Human serum albumin fusion protein as therapeutics for targeting amyloid beta in Alzheimer's diseases.

Alzheimer's disease (AD) is characterized by amyloid beta (Aß) plaques and neurofibrillary tangles. AD drug development has been limited due to the presence of the blood-brain barrier (BBB), which prevents efficient uptake of therapeutics into the brain. To solve this problem, we used trans-activator of transcription (TAT)-transducing domain and added the human serum albumin (HSA) carrier to increase the half-life of the drug within the body. In addition, we included the protein of interest for lowering Aß deposition and/or neurofibrillary tangles. We made HSA fusion protein (designated AL04) which contains Cystatin C (CysC) as core mechanism of action moiety in the construct containing tandem repeat TAT (dTAT). After purification of 80KDa AL04, we investigate the therapeutic potential of AL04 in vitro and AD mouse model Tg2576. We evaluated the permeability of AL04 through the BBB using a cell-basedhuman BBB model and show that dTAT plays a role in facilitating the delivery of 80 kDa protein. We found out that AL04 attenuates Aß-induced neurotoxicity in PC12 cells. In Tg2576 mice brain, Aß plaques were dramatically reduced in AL04 treated mice. These data suggest that BBB-crossing albumin fusion protein AL04 with CysC active moiety can be a disease modifying treatment for AD.

Type II collagen and glycosaminoglycan expression induction in primary human chondrocyte by TGF-ß1.

BACKGROUND: A localized non-surgical delivery of allogeneic human chondrocytes (hChonJ) with irradiated genetically modified chondrocytes (hChonJb#7) expressing transforming growth factor-ß1 (TGF-ß1) showed efficacy in regenerating cartilage tissue in our pre-clinical studies and human Phase I and II clinical trials. These previous observations led us to investigate the molecular mechanisms of the cartilage regeneration. METHODS: Genetically modified TGF-ß1preprotein was evaluated by monitoring cell proliferation inhibition activity. The effect of modified TGF-ß1 on chondrocytes was evaluated based on the type II collagen mRNA levels and the amount of glycosaminoclycan (GAG) formed around chondrocytes, which are indicative markers of redifferentiated chondrocytes. Among the cartilage matrix components produced by hChonJb#7 cells, type II collagen and proteoglycan, in addition to TGF-ß1, were also tested to see if they could induce hChonJ redifferentiation. The ability of chondrocytes to attach to artificially induced defects in rabbit cartilage was tested using fluorescent markers. RESULTS: Throughout these experiments, the TGF-ß1 produced from hChonJb#7 was shown to be equally as active as the recombinant human TGF-ß1. Type II collagen and GAG production were induced in hChonJ cells by TGF-ß1 secreted from the irradiated hChonJb#7 cells when the cells were co-cultured in micro-masses. Both hChonJ and hChonJb#7 cells could attach efficiently to the defect area in the rabbit cartilage. CONCLUSIONS: This study suggests that the mixture (TG-C) of allogeneic human chondrocytes (hChonJ) and irradiated genetically modified human chondrocytes expressing TGF-ß1 (hChonJb#7) attach to the damaged cartilage area to produce type II collagen-GAG matrices by providing a continuous supply of active TGF-ß1.

BMP2 shows neurotrophic effects including neuroprotection against neurodegeneration.

In this study, we found that BMP2 exerts neurotrophic effects, including a neuroprotective effect against nocodazole-induced neuritic degeneration, on neuronal cells. We also found that BMP2-induced neurotrophic effects are directly involved in Smad-dependent signaling as well as PI3K/PTEN-Akt-mTOR signaling. Moreover, BMP2-induced neurotrophic effects occur by stabilization of neuronal microtubules. Thus, these findings suggest that BMP2 can be a potential therapeutic target for nerve injury treatment.

Current advances in retroviral gene therapy.

There have been major changes since the incidents of leukemia development in X-SCID patients after the treatments using retroviral gene therapy. Due to the risk of oncogenesis caused by retroviral insertional activation of host genes, most of the efforts focused on the lentiviral therapies. However, a relative clonal dominance was detected in a patient with ß-thalassemia Major, two years after the subject received genetically modified hematopoietic stem cells using lentiviral vectors. This disappointing result of the recent clinical trial using lentiviral vector tells us that the current and most advanced vector systems does not have enough safety. In this review, various safety features that have been tried for the retroviral gene therapy are introduced and the possible new ways of improvements are discussed. Additional feature of chromatin insulators, co-transduction of a suicidal gene under the control of an inducible promoter, conditional expression of the transgene only in appropriate target cells, targeted transduction, cell type-specific expression, targeted local administration, splitting of the viral genome, and site specific insertion of retroviral vector are discussed here.

