安部 貴大

BACKGROUND: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and irradiation of a specific wavelength to exert cytotoxic effects. To enhance the antitumor effect against head and neck squamous cell carcinoma (HNSCC), we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin (IT) and a PS for PDT and light irradiation. In our prior study, we demonstrated that an immunotoxin (IT) consisting of an anti-ROBO1 antibody conjugated to saporin, when used in combination with the photosensitizer (PS) disulfonated aluminum phthalocyanine (AlPcS2a) and irradiated with light at the appropriate wavelength, resulted in increased cytotoxicity against head and neck squamous cell carcinoma (HNSCC) cells. ROBO1 is a receptor known to be involved in the progression of cancer. In this study, we newly investigate the iTAP targeting epidermal growth factor receptor (EGFR) which is widely used as a therapeutic target for HNSCC. METHODS: We checked the expression of EGFR in HNSCC cell lines, SAS, HO-1-u-1, Sa3, and HSQ-89. We analyzed the cytotoxicity of saporin-conjugated anti-EGFR antibody (cetuximab) (IT-Cmab), mono-L-aspartyl chlorin e6 (NPe6, talaporfin sodium), and light (664 nm) irradiation (i.e., iTAP) in SAS, HO-1-u-1, Sa3, and HSQ-89 cells. RESULTS: EGFR was expressed highly in Sa3, moderately in HO-1-u-1, SAS, and nearly not in HSQ-89. Cmab alone or IT-Cmab alone did not show cytotoxic effects in Sa3, HO-1-u-1, and HSQ-89 cells, which have moderate or low expression levels of EGFR protein. However, the iTAP method enhanced the cytotoxicity of IT-Cmab by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression. CONCLUSION: Our study is the first to report on the iTAP method using IT-Cmab and NPe6 for HNSCC. The cytotoxic effects are enhanced in cell lines with moderate levels of EGFR protein expression, but not in nonexpressing cell lines, which is expected to expand the range of therapeutic windows and potentially reduce complications.

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare autosomal dominant disorder characterized by congenital skeletal malformation and progressive heterotopic ossification. In the oral and maxillofacial region, deformity of the temporomandibular joint is a common feature of FOP, as well as restricted mouth opening derived from heterotopic ossification in the masticatory muscles. Since surgical procedures are generally not recommended because of the risk of flare-ups and increased heterotopic ossification, reports of tooth extractions and their outcomes in patients with FOP are limited. The present article reports the long-term oral outcomes of three Japanese patients with FOP, in whom the teeth were deliberately extracted to avoid the risk of oral inflammation causing further heterotopic ossification. The extractions were conducted under local or general anesthesia, and healing of sockets was nonproblematic with the formation of new bone. Undesirable events, including progression of heterotopic ossification in the oral and maxillofacial region and further restriction of mouth opening, were not apparent. The extractions also alleviated the existing inflammation, contributing to maintaining their oral hygiene. These cases suggest that deliberate planning and judicious surgery could induce favorable healing after tooth extractions in patients with FOP, leading to long-term stability of their oral health status.

BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of cancer worldwide. Our study focused on the axon guidance receptor roundabout guidance receptor 1 (ROBO1) as a target for monoclonal antibody therapy of HNSCC. We previously showed that saporin-conjugated anti-ROBO1 (B5209B) immunotoxin (IT-ROBO1) enhanced cytotoxic effects on HNSCC cells in combination with the photosensitizer aluminum phthalocyanine disulphonate (AlPcS2a) and illumination. We examined the effects of this combination therapy in a mouse xenograft model. MATERIALS AND METHODS: IT-ROBO1 was intraperitoneally administered to HSQ-89 (derived from Japanese maxillary sinus squamous carcinoma, RCB0789; RIKEN, Tsukuba, Japan) xenografted mice. After 3 days, AlPcS2a was injected subcutaneously around the tumor and the area was illuminated at 650 nm for 30 min. The growth of the tumor was evaluated and the effects on the tumor were examined. RESULTS: Pronounced anti-tumor effects were elicited by the administration of IT-ROBO1 and AlPcS2a with light illumination on tumor size and pathological characteristics. CONCLUSION: The results showed that photosensitizer treatment with illumination robustly enhanced the antitumor effect of the IT-ROBO1 immunotoxin.

