Near-Infrared Photoimmunotherapy Using a Protein Mimetic for EGFR-Positive Salivary Gland Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer (IR700Dye). The conjugate can be activated by near-infrared light irradiation, causing necrotic cell death with high selectivity. In this study, we investigated NIR-PIT using a small protein mimetic (6-7 kDa, Affibody) which has more rapid clearance and better tissue penetration than mAbs for epidermal growth factor receptor (EGFR)-positive salivary gland cancer (SGC). The level of EGFR expression was examined in vitro using immunocytochemistry and Western blotting. Cell viability was analyzed using the alamarBlue assay. In vivo, the volume of EGFR-positive tumors treated with NIR-PIT using the EGFR Affibody-IR700Dye conjugate was followed for 43 days. It was found that NIR-PIT using the EGFR Affibody-IR700Dye conjugate induced the selective destruction of EGFR-positive SGC cells and restricted the progression of EGFR-positive tumors. We expect that NIR-PIT using the EGFR Affibody-IR700Dye conjugate can efficiently treat EGFR-positive SGC and preserve normal salivary function.

Study of clinical factors, focus score, lymphocyte type and NF-κB pathway in Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease that occurs predominantly in middle-aged and older women. Although focus score (FS) and lesion grade are determined at pathological diagnosis, few reports have examined whether these results reflect clinical symptoms. In this study, we examined and compared the results of comprehensive immunohistochemical staining of lymphocytes and NF-κB pathway in labial gland biopsies, clinical test data, and radionuclide imaging findings. One hundred labial gland biopsy specimens obtained from 20 female patients with primary SS (5 specimens per patient) were studied. Hematoxylin-eosin-stained specimens were reviewed and FS were calculated. Immunohistochemical staining of CD4, CD8, CD20, CD25, Foxp3, NF-κB, TNFAIP3 and IκBα was performed, and the results were compared with anti-SS-A/Ro (SS-A), anti-SS-B/La (SS-B) and antinuclear antibodies (ANA), and salivary gland scintigraphy findings. FS were significantly higher in the SS-A-, SS-B- and ANA-positive groups than in the respective -negative groups (p < 0.05). Of eight SS-A-positive and SS-B-negative cases, mean FS was 1.9 (seven cases: FS ≥ 1.0) and six cases were ANA-positive. In four SS-A-positive and SS-B-positive cases, mean FS was 3.2 (all cases: FS ≥ 1.0) and all cases were ANA-positive. In immunohistochemical staining, CD4-positive T cells tended to be more abundant than CD8-positive T cells. Small numbers of Foxp3-positive cells were found in all cases. NF-κB, TNFAIP3 and IκBα were positive in the acini, ductal epithelium, and lymphocytes in all cases. The above findings indicated a relationship between FS and clinical test data, and the association of NF-κB pathway with the pathophysiology of primary SS.

Combination of Near-Infrared Photoimmunotherapy Using Trastuzumab and Small Protein Mimetic for HER2-Positive Breast Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6-7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.

Histopathological evaluation of minor salivary gland papillary-cystic tumours: focus on genetic alterations in sialadenoma papilliferum and intraductal papillary mucinous neoplasm.

AIMS: Minor salivary gland tumours showing a predominant papillary-cystic structure are rare, and constitute a mixture of various types of neoplasm; thus, the histopathological assessment of these tumours poses a significant diagnostic challenge. We aimed to delineate the histological characteristics of these tumours and further mutational aspects with a particular focus on sialadenoma papilliferum (SP) and intraductal papillary mucinous neoplasm (IPMN). METHODS AND RESULTS: We retrieved 28 papillary-cystic tumours of the minor salivary glands, and performed histological re-evaluation and mutation analyses of several key oncogenes. The histological classifications were as follows: SP (n = 10), SP-like intraductal papillary tumour (SP-IPT) (n = 2), IPMN (n = 9), intraductal papilloma, cystadenoma, and cystadenocarcinoma (two, three and two respectively). Whereas SP typically consisted of a combination of exophytic squamous epithelium and endophytic intraductal papillary infoldings, SP-IPT lacked the exophytic component. SP and SP-IPT frequently harboured BRAF V600E mutations (75.0%), which were identified in both squamous and ductal components. IPMN was characterised by a well-demarcated cystic lesion filled exclusively with a papillary proliferation of mucinous cells and a high rate of AKT1 E17K mutations (88.9%). Intraductal papillomas were unilocular cystic lesions with intraluminal papillary growth of bland columnar cells. In contrast, both cystadenomas and cystadenocarcinomas showed a multicystic appearance with a papillary configuration. Cystadenocarcinomas invaded the surrounding tissue and were composed of markedly atypical tumour cells. CONCLUSION: The appropriate interpretation of histological findings and specific genetic alterations (e.g. BRAF V600E and AKT1 E17K in SP and IPMN) would be useful for the correct diagnosis of minor salivary gland papillary-cystic tumours.

Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and Possibility of a Theranostic Approach.

The accurate detection of lymph node metastases is essential for treatment success in early-stage malignant cancer. Sentinel lymph node (SLN) biopsy is the most effective procedure for detecting small or micrometastases that are undetectable by conventional imaging modalities. To demonstrate a new approach for developing a more efficient SLN biopsy procedure, we reported a two-stage imaging method combining lymphoscintigraphy and near-infrared (NIR) fluorescence imaging to depict metastatic cancer cells in SLNs in vivo. Furthermore, the theranostic potential of the combined procedure was examined by cell culture and xenograft mouse model. Anti-HER2 and anti-epidermal growth factor receptor (EGFR) affibody probes were used for NIR fluorescence imaging. Strong NIR fluorescence signal intensity of the anti-EGFR affibody probe was observed in SAS cells (EGFR positive). Radioactivity in the SLNs was clearly observed in the in vivo studies. High anti-EGFR affibody NIR fluorescence intensity was observed in the metastatic lymph nodes in mice. The addition of the IR700-conjugated anti-EGFR affibody to the culture medium decreased the proliferation of SAS cells. Decreased proliferation was shown in Ki-67 immunohistochemistry in xenograft tumors. Our data suggest that a two-stage combined imaging method using lymphoscintigraphy and affibody probes may offer the direct visualization of metastatic lymph nodes as an easily applied technique in SLN biopsy. Although further animal studies are required to assess the effect of treating lymphatic metastasis in this approach, our study results provide a foundation for the further development of this promising imaging and treatment strategy for earlier lymph node metastasis detection and treatment.

Study of MYB-NFIB chimeric gene expression, tumor angiogenesis, and proliferation in adenoid cystic carcinoma of salivary gland.

Adenoid cystic carcinoma (ACC) is one of the common malignant tumors in salivary glands, and the clinical prognosis is poor with frequent distant metastasis which may lead to death. Expression of the MYB-NFIB chimeric gene in ACC has been reported recently. MYB is an oncogene with transcription regulating functions, and NFIB encodes nuclear transcription factor although detailed functions are unknown. This study investigated whether MYB-NFIB chimeric gene expression affects tumor angiogenesis and proliferation in salivary gland ACC. In 26 salivary gland ACC cases, MYB-NFIB chimeric gene expression was analyzed by RT-PCR and direct sequencing. Immunohistochemical studies for CD31, vascular endothelial growth factor (VEGF) and Ki-67 were performed. Tumor angiogenesis was evaluated by blood vessel (CD31-positive) density and tumor proliferation by Ki-67 labeling index, and the relationship with MYB-NFIB chimeric gene expression was analyzed. MYB-NFIB chimeric gene expression was detected in nine of 26 ACC cases. Blood vessel density was significantly higher in chimeric gene-expressing cases compared to non-expressing cases. VEGF score tended to be higher in chimeric gene-expressing cases than in non-expressing cases, while Ki-67 labeling index was not significantly different. The number of chimeric gene-expressing cases increased with age, peaking in the sixties age group and declining thereafter, while the number of non-expressing cases increased with age continuously. In ACC, blood vessel density was significantly higher in MYB-NFIB chimeric gene-expressing cases compared to non-expressing cases, which may be due to higher VEGF production capability. MYB-NFIB chimeric gene expression may also be related to the onset age of ACC.

HER2-Targeted Multifunctional Silica Nanoparticles Specifically Enhance the Radiosensitivity of HER2-Overexpressing Breast Cancer Cells.

