Laser-based angle-resolved photoemission spectroscopy with micrometer spatial resolution and detection of three-dimensional spin vector.

We have developed a state-of-the-art apparatus for laser-based spin- and angle-resolved photoemission spectroscopy with micrometer spatial resolution (µ-SARPES). This equipment is realized by the combination of a high-resolution photoelectron spectrometer, a 6 eV laser with high photon flux that is focused down to a few micrometers, a high-precision sample stage control system, and a double very-low-energy-electron-diffraction spin detector. The setup achieves an energy resolution of 1.5 (5.5) meV without (with) the spin detection mode, compatible with a spatial resolution better than 10 µm. This enables us to probe both spatially-resolved electronic structures and vector information of spin polarization in three dimensions. The performance of µ-SARPES apparatus is demonstrated by presenting ARPES and SARPES results from topological insulators and Au photolithography patterns on a Si (001) substrate.

High-power laser experiment forming a supercritical collisionless shock in a magnetized uniform plasma at rest.

We present an experimental method to generate quasiperpendicular supercritical magnetized collisionless shocks. In our experiment, ambient nitrogen (N) plasma is at rest and well magnetized, and it has uniform mass density. The plasma is pushed by laser-driven ablation aluminum (Al) plasma. Streaked optical pyrometry and spatially resolved laser collective Thomson scattering clarify structures of plasma density and temperatures, which are compared with one-dimensional particle-in-cell simulations. It is indicated that just after the laser irradiation, the Al plasma is magnetized by a self-generated Biermann battery field, and the plasma slaps the incident N plasma. The compressed external field in the N plasma reflects N ions, leading to counterstreaming magnetized N flows. Namely, we identify the edge of the reflected N ions. Such interacting plasmas form a magnetized collisionless shock.

Contour prognostic model for predicting survival after resection of colorectal liver metastases: development and multicentre validation study using largest diameter and number of metastases with RAS mutation status.

BACKGROUND: Most current models for predicting survival after resection of colorectal liver metastasis include largest diameter and number of colorectal liver metastases as dichotomous variables, resulting in underestimation of the extent of risk variation and substantial loss of statistical power. The aim of this study was to develop and validate a new prognostic model for patients undergoing liver resection including largest diameter and number of colorectal liver metastases as continuous variables. METHODS: A prognostic model was developed using data from patients who underwent liver resection for colorectal liver metastases at MD Anderson Cancer Center and had RAS mutational data. A Cox proportional hazards model analysis was used to develop a model based on largest colorectal liver metastasis diameter and number of metastases as continuous variables. The model results were shown using contour plots, and validated externally in an international multi-institutional cohort. RESULTS: A total of 810 patients met the inclusion criteria. Largest colorectal liver metastasis diameter (hazard ratio (HR) 1.11, 95 per cent confidence interval 1.06 to 1.16; P < 0.001), number of colorectal liver metastases (HR 1.06, 1.03 to 1.09; P < 0.001), and RAS mutation status (HR 1.76, 1.42 to 2.18; P < 0.001) were significantly associated with overall survival, together with age, primary lymph node metastasis, and prehepatectomy chemotherapy. The model performed well in the external validation cohort, with predicted overall survival values almost lying within 10 per cent of observed values. Wild-type RAS was associated with better overall survival than RAS mutation even when liver resection was performed for larger and/or multiple colorectal liver metastases. CONCLUSION: The contour prognostic model, based on diameter and number of lesions considered as continuous variables along with RAS mutation, predicts overall survival after resection of colorectal liver metastasis.

Multicentre study of perioperative versus adjuvant chemotherapy for resectable colorectal liver metastases.