Tissue reactions after transplantation can affect the maturation and prognosis of the transplanted engineered tissue in regenerative medicine. Since macrophages are broadly subdivided into two major phenotypes, inflammatory (M1) and anti-inflammatory/wound healing (M2), in this study, we examined the properties of macrophages in transplantation of tissue-engineered cartilage, to clarify their effects on cartilage maturation. Human chondrocytes were embedded in a poly-L-lactic acid scaffold, which was transplanted subcutaneously on the back in athymic mice. When the constructs were analyzed by real-time polymerase chain reaction, interleukin 1 expression was detectable at 4 days, and it reached a peak at 7 days. Interleukin 6 expression was increased at 7 to 11 days, suggesting that M1 macrophages were abundant around this time. On the other hand, expression of markers for M2 macrophages occurred rather later, with Fizz and Ym1 expression peaking at around 11 to 14 days, possibly indicating that polarization of macrophages in tissue-engineered cartilage could shift from M1 to M2 around 11 days after transplantation. When cultured by using the conditioned medium of M2 macrophages, chondrocytes showed significantly increased expression of type 2 collagen, suggesting that M2 macrophages could enhance the maturation of tissue-engineered cartilage. Also, by partially depleting macrophages with clodronate liposomes in the initial period, during which M1 macrophages were dominant, more cartilage matrix accumulated in transplanted constructs at 2 weeks. It was suggested that polarization of macrophages shifted from M1 to M2 in the transplantation of tissue-engineered cartilage, and controlling the polarization could be advantageous for the maturation of transplanted engineered tissues. Impact statement In transplantation of engineered tissues, it is imperative for immune reactions to proceed in a proper and timely manner. In this study, we transplanted tissue-engineered cartilage consisting of a biodegradable polymer scaffold and chondrocytes, and examined the properties of macrophages. It was shown that the polarization of macrophages shifted from inflammatory (M1) to anti-inflammatory/wound healing (M2) around 11 days after transplantation. Partial suppression of macrophages at the early stage of transplantation, which were mainly M1 macrophages, promoted more accumulation of cartilage matrix. This study indicates a possible approach to facilitate cartilage maturation by intervening in the polarity of macrophages.

To obtain stable outcomes in regenerative medicine, the quality of cells for transplantation is of great importance. Cellular stress potentially results in the release of damage-associated molecular patterns (DAMPs) and activates immunological responses, affecting the outcome of transplanted tissue. In this study, we intentionally prepared necrotic chondrocytes that would gradually die and release DAMPs and investigated how the maturation of tissue-engineered cartilage was affected. Necrotic chondrocytes were prepared by a conventional heat-treatment method, by which their viability started to decrease after 24 h. When tissue-engineered cartilage containing necrotic chondrocytes was subcutaneously transplanted into C57BL/6J mice, accumulation of cartilage matrix was decreased compared to the control. Meanwhile, immunohistochemical staining demonstrated that localization of macrophages and neutrophils was more apparent in the constructs of necrotic chondrocytes, suggesting that DAMPs from necrotic chondrocytes could prompt migration of more immune cells. Two-dimensional electrophoresis and mass spectrometry identified prelamin as a significant biomolecule released from necrotic chondrocytes. Also, when prelamin was added to a culture of RAW264, Inos and Il1b were increased in accordance with the content of added prelamin. It was suggested that DAMPs from dying chondrocytes could induce inflammatory properties in surrounding macrophages, impairing the maturation of tissue-engineered cartilage. In conclusion, maturation of tissue-engineered cartilage was hampered when less viable chondrocytes releasing DAMPs were included.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide. The standard treatment of surgery, chemotherapy, and radiotherapy can result in long-term complications which lower the patient's quality of life, such as eating disorders, speech problems, and disfiguring or otherwise untoward cosmetic issues. Antibody therapy against cancer-specific antigens is advantageous in terms of its lesser side effects achieved by its greater specificity, though the antitumor activity is still usually not enough to obtain a complete cure. Robo1, an axon guidance receptor, has received considerable attention as a possible drug target in various cancers. We have shown previously the enhanced cytotoxic effects of saporin-conjugated anti-Robo1 immunotoxin (IT-Robo1) on the HNSCC cell line HSQ-89 in combination with a photochemical internalization technique. Considering the light source, which has only limited tissue penetrance, we examined the drug internalization effect of saponin. Treatment with saponin facilitated significant cytotoxic effects of IT-Robo1 on HSQ-89 cells. Saponin exerts its own nonspecific cytotoxicity, which may cover the actual extent of the internalization effect. We thus examined whether a flashed treatment with saponin exerted a significant specific cytotoxic effect on cancer cells. The combination of an immunotoxin with saponin also exhibited a significant tumor-suppressive effect on mice HSQ-19 xenografts. These results suggest the utility of saponin treatment as an enhancer of immunotoxin treatment in cancer.

