NK Cells Can Preferentially Target Prostate Cancer Stem-like Cells via the TRAIL/DR5 Signaling Pathway.

BACKGROUND: The occurrence of androgen-dependent prostate cancer mainly depends on prostate cancer stem cells. To reduce the risk of androgen-dependent prostate cancer, the direct elimination of prostate cancer stem cells is important, but an elimination strategy has not yet been established. A previous study showed that natural killer (NK) cells can preferentially target cancer stem cells in several solid tumors except prostate cancer. In this context, this study was undertaken to investigate if NK cells can selectively attack androgen-dependent prostate cancer stem cells. METHODS: Prostate cancer stem-like cells were separated from an androgen-dependent prostate cancer cell line (LNCaP) using a three-dimensional culture system. LNCaP stem-like cells or LNCaP cells were co-cultured with human NK cells (KHYG-1) for 24-72 h, and cell viability was determined using the WST-8 method. The expression of each protein in the cell membrane was evaluated through FACS analysis, and mRNA levels were determined using real-time PCR. RESULTS: KHYG-1 cells had more potent cytotoxicity against LNCaP stem-like cells than LNCaP cells, and the potency of the cytotoxicity was strongly related to the TRAIL/DR5 cell death pathway. CONCLUSION: NK cells can preferentially target prostate cancer stem-like cells via the TRAIL/DR5 pathway.

[Locally Advanced Gastric Cancer Responding to Neoadjuvant Chemotherapy with Ramucirumab plus Paclitaxel-Case Report].

Neoadjuvant chemotherapy(NAC)is a promising approach for the improvement of gastric cancer treatment outcome. S-1 plus cisplatin(SP)or S-1 plus oxaliplatin (SOX)is generallythe first choice of NAC regimen. We experienced that NAC with ramucirumab(RAM)plus paclitaxel(PTX)was effective in locallyadvanced gastric cancer, but that with SOX was ineffective. A 68-year-old man developed locallyadvanced gastric cancer and received NAC with SOX, which was stopped because of tumor enlargement. The patient was then given NAC with RAM plus PTX, which was effective and enabled radical excision. Anti-angiogenic agents maycause wound healing complications, which mayincrease the risk of leakage. However, he was discharged without postoperative complications. Therefore, RAM plus PTX can be a promising NAC regimen for locallyadvanced gastric cancer.

Clotrimazole inhibits the Wnt/ß-catenin pathway by activating two eIF2α kinases: The heme-regulated translational inhibitor and the double-stranded RNA-induced protein kinase.

The Wnt/ß-catenin signaling pathway controls cell proliferation and differentiation, and therefore, when this pathway is excessively activated, it causes tumorigenesis. Our chemical suppressor screening in zebrafish embryos identified antifungal azoles including clotrimazole, miconazole, and itraconazole, as Wnt/ß-catenin signaling inhibitors. Here we show the mechanism underlying the Wnt/ß-catenin pathway inhibition by antifungal azoles. Clotrimazole reduced ß-catenin revels in a proteasome-independent fashion. By gene knockdown of two translational regulators, heme-regulated translational inhibitor and double-stranded RNA-induced protein kinase, we show that they mediate the clotrimazole-induced inhibition of the Wnt/ß-catenin pathway. Thus, clotrimazole inhibits the Wnt/ß-catenin pathway by decreasing ß-catenin protein levels through translational regulation. Antifungal azoles represent genuine candidate compounds for anticancer drugs or chemopreventive agents that reduce adenomatous polyps.

Tetraalkoxyphenanthrene-Fused Hexadecadehydro[20]- and Tetracosadehydro[30]annulenes: Syntheses, Aromaticity/Antiaromaticity, Electronic Properties, and Self-Assembly.

