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Problem: What are the pregnancy outcomes after the OPtimization of Thyroid function, Immunity, and Uterine Milieu (OPTIMUM) treatment strategy in patients with repeated implantation failure (RIF)?, Method of Study: Infertile women with a history of RIF after more than three embryo transfer (ET) cycles underwent implantation testing, including a hysteroscopy, endometrial biopsy for CD138 immunostaining and bacterial culture, and serum 25-hydroxyvitamin D 3 , interferon-γ-producing helper T (Th1) cell, IL-4-producing helper T (Th2) cell, thyroid-stimulating hormone, thyroid peroxidase antibody, and thrombophilia screening between April 2017 and August 2018. We treated chronic endometritis with antibiotics, aberrant high Th1/Th2 cell ratios with vitamin D and/or tacrolimus intake, overt/subclinical hypothyroidism with levothyroxine, and thrombophilia with low-dose aspirin. Of the 116 RIF women, 88 women with 133 ET cycles were recruited from a questionnaire-based survey regarding pregnancy outcomes. Fifty-nine consecutive RIF patients without the OPTIMUM treatment strategy were also recruited as a control., Results: The 116 women with RIF after the OPTIMUM treatment strategy were 38.3 ± 3.8 years old and had an implantation failure history over 5 (3-19) ET cycles. Implantation testing identified impaired intrauterine circumstances in 75 women (64.7%), an aberrant elevated Th1/Th2 cell ratio in 56 women (48.3%), and thyroid abnormalities in 33 women (28.4%). Cumulative ongoing pregnancy rates including spontaneous pregnancy in the patients aged < 40 and ≥ 40 years were 72.7% and 45.5% within two ET cycles, respectively. The pregnancy outcomes in the OPTIMUM group were significantly higher than those in the control., Conclusions: The OPTIMUM treatment strategy improved pregnancy outcomes in patients with RIF., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Ultrasound-mediated destruction of microbubbles has been proposed as an innovative non-invasive drug delivery system for cancer therapy. We developed a specific drug delivery system for squamous cell carcinoma that uses sonoporation with the anti-epidermal growth factor receptor (EGFR) antibody. Administration of a low dose of bleomycin (BLM) by sonoporation with the anti-EGFR antibody produced a marked growth inhibition of Ca9-22 cells in vitro. In addition, scanning electron microscopic analysis revealed apparent surface deformation of Ca9-22 cells treated with sonoporation in the presence of the antibody. Interestingly, the population of apoptotic cells was remarkably increased when a low dose of BLM was delivered using sonoporation with the Fab fragment of the anti-EGFR antibody. These findings indicate that sonoporation with the Fab fragment makes it possible to administer drugs into cells more efficiently and specifically, suggesting a novel application for chemotherapy and gene therapy treatments for oral squamous cell carcinoma.

Background: There is limited knowledge relating to the influence of mechanical stress on the temporomandibular joint (TMJ) and the degree of damage this stress can induce in the case of an altered occlusal plane on the extracellular matrix cartilage and chondrocytes. The aim of this study was to examine the histopathological influence of an altered occlusal plane on rabbit TMJ cartilage., Methods: Fifteen male Japanese white rabbits were used in this study. In 12 cases, the upper right and lower left teeth were ground to their cervical level so that the occlusal plane was tilted right side up. Their temporomandibular joints (TMJs) harvested at 3 days, 1, 2 and 4 weeks were analyzed immunocytochemically and immunohistochemically. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick endlabeling (TUNEL) method was used for immunocytochemical study to detect DNA fragmentation in chondrocytes. An immunohistochemical study was also performed to evaluate the metabolism of type 2 collagen in the extracellular matrix in the TMJ cartilage., Results: The number of apoptotic cells increased in all experimental animals. More apoptotic cells were observed in the articular eminences than in the condyles. The type 2 collagen-positive area in the condyles was reduced at the beginning but recovered within 4 weeks, and remained small throughout this experimental period in the articular eminences., Conclusions: These findings suggest that histopathological changes occurred in response to alteration of the occlusal plane. The influence decreased as the occlusal plane recovered in the condyles, but the immunocytochemical and immunohistochemical influence continued in the articular eminences.

