Circulating tumor necrosis factor-related biomarkers predict kidney function decline in Japanese patients with diabetes: An observational cohort study.

AIMS: Tumor necrosis factor (TNF) receptors (TNFRs: TNFR1 and, TNFR2) are reportedly associated with chronic kidney disease (CKD) progression chiefly in Caucasian patients with diabetes. We assessed the prognostic value of TNF-related biomarkers for CKD progression in Japanese patients with diabetes. METHODS: We estimated TNF-related biomarkers using an enzyme-linked immunosorbent assay in 640 patients with diabetes. Cox proportional hazards analysis was performed to estimate hazard ratios (HRs) per one standard deviation (SD) increase in a log-transformed biomarker. The kidney and the composite outcome were defined as a 30% reduction in estimated glomerular filtration rate (eGFR) from baseline, and kidney outcome plus death before kidney outcome, respectively. RESULTS: During the median follow-up of 5.4 years, 75 (11.7%) patients reached the kidney outcome and 37 (5.8%) died before reaching the kidney outcome. Each SD increase in baseline circulating TNFR1, TNFR2, and ephrin type-A receptor 2 (EphA2) was associated with a higher risk of the kidney outcome independently from baseline eGFR and urine albumin-to-creatinine ratio. However, circulating osteoprotegerin was associated with the composite outcome only. CONCLUSIONS: Elevated TNFR1, TNFR2, and EphA2 were associated with both kidney and composite outcomes in Japanese patients with diabetes.

Novel [ 111 In]In-BnDTPA-EphA2-230-1 Antibody for Single-Photon Emission Computed Tomography Imaging Tracer Targeting of EphA2.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) is overexpressed in cancer cells and causes abnormal cell proliferation. Therefore, it has attracted attention as a target for diagnostic agents. In this study, the EphA2-230-1 monoclonal antibody (EphA2-230-1) was labeled with [111In]In and evaluated as an imaging tracer for single-photon emission computed tomography (SPECT) of EphA2. EphA2-230-1 was conjugated with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-BnDTPA) and then labeled with [111In]In. [111In]In-BnDTPA-EphA2-230-1 was evaluated in cell-binding, biodistribution, and SPECT/computed tomography (CT) studies. The cellular uptake ratio of [111In]In-BnDTPA-EphA2-230-1 was 14.0 ± 2.1%/mg protein at 4 h in the cell-binding study. In the biodistribution study, a high uptake of [111In]In-BnDTPA-EphA2-230-1 was observed in tumor tissue (14.6 ± 3.2% injected dose/g at 72 h). The superior accumulation of [111In]In-BnDTPA-EphA2-230-1 in tumors was also confirmed using SPECT/CT. Therefore, [111In]In-BnDTPA-EphA2-230-1 has potential as a SPECT imaging tracer for EphA2.

Development of an In-House EphA2 ELISA for Human Serum and Measurement of Circulating Levels of EphA2 in Hypertensive Patients with Renal Dysfunction.

Identifying novel biomarkers of kidney function in patients with chronic kidney disease (CKD) has strong clinical value as current measures have limitations. This study aims to develop and validate a sensitive and specific ephrin type-A receptor 2 (EphA2) enzyme-linked immunosorbent assay (ELISA) for human serum, and determine whether its results correlate with traditional renal measures in patients with hypertension. The novel ELISA of the current study was validated and used to measure circulating EphA2 levels in 80 hypertensive patients with and without kidney function decline (eGFR less than 60 mL/min/1.73 m2). Validation of the EphA2 ELISA showed good recovery (87%) and linearity (103%) and no cross-reactivity with other Eph receptors. Patients with kidney function decline had lower diastolic blood pressure, and higher UPCR and EphA2 than those without kidney function decline. The association of age and eGFR with EphA2 was maintained in the stepwise multiple regression analysis. In a multivariate logistic model, EphA2 was associated with a lower eGFR (<60 mL/min/1.73 m2) after adjustment for age, sex, and UPCR. High circulating EphA2 levels have potential application as a clinical biomarker for the presence of CKD in patients with hypertension.

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.

Altered binding avidities and improved growth inhibitory effects of novel anti-HER3 mAb against human cancers in the presence of HER1-or HER2-targeted drugs.

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (KA) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The KA of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of KA, and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high KA by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI-H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high KA by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI-H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI-H1838, the reactivity and KA of Ab4 increased compared with in parent NCI-H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of KA with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116.

Serum levels of galectin-3 in idiopathic inflammatory myopathies: a potential biomarker of disease activity.

OBJECTIVES: Galectin-3 is involved in various biological activities, including immune activations and fibrosis. Idiopathic inflammatory myopathies (IIMs) are autoimmune diseases of unknown aetiology, often complicated by interstitial lung disease (ILD). The aim of this study was to evaluate the expression of galectin-3 in sera and tissues of patients with IIM and assess the associations of galectin-3 with patient characteristics and disease activity. RESULTS: Serum galectin-3 levels were significantly higher in IIM patients than in healthy controls. The serum galectin-3 levels positively correlated with serum levels of inflammatory markers and proinflammatory cytokines/chemokines and the Myositis Intention-to-Treat Activity Index. Stratification analysis revealed that patients with IIM-associated ILD (IIM-ILD) had significantly higher levels of serum galectin-3 than those without IIM-ILD. In addition, patients with acute/subacute interstitial pneumonia had significantly higher levels of serum galectin-3 than those with chronic interstitial pneumonia. Furthermore, serum galectin-3 levels in IIM-ILD patients correlated with the radiological assessments of parenchymal lung involvement and treatment response. Immunohistochemical analysis revealed that galectin-3 was expressed in inflammatory cells of myositis and dermatitis sections, whereas in ILD sections, galectin-3 was expressed in interstitial fibrosis and inflammatory cells. CONCLUSION: Galectin-3 may be involved in the pathogenesis of inflammatory and fibrotic conditions in IIM and can serve as a potential biomarker of disease activity, especially in patients with IIM-ILD.

Habitual cigarette smoking attenuates shear-mediated dilation in the brachial artery but not in the carotid artery in young adults.

In the present study, we hypothesized that habitual cigarette smoking attenuates endothelial function in the cerebral circulation as well as that of the peripheral circulation in young adults. To test this hypothesis, we measured cerebrovascular and peripheral flow-mediated dilation (FMD) in young smokers and nonsmokers in the present study. Ten healthy nonsmokers and 10 smokers participated in the study. We measured blood velocity and diameter in the brachial artery and internal carotid artery (ICA) using Doppler ultrasound. We identified shear-mediated dilation in the brachial artery and ICA by the percentage change in peak diameter during hyperemia stimulation (reactive hyperemia and hypercapnia). We measured the baseline diameter and the shear rate area under the curve from the onset of hyperemia to peak dilation in the brachial artery and ICA, finding the measurements of the smokers and those of the nonsmokers did not differ (p > .05). In contrast to brachial FMD (5.07 ± 1.79% vs. 7.92 ± 3.01%; smokers vs. nonsmokers, p = .019), FMD in the ICA was not attenuated in the smokers compared with that of the nonsmokers (5.46 ± 2.32% vs. 4.57 ± 2.70%; p = .442). These findings indicate that in young healthy smokers, cerebral endothelial function was preserved, and the response of cerebral endothelial function to smoking was different from that of peripheral vasculature.

Combination Effect of Bowman-Birk Inhibitor and α-Tocopheryl Succinate on Prostate Cancer Stem-Like Cells.

The reoccurrence of androgen-dependent prostate cancer after anti-androgen therapy mainly depends on prostate cancer stem-like cells. To reduce the risk, it is important to delete the cancer stem-like cells. Furthermore, to induce differentiation of cancer stem-like cells is critical to abrogate stemness of the cells. Therefore, we tried to investigate a possibility on the establishment of a new effective therapy to eradicate the cancer stem-like cells via the induction of differentiation in this study. Prostate cancer stem-like cells from an androgen-dependent prostate cancer cell line (LNCaP cell) had severe resistance against an anti-androgen therapeutic agent. We selected Bowman-Birk inhibitor (BBI) from soybeans reported as a chemopreventive agent in prostate cancer to differentiate the caner stem-like cells and α-tocopheryl succinate (TOS) known as a mitocan to induce effectively cytotoxic effect against the cancer stem-like cells. In fact, only TOS treatment had cytotoxic effect against the cancer stem-like cells, but the addition of BBI treatment to the cells treated with TOS reinforced TOS-mediated cytotoxicity in the cancer stem-like cells. This reinforcement coincided with the combination-enhanced apoptosis in the stem-like cells. Also, we confirmed caspase9-caspase3 cascade mainly contributed to the enhancement of the cytotoxicity in the stem-like cells caused by the combination, indicating that the reinforcement of BBI on TOS-mediated apoptosis via mitochondria related to the enhancing cytotoxic effect of the combination on the prostate cancer stem-like cells. Overall, it seems that the combination is an effective new approach to reduce the reoccurrence of prostate cancer targeting prostate cancer stem cells.

Advanced microscopic evaluation of parallel type I and type II cell deaths induced by multi-functionalized gold nanocages in breast cancer.

Despite aggressive surgical resections and combinatorial chemoradiations, certain highly malignant populations of tumor cells resurrect and metastasize. Mixed-grade cancer cells fail to respond to standard-of-care therapies by developing intrinsic chemoresistance and subsequently result in tumor relapse. Macroautophagy is a membrane trafficking process that underlies drug resistance and tumorigenesis in most breast cancers. Manipulating cellular homeostasis by a combinatorial nanotherapeutic model, one can evaluate the crosstalk between type I and type II cell death and decipher the fate of cancer therapy. Here, we present a multi-strategic approach in cancer targeting to mitigate the autophagic flux with subcellular toxicity via lysosome permeation, accompanied by mitochondrial perturbation and apoptosis. In this way, a nanoformulation is developed with a unique blend of a lysosomotropic agent, an immunomodulating sulfated-polysaccharide, an adjuvant chemotherapeutic agent, and a monoclonal antibody as a broad-spectrum complex for combinatorial nanotherapy of all breast cancers. To the best of our knowledge, this manuscript illustrates for the first time the applications of advanced microscopic techniques such as electron tomography, three-dimensional rendering and segmentation of subcellular interactions, and fate of the multifunctional therapeutic gold nanocages specifically targeted toward breast cancer cells.

An Agonistic Antibody to EPHA2 Exhibits Antitumor Effects on Human Melanoma Cells.

BACKGROUND/AIM: EPH receptor A2 (EPHA2) is highly expressed in aggressive types of human cancer, and is expected to be an excellent target molecule for antibody treatments. In this study, we investigated the therapeutic potential of antibody to EPHA2 against melanoma in vitro. MATERIALS AND METHODS: We generated three monoclonal antibodies (mAbs) to EPHA2 and examined cell-surface expression by flow cytometry. To investigate the ability to inhibit tumor cell migration therapy with mAbs to EPHA2, we performed a wound scratch assay and invasion assay. We investigated the therapeutic effects of immunotoxins consisting of toxin-conjugated EPHA2 mAbs. RESULTS: All human melanoma cell lines studied expressed EPHA2. Like natural ligand ephrin-A1, one of EPHA2 mAbs, SHM16, inhibited metastatic behavior of cells, such as migration and invasion. In addition, drastic growth inhibition and cytotoxicity were found using immunotoxin-conjugated SHM16. CONCLUSION: These observations indicate a promising role for EPHA2 as a target in antibody treatments for melanoma, and demonstrate the potential therapeutic effects of an agonistic antibody to EPHA2.

Trapping and proliferation of target cells on C60 fullerene nano fibres.

The ratio of the surface area to the volume of materials increases in inverse proportion to their size and therefore the surface area of nanostructures and nanomaterials is extremely large compared to that of macroscopic materials of the same volume, thanks to which it is supposed that chemical and biochemical reactions may be greatly enhanced and target molecules and cells may be efficiently trapped on the surface of nanomaterials. It is well known that C60 molecules are stable both physically and chemically and the affinity of C60 molecules with biomolecules is rather high. Here, we synthesise fibres composed of C60 and sulphur and immobilise the surface of the fibres with the primary antibody; i.e., epithelial cell adhesion molecules (anti-EpCAM), to trap target cells. The primary antibody is evenly immobilised on the fibres confirmed by a fluorescent secondary antibody attached to the primary one and then TE2 esophageal and DLD-1 colon cancer cells are successfully trapped by the primary antibody immobilised on the fibres thanks to its high affinity with TE2 and DLD-1 cells, whereas few IM9 B lymphoblast cells are captured on the fibres since the affinity of the primary antibody with IM9 cells is extremely low. Furthermore, those cells trapped by the primary antibody immobilised on the fibres proliferate faster than native cells thanks to the primary antibody acting as a growth factor. The present result suggests that different types of cells can be trapped and grown on nano fibres by immobilising appropriate antibody molecules on the surface of the fibres. Even an extremely small number of cells in sample fluids may be analysed and characterised for the detection of diseases such as cancer in the early stage by trapping and proliferating target cells on the fibres.

Elevated serum levels of soluble CD146 in patients with systemic sclerosis.

CD146, a transmembrane glycoprotein member of the immunoglobulin superfamily, acts as an adhesion molecule that helps maintain the cell monolayer. Human endothelial cells expressing CD146 are involved in angiogenesis and inflammation. Recently, we developed a sandwich ELISA for detecting soluble CD146 (sCD146) in human serum specimens. The aim of this study is to determine serum levels of sCD146 in patients with systemic sclerosis (SSc) and to examine the relationship between sCD146 levels and clinical manifestations. We quantified serum sCD146 levels in 47 serum samples from patients fulfilling criteria for SSc, 23 serum samples from patients fulfilling criteria for rheumatoid arthritis (RA), and 25 healthy controls. We also investigated the relationship between sCD146 levels and various clinical characteristics with SSc patients. Levels of sCD146 were significantly higher in the 47 patients with SSc than in the 25 healthy controls and 23 patients with RA (12.50 vs. 6.91 vs. 9.95 ng/ml; p < 0.001). Serum sCD146 levels in SSc patients with pulmonary arterial hypertension (PAH) were lower than in SSc patients without PAH (10.12 vs.13.17 ng/ml; p < 0.01). The serum levels of sCD146 were elevated in patients with SSc. However, decreased sCD146 levels were observed in SSc patients with PAH. Further studies are necessary to elucidate the sources and the mechanisms.

Cell Surface Antibody Retention Influences In Vivo Antitumor Activity Mediated by Antibody-dependent Cellular Cytotoxicity.

BACKGROUND: Multiple factors affect the in vivo antitumor activity of antibody-based therapeutics; however, the influence of cell surface retention on antibody-dependent cellular cytotoxicity (ADCC) is not fully understood. Here we evaluated the importance of cell surface antibody retention in antitumor activity mediated by ADCC in vivo. MATERIALS AND METHODS: Two mAbs against tumor-associated calcium signal transducer 2 (TACSTD2/TROP2), AR47A6.4.2 and Pr1E11, were used. Antitumor activities against BxPC3 and Colo205 cells were investigated through in vitro and in vivo assays. RESULTS: Pr1E11 showed better cell surface retention than AR47A6.4.2 in vitro although Pr1E11 and AR47A6.4.2 showed equivalent ADCC activity. Complement-dependent cytotoxicity and antiproliferative activity were not observed for either antibody. Pr1E11 exhibited higher antitumor activity than AR47A6.4.2 in vivo. CONCLUSION: Our results suggest that high cell surface retention can result in potent ADCC activity in vivo. This observation could provide novel insight into how effectively screen for antibodies with strong in vivo antitumor activity.

Aminopeptidase N (APN/CD13) as a target molecule for scirrhous gastric cancer.

BACKGROUND: Scirrhous gastric cancer is associated with peritoneal dissemination and advanced lymph node metastasis from an early stage, and the prognosis is still poor. In this study, we aimed to analyze candidate molecules for targeted therapy of scirrhous gastric cancer. We searched for molecules/metabolic activity that might be predominantly expressed in a subpopulation of scirrhous gastric cancer cells and might function as cancer stem cell markers. RESULTS: For this purpose, we investigated the expression of various cell surface markers and of aldehyde dehydrogenase (ALDH) activity. These analyses showed that the scirrhous gastric cancer cell lines HSC-58 and HSC-44PE heterogeneously expressed CD13, while CD44, CDCP1, EpCAM and ABCG2 were expressed uniformly. Moreover, 10% of the total HSC-58 cell population expressed ALDH enzyme activity. A subpopulation of cells strongly positive for ALDH also expressed high levels of CD13, both of which are known as cancer stem cell markers. HSC-58 cells expressing high levels of CD13 showed lower sensitivity to a cancer drug cisplatin than cells with low levels of CD13. In contrast, CD13(-high) subpopulation of HSC-58 was more sensitive to an aminopeptidase N inhibitor bestatin. In terms of antibody-drug therapy, anti-CD13-immunotoxin was highly cytotoxic towards HSC-58 cells and was more cytotoxic than anti-EpCAM-immunotoxin. CONCLUSION: These data suggest that CD13 is a suitable cell surface candidate for targeted antibody-drug therapy of scirrhous gastric cancer.

Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope.

TROP-2 is a type Ⅰ transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments.

[A novel screening method to establish tumor-targeting antibodies reliable for drug delivery system].

Establishment of a system that allows selective drug delivery and gene silencing to a tumor is expected to enable targeted therapy. We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33 (Adv-FZ33). By cross-linking the Adv-FZ33 virus and the surface antigen molecules with the targeting monoclonal antibodies (mAbs), we attained highly enhanced gene deliveries into the respective antigen-positive cancer cells. Therefore, we aimed to establish a systematic screening method to search for antibody and cell surface target candidates that would provide highly selective anti-cancer reagents to malignant tumors. Using an Adv-FZ33, hybridoma libraries producing a variety of mAbs for human pancreatic, prostate, lung or ovarian carcinoma cells were screened, and we were able to selectively obtain several mAbs which had potent high affinity and recognized antigens of high structure. Within these mAbs, we have identified tumor cell target molecules including not only carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), prostate specific membrane antigen (PSMA) but also novel tumor surface target molecules such as phosphatidic acid phosphatase type 2a (PAP2a) and interleukin-13 receptor variant α2 (IL-13Rα2) as tumor antigens. Overall, these results indicate that this type of inductive method approach is a reliable strategy for screening in antibody therapy on par with antibody-dependent drug-delivery system.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines.

In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.

Elicitation of both anti HIV-1 Env humoral and cellular immunities by replicating vaccinia prime Sendai virus boost regimen and boosting by CD40Lm.