Introduction: The central regulatory system that generates biological rhythms is regulated by circadian clock genes expressed by cells in the suprachiasmatic nucleus. Signals from this system are converted to adrenocortical hormones through the sympathetic nervous system and transmitted to peripheral organs. Another system releases glucocorticoids (GCs) in response to stress through the HPA-axis. Here we investigated the second messenger GC, which is shared by these systems and influences the expression of circadian clock genes of cells of the musculoskeletal system and in viable bone tissue. Methods: We used mouse-derived cell lines, which differentiate into osteoblasts (MC3T3-E1, C2C12, and 10T1/2) as well as primary cultures of mouse osteoblasts to determine the expression levels of circadian clock genes that respond to GC. Mice (mPer2m/m) with an inactivating mutation in the period circadian clock 2 gene (Per2) exhibit marked dysrhythmia. Here we compared the bone morphologies of mPer2m/m mice with those of wild-type (WT) mice. Results: The expression of major circadian clock genes was detected in each cell line, and their responsiveness to GC was confirmed. We focused on Per2, a negative regulator of the circadian clock and found that a Per2-loss-of-function mutation increased the proliferative capacity of osteoblasts. Treatment of mutant mice with slow-release GC and bisphosphonate affected the maturation of bone tissue, which reflects a tendency to retard calcification. Conclusion: Our investigations of the mechanisms that regulate circadian rhythm function in tissues of the musculoskeletal system that respond to the stress hormone GC, reveal that Per2 is required for the maturation of bone tissue. Thus, the influences of the systems that control circadian rhythms and the responses to stress by regenerating tissue used for regenerative medicine must be considered and studied in greater detail.

BACKGROUND: Cancers of unknown primary origin (CUPs) are reported to be the 3-4th most common causes of cancer death. Recent years have seen advances in mutational analysis and genomics profiling. These advances could improve accuracy of diagnosis of CUPs and might improve the prognosis of patients with CUPs. CASE PRESENTATION: A 76-year old male with an adenocarcinoma of unknown primary origin in the lung presented with another tumor of the palate mucosa. The tumor cells in the pleural effusion were all negative for immunohistochemical markers (TTF-1 and Napsin A) and lung-specific oncogenic driver alterations (EGFR mutation and ALK translocation). The tumor of the palate mucosa was likewise identified as an adenocarcinoma, and the cells showed cytological similarities with the tumor cells in the pleural effusion; TTF-1, Napsin A, EGFR mutation and ALK translocation were all negative. This result suggested that origins of the tumors of the palate mucosa and in the lung were the same, even though the origin had not yet been determined. Next, we addressed whether the tumor of the palate mucosa was a primary tumor or not. Secretory carcinoma (SC), which is a common type of minor salivary gland tumor (MSGT), was suspected; however, mammaglobin was negative and ETV6-NTRK3 (EN) fusion was not observed. Other MSGTs were excluded based on histological and immunohistochemical findings. Furthermore, an additional examination demonstrated an oncogenic KRAS mutation at codon 12 (p.G12D) in both palate tumor and in pleural effusion. KRAS mutation is known to exist in one-third of lung adenocarcinomas (LUADs), but quite rare in MSGTs. The possibility of metastasis from other organs was considered unlikely from the results of endoscopic and imaging studies. This result indicated that the primary site of the CUP was indeed the lung, and that the tumor of the palate mucosa was a metastasis of the LUAD. CONCLUSIONS: A tumor of the palate mucosa that showed diagnostic difficulties was determined to be a metastatic LUAD by genomic alterations and histopathological findings.

