Background: Oral mucositis is the most common adverse event seen in chemotherapy patients. While it is widely accepted that salivary leucocytes contribute to maintain oral mucosal health, their association with oral mucositis occurred in patients during chemotherapy is unknown.

Aim: As a preliminary study, we examined the reliability and applicability of salivary leukocyte assays with a commercially available testing device （SillHa, Arkrey, Japan） in healthy volunteers. In subsequent clinical observational study using the testing device, we aimed primarily to elucidate the association of the occurrence of oral mucositis with changes in leukocyte levels of saliva and peripheral blood in chemotherapy patients.

Methods: SillHa （Arkrey, Japan） was used as a commercially available testing device. Leukocyte levels in saliva samples from 50 healthy adult volunteers were determined both with SillHa and CD45 positive cell counts by flow cytometry. For subsequent clinical longitudinal study with the occurrence of oral mucositis as the primary outcome, thirty-one patients undergoing chemotherapy at Iwate Medical University Hospital participated in the study after providing informed consent for use of the findings in an anonymous manner. Data regarding attributes, lifestyle factors, and treatments received for the primary disease were obtained from medical records. Each underwent an oral examination before starting chemotherapy （baseline）. Thereafter, leukocyte levels in saliva and peripheral blood were determined approximately every two days. This study followed the Declaration of Helsinki and STROBE statement for reports of observational studies.

Results: The correlation coefficient between measurements by SillHa and flow cytometry showed high value （r=0.818, p<0.001）. In the patients undergoing chemotherapy, oral mucositis occurred in 41.9% participants during the observation periods. Among the data analyzed in this study, the strongest relationship with occurrence of oral mucositis was found to be number of days from lowest recorded salivary leucocyte count to recovery to more than 50% of the baseline value. In ROC analysis, the area under the curve was 0.771, indicating a high capacity to discriminate between patients with and without occurrence of oral mucositis.

Conclusion: The present findings indicate that changes in salivary leukocyte levels measured with a commercially testing device is useful to predict occurrence of oral mucositis in patients receiving chemotherapy.

The immunomodulatory ability of mesenchymal stem cells（ MSCs） is synergistically regulated through cell contact-dependent mechanisms and secretion of soluble factors. TGF-β1 plays an important role in immunosuppression. In this study, the expression of TGF-β1-induced inflammation-related cytokines, chemokines, and growth factors in the human MSC line, β1 stimulation. TGF-β1-induced expression of IL-6 was attenuated by a TGF-β receptor inhibitor and an MEK inhibitor. These results indicate that TGF-β1 increases IL-6 expression via the MEK pathway in human MSCs. Considering that TGF-β1 increased the expression levels of both NGF and IL-6 in the MSCs, we further investigated the effect of the cytokines secreted from UE7T-13 cells on neurite extension of neuronal PC12 cells. We found that the neurite extension in PC12 cells was significantly enhanced in the conditioned medium derived from TGF-β1-pretreated UE7T-13 cells. In addition, it was significantly enhanced by the indirect co-culture of PC12 cells and TGF-β1-stimulated UE7T13 cells under transwell conditions. Interestingly, these enhancements of neurite extension mediated using the conditioned medium or indirect co-culture were negated by the addition of a neutralizing antibody against the soluble IL-6 receptor in the culture media. We also confirmed that the administration of both IL-6 and soluble IL-6 receptors to PC12 cells did not promote these neurite extensions. Overall, these results suggest that NGF secreted from TGF-β1-stimulated MSCs induces neuronal differentiation of PC12 cells, which is further enhanced by IL-6 secreted from MSCs.