We investigated the effects of targeted functionalized silica nanoparticles on the radiosensitivity of cancer cells. Better control of the local concentration of silica nanoparticles may facilitate their use as an adjuvant in conjunction with ionizing radiation to target cancer cells while preventing damage to normal cells. Hyperbranched polyamidoamine (PAMAM) was grafted onto the surface of amorphous silica nanoparticles to functionalize them. The PAMAM-coated silica nanoparticles (PCSNs) were then conjugated with fluorescent dyes. Anti-HER2 antibodies were covalently attached to the labeled PCSNs. The HER2-overexpressing SK-BR3 breast cancer cell line was incubated in medium containing the PCSN probes. After incubation; the cells were exposed to X-ray radiation. Cells were counted in all samples using cell proliferation assays; and apoptotic cells were detected. The cell survival results showed that the combination of the targeted PCSN probes and radiation reduced the survival rate of SK-BR3 cells to a greater extent than when either PCSN probes, PCSNs or radiation were applied individually. The results also showed an increase in apoptosis in the SK-BR3 cells that internalized the PCSN probes and were then irradiated. Based on these data, PCSN probes act as specific radiosensitizing agents for HER2-overexpressing cells.

Developmental Anomalies in Human Teeth: Odontoblastic Differentiation in Hamartomatous Calcifying Hyperplastic Dental Follicles Presenting with DSP, Nestin, and HES1.

Hyperplastic dental follicles (HDFs) represent odontogenic hamartomatous lesions originating from the pericoronal tissues and are often associated with impacted or embedded teeth. These lesions may occasionally feature unique calcifying bodies, known as calcifying whorled nodules (CWNs), characterized by stromal cells arranged in a whorled or spiral fashion. CWNs are typically observed in multiple calcifying hyperplastic dental follicles or regional odontodysplasia. In our study, we examined 40 cases of HDFs, including nine instances with characteristics of CWNs, referred to as calcifying hyperplastic dental follicles (CHDFs), which are infrequently accompanied by odontodysplasia. The median ages of the HDFs and CHDFs were 16 (ranging from 3 to 66) and 15 (ranging from 11 to 50) years, respectively. The lower third molars were the most frequently affected by HDSFs and CHDFs, followed by the upper canines. A histological examination was conducted on all 40 cases, with an immunohistochemical analysis performed on 21 of them. Among the cases with CWN, nine affected a single embedded tooth, with one exception. CWNs exhibited diverse calcifications featuring sparse or entirely deposited psammoma bodies, and some displayed dentinoid formation. Immunohistochemically, the stromal cells of HDFs were frequently positive for CD56 and nestin. By contrast, CWNs were negative for CD56 but positive for nestin as well as hairy and enhancer split 1 (HES1), with a few dentin sialoprotein (DSP)-positive calcified bodies. Our results revealed that hamartomatous CHDFs can impact multiple and single-embedded teeth. CWNs composed of nestin and HES1-positive ectomesenchymal cells demonstrated the potential to differentiate into odontoblasts and contribute to dentin matrix formation under the influence of HES1. This study is the first report documenting odontoblastic differentiation in HDFs. The rare occurrence of HDFs and CHDFs contributes to limited comprehension. To prevent misdiagnosis, a better understanding of these conditions is necessary.

A case of radicular cyst on deciduous tooth in a 7-year-old child.

Radicular cysts are the most common cystic lesions in the oral cavity, and have a rare occurrence in the primary dentition. We report a case of radicular cyst of mandible in child by multimodal imaging including panoramic radiography, CT, and MR imaging. A 7-year-old girl presented with swelling and without pain, and hypoesthesia on the right side of the mandible. On clinical examination, an expansive lesion with undulation was found to the buccal cortex of the right side of the mandible. Panoramic radiograph showed a unilocular radiolucency with well-defined margin, displaced tooth, and root resorption in the right mandible. Regarding CT imaging, axial soft tissue algorithm CT and bone tissue algorithm CT showed a low-attenuation internal structure and expansion of the buccal cortex of the right side of the mandible. Three-dimensional-CT showed expansion of the buccal cortex of the right side of the mandible. Multiplanar reformation imaging showed displaced tooth, root resorption, and expansion of the buccal cortex of the right side of the mandible. On T1-weighted image, the expansive lesion showed low signal intensity, and T2-weighted and STIR images revealed high signal intensity. A partial biopsy of the mandibular region was performed. Histopathological diagnosis was radicular cyst caused by apical periodontitis with abscess. This case suggests that multimodal imaging, especially CT and MR imaging, could be effective for evaluating mandibular lesions in child.