Tetraalkoxyphenanthrene-fused hexadecadehydro[20]- and tetracosadehydro[30]annulenes possessing octatetrayne linkages were synthesized and their properties together with those of phenanthrene-fused octadehydro[12]- and dodecadehydro[18]annulenes have been investigated. Various spectroscopic and electrochemical measurements as well as quantum chemical calculations support that planar [20]- and [30]annulenes are weakly antiaromatic and nonaromatic, respectively. The detailed concentration- and temperature-dependent 1H NMR and UV-vis data of present dehydroannulenes provided evidence for the enhancement of π-π stacking interactions by extension of the acetylenic linkages. Dehydroannulenes formed self-assembled clusters and their morphology and crystallinity proved to depend on the length of acetylenic linkages, the shape of dehydroannulene core, and the bulkiness of alkoxy groups appended to the phenanthrene moieties.

Role of interleukin-32 in the mechanism of chronic inflammation in IgG4-related disease and as a predictive biomarker for drug-free remission.

OBJECTIVES: In immunoglobulin (Ig) G4-related disease (IgG4-RD), the mechanism of chronic inflammation and predictive factors for drug-free remission is still unclear. To examine the issues, we focused on tuberculosis, a chronic infection, and on the role of interleukin (IL)-32. METHODS: We examined the positive rate of QuantiFERON TB-2G (QFT-2G) in 126 patients with IgG4-RD, and compared with the rate in the general population. Furthermore, specimens of submandibular glands from the maintenance treatment group and drug-free group of IgG4-RD and specimens of small salivary glands from primary Sjögren's syndrome (SS) were stained with anti-IL-32 antibody and anti-protease-activated receptor 2 antibody, and the number of positive cells was compared between these groups. RESULTS: The positive rate of QFT-2G was 19.8% in IgG4-RD patients, which is higher than in the general population. The expression of IL-32 and PAR2 in the submandibular glands of the maintenance treatment group of IgG4-RD was significantly greater than that of the drug-free remission group and SS patients. CONCLUSIONS: This study indicates the possibility that IL-32 is associated with chronic inflammation and that it can be a predictive factor for drug-free remission in IgG4-RD.

Loss of interleukin-21 leads to atrophic germinal centers in multicentric Castleman's disease.

Both multicentric Castleman's disease (MCD) and immunoglobulin (Ig)G4-related disease (IgG4-RD) are systemic diseases, presenting with hypergammaglobulinemia and elevated serum levels of IgG4. However, with regard to histopathological findings, MCD shows atrophic germinal centers. On the other hand, expanded germinal centers are detected in IgG4-RD. We extracted germinal centers from specimens of each disorder by microdissection and analyzed the expression of mRNAs by real-time polymerase chain reaction to clarify the mechanisms underlying atrophied germinal centers in MCD. This analysis disclosed loss of interleukin (IL)-21 and B cell lymphoma (Bcl)-6 in the germinal centers of MCD. Loss of IL-21 is considered to be involved in the disappearance of Bcl-6 and leads to atrophied germinal centers in MCD.

CCAAT/enhancer binding protein α (C/EBPα)(+) M2 macrophages contribute to fibrosis in IgG4-related disease?

IgG4-related disease (IgG4-RD) is a new disease entity characterized by type 2 helper T (Th2)-dominant inflammation and progressive fibrosis. We found the infiltration of strange cell populations in the fibrotic lesions of submandibular gland specimens obtained from 15 patients with IgG4-RD. These cells expressed CCAAT/enhancer binding protein a (C/EBPα). Many of the cell populations were identified with M2 macrophages. The degrees of infiltration of C/EBPα(+)M2 macrophages and the ratio of fibrotic lesions in the specimens were correlated (r(2) = 0.83, p < 0.01). We also analyzed the expression of C/EBPα in other chronic inflammatory disorders: synovium in rheumatoid arthritis (RA), liver tissue in chronic viral hepatitis, and mucosa in ulcerative colitis. The specimens from RA and chronic viral hepatitis showed infiltration of C/EBPα(+) cells, but there were few C/EBPα-positive cells in ulcerative colitis. Fibrosis is not a major issue in ulcerative colitis. In conclusion, we found the remarkable infiltration of C/EBPα(+)M2 macrophages in cases of chronic inflammation with fibrosis, including IgG4-RD. This primitive study also disclosed that most of C/EBPα(+)M2 macrophages localized in fibrotic lesions, and the degree of the infiltration and the ratio of fibrotic area were correlated.

