Klf5 down-regulation induces vascular senescence through eIF5a depletion and mitochondrial fission.

Although dysregulation of mitochondrial dynamics has been linked to cellular senescence, which contributes to advanced age-related disorders, it is unclear how Krüppel-like factor 5 (Klf5), an essential transcriptional factor of cardiovascular remodeling, mediates the link between mitochondrial dynamics and vascular smooth muscle cell (VSMC) senescence. Here, we show that Klf5 down-regulation in VSMCs is correlated with rupture of abdominal aortic aneurysm (AAA), an age-related vascular disease. Mice lacking Klf5 in VSMCs exacerbate vascular senescence and progression of angiotensin II (Ang II)-induced AAA by facilitating reactive oxygen species (ROS) formation. Klf5 knockdown enhances, while Klf5 overexpression suppresses mitochondrial fission. Mechanistically, Klf5 activates eukaryotic translation initiation factor 5a (eIF5a) transcription through binding to the promoter of eIF5a, which in turn preserves mitochondrial integrity by interacting with mitofusin 1 (Mfn1). Accordingly, decreased expression of eIF5a elicited by Klf5 down-regulation leads to mitochondrial fission and excessive ROS production. Inhibition of mitochondrial fission decreases ROS production and VSMC senescence. Our studies provide a potential therapeutic target for age-related vascular disorders.

[Experimental treatment of pulmonary interstitial fibrosis with human umbilical cord blood mesenchymal stem cells].

OBJECTIVE: To investigate the therapeutic effect and possible action mechanism of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in the treatment of bleomycin-induced pulmonary fibrosis in rats. METHODS: The second generation of hUCB-MSCs was cultured to the fourth generation. Sixty healthy male Sprague-Dawley rats (clean grade) were randomly and equally divided into 4 groups: bleomycin group, stem cell treatment group, dexamethasone treatment group, and negative control group. A pulmonary fibrosis model was established by intratracheal instillation of bleomycin in the bleomycin group, stem cell treatment group, and dexamethasone treatment group. The stem cell treatment group was injected with stem cells labeled with 5-bromo-2-deoxyuridine (Brdu) via the caudal vein immediately after the model was established. The dexamethasone treatment group was intraperitoneally injected with dexamethasone for 7 d from the next day after the model was established. The negative control group was given an equal volume of normal saline by intra-tracheal instillation. In each group, 5 rats were sacrificed in the 7th, 14th, and 28th days. The expression of transforming growth factor ß1 (TGF-ß1) and Brdu-labeled stem cells were observed by HE and Masson staining and immunohistochemistry. Lung hydroxyproline content was determined by acid hydrolysis. RESULTS: The stem cell treatment groups had Brdu-labeled stem cells seen in lung tissue in the 7th, 14th, and 28th days. Compared with the negative control group, the bleomycin group, stem cell treatment group, and dexamethasone treatment group had significantly increased scores of alveolitis and pulmonary fibrosis (P < 0.05). In the 7th, 14th, and 28th days, the scores of alveolitis in stem cell treatment group and dexamethasone treatment group were significantly lower than those in bleomycin group (P < 0.05); in the 28th day, the scores of pulmonary fibrosis in stem cell treatment group and dexamethasone treatment group were significantly lower than that in bleomycin group (P < 0.05). There were no significant differences in scores of alveolitis and pulmonary fibrosis between the dexamethasone treatment group and stem cell treatment group (P > 0.05). Compared with the bleomycin group, the stem cell treatment group and dexamethasone treatment group had significantly decreased number of TGF-ß1-positive cells and hydroxyproline content in lung tissue at all time points (P < 0.05). There were no significant differences in number of TGF-ß1-positive cells and hydroxyproline content in lung tissue between the stem cell treatment group and dexamethasone treatment group (P > 0.05). CONCLUSION: hUCB-MSCs can be transplanted into damaged lung tissue and effectively reduce alveolitis and pulmonary fibrosis in the early stage of pulmonary fibrosis. The action mechanism of hUCB-MSCs may involve inhibiting the expression of TGF-ß1 and reducing the formation of collagen.

[Expression of neuropilin 2 in human colorectal carcinoma and its correlation with lymphangiogenesis and lymphatic metastasis of tumor].