A case of odontogenic keratocyst in the buccal space: characterization by multimodality imaging including computed tomography, diffusion-weighted magnetic resonance imaging, and ultrasonography.

Odontogenic keratocyst (OKC) is a relatively common non-inflammatory jaw lesion. OKC is known to occur most often in the mandibular angle and mandibular ramus, but rarely outside the bone. In this report, we describe characteristic multimodality imaging of OKC in the buccal space, especially diffusion-weighted MR imaging (DWI) with apparent diffusion coefficient (ADC) mapping, extra-oral and intra-oral ultrasonography. On clinical examination, an approximately 20 mm in diameter mass with elastic hardness was found the left side of the buccal area. Contrast-enhanced CT showed areas of internal non-contrast lesions in the left buccal space. On T1-weighted image, the mass showed multilocular high signal intensity, and homogeneous internal. T2-weighted images revealed high signal at the marginal part and slightly median signal in the internal part. STIR images revealed a heterogeneous high signal in the interior. Furthermore, DWI and ADC map showed high signal and moderate-to-low signal intensity, respectively. ADC value of the lesion was 1.55 × 10-3 mm2 s-1. On extra-oral ultrasonography, the tumor showed clear boundary, hypoechoic, homogeneous internal architecture and vascular signals, and heterogeneous hard of the lesion. On intra-oral ultrasonography also showed clear boundary, hypoechoic, homogeneous internal architecture, heterogeneous hard of the tumor, and back echo enhance. The histopathologic diagnosis based on a full excisional specimen was odontogenic keratocyst. This case suggests that multimodality imaging, especially MR imaging with ADC and DWI, and extra and intra-oral ultrasonography with color Doppler imaging and elastography, could be effective for evaluating buccal lesions.

The effect of intentionally perforating the floor of the pulp chamber on pulpal healing after tooth replantation in mice.

OBJECTIVES: Shortening the root of a mouse molar prior to tooth replantation results in early revascularization in the pulp cavity and activation of the dental pulp quiescent stem cells. This study aimed to validate the effects of pulp chamber floor perforation on pulpal healing after tooth replantation as a strategy to promote early revascularization into the pulp. METHODS: The maxillary first molars of three-week-old Crlj:CD1 mice were extracted and repositioned into the original socket: the left teeth were immediately replanted (control group: CG), whereas the floor of the pulp chamber of the right teeth was perforated with a tungsten carbide bur before tooth replantation (experimental group: EG). The samples were collected from three days to eight weeks postoperatively. In addition to the TUNEL assay, immunohistochemistry for Nestin, CK14, and Ki-67 was conducted. RESULTS: In the EG, early revascularization occurred with a decrease in apoptosis and an increase in cell proliferation, facilitating pulpal healing, compared with the CG. The rate of Nestin-positive perimeter in the distal root significantly increased on days 5 and 14 and the amount of Nestin-positive hard tissue increased on day 14. However, on day 7, the number of epithelial cell rests of Malassez in the EG significantly decreased, making the EG susceptible to ankylosis at the floor. CONCLUSIONS: Intentionally perforating the floor of the pulp chamber provides a route for early revascularization, resulting in better pulpal healing after tooth replantation.

Characteristic multimodal imaging of palatal follicular lymphoma: a case report on effectiveness of CT, diffusion-weighted MR imaging and intraoral ultrasonography.

Oral lymphomas are relatively uncommon. Follicular lymphoma is the second most common subtype of non-Hodgkin lymphoma. We report characteristic multimodal imaging of palatal follicular lymphoma, especially CT, diffusion-weighted MR imaging (DWI) and intraoral ultrasonography. A 67-year-old woman presented with swelling on the right side of the palate within 2 months. On clinical examination, an approximately 35 × 20 mm mass lesion with elastic soft was found to overlay the right side of the palate. Contrast-enhanced CT image showed a mass with homogeneous enhancement on the right side of the palate, and bone tissue algorithm CT showed focal erosion of the right posterior maxilla. Regarding MR imaging, on T1-weighted image, the mass showed low signal intensity and homogeneous enhancement, and T2-weighted and STIR images revealed intermediate and high signal intensity, respectively. Furthermore, DWI and apparent diffusion coefficient (ADC) map showed high and low signal intensity, respectively. ADC value of the mass was 0.60 × 10-3 mm2s-1. On intraoral ultrasonography, the mass showed clear boundary, hypoechoic echogenicity, homogeneous internal architecture, vascular signals using color Doppler imaging and heterogeneous hard using strain elastography. A partial biopsy of the palatal region was performed. Histopathological diagnosis was follicular lymphoma. This case suggests that multimodal imaging, especially CT, DWI with ADC map and intraoral ultrasonography with color Doppler imaging and strain elastography, could be effective for evaluating palatal lesions.

