Personal immune profiles: Diversity and prognostic value for oral tongue squamous cell carcinoma evaluated by comprehensive immune parameter analyses with multiplex immunofluorescence.

OBJECTIVES: Understanding the tumor immune microenvironment is becoming increasingly necessary for risk prediction and treatment selection. In particular, oral cancer has various immunosuppressive characteristics in the tumor microenvironment. Therefore, we comprehensively assessed the immune profiles of oral tongue squamous cell carcinoma (OTSCC). MATERIALS AND METHODS: Multiplex immunofluorescence and tissue imaging analyses were performed to evaluate immune profiles at the invasive tumor front of 60 OTSCC surgical specimens. We analyzed 58 immune parameters including the density and proportion (%) of total leukocytes (Leu) and T cells, six subsets of T and myeloid cells, and the expression of programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1). RESULTS: The density, proportion, and location of CD45+ Leu, three T cell subsets (CD8+, Foxp3-CD4+ conventional, and Foxp3+CD4+ regulatory T cells), CD163-CD68+ M1 and CD163+CD68+ M2 macrophages, and neutrophils were highly variable at the individual level. The density and proportion of M2 macrophages were significantly lower in the T1 stage group. Risk prediction analyses for recurrence and/or metastasis (R/M) showed that R/M (+) T1 cases had significantly higher M2 density and percentages. CONCLUSIONS: The immune profiles of OTSCC patients are diverse and cannot be predicted from clinicopathological information alone. The M2 macrophage abundance is a potential candidate biomarker for R/M in the early stage of OTSCC. Personal immune profiling may provide beneficial information for risk prediction and treatment selection.

A Novel, Tumor-Induced Osteoclastogenesis Pathway Insensitive to Denosumab but Interfered by Cannabidiol.

Bone metabolism is strictly regulated, and impaired regulation caused by hormonal imbalances induces systemic bone loss. Local bone loss caused by tumor invasion into bone is suggested to be induced by the generation of cytokines, which affect bone metabolism, by tumor cells. The major cause of systemic and local bone losses is excess bone resorption by osteoclasts, which differentiate from macrophages by receptor activator of nuclear factor kappa-B ligand (RANKL) or tumor necrosis factor-alpha (TNF-α). We previously found a novel pathway for tumor-induced osteoclastogenesis targeting osteoclast precursor cells (OPCs). Tumor-induced osteoclastogenesis was resistant to RANKL and TNF-α inhibitors. In the present study, we confirmed that exosomes derived from oral squamous cell carcinoma (OSCC) cells induced osteoclasts from OPCs. We also showed that the depletion of exosomes from culture supernatants of OSCC cells partially interfered with osteoclastogenesis, and cannabidiol, an innoxious cannabinoid without psychotropic effects, almost completely suppressed tumor-induced osteoclastogenesis. Osteoclastogenesis and its interference by cannabidiol were independent of the expression of nuclear factor of T cell c1 (NFATc1). These results show that osteoclastogenesis induced by OSCC cells targeting OPCs is a novel osteoclastogenic pathway independent of NFATc1 expression that is partially caused by tumor-derived exosomes and suppressed by cannabidiol.

Inflammasome Activation Induced by Perfringolysin O of Clostridium perfringens and Its Involvement in the Progression of Gas Gangrene.

Clostridium perfringens (C. perfringens) is Gram-positive anaerobic, spore-forming rod-shaped bacterial pathogen that is widely distributed in nature. This bacterium is known as the causative agent of a foodborne illness and of gas gangrene. While the major virulence factors are the α-toxin and perfringolysin O (PFO) produced by type A strains of C. perfringens, the precise mechanisms of how these toxins induce the development of gas gangrene are still not well understood. In this study, we analyzed the host responses to these toxins, including inflammasome activation, using mouse bone marrow-derived macrophages (BMDMs). Our results demonstrated, for the first time, that C. perfringens triggers the activation of caspase-1 and release of IL-1ß through PFO-mediated inflammasome activation via a receptor of the Nod-like receptor (NLR) family, pyrin-domain containing 3 protein (NLRP3). The PFO-mediated inflammasome activation was not induced in the cultured myocytes. We further analyzed the functional roles of the toxins in inducing myonecrosis in a mouse model of gas gangrene. Although the myonecrosis was found to be largely dependent on the α-toxin, PFO also induced myonecrosis to a lesser extent, again through the mediation of NLRP3. These results suggest that C. perfringens triggers inflammatory responses via PFO-mediated inflammasome activation via NLRP3, and that this axis contributes in part to the progression of gas gangrene. Our findings provide a novel insight into the molecular mechanisms underlying the pathogenesis of gas gangrene caused by C. perfringens.