OBJECTIVE: To explore the relationship between neuropilin 2 (NRP-2) and lymphangiogenesis and lymphatic metastasis of human colorectal carcinoma (CRC), as well as the expression of NRP-2 in CRC tissues. METHODS: A total of 55 cases of CRC, adjacent and normal tissues of surgical resection were randomly selected at our hospital from March 2010 to January 2012. All pathological findings were confirmed by histopathology. The expression of NRP-2 was detected with reverse transcription (RT)-PCR and immunohistochemistry in parenchymatous and surrounding malignant tissues. Then lymphangiogenesis was marked with D2-40 monoclonal antibody and microlymphatic density (MLD) counted. RESULTS: Significant differences of MLD existed between those of tumor region (39 ± 19) and tumor margin (53 ± 26, P < 0.01). Both the number and shape of lymphangiogenesis were different between the parenchymatous and surrounding tissues. The expression of NRP-2 had a positive correlation with MLD both at the protein level (r = 0.325, P < 0.05) and at the gene level (r = 0.545, P = 0.000). And it was also correlated with the differentiation degree, infiltrative degree, lymphovascular invasion, lymph node metastasis and Dukes tumor staging (all P < 0.05). CONCLUSION: The expression of NRP-2 may regulate lymphangiogenesis and it may play an important role in the incidence and development of CRC.

OC-STAMP Overexpression Drives Lung Alveolar Epithelial Cell Type II Senescence in Silicosis.

Cellular senescence has been considered an important driver of many chronic lung diseases. However, the specific mechanism of cellular senescence in silicosis is still unknown. In the present study, silicotic rats and osteoclast stimulatory transmembrane protein (Ocstamp) overexpression of MLE-12 cells were used to explore the mechanism of OC-STAMP in cellular senescence in alveolar epithelial cell type II (AEC2). We found an increasing level of OC-STAMP in AEC2 of silicotic rats. Overexpression of Ocstamp in MLE-12 cells promoted epithelial-mesenchymal transition (EMT), endoplasmic reticulum (ER) stress, and cellular senescence. Myosin heavy chain 9 (MYH9) was a potential interacting protein of OC-STAMP. Knockdown of Ocstamp or Myh9 inhibited cellular senescence in MLE-12 cells transfected with pcmv6-Ocstamp. Treatment with 4-phenylbutyrate (4-PBA) to inhibit ER stress also attenuated cellular senescence in vitro or in vivo. In conclusion, OC-STAMP promotes cellular senescence in AEC2 in silicosis.

Upregulation of aurora kinase A promotes vascular smooth muscle cell proliferation and migration by activating the GSK-3ß/ß-catenin pathway in aortic-dissecting aneurysms.

AIMS: Aurora kinase A (AURKA) is a mitotic serine/threonine kinase that contributes to the regulation of cell-cycle progression. AURKA has been shown to further enhance Wnt/ß-catenin signaling; however, in the context of driving aortic-dissecting aneurysm (ADA), the molecular details of this phenomenon remain poorly understood. MATERIALS AND METHODS: A total of 43 specimens of ADA tissues and eleven healthy aortic tissues as controls were collected from the hospital. Pathological changes were observed by hematoxylin and eosin staining. AURKA expression in aortic tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunohistochemistry staining. The proliferative and migratory effects of AURKA were observed in cultured vascular smooth muscle cells (VSMCs). KEY FINDINGS: AURKA expression was significantly increased in aorta samples from ADA patients relative to those from normal donors, and the expression was even higher in ruptured ADA tissues. AURKA overexpression promoted and AURKA knockdown inhibited, respectively, the proliferation, and migration of VSMCs. Angiotensin II (AngII) treatment of VSMCs significantly increased AURKA expression. The knockdown of AURKA partially reversed AngII-induced VSMC proliferation and migration. Finally, the downregulation of AURKA inhibited cell proliferation by inactivating the p-GSK-3ß/ß-catenin pathway. SIGNIFICANCE: The GSK-3ß/ß-catenin signaling pathway participates in the AURKA-regulated proliferation and migration of VSMCs. The expression of AURKA may be involved in the phenotypic conversion of VSMC and the occurrence and development of ADA and could be a potential molecular target for ADA therapy.

[Expressions of cadherin molecules CDH18 and PCDH17 in human azoospermic testes].