The effect of the formalin-fixed paraffin-embedded process on salivary microbiota profiling.

In recent years, bacterial DNA in formalin-fixed paraffin-embedded (FFPE) samples has been recognized as a valuable bioresource for microbiota studies. This study aimed to examine the effect of the FFPE process on microbiota profiling to evaluate whether FFPE samples could serve as an alternative bioresource to fresh samples in oral microbiota studies. Fresh saliva was collected from nine subjects. The pellets obtained by centrifuging the collected saliva were fixed in formalin, then dehydrated and embedded in paraffin to prepare FFPE samples. The abundance of the hypervariable regions V1-9, V1-2, and V3-4 of the 16S rRNA gene in fresh and FFPE samples was relatively compared. In addition, microbiota profiling was performed to compare the results between the two sample types. The results showed that the FFPE process resulted in a certain degree of fragmentation of the 16S rRNA gene. However, the V1-2 region was relatively well-preserved compared to the V1-9 and V3-4 regions, suggesting that short regions are suitable targets for oral microbiota analysis. Importantly, there were no significant differences in alpha and beta diversity of microbiota between fresh and FFPE samples, and microbiota profiles were similar between the two sample types, suggesting that FFPE samples could be a valuable bioresource for oral microbiota studies.

Early revascularization activates quiescent dental pulp stem cells following tooth replantation in mice.

Introduction: The intentional perforation of the pulp chamber floor before tooth replantation promotes pulpal healing by facilitating the revascularization of the pulp cavity. This study aimed to elucidate the effects of this method on the dynamics of quiescent dental pulp stem cells (DPSCs). Methods: The right and left maxillary first molars of Crlj:CD1 mice and TetOP-histone 2B (H2B)-green fluorescent protein (GFP) mice were extracted. The left molars were immediately replanted as the control group (CG), whereas the pulp chamber floor of the right molars were perforated before the tooth was replanted as the experimental group (EG). Immunohistochemistry for Nestin and GFP, and quantitative RT-PCR for Nestin, Opn, CD11c, and Oct3/4 mRNA were performed. Results: The rate of Nestin-positive perimeter along the pulp-dentin border in the EG tended to be higher than that of the CG at days 5 and 7 and was significantly increased between days 3 and 7. The rate of GFP-positive cells in the EG was significantly higher than that of the CG at days 5 and/or 7 in the mesial and middle coronal pulp. CD11c mRNA in the EG at day 5 was significantly higher than that of the CG and tended to be higher than that of the CG during the observation period. Oct3/4 mRNA expression in the EG was significantly higher than that of the CG at day 7. Conclusions: The current experimental model demonstrated the promotion of the survival of DPSCs and their differentiation into odontoblast-like cells (OBLCs). Thus, the use of this model is expected to clarify the crosstalk mechanism between immune cells, including macrophages and dendritic cells, and DPSCs with regards to pulpal healing after tooth replantation. It also provides insight into the differentiation process of DPSCs into OBLCs.

Medication-related osteonecrosis of the jaw in a patient with multiple myeloma: an unusual case with tumor in the surgical specimen.