Background: It is not known whether perioperative chemotherapy, compared with adjuvant chemotherapy alone, improves disease-free survival (DFS) in patients with upfront resectable colorectal liver metastases (CLM). The aim of this study was to estimate the impact of neoadjuvant 5-fluorouracil, leucovorin and oxaliplatin (FOLFOX) on DFS in patients with upfront resectable CLM. Methods: Consecutive patients who presented with up to five resectable CLM at two Japanese and two French centres in 2008-2015 were included in the study. Both French institutions favoured perioperative FOLFOX, whereas the two Japanese groups systematically preferred upfront surgery plus adjuvant chemotherapy. Inverse probability of treatment weighting (IPTW) and Cox regression multivariable models were used to adjust for confounding. The primary outcome was DFS. Results: Some 300 patients were included: 151 received perioperative chemotherapy and 149 had upfront surgery plus adjuvant chemotherapy. The weighted 3-year DFS rate was 33·5 per cent after perioperative chemotherapy compared with 27·1 per cent after upfront surgery plus adjuvant chemotherapy (hazard ratio (HR) 0·85, 95 per cent c.i. 0·62 to 1·16; P = 0·318). For the subgroup of 165 patients who received adjuvant FOLFOX successfully (for at least 3 months), the adjusted effect of neoadjuvant chemotherapy was not significant (HR 1·19, 0·74 to 1·90; P = 0·476). No significant effect of neoadjuvant chemotherapy was observed in multivariable regression analysis. Conclusion: Compared with adjuvant chemotherapy, perioperative FOLFOX does not improve DFS in patients with resectable CLM, provided adjuvant chemotherapy is given successfully.

Antecedentes: Se desconoce si la quimioterapia perioperatoria en comparación con la quimioterapia adyuvante sola mejora la supervivencia libre de enfermedad (disease­free survival, DFS) en pacientes con metástasis hepáticas de origen colorrectal (colorectal liver metastases, CLM) resecables de inicio. El objetivo de este estudio fue estimar el impacto de la neoadyuvancia con 5­fluorouracilo, leucovorina y oxaliplatino (FOLFOX) sobre la DFS en pacientes con CLM resecables desde el principio. Métodos: Se incluyeron pacientes consecutivos que presentaban hasta cinco CLM resecables en dos centros japoneses y dos centros franceses entre 2008 a 2015. Ambas instituciones francesas favorecían FOLFOX perioperatorio, mientras que los dos grupos japoneses utilizaban sistemáticamente la cirugía de entrada y quimioterapia adyuvante. Se utilizaron la probabilidad inversa del tratamiento ponderado (Inverse Probability of Treatment Weighting, IPTW) y el modelo multivariable de regresión de Cox para ajustar por factores de confusión. El resultado primario fue la DFS. Resultados: Se incluyeron 300 pacientes (grupo de quimioterapia perioperatoria n = 151 y grupo de cirugía de entrada más quimioterapia adyuvante n = 149). La DFS a los 3 años ponderada fue del 33% después de quimioterapia perioperatoria versus 27% tras cirugía de entrada (cociente de riesgos instantáneos, hazard ratio HR: 0,85; i.c. del 95% (0,62­1,16); P = 0,32). Cuando se consideró el subgrupo de pacientes que (n = 165) de manera efectiva (al menos 3 meses) recibieron FOLFOX adyuvante, el efecto ajustado de la quimioterapia neoadyuvante no fue significativo (HR: 1,19 (0,74­1,90); P = 0,48). No se observó un efecto significativo de la quimioterapia neoadyuvante en el análisis de regresión multivariable. Conclusión: En comparación con la quimioterapia adyuvante, el FOLFOX perioperatorio no mejora la DFS en CLM resecables siempre y cuando la quimioterapia adyuvante se administre de forma efectiva.

Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

Magnetic field effects of photocarrier generation in bulk heterojunctions at low temperature.