Changes in oxidative stress biomarker and gene expression levels in workers exposed to volatile organic compounds.

Exposure to volatile organic compounds (VOCs) was known to result in immunologic, respiratory, carcinogenic, reproductive, neurologic, and cardiovascular effects. However, the mechanisms by which VOCs induce these adverse health effects are not well understood. To evaluate the change of oxidative stress biomarker and gene expression levels in workers exposed to VOCs, we obtained urine and blood samples from 21 subjects before and after occupational exposure to VOCs. We measured levels of muconic acid (MuA), hippuric acid (HA), mandelic acid (MaA), and methyl hippuric acid (MHA) as urinary exposure biomarkers for benzene, toluene, ethylbenzene, and xylene (collectively BTEX), and malondialdehyde (MDA) and 8-hydroxydeoxyguanine (8-OHdG) as oxidative stress biomarkers in all subjects. We also evaluated BTEX-mediated RNA expression using cDNA microarray in 14 subjects. HA and MHA levels were higher following occupational exposure to VOCs (p < 0.01). In the linear regression analysis, HA ratios of after- and before-exposure were found to be significantly associated with increase of MDA ratios of after- and before-exposure after controlling for age, body mass index, and smoking (ß = 0.06, p = 0.031). Evaluation of the gene expressions by HA showed that 23 gene expressions were found to be significantly associated with HA levels after adjusting for age, body mass index, and smoking (p < 0.001). In particular, expressions of ENO3 and CDNA FLJ39461 fis among the 23 genes were significantly associated with the change in MDA level (p < 0.05). Our study results suggest that exposure to VOCs, specifically toluene, induces oxidative stress and various gene expression change of which some may be responsible for oxidative stress.

TLR4-mediated activation of mouse macrophages by Korean mistletoe lectin-C (KML-C).

Korean mistletoe lectin (KML-C) is an adjuvant that activates systemic and mucosal immune cells to release cytokines including TNF-alpha, which induces immunity against viruses and cancer cells. Although the immunomodulatory activity of KML-C has been well established, the underlying mechanism of action of KML-C has yet to be explored. When mouse peritoneal macrophages were treated with KML-C, both transcription and translation of TLR4 were upregulated. KML-C-induced TLR4 downstream events were similar to those activated by LPS: the upregulation of interleukin-1 receptor-associated kinase-1 (IRAK1); resulting in macrophage activation and TNF-alpha production. When TLR4 was blocked using a TLR4-specific neutralizing antibody, TNF-alpha production from the macrophages was significantly inhibited. Moreover, TLR4-deficient mouse macrophages treated with KML-C also secreted greatly reduced level of TNF-alpha secretion. Finally, TLR4 molecules were co-precipitated with KML-C, to which agarose beads were conjugated, indicating that those molecules are associated. These data indicate that KML-C activates mouse macrophages to secrete TNF-alpha by interacting with the TLR4 molecule and activating its signaling pathways.

Pre-clinical studies of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 (TG-C).

BACKGROUND AIMS: The aim was to evaluate cartilage regeneration in animal models involving induced knee joint damage. Through cell-mediated gene therapy methods, a cell mixture comprising a 3:1 ratio of genetically unmodified human chondrocytes and transforming growth factor beta-1 (TGF-beta1)-secreting human chondrocytes (TG-C), generated via retroviral transduction, resulted in successful cartilage proliferation in damaged regions. METHODS: Non-clinical toxicology assessments for efficacy, biodistribution and local/systemic toxicity of single intra-articular administration of the cell mixture in mice, rabbits and goats was conducted. RESULTS: Administration of the mixture was tolerated well in all of the species. There was evidence of cartilage proliferation in rabbits and goats. As an additional precautionary step, the efficacy of TGF-beta1 secretion in irradiated human chondrocytes was also demonstrated. CONCLUSIONS: Four studies in rabbits and goats demonstrated the safety and efficacy of TG-C following direct intra-articular administration in animal models involving induced knee joint damage. Based on these pre-clinical studies authorization has been received from the USA Food and Drug Administration (FDA) to proceed with an initial phase I clinical study of TG-C for degenerative arthritis.