Squamous cell carcinoma （SCC） is the most common cancer in the oral cavity. Epithelial–mesenchymal transition （EMT） plays an important role in SCC cell metastasis process. We have previously demonstrated that transforming growth factor-β1 （TGF-β1） induces EMT in HSC-4, a human oral SCC （hOSCC） cell line. However, the molecular mechanisms of metastasis after EMTpromoted cancer are poorly understood. On the other hand, tumor-associated macrophages （TAMs）that coexist with cancer tissue participate in permeation metastasis. However, whether TAMs affect hOSCC cell metastasis in the tumor microenvironment （TME） remains uncertain. Here, we investigated the expression status of inflammatory and anti-inflammatory cytokines and M2 polarization status in macrophages （Mφs） cocultured with EMT-promoted HSC-4 cells. Under coculture of EMT-promoted HSC-4 cells and M1-Mφs, we found that the expression of tumor necrosis factor-α （TNF-α）, an M1-polarization inducer, was clearly suppressed in M1-Mφ, while the expression of M2-polarization promoter TGF-β1 and M2-Mφ marker CD163 was significantly reinforced. These results indicate that M1-Mφ polarization into M2-Mφ was promoted by coculture with EMT-promoted HSC-4 cells. In the next step, we tried to identify the factors derived from EMTpromoted HSC-4 cells that promoted M2-Mφ polarization. We found that 1） TGF-β1 induces sclerostin expression through the Smad signal transduction pathway in HSC-4 cells, 2） sclerostin reinforces interleukin-6 （IL-6）, TGF-β1, and IL-17A expression in M1-Mφs, 3） IL-6 promotes IL-17A expression in M1-Mφs and M1-Mφ polarization into M2-Mφ, and 4） IL-17A reinforces M1-Mφ polarization into M2-Mφ. These results strongly suggest that the EMT-promoted hOSCC cells retained the ability to promote polar changes from M1-Mφs to M2-Mφs through sclerostin production in the TME.

Child abuse is a serious problem worldwide, and saving children from abuse is an important mission. Child abuse-induced psychological stress triggers diseases such as adjustment disorders and depression. Therefore, there is a strong demand for the practical application of biomarkers that can objectively identify child abuse. However, no objective index or quantitative method for abuse or neglect-induced mental stress has been established. In this study, we compared the expression levels of saliva-derived microRNAs（ miRNAs） in abused children with those in nonabused children and assessed their effectiveness as biomarkers for child abuse. Salivary miRNAs were extracted from 52 non-abused children（ CG） and 52 children stressed by abuse（SG ）. The expression levels were compared using microarray and quantitative reverse transcription-polymerase chain reaction（ RT-PCR）. In primary screening, several miRNAs were found to be expressed more than double in SG when compared with CG. In the quantitative RT-PCR analysis, the expression levels of hsa-miRNA-223-5p, hsa-miRNA-181a-5p, and hsa-miRNA-30b-5p in SG were significantly higher than those in CG. Receiver operating characteristic curve showed that three types of miRNAs possessed excellent sensitivity and specificity. Two miRNAs, miRNA 223-5p and miRNA 30b-5p, were considered to be desirable biomarkers because of their extremely high expression levels. We speculate that the expression levels of hsa-miRNA-223-5p and hsa-miR-30b-5p are upregulated during child abuse and hence can be used as biomarkers to determine child abuse-related psychological stress.

This case accumulation study enrolled the subset of ten patients with pathological mandibular fractures diagnosed in our clinic during the period from January 2015 to December 2020. The objective of this study is to evaluate the clinical conditions for pathological mandibular fractures. In 5 years, 10 cases were diagnosed with pathological mandibular fractures. In these 10 cases, the primary diseases were malignant tumor in 5 cases, radiation osteomyelitis in 2 cases, cyst in 2 cases and benign tumor in 1 case, In 5 of the malignant tumors, 1 had pathological fractures from the first visit, 2 experienced fractures during surgery, and 2 fractures occurred after surgery. The two cases of radiation osteomyelitis received radiotherapy of over 40 Gy. The two cases of cysts were identified as being dental cysts of the wisdom teeth, and fractures were observed during the surgery. In the case with the benign tumor, the maxillary bone defect was large after the first operation; thus, the patient waited for second-stage reconstruction. Although this study enrolled small number of cases, conservative treatment failed for patients with occlusal deviation. Therefore, careful consideration of surgical treatment is necessary for these cases.