We report an experimental investigation of the magnetic field effect (MFE) in polymer bulk heterojunction devices at temperatures below 10 K using photocarrier extraction by linearly increasing voltages. The examined devices were composed of an active layer of poly(3-hexylthiophene) and [6,6]-phenyl-C61-butyric acid methyl ester. In the experiments, the delay time (td) dependence of the MFE was investigated in detail. For td < 80 µs, a positive MFE was observed in the field region B < 0.1 T and a negative MFE was observed for B > 0.2 T. For td > 8 ms, only a positive MFE proportional to B2 was observed. For the photocurrent pulse detected immediately after light irradiation, the MFE was negligibly small. In a high magnetic field of 15 T, a significant MFE exceeding 80% was observed at 1.8 K for td = 800 ms. We discuss the results based on a model of triplet-singlet (or singlet-triplet) conversion in the magnetic field and estimate the exchange integral for the charge-transfer exciton in this photovoltaic cell.

Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations.

Despite initial dramatic response, epidermal growth factor receptor (EGFR) mutant lung cancer patients always acquire resistance to EGFR-tyrosine kinase inhibitors (TKIs). Gatekeeper T790M mutation in EGFR is the most prevalent genetic alteration underlying acquired resistance to EGFR-TKI, and EGFR mutant lung cancer cells are reported to be addictive to EGFR/Akt signaling even after acquired T790M mutation. Here, we focused on Akt kinase-interacting protein1 (Aki1), a scaffold protein of PI3K (phosphoinositide 3-kinase)/PDK1 (3-phosphoinositide-dependent protein kinase)/Akt that determines receptor signal selectivity for non-mutated EGFR, and assessed its role in EGFR mutant lung cancer with or without gatekeeper T790M mutation. Cell line-based assays showed that Aki1 constitutively associates with mutant EGFR in lung cancer cells with (H1975) or without (PC-9 and HCC827) T790M gatekeeper mutation. Silencing of Aki1 induced apoptosis of EGFR mutant lung cancer cells. Treatment with Aki1 siRNA dramatically inhibited growth of H1975 cells in a xenograft model. Moreover, silencing of Aki1 further potentiated growth inhibitory effect of new generation EGFR-TKIs against H1975 cells in vitro. Aki1 was frequently expressed in tumor cells of EGFR mutant lung cancer patients (53/56 cases), including those with acquired resistance to EGFR-TKI treatment (7/7 cases). Our data suggest that Aki1 may be a critical mediator of survival signaling from mutant EGFR to Akt, and may therefore be an ideal target for EGFR mutant lung cancer patients, especially those with acquired EGFR-TKI resistance due to EGFR T790M gatekeeper mutation.

Nerve growth factor derived from bronchial epithelium after chronic mite antigen exposure contributes to airway hyperresponsiveness by inducing hyperinnervation, and is inhibited by in vivo siRNA.

BACKGROUND: Bronchial asthma is a chronic allergic airway inflammatory disease. Neurotrophins, including nerve growth factor (NGF), play an important role in the pathogenesis of asthma. However, the effects of NGF derived from epithelium on airway hyperresponsiveness (AHR) after antigen sensitization/exposure remain uncertain. OBJECTIVE: In this study, we examined the role of NGF on AHR after chronic antigen exposure and the effect of inhibiting NGF by in vivo siRNA on AHR exacerbation. METHODS: We generated chronic mouse models of bronchial asthma using house-dust mite antigen (Dermatophagoides pteronyssinus; Dp). NGF concentrations in bronchoalveolar lavage fluid (BALF), lung histopathology, hyperresponsiveness, and related neuronal peptides and cytokines in supernatants of lung homogenates were determined. RESULTS: NGF in BALF was increased in a dose- and time-dependent manner, and was expressed primarily in bronchial epithelium. Nerve fibres and substance P-positive fibres were detected in subepithelium of Dp-sensitized and challenged mice over 4 weeks of mite antigen exposure. AHR was positively correlated with NGF concentration and nerve fibre innervation. AHR, modulation of innervation, and increased substance P were inhibited by in vivo administration of siRNA that targeted NGF, although the inhibition of NGF did not affect allergic inflammation and subepithelial fibrosis. CONCLUSION AND CLINICAL RELEVANCE: These findings suggest that NGF derived from bronchial and alveolar epithelium plays an important role in AHR after chronic exposure to mite antigen. NGF inhibition could potentially manage bronchial asthma, including AHR.