Differential oxidative stress response in young children and the elderly following exposure to PM(2.5).

OBJECTIVES: The mechanism of the adverse health effects of ambient particulate matter on humans has not been well-investigated despite many epidemiologic association studies. Measurement of personal exposure to particulate pollutants and relevant biological effect markers are necessary in order to investigate the mechanism of adverse health effects, particularly in fragile populations considered to be more susceptible to the effects of pollutants. METHODS: We measured personal exposure to PM(2.5) and examined oxidative stress using urinary malondialdehyde three times in 51 preschoolers and 38 elderly subjects. A linear mixed-effects model was used to estimate PM(2.5) effects on urinary MDA levels. RESULTS: Average personal exposure of the children and elderly to PM(2.5) was 80.5 +/- 29.9 and 20.7 +/- 12.7 mug/m(3), respectively. Mean urinary MDA level in the children and the elderly was 3.6 +/- 1.9 and 4.0 +/- 1.6 mumol/g creatinine. For elderly subjects the PM(2.5) level was significantly associated with urinary MDA after adjusting for age, sex, BMI, passive smoking, day-care facility site, alcohol consumption, cigarette smoking, and medical history (heart disease, hypertension and bronchial asthma). However, there was no significant relationship for children. CONCLUSIONS: The elderly were more susceptible than young children to oxidative stress as a result of ambient exposure to PM(2.5). Identification of oxidative stress induced by PM(2.5) explains the mechanism of adverse health effects such as cardiovascular or respiratory diseases, particularly in the elderly.

Irradiated human chondrocytes expressing bone morphogenetic protein 2 promote healing of osteoporotic bone fracture in rats.

Bone morphogenetic protein 2 (BMP2) was selected as a transgene to regenerate osteoporotic bone defects after several BMPs were tested using a bone formation study in nude mice. Human chondrocytes were transduced with a BMP2-containing retroviral vector, and single clones were selected. The cells were characterized over numerous passages for growth and BMP2 expression. The single clones were irradiated and tested for viability. BMP2 expression lasted for 3 weeks before dying off completely after approximately 1 month. Irradiated and non-irradiated transduced chondrocytes successfully healed fractures in osteoporotic rats induced by ovariectomy. The osteoinducing effect of irradiated cells was better than that of their non-irradiated counterparts or a chondrocytes-only control. This study showed that delivering BMP2 from the transduced and irradiated chondrocytes could be an effective and safe method of repairing osteoporotic bone fractures.

Community level exposure to chemicals and oxidative stress in adult population.

Little information is available on the role of environmental chemical exposure in oxidative stress. This study was designed to investigate whether exposure to environmental chemicals, such as polycyclic aromatic hydrocarbons, volatile organic compounds, bisphenol A or phthalates, induces oxidative stress in urban adult populations. A total of 960 adults dwelling in urban areas were evaluated between April and December 2005. To assess environmental chemical exposure, we measured urinary levels of 1-hydroxypyrene, 2-naphthol, hippuric acid, methyl hippuric acid, mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, and mono-butyl phthalate and bisphenol A. Urinary malondialdehyde and 8-hydroxydeoxyguanosine were also measured to evaluate oxidative stress. Significant dose-responsive relationship was found between urinary concentrations of the chemical exposure biomarkers and oxidative stress levels in simple regression analyses (P<0.05). Regression coefficients of these exposure biomarkers except bisphenol A remained significantly in the multiple regression models after controlling for age, sex, weight, smoking, and exercise for at least one of the two oxidative stress biomarkers (P<0.05). The oxidative stress biomarkers significantly affected the indicators of insulin resistance, particularly glucose level. This study indicates that environmental chemical exposure is associated with oxidative stress in urban adult populations and suggests that exposure to certain environmental chemicals might contribute to insulin resistance.

Maternal exposure to environmental tobacco smoke, GSTM1/T1 polymorphisms and oxidative stress.

Environmental tobacco smoking (ETS) is known to be associated with adverse pregnancy outcomes. The purpose of this study was to investigate the relationship between maternal exposure to ETS and oxidative stress for neonates, as well as the effect of maternal genetic polymorphisms, glutathione-S-transferase M1 (GSTM1) and GSTT1, on this relationship. We used the radioimmunoassay to measure the urinary concentration of cotinine in 266 pregnant women who denied smoking cigarettes during pregnancy and in their singleton babies. In addition, the urinary concentration of malondialdehyde (MDA) and 8-hydroxy-2-deoxyguanosine (8-OH-dG) were assessed using high-performance liquid chromatography and enzyme-linked immunosorbent assay, respectively. We also extracted DNA from whole blood obtained from the mothers and then conducted polymerase chain reaction on the samples to determine the GSTM1 and GSTT1 genotypes. The maternal cotinine concentration was found to be significantly associated with the fetal cotinine concentration, particularly for mothers whose urine cotinine concentrations were above 120 microg/gcr (p<0.01). The fetal urine cotinine concentration was also found to be significantly associated with the fetal urine MDA concentration (p<0.01). When the null type maternal GSTM1 or the wild type GSTT1 was present, the maternal oxidative stress level increased significantly as the maternal continine concentration increased (MDA: p<0.01; 8-OH-dG: p<0.01). No significant relationships were found between maternal cotinine and fetal oxidative stress markers, however, the fetal MDA levels increased significantly as fetal cotinine levels increased. These results suggest that the maternal exposure to ETS affects the fetal urine cotinine concentration and induces production of maternal oxidative stress. In addition, maternal genetic polymorphisms of GSTM1 and GSTT1 may modify the oxidative stress by maternal exposure to ETS.

Korean mistletoe lectin (KML-IIU) and its subchains induce nitric oxide (NO) production in murine macrophage cells.

Synthesis of nitric oxide (NO) is one of the important effector functions of innate immune cells. Although several reports have indicated mistletoe lectins induce immune cells to produce cytokines, studies regarding the activities of the lectins in the production of NO have been very limited. Here, we report on the induction of NO synthesis in a murine macrophage cell line, RAW264.7, by Korean mistletoe lectin (KML-IIU). When the macrophage cells were treated with KML-IIU in the presence of a suboptimal concentration of IFN-gamma, NO production was induced in a concentration-dependent manner. Significantly higher levels of NO were induced by subchains of the KML-IIU (A and B), which have lower toxicities, as compared to the hololectin. Furthermore, expression of the inducible nitric oxide synthase (iNOS) gene was elevated in accordance with the level of NO production. When the synthase was inhibited by iNOS inhibitors (L-NIL and L-NAME), NO production was specifically reduced in a concentration-dependent manner. Our studies demonstrate that the KML-IIU and its subchains induce NO production in murine macrophage cells via activation of the iNOS gene expression, suggesting that the KML-IIU subchains may be used as an immunomodulator to enhance the effector functions of innate immune cells.

Isolation and characterization of two Korean mistletoe lectins.

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.

GSTM1 and TNF-alpha gene polymorphisms and relations between blood lead and inflammatory markers in a non-occupational population.

Inflammation is known to be an important underlying condition in the development of a variety of diseases. To investigate whether blood lead induces inflammatory reactions in non-occupationally exposed adults and the effects of genetic susceptibility associated with GSTM1 and TNF-alpha gene polymorphisms on this inflammatory response, we measured blood lead levels in 300 healthy university students. Total serum TNF-alpha and IL-6 levels and WBC counts were determined to evaluate the inflammatory response. Allelic loss of GSTM1 and the TNF-alpha-308 G>A polymorphism were determined by PCR and RFLP. Positive relations between blood lead and three inflammation biomarkers were shown in male subjects with blood lead > or =2.51microg/dl (median value) (TNF-alpha, p=0.015; IL-6, p=0.082; and WBC, p=0.044). However, subgroup analysis by genotype showed an effect of blood lead on the three biomarkers only in individuals with the GSTM1 null (TNF-alpha, p=0.020; IL-6, p=0.096; and WBC, p=0.017) or TNF-alpha GG (TNF-alpha, p=0.017; IL-6, p=0.088; and WBC, p=0.095) genotype, and not in individuals with GSTM1 present (all three inflammatory biomarkers, p>0.1) or the TNF-alpha GA or AA (all three biomarkers, p>0.1) genotype. These results suggest that blood lead affects the inflammatory response and that GSTM1 and TNF-alpha gene polymorphisms are genetic factors associated with lead-induced inflammatory response.

Aryl hydrocarbon receptor gene polymorphisms affect lung cancer risk.

To evaluate the role of genetic polymorphisms of AhR related to the carcinogen metabolism and cell proliferation, genotypes of three AhR polymorphisms Ex1+185A>G, IVS7+33T>G and Ex10+501G>A were determined in 616 lung cancer cases and 616 lung cancer-free controls. When the effect of each AhR allele on lung cancer risk was evaluated, any AhR genotype did not show the association with lung cancer risk. However, when haplotypes were composed of three AhR SNP sites, non-smokers with GGG haplotype (adjusted OR=1.7, 95% CI, 1.06-2.71) and smokers without GGG haplotype (adjusted OR=2.5, 95% CI, 1.64-3.74) showed significantly increased risk of lung cancer compared to non-smokers without GGG haplotype. Moreover, smokers with GGG haplotype showed the highest risk (adjusted OR=3.2, 95% CI, 2.10-4.74). Particularly, the synergistic effect between AhR haplotype and smoking was more apparent in squamous cell carcinoma (adjusted OR=6.1, 95% CI, 2.53-14.68). This result suggests that haplotypes of AhR gene play an important role in the development of lung cancer and there is a synergistic interaction between AhR gene and smoking for lung cancer risk.

GSTM1 and GSTP1 polymorphisms as potential factors for modifying the effect of smoking on inflammatory response.

Inflammation has been known to be an important underlying condition for development of various diseases including cancer. The aims of this study were to investigate whether tobacco smoke exposure increases the level of inflammation biomarkers and the GSTM1 and GSTP1 gene polymorphisms are associated with inflammatory response due to tobacco smoke exposure. We measured urinary cotinine level in 300 healthy university students. Total serum TNF-alpha levels and blood WBC counts were determined to evaluate inflammatory response. Allelic loss of the GSTM1 and the GSTP1 (Ile105Val) polymorphism were determined by PCR and RFLP. Tobacco smoke exposure was found to be associated with increase of both TNF-alpha level and WBC count. Particularly, smokers with combination of GSTM1 null and GSTP1 AG or GG genotypes showed higher TNF-alpha level than those with the other genotype combinations (p=0.07). This result suggests that smoking may induce inflammation measured as TNF-alpha level or WBC count and combinations of the GSTM1 and GSTP1 polymorphisms may modify the effect of smoking on serum TNF-alpha level.

Interactive effect of genetic polymorphism of glutathione S-transferase M1 and smoking on squamous cell lung cancer risk in Korea.

To evaluate the role of the genetic polymorphisms of CYP2E1, GSTM1 and GSTT1, and their interaction with smoking in lung cancer development in Korean males, a hospital-based case-control study was conducted. Histologically confirmed male lung cancer patients (n=171) and male patients with no present or previous history of systemic illness who visited the urology department (n=196) were recruited from Seoul National University Hospital, Korea (1998-1999). CYP2E1 genotypes were determined by PCR-RFLP using RsaI digestion and GSTM1 and T1 genotypes were determined by multiplex PCR. Risks were estimated as odds ratios (ORs) and 95% confidence intervals (CIs) using a logistic regression model adjusting for age and pack-year. Smoking was a significant risk factor for lung cancer (P<0.001). Although genetic polymorphisms of CYP2E1, GSTM1 and T1 were not associated with the overall risk of lung cancer, the GSTM1 null genotype significantly increased the risk of squamous cell lung cancer (OR=1.9, 95% CI=1.04-3.60). An interactive effect between the GSTM1 null genotype and smoking was observed (P=0.04). These results suggest that the GSTM1 null genotype is associated with squamous cell lung cancer and modifies the effect of smoking on squamous cell lung cancer development in Korean males.

Effect of genetic polymorphisms of MnSOD and MPO on the relationship between PAH exposure and oxidative DNA damage.