OBJECTIVE: To investigate the expressions of cadherin molecules CDH18 and PCDH17 in normal and azoospermic human testes and their significance. METHODS: We studied the routine pathological slices of normal and non-obstructive azoospermic human testis tissues for changes in the tight junction of Sertoli-germ cells, and identified the differential gene expression profiles of the normal and azoospermic testis tissues using cDNA microarrays containing multiple cadherin molecules. The results were confirmed by Western blot. RESULTS: Abnormal tight junction of the Sertoli-germ cells was observed in 37.5% of the azoospermic testis samples, and obvious changes were seen in the expressions of some cadherin molecules, with down-regulation of CDH18 and PCDH17. CONCLUSION: Cadherin molecules such as CDH18 and PCDH17 may play a certain role in the development and progression of azoospermia, which might be related with the abnormal tight junction of the Sertoli-germ cells.

[Expression of COX10 in human non-obstructive azoospermia testes].

OBJECTIVE: To evaluate the expression of COX10 mRNA in the testes of non-obstructive azoospermia patients and normal men. METHODS: A cDNA microarray containing COX10 and some other genes as RBM and EIF1AY was used to identify the differential gene expression profiles in the normal and azoospermic testes. The cDNA probes were prepared by labeling mRNA from azoospermic and normal testis tissues with Cy5-dUTP and Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with cDNA microarray. Later the fluorescent signals were scanned and the values of Cy5-dUTP and Cy3-dUTP on each spot were calculated and analyzed. After that an ISH was employed to detect the expression of COX10 mRNA in 10 fertile and 39 non-obstructive azoospermic testes, and the expression levels were compared to evaluate the significance. RESULTS: We obtained 128 differentially expressed genes that might be related with azoospermia, among which 56 were up-regulated and 72 down-regulated, with the expression of COX10 significantly decreased. In situ hybridization confirmed that the mRNA expression of COX10 was stronger in the spermatogenic cells of the normal fertile than the azoospermic testes. CONCLUSION: COX10 may play a certain role in the development and progression of azoospermia. The technique of cDNA microarray can be applied to further studies of screening non-obstructive azoospermia associated genes.

Spatial analysis of tuberculosis cases in migrants and permanent residents, Beijing, 2000-2006.

To determine the role of the migrant population in the transmission of tuberculosis (TB), we investigated the distribution and magnitude of TB in permanent residents and migrant populations of Beijing, People's Republic of China, from 2000 through 2006. An exploratory spatial data analysis was applied to detect the "hot spots" of TB among the 2 populations. Results, using the data obtained from 2004-2006, showed that people who migrated from the western, middle, and eastern zones of China had a significantly higher risk of having TB than did permanent residents. These findings indicate that population fluctuations have affected the rate of TB prevalence in Beijing, and interventions to control TB should include the migrant population.

[Association of surfactant protein D gene polymorphisms at rs3088308 and rs721917 with susceptibility to silicosis].

OBJECTIVE: To investigate the relationship between polymorphisms of surfactant protein D (rs3088308 and rs721917) and the susceptibility to silicosis. METHODS: This case-control study included 125 silicosis patients and 125 individuals exposed to industrial dust but without silicosis (control group), who were strictly matched with the case group for age, gender, work type and cumulative length of dust exposure. The rs3088308 and rs721917 polymorphisms of surfactant protein-D were detected in all the participants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequencies of T/T, T/A and A/A genotypes of surfactant protein-D rs3088308 locus were 22.2%, 71.2% and 5.6% in the case group, significantly different from the frequencies of 17.6%, 58.4% and 24.0% in the control group, respectively (P<0.05). The frequencies of C/C, C/T and T/T genotypes of rs721917 locus were 17.6%, 56.8% and 25.6% in the case group, similar to the frequencies of 15.2%, 60.0% and 24.8% in the control group, respectively (P>0.05). CONCLUSION: Surfactant protein-D rs3088308 polymorphism is significantly associated with silicosis, and the T allele may be a risk factor for silicosis in individuals exposed to industrial dust.

Expression and significance of Rap1A in testes of azoospermic subjects.