Do biologics-naïve patients with rheumatoid arthritis respond better to tocilizumab than patients for whom anti-TNF agents have failed? A retrospective study.

OBJECTIVES: To determine responses to tocilizumab between patients with rheumatoid arthritis (RA) who switched to anti-TNF agents and those who are biologics-naïve. METHODS: This retrospective study investigated 107 patients with RA who were treated with tocilizumab. At baseline, 61 of them had already been treated with anti-TNF agents (switched group; 46 for inefficacy and 15 for adverse events), and 46 were biologics-naïve (naïve group). Treatment responses to tocilizumab at week 12 and 24 were compared between the switched and naïve groups using the disease activity score 28 (DAS28). RESULTS: Forty-two (91.3%) and 50 (82.0%) patients in the naïve and switched groups, respectively, completed 24 weeks of tocilizumab treatment. The DAS28-ESR and DAS28-CRP values (means±SD) at weeks 12 and 24 compared to baseline decreased significantly for the naïve and switched groups. The DAS28-ESR and DAS28-CRP values at weeks 12 and 24 were significantly decreased in the naïve group, compared to the switched group. Disease activity was improved in the naïve patients compared to the switched patients. CONCLUSIONS: Tocilizumab was safe, tolerable, and clinically effective for patients with inadequate responses to anti-TNF therapy and for those who were biologics-naïve, and it was more effective among the latter.

Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.

IκB kinase ß inhibitor IMD-0354 suppresses airway remodelling in a Dermatophagoides pteronyssinus-sensitized mouse model of chronic asthma.

BACKGROUND: Nuclear factor (NF)-κB is a transcription factor that regulates cytokine and chemokine production in various inflammatory diseases, including bronchial asthma. IκB kinase (IKK) ß is important for NF-κB activation in inflammatory conditions, and is possibly related to airway remodelling. Thus, inhibition of the IKKß-NF-κB pathway may be an ideal strategy for the management of airway remodelling. OBJECTIVE: We examined the effects of a newly synthesized IKKß inhibitor, IMD-0354, in a chronic allergen exposure model of bronchial asthma in mice. METHODS: A chronic mouse model was generated by challenge with house dust mite antigen (Dermatophagoides pteronyssinus). IMD-0354 was administrated intraperitoneally in therapeutic groups. Lung histopathology, hyperresponsiveness and the concentrations of mediators and molecules in supernatants of lung homogenates were determined. RESULTS: NF-κB activation was inhibited by prolonged periods of IMD-0354 administration. IMD-0354 reduced the numbers of bronchial eosinophils. IMD-0354 also inhibited the pathological features of airway remodelling, including goblet cell hyperplasia, subepithelial fibrosis, collagen deposition and smooth muscle hypertrophy. Inhibition of these structural changes by IMD-0354 was the result of the suppressing the production and activation of remodelling-related mediators, such as TGF-ß, via inhibition of IKKß. IMD-0354 inhibited IL-13 and IL-1ß production, and it restored the production of IFN-γ. It also ameliorated airway hyperresponsiveness. CONCLUSION: IKKß plays crucial roles in airway inflammation and remodelling in a chronic mouse model of asthma. A specific IKKß inhibitor, IMD-0354, may be therapeutically beneficial for treating airway inflammation and remodelling in chronic asthma.

A new method of preparing TRH derivative-loaded poly(dl-lactide-coglycolide) microspheres based on a solid solution system.