Multiple myeloma is characterized by a neoplastic proliferation of plasma cells primarily in the bone marrow. Neoplastic plasma cells stimulated osteoclasts, and destroy bone tissue, causing bone pain, pathological fractures, paralysis due to spinal cord compression, and hypercalcemia. Bisphosphonates are used as supportive therapy in the management of multiple myeloma. Medication-related osteonecrosis of the jaw (MRONJ) is a well-known complication of treatment with bisphosphonates, denosumab, and other drugs, such as anti-angiogenic agents and novel anti-cancer drugs. We report MRONJ in a patient with multiple myeloma, especially an unusual case with tumor in the surgical specimen. A 73-year-old woman presented with pain on the left side of the mandible within 3 months. On clinical examination, an exposed bone without purulent drainage presented on the left side of the mandible. Before 2 years, she received chemotherapy of zoledronate for multiple myeloma at another hospital. Panoramic imaging showed radiopacities of bone in the left side of the mandibular molar area. Multidetector computed tomography (MDCT) with axial, multiplanar reformation (MPR) and three-dimensional (3D) images showed the sequestrum without periosteal reaction. She was diagnosed as MRONJ, and underwent surgery. Finally, the surgical specimen was diagnosed as multiple myeloma in the sequestrum. This case suggests that the evaluation of the surgical specimen of MRONJ could be essential for detection of primary tumor.

[Insanity and suicide, as described in the "Okuni-nikki (Diaries of our clan)" of the Hirosaki Clan Government].

The "Okuni-nikki (Diaries of our Clan)" of the Hirosaki Clan Government covers the years 1661-1867. Akitomo Matsuki and Yoichi Hanada picked up medical topics from these diaries, and edited them in two volumes of "Tsugaru-ijibunka-shiryoshusei: Okuni-nikki" (1993, 1994). I selected cases of insanity and suicide from these volumes. Selected were (1) 12 cases of insanity (in its wider sense) (5 cases of insanity, 2 cases of depression, a case of mania, a case of hypochondriasis, a case of epilepsy, a case of pavor nocturnus, and a case of insomnia), (2) 16 cases of suicide and self injury, cased by insanity (in its wider sense) (11 cases of suicide, a case of suicide and injuring another person, a case of attempted suicide, 2 cases of lovers' attempted suicide, and a case of self injury; 8 cases of them were caused by insanity), (3) 3 cases of killing and injuring others, caused by insanity (a case of killing another person, and 2 cases of injuring others), (4) a drunk case of injuring another person, and (5) 13 cases of suicide and self injury, without description of insanity (9 cases of suicide, 2 cases of attempted suicide, a case of lovers' suicide, and a case of penis cutting off). There were no described cases of possession or alcoholism. In the Edo Era, in several provinces and clans had legal procedure to confine insane patients. As for the Hirosaki Clan, I could not find clues to suggest the existence of such legal or customary procedures.

Pathogenesis of taste impairment and salivary dysfunction in COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is a highly transmissible pandemic disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The characteristics of the disease include a broad range of symptoms from mild to serious to death, with mild pneumonia to acute respiratory distress syndrome and complications in extrapulmonary organs. Taste impairment and salivary dysfunction are common early symptoms in COVID-19 patients. The mouth is a significant entry route for SARS-COV-2, similar to the nose and eyes. The cells of the oral epithelium, taste buds, and minor and major salivary glands express cell entry factors for SARS-COV-2, such as ACE2, TMPRSS2, and Furin. We describe the occurrence of taste impairment and salivary dysfunction in COVID-19 patients and show immunohistochemical findings regarding the cell entry factors in the oral tissue. We review and describe the pathogeneses of taste impairment and salivary dysfunction. Treatment for the oral disease is also described. Recently, it was reported that some people experience persistent and prolonged taste impairment and salivary dysfunction, described as post-COVID-19 syndrome or long COVID-19, after the acute illness of the infection has healed. To resolve these problems, it is important to understand the pathogenesis of oral complications. Recently, important advances have been reported in the understanding of gustatory impairment and salivary dysfunction. Although some progress has been made, considerable effort is still required for in-depth elucidation of the pathogenesis.

CBCT imaging and histopathological characteristics of osteoradionecrosis and medication-related osteonecrosis of the jaw.