Problem: To investigate the role of platelet-activating factor (PAF) in human ovulation, we studied the regulation of interleukin (IL)-8 and growth-regulated oncogene (GRO) alpha in cultured human immortalized granulosa cell line (GC1a)., Method of Study: GC1a was cultured in serum-free medium, and incubated with carbamyl-PAF (C-PAF) and/or PAF receptor antagonist (WEB 2086). The supernatants were collected, and IL-8 and GRO alpha were measured by enzyme-linked immunosorbent assay., Results: After treatment with C-PAF, the levels of IL-8 and GROalpha increased in a time-dependent manner. The levels of IL-8 and GROalpha were significantly increased after treatment with C-PAF in a dose-dependent manner. However, the levels of IL-8 and GROalpha were significantly decreased by treatment with C-PAF and with increasing concentrations of WEB 2086., Conclusion: Our data indicated that IL-8 and GROalpha were regulated by C-PAF. The results suggested that PAF may play an important role in human pre-ovulatory processes involving IL-8 and GROalpha production.

Background: Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) staining is a widely accepted method for the detection of DNA fragmentation in nuclei of apoptotic cells. Tumor necrosis factor (TNF)-alpha is closely associated with changes in condylar cartilage and modulates apoptosis in various tissues including cartilage. The aim of this study was to investigate the relationship between apoptotic chondrocytes and TNF-alpha in a rabbit model of arthritis., Method: Unilateral temporomandibular joint (TMJ) arthritis was induced in 20 adult New Zealand White rabbits. From 1 day to 6 weeks after the induction of arthritis, immunohistochemical analysis for TNF-alpha and TUNEL was performed., Results: In condylar cartilage, TNF-alpha-positive cells and TUNEL-positive cells were localized together. TNF-alpha-positive chondrocytes seemed to precede TUNEL-positive cells., Conclusions: The results of the present study suggest that TNF-alpha may be involved in apoptosis and/or apoptotic necrosis of chondrocytes as TMJ arthritis progresses from the acute to chronic stage.

The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans consists of three proteins, CdtA, CdtB, and CdtC, which are responsible for cell cycle arrest and apoptosis. In the present study, local delivery systems of recombinant CdtB and CdtB-expressing plasmid were established using Ca9-22, human gingival squamous cell carcinoma cell line. When CdtB was delivered to Ca9-22 cells using a BioPORTER, a 32-kDa protein was detected by Western blotting, and G2 cell cycle arrest and apoptosis occurred. In addition, the CdtB delivered upregulated the expression of phosphorylated p53 and the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) in Ca9-22 cells, suggesting that these intracellular molecules might contribute to the induction of G2 cell cycle arrest and apoptosis. When the CdtB-expressing plasmid was transfected into Ca9-22 cells by lipofection or electroporation, CdtB (32 kDa) was clearly detected. Further, TdT-mediated dUTP nick end labeling positive cells were observed after transfection of the CdtB-expressing plasmid. These findings indicated that delivery of the CdtB protein and transfection of the cdtB gene induced cell cycle arrest and apoptosis in Ca9-22 cells in vitro, and we conclude that it may be possible to induce apoptosis in human gingival squamous cell carcinoma by electroporation of the cdtB gene.

Problem: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein -1 (MCP-1) in human ovulation, we studied the regulation of M-CSF and MCP-1 in cultured human granulosa cells., Method of Study: Immortalized granulosa cells (GC1a) were cultured in serum-free medium, and incubated with interleukin (IL)-1alpha, IL-1 receptor antagonist (ra) and tumor necrosis factor (TNF)-alpha. The supernatants were collected, and M-CSF and MCP-1 were measured by ELISA., Results: The levels of M-CSF and MCP-1 were increased after treatment with IL-1alpha (1 nm) and TNF-alpha (1 nm) in a time-dependent manner. The levels of M-CSF and MCP-1 were significantly increased after treatment with IL-1alpha and TNF-alpha in a dose-dependent manner. However, the levels of M-CSF and MCP-1 were significantly decreased by treatment with IL-1alpha (1 nm) and/or increasing concentrations of IL-1 ra., Conclusions: Our data indicated that M-CSF and MCP-1 were regulated by IL-1alpha and TNF-alpha. It was suggested that M-CSF and MCP-1 may play an important role in human preovulatory processes.