Everyday clinical practice in IgG4-related dacryoadenitis and/or sialadenitis: results from the SMART database.

Abstract Objective. Immunoglobulin (Ig)G4-related disease (IgG4-RD) is a new disease entity that has only been identified this century. Clinical information is thus lacking. We established the Sapporo Medical University and Related Institutes Database for Investigation and Best Treatments of IgG4-related Disease (SMART) to clarify the clinical features of IgG4-RD and provide useful information for clinicians. Methods. Participants comprised 122 patients with IgG4-related dacryoadenitis and/or sialadenitis (IgG4-DS), representing lacrimal and/or salivary lesions of IgG4-RD, followed-up in December 2013. We analyzed the sex ratio, mean age at onset, organ dysfunction, history or complications of malignancy, treatments, rate of clinical remission, and relapse. Results. The sex ratio was roughly equal. Mean age at diagnosis was 59.0 years. Positron emission tomography revealed that the ratio of other organ involvements was 61.4%. Complications of malignancy were observed in 7.4% of cases. Glucocorticoid was used to treat 92.1% of cases, and the mean maintenance dose of prednisolone was 4.8 mg/day. Rituximab was added in three cases, and showed good steroid-sparing effect. The clinical remission rate was 73.8%, and the annual relapse rate was 11.5%. Half of the cases experienced relapses within 7 years of initial treatment. Conclusion. We analyzed the clinical features and treatments of IgG4-DS using SMART, providing useful information for everyday clinical practice.

Identification of relapse predictors in IgG4-related disease using multivariate analysis of clinical data at the first visit and initial treatment.

OBJECTIVES: Inducting clinical remission by glucocorticoid treatment is relatively easy in IgG4-related disease (IgG4-RD), but relapse also occurs easily with tapering of the steroid dose. The present study tried to analyse the cases to extract predictors of relapse present at the diagnosis of IgG4-RD. METHODS: Subjects comprised 79 patients with IgG4-related dacryoadenitis and sialadenitis, known as Mikulicz's disease, who were diagnosed between April 1997 and October 2013 and followed-up for >2 years from the initial induction treatment. They were applied to Cox proportional hazard modelling, based on the outcome of interval to relapse. We performed multivariate analysis for the clinical factors of these cases and identified predictors of relapse. RESULTS: Identified factors were male sex and younger onset in cases without organ involvement at diagnosis and low levels of serum IgG4 in cases with organ dysfunction at diagnosis. Complication with autoimmune pancreatitis and low steroid dose at initial treatment also tended to be associated with recurrence. CONCLUSION: Follow-up is important in cases with recognized risk factors for relapse, including male sex and younger onset in cases without organ damage.

The role of cytotoxic T cells in IgG4-related dacryoadenitis and sialadenitis, the so-called Mikulicz's disease.

OBJECTIVES: Immunoglobulin (Ig) G4-related dacryoadenitis and sialadenitis, the so-called Mikulicz's disease (MD), is a chronic inflammatory disease. However, little is known about its pathogenesis and pathological condition. In the present study, we used immunohistological techniques to compare the roles of cytotoxic T lymphocytes (CTLs) in MD and primary Sjogren's syndrome (SS). We examined the state of CTLs [cytotoxic granule-positive rate and programmed death-1 (PD-1) expression rate] in the salivary glands. METHODS: The study samples comprised 12 submaxillary glands from untreated MD patients and 12 labial glands from SS patients. We performed immunofluorescence and multicolor immunofluorescence to stain CD8, perforin (PRF), granzyme B (GZMB), and PD-1. We measured the total number of CTLs as well as the PRF(+)CTLs, GZMB(+)CTLs, and PD-1(+)CTLs. RESULTS: We found that the degree of infiltration of CTLs was equal in MD and SS, but the rate of CTLs with cytotoxic granules, especially PRF, in MD was less than in SS. In addition, the frequency of PD-1(+)CTLs in MD was higher than that in SS. CONCLUSIONS: Cytotoxic granule-positive CTLs were in the minority in D salivary glands, and this regulation might relate to PD-1 signals like the state of exhaustion and anergy.

