively), but only in the chemotherapy with Endostar group pre-therapy (P = 0.030) rather than post-therapy. A positive correlation was found between the decreased activated CECs after therapy and time to progression (TTP) (r = 0.322, P = 0.012); a negative correlation was found between the amount of survivin mRNA in serum post-therapy and TTP (r = -0.291, P = 0.048). CONCLUSIONS: Activated CECs and survivin may be ideal markers forecasting efficacy and prognosis of NSCLC. The former can reflect more sensitively antiangiogenic efficacy and the latter is more sensitive to shrinkage or swelling of tumors. Their combination can evaluate more accurately the efficacy of antiangiogenic therapy of NSCLC.