A high α1-antitrypsin/interleukin-10 ratio predicts bacterial pneumonia in adults with community-acquired pneumonia: a prospective cohort study.

BACKGROUND: Current microbiological tests fail to identify the causative microorganism in more than half of all pneumonia cases. We explored biomarkers that could be used for differentiating between bacterial and viral pneumonia in patients with community-acquired pneumonia (CAP). METHODS: In this prospective cohort study conducted in Japan, data obtained from adult patients with bacterial pneumonia, including bacterial and viral coinfections (bacterial pneumonia [BP] group), and purely viral pneumonia (VP group) at diagnosis were analyzed using multivariate logistic regression analysis to identify predictors of bacterial pneumonia. Furthermore, a decision tree was developed using the predictors. RESULTS: A total of 210 patients were analyzed. The BP and VP groups comprised 108 and 18 patients, respectively. The other 84 patients had no identified causative microorganism. The two groups shared similar characteristics, including disease severity; however, a significant difference (p < 0.05) was observed between the two groups regarding sputum type; sputum volume score; neutrophil counts; and serum levels of interleukin (IL)-8, IL-10, and α1-antitrypsin (AAT). Sputum volume score (p < 0.001), IL-10 (p < 0.001), and AAT (p = 0.008) were ultimately identified as predictors of BP. The area under the curve for these three variables on the receiver operating characteristic (ROC) curve was 0.927 (95% confidence interval [CI]: 0.881-0.974). The ROC curve for sputum volume score and an AAT/IL-10 ratio showed a diagnostic cutoff of 1 + and 65, respectively. Logistic regression analysis using dichotomized variables at the cutoff values showed that the odds ratios for the diagnosis of BP were 10.4 (95% CI: 2.2-50.2) for sputum volume score (absence vs. presence) and 19.8 (95% CI: 4.7-83.2) for AAT/IL-10 ratio (< 65 vs. ≥ 65). CONCLUSIONS: Considering that obtaining a definitive etiologic diagnosis with the current testing methods is difficult and time consuming, a decision tree with two predictors, namely sputum volume and the AAT/IL-10 ratio, can be useful in predicting BP among patients diagnosed with CAP and facilitating the appropriate use of antibiotics. TRIAL REGISTRATION: UMIN000034673 registered on November 29, 2018.

Dabrafenib and trametinib therapy in an elderly patient with non-small cell lung cancer harboring the BRAF V600E mutation.

Dabrafenib and trametinib therapy for BRAF V600E-mutant non-small cell lung cancer (NSCLC) has demonstrated strong antitumor effects in clinical trials and has been approved for use in clinical practice. However, the efficacy and safety of this combination therapy in elderly patients remain unclear. An 86-year-old male patient, who had been diagnosed with lung adenocarcinoma with the BRAF V600E mutation, received dabrafenib and trametinib combination chemotherapy. The tumor shrunk rapidly; however, therapy was discontinued after 40 days because adverse events (hypoalbuminemia, peripheral edema, and pneumonia) developed. Although this targeted combination therapy seemed to cause relatively severe adverse events compared with single-agent targeted therapy in this "oldest old" elderly patient, the marked tumor shrinkage prolonged the patient's life and helped him to maintain a good general condition. Active targeted therapy may therefore be considered with appropriate drug dose reduction instead of conservative treatment, even if a patient is extremely old.

Effect of long-term clarithromycin therapy on prevention of pneumonia in older adults: A randomized, controlled trial.

AIM: Pneumonia in older adults is increasingly recognized as a healthcare issue in countries with an aging population. Long-term macrolide therapy reduces exacerbations of chronic respiratory diseases, but its effects on the prevention of pneumonia have not been determined. METHODS: We carried out a randomized, controlled trial to test the effect of long-term clarithromycin therapy on the prevention of pneumonia among older adults. People aged ≥65 years who had recovered from pneumonia within the previous 3 months were recruited and randomly allocated to a long-term, low-dose clarithromycin (CAM) therapy group (n = 13) or a control group (n = 15). RESULTS: Both groups were followed up until recurrence of pneumonia. The median follow-up period was 251 days (95% CI 171-330) in the CAM group and 132 days (95% CI 67-196) in the control group (P = 0.627). The recurrence rate of pneumonia was two out of 13 (15%) in the CAM group and five out of 15 (33%) in the control group (P = 0.268). The median time to recurrence of pneumonia was 315 days (95% CI 249-382) in the CAM group and 260 days (95% CI 184-335) in the control group (P = 0.260). None of the differences between groups were statistically significant. CONCLUSIONS: No statistically significant suppressive effects of long-term, low-dose macrolide therapy on the development of pneumonia among older people were found in this small sample. A large-scale, randomized, controlled study is required. Geriatr Gerontol Int 2019; 19: 1006-1009.