Progressive Encephalomyelitis with Rigidity and Myoclonus Resolving after Thymectomy with Subsequent Anasarca: An Autopsy Case.

Progressive encephalomyelitis with rigidity and myoclonus (PERM) is an autoimmune disorder involving the brainstem and spinal cord and is sometimes associated with thymoma. We encountered a 75-year-old woman with typical PERM features, glycine receptor antibody, and thymoma. Her neurologic symptoms improved after thymectomy, but she unexpectedly developed anasarca with massive pleural effusions and hypoalbuminemia and finally succumbed to death. The autopsy showed edema and mononuclear infiltration in the pleura but no neuropathological findings typical of PERM. Effective treatment of PERM can reverse the neuropathological signs of encephalomyelitis. The autoimmune nature of anasarca is possible but not proven.

Establishment of a xenograft model to explore the mechanism of bone destruction by human oral cancers and its application to analysis of role of RANKL.

BACKGROUND: The molecular mechanism underlying bone invasion caused by oral squamous cell carcinoma (OSCC) is not well understood. To elucidate the molecular mechanism, the development of more suitable xenograft models mimicking human mandibular bone destruction by OSCC has been required. MATERIALS AND METHODS: Human OSCC cell lines, HSC3, HSC3-C1, and HSC3-R2, were injected in the periosteal region of the mandible in athymic mice, and the bone destruction was analyzed. Receptor activators of nuclear factor κ-B ligand (RANKL) mRNA and protein expression levels were measured in the OSCC cell lines. Antibody that specifically neutralizes mouse RANKL and human RANKL, respectively, was injected into HSC3-cell-transplanted mice. RESULTS: Transplantation of HSC3 cells induced mandibular bone destruction. Histological examination revealed numerous osteoclasts on the bone destruction surface. Fibroblastic cell intervention between the cancer nests and resorbing bone surface was observed in a similar fashion to those observed in human OSCC cases. The number of osteoclasts and fibroblasts was significantly correlated. Bone destruction induced by the transplantation of HSC3 cells was reduced by injection of an antibody that specifically neutralizes mouse RANKL. Transplantation of HSC3-R2 cells, which overexpresses RANKL, induced advanced bone destruction compared to that of HSC3-C1 cells, which only overexpress the empty vector. CONCLUSIONS: We established a useful xenograft model for investigating the molecular mechanism underlying the bone destruction induced by OSCC in the jaw. This model will be used to investigate the precise roles of several cytokines synthesized by both cancer cells and fibroblastic cells in OSCC-associated bone destruction in the jaw.

Spatiotemporal disorder in the axial skeleton development of the Mesp2-null mouse: a model of spondylocostal dysostosis and spondylothoracic dysostosis.

Spondylocostal dysostosis (SCDO) is a genetic disorder characterized by severe malformation of the axial skeleton. Mesp2 encodes a basic helix-loop-helix type transcription factor that is required for somite formation. Its human homologue, Mesp2, is a gene affected in patients with SCDO and a related vertebral disorder, spondylothoracic dysostosis (STDO). This work investigated how the loss of Mesp2 affects axial skeleton development and causes the clinical features of SCDO and STDO. We first confirmed, by three-dimensional computed tomography scanning, that Mesp2-null mice exhibited mineralized tissue patterning resembling the radiological features of SCDO and STDO. Histological observations and in situ hybridization probing for extracellular matrix molecules demonstrated that the developing vertebral bodies in Mesp2-null mice were extensively fused with rare insertions of intervertebral tissue. Unexpectedly, the intervertebral tissues were mostly fused longitudinally in the vertebral column, instead of exhibiting extended formation, as was expected based on the caudalized properties of Mesp2-null somite derivatives. Furthermore, the differentiation of vertebral body chondrocytes in Mesp2-null mice was spatially disordered and largely delayed, with an increased cell proliferation rate. The quantitative three-dimensional immunofluorescence image analyses of phospho-Smad2 and -Smad1/5/8 revealed that these chondrogenic phenotypes were associated with spatially disordered inputs of TGF-ß and BMP signaling in the Mesp2-null chondrocytes, and also demonstrated an amorphous arrangement of cells with distinct properties. Furthermore, a significant delay in ossification in Mesp2-null vertebrae was observed by peripheral quantitative computed tomography. The current observations of the spatiotemporal disorder of vertebral organogenesis in the Mesp2-null mice provide further insight into the pathogenesis of SCDO and STDO, and the physiological development of the axial skeleton.

