Takeaki Hiratsuka

Increased concentrations of human beta-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis.

Background: Human β-defensin (HBD)-1 and -2 are antimicrobial peptides present in the respiratory tract. Recent reports have indicated reduced activity of β-defensins in cystic fibrosis, suggesting that β-defensins may play an important role in the pathological process of chronic respiratory tract infection. Diffuse panbronchiolitis (DPB) is a progressive disease characterised by frequent episodes of superimposed infection, typically caused by Pseudomonas aeruginosa. The aim of this study was to elucidate the role of these antimicrobial peptides in this disease. Methods: The concentrations of HBD-1 and HBD-2 in plasma and bronchoalveolar lavage (BAL) fluid from 33 patients with DPB and 30 normal adults were measured by radioimmunoassay. Localisation of HBD-2 was investigated immunohistochemically in an open lung biopsy specimen obtained from a patient with DPB. Results: High concentrations of HBD-1 and HBD-2 were noted in BAL fluid from DPB patients. Increased plasma concentrations of HBD-2, but not HBD-1, were found in patients with DPB compared with control subjects. In patients with DPB the HBD-2 concentration in BAL fluid correlated significantly with the numbers of cells recovered from the BAL fluid (total cells, neutrophils, and lymphocytes) and with the BAL fluid concentration of IL-1β. Synthetic HBD-2, but not HBD-1, had dose dependent bactericidal activity against P aeruginosa. Treatment of 14 patients with macrolides significantly reduced BAL fluid concentrations of HBD-2 but not HBD-1 or plasma concentrations of HBD-1 and HBD-2. Immunohistochemistry of lung tissue showed localisation of HBD-2 in the epithelia of the distal bronchioles. Conclusions: These results indicate that β-defensins, particularly HBD-2, participate in antimicrobial defence in the respiratory tract in DPB, and that the BAL fluid concentration of HBD-2 may be a useful marker of airway inflammation in patients with DPB.

Raised plasma concentrations of α-defensins in patients with idiopathic pulmonary fibrosis

Background: Neutrophils are thought to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Human neutrophils contain antimicrobial and cytotoxic peptides in the azurophil granules which belong to a family of mammalian neutrophil peptides named α-defensins. A study was undertaken to investigate the role of α-defensins in the pathogenesis of IPF. Methods: The concentrations of α-defensins (human neutrophil peptides (HNPs) 1, 2, and 3) in plasma and bronchoalveolar lavage (BAL) fluid of 30 patients with IPF and 15 healthy subjects were measured by radioimmunoassay. Results: The concentrations of α-defensins in plasma, but not in BAL fluid, were significantly higher in IPF patients than in controls. BAL fluid concentrations of interleukin (IL)-8 in patients with IPF, which were significantly higher than in controls, correlated with those of α-defensins. An inverse relationship was seen between plasma α-defensin levels and the arterial oxygen tension (Pao2) and pulmonary function (vital capacity (%VC), forced expiratory volume in 1 second (FEV1), and carbon monoxide transfer factor (%Tlco)) in patients with IPF. Plasma levels of α-defensins also correlated with the clinical course in IPF patients with an acute exacerbation. Immunohistochemically, positive staining was observed inside and outside neutrophils in the alveolar septa, especially in dense fibrotic areas. Conclusion: These findings suggest that α-defensins play an important role in the pathogenesis of IPF, and that the plasma α-defensin level may be a useful marker of disease severity and activity.

Structural Analysis of Human β-Defensin-1 and Its Significance in Urinary Tract Infection

Human β-defensin-1 (hBD-1) is a 36-amino-acid antimicrobial peptide that functions in the host innate defense. We developed a highly sensitive radioimmunoassay for hBD-1 and identified several hBD-1 peptides in human kidney, urine, and plasma by amino acid sequencing and mass spectrometry. Large quantities of hBD-1 peptides are produced in the kidney, are released into the tubular lumen as 47-amino-acid pro-hBD1, and then undergo proteolytic processing and generate multiple truncated forms. The respective urine and plasma concentrations of hBD-1 in patients with pyelonephritis are 48.1 ± (SEM) 15.7 pmol/mg creatinine and 2.66 ± 0.41 pmol/ml, 3.1-fold and 1.8-fold those of normal individuals. hBD-1 is thought to contribute to mucosal defense in the urinary tract. Our findings provide a better understanding of the biosynthesis of this peptide and its pathophysiological significance in infectious diseases.

