Takeshi Umino

Contraction of fibroblast-containing collagen gels: Initial collagen concentration regulates the degree of contraction and cell survival

SummaryRemodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentration (0.75–2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P <0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P<0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 μg/gel, P<0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P<0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.

A clinical study of hypersensitivity pneumonitis presumably caused by feather duvets

BACKGROUND Bird fanciers lung (BFL) is a type of hypersensitivity pneumonitis induced by the inhalation of bird-related antigens. The BFL induced by feathers is difficult to diagnose because feathers are generally unrecognized as a causative antigen. OBJECTIVE To determine the clinical features of BFL presumably induced by feather duvets (feather duvet lung) to provide clues for diagnosis. METHODS We performed a retrospective review of the medical records of patients with feather duvet lung evaluated between April 1, 2000, and June 30, 2003, at the Tokyo Medical and Dental University Hospital in Japan. RESULTS Seven patients with feather duvet lung were included in this study; 4 patients had acute disease and 3 had chronic BFL. Duration of contact with feather duvets was 1 month to 10 years. Serum KL-6 and surfactant protein D levels were elevated in all the patients. Specific antibodies against avian antigens were positive in acute BFL but negative in chronic BFL. Antigen-induced lymphocyte proliferation in peripheral blood or bronchoalveolar lavage cells was positive in all the patients. The diagnosis was confirmed by an environmental or inhalation provocation test. CONCLUSIONS Feather duvets can induce acute and chronic BFL. Physicians should be aware of feather duvets as a cause of BFL because feather duvets are becoming more prevalent.

Human bronchial epithelial cells can contract type I collagen gels

Fibroblasts can contract collagen gels, a process thought to be related to tissue remodeling. Because epithelial cells are also involved in repair responses, we postulated that human bronchial epithelial cells (HBECs) could cause contraction of collagen gels. To evaluate this, HBECs were plated on the top of native type I collagen gels and were incubated for 48 h. After this, the gels were released and floated in LHC-9-RPMI 1640 for varying times, and gel size was measured with an image analyzer. HBECs caused a marked contraction of the gels within 24 h; the area was reduced by 88 +/- 4% (P < 0.01). The degree of gel contraction was dependent on cell density; 12,500 cells/cm2 resulted in maximal contraction, and half-maximal contraction occurred at 7,500 cells/cm2. Contraction varied inversely with the collagen concentration (91 +/- 1% with 0.5 mg/ml collagen vs. 43 +/- 5% with 1.5 mg/ml collagen). In contrast to fibroblasts that contract gels most efficiently when cast into the gel, HBEC-mediated contraction was significantly (P < 0.01) more efficient when cells were on top of the gels rather than when cast into the gels. Anti-beta 1-integrin antibody blocked HBEC-mediated contraction by > 50%, whereas anti-alpha 2-, anti-alpha 3-, anti-alpha v-, anti-alpha v beta 5-, anti-beta 2-, or anti-beta 4-integrin antibody was without effect. The combination of anti-beta 1-integrin antibody and an anti-alpha-subfamily antibody completely blocked gel contraction induced by HBECs. In contrast, anti-cellular fibronectin antibody did not block HBEC-induced gel contraction, whereas it did block fibroblast-mediated gel contraction. In summary, human airway epithelial cells can contract type I collagen gels, a process that appears to require integrins but may not require fibronectin. This process may contribute to airway remodeling.Fibroblasts can contract collagen gels, a process thought to be related to tissue remodeling. Because epithelial cells are also involved in repair responses, we postulated that human bronchial epithelial cells (HBECs) could cause contraction of collagen gels. To evaluate this, HBECs were plated on the top of native type I collagen gels and were incubated for 48 h. After this, the gels were released and floated in LHC-9-RPMI 1640 for varying times, and gel size was measured with an image analyzer. HBECs caused a marked contraction of the gels within 24 h; the area was reduced by 88 ± 4% ( P < 0.01). The degree of gel contraction was dependent on cell density; 12,500 cells/cm2 resulted in maximal contraction, and half-maximal contraction occurred at 7,500 cells/cm2. Contraction varied inversely with the collagen concentration (91 ± 1% with 0.5 mg/ml collagen vs. 43 ± 5% with 1.5 mg/ml collagen). In contrast to fibroblasts that contract gels most efficiently when cast into the gel, HBEC-mediated contraction was significantly ( P < 0.01) more efficient when cells were on top of the gels rather than when cast into the gels. Anti-β1-integrin antibody blocked HBEC-mediated contraction by >50%, whereas anti-α2-, anti-α3-, anti-αv-, anti-αvβ5-, anti-β2-, or anti-β4-integrin antibody was without effect. The combination of anti-β1-integrin antibody and an anti-α-subfamily antibody completely blocked gel contraction induced by HBECs. In contrast, anti-cellular fibronectin antibody did not block HBEC-induced gel contraction, whereas it did block fibroblast-mediated gel contraction. In summary, human airway epithelial cells can contract type I collagen gels, a process that appears to require integrins but may not require fibronectin. This process may contribute to airway remodeling.