Background: The molecular weight (MW) of hyaluronic acid (HA) in joints generally declines in inflammatory arthritis. As inflammation exists in certain temporomandibular disorders (TMD), we measured the MW of HA in synovial fluid (SF) recovered from patients with TMD and compared it with that from normal controls., Methods: Synovial fluid was obtained from patients with TMD (21 TMJs) and normal controls (5 TMJs). The MW of HA was measured using high-performance liquid chromatography (HPLC)., Results: In the controls, the MW of HA in SF exceeded the detection limit, 3000 kDa. In contrast, 19 (90.5%) of 21 SF samples from patients showed a decreased MW of HA. The median MW of HA (1570 kDa) in TMD was significantly lower than that in the controls (P < 0.01)., Conclusion: The MW of HA in SF from patients with TMD is decreased.

Background: In temporomandibular joint (TMJ) arthritis, knowledge is limited about the source of the inflammatory mediators. The aim of this study was to investigate the immunohistochemical role of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) in the development of the antigen-induced arthritis of the rabbit TMJ., Methods: Unilateral TMJ arthritis was induced in 28 adult rabbits. From 6 h to 6 weeks after induction of arthritis, the topology of TNF-alpha and IL-8 was observed., Results: Positive reaction for TNF-alpha of synovial cells was observed within 3 days after induction and at 3 weeks after induction. TNF-alpha positive vascular endothelial cells and chondrocytes were identified throughout the observation period. IL-8 was detected only during the acute stage., Conclusions: The cytokines TNF-alpha and IL-8 were observed in specific cells depending on the stage. TNF-alpha was particularly related with angiogenesis and cartilage destruction and IL-8 was involved in the acute stage of inflammation.

Problem: Interleukin-8 (IL-8) and growth-regulated oncogene-alpha (GRO-alpha) have been proved to be important modulators of leukocyte chemotaxis in the mechanism of human ovulation. This study investigated the possible effects of IL-1alpha, tumor necrosis factor-alpha (TNF-alpha), protein kinase C (PKC) activators (TPA), and db-cyclic adenosine monophosphate (cAMP) on IL-8 and GRO-alpha production by immortalized GC1a and granulosa-lutein cells., Method of Study: Confluent granulosa-lutein cells were placed in serum-free medium before incubated for 8 hr with the above-mentioned test agents. Finally, we measured IL-8 and GRO-alpha levels in the culture media using an enzyme-linked immunosorbent assay (ELISA)., Results: Treatment of granulosa-lutein cells with of IL-1alpha (1 nM), TNF-alpha (1 nM), TPA (1 nM) and db-cAMP (100 microM) produced higher levels of IL-8 than untreated cells by 8 hr (2274.7 +/- 146.3, 1489.8 +/- 190.1, 1452.9 +/- 152.7, 1313.6 +/- 48.4 pg/mL, respectively; control = 457.7 +/- 38.2 pg/mL; P < 0.001). Treatment of granulosa-lutein cells with 1 nM of IL-1alpha, TNFalpha, TPA, and db-cAMP (100 microM) resulted in higher levels of GRO-alpha than untreated cells by 8 hr (993.7 +/- 9.5, 171.4 +/- 6.5, 147.5 +/- 6.7, 472.4 +/- 16.2 pg/mL respectively; control = 73.8 +/- 8.2 pg/mL; P < 0.001)., Conclusions: Our data strongly suggests roles for IL-1alpha, TNFalpha, and PKC activators in the inflammation-like mechanism of human ovulation. Furthermore, our study suggests a positive, but still debatable, role for cAMP in the same mechanism.