We investigated a new method of preparing peptide-loaded poly(dl-lactide-co-glycolide) microspheres with high encapsulation efficiency, low initial burst, and long-term sustained release by dissolving a peptide in a polymer by applying a solid solution system to the preparation of an oil phase. Solid solutions were prepared by dissolving a polymer (poly(dl-lactide-co-glycolide)) and a peptide (TRH derivative) in mixed solvents and then evaporating the solvents. Microspheres were prepared by an O/W emulsion solvent evaporation method, using the solution of the solid solution in dichloromethane as an oil phase. The state of the peptide in the solid solution and in the microspheres was evaluated by X-ray diffraction analysis. Release of the peptide from the microspheres was evaluated by an in vitro drug release test. Observation of the oil phase, X-ray diffraction analysis, and DSC analysis revealed that the peptides were dispersed in a molecular state in the solid solution and in microspheres with peptide loading of up to 15%. Encapsulation efficiency was over 90% for microspheres with peptide loading of up to 15%. The release of the peptide from the microspheres lasted over 21 days at least with the limited initial burst in vitro. High encapsulation efficiency, low initial burst, and long-term sustained release can be accomplished with microspheres prepared by a method based on a solid solution system.

CXCL9 and 11 in patients with pulmonary sarcoidosis: a role of alveolar macrophages.

Interferon-inducible protein-10 (IP-10)/CXCL10, which is a ligand for CXC chemokine receptor 3 (CXCR3), is known to be involved in the pathogenesis of pulmonary sarcoidosis. However, the roles of monokine induced by interferon gamma (Mig)/CXCL9 and interferon-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, which are also CXCR3 ligands, remain unclear. Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in both bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoidosis were measured by enzyme-linked immunosorbent assay (ELISA). The expression of these chemokines in alveolar macrophages was examined using ELISA, quantitative real-time polymerase chain reaction and immunostaining. In BALF, Mig/CXCL9 and IP-10/CXCL10 were significantly elevated in stage II sarcoidosis as compared with the levels in healthy volunteers. In serum, Mig/CXCL9 and I-TAC/CXCL11 were increased in stage II of the disease. The levels of all CXCR3 ligands in BALF were correlated with the numbers of both total and CD4(+) lymphocytes. Alveolar macrophages were stained positive for all CXCR3 ligands and produced increased amounts of these chemokines. Positive staining of the three chemokines was also observed in the epithelioid and giant cells in the sarcoid lungs. These findings suggest that Mig/CXCL9 and I-TAC/CXCL11 as well as IP-10/CXCL10 play important roles in the accumulation of Th1 lymphocytes in sarcoid lungs.

A case of chromomycosis caused by Fonsecaea pedrosoi and a review of reported cases of dematiaceous fungal infection in Japan.

We report a case of chromomycosis caused by Fonsecaea pedrosoi that developed in the left buttock of a 63-year-old female farmer. About 4 years ago, the patient developed erythema in the left buttock, which gradually spread. At the first consultation, we noted a well-defined, red-brown, infiltrated erythematous plaque (8 x 6 cm). Histopathological examination revealed a granulomatous lesion, containing sclerotic cells, associated with giant cells in the upper dermis. The causative fungus was difficult to identify due to low conidiogenesis, but was eventually identified by slide culture as F. pedrosoi. Excision and skin graft were performed, and no recurrence has been observed after 2 years. In Japan, 212 cases of dematiaceous fungal infection were reported in the period from 1982 to 2001. The causative fungus was F. pedrosoi in the majority of cases (126/212; 66%), followed by Exophiala jeanselmei (36/212; 19%). Similar incidence of dematiaceous fungal infection was reported in male and female patients. The upper limbs were affected most frequently in both male and female patients. Ten cases were associated with visceral lesions.

Adenoviral interleukin-12 gene transduction into human bronchial epithelial cells: up-regulation of pro-inflammatory cytokines and its prevention by corticosteroids.

