Naoki Nishimura

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‐γ. 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.

Enhanced efficiency by centrifugal manipulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells.

Transduction of dendritic cells (DCs) with genes encoding tumor-associated antigen or with other genes that enhance immune reaction has been theorized to be potentially useful for enhancing the efficiency of DC-based immunotherapy. However, gene transduction of DCs generated from human peripheral blood monocytes has been of limited use because of the low efficiency. Here, we report that the efficiency of in vitro adenovirus-mediated gene transduction into human monocyte-derived DCs can be dramatically enhanced by centrifugation. The best conditions for centrifugal gene transduction were determined to be as follows: 2000 x g at 37 degrees C for 2 hr at a multiplicity of infection (MOI) of 10 or greater. By this centrifugal method, approximately 88 and 70% of DCs were gene transducible at an MOI of 50 and 10, respectively. Functional analysis showed that DCs transduced with human interleukin 12 (IL-12)-expressing adenoviral vector under the optimal conditions of centrifugation stably produced IL-12 protein at high levels (8.1 ng/10(6) cells/48 hr). IL-12 gene-modified DCs (DC/IL-12) displayed a more mature phenotype than nontransduced DCs, as judged by decreased expression of CD1a and increased expression of CD83, B7.1 (CD80), B7.2 (CD86), and MHC class I and II molecules. DC/IL-12 showed a high phagocytic ability similar to nontransduced DCs and were significantly superior to control DCs in the stimulation of autologous and allogeneic T lymphocyte responses. The centrifugal transduction method with adenoviral vector might be useful for efficient generation of gene-modified DCs because it is very simple, highly efficient, reproducible, and not cytopathic. IL-12 gene-modified human DCs may be therapeutically useful as a good adjuvant in DC-based immunotherapy.

Cytolysis of human dendritic cells by autologous lymphokine-activated killer cells: participation of both T cells and NK cells in the killing

Dendritic cells (DC) play a key role in the initiation of immune response by stimulating the naive T cells. The fate of DC after the initiation of immune response is not clearly understood. Although there are few reports implicating natural killer (NK) cells in the elimination of DC, killing of DC by LAK cells, and specifically by T cells, has not been studied. In this study, we observed that DC, generated from monocytes, in vitro in the presence of granulocyte‐macrophage colony‐stimulating factor, interleukin‐4 (IL‐4), and tumor necrosis factor α were susceptible to cytolysis by lymphokine‐activated killer (LAK) cells induced in the presence of IL‐2 and IL‐15 but not IL‐12 alone. However, LAK cells induced by a combination of IL‐12 and suboptimal dose of IL‐2 were cytotoxic to DC. When purified lymphocytes were activated with IL‐2, the CD8+/CD57− fraction (T‐LAK), but not the CD8−/CD57+ fraction (NK‐LAK) was cytotoxic to autologous DC. However, when unseparated peripheral blood mononuclear cells were used to generate LAK cells, both T‐LAK and NK‐LAK fractions showed equal cytotoxicity against autologous DC. Monoclonal antibodies against CD54, CD11a, and CD18 significantly inhibited the cytolysis, indicating that the killing involves the engagement of CD54 with its ligands. J. Leukoc. Biol. 65: 764–770; 1999.

Adrenal function in patients with community-acquired pneumonia

Adrenal insufficiency is believed to occur frequently in severe sepsis and septic shock. The aim of the present study was to determine whether adrenal function is also related to the severity of community-acquired pneumonia (CAP). In total, 64 Japanese patients with CAP were consecutively enrolled in the present study, which was carried out during 2005–2006. Serum adrenocorticotropic hormone (ACTH) and cortisol were measured in each subject, as was the response of cortisol secretion when 250 μg of cosyntropin was administered. Analyses were performed comparing these values with the score calculated according to the Pneumonia Patient Outcomes Research Team (PORT) cohort study, the number of in-hospital deaths and the length of hospital stay. As the PORT score increased, serum ACTH and cortisol also increased, while the response of cortisol secretion to the administration of cosyntropin decreased. In the analysis by receiver operating characteristic curves, adrenal dysfunction was related significantly to both the number of in-hospital deaths and the length of hospital stay. Adrenal dysfunction was shown to correlate with the Pneumonia Patient Outcomes Research Team score and the clinical outcomes, while adrenal insufficiency defined by the cosyntropin stimulation test was rare in the present study.

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.

Transduction of KAI1/CD82 cDNA promotes hematogenous spread of human lung-cancer cells in natural killer cell–depleted SCID mice

KAI1, which is identical to CD82, was initially identified as a metastasis‐suppressor gene for human prostate cancer, and its expression is reported to be a favorable prognostic factor for operable human lung cancer. In this study, we examined the functional role of KAI1/CD82 in the late phase of metastatic spread of human lung‐cancer cells. For this, KAI1/CD82 cDNA was introduced into KAI1/CD82 low‐expressing human lung‐cancer cell lines, SBC‐3 and PC‐14, and then the metastatic potential of the transformants was analyzed by i.v. inoculation of KAI1/CD82‐transduced cells, SBC‐3/KAI1 and PC‐14/KAI1, into NK cell–depleted SCID mice. Contrary to our expectations, KAI1/CD82 gene transfer promoted multiorgan metastasis of i.v.‐inoculated human lung‐cancer cells, while s.c. tumor growth was unaffected. Cancer cells from metastatic tumors of NK cell–depleted SCID mice injected i.v. with SBC‐3/KAI1 expressed appreciable cell‐surface KAI1/CD82, and cells not expressing KAI1/CD82 (revertants) were not detected in the tumors. Our findings indicate that under conditions where the hosts natural cytotoxicity is suppressed, KAI1/CD82 may enhance the formation of tumors by circulating lung‐cancer cells at metastatic sites.