Ectopic production of hair keratin constitutes Rushton's hyaline bodies in association with hematogenous deposits.

A Rushton's hyaline body (HB) is a concretion occasionally found in odontogenic cysts. Unspecified substances produced by the lining epithelium or derived from blood components have been suggested as possible causes of HB formation, but the origin of HBs is still elusive. This study aimed to clarify the origin of HBs. Ten specimens with HBs were obtained from 400 odontogenic cysts. HBs were stained by orcein and Congo red. Immunohistochemical examination revealed that HBs were positive for hair keratin and keratin 17. Hair keratin was concentrated in HBs, and cells with hair keratin expression were hardly seen, while cells with keratin 17 expression were observed near HBs. HBs were also positive for hemoglobin alpha chain. The presence of hair keratin in HBs was confirmed by Western blot analysis. The present study suggests that HBs are formed as a consequence of two independent events: unusual alteration of epithelial differentiation so as to provide hair keratin, and hemorrhage so as to provide erythrocytic substances. Although the ectopic production of hair keratin appears more essential, our results reconcile the long-standing debate between two theories, the keratin theory versus the hematogenous theory, concluding that both substances are required for the genesis of HBs, and also suggesting that they might be novel non-pathological amyloidogenic proteins.

Comparative morphology of the osteocyte lacunocanalicular system in various vertebrates.

Osteocytes are embedded in the bone matrix, and they communicate with adjacent osteocytes, osteoblasts, and osteoclasts through the osteocyte lacunocanalicular system. Osteocytes are believed to be essential for the maintenance of bone homeostasis because they regulate mechanical sensing and mineral metabolism in mammalian bones; however, osteocyte morphology in other vertebrates has not been well documented. We conducted a comparative study on the morphology of osteocytes and the lacunocanalicular system of the following vertebrates: two teleost fishes [medaka (Oryzias latipes), and zebrafish (Danio rerio)], three amphibians [African clawed frog (Xenopus laevis), black-spotted pond frog (Rana nigromaculata), and Japanese fire-bellied newt (Cynops pyrrhogaster)], two reptiles [four-toed tortoise (Testudo horsfieldii) and green iguana (Iguana iguana)], and two mammals (laboratory mouse C57BL6 and human). The distribution of the osteocyte lacunocanalicular system in all these animals was investigated using the modified silver staining and the fluorescein-conjugated phalloidin staining methods. Bones of medaka had few osteocytes (acellular bone). Bones of zebrafish contained osteocytes (cellular bone) but had a poorly developed osteocyte lacunocanalicular system. Bones of Xenopus laevis, a freshwater species, and of other amphibians, reptiles, and mammals contained numerous osteocytes and a well-developed lacunocanalicular system. The present study indicates that development of the osteocyte lacunocanalicular system differs between teleost fishes and land vertebrates, but this pattern is not directly related to aquatic habitat.

Oral pseudotumor: benign polypoid masses following radiation therapy.

BACKGROUND: The current study evaluated postirradiation pseudotumor in the oral cavity to investigate clinical and pathologic characteristics and incidence rates. METHODS: Between 1960 and 1999, 2719 patients with squamous cell carcinoma of the oral cavity were treated with radiation therapy at Tokyo Medical and Dental University Hospital. Of these, six patients developed clinically polypoid tumors pathologically diagnosed as pseudotumors. Histopathology, immunohistology, and the clinical courses of these lesions were investigated. RESULTS: Spindled fibroblastic cells arranged in whorled patterns were common pathologic features. Immunohistochemical results showed reactivity for vimentin and smooth muscle actin and nonreactivity for cytokeratin. The latent period from the initial radiation therapy to the development of the pseudotumor ranged from 45 to 145 months (median, 79 months). The overall cumulative incidence of pseudotumor was 0.22% during the 40 year period. All six patients were cured with simple excisions. CONCLUSIONS: Oral carcinoma patients treated with radiation therapy may develop pseudotumor in a low frequency with a shorter period than that of radiation induced carcinoma/sarcoma. Pseudotumor was curable with surgery. Pseudotumor should be added to the list of differential diagnosis for postirradiation oral polypoid masses.