BACKGROUND: One of the potential effects of IL-12 is to restore Th1/Th2 balance. Therefore, we investigated the possibility of developing a system for local delivery of IL-12 into the airways by examining protein expression in a human bronchial epithelial cell line (BEAS-2B) after adenoviral IL-12 gene transduction. The effects of dexamethasone on the gene-modified cells were also examined. METHODS: Adenoviral vectors AxCAegfp and Ax1CIhp40ip35 were used to transduce enhanced green fluorescence protein and IL-12 genes, respectively, into BEAS-2B cells. Wild-type and IL-12 gene-transduced BEAS-2B cells were then incubated with or without dexamethasone, and concentrations of IL-12, IFN-gamma, IL-6, IL-8, granulocyte macrophage-colony stimulating factor and chemokines (TARC and RANTES) in the supernatant were measured by ELISA. IL-12 receptor expression was analysed by flow cytometry and RT-PCR. RESULTS: The efficiency of transgene expression in BEAS-2B cells at a multiplicity of infection of 30 was approximately 80%. Gene-modified BEAS-2B cells produced biologically active IL-12, regardless of dexamethasone treatment. While IL-12 gene transduction led to increased production of IL-6 and IL-8 by BEAS-2B cells, expressions of these proteins were suppressed by dexamethasone. Addition of exogenous IL-12 failed to augment BEAS-2B cell IL-6 and IL-8 production, and IL-12 receptor expression by BEAS-2B cells was not detected. CONCLUSIONS: Our findings suggest that adenoviral IL-12 gene transduction may be effective in inducing IL-12 expression in the airways, and could be a potential approach in the management of bronchial asthma.

Efficient generation of dendritic cells from alveolar and pleural macrophages as well as blood monocytes in patients with lung cancer.

In this study, we investigated the generation of dendritic cells (DCs) from blood monocytes and mature macrophages from untreated primary lung cancer patients. Blood monocytes were separated by adherence from blood mononuclear cells (MNC) from ten lung cancer patients and ten control subjects, and cultured for 7 days in medium with granulocyte/macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL-) 4. In all cases examined, DCs with typical characteristics were obtained even in lung cancer patients after 7 days culture with these cytokines, and there was no significant difference in phenotype and stimulatory activity in allogeneic lymphocyte proliferation between DCs derived from monocytes from lung cancer patients and those from control subjects. Next, we examined whether alveolar and pleural macrophages in malignant pleural effusion separated by magnetic beads could differentiate to immunostimulatory DCs. Conventional culture conditions with GM-CSF and IL-4 did not induce efficient numbers of DCs from mature macrophages, whereas the addition of tumor necrosis factor-alpha (TNF-alpha) to GM-CSF and IL-4 effectively contributed to generate DCs. These findings suggest that both mature macrophages and blood monocytes from lung cancer patients could differentiate to DCs, and might be a useful source of DCs for immunotherapy.

Human monocyte-derived and CD83(+) blood dendritic cells enhance NK cell-mediated cytotoxicity.

Dendritic cells (DC) are known to be the most potent APC and to stimulate antigen-specific T cell responses. Recently it was reported that murine DC were also capable of modulating the innate immunity by stimulating NK cells through cell-to-cell contact. In the present study, we examined whether human DC could affect NK activity. Both monocyte-derived and CD83(+) blood DC were tested. The addition of DC to cultures of CD56(+) cells resulted in the significant dose-dependent enhancement of the killing activity against various NK-sensitive targets. The resultant activity was comparable to that induced by optimal concentrations of various cytokines, including IL-2, IL-12, IL-15 and IFN-gamma. Interestingly, DC enhanced the cytotoxicity of CD3(-)CD56(+) NK cells, but not that of CD3(+)CD56(+) T cells. Experiments using transwells clearly demonstrated that the enhancement of NK activity by DC was mediated by soluble factors produced by DC. The culture supernatants of DC also stimulated NK activity. The treatment of both DC and their supernatants with anti-human IL-12 or IL-18 antibodies did not block the enhancement of NK cell-mediated cytolysis by DC, indicating that other factor(s) produced by DC were responsible for the enhancement of NK activity. These results suggest that human myeloid DC can modulate innate immunity by enhancing NK activity.

A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells.

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.

Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen (TAA) or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection (MOI). In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein (EGFP)-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 x g at 37 degrees C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity (MFI) of EGFP expression was about 150 (at 37 degrees C for 2 h). With centrifugal transduction (2000 x g at an MOI of 50 at 37 degrees C for 2 h), 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced (MFI: about 3100 or greater). Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction (MLR). The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC.