Akihiko Ohwada

Synergistic actions of antibacterial neutrophil defensins and cathelicidins.

Abstract.Objective: Activated neutrophils extracellularly release antibacterial defensins and cathelicidins from the granules. In this study, to elucidate the interactions between defensins and cathelicidins in the extracellular environment, we evaluated the individual and synergistic actions of defensins and cathelicidins in the presence of physiological concentration of NaCl (150 mM).¶Materials and Methods: Antibacterial activities against Escherichia coli and Staphylococcus aureus were assessed using human and guinea pig defensins and cathelicidins. Furthermore, the effect of defensins and cathelicidins on membrane permeabilization was examined using E. coli ML-35p, as a target organism.¶Results: In the absence of NaCl, human defensin (HNP-1) and guinea pig defensins (GNCPs) exhibited the antibacterial activities in a dose-dependent manner (0.1-10 μg/ml); however, their activities were completely lost in the presence of 150mM NaCl. In contrast, the antibacterial activities of human cathelicidin (CAP18/LL-37) and guinea pig cathelicidin (CAP11) were resistant to NaCl. Interestingly, HNP-1 and GNCPs synergized with CAP18/LL-37 and CAP11 to enhance the antibacterial activities against E. coli and S. aureus in the presence of 150 mM NaCl (p<0.05). Similarly, HNP-1 and GNCPs were synergistic with CAP18/LL-37 and CAP11 to potentiate the outer and inner membrane permeabilization of E. coli ML-35p (p<0.05).¶Conclusions: Together these observations indicate that when extracellularly released from neutrophils, defensins cannot function as antibacterial molecules by themselves, but can synergistically work with cathelicidins to exert the antibacterial activity in the extracellular milieu by augmenting the membrane permeabilization of target cells.

Coadministration of an Interleukin-12 Gene and a Trypanosoma cruzi Gene Improves Vaccine Efficacy

ABSTRACT We tested the immunogenicity of two Trypanosoma cruzi antigens injected into mice in the form of DNA vaccine. Immunization with DNA encoding dihydroorotate dehydrogenase did not confer protective immunity in all mouse strains tested. Immunization with DNA encoding trans-sialidase surface antigen (TSSA) protected C57BL/6 (H-2b) mice but not BALB/c (H-2d) or C3H/Hej (H-2k) mice against lethal T. cruzi infection. In vivo depletion of CD4+ or CD8+ T cells abolished the protective immunity elicited by TSSA gene in C57BL/6 mice. Enzyme-linked immunospot assay with splenocytes from T. cruzi-infected mice or TSSA gene-vaccinated mice identified an H-2Kb-restricted antigenic peptide, ANYNFTLV. The CD8+-T-cell line specific for this peptide could recognize T. cruzi-infected cells in vitro and could protect naive mice from lethal infection when adoptively transferred. Coadministration of the interleukin-12 (IL-12) gene with the TSSA gene facilitated the induction of ANYNFTLV-specific CD8+ T cells and improved the vaccine efficacy against lethal T. cruzi infection. These results reinforced the utility of immunomodulatory adjuvants such as IL-12 gene for eliciting protective immunity against intracellular parasites by DNA vaccination.

Immune Responses against a Single CD8+-T-Cell Epitope Induced by Virus Vector Vaccination Can Successfully Control Trypanosoma cruzi Infection

ABSTRACT In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFκB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.

Serum vascular endothelial growth factor as a possible prognostic indicator in sarcoidosis.

Sarcoidosis is a systemic granulomatous disorder of unknown etiology, which involves the lung, eye, liver, and other organs. Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis involved in an important role in the development of granuloma. However, only a limited number of studies have reported on the relationship between serum VEGF values and the clinical status of sarcoidosis. Concentrations of serum VEGF were determined in 33 patients with sarcoidosis. We investigated the correlation between serum VEGF values and extent of disease, prognosis, and radiographic stage compared with serum angiotensin converting enzyme (ACE) values as another candidate. Concentrations of serum VEGF in patients who received corticosteroid treatment were significantly higher than those of patients with spontaneous remission (p < 0.05). In addition, serum VEGF values in patients with extrathoracic involvements were significantly higher than those of patients with sarcoid lesions limited to the thoracic space (p < 0.05), accompanied by a tendency to increase the number of organs involved. The values of serum ACE revealed no relationship to the values of serum VEGF, administration of corticosteroid, or extrathoracic involvements. We concluded that serum VEGF values in patients with sarcoidosis is a predictive factor in determining extrathoracic organ involvements and as a parameter for deciding the necessity of treatment with corticosteroid. Serum VEGF might be a useful marker as a prognostic indicator in sarcoidosis.

VEGF regulates the proliferation of acid-exposed alveolar lining epithelial cells

Background: Acid induced pneumonitis resulting in acute respiratory distress syndrome (ARDS) is characterised by increased alveolar permeability and accumulation of neutrophils. It is hypothesised that vascular endothelial growth factor (VEGF) is involved in the development of lung oedema. Furthermore, lower levels of VEGF are detected in bronchoalveolar lavage fluid from patients with ARDS than from non-ARDS patients. We hypothesised that VEGF acts cytoprotectively and have investigated this possibility in vitro with A549 cells. Methods: A549 cells were incubated in 24 well culture dishes 24 hours before exposure to acid, then incubated with serum free medium containing various concentrations of HCl for 30 minutes at 37°C in 5% CO2. The acidified medium was changed to normal complete medium; at specified incubation periods the supernatants were collected and the VEGF concentration measured and the number of adherent cells counted. Results: Proliferation of A549 cells and VEGF production were suppressed for at least 48 hours in HCl at a concentration of 50 mM. Restoration of cellular proliferation occurred following exogenous administration of VEGF (concentration of 1–250 ng/ml) and was inhibited by co-incubation with neutralising anti-VEGF antibody, indicating an interaction between VEGF molecules and A549 cells. Control cells were not influenced by administration of exogenous VEGF or anti-VEGF antibody. Treatment with neutralising anti-VEGF receptor (VEGFR) antibodies against VEGFR-1 and VEGFR-2 suppressed proliferation of acid exposed A549 cells but had no effect on control cells. Conclusions: Exogenous VEGF interacts with VEGFR-1 and VEGFR-2 on the surface and regulates the proliferation of injured alveolar lining epithelial cells in an autocrine or paracrine fashion.

Ultrasonographic evaluation of the diaphragm in patients with amyotrophic lateral sclerosis.

Abstract:  Real‐time diaphragmatic movement was evaluated with ultrasonography in three patients with amyotrophic lateral sclerosis (ALS). The initial complaint of two patients was weakness of the extremities followed by dyspnoea later in the disease course, while the third patient had dyspnoea as the initial symptom. Ultrasonographic analyses revealed that the contractile function of the diaphragm was not maintained during maximum inspiratory effort, with unsatisfactory diaphragmatic excursion and no change in diaphragmatic thickness during respiration, indicating diaphragmatic paralysis. Ultrasonography may be useful for the diagnosis and follow up of diaphragmatic involvement with amyotrophic lateral sclerosis and other motor‐neuron diseases.

Effect of cigarette smoke on the mRNA and protein expression of carcinoembryonic antigen (CEA), a possible chemoattractant for neutrophils in human bronchioloalveolar tissues.

BACKGROUND--The concentration of carcinoembryonic antigen (CEA), known as a marker of malignant transformation and chronic inflammation, is increased in bronchoalveolar lavage fluid obtained from smokers compared with fluid from non-smokers. This study investigated the mechanism and biological significance of CEA production in the lungs of smokers by evaluating protein and mRNA expression in non-carcinomatous lung parenchymal tissues and in cell lines derived from human fetal lung. METHODS--Lung parenchymal tissue free from cancer or an inflammatory lesion was obtained from five non-smokers (four with lung cancer, one with pulmonary mycetoma), five ex-smokers (all with lung cancer except for one with mesothelioma), and 14 smokers (nine with lung cancer, five with emphysema) at surgery or necropsy. Cancer tissue was also collected simultaneously from the subjects with lung cancer. CEA protein in the tissue homogenates was measured by enzyme linked immunoassay. CEA mRNA expression in the non-carcinomatous parenchymal tissue and cancer tissue was evaluated by in situ hybridisation using CEA specific riboprobe and was semiquantitated by counting the number of silver grains per cell. CEA mRNA expression was also compared in three cell lines derived from human fetal lung (IMR-90, MRC-9, and CCD-14Br) after in vitro stimulation with medium exposed to cigarette smoke or air. Chemoattractant activity of purified CEA for neutrophils and monocytes was also studied in vitro. RESULTS--CEA content in non-carcinomatous lung tissue was increased in smokers with emphysema (mean (SD) 38.0 (9.2) ng/mg protein) or with lung cancer (38.2 (21.6)) compared with non-smokers (11.0 (5.4)) or ex-smokers (5.9 (2.2)). CEA mRNA expression in non-carcinomatous tissue, expressed by average number of grains per cell, was also increased in smokers with emphysema (mean (SD) 11.2 (4.1)) or with lung cancer (14.0 (8.4)) compared with non-smokers (3.1 (0.6)) or ex-smokers (4.0 (1.7)). CEA content in carcinomatous tissues was 42.8 (37.3) for non-smokers, 38.2 (42.4)) for ex-smokers, and 59.0 (22.5) for smokers. The CEA content in carcinomatous tissue was higher than in non-carcinomatous tissue, but there was no difference between non-smokers, ex-smokers, and smokers. The numbers of grains per cell in carcinomatous tissue were higher than in non-carcinomatous tissues, but not different among non-smokers (30.3 (3.9)), ex-smokers (38.3 (13.8)), and smokers (44.3 (5.2)). CEA mRNA expression in the cell lines was upregulated after the incubation with smoke-treated medium. Purified CEA was chemoattractant for neutrophils but not for monocytes in vitro. CONCLUSIONS--mRNA and protein expression of CEA were increased in the normal lung tissue from smokers compared with non-smokers or ex-smokers. Since CEA content and mRNA expression were no different between smokers with non-small cell lung cancer and those with non-carcinomatous disease, it is unlikely that CEA expression in non-carcinomatous lung parenchymal tissue was influenced by the presence of the tumour and is consistent with the effect of smoking. This is supported by in vitro studies which show that cigarette smoke could induce CEA mRNA expression in fetal lung derived cells. In addition, CEA might play a part in recruitment of neutrophils into the lower respiratory tract.

