Minoru Kanazawa

Clinical and immunoregulatory effects of roxithromycin therapy for chronic respiratory tract infection.

The clinical and immunoregulatory effects of long-term macrolide antibiotic therapy for patients with chronic lower respiratory tract infections (CLRTI) were investigated. Clinical parameters and neutrophil chemotactic mediators in the epithelial lining fluid (ELF) of CLRTI patients (n = 10) were examined before and after 3 months oral administration of roxithromycin (RXM). The in vitro effects of RXM were also examined on the release of these mediators from alveolar macrophages (AM) and neutrophils. Arterial oxygen tension (p<0.05), vital capacity (VC) (p<0.001), %VC (p<0.05) and forced expiratory volume in one second (p<0.01) were improved after RXM treatment, but airway bacteria were not eradicated. Among the mediators, the levels of interleukin (IL)-8, neutrophil elastase (NE) and leukotriene B4 (LTB4) were higher in ELF than in plasma of CLRTI patients and they decreased after RXM treatment (n = 7, p<0.05 for each). RXM concentrations were significantly increased in the bronchoalveolar lavage cells of the treated patients. In in vitro experiments, RXM showed inhibitory effects on IL-8 release from AM and neutrophils. In conclusion, interleukin-8, neutrophil elastase and leukotriene B4 contribute to the neutrophilic inflammation in the airways of chronic lower respiratory tract infection patients and the clinical effects of roxithromycin may, in part, be attributable to the suppression of excess release of the chemotactic mediators from inflammatory cells.

Genetic polymorphisms of CC chemokine receptor 3 in Japanese and British asthmatics

Whole genome scan analyses have revealed that chromosomal region 3p21-24, which contains a gene cluster of CC chemokine receptors such as CCR3, is possibly linked to asthma. Because CCR3 ligands play a pivotal role in the selective recruitment and activation of inflammatory cells in the asthmatic airway, the authors examined whether there is any association between asthma and the CCR3 gene polymorphisms. Three polymorphisms were identified using the single stranded conformational polymorphism method in Japanese (Asian) and British (Caucasian) subjects; one silent mutation T51C and two missense mutations G824A and T971C. These polymorphisms were examined in 391 Japanese subjects (210 asthmatics and 181 nonasthmatic controls) and 234 British subjects (142 asthmatics and 92 nonasthmatic controls). Asthma diagnosis was based on episodic symptoms, documented wheeze, and the presence of reversible airflow limitation. CCR3 T51C demonstrated a significant association with the diagnosis of asthma in the British population (odds ratio 2.35, p<0.01), but not in the Japanese population. Multiple logistic regression analysis also showed that CCR3 T51C was associated with asthma (odds ratio 2.83, p < 0.02), independent of atopic phenotypes such as high levels of total or house dust mite-specific immunoglobulin-E in serum. In conclusion, a significant association between asthma and CCR3 T51C polymorphism localized on chromosome 3p21 was found.

Mycobacterium avium-intracellulare pleuritis with massive pleural effusion

Atypical mycobacterial infection is seldom accompanied by pleural involvement. We report a very rare case of Mycobacterium avium-intracellulare pleuritis with massive pleural effusion. The patient was a non-immunocompromised 35-year-old Japanese male with insidious onset of fever, chest pain and anorexia. The pleural effusion gradually resolved with empirical antimycobacterial treatment, leaving considerable pleural adhesion and thickening.

Attenuation by intravenous 2-chloroadenosine of acute lung injury induced by live Escherichia coli or latex particles added to endotoxin in the neutropenic state