Aims: To study the effects of rifampicin on the pharmacokinetics and pharmacodynamics of nilvadipine., Methods: Five healthy adult volunteers received nilvadipine (4 mg) orally before and after a 6 day treatment with rifampicin. Blood and urine were collected and assayed for plasma nilvadipine and urinary 6beta-hydroxycortisol and cortisol., Results: The treatment with rifampicin reduced the mean (+/- s.d.) AUC of nilvadipine from 17.4 +/- 8.4 to 0.6 +/- 0.4 microg l-1 h (mean difference -16.8 microg l-1 h, 95% CI -9.4, 24.2 microg l-1 h). While the administration of nilvadipine alone elicited a significant (P < 0.05) hypotensive (mean difference for diastolic blood pressure -8 mmHg, 95% CI -4, -12 mmHg) and reflex tachycardia (mean difference 5 beats min-1, 95% CI 1, 9 beats min-1), the treatment with rifampicin abolished these responses. The urinary 6beta-hydroxycortisol/cortisol ratio showed a significant (P < 0.05) increase from 10.3 +/- 4.0 to 50.3 +/- 24.6 by rifampicin: mean difference 40.1, 95% CI 20.4, 59.8., Conclusions: Because rifampicin may greatly decrease the oral bioavailability of nilvadipine, caution is needed when these two drugs are to be coadministered.

Background: In temporomandibular joint (TMJ) arthritis, there is limited knowledge of the relationship between interleukin-1beta (IL-1beta) and interleukin-1 receptor antagonist (IL-1ra), as well as the source of these cytokines. We investigated the development of an antigen-induced arthritis in the rabbit TMJ immunohistochemically., Methods: Unilateral TMJ arthritis was induced in 32 adult New Zealand White rabbits. From 6 h to 12 weeks after induction of arthritis, topology of IL-1beta and IL-1ra were observed., Result: The acute stage of induced arthritis lasted for one week after induction, thereafter it became chronic. In the early phase of the acute stage, infiltrating inflammatory cells, as well as synovial cells, produced IL-1beta and IL-1ra. In the late phase of the acute stage, the main source of these cytokines was subsynovial fibroblasts. In this phase of arthritis, IL-1beta and IL-1ra did not appear to be produced by synovial cells. From the early to intermediate phase of the chronic stage, proliferating synovial cells produced IL-1beta and IL-1ra. In this phase of the arthritis, these cytokines were also observed in a cluster formation in chondrocytes., Conclusion: This arthritis model shows a staging of the joint inflammation process with time. IL-1beta and IL-1ra are produced by a certain kind of cells depending on the stage of inflammation.

Problem: Prostaglandins (PGs) are synthesized in the endometrium. Our objective was to evaluate interleukin (IL)-1alpha-induced production of PGE2 and PGF2alpha in endometrial stromal cells (ESC) following treatment with ceramide analogues., Methods of Study: ESC were obtained from human uterine endometrium by enzymic digestion and filtration. ESC were treated with IL-1alpha, IL-1 receptor antagonist (ra), C2-ceramide and C6-ceramide. The concentrations of PGE2 and PGF2alpha in media were determined using ELISA. The induction of prostaglandin H synthase (PGHS)-2 mRNA was also ascertained by reverse transcription-polymerase chain reaction (RT-PCR)., Results: The production of PGE2 and PGF2alpha was significantly increased by IL-lalpha and suppressed by IL-1 ra, in a dose-dependent manner. PGF2alpha production was further increased by treatment with the combination of IL-1alpha and C2-ceramide as compared with IL-1alpha treatment alone. There was no significant difference in PGE2 production between cells treated with IL-1alpha and C2-ceramide and those treated with IL-1alpha alone. Both PGE2 and PGF2alpha production were significantly increased by treatment with IL-1alpha and C6-ceramide as compared with IL-1alpha treatment alone. Treatment of ESC with IL-1alpha stimulated PGHS-2 mRNA. PGHS-2 mRNA was decreased when IL-1 ra was added to the IL-1alpha-stimulated cells., Conclusions: These results suggest that IL-1alpha stimulates the production of PGE2 and PGF2alpha by a mechanism that involves the sphingomyelin-ceramide system, and thus that ceramide may be important in increasing the production of PGE2 and PGF2alpha in the human endometrium.