Identification of hBD-3 in respiratory tract and serum: the increase in pneumonia

Human β-defensin (hBD)-3, a 45 amino acid antimicrobial peptide, was originally isolated from human skin. hBD-3 mRNA has also been detected in the airways by RT-PCR. While hBD-3 may be involved in antimicrobial defences within the respiratory tract, the presence of hBD-3 peptide in the respiratory system has not yet been confirmed. The antimicrobial activity of the synthesised hBD-3 peptide was measured by a radial diffusion assay and a colony count assay. The present authors confirmed the presence of hBD-3 peptide in homogenates of human lung and serum using reverse-phase HPLC coupled with a highly sensitive RIA. The localisation of the hBD-3 peptide was investigated by immunohistochemistry. In addition, the serum concentrations of hBD-3 were measured by RIA. hBD-3 exhibited a strong antimicrobial activity, which was unaffected by increasing salt concentrations. Immunohistochemically, the current authors observed the expression of hBD-3 in bronchial and bronchiolar epithelial cells. The mean±sd serum concentration of hBD-3 in patients with bacterial pneumonia was 239.4±17.8 pg·mL−1 in the acute phase and, decreased to 159.3±20.1 pg·mL−1 after the completion of therapy. In conclusion, these findings will help elucidate the role of human β-defensin-3 in host immune responses and identify the pathophysiological significance of this molecule in respiratory infections.

Nucleotide Sequence and Expression of Rat β-Defensin-1: Its Significance in Diabetic Rodent Models

β-Defensins are epithelium-derived antimicrobial peptides that function in the host’s innate defense. We identified the first member of the rat β-defensin family, β-defensin-1 (BD-1), in the kidney and determined its nucleotide sequence. It was predicted to be a 37-amino-acid peptide. Rat BD-1 mRNA was expressed most abundantly in the kidney, next in skin, tongue, esophagus, and uterus, followed (at low levels) by brain, trachea, stomach, urinary bladder, and ovary. BD-1 gene expression in rat kidney was not increased by lipopolysaccharide administration. BD-1 gene expressions in the kidneys of diabetic rodent models, cholecystokinin-insensitive Otsuka Long-Evans Tokushima Fatty rats, leptin-insensitive obese (fa/fa) Wistar rats, and db/db mice, were significantly lower than those of their lean littermates. BD-1 reduction may be in part responsible for the high incidence of urinary tract infections in diabetes mellitus.

新たな抗菌ペプチドhuman β-defensin-1と-2の気道感染防御に関する検討

Human beta-defensin-1 (hBD-1) and human beta-defensin-2 (hBD-2) are new members of the defensin family. In this study, we investigated their gene expressions in the respiratory epithelial surface of the human lung and their bactericidal activities against Escherichia coli. Both hBD-1 and hBD-2 gene transcripts were detected in human lung tissue and cellular component of bronchoalveolar lavage fluid by reverse transcription-polymerase chain reaction. Synthetic hBD-1 and hBD-2 had dose-dependent bactericidal activities against E. coli. The concentration for the 50% colony reduction of hBD-2 was 0.46 nmol/ml, that for HNP-1 2.15 nmol/ml, and hBD-1 99.3 nmol/ml under conditions nearly the same as in human bronchial airway surface liquid. We prepared an antiserum against hBD-2 and established a highly sensitive radioimmunoassay and quantified plasma hBD-2 concentrations in normal subjects and patients with pneumonia. The plasma concentration of hBD-2 in normal individuals was 8.3 +/- 0.9 fmol/ml (mean +/- SE). The mean hBD-2 plasma concentration for the 12 patients with bacterial pneumonia in the acute stage was 34.2 +/- 3.4 fmol/ml, significantly higher than normal individuals, and returned to the normal range after recovery. The presence of hBD-1 and hBD-2 in the airway tract and their bactericidal activity suggest that they function in innate airway mucosal defense.

Effect of different dialyzers on defensins during hemodialysis

AbstractBackground. Defensins are antimicrobial peptides that constitute 30% to 50% of total azurophil granule proteins. Although the activation of azurophils during hemodialysis has been reported, including the release of granulocyte lactoferrin, myeloperoxidase, and elastase, the release of defensins during hemodialysis has not been investigated. The purpose of this study was to determine the effects of hemodialysis on defensin release. Methods. Plasma defensin levels and excreted defensins in dialyzer eluates and dialysates during hemodialysis were determined by radioimmunoassay. Thirty hemodialysis patients were divided into three groups, each group using a different dialyzer membrane: cuprophan (CU), polymethylmethacrylate (PMMA), or polysulfone (PS). Results. The postdialysis plasma defensin level in the CU group was significantly increased compared with the predialysis level. In contrast, defensin levels in the PMMA and PS groups showed significant decreases postdialysis. The predialysis neutrophil count and defensin level exhibited a significant positive correlation in all groups. However, postdialysis, a significant positive correlation was found only in the CU group. Interestingly, defensin excretion in the dialyzer eluates of the PMMA group and in the dialysates of the PS group was higher than that in each of the other two groups respectively. Conclusions. No significant statistical correlation between postdialysis neutrophil count and defensin level was observed in the PMMA and PS groups, a finding that may be explained by the increased excretion of defensins during hemodialysis in these groups. These results suggested that plasma defensin level may be a marker for granulocyte activation in patients dialyzed with membranes showing lower levels of excreted defensins. This study also revealed the importance of determining both plasma changes and excreted volumes of granule proteins to define neutrophil degranulation during hemodialysis.