Glycosylation profiles of breast cancer cells may represent clonal variations of multiple organ metastases.

Glycosylation changes of cancer cells are known to be associated with malignant progression and metastases and potentially determine the organ-selective nature of metastasis as theorized by Paget (Lancet 1:571-573, 1889). Cellular glycans play a variety of roles in the processes of metastasis and may be unique to the cells that metastasize to different organs. We analyzed the glycosylation profiles of the primary tumor and tumors metastasized to lymph node, liver, lung, brain, bone, thyroid, kidney, adrenal, small intestine and pancreas in an autopsy case of breast cancer employing a lectin microarray with 45 lectins. Clustering analysis of the data revealed that metastatic breast cancer cells were categorized into several clusters according to their glycosylation profiles. Our results provide a biological basis to understand differential phenotypes of metastatic breast cancer cells potentially reflecting clonal origin, which does not directly reflect genomic or genetic changes or microenvironmental effects but connects to glycosylation profiles.

Serum Levels of N- and C-ERC/Mesothelin and Clinicopathological Factors in Mesothelioma Patients and Those without Mesothelioma.

Objectives: ERC/mesothelin is a glycosylphosphatidylinositol (GPI)-anchor protein expressed in mesothelioma. A precursor protein is cleaved by proteases and an N-terminal fragment (N-ERC) is extracellularly secreted. A remaining C-terminal fragment (C-ERC) is tethered on cellular membranes by the GPI-anchor, but C-ERC is also released after cleavage by proteases. We and other groups reported that serum N-/C-ERC levels are associated with stages of mesothelioma and suggested the possibility of their usefulness as diagnostic markers. However, the N-ERC level is also influenced by renal functions that are not directly associated with conditions of mesothelioma. It is not known whether other clinical factors influence serum N-/C-ERC values. Furthermore, their relationship to the amount of ERC/Mesothelin in mesothelioma is not yet validated. The objective of this study is to clarify the relationship of serum N-/C-ERC levels and the status of mesothelioma and several clinical factors. Materials and Methods: We analyzed relations of serum N-/C-ERC levels and ages, gender and other clinical factors in 522 patients without mesothelioma and examined their relation to the amount of ERC/Mesothelin in mesothelioma tissues in 13 mesothelioma cases. Results: Serum N-ERC levels were influenced by renal functions. On the contrary, those of C-ERC were not influenced by any clinical factors examined in this study and were significantly correlated with the amount of ERC/Mesothelin in mesothelioma. Conclusion: Although both markers are good indicators of treatment-responses in individual patients with mesothelioma, only C-ERC reflected the amount of ERC/Mesothelin in mesothelioma among multiple patients, possibly because N-ERC was influenced by renal functions.

The Inhibitory Effects of Anti-ERC/Mesothelin Antibody 22A31 on Colorectal Adenocarcinoma Cells, within a Mouse Xenograft Model.

The expression of Renal Carcinoma (ERC)/mesothelin is enhanced in a variety of cancers. ERC/mesothelin contributes to cancer progression by modulating cell signals that regulate proliferation and apoptosis. Based on such biological insights, ERC/mesothelin has become a molecular target for the treatment of mesothelioma, pancreatic cancer, and ovarian cancer. Recent studies revealed about 50-60% of colorectal adenocarcinomas also express ERC/mesothelin. Therefore, colorectal cancer can also be a potential target of the treatment using an anti-ERC/mesothelin antibody. We previously demonstrated an anti-tumor effect of anti-ERC antibody 22A31 against mesothelioma. In this study, we investigated the effect of 22A31 on a colorectal adenocarcinoma cell line, HCT116. The cells were xenografted into BALB/c nu/nu mice. All mice were randomly allocated to either an antibody treatment group with 22A31 or isotype-matched control IgG1κ. We compared the volume of subsequent tumors, and tumors were pathologically assessed by immunohistochemistry. Tumors treated with 22A31 were significantly smaller than those treated with IgG1κ and contained significantly fewer mitotic cells with Ki67 staining. We demonstrated that 22A31 exhibited a growth inhibitory property on HCT116. Our results implied that ERC/mesothelin-targeted therapy might be a promising treatment for colorectal cancer.

Effluent N-terminal expressed in renal cell carcinoma/mesothelin predicts increased peritoneal permeability in patients undergoing peritoneal dialysis.