To investigate the effect of genetic polymorphisms on the oxidative damage caused by PAH exposure, we measured urinary 1-hydroxypyrene (1-OHP) and 8-hydroxydeoxyguanosine (8-OHdG) levels to determine exposure and oxidative injury in university students. After examining myeloperoxidase (MPO) and manganese superoxide dismutase (MnSOD) genotypes by PCR and RFLP, we evaluated the effects of these polymorphisms on the relationship between the urinary levels of 1-OHP and 8-OHdG. No significant relation was observed between log 1-OHP and 8-OHdG concentrations in the whole study group (p=0.182), or between urinary 8-OHdG levels and polymorphisms of MnSOD or MPO (p=0.539 and 0.993, respectively). However, significant differences of regression coefficient were found for the relation between urinary log 1-OHP and urinary 8-OHdG concentrations in the presence of different MnSOD or MPO genotypes by multiple regression after controlling for age, sex, body mass index, cotinine, and smoking. In those with the MnSOD Val/Ala or Ala/Ala genotypes this regression coefficient was 1.480 (p=0.040), whereas for the MnSOD Val/Val genotype it was 0.088 (p=0.859). The higher regression coefficient was obtained for the subject group with the MnSOD Val/Ala or Ala/Ala genotype in combination with the MPO G/G genotype (p=0.012). We suggest that the oxidative injury caused by PAH exposure is modulated by genetic polymorphisms such as MnSOD and MPO.

Hyaline cartilage regeneration using mixed human chondrocytes and transforming growth factor-beta1- producing chondrocytes.

The purpose of this study was to investigate the efficacy of cartilage regeneration when using a mixture of transforming growth factor-beta1 (TGF-beta1)-producing human chondrocytes (hChon-TGF-beta1) and primary human chondrocytes (hChon) ("mixed cells"), compared with either hChon-TGF-beta1 or hChon cells alone. Specifically, mixed cells or hChon cells were first injected intradermally into the backs of immune-deficient nude mice to test the feasibility of cartilage formation in vivo. Both the mixed cells and the hChon-TGF-beta1 cells alone induced cartilage formation in nude mice, whereas hChon cells alone did not. To further test the efficacy of the cells in generating cartilage, an artificially induced partial thickness defect of the femoral condyle of a rabbit knee joint was loaded with hChon-TGF-beta1 cells with or without mixing additional untransduced hChon cells, and hyaline cartilage regeneration was observed at 4 or 6 weeks. The efficiency of complete filling of the defect and the quality of tissue generated after implanting were evaluated on the basis of a histological grading system modified from O'Driscoll et al. (J. Bone Joint Surg. 70A, 595, 1988). Significantly, mixed cells (14.2 +/- 0.9) produced significantly better results than hChon-TGF-beta1 (9.0 +/- 1.7) or hChon (8.0 +/- 1.8) cells alone. Histological and immunohistochemical staining of the newly repaired tissues produced after treatment with either mixed cells or hChon-TGF-beta1 cells alone showed hyaline cartilage- like characteristics. These results suggest that the implantation of mixed cells may be a clinically efficient method of regenerating hyaline articular cartilage.

Asthma attack associated with oxidative stress by exposure to ETS and PAH.

UNLABELLED: Asthma is primarily an airways inflammatory disease, and the bronchial airways have been shown to be particularly susceptible to oxidant-induced tissue damage. OBJECTIVE: The purpose of this study was to investigate whether pulmonary inflammation in asthma is associated with exposure to environmental oxidants such as polycyclic aromatic hydrocarbon (PAH) and environmental tobacco smoke (ETS). METHOD: We assessed the exposure level of PAH and ETS by using urinary 1-hydroxypyrene glucuronide (1-OHPG) and cotinine. We estimated oxidative damage and inflammatory cytokine levels from 16 asthma patients and 16 patients in stable conditions 1 to 2 months later. RESULTS: Our study showed that the levels of oxidative damage, as measured by malondialdehyde (MDA), were significantly increased (p = 0.006) during the asthma attacks. Proinflammatory and anti-inflammatory cytokines were both increased during the asthma attacks compared to the stable conditions at follow-up. Interleukin (IL-6) and IL-10 were especially increased significantly (p = 0.015 and p < 0.001, respectively). Correlations were observed between inflammatory cytokines such as IL-6 and IL-1beta (p = 0.034). CONCLUSION: This study supports the results of in vitro studies that oxidative stress, specifically lipid peroxidation, contributes to the pathophysiology of asthma. Therefore, environmental interventions based on this better understanding are needed to significantly reduce oxidant stress and prevent or minimize the development of asthmatic symptoms.