Elevated Ozone Deteriorates Grain Quality of Japonica Rice cv. Koshihikari, Even if it Does Not Cause Yield Reduction.

BACKGROUND: It is becoming clear that ozone affects not only grain yield but also grain quality in rice. However, the biochemical mechanisms responsible for ozone-induced changes in appearance quality or components are poorly understood. We analyzed appearance quality and starch composition in the rice cultivars "Koshihikari" (japonica) and "Kasalath" (indica) grown under elevated ozone conditions. RESULTS: Elevated ozone significantly increased the proportion of immature (mainly chalky) kernels in "Koshihikari" but not in "Kasalath". Scanning electron microscopy of transverse sections of kernels showed that endosperm starch granules of "Koshihikari" ripened under elevated ozone were loosely packed with large spaces and contained irregular rounded granules. Amylose content was increased in "Koshihikari" kernels with ozone exposure, but was unchanged in "Kasalath" kernels. Distribution analysis of amylopectin chain length showed that ozone induces a decrease of long-side chains and alterations of short side-chains in "Koshihikari" kernels. Furthermore, Starch Synthase (SS) IIIa transcript levels in "Koshihikari" caryopses were decreased by elevated ozone. CONCLUSIONS: The japonica cultivar "Koshihikari" showed significant deterioration in appearance quality of kernels caused by abnormal starch accumulation due to exposure to ozone. The alteration patterns of amylose and amylopectin in ozone-exposed rice kernels are similar to those in rice kernels harvested from SSIIIa-deficient mutants. These findings suggest that the increase of chalky kernels in ozone-treated "Koshihikari" is partly attributable to the repressed expression of SSIIIa involved in amylopectin side-chain elongation with ozone exposure. Elevated ozone reduced appearance quality in "Koshihikari" although it did not impair starch properties contributing to the eating quality of cooked rice.

Epidemiology and clinical features of pulmonary nontuberculous mycobacteriosis in Nagasaki, Japan.

BACKGROUND AND OBJECTIVES: Recent reports indicate that the incidence of nontuberculous mycobacterial-lung disease (NTM-LD) is increasing. This study aimed to investigate the epidemiology and clinical features of NTM-LD patients in Nagasaki prefecture, Japan to identify the negative prognostic factors for NTM-LD in Japan. METHODS: The medical records of patients newly diagnosed with NTM-LD in eleven hospitals in Nagasaki prefecture between January 2001 and February 2010 were reviewed. Data regarding the annual population of each region and the incidence of all forms of tuberculosis were collected to assess geographic variations in NTM-LD incidence, isolates, and radiological features. RESULTS: A total 975 patients were diagnosed with NTM-LD. The incidence increased over the study period and reached 11.0 and 10.1 per 100,000 population in 2008 and 2009, respectively. M. intracellulare was the most common pathogen in the southern region, and M. avium most common in other regions. The most common radiographic pattern was the nodular-bronchiectatic pattern. Age >60 years, body mass index <18.5 kg/m2, underlying lung disease, and cavitary pattern were the negative prognostic factors at the 1-year follow-up. CONCLUSIONS: The incidence of NTM-LD has been increasing in Nagasaki prefecture. The isolates and radiographic features of patients vary markedly by region.

Ozone-Induced Rice Grain Yield Loss Is Triggered via a Change in Panicle Morphology That Is Controlled by ABERRANT PANICLE ORGANIZATION 1 Gene.

Rice grain yield is predicted to decrease in the future because of an increase in tropospheric ozone concentration. However, the underlying mechanisms are unclear. Here, we investigated the responses to ozone of two rice (Oryza Sativa L.) cultivars, Sasanishiki and Habataki. Sasanishiki showed ozone-induced leaf injury, but no grain yield loss. By contrast, Habataki showed grain yield loss with minimal leaf injury. A QTL associated with grain yield loss caused by ozone was identified in Sasanishiki/Habataki chromosome segment substitution lines and included the ABERRANT PANICLE ORGANIZATION 1 (APO1) gene. The Habataki allele of the APO1 locus in a near-isogenic line also resulted in grain yield loss upon ozone exposure, suggesting APO1 involvement in ozone-induced yield loss. Only a few differences in the APO1 amino acid sequences were detected between the cultivars, but the APO1 transcript level was oppositely regulated by ozone exposure: i.e., it increased in Sasanishiki and decreased in Habataki. Interestingly, the levels of some phytohormones (jasmonic acid, jasmonoyl-L-isoleucine, and abscisic acid) known to be involved in attenuation of ozone-induced leaf injury tended to decrease in Sasanishiki but to increase in Habataki upon ozone exposure. These data indicate that ozone-induced grain yield loss in Habataki is caused by a reduction in the APO1 transcript level through an increase in the levels of phytohormones that reduce leaf damage.

Comparative analysis of seed transcriptomes of ambient ozone-fumigated 2 different rice cultivars.

High ozone (O3) concentrations not only damage plant life but also cause considerable losses in plant productivity. To screen for molecular factors usable as potential biomarkers to identify for O3-sensitive and -tolerant lines and design O3 tolerant crops, our project examines the effects of O3 on rice, using high-throughput omics approaches. In this study, we examined growth and yield parameters of 4 rice cultivars fumigated for a life-time with ambient air (mean O3: 31.4-32.7 ppb) or filtered air (mean O3: 6.6-8.3 ppb) in small open-top chambers (sOTCs) to select O3-sensitive (indica cv Takanari) and O3-tolerant (japonica cv Koshihikari) cultivars for analysis of seed transcriptomes using Agilent 4 × 44K rice oligo DNA chip. Total RNA from dry mature dehusked seeds of Takanari and Koshihikari cultivars was extracted using a modified protocol based on cethyltrimethylammonium bromide extraction buffer and phenol-chloroform-isoamylalcohol treatment, followed by DNA microarray analysis using the established dye-swap method. Direct comparison of Koshihikari and Takanari O3 transcriptomes in seeds of rice plants fumigated with ambient O3 in sOTCs successfully showed that genes encoding proteins involved in jasmonic acid, GABA biosynthesis, cell wall and membrane modification, starch mobilization, and secondary metabolite biosynthesis are differently regulated in sensitive cv Takanari and tolerant cv Koshihikari. MapMan analysis further mapped the molecular factors activated by O3, confirming Takanari is rightly classified as an O3 sensitive genotype.

Analysis of proteins associated with ozone stress response in soybean cultivars.

Ozone stress induces leaf injuries and yield losses in soybean. To identify the proteins associated with the stress response, ozone tolerant and sensitive cultivars were analyzed by 2D-PAGE. After 11 days of ozone stress unifoliate leaves of tolerant soybean cv. Enrei developed 15% and sensitive cv. Nakasennari developed 52% leaf injury symptoms. Analysis of proteins in the unifoliate leaves identified six proteins in tolerant cv. Enrei and three proteins in sensitive cv. Nakasennari, responded significantly to ozone stress. The significantly responded proteins identified in this study were ATP synthase α-subunit, ATP synthase ß-subunit, phosphoglycerate kinase, aldo/ketoreductase, rubiscoactivase and glutamine synthetase. To understand that the differences in response of ATP synthase proteins were ultimately associated with extracellular ATP signaling response, ozone sensitive cv. Nakasennari was treated with 1 mM ATP. Unifoliate leaves of ATP treated plants have significantly reduced injury symptoms (20%) under ozone stress compare to control plants (47%). This confirms that extracellular ATP signaling in ATP synthase dependent manner is playing a pivotal role in inducing ozone stress tolerance and preventing injuries in soybean cultivars.

Genome-wide mapping of the ozone-responsive transcriptomes in rice panicle and seed tissues reveals novel insight into their regulatory events.

The 'ozone (O3)-responsive transcriptome' behavior in the panicles and grains of rice plant was studied individually through high-throughput oligo-DNA microarray technique. O3 differentially and separately regulated 620 and 130 genes in the panicles and grains. Among the O3-responsive genes, 176 and 444 genes were up- and down-regulated in panicle compared to 24 and 106 genes in grain, respectively. Further mapping revealed that the majority of differentially expressed genes were mainly involved in signaling, hormonal, cell wall, transcription, proteolysis, and defense events. Many previously unknown O3-responsive novel genes were identified. Inventory of 745 O3-responsive genes and their mapping will expand our knowledge on novel regulatory processes in both panicles and grains of rice; and, serve as a resource towards the designing of rice crops for future high-O3world. PURPOSE OF WORK: Tropospheric ozone (O3) severely affects agricultural production worldwide. Present study aims to reveal a detailed O3 responsive gene network in panicle and grains of rice plants through transcriptomics approach. Our results provide an insight into the basis of O3-response in rice plants, and will help to develop suitable rice genotype for future high O3- world.

Indirect prediction of surface ozone concentration by plant growth responses in East Asia using mini-open top chambers.

We developed small and mobile open top chambers (mini-OTC) measuring 0.6 m (W) × 0.6 m (D) × 1.2 m (H) with an air duct of 0.6 m (W) × 0.23 m (D) × 1.2 m (H). The air duct can be filled with activated charcoal to blow charcoal filtered air (CF) into the chamber, as opposed to non-filtered ambient air (NF). Ozone sensitive radish Raphanus sativus cv. Red Chime and rosette pakchoi Brassica campestris var. rosularis cv. ATU171 were exposed to NF and CF in mini-OTCs at different locations in East Asia. A total of 29 exposure experiments were conducted at nine locations, Shanghai, China, Ha Noi, Vietnam, Lampang, Phitsanulok and Pathumtani, Thailand, and Hiratsuka, Kisai, Abiko and Akagi, Japan. Although no significant relationships between the mean concentrations of ambient O(3) during the experimental period and the growth responses were observed for either species, multiple linear regression analysis suggested a good relationship between the biomass responses in each species and the O(3) concentration, temperature, and relative humidity. The cumulative daily mean O(3) (ppb/day) could be indirectly predicted by NF/CF based on the dry weight ratio of biomass, mean air temperature, and relative air humidity.

Leaf ascorbic acid level--is it really important for ozone tolerance in rice?

Leaf ascorbic acid (ASA) level is thought to be an important trait conferring stress tolerance in plants, but definite evidence regarding its effectiveness in the breeding of stress tolerant crops is lacking. Therefore, the stress response of a rice TOS17 insertion mutant (ND6172) for a GDP-D-mannose-3',5'-epimerase gene, which is involved in ASA biosynthesis, was tested. Two fumigation experiments were conducted, in which rice plants (Oryza sativa L.) were exposed to (i) high ozone for ten days at the tillering stage (100 ppb, 7 h day⁻¹); and (ii) to four different ozone concentrations ranging from charcoal filtered air to 2.5 times the ambient concentration for the entire growth season. The mutant ND6172 had around 20-30% lower ASA level than the wild-type (Nipponbare), and exhibited a moderately higher level of visible leaf symptoms due to ozone exposure. Differences in ASA level between ND6172 and Nipponbare led to differential responses of the glutathione level, and the activities of glutathione reductase, ascorbate peroxidase, and dehydroascorbate reductase. With season-long ozone fumigation, yields and yield components were not negatively affected at ambient ozone level in both genotypes, but showed stronger decreases in ND6172 at higher ozone levels, especially at 2.5 times the ambient level. Similarly, the mature straw of ND6172 exhibited a higher degree of lignification at the 2.5 times ambient ozone level. In conclusion, a difference in leaf ASA level of around 20-30% is relevant for ozone tolerance in rice at levels exceeding the current ambient ozone concentrations.

The response of rice grain quality to ozone exposure during growth depends on ozone level and genotype.

The effects of ozone exposure during the cropping season on rice grain quality were investigated in chamber experiments employing four ozone treatments (charcoal filtered air, ambient, 2× ambient, and 2.5× ambient concentration) and six genotypes. The concentrations of protein and lipids in brown rice increased significantly in response to ozone, while starch concentration and thousand kernel mass decreased. Other parameters, including the concentrations of iron, zinc, phenolics, stickiness and geometrical traits did not exhibit significant treatment effects. Total brown rice yield, protein yield, and iron yield were negatively affected by ozone. Numerous genotypic differences occurred in the response to ozone, indicating the possibility of optimizing the grain quality in high ozone environments by breeding. It is concluded that although the concentrations of two important macronutrients, proteins and lipids, increased in ozone treated grains, the implications for human nutrition are negative due to losses in total grain, protein and iron yield.

Eosinophilic pneumonia due to visceral larva migrans possibly caused by Ascaris suum: a case report and review of recent literatures.

We report the case of a 62-year-old man who developed eosinophilic pneumonia due to visceral larva migrans (VLM) that was possibly caused by Ascaris suum. The patient, a resident of the middle Kyushu area who was found of eating raw porcine liver, complained of dry cough without dyspnea. The chest radiography showed a migration of infiltrative shadow. Transbronchial lung biopsy of the right middle lobe revealed massive infiltration of eosinophils. The multi-dot enzyme-linked immunosorbent assay (ELISA) and microtiter plate ELISA showed positive results for A. suum; therefore, the patient was diagnosed with VLM caused by A. suum. The patient was administered albendazole (600 mg/day) for 28 days; he recovered successfully with no adverse effects except mild liver dysfunction. Several cases of VLM caused by A. suum have been reported in Japan, with a majority of the cases being reported in Kyushu. Careful history taking of the patient's area of residence and dietary habit is essential for the diagnosis of this parasitic disease with underestimated prevalence.

Outpatients flow management and ophthalmic electronic medical records system in university hospital using Yahgee Document View.

General electronic medical records systems remain insufficient for ophthalmology outpatient clinics from the viewpoint of dealing with many ophthalmic examinations and images in a large number of patients. Filing systems for documents and images by Yahgee Document View (Yahgee, Inc.) were introduced on the platform of general electronic medical records system (Fujitsu, Inc.). Outpatients flow management system and electronic medical records system for ophthalmology were constructed. All images from ophthalmic appliances were transported to Yahgee Image by the MaxFile gateway system (P4 Medic, Inc.). The flow of outpatients going through examinations such as visual acuity testing were monitored by the list "Ophthalmology Outpatients List" by Yahgee Workflow in addition to the list "Patients Reception List" by Fujitsu. Patients' identification number was scanned with bar code readers attached to ophthalmic appliances. Dual monitors were placed in doctors' rooms to show Fujitsu Medical Records on the left-hand monitor and ophthalmic charts of Yahgee Document on the right-hand monitor. The data of manually-inputted visual acuity, automatically-exported autorefractometry and non-contact tonometry on a new template, MaxFile ED, were again automatically transported to designated boxes on ophthalmic charts of Yahgee Document. Images such as fundus photographs, fluorescein angiograms, optical coherence tomographic and ultrasound scans were viewed by Yahgee Image, and were copy-and-pasted to assigned boxes on the ophthalmic charts. Ordering such as appointments, drug prescription, fees and diagnoses input, central laboratory tests, surgical theater and ward room reservations were placed by functions of the Fujitsu electronic medical records system. The combination of the Fujitsu electronic medical records and Yahgee Document View systems enabled the University Hospital to examine the same number of outpatients as prior to the implementation of the computerized filing system.

Ozone stress-induced proteomic changes in leaf total soluble and chloroplast proteins of soybean reveal that carbon allocation is involved in adaptation in the early developmental stage.

Considerable soybean yield losses caused by ozone (O3) stress have been demonstrated by large-scale meta-analyses of free-gas concentration enrichment systems. In this study, comparative proteomic approach was employed to explore the differential changes of proteins in O3 target structures such as leaf and chloroplasts of soybean seedlings. Acute O3 exposure (120 parts-per-billion) for 3 days did not cause any visible symptoms in developing leaves. However, higher amounts of ROS and lipid peroxidation indicated that severe oxidative burst occurred. Immunoblot analysis of O3-induced known proteins revealed that proteins were modulated before symptoms became visible. Proteomic analysis identified a total of 20 and 32 differentially expressed proteins from O3-treated leaf and chloroplast, respectively. Proteins associated with photosynthesis, including photosystem I/II and carbon assimilation decreased following exposure to O3. In contrast, proteins involved in antioxidant defense and carbon metabolism increased. The activity of enzymes involved in carbohydrate metabolism increased following exposure to O3, which is consistent with the decrease in starch and increase in sucrose concentrations. Taken together, these results suggest that carbon allocation is tightly programmed, and starch degradation probably feeds the tricarboxylic acid cycle while the photosynthesis pathway is severely affected during O3 stress.

[A case of cardiac tamponade caused by tuberculous pericarditis].

An 86-year-old male was admitted to Izumikawa Hospital complaining of fever and chest pain. Electrocardiography revealed low-voltage, atrial fibrillation and QRS complexes. The chest PA-view showed an increased cardiothoracic ratio (65.9%) and an infiltrative shadow in the left lower lung field. Computed tomography revealed copious pericardial and bilateral pleural effusion. Pericardiocentesis was performed immediately after admission, and 80 ml of hemorrhagic fluid was aspirated. The adenosine deaminase activity of the pericardial fluid was 77.2 IU/l, and testing for tuberculous bacilli by polymerase chain reaction was positive. As these parameters strongly suggested tuberculous pericarditis, pericardial drainage was continued for another two weeks, and a delayed combination therapy with isoniazid, rifampicin, streptomycin, and a high dose of prednisolone was initiated. Two weeks later, the symptoms were relieved and the pericardial effusion had also decreased.

In vitro and in vivo activities of novel fluoroquinolones alone and in combination with clarithromycin against clinically isolated Mycobacterium avium complex strains in Japan.

The recommended treatments for Mycobacterium avium complex (MAC) infectious disease are combination regimens of clarithromycin (CLR) or azithromycin with ethambutol and rifamycin. However, these chemotherapy regimens are sometimes unsuccessful. Recently developed antimicrobial agents, such as newer fluoroquinolones (FQs) containing C-8 methoxy quinolone (moxifloxacin [MXF] and gatifloxacin [GAT]), are expected to be novel antimycobacterial agents. Here, we evaluated the in vitro and in vivo antimycobacterial activities of three FQs (MXF, GAT, and levofloxacin) and CLR against clinically isolated MAC strains. Subsequently, the in vitro and in vivo synergic activities of FQ-CLR combinations against MAC strains were investigated. CLR and the individual FQs alone showed promising activity against MAC strains in vitro, and the bacterial counts in organs (lungs, liver, and spleen) of MAC-infected mice treated with single agents were significantly reduced compared to control mice. CLR showed the best anti-MAC effect in vivo. When the three FQs were individually combined with CLR in vitro, mild antagonism was observed for 53 to 57% of the tested isolates. Moreover, mice were infected with MAC strains showing mild antagonism for FQ-CLR combinations in vitro, and the anti-MAC effects of the FQ-CLR combinations were evaluated by counting the viable bacteria in their organs and by histopathological examination after 28 days of treatment. Several FQ-CLR combinations exhibited bacterial counts in organs significantly higher than those in mice treated with CLR alone. Our results indicate that the activity of CLR is occasionally attenuated by combination with an FQ both in vitro and in vivo and that this effect seems to be MAC strain dependent. Careful combination chemotherapy using these agents against MAC infectious disease may be required.

Effects of elevated carbon dioxide and ozone on foliar proanthocyanidins in Betula platyphylla, Betula ermanii, and Fagus crenata seedlings.

Proanthocyanidins (PAs) or condensed tannins are a major group of phenolic compounds in the leaves of birch trees and many other woody and herbaceous plants. These compounds constitute a significant allocation of carbon in leaves and are involved in plant responses to environmental stress factors, such as pathogens or herbivores. In some plants, PA concentrations are affected by atmospheric carbon dioxide (CO(2)) and ozone (O(3)) levels that may influence, for example, species fitness, community structure, or ecosystem nutrient cycling. Therefore, a study on the quantitative response of PAs to elevated concentrations of carbon dioxide (CO(2)) and ozone (O(3)) was undertaken in seedlings of Betula platyphylla, Betula ermanii, and Fagus crenata. Seedlings were exposed to ambient or elevated O(3) and CO(2) levels during two growing seasons in the Kanto district in Japan. Ten open-top chambers were used for five different treatments with two replicates: filtered air (FA), ambient air (ambient O(3), 43 ppb; ambient CO(2), 377 ppm), elevated O(3) (1.5 x ambient O(3), 66 ppb), elevated CO(2) (1.5 x ambient CO(2), 544 ppm), and elevated O(3) and CO(2) combined. In addition, seedlings growing in natural conditions outside of chambers were studied. Leaf samples were analyzed for total PA concentrations by butanol-HCl assay and for polymeric PA concentrations by normal-phase high-performance liquid chromatography. Total PA concentrations in leaves of all species were similarly affected by different treatments. They were significantly higher in seedlings treated with elevated CO(2) and O(3) combined, and in seedlings growing outside chambers compared with the FA controls. F. crenata contained only traces of polymeric PAs, but significant species x treatment interaction was observed in the polymeric PA concentrations in B. ermanii and B. platyphylla. In B. platyphylla, leaves treated with elevated CO(2) + O(3) differed significantly from all other treatments. It was suggested that the strongest effect of elevated CO(2) and O(3) combined on leaf PA contents resulted from the additive effect of these environmental factors on phenolic biosynthesis.