INTRODUCTION: We investigated whether N-terminal and C-terminal products of expressed in renal cell carcinoma/mesothelin (N-ERC and C-ERC) in peritoneal effluent can predict peritoneal permeability in patients undergoing peritoneal dialysis (PD). METHODS: Thirty-seven peritoneal effluent samples were obtained from 26 PD patients. High transport status was determined by the peritoneal equilibration test as a dialysate/plasma ratio of creatinine (D/P Cr) ≥ 0.81. Effluent cancer antigen 125 (CA125) was used as a reference. RESULTS: Effluent N-ERC concentration was better correlated with D/P Cr than effluent C-ERC or CA125 concentration. In multivariate analyses, effluent N-ERC and C-ERC, but not CA125, were significant predictors of high transport status after adjusting for age, PD duration, and residual renal Kt/V. ROC analysis showed that effluent N-ERC was the best predictor of high transport status among those three biomarkers. CONCLUSION: Effluent N-ERC predicts increased peritoneal permeability in patients undergoing PD.

A Novel Therapeutic Strategy Targeting the Mesenchymal Phenotype of Malignant Pleural Mesothelioma by Suppressing LSD1.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial-mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK-AKT-GSK3ß pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. IMPLICATIONS: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Bisecting-GlcNAc on Asn388 is characteristic to ERC/mesothelin expressed on epithelioid mesothelioma cells.

Mesothelioma is a highly aggressive tumour associated with asbestos exposure and is histologically classified into three types: epithelioid-type, sarcomatoid-type and biphasic-type. The prognosis of mesothelioma patients is poor and there is no effective molecular-targeting therapy as yet. ERC/mesothelin is a glycoprotein that is highly expressed on several types of cancers including epithelioid mesothelioma, but also expressed on normal mesothelial cells. This is a predicted reason why there is no clinically approved therapeutic antibody targeting ERC/mesothelin. In the present study, we focussed on the differential glycosylation between ERC/mesothelin present on epithelioid mesothelioma and that on normal mesothelial cells and aimed to reveal a distinct feature of epithelioid mesothelioma cells. Lectin microarray analysis of ERC/mesothelin using cells and patient specimens showed significantly stronger binding of PHA-E4 lectin, which recognizes complex-type N-glycans having a so-called bisecting-GlcNAc structure, to ERC/mesothelin from epithelioid mesothelioma cells than that from normal mesothelial cells. Further, liquid chromatography/mass spectrometry analysis on ERC/mesothelin from epithelioid mesothelioma cells confirmed the presence of a bisecting-GlcNAc attached to Asn388 of ERC/mesothelin. These results suggest that this glycoproteome could serve as a potential target for the generation of a highly selective and safe therapeutic antibody for epithelioid mesothelioma.

A New CD10 Antibody Inhibits the Growth of Malignant Mesothelioma.

Malignant mesotheliomas (MMs) are aggressive therapy-resistant tumors that generally have a poor prognosis. We previously reported the establishment of four new monoclonal antibodies (mAbs) for the diagnosis and treatment of MM. In this report, we characterized one of these antibodies, JMAM-1. The molecules whose antibodies were calibrated were picked up, transfected assuming CD10, and elucidated by fluorescence activated cell sorter. Survival experiments were performed using tumor-bearing mice model. JMAM-1 mAb was found to bind with CD10 antigen. The Kaplan-Meier survival curve showed a small but prolonged survival effect. JMAM-1 mAb-treated MSTO-211H cells showed increased cell cycle arrest involved by cyclin-dependent-kinase. JMAM-1 antibody has cytostatic effect and may be a candidate for the treatment of MM. Among mesothelioma, CD10-positive cases have been reported to have a poorer prognosis than negative cases, which can be used as a tool for diagnosis.

Dinuclear Triple-Stranded Helicates Composed of Tetradentate Ligands with Aluminum(III) Chromophores: Optical Resolution and Multi-color Circularly Polarized Luminescence Properties.