Problem: The aim of this study was to investigate the expression of chaperonin (cpn) 10 and cpn 60 mRNA in oocytes or embryos, and to further explore the possibility that early pregnancy factor (EPF) is identical with cpn 10., Method of Study: The expressions of cpn 10 and cpn 60 mRNA in oocytes and embryos at the different stages (1-cell, 2-cell, 8-cell, and morula) were examined by polymerase chain reaction techniques. The EPF activity of native rat cpn 10 isolated from rat livers was evaluated by the rosette inhibition test., Results: Similar levels of mRNA of cpn 10 and cpn 60 were detected in oocytes and embryos at every stage. There were no detectable EPF activities in the native cpn 10. Immunoprecipitation using polyclonal antibodies against cpn 10 did not affect the activity of EPF in the pregnant rat serum., Conclusion: Our results do not support the hypothesis that cpn 10 is identical with EPF.

Fas antigen is a cell surface protein that mediates apoptosis via signal transduction from the plasma membrane. Using reverse transcriptase-polymerase chain reaction (RT-PCR), messenger RNA for Fas antigen was detected in the human oral squamous cell carcinoma cell line, SCC-25. In serum-free medium, a monoclonal anti-Fas antibody (CH-11) induced Fas antigen expression in SCC-25 cells, as determined by immunocytochemistry and Western blotting, using an anti-Fas polyclonal antibody (Fas D) as primary antibody. Fas antigen was localized to the cytoplasm and the cell membrane. The molecular weight of the protein recognized by Western blot analysis was 35,000, consistent with the value reported for the Fas antigen. The CH-11 antibody did not induce Fas antigen expression in serum-containing medium. To determine whether CH-11 could induce apoptosis in oral squamous cell carcinoma, we examined its effects on the survival of cultured SCC-25 cells. Anti-Fas monoclonal antibody in serum-free medium induced cytotoxicity in SCC-25 cells in a time-dependent manner up to 8 h, as determined by phase-contrast microscopy and WST-1 assay. Marked nuclear condensation and fragmentation of chromatin were observed in the CH-11-treated cells using Hoechst 33342 staining. This anti-Fas monoclonal antibody also induced DNA ladder formation in SCC-25 cells in a time-dependent manner. The present results indicate that the anti-Fas monoclonal antibody (CH-11) may mediate apoptosis by binding to the Fas antigen expressed in SCC-25 cells.

A monospecific and high titer polyclonal antibody, designated as Fas D, raised against synthetic polypeptides selected from a part of the human Fas antigen (aa 104-114), was used to identify the Fas antigen in human oral epithelia in normal and pathological states. The human gingival proteins had been extracted and analysed by an ABC (avidin-biotin complex) immunoblotting technique. The antibody interacted with a single band in gingival proteins with an estimated molecular weight of 35,000, which is in good agreement with that calculated from amino acid sequences of the human Fas antigen. Using an indirect immunohistochemical method, the antibody localized on the stratum spinosum and the basal part of the stratum corneum of normal human gingiva. Specimens obtained from patients with odontogenic keratocysts, leukoplakia, lichen planus, and squamous cell carcinoma were also stained with the antibody. The pattern of the Fas antigen distribution in oral stratified squamous epithelia was, with some overlapping, characteristic for each disease.

Malignant lymphomas arising from the nasal cavity have been considered to be derived from T cells, but recent surface marker studies suggest that more than half of the lymphomas are derived from natural killer (NK) cells. Here we describe a case of nasal lymphoma whose lymphoma cells were identified as NK cells by morphological, phenotypic, immunogenotypic, and functional studies. We believe this is the first study with functional evidence of NK activity. When compared with normal freshly isolated NK cells or activated NK cells, the surface phenotypes and NK activity of the patient tumour cells were those of the activated, but not resting, NK cells. Also, Epstein-Barr virus (EBV) was detected in the tumour cells and the lymphoma cells were found to be monoclonally expanded. The patient's lymphoma cells also expressed EBV receptor CD21 (CR2) and CD30 (Ki-1) that have not been described on normal NK cells. We therefore examined highly enriched NK cells of normal donors, and found that some resting and/or activated NK cells express these antigens.