Evaluation of subclinical respiratory tract inflammation in heavy smokers who switch to a cigarette-like nicotine delivery device that primarily heats tobacco

Cigarette smoking remains a major public health problem. For smokers who cannot or do not wish to quit, few options exist to reduce health risks. A cigarette-like nicotine delivery device that heats rather than burns tobacco might deliver nicotine with fewer toxins. The current study was designed to determine whether asymptomatic heavy smokers who did not wish to quit had improvement in lower respiratory tract inflammation after switching to Eclipse, a cigarette-like nicotine delivery device that primarily heats rather than burns tobacco. Twelve smokers of at least 40 cigarettes daily, asymptomatic and in good health, underwent paired bronchoscopies, bronchoalveolar lavages and endobronchial biopsies before and after 2 months of using Eclipse. Eight normal non-smoking individuals were evaluated on one occasion for comparison. Inflammation was assessed by direct inspection and by cytological parameters. Goblet cell metaplasia was assessed histologically. Compared to non-smokers, smokers had increased visible inflammation, increased recovery of inflammatory cells and increased percentage of goblet cells. There were significant reductions in all these parameters following a switch to Eclipse use, although the improvement did not reach the normal range. No significant differences were observed in peripheral blood measures. Nicotine levels were generally maintained, and exhaled carbon monoxide (CO) levels trended strongly upward. One individual experienced a transient twofold increase in CO and concurrently experienced transient headaches. Eclipse use may be a strategy to reduce the health risks for heavy smokers unwilling or unable to quit.

Expression of epithelial markers in nocturnal asthma

BACKGROUND Although the airway epithelium participates in inflammation and repair, the circadian expression of epithelial cell markers involved in these processes has not been investigated. OBJECTIVE We sought to determine whether expression of CD51 (vitronectin and fibronectin receptor), CD54 (intercellular adhesion molecule-1), HLA-DR (activation marker), CD29 (beta1 integrin), CD49b (collagen receptor), and CD11b (complement receptor) exhibit a circadian rhythm in asthma. METHODS Eleven patients with nocturnal asthma (NA), 9 subjects with nonnocturnal asthma (NNA), and 10 control subjects underwent bronchoscopy at 4 PM and 4 AM in a random order 1 week apart, with brushing of the proximal and distal airways. The percentage of cells staining for a particular marker was determined. RESULTS At 4 PM, HLA-DR in the proximal airways and CD54 in the distal airways was significantly greater in control subjects as compared with asthmatic subjects (HLA-DR, control subjects: 10.0% [range, 5.0% to 21.0%]; NNA: 8.0% [range, 4.0% to 14.5%] NA: 3.5% [range, 2.0% to 6.0%], P = .01; CD54, control subjects: 17.0% [range, 8.0% to 25.0%], NNA: 8.0% [range, 5.3% to 11.5%], NA: 7.0% [range, 4.0% to 15.0%], P = .O;). At 4 AM, CD51 in the distal airways was significantly greater in patients with NA as compared with patients with NNA and control subjects (control subjects, 23.0% [range, 13.8% to 30.5%]; NNA, 32.0% [range, 13.0% to 35.0%]; NA, 40.0% [range, 23.0% to 50.0%], P = .05). Expression of CD51 in the distal airways correlated with the degree of airway obstruction (r = -0.57, P = .001). Control subjects exhibited significant circadian variation in the expression of HLA-DR in the proximal airways and CD54 in the distal airways. CONCLUSION The increased CD51 at night in patients with NA may be related to increased airway inflammation and repair processes in response to injury. The circadian changes in CD54 and HLA-DR in control subjects require further study to determine their significance. (J Allergy Clin

The Effect of Seratrodast on Eosinophil Cationic Protein and Symptoms in Asthmatics

Thromboxane A2 (TXA2), an arachidonate derivative, is a potent bronchoconstrictor; therefore, blocking TXA2 should attenuate airway narrowing. Seratrodast, a TXA2 receptor antagonist, is expected to be a potent antiasthmatic. It was reported that seratrodast reduced bronchial hyperresponsiveness. However, it is controversial whether it reduces airway inflammation. We studied some additional effects of oral seratrodast to inhaled corticosteroids on 10 adult asthmatics in an open-label, crossover design study. Eosinophil cationic protein (ECP) levels in serum and sputum, peak expiratory flow rate (PEF), clinical symptoms, and airway responsiveness were evaluated. Clinical symptom scores were improved by administration of seratrodast (p<0.05). The addition of seratrodast to asthmatic patients significantly improved mean PEF (p<0.05). In addition, withdrawal of seratrodast resulted in deterioration of PEF. Airway hyperresponsiveness to acetylcholine measured by Astograph was improved by administration of seratrodast (p<0.01), and returned to the level of “run-in period” after withdrawal. Administration of seratrodast decreased the concentration of ECP in sputum significantly (p<0.05), and sputum ECP significantly increased again after withdrawal of (p<0.05). These results suggest that seratrodast improves clinical symptoms and airway hyperresponsiveness by reducing airway inflammation. Seratrodast may be useful as an anti-inflammatory agent and beneficial when added to inhaled corticosteroids in the treatment of bronchial asthma.