New chiroptical chromophores, dinuclear triple-stranded helicates, composed of tetradentate ligands with aluminum(III) ions, are described. These are synthesized in two steps using inexpensive pyrrole derivatives, hydrazine, and aluminum chloride. These molecular architectures (ALPHY) show multi-color (cyan, yellow, and orange) photoluminescence in solution and in the solid-state, which depends on the substituents of the ligands. The photoluminescence quantum yields of helicates were up to 54 %. The right-handed (P) and left-handed (M) helicates are so stable that they do not undergo racemization in some solvents and are mirror images according to circular dichroism and circularly polarized luminescence (CPL) with an absolute luminescence dissymmetry factor (glum ) of up to the 10-3 order. Mixing the different helicates produces white-light emission with CPL characters. This study offers a glimpse into the potential applications of chromophores with diverse photophysical properties.

Expression status of PD-L1 and B7-H3 in mesothelioma.

Mesothelioma is a rare, aggressive malignancy with poor outcome, and has limited treatment options. The aim of this study was to perform a comprehensive analysis of programmed death ligand 1 (PD-L1) and B7 homolog 3 (B7-H3) expression in mesothelioma. We investigated the protein expression of PD-L1 and B7-H3 and their potential correlation with histological subtype, which might help to develop new therapies targeting these immune checkpoint molecules. Expression analysis of PD-L1 and B7-H3 was performed by immunohistochemistry using serial tissue sections of specimens obtained from 31 patients with mesothelioma. Tumors were classified into 22 epithelioid, 6 sarcomatoid, and 3 biphasic types. Of the 31 patients, 13 (41.9%) were positive for PD-L1 and 28 (90.3%) were B7-H3 positive. Twelve of the 13 PD-L1 positive patients were positive for B7-H3. PD-L1 and B7-H3 were widely co-expressed in biphasic and sarcomatoid type tumor cells. These findings might provide a rationale for the use of combination therapy for mesothelioma by targeting PD-L1 and B7-H3, as well as the development of anti-B7-H3 or anti-PD-L1 single agents.

Possible reversibility between epithelioid and sarcomatoid types of mesothelioma is independent of ERC/mesothelin expression.

BACKGROUND: Mesothelioma is histologically divided into three subgroups: epithelioid, sarcomatoid, and biphasic types. The epithelioid or sarcomatoid type is morphologically defined by polygonal or spindle-like forms of cells, respectively. The biphasic type consists of both components. It is not yet understood how histological differentiation of mesothelioma is regulated. ERC/mesothelin is expressed in most cases of the epithelioid type, but not in the sarcomatoid type of mesothelioma. Consequently, its expression is well correlated to the histological subtype. We hypothesized that ERC/mesothelin expression influences the histological differentiation of mesothelioma, and tested this hypothesis. METHODS: We performed studies using the overexpression or knockdown of ERC/mesothelin in mesothelioma cells to examine its effect on cellular morphology, growth kinetics, or migration/invasion activity, in vitro. We then transplanted ERC/mesothelin-overexpressing and control cells into the intraperitoneal space of mice. We examined the effect of ERC/mesothelin overexpression on mouse survival and tumor phenotype. RESULTS: In vitro cell culture manipulations of ERC/mesothelin expression did not affect cellular morphology or proliferation, although its overexpression enhanced cellular adhesion and the migration/invasion activity of mesothelioma cells. The survival rate of mice following intraperitoneal transplantation of ERC/mesothelin-overexpressing mesothelioma cells was significantly lower than that of mice with control cells. The histological evaluation of the tumors, however, did not show any morphological difference between two groups, and our hypothesis was not validated. Unexpectedly, both groups (ERC/mesothelin-overexpressing and control) of mesothelioma cells that were morphologically monophasic and spindle-like in vitro differentiated into a biphasic type consisting of polygonal and spindle-like components in the transplanted tumor, irrespective of ERC/mesothelin expression. CONCLUSIONS: These results suggested that the histological transition of mesothelioma between epithelioid and sarcomatoid types may be reversible and regulated not by ERC/mesothelin, but by other unknown mechanisms.

Detachment and recovery of an X-ray opaque tip of a disposable guide sheath kit: A rare complication of endobronchial ultrasound-guided transbronchial biopsy.