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans.

Morbillivirus infection is a severe threat to marine mammals. Mass die-offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain-like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three-dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.

Necessity of early intervention for IgG4-related disease--delayed treatment induces fibrosis progression.

OBJECTIVE: Despite ongoing research, the clinical and histopathological natural history of immunoglobulin (Ig) G4-related disease (IgG4-RD) remains unclear and the optimal time to initiate treatment is unknown. A focus on clinical symptoms rather than image finding is recommended for therapeutic initiation in autoimmune pancreatitis, but evidence for this approach is lacking. We aimed to retrospectively analyse disease duration, efficacy of treatment with glucocorticoids and results of histopathological examination of submandibular gland specimens to clarify the necessity for early intervention in IgG4-RD. METHODS: Salivary secretions were assessed before and after treatment in 26 cases of IgG4-related Mikulicz's disease (IgG4-MD). Relationships between disease duration, amount of salivary secretion before treatment, improvement of salivary secretion and ratios of areas of residual acini, fibrosis and lymphoid follicles in the involved submandibular gland specimens were analysed. RESULTS: Salivary secretions were significantly reduced in cases with illness of >2 years (P < 0.05). An inverse correlation was seen between improved amount of salivary secretion and amount of salivary secretion before treatment (r = -0.60). Improved amount of salivary secretion was also associated with each histological factor (acini, r = 0.29; fibrosis, r = -0.23; lymphoid follicles, r = -0.31), which showed interrelationships (acini and lymphoid follicles, r = -0.23; acini and fibrosis, r = 0.42; lymphoid follicles and fibrosis, r = 0.30). CONCLUSION: Salivary secretion can be improved even in cases with lower levels of salivary secretion before treatment in IgG4-RD, but improvements in the amount of salivary secretion decrease with histological changes with delayed therapeutic intervention. These data suggest that early intervention is needed to improve outcomes in patients with IgG4-MD.

Evaluation and Clinical Validity of a New Questionnaire for Mikulicz's Disease.

Objectives. The characteristic features of Mikulicz's disease (MD) are diffuse enlargement of the lacrimal and submandibular glands, elevated levels of serum immunoglobulin (Ig)G4, and abundant infiltration of IgG4-positive plasmacytes into both glands. No disease index is available to properly evaluate MD, so we developed a functional assessment of MD, the Mikulicz's disease activity questionnaire (MAQ), and evaluated its clinical efficacy. Methods. We selected 18 patients who were either being treated for MD or who had presented with recurrence. The patients completed a self-assessment and were scored according to the MAQ sheet during each visit between December 2009 and August 2011. Assessment items were in regard to increases or decreases in lacrimal and salivary gland enlargement and severity of sicca symptoms. Results. On the first visits, MAQ scores were high, but scores decreased rapidly as treatment progressed. When doses of glucocorticoid were reduced, some patients showed increased scores. Dry-symptom scores increased initially. MAQ scores for patients with recurrent MD gradually increased over several months before relapse. However, some patients displayed no elevation in MAQ scores due to relapses at other sites. Conclusion. MAQ score can be used to quantify flares and treatment response and is useful for functional assessment of MD.

[Clinical efficacy of adalimumab for a postoperative marginal ulcer in gastrointestinal Behçet disease].

A 63-year-old woman with Behçet disease presented with epigastric pain due to refractory gastric ulcers. Examinations indicated that these ulcers were caused by gastrointestinal Behçet disease. Steroid therapy proved ineffective, so we gave 5mg/kg of infliximab. However, since the patient responded poorly to the treatment the infliximab was discontinued and a total gastrectomy was performed. After surgery, a marginal ulcer developed and infliximab was again administered. Although this brought about improvement in the conditions of the marginal ulcer, infusion-related hypersensitivities in the patient caused polyarthralgia. We therefore discontinued the infliximab treatment and began 40 mg of adalimumab every other week. After 3 months of the new treatment, the patient's marginal ulcer completely healed and her epigastric pain disappeared. This case suggests that adalimumab may be as useful as infliximab for treating refractory gastrointestinal Behçet disease.