The authors aimed to assess the factors that impair cell proliferation in the granulation tissue of pressure ulcers using immunohistochemistry for the cell proliferation marker Ki-67. This was a single center, cross-sectional study. The study included 86 patients with stage III or IV pressure ulcers. Two granulation tissue biopsy specimens were obtained from 86 patients. The specimens were used for histological examination, Ki-67 immunohistochemistry, and bacterial count assessment. The % of Ki-67-stained cells was considered as the Ki-67 index. Pearson's product-moment correlation coefficient (r) was used to assess the relationship between the Ki-67 index and other quantitative variables, including age, body mass index, bacterial count (Log10 CFU/g), serum albumin level, hemoglobin level, white blood cell count, and C-reactive protein level. The Mann-Whitney U test was used to compare the mean Ki-67 index according to gender, diabetes, smoking status, and wound culture. Univariate and multivariate linear regression analyses were used to assess the association between the Ki-67 index and other parameters. The Mann-Whitney U test revealed that the bacteria-positive group had a lower Ki-67 index (p = 0.045). Bacterial count demonstrated a significant negative correlation with the Ki-67 index (r = -0.325, p = 0.002). Multivariate linear regression analysis showed that bacterial count was a significant predictor of the Ki-67 index. The adjusted β-coefficient was -1.34 (95% confidence interval, -2.01 to -0.66, p < 0.001). Among the isolated bacteria, Corynebacterium spp. and Staphylococcus aureus were significantly associated with a low Ki-67 index, but Pseudomonas aeruginosa was not. These results suggest a negative relationship between bacterial count and cell proliferation in pressure ulcer granulation tissue, as indicated by the Ki-67 index. Granulation tissue formation in pressure ulcers may be accelerated if high bacterial load is treated appropriately.

BACKGROUND: Infections in the oral and maxillofacial region can sometimes extend beyond the oral cavity, with serious consequences. Most oral infections are odontogenic, occurring through the root apex of the tooth or the periodontal pocket. It thus makes sense that edentulous patients have a much lower risk of oral bacterial infection. For this reason, while there are many reports on systemic infections caused by oral infections, few of these describe such infections in edentulous patients. CASE PRESENTATION: We present a case of oral and maxillofacial cellulitis followed by sepsis due to Streptococcus pyogenes infection in an 89-year-old Japanese edentulous woman. S. pyogenes was detected in the wound of left maxilla and the blood sample. S. pyogenes has been reported to be one of the most common and influential aerobic bacteria associated with deep neck infection and subsequent systemic infection. Left maxillary sinusitis was observed, and this could be the origin of the S. pyogenes infection. S. pyogenes derived from the sinusitis and leaked to the oral cavity might have caused systemic infection through wounding of the oral mucosa. Fortunately, intensive antibiotic therapy was effective, and the patient recovered without any surgical procedures. CONCLUSIONS: We experienced a rare case of oral and maxillofacial cellulitis followed by sepsis due to a Streptococcus pyogenes infection in an old edentulous woman. This result indicated that, while edentulous patients are considered to have no risk of odontogenic infection, they still carry a risk of bacterial infection.

Odontogenic infection in immunocompromised patients tends to extend systemically beyond the oral cavity. Our case report presents a patient with sepsis due to a Streptococcus constellatus (S. constellatus) odontogenic infection in a 64-year-old-immunocompromised woman with Cogan's syndrome. She had been suffering from chronic mandibular osteomyelitis which was thought to have been caused by dental caries and/or chronic periodontitis with furcation involvement of the left mandibular first molar. We suspect that the acute symptoms of the chronic osteomyelitis due to S. constellatus led to the systemic infection. This infection could be accelerated by the use of a corticosteroid and an alendronate. This is the first report which represents the potential association between odontogenic infection and Cogan's syndrome.

Many studies on the cholinergic pathway have indicated that cholinergic receptors, which are widely expressed in various cells, play an important role in all body organs. In this review, we present the concept that cholinergic responses are regulated through a neuronal or non-neuronal mechanism. The neuronal mechanism is a system in which acetylcholine binds to cholinergic receptors on target cells through the nerves. In the non-neuronal mechanism, acetylcholine, produced by neighboring cells in an autocrine/paracrine manner, binds to cholinergic receptors on target cells. Both mechanisms subsequently lead to physiological and pathophysiological responses. We also investigated the subunits/subtypes of cholinergic receptors on target cells, physiological and pathophysiological responses of the organs via cholinergic receptors, and extracellular factors that alter the subtypes/subunits of cholinergic receptors. Collectively, this concept will elucidate how cholinergic responses occur and will help us conduct further experiments to develop new therapeutic agents.