A Case of Pulmonary Tumor Thrombotic Microangiopathy Diagnosed by Transbronchial Lung Biopsy and Treated with Chemotherapy and Long-Term Oxygen and Anticoagulation Therapies

A 41-year-old woman, who underwent breast resection for cancer of the right breast and adjuvant chemotherapy 2 years ago, was admitted to our hospital due to shortness of breath upon exertion. High-resolution computed tomography of the chest showed small nodular opacities in the peribronchiolar area in both lungs, as well as mediastinal and hilar lymphadenopathy. A transbronchial lung biopsy revealed breast cancer metastasis and pulmonary tumor thrombotic microangiopathy (PTTM). Treatment of PTTM is rarely reported due to the difficulty of antemortem diagnosis; however, the patient was effectively treated with chemotherapy and oxygen and anticoagulation therapies for 3 months.

Effect of Switching Tiotropium HandiHaler® to Respimat® Soft Mist™ Inhaler in Patients with COPD: The Difference of Adverse Events and Usability Between Inhaler Devices

BACKGROUND Recently, tiotropium Respimat® Soft Mist™ Inhaler has been developed. Respimat is a multidose and propellant-free kit. The aerosol generated from Respimat improved lung drug deposition and required a lower dose of drug than HandiHaler®. The aim of this study is to assess the effect of switching from tiotropium HandiHaler to Respimat in patients with chronic obstructive pulmonary disease (COPD). METHODS Thirty-four patients with COPD who received 18 μg of tiotropium delivered by the HandiHaler once daily were enrolled in this study between May and September 2010. Symptoms, adverse events, pulmonary functions, and usability of inhaler devices were assessed before and 12 weeks after switching from HandiHaler to 5 μg of tiotropium delivered by the Respimat. Symptoms and adverse events were also assessed 4 and 12 weeks after switching. Dyspnea was evaluated using the British Medical Research Council dyspnea scale. The usability of inhaler devices was scored using a 12-step checklist. RESULTS Twenty-nine patients were followed until 12 weeks after switching. The median FEV1 (forced expiratory volume in 1 sec) values before and 12 weeks after switching to Respimat were 1.41 L and 1.60 L, respectively. Dry mouth appeared to improve after switching to Respimat. Cough just after inhalation was observed in seven patients until 4 weeks after switching. However, six patients overcame cough as they got used to Respimat. Regarding the handling of inhaler devices, patients were not good at breathing out before inhalation and holding their breath just after inhalation both with HandiHaler and with Respimat. However, in general, both inhalers were considered to be easy to use. Twenty-one patients replied that handling of Respimat was easier than that of HandiHaler. CONCLUSIONS There was no major problem in switching from tiotropium HandiHaler to Respimat. Respimat and HandiHaler showed similar effects and usability. However, we should be aware of cough just after inhalation with Respimat.

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.

Combined therapy with anti-p-glycoprotein antibody and macrophage colony-stimulating factor gene transduction for multiorgan metastases of multidrug-resistant human small cell lung cancer in nk cell-depleted scid mice

Our aim was to determine the antimetastatic potential of anti‐P‐glycoprotein (P‐gp) antibodies (Abs) against multidrug‐resistant (MDR) human small cell lung cancer (SCLC) cells expressing P‐gp. Human SCLC cells H69 (P‐gp negative) and its etoposide‐resistant variant H69/VP (P‐gp positive) were used. H69 and H69/VP cells injected i.v. metastasized to the liver, kidneys and systemic lymph nodes of NK cell‐depleted severe combined immunodeficient (SCID) mice. H69/VP cells, but not H69 cells, were resistant to treatments with vindesine. Treatment with mouse‐human chimeric anti‐P‐gp Ab (MH162) and its mouse counterpart (MRK‐16) reduced metastasis of H69/VP cells in various organs and prolonged the survival of tumor‐bearing mice, although they were less effective if injected at late times (after 28 days). Treatment with another mouse anti‐Pgp Ab, MRK‐17, was effective only against liver metastasis. MH162 and MRK‐16 efficiently induced Ab‐dependent cellular cytotoxicity (ADCC) by peritoneal macrophages against H69/VP cells in vitro, but MRK‐17 was less effective, in accordance with their in vivo antimetastatic potential. Gene transfection of macrophage colony‐stimulating factor (M‐CSF) into H69/VP cells to augment macrophage‐mediated ADCC resulted in inhibition of metastasis to the liver and lymph nodes, but not kidneys. Combined treatment with a low dose of MRK‐16 completely cured metastasis of M‐CSF transfectant, but not of the mock transfectant. Our findings suggest that while anti‐P‐gp Abs had antimetastatic potential against SCLC cells expressing P‐gp, combined treatment with M‐CSF gene transduction to augment the therapeutic efficacy of anti‐P‐gp Abs may be beneficial for eradicating metastatic MDR SCLC in humans. Int. J. Cancer 82:105–111, 1999.