For protection from HIV-1 infection, a vaccine should elicit both humoral and cell-mediated immune responses. A novel vaccine regimen and adjuvant that induce high levels of HIV-1 Env-specific T cell and antibody (Ab) responses was developed in this study. The prime-boost regimen that used combinations of replication-competent vaccinia LC16m8Δ (m8Δ) and Sendai virus (SeV) vectors expressing HIV-1 Env efficiently produced both Env-specific CD8(+) T cells and anti-Env antibodies, including neutralizing antibodies (nAbs). These results sharply contrast with vaccine regimens that prime with an Env expressing plasmid and boost with the m8Δ or SeV vector that mainly elicited cellular immunities. Moreover, co-priming with combinations of m8Δs expressing Env or a membrane-bound human CD40 ligand mutant (CD40Lm) enhanced Env-specific CD8(+) T cell production, but not anti-Env antibody production. In contrast, priming with an m8Δ that coexpresses CD40Lm and Env elicited more anti-Env Abs with higher avidity, but did not promote T cell responses. These results suggest that the m8Δ prime/SeV boost regimen in conjunction with CD40Lm expression could be used as an immunization platform for driving both potent cellular and humoral immunities against pathogens such as HIV-1.

Heme oxygenase-1 (HO-1) is constitutively up-regulated in top alpinists.

Alpinists who challenge Mt. Everest need adaptation to hypoxia before the attack of Mt. Everest. Although this adaptation is important for the success of climbing Mt. Everest, the molecular mechanism on the adaptation to hypoxia is not well understood. In order to clarify this mechanism, we investigated hypoxia-induced gene expressions specific for top alpinists using microarray analyses. We report here that heme oxygenase-1 (HO-1) is significantly higher in the blood of top alpinist compared with non-alpinists. Although HO-1 expression of non-alpinists is also up-regulated in response to hypoxia, HO-1 level of the top alpinists are constitutively higher than that of non-alpinists. Serial examinations of HO-1 in one top alpinist revealed that the higher expression of HO-1 is maintained in high-level several months after the attack of top mountains. Taken together with the biochemical function of HO-1 that catalyzes heme into CO and bilirubin, HO-1 expression may improve the circulation and compensate with oxidative tissue damages induced by hypoxia. These data also suggest that peripheral blood has the memory on hypoxia independent of antigens by maintaining the high-level of HO-1 expression in top alpinists, which merits the rapid adaptation to hypoxia for 8000m climbing.

Epigenetic modulation enhances the therapeutic effect of anti-IL-13R(alpha)2 antibody in human mesothelioma xenografts.

PURPOSE: The interleukin-13 receptor α2 (IL-13Rα2) is expressed by a variety of human malignant cells. Here, we have examined the constitutive surface expression and the epigenetic regulation of IL-13Rα2 by human mesothelioma. We have also investigated the therapeutic effect of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) and anti-IL-13Rα2 monoclonal antibody on mesothelioma xenografts. EXPERIMENTAL DESIGN: Cell surface expression of IL-13Rα2 by various lung carcinomas was analyzed using flow cytometry. Therapeutic effects of anti-IL-13Rα2 and 5-aza-dC were investigated using antibody-dependent cellular cytotoxicity and proliferation assays and by monitoring the survival of mesothelioma-bearing mice. RESULTS: We found that human malignant mesotheliomas expressed surface IL-13Rα2 on their surface and that it was upregulated by treatment with 5-aza-dC. This augmented expression of IL-13Rα2 resulted in growth inhibition of the mesothelioma cells when cocultured with anti-IL-13Rα2 and effector cells, such as splenocytes and peritoneal exudate cells. The growth inhibition of mesothelioma cells was mediated by IFN-γ that was only detected in the supernatant when effector cells were exposed to 5-aza-dC-treated tumors in the presence of anti-IL-13Rα2. Compared with the control or either regimen alone, in vivo administration of anti-IL-13Rα2 in combination with 5-aza-dC significantly prolonged the survival of mice with mesothelioma xenografts. CONCLUSIONS: These observations indicate a promising role for IL-13Rα2 as a target for antibody treatment in malignant mesothelioma, and, in combination with epigenetic regulation by a DNA methylation inhibitor, suggest the potential for a novel strategy to enhance therapeutic potency.