Endobronchial ultrasound with a guide sheath (EBUS-GS)-guided transbronchial biopsy offers advantages and is frequently used for the diagnosis of peripheral pulmonary lesions. A previously healthy 75-year-old man was hospitalized to undergo bronchoscopy for the diagnosis of a mass with a diameter of about 40 mm in right S3 area. The mass was detected during a regular medical check-up, and lung cancer was suspected. EBUS-GS guided transbronchial biopsy was performed through the right B3. Following the bronchoscopic procedure and removal of the GS, we observed that an X-ray opaque tip attached to the point of the GS was missing. We examined the lung field through X-ray fluoroscopy and found that the detached opaque tip was located in the right middle lung field. We re-inserted the bronchoscope, and successfully recovered it using transbronchial biopsy forceps. The rate of complications in EBUS-GS is low, and the complication presented in this report is rare. Physicians should exercise caution when performing this procedure and carefully check the condition of the kit to reduce the risk of such complications.

Stromal fibroblasts induce metastatic tumor cell clusters via epithelial-mesenchymal plasticity.

Emerging evidence supports the hypothesis that multicellular tumor clusters invade and seed metastasis. However, whether tumor-associated stroma induces epithelial-mesenchymal plasticity in tumor cell clusters, to promote invasion and metastasis, remains unknown. We demonstrate herein that carcinoma-associated fibroblasts (CAFs) frequently present in tumor stroma drive the formation of tumor cell clusters composed of two distinct cancer cell populations, one in a highly epithelial (E-cadherinhiZEB1lo/neg: Ehi) state and another in a hybrid epithelial/mesenchymal (E-cadherinloZEB1hi: E/M) state. The Ehi cells highly express oncogenic cell-cell adhesion molecules, such as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 that associate with E-cadherin, resulting in increased tumor cell cluster formation and metastatic seeding. The E/M cells also retain associations with Ehi cells, which follow the E/M cells leading to collective invasion. CAF-produced stromal cell-derived factor 1 and transforming growth factor-ß confer the Ehi and E/M states as well as invasive and metastatic traits via Src activation in apposed human breast tumor cells. Taken together, these findings indicate that invasive and metastatic tumor cell clusters are induced by CAFs via epithelial-mesenchymal plasticity.

In commemoration of the 2018 Mataro Nagayo Prize: A road to early diagnosis and monitoring of asbestos-related mesothelioma.

Primarily caused by exposure to asbestos, mesothelioma is a typical occupational disease. The latency of mesothelioma is as long as 20-40 years, and the cancer initially progresses mainly along the surfaces of pleura or peritoneum without forming masses. As symptoms do not develop until late stages, it has been challenging to diagnose this disease in its early stages and to carry out complete surgical removal. In responding to Japan's asbestos crisis in the mid-2000s, we have developed and improved ERC/MSLN-based serum and radiological markers and pioneered the use of an N-ERC ELISA kit for screening populations at risk for asbestos exposure. In the present article, we review our research toward early diagnosis of asbestos-related mesothelioma before symptoms develop and share our clinical experience of screening, diagnosing and monitoring of this disease. This paper is dedicated to the author (Dr Okio Hino) to commemorate the honor bestowed upon him as the recipient of the Mataro Nagayo Prize in 2018.

Biomarkers for differentiation of patients with asthma and chronic obstructive pulmonary disease.

OBJECTIVE: Asthma and chronic obstructive pulmonary disease (COPD) are airflow limitation diseases with similar clinical manifestations but different pathophysiologic mechanisms. To implement the appropriate treatment, it is important to distinguish between asthma and COPD which sometimes might result difficult in clinical practice. We evaluated biomarkers to distinguish between asthma and COPD. METHODS: Blood eosinophil counts and fractional exhaled nitric oxide (FeNO) levels were analyzed. Serum periostin, interleukin-25 (IL-25), and immunoglobulin E (IgE) concentrations were compared between patients with asthma (n = 60), including atopic-asthma (n = 30) and non-atopic asthma (n = 30), and patients with COPD (n = 30). RESULTS: Significantly higher peripheral blood eosinophil counts (p < 0.001), FeNO levels (p < 0.001), and total serum IgE (P = 0.003) concentrations, but not serum periostin (p = 0.584) or serum IL-25 (p = 0.085) concentrations, were detected in patients with asthma compared to patients with COPD. Serum periostin and IgE concentrations were increased in patients with atopic-asthma compared with those with non-atopic asthma and COPD (p < 0.05). The FeNO levels were significantly correlated with the peripheral blood eosinophil counts (r = 0.430, p = 0.001) and serum IL-25 concentrations (r = 0.338, p = 0.009) in patients with asthma. The serum periostin concentrations were also correlated with the serum IgE concentrations (r = 0.375, p = 0.003)and FeNO levels (r = 0.291, p = 0.024) in patients with asthma. Asthma patients were effectively differentiated from COPD patients based on the FeNO levels (p < 0.001) and peripheral blood eosinophil counts (p < 0.001). CONCLUSIONS: FeNO levels and peripheral blood eosinophil counts were useful biomarkers for distinguishing between patients with asthma and COPD. Serum periostin and IgE concentrations could be biomarkers for atopic asthma.

