Development of a complete human IgG monoclonal antibody to transferrin receptor 1 targeted for adult T-cell leukemia/lymphoma.

Iron is an essential nutrient for normal cell growth, and reprogramming of iron metabolism is essential to tumor cell survival and progression. HTLV-1-associated adult T-cell leukemia/lymphoma (ATLL) has no effective therapy and high levels of cell surface transferrin receptor 1 (TFR1) expression have been reported in ATLL by us and other groups. In this study, to develop a novel molecular-targeted therapy against TFR1 to modulate iron metabolism, we initially determined the expression pattern of several iron-related genes along with TFR1 and found that ATLL cells presented characteristic of an iron-deficiency state such as high expression of iron-regulatory protein 2 (IRP2) and low expression of its E3 ubiquitin-ligase, FBXL5. Therefore, we developed human IgG monoclonal antibodies to human TFR1 using a phage display method (ICOS method) to block the incorporation of the transferrin (TF)-iron complex into ATLL cells for inhibiting cell growth. One of the mAbs, JST-TFR09, presented its greater affinity to TFR1 on ATLL cells in flow cytometry (FCM) analysis than those of commercially available anti-TFR1 antibodies and identified high expression of TFR1 in most of the acute-type ATLL cells. Moreover, JST-TFR09 could interfere with binding between TFR1 and TF, which resulted in effective blockade of TFR1 internalization and induction of cell apoptosis by the treatment of ATLL cells with JST-TFR09. JST-TFR09 showed dual activities through direct cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), and the treatment of JST-TFR09 significantly suppressed cell growth of ATLL cells with induction of apoptosis in in vitro and in vivo experiments. Thus, JST-TFR09 described here may become a promising therapeutic antibody for the treatment of ATLL.

Development of a new minimally invasive phototherapy for lung cancer using antibody-toxin conjugate.

BACKGROUND: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and laser irradiation. The effectiveness of current PDT using red light for advanced cancers is limited, because red light can only reach depths within a few millimeters. To enhance the antitumor effect for lung cancers, we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin and a PS, mono-L-aspartyl chlorin e6 (NPe6). METHODS: We examined whether cetuximab encapsulated in endosomes was released into the cytosol by PS in PDT under light irradiation. A431 cells were treated with fluorescein isothiocyanate-labeled cetuximab, NPe6, and light irradiation and were observed with fluorescence microscopy. We analyzed the cytotoxicity of saporin-conjugated cetuximab (IT-cetuximab) in A431, A549, and MCF7 cells and the antitumor effect in model A549-bearing mice in vivo using the iTAP method. RESULTS: Fluorescent microscopy analysis showed that the photodynamic effect of NPe6 (20 µM) and light irradiation (37.6 J/cm2 ) caused the release of cetuximab from the endosome into the cytosol. In vitro analysis demonstrated that the iTAP method enhanced the cytotoxicity of IT-cetuximab by the photodynamic effect. In in vivo experiments, compared with IT-cetuximab alone or PDT alone, the iTAP method using a low dose of IT-cetuximab showed the greatest enhancement of the antitumor effect. CONCLUSIONS: Our study is the first report of the iTAP method using NPe6 for lung cancer cells. The iTAP method may become a new, minimally invasive treatment superior to current PDT methods.

The Human Anatomic Gene Expression Library (H-ANGEL), the H-Inv integrative display of human gene expression across disparate technologies and platforms.

The Human Anatomic Gene Expression Library (H-ANGEL) is a resource for information concerning the anatomical distribution and expression of human gene transcripts. The tool contains protein expression data from multiple platforms that has been associated with both manually annotated full-length cDNAs from H-InvDB and RefSeq sequences. Of the H-Inv predicted genes, 18 897 have associated expression data generated by at least one platform. H-ANGEL utilizes categorized mRNA expression data from both publicly available and proprietary sources. It incorporates data generated by three types of methods from seven different platforms. The data are provided to the user in the form of a web-based viewer with numerous query options. H-ANGEL is updated with each new release of cDNA and genome sequence build. In future editions, we will incorporate the capability for expression data updates from existing and new platforms. H-ANGEL is accessible at http://www.jbirc.aist.go.jp/hinv/h-angel/.

[A case of pulmonary tuberculosis complicated with tuberculosis of bilateral cervical lymph nodes and exacerbated pericostal abscess].

A 23-year-old man was admitted to our hospital because of cough and sputum in April 2001. A chest roentgenogram revealed infiltrative shadow with cavity formation in the bilateral lung fields. He was treated with sensitive antituberculous drugs. After starting the antituberculous therapy with INH, RFP, EB and PZA, bilateral cervical lymphadenopathy developed. Three months later, pericostal abscess appeared in the left anterior chest wall. Microscopic examination of the specimen obtained by needle aspiration biopsy disclosed positive for acid-fast bacilli. Smears of the pus showed acidfast bacilli identified as Mycobacterium tuberculosis by DNA-DNA PCR method. He developed tuberculous bilateral cervical lymphadenopathy and pericostal abscess during the course of antituberculosis chemotherapy. Drug sensitivity test revealed that tubercle bacilli in this case were sensitive. One year after the administration of chemotherapy, cervical lymphadenopathy and pericostal abscess were improved. Both masses were discontinuous with pulmonary tuberculosis and the possibility of lymphogenous spread of organism was speculated as its etiology. We assumed that both masses were due to paradoxical response to the antituberculosis chemotherapy.

Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC.

Pentraxin-3 in chronic heart failure: the CORONA and GISSI-HF trials.

AIMS: Pentraxin-3 (PTX3) is a component of the humoral arm of innate immunity which can regulate inflammatory processes. Since the role of inflammation in the progression of chronic heart failure (HF) is debated, we investigated the prognostic value of PTX3 and the effect of a statin in two large populations of patients with HF. METHODS AND RESULTS: Plasma levels of PTX3 were measured at randomization and after 3 months in 1457 patients enrolled in the Controlled Rosuvastatin Multinational Trial in HF (CORONA) and 1233 patients enrolled in the GISSI-Heart Failure trial (GISSI-HF). The relationships between baseline PTX3 levels or their changes over time and mortality were evaluated with multivariable Cox proportional hazard models including clinical factors, high sensitivity C-reactive protein (hsCRP), and N-terminal pro brain natriuretic peptide (NT-proBNP). PTX3 concentration [median (Q1-Q3) = 5.34 (3.55-7.64) ng/mL, n = 2690] was higher in females, in older patients, and those with lower body mass index. Baseline elevated PTX3 was associated with a higher risk of all-cause mortality [759 events, hazard ratio (HR) for 1 SD increase 1.20, 95% confidence interval (CI) 1.12-1.30, P < 0.0001], cardiovascular mortality (587 events, HR 1.27, 95% CI 1.17-1.38, P < 0.0001), or hospitalization for worsening HF (720 events, HR 1.21, 95% CI 1.12-1.30, P < 0.0001), and marginally improved discrimination. Three-month changes in PTX3 were associated with fatal events after adjustment for hsCRP or NT-proBNP. Rosuvastatin lowered hsCRP levels but significantly raised PTX3. CONCLUSION: In two independent clinical trials that enrolled patients with chronic HF, PTX3 was consistently associated with outcomes. The opposite effects of a statin on hsCRP and PTX3 call for further investigation. TRIAL REGISTRATION: NCT00336336 (GISSI-HF), NCT00206310 (CORONA).

Influence of pentraxin 3 (PTX3) genetic variants on myocardial infarction risk and PTX3 plasma levels.

PTX3 is a long pentraxin of the innate immune system produced by different cell types (mononuclear phagocytes, dendritic cells, fibroblasts and endothelial cells) at the inflammatory site. It appears to have a cardiovascular protective function by acting on the immune-inflammatory balance in the cardiovascular system. PTX3 plasma concentration is an independent predictor of mortality in patients with acute myocardial infarction (AMI) but the influence of PTX3 genetic variants on PTX3 plasma concentration has been investigated very little and there is no information on the association between PTX3 variations and AMI. Subjects of European origin (3245, 1751 AMI survivors and 1494 controls) were genotyped for three common PTX3 polymorphisms (SNPs) (rs2305619, rs3816527, rs1840680). Genotype and allele frequencies of the three SNPs and the haplotype frequencies were compared for the two groups. None of the genotypes, alleles or haplotypes were significantly associated with the risk of AMI. However, analysis adjusted for age and sex indicated that the three PTX3 SNPs and the corresponding haplotypes were significantly associated with different PTX3 plasma levels. There was also a significant association between PTX3 plasma concentrations and the risk of all-cause mortality at three years in AMI patients (OR 1.10, 95% CI: 1.01-1.20, p = 0.02). Our study showed that PTX3 plasma levels are influenced by three PTX3 polymorphisms. Genetically determined high PTX3 levels do not influence the risk of AMI, suggesting that the PTX3 concentration itself is unlikely to be even a modest causal factor for AMI. Analysis also confirmed that PTX3 is a prognostic marker after AMI.

Effects of direct and indirect activation of G protein of adenylyl cyclase on the subsequent response to beta-adrenergic receptor agonists in human trachealis.

To examine the response to beta-adrenergic receptor agonists (beta-agonists) following prolonged activation of the stimulatory G protein of adenylyl cyclase (Gs), relaxation by isoproterenol (isoprenaline, CAS 949-36-0) and formoterol (CAS 73573-87-2), a long-acting beta-agonist, after exposure to formoterol was measured in human tracheal smooth muscle, using isometric tension records. Prior exposure to formoterol (0.3-30 nmol/l) for 45 min reduced the subsequent relaxation induced by this drug in a concentration-dependent manner, but only modestly reduced that induced by isoproterenol. Next, the effects of cholera toxin (CTX, CAS 9012-63-9) an irreversible direct activator of Gs and formoterol on the reduced responsiveness to isoproterenol after continuous and repeated exposure to isoprotenerol were examined. Preincubation with cholera toxin (0.02-2 micrograms/ml) caused concentration-dependent inhibition of the desensitization induced by isoproterenol, but preincubation with formoterol did not. These results indicate that prolonged activation of Gs via beta-adrenergic receptors does not cause cross-desensitization to short-acting beta-agonists. However, it also fails to inhibit the desensitization of beta-adrenergic receptors after excessive exposure to short-acting beta-agonists. Activation of Gs via a pathway that bypasses the receptors may be beneficial for the prevention of this phenomenon.