Although neutrophil depletion can reduce the level of acute lung injury (ALI) induced by Escherichia coli endotoxin, that induced by live E coli cannot be attenuated even in neutropenia. This suggests that live E coli cause ALI by way of an mechanism independent of circulating neutrophil. Tumor necrosis factor-alpha (TNF-alpha), which is released from monocytes and macrophages, is a proinflammatory cytokine that is recognized as a central mediator of several forms of inflammation. In this controlled experimental study, we examined the effects of an adenosine-receptor agonist, 2-chloroadenosine (2CA), that has suppressive effects on various cell types and TNF-alpha, on endotoxin plus latex particles, and on ALI induced by live E coli in the neutropenic state. We studied 42 guinea pigs rendered neutropenic by means of intraperitoneal cyclophosphamide administration. Experimental groups consisted of (1) a saline-solution control group; (2) an endotoxin (0.2 mg/kg)-treated group; (3) a group treated with endotoxin plus 2CA (10 micro g/kg); (4) a group treated with latex (2 x 10(9)/kg); (5) a group exposed to endotoxin and latex; (6) a group exposed to endotoxin, latex, and 2CA; (7) a group exposed to E coli (2 x 10(9)/kg); and (8) a group exposed to E coli and 2CA. The injection of endotoxin alone in neutropenic animals did not increase the indexes of ALI (lung tissue/plasma ratio [T/P] and lung wet weight/dry weight ratio [W/D], calculated with the use of iodine 125-labeled albumin). In contrast, these indexes were increased in the endotoxin-and-latex groups compared with those of the control group. ALI in the endotoxin-and-latex group was attenuated by intravenous 2CA. The intravenous injection of live E coli also caused increases in T/P, W/D, and plasma TNF-alpha, but thse were limited by 2CA. In summary, ALI induced by latex particles added to endotoxin and live E coli in the neutropenic state was attenuated by 2CA, suggesting a partial contribution of various cell types or humoral mediators as a neutrophil-independent pathway in its pathogenesis.

Flow cytometric detection of cell-associated cytokines in alveolar macrophages

To elucidate the cytokine-producing capacity of alveolar macrophages (AMs), we have introduced a method of flow cytometry combined with saponin treatment to detect the cell-associated cytokines. We studied bronchoalveolar lavage fluid cells from six patients with sarcoidosis (SAR) and six control (CTL) subjects. Cells were either left uncultured, or cultured with and without lipopolysaccharide (LPS), then treated with paraformaldehyde and saponin and analysed for cell-associated interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) by flow cytometry. Cell-associated IL-1 beta and TNF-alpha were also analysed by immunoassays. The flow cytometric cytokine values were correlated with the immunoreactive cell-associated cytokines (IL-1 beta: r = 0.45, p < 0.05; TNF-alpha: r = 0.82, p < 0.001). The histograms of cell-associated IL-1 beta yielded a single peak both in the patients and controls, whereas the histograms of cell-associated TNF-alpha exhibited two peaks in SAR patients, but just a single peak in the CTL subjects. The mean value of the cell-associated TNF-alpha in LPS+ AMs was higher in the SAR patients than in the CTL subjects (p < 0.001). In conclusion, the flow cytometric method can be applied to the semiquantitative detection of cell-associated cytokines in alveolar macrophages at the single cell level.

Cell‐associated IL‐8 in human blood monocytes: Analysis by flow cytometry

Several cell-associated cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor, exist on the cell surface and are biologically active. Although extracellular IL-8, a potent chemotactic factor for primarily neutrophils, has been studied extensively, cell-associated IL-8 has barely been studied. In this study, we analyzed the intracellular and cell-surface IL-8 in human blood monocytes in vitro by using flow cytometry and predicted the biological activity of the cell-associated IL-8 in vivo. After fixation with paraformaldehyde, mononuclear cells were divided into two subgroups. One subgroup was left untreated to study cell-associated antigens, and the other subgroup was permeabilized with saponin to detect intracellular antigens. In lipopolysaccharide (LPS)-stimulated monocytes, IL-8 was detected solely intracellularly, whereas both the intracellular and cell-surface IL-1 beta was detectable. In a time-course study, the intracellular IL-8 increased in response to LPS stimulation, but the cell-surface IL-8 was undetectable throughout the course. In an LPS-stimulated monocytic cell line, both ELISA and flow cytometry detected the quantitative change of the intracellular IL-8. The dissimilar localization between IL-8 and IL-1 beta within cells was confirmed by the immunohistochemical analysis. In summary, LPS stimulation induced a time-dependent increase in intracellular but not cell-surface IL-8 in monocytes. Thus, it is unlikely that the cell-associated IL-8 is functioning physiologically. The semiquantitative flow cytometric procedure may be useful for simultaneous examination for cell-surface and intracellular cytokines.

Attenuation of live E. coli-induced acute lung injury by X-ray irradiation in guinea pigs.

Abstract. Irradiation is suspected to injure inflammatory cells, such as neutrophils and mononuclear phagocytes, cells known to contribute to the development of acute lung injury (ALI). This study examined whether preexposure to x-ray irradiation modifies ALI induced by E. coli injected intravenously in guinea pig. Thirty animals were divided into two control and two irradiated subgroups: the first control group received saline only (n= 8), and the second control group received E. coli, 2 × 109/kg body weight, suspended in saline (n= 6), IV. The first irradiated group received a single 12-Gy dose + saline (n= 6), and the second irradiated group received a single 12-Gy dose +E. coli (n= 10). The lung wet-to-dry-weight ratio (W/D) and 125I-albumin lung tissue/plasma ratio (T/P) were measured as markers of lung injury. W/D was significantly higher in the control E. coli group than in the other groups. T/P in the control E. coli group was also increased compared with T/P measured in the other groups. In the control E. coli group, a marked increase in bronchoalveolar lavage (BAL) neutrophils was observed compared with the control saline group. However, no significant difference in BAL neutrophil counts was observed between the control and irradiated E. coli groups. In contrast, BAL macrophages were significantly reduced in the irradiated E. coli groups compared with the control E. coli group. These findings suggest that x-ray irradiation attenuates E. coli–induced ALI in guinea pigs, an effect explained, at least in part, by a reduction in the number of alveolar macrophages.

Immunohistochemical analysis of rat and human respiratory cilia with anti-dynein antibody: comparison between normal cilia and pathological cilia in primary ciliary dyskinesia

Wistar Imamichi rat and human respiratory cilia were examined with anti-dynein antibody (AD2), which is specific for sea urchin sperm flagellar dynein. AD2-labelled fresh-frozen normal rat and human cilia stained clearly by immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. On immunoelectron microscopy, AD2 labelled the outer dynein arms of normal human cilia. Paraffin-embedded normal human cilia also stained by immunofluorescence, although not always clearly. Neither the cilia of WIC-Hyd male rats, an animal model of Kartageners syndrome, nor human cilia from patients with primary ciliary dyskinesia (PCD) reacted positively by the immunofluorescence or PAP technique. Western blots of normal rat cilia yielded a single band of about 450 kDa. In conclusion, AD2 recognizes the outer arm dynein heavy chains of healthy cilia and may be useful in diagnosing and classifying PCD light microscopically especially when only paraffin-embedded specimens are available. This approach may be of potential use for better defining and classifying PCD.

Differential expression of CCR3 ligand mRNA in guinea pig lungs during allergen-induced inflammation

Abstract.Objective and design: The gene expression profile of CCR3 ligands, eotaxin, RANTES, and monocyte chemoattractant protein-3 (MCP-3), was examined in normal and inflamed guinea pig lungs.¶Material: Male Hartley guinea pigs (n = 49).¶Methods: Pulmonary mRNA was obtained from naive animals, animals treated with intravenous lipopolysaccharide administration, and animals repeatedly exposed to aerosolized allergen (ovalbumin). Northern analysis was performed to quantify pulmonary expression of eotaxin, RANTES, and MCP-3 mRNA. Pulmonary eosinophil peroxidase (EPO) activity was measured to quantify eosinophil accumulation.¶Results: Eotaxin and RANTES mRNAs, but not MCP-3 mRNA, were constitutively expressed in guinea pig lungs. Lipopolysaccharide treatment increased MCP-3 mRNA expression, but not eotaxin or RANTES mRNA. In contrast, allergen exposure in sensitized animals caused an increase in eotaxin mRNA, which demonstrated good temporal and quantitative correlation with pulmonary EPO activity, but not in MCP-3 or RANTES mRNA.¶Conclusions: Guinea pig CCR3 ligands demonstrated different gene expression profiles in normal and inflamed airways, suggesting that they play different physiological and pathophysiological roles in the airway.

Acute Septic Shock during Thrombolysis in a Patient with Acute Pulmonary Thromboembolism

A 65-year-old man developed severe septic shock during thrombolytic treatment with urokinase for pulmonary thromboembolism associated with deep venous thrombosis of the right lower limb. Blood cultures were positive forKlebsiella pneumoniae and treatment was successful only when the antibiotics were infused via the same route as the urokinase.