Possible Role of IL-25 in Eosinophilic Lung Inflammation in Patients with Chronic Eosinophilic Pneumonia.

PURPOSE: Interleukin (IL)-25 and IL-33 induce IL-5 production by various types of cells, such as type 2 helper T (Th2) cells and type 2 innate lymphoid cells. The number of Th2 cells and concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) are increased in patients with eosinophilic pneumonia (EP). To examine the contribution of IL-25 and IL-33 to eosinophilic inflammation of the lung in humans, we evaluated IL-5, IL-25 and IL-33 levels in the BALF of patients with EP. METHODS: IL-5, IL-25, and IL-33 concentrations in the BALF were measured by enzyme-linked immunosorbent assay in patients with acute eosinophilic pneumonia (AEP), chronic eosinophilic pneumonia (CEP), idiopathic pulmonary fibrosis (IPF), and sarcoidosis. RESULTS: The absolute number of eosinophils, and IL-5 levels, but not IL-33 levels, in the BALF were significantly higher in patients with EP than in patients with IPF and sarcoidosis. IL-25 levels in the BALF were significantly higher in patients with CEP, but not in patients with AEP, than in patients with IPF and sarcoidosis. The absolute number of eosinophils was significantly correlated with the IL-5 concentration in the BALF of patients with EP. IL-5 concentrations were significantly correlated with IL-25 concentrations in the BALF of patients with CEP, but not in patients with AEP. IL-5 levels were not correlated with IL-33 levels in the BALF of patients with EP. CONCLUSIONS: Our findings suggest that IL-25 plays an important role via IL-5 in eosinophilic lung inflammation in patients with CEP.

Efficacy of Varenicline for Cigarette Reduction Before Quitting in Japanese Smokers: A Subpopulation Analysis of the Reduce to Quit Trial.

PURPOSE: This prospective analysis of the Japanese subpopulation of the varenicline reduce to quit study was conducted to evaluate whether results for Japanese participants were consistent with the full study population. METHODS: Patients received varenicline or placebo for a 24-week treatment period (12-week smoking reduction phase then a 12-week smoking abstinence phase) followed by a 28-week nontreatment, follow-up phase. Participants were to reduce the daily number of cigarettes smoked by at least 50% by week 4 and by a further 50% by week 8, with the goal of achieving complete abstinence by week 12. The primary efficacy end point was the carbon monoxide-confirmed continuous abstinence during weeks 15 to 24. FINDINGS: Overall, 210 Japanese patients were randomly assigned to 1 of the 2 study groups (varenicline, 107; placebo, 103). Continuous abstinence rates for weeks 15 to 24 were higher for participants in the varenicline group versus the placebo group (46.7% vs 12.6%; odds ratio = 14.68; 95% CI, 5.38-40.05), and the 7-day point prevalence of abstinence rates were higher for varenicline versus placebo at week 12 (odds ratio = 13.76; 95% CI, 5.28-35.86). The number of participants with a ≥50% reduction in the number of daily cigarettes smoked from baseline to week 4 and a ≥75% reduction by week 8 was greater in the varenicline group versus the placebo group (week 4: 59.8% vs 30.1%; week 8: 38.3% vs 12.6%). Serious adverse events were reported in 3.7% of varenicline participants and 1.0% of placebo participants. IMPLICATIONS: The efficacy and tolerability results of this analysis are consistent with those of the full varenicline reduce to quit study. Varenicline treatment and cigarette reduction before quitting may provide an alternative approach to smoking cessation in Japanese smokers who are not ready to quit immediately. ClinicalTrials.gov identifier: NCT01370356.

Conformational Change in Molecular Assembly of Nickel(II) Tetra(n-propyl)porphycene Triggered by Potential Manipulation.