AIM: To evaluate the Rap1A mRNA expression and its significance in the testes of normal and azoospermic subjects. METHODS: A cDNA microarray that contained Rap1A and some other genes such as RBM, EIF1AY was used to identify the differential gene expression profiles between the normal and azoospermic testes. cDNA probes were prepared by labeling mRNA from azoospermic and normal testicular tissues through reverse transcription with Cy5-dUTP and Cy3-dUTP, respectively. The mixed cDNA probes were then hybridized with cDNA microarray (each containing 4096 unique human cDNA sequences). The fluorescent signals were scanned and the values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated. In situ hybridization was employed to detect the expression of Rap1A in the testes of 10 fertile and 39 azoospermic subjects. RESULTS: One hundred and twenty-eight differentially expressed genes were found to be possibly related to azoospermia, of which 56 were up-regulated and 72, down-regulated genes. The mRNA expression of Rap1A in the spermatogenic cells of azoospermic was stronger than that in those of the fertile testes. CONCLUSION: Rap1A may play certain roles in the development of azoospermia.

[Changes of carbon monoxide, nitric oxide levels and heme oxygenase system in acute respiratory distress syndrome induced by oleic acid].

OBJECTIVE: To investigate possible role of carbon monoxide (CO) and heme oxygenase (HO) in the pathogenesis of acute respiratory distress syndrome (ARDS) induced by oleic acid (OA) and to compared with that induced by nitric oxide (NO). METHODS: ARDS model was established in rats by oleic acid injection and concentrations of CO and NO in pulmonary arterial, carotid jugular blood and bronchoalveolar lavage fluid (BALF) were measured sequentially. Immunohistochemical method was used to determine the expression of HO in the lung. RESULTS: Pulmonary arterial pressure in ARDS rats elevated 10 min after OA injection [(13.80 +/- 1.87) mm Hg to (19.51 +/- 5.02) mm Hg]. At 0.5 h after OA injection, concentration of CO in pulmonary artery began to increase and was markedly higher at 2 h than that in control rats [(0.135 +/- 0.010) g/L versus (0.116 +/- 0.005) g/L] (P < 0.01), also higher than that in carotid artery [(0.117 +/- 0.013) g/L] and in jugular vein [(0.107 +/- 0.018) g/L] in the same group, and maintained at a relatively high level thereafter. Concentration of CO in BALF also increased at 0.5 - 24 h and diminished at 72 h, as compared with that in controls. Concentration of NO in blood of pulmonary and systemic circulation all elevated markedly at 0.5 h and 2 h after OA injection, and then declined to normal at 12 h. Concentration of NO in BALF was significantly higher than that in controls. Arterial blood gas analysis showed that PaO2 markedly decreased in ARDS rats, especially at 2 h after OA injection. HO-2 could be expressed in the lung tissues of normal rats with immunohistochemical method, the strongest in epithelial cells of the bronchi, and HO-1 could only be expressed in pulmonary blood vessel walls, bronchial epithelial cells, alveolar epithelial cells and inflammatory cells of ARDS rats, lasting for 72 h after OA injection, consistent with that of CO level. CONCLUSION: ARDS rats showed a lastecl increase of CO level in pulmonary blood circulation, suggesting CO/HO system might play a more important role in modulation of blood vessel tension than NO might do in pathogenesis of ARDS.

[Protective effect of nitric oxide synthase inhibitor (L-NAME) on germ cell apoptosis in experimentally cryptorchid rats].

OBJECTIVE: To explore the protective effect of nitric oxide synthase inhibitor (L-NAME) on the germ cell apoptosis in the rat cryptorchid. METHODS: Immature rats (22 day-old Sprague Dawley) were subjected to unilateral cryptorchid. Thirty rats were divided into three groups: sham operation group (testes still in the scrotum after operation); operation group; operation + L-NAME group(given L-NAME 10 mg/kg after operation, dip). Seven days after operation germ cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated-dUTP nick end labeling(TUNEL). Biochemical parameters (NO, NOS) were evaluated with spectrophotometric determination. RESULTS: At the 7th day after the operation, compared with the control, the number of apoptotic germ cells in the cryptorchid testis was increased significantly, but the testis weight was decreased predominantly(P < 0.01). The levels of NO and NOS in the cryptorchid were significantly higher than the control. CONCLUSIONS: The levels of NO and NOS might be involved in the germ cell apoptosis in the cryptorchid; L-NAME could protect the germ cell from apoptosis in experimentally cryptorchid rats by reducing the activity of NOS and reducing the level of NO in the testis.

[The relationships of PSA, FPSAR, clinical and pathological stage in patients with prostate cancer].