Increased circulating CD16+ CD14dim monocytes in a patient with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveoli with a periodic acid‐Schiff‐positive proteinaceous material. Although the pathogenesis of primary or idiopathic PAP remains unknown, it has been proposed that a deficiency or loss of responsiveness of the monocyte/macrophage lineage to granulocyte–macrophage colony stimulating factor (GM‐CSF) is involved in PAP. Secondary PAP is associated with haematological malignancies, especially in myeloid disorders. Herein, we report on an adult with PAP associated with myelodysplastic syndrome (MDS). The CD16+ CD14dim monocytes comprise 5–10% of circulating monocytes in healthy volunteers. Flow cytometric analysis of the patient in the present study revealed increased CD16+ CD14dim monocytes in the peripheral blood. It has been demonstrated that the expression of CD16 and CD14 is regulated by macrophage colony stimulating factor (M‐CSF) and GM‐CSF. Hence, serum cytokines were analysed in our patient and the concentration of serum GM‐CSF was found to be less than the lower limit of the assay. In addition, serum M‐CSF and granulocyte colony stimulating factor levels were only slightly increased above the normal range. These results suggest that the increase in the CD16+ CD14dim subpopulation in the circulation of our patient indicates another pathogenetic mechanism for secondary PAP, such as hyperresponsiveness of the monocyte/ macrophage lineage to these cytokines.

Adenovirus vector-mediated transfer of 9 kDa granulysin induces DNA fragmentation in HuD antigen-expressing small cell lung cancer murine model cells

Objective: Granulysin is a tumoricidal molecule secreted by cytotoxic T cells (CTL) and natural killer (NK) cells, that induces apoptotic cell death in tumour cells. It has been demonstrated that small cell lung cancer (SCLC) cell lines are susceptible to NK cells and lymphokine activated killer (LAK) cells, and HuD antigen is assumed to be a target molecule on SCLC cells for host cellular immunity.

Concave Pattern of a Maximal Expiratory Flow-Volume Curve: A Sign of Airflow Limitation in Adult Bronchial Asthma

Background. In patients with bronchial asthma, spirometry could identify the airflow limitation of small airways by evaluating the concave shape of the maximal expiratory flow-volume (MEFV) curve. As the concave shape of the MEFV curve is not well documented, we reevaluated the importance of this curve in adult asthmatic patients. Methods. We evaluated spirometric parameters, the MEFV curve, and its concave shape (scoop between the peak and endpoint of expiration) in 27 nonsmoking asthmatic patients with physician-confirmed wheeze and positive bronchial reversibility after a short-acting β2-agonist inhalation. We also calculated angle β and shape factors (SF25% and SF50%) to quantitate the curvilinearity of the MEFV curve. Results. The MEFV curve was concave in all patients. Along with improvements in standard spirometric parameters, curvilinear parameters, angle β, SF25%, and SF50% were significantly improved after bronchodilator inhalation. There were significant correlations between improvements in angle β, and FEF50%, and FEF25-75%, and between improvements in SF25%, and SF50%, and FEF75%. Conclusions. The bronchodilator greatly affected the concave shape of the MEFV curve, correlating with spirometric parameters of small airway obstructions (FEF50%, FEF75%, and FEF25-75%). Thus, the concave shape of the MEFV curve is an important indicator of airflow limitation in adult asthmatic patients.