Metal-coordinated porphyrin and related compounds are important for developing molecular architectures that mimic enzymes. Porphycene, a structural isomer of porphyrin, has shown unique properties in semiartificial myoglobin. To explore its potential as a molecular building block, we studied the molecular assembly of nickel(II) tetra(n-propyl)porphycene (NiTPrPc), a metalloporphycene with introduced tetra n-propyl moieties, on the Au(111) electrode surface using in situ scanning tunneling microscopy. Because of the low molecular symmetry of NiTPrPc, the molecular assembly undergoes unique phase transitions due to conformational change of the n-propyl moieties. The phase transitions can be precisely controlled by the electrode potential, demonstrating that the latter can play an important role in the porphycene molecular assembly on Au surface. This new discovery indicates possible uses of this porphycene framework in molecular engineering.

Establishment of anti-mesothelioma monoclonal antibodies.

BACKGROUND: Mesotheliomas are aggressive, therapy-resistant tumors that are predicted to increase in incidence at least until 2020. The prognosis of patients with mesothelioma is generally poor because they are typically diagnosed at a late stage and their tumors are resistant to current conventional therapies. For these reasons, improved diagnosis and therapy are urgently required. To address these issues, the aim of our research was to develop novel mesothelioma-specific monoclonal antibodies (mAbs) as diagnostic and therapeutic agents. METHODS: To develop anti-mesothelioma mAbs useful for diagnosis and therapy, we repeatedly immunized a BALB/c mouse with viable mesothelioma cells, alternating between those from three mesothelioma cell lines. We hybridized the spleen cells from this immunized mouse with P3U1 myeloma cells. We then screened supernatants harvested from the hybridoma clones by assessing whether they bound to a mesothelioma cell line not used for immunization and altered its morphology. We designed this developmental strategy to reduce the risk of obtaining clonotypic mAbs against a single mesothelioma cell line. RESULTS: Our newly generated mouse anti-human mAbs immunostained clinical samples of mesotheliomas. One of the newly generated mAbs did not react with any other tumor cell line tested. Two other mAbs significantly inhibited the proliferation of mesothelioma cells. CONCLUSION: These newly generated anti-mesothelioma mAbs are potentially useful as diagnostic and therapeutic agents for mesothelioma. Moreover, our novel strategy for establishing antitumor mAbs may facilitate the development of new diagnostic and therapeutic techniques for mesotheliomas and other malignancies.

Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death via Large Pore Formation.

To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

Increased Galectin-9 Concentration and Number of CD4+Foxp3high+Cells in Bronchoalveolar Lavage Fluid of Patients with Cryptogenic Organizing Pneumonia.

PURPOSE: Galectin-9 (Gal-9) is a ß-galactoside-binding protein that exhibits various biological reactions, such as chemoattraction, cell aggregation, and apoptosis. Recent studies demonstrated that Gal-9 has a role as an immunomodulator in excessive immunological reactions by expanded regulatory T cells (Tregs). We examined the role of Gal-9 in the pathogenesis of one of the major idiopathic interstitial pneumonias, cryptogenic organizing pneumonia (COP) as compared with idiopathic pulmonary fibrosis (IPF). METHODS: Gal-9, transforming growth factor-ß1, and interleukin (IL)-10 levels in the bronchoalveolar lavage fluid (BALF) of patients with COP and IPF were estimated by enzyme-linked immunosorbent assay. Forkhead box protein 3 (Foxp3) expressing Tregs were evaluated by flow cytometry. The effect of Gal-9 on interactions between human lung fibroblast cells and hyarulonan was assessed in vitro. RESULTS: Gal-9 and IL-10 levels in the BALF were significantly higher in patients with COP than in patients with IPF. The number of CD4+Foxp3high+cells was significantly higher in the BALF of patients with COP than in those with IPF. Gal-9 levels significantly correlated with the absolute number of CD4+CD25+Foxp3+cells or CD4+Foxp3high+cells, but not with the absolute number of CD4+CD25+Foxp3-cells, in the BALF of patients with COP. Gal-9 suppressed the CD44-dependent interaction of human lung fibroblast cells with hyarulonan in a dose-dependent manner. CONCLUSIONS: Our findings suggest that increased Gal-9 levels in the lung have a protective role against lung inflammation and fibrosis in patients with COP through the induction of Tregs in the lung and CD44-dependent inhibitory effects on lung fibroblast cells.