PURPOSE: The purpose of this study was to evaluate the cone-beam computed tomographic (CBCT) imaging and histopathological characteristics of osteoradionecrosis (ORN) and medication-related osteonecrosis of the jaw (MRONJ). MATERIALS AND METHODS: Ten surgical specimens from segmental mandibulectomy (3 ORN and 7 MRONJ) were analyzed using CBCT. The CBCT parameters were as follows: high-resolution mode (tube voltage, 90.0 kV; tube current, 4.00 mA; rotation time, 16.8 s; field of view, 56 mm×56 mm; thickness, 0.099 mm). Histopathological characteristics were evaluated using histological slides of the surgical specimens. The Pearson chi-square test was used to compare ORN and MRONJ in terms of CBCT findings (internal texture, sequestrum, periosteal reaction and cortical perforation) and histopathological characteristics (necrotic bone, inflammatory cells, reactive bone formation, bacteria, Actinomyces, and osteoclasts). A P value less than 0.05 was considered to indicate statistical significance. RESULTS: MRONJ showed periosteal reaction on CBCT more frequently than ORN (7 of 7 [100%] vs. 0 of 3 [0%], P<0.05). Regarding histopathological characteristics, MRONJ showed osteoclasts more frequently than ORN (6 of 7 [85.7%] vs. 0 of 3 [0%], P<0.05). CONCLUSION: This study evaluated the CBCT imaging and histopathological characteristics of ORN and MRONJ, and the findings suggest that CBCT could be useful for the evaluation of ORN and MRONJ.

Relationships of cervical lymph node metastasis to histopathological malignancy grade, tumor angiogenesis, and lymphatic invasion in tongue cancer.

Cervical lymph node (CLN) metastasis from oral cancer correlates with poor prognosis. Therefore, accurate assessment of CLN status is crucial in treatment planning. However, there are few reports focusing on CLN metastasis from tongue cancer. Further, the growth and progress of the tumor are known to be profoundly related to histological malignancy, tumor angiogenesis, and lymphangiogenesis. Thus, this study aimed to identify predictive factors for CLN metastasis in tongue squamous cell carcinoma (SCC). Initial biopsy specimens obtained from 30 patients with tongue SCC were examined to evaluate histological malignancy according to Anneroth's classification. In addition, blood vessel density, lymph vessel density, and lymphatic invasion in the tumor were evaluated immunohistochemically using CD31, CD34, D2-40, and AE1/AE3, and then the relationships of CLN metastasis to these parameters were investigated. Histological malignancy grade, blood vessel density, and lymphatic invasion were significantly related to CLN metastasis (P < 0.05), but there was no relationship between lymph vessel density and CLN metastasis. However, double immunostaining showed that lymphatic invasion by tumor cells was significantly related to CLN metastasis. The results indicate that Anneroth's histological malignancy grade of 16 or more, tumor blood vessel density of more than 37, and the presence of lymphatic invasion by tumor cells can be predictive factors for CLN metastases in tongue SCC.

Experimental study of antiangiogenic gene therapy targeting VEGF in oral cancer.

It is well known that tumor angiogenesis plays an important role in local growth and metastasis of oral cancer; therefore, inhibiting angiogenesis is considered to be effective for treating oral cancer. This study aimed to investigate the effectiveness of systemically available antiangiogenic gene therapy targeting vascular endothelial growth factor (VEGF), which is one of the most important angiogenesis accelerators. We administered a soluble form of VEGF receptor-expressing gene incorporated into adenovirus (AdVEGF-ExR) intraperitoneally to nude mice to which oral cancer cell lines (SAS, HSC-3, and Ca9-22) had been transplanted subcutaneously in vivo to inhibit angiogenesis and tumor proliferation. Then, we measured tumor volumes over time, and tumors were enucleated and examined histopathologically and immunohistologically at 28 days after AdVEGF-ExR administration. Compared to the controls to which we administered AdLacZ or saline, significant antiproliferative effects were observed (P < 0.05) in the AdVEGF-ExR administration group, and extensive tumor necrosis was found histopathologically. Immunohistochemical analysis with CD34 (NU-4A1) revealed tumor angiogenesis was suppressed significantly (P < 0.05), and that with ssDNA revealed apoptosis induction was significantly high (P < 0.05) in the AdVEGF-ExR group. However, analysis with Ki-67 (MIB-1) revealed tumor proliferative capacity was not significantly different between the groups. Consequently, we consider that AdVEGF-ExR administration achieved tumor growth suppression by inhibiting angiogenesis and inducing apoptosis, but not by inhibiting the proliferative capacity of tumor cells. Neither topical administration of a soluble form of VEGF receptor (sVEGFR) to the tumor nor a megadose was needed to achieve this inhibition effect. These results suggest gene therapy via sVEGFR would be an effective oral cancer therapy and benefit future clinical applications.