OBJECTIVES: To investigate the relationship between clinical and pathological stage, serum prostate specific antigen (PSA) concentration and free-to-total PSA ratio (FPSAR) in patients with prostate cancer. METHODS: Clinical and pathological stage were determined on the basis of pathological examination and clinic material in 42 prostate cancer patients treated by prostatectomy. PSA and FPSAR were measured before the operation. Spearman rank correlation was applied to evaluate the relationship between clinical and pathological stage, serum PSA concentration and FPSAR. RESULTS: Serum PSA concentration was significantly positively correlated with pathological stage(P < 0.05) but not correlated with clinical stage (P > 0.05) in prostate cancer patients. FPSAR was significantly correlated with pathological stage and negatively correlated with clinical stage in prostate cancer patients (P < 0.05). CONCLUSIONS: FPSAR is a more powerful predictor of clinical stage, pathological stage and prognosis than PSA.

[A study of aspermia-related genes by genchips and analysis of the RAP1A gene].

OBJECTIVE: To study the differential gene expression profiles between the normal and aspermia human testes by genechips. METHODS: Probes were prepared from mRNA extracted from both normal and aspermia testes and employed on Biostar H-40s genechips to detect the differential gene expression profiles. A distinctly up-regulated gene RAP1A was analyzed by bibliogrphic retrieval. RESULTS: Six hundred and twenty-three differential expressed genes were found, among which the distinctly up-regulated gene RAP1A was closely related to human sperm regulation. CONCLUSIONS: Screening the differential gene expression profiles between the normal and aspermia human testes by genechips can be used in the study of aspermia-related genes.

[Study on the changes in circulating endothelial cells and hemorheology of lung in rats with acute lung injury by chemicals].

OBJECTIVE: To investigate the mechanism of pulmonary circulation obstacle in chemicals-induced acute lung injury and its clinical significance. METHOD: Pulmonary arterial intubation, circulating endothelial cells (CEC) isolation and hemorheology detection technique were used to observe the changes of CEC numbers and hemorheology in rat pulmonary vascular system during oleic acid-induced acute lung injury (ALI). RESULTS: The numbers of CEC were obviously increased even in the early phase of ALI [from (2.06 +/- 0.48)/0.9 micro l to (5.50 +/- 0.54)/0.9 micro l]; there was no obvious change in whole blood viscosity under high shear rate (200 s(-1), 30 s(-1)) but the whole blood viscosity and hematocrit were remarkably increased in pulmonary artery blood at low shear rate (5 s(-1), 1 s(-1)) (P < 0.05). Erythrocytes had increasing tendency, whereas platelets were also decreased but there was no statistical significance. CONCLUSION: The deterioration of pulmonary circulation may be the key point in the pathogenesis of ALI; the injury and dysfunction of pulmonary capillary endothelial cells (PCEC) may be the common starting phase in the pathological processes of ALI; the detection of CEC may offer a new valuable and sensitive index for diagnosis of ALI.

Expression of MSP58 in human colorectal cancer and its correlation with prognosis.

We had reported that MSP58 regulates colorectal cancer cell proliferation, development, and apoptosis, by the cyclin D1-cyclin-dependent kinase 4-p21 pathway. In this study, MSP58 protein expression was examined by immunohistochemistry in 499 specimens of CRC. The relationship between various clinicopathological features and overall patient survival rate was analyzed. The association of MSP58 expression with the 499 CRC patients' survival rate was assessed by Kaplan-Meier and Cox regression. Using ROC curve to provide sensitivity and specificity of the score of MSP58 predicts local recurrence and survival of CRC patients. The expression of MSP58 was positively correlated with the depth of invasion (P < 0.001), local recurrence (P = 0.008), tumor grade (P = 0.002), and UICC stage (P < 0.001). The Kaplan-Meier survival analysis demonstrated that the survival time of CRC patients with low expression of MSP58 was longer than those with high expression during the 5-year follow-up period (P < 0.001). COX regression analysis indicated that high expression of MSP58 (P < 0.001), depth of invasion >pT(1) (P = 0.008), distant organ metastasis (pM(1)) (P < 0.001), regional lymph node metastasis (≥ pN(1)) (P < 0.001), and local recurrence (Yes) (P = 0.007) were independent, poor prognostic factors of CRC. ROC curve showed the score of MSP58 expression level did provide a maximal sensitivity and specificity to predict local recurrence and survival of CRC patients. Our results demonstrated MSP58 might serve as a novel prognostic marker that is independent of, and additive to, the UICC staging system.

[Expression of cell cycle molecules in human azoospermic testes].

AIM: To evaluate the expression and significance of cell cycle molecules in human normal and azoospermia testes. METHODS: A cDNA microarray containing cDNA of some cell cycle molecules was used to identify the differential gene expression profiles between normal and azoospermic testes. cDNA probes were prepared by labeling mRNA from normal and testis tissues with Cy5-dUTP and Cy3-dUTP, respectively, through reverse transcription. Then the mixed cDNA probes were hybridized with cDNA microarray. The fluorescent signals were scanned, and the values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated. After that an ISH was employed to detect the expression of CDC10 mRNA in ten fertility and thirty-nine azoospermic testes, whose expression level was compared to evaluate the significance. RESULTS: The genes which were differentially expressed in azoospermic testes were found, among which the expression of CDC7L1 and CDC10 was up-regulated but the expression of CDK9, CDC20 and CLK3 was down- regulated. The mRNA expression of CDC10 was confirmed to be stronger in spermatogenic cells of normal fertility compared with that of azoospermic testes by in situ hybridization. CONCLUSION: The cell cycle molecules such as CDC10, CDC7L1, CDK9, CDC20 and CLK3 may play a role in the development and progression of azoospermia.

Enhanced nitric oxide production associated with airway hyporesponsiveness in the absence of IL-10.

Interleukin (IL)-10 is an anti-inflammatory cytokine implicated in the regulation of airway inflammation in asthma. Among other activities, IL-10 suppresses production of nitric oxide (NO); consequently, its absence may permit increased NO production, which can affect airway smooth muscle contractility. Therefore, we investigated airway reactivity (AR) in response to methacholine (MCh) in IL-10 knockout (-/-) mice compared with wild-type C57BL/6 (C57) mice, in which airway NO production was measured as exhaled NO (E(NO)), and NO production was altered with administration of either NO synthase (NOS)-specific inhibitors or recombinant murine (rm)IL-10. AR, measured as enhanced pause in vivo, and tracheal ring tension in vitro were lower in IL-10(-/-) mice by 25-50%, which was associated with elevated E(NO) levels (13 vs. 7 ppb). Administration of NOS inhibitors N(G)-nitro-L-arginine methyl ester (8 mg/kg ip) or L-N(6)-(1-iminoethyl)-lysine (3 mg/kg ip) to IL-10(-/-) mice decreased E(NO) by an average of 50%, which was associated with increased AR, to levels similar to C57 mice. E(NO) in IL-10(-/-) mice decreased in a dose-dependent fashion in response to administered rmIL-10, to levels similar to C57 mice (7 ppb), which was associated with a 30% increment in AR. Thus increased NO production in the absence of IL-10, decreased AR, which was reversed with inhibition of NO, either by inhibition of NOS, or with reconstitution of IL-10. These findings suggest that airway NO production can modulate airway smooth muscle contractility, resulting in airway hyporesponsiveness when IL-10 is absent.

Alterations in nitric oxide and cytokine production with airway inflammation in the absence of IL-10.

IL-10 is an anti-inflammatory cytokine that suppresses NO synthase (NOS) and production of NO; its lack may promote NO production and alterations in cytokines modulated by NO with allergic airway inflammation (AI), such as IL-18 and IL-4. Therefore, we induced AI in IL-10 knockout ((-/-)) and IL-10-sufficient C57BL/6 (C57) mice with inhaled OVA and measured airway NO production, as exhaled NO (E(NO)) and bronchoalveolar lavage fluid nitrite levels. E(NO) and nitrite levels were elevated significantly in naive IL-10(-/-) mice as compared with C57 mice. With AI, E(NO) and nitrite levels increased in C57 mice and decreased in IL-10(-/-) mice. IL-18 production fell with both AI and addition of S-nitroso-N-acetyl-d,l-penicillamine (a NO donor) but was not significantly increased by chemical NOS inhibition by l-N(5)-(1-iminoethyl)-ornithine. IL-4 AI was increased significantly (up to 10-fold greater) in the absence of IL-10 but was reduced significantly with chemical inhibition of NOS. Airway responsiveness was lower in IL-10(-/-) mice and was associated with alteration in production of NO and IL-4. Thus, IL-4 production was increased, and likely decreased NO production, in a way not predicted by the absence of IL-10. Inhibition of IL-4 production, with inhibition of NOS in the absence of IL-10, demonstrated the importance of a NO and IL-4 feedback mechanism regulating this interaction.