Changes In Bronchoalveolar Lavage Fluid Research Articles

Targeting the translationally controlled tumor protein by a monoclonal antibody improves allergic airway inflammation in mice

Secretion of translationally controlled tumor protein (TCTP) was found in body fluids during the late phase of allergic reactions, implicating TCTP in allergic diseases. Furthermore, blocking TCTP has been shown to be helpful in treating asthma and allergies in animal models. The objectives of this study were to produce anti-TCTP monoclonal antibodies (mAbs), test their ability to inhibit the cytokine-like function of dimeric TCTP (dTCTP) in vitro and to assess their therapeutic effects in a murine model of ovalbumin (OVA)-induced airway inflammation. We first verified the inhibitory effects of 4 anti-TCTP mAbs on dTCTP-induced secretion of IL-8 in BEAS-2B cells. To investigate the anti-inflammatory effect of anti-TCTP mAbs on allergic airway inflammation, we treated OVA-sensitized mice with anti-TCTP mAbs before OVA challenge. The changes in bronchoalveolar lavage fluid (BALF) cells, IL-4, IL-5, and IL-13 levels in both BALF and lung homogenates, plasma levels of OVA-specific IgE, and lung tissues were analyzed. We found that JEW-M449 anti-TCTP mAb bound to the flexible loop of TCTP and significantly inhibited dTCTP-induced IL-8 release, making it the most effective inhibitor in our study. We also found that treatment with JEW-M449 significantly reduced the infiltration of inflammatory cells and suppressed the OVA-induced upregulation of type 2 cytokines in both BALF and lung homogenates in a dose-dependent manner. In addition, JEW-M449 significantly attenuated the degree of goblet cell hyperplasia and mucus secretion. Our results demonstrate that specific targeting of the flexible loop of TCTP is a potent strategy for treating airway inflammatory diseases.

The amino acid and carnitine concentration changes in bronchoalveolar lavage fluid from lung cancer patients

ObjectiveTo screen out potential biomarkers by analyzing fundamental nutrients in the bronchoalveolar lavage fluid (BALF) before confirming the lung cancer.MethodsIn this study, 44 patients were enrolled with clinical information. The concentrations of 23 amino acids and 35 carnitines in their BALF were detected with the high-performance liquid chromatography–mass spectrometry (HPLC–MS). Combined with clinicopathological diagnosis, the patients were divided into the lung cancer group (grades I & II and III & IV) and the non-cancer group for standard statistical analysis.ResultsThe partial least squares-discriminant analysis (PLS-DA), the Shapiro–Wilk test, and the Bonferroni correction results showed that the serine concentration was higher and the butane-diacyl-carnitine (C4DC) concentration was lower in the lung cancer group, further showing the same changing trend continuously through the non-cancer stage, grades I & II stage and grades III & IV stage. Those two potential biomarkers have been identified.ConclusionThe HPLC–MS target detection in clinic for nutrient concentration levels is a promising technique to find the changing concentration of serine and C4DC in BALF, which provides an economical and practical way for early warning of lung cancer.

Integrated Single Cell Analysis of Human Lung Fibrosis Resolves Cellular Origins of Predictive Protein Signatures in Body Fluids

Single cell genomics enables characterization of disease specific cell states, while improvements in mass spectrometry bring the clinical use of body fluid proteomics within reach. The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we leverage single-cell RNA-seq and proteomic analysis of large pulmonary fibrosis patient cohorts to identify disease specific changes on the cellular level and their corresponding reflection in body fluid proteomes. We discovered and validated transcriptional changes in 45 cell types across three patient cohorts that translated into distinct changes in bronchoalveolar lavage fluid and plasma proteomes. These protein signatures correlated with diagnosis, lung function, smoking and injury status. Specifically, the altered expression of a novel marker of lung health, CRTAC1, in alveolar epithelium is robustly reflected in patient plasma. Our findings have direct implications for future non-invasive prediction and monitoring of pathological cell state changes in patient organs.

17β-Estradiol affects lung function and inflammation following ozone exposure in a sex-specific manner.

Inflammatory lung diseases affect men and women disproportionately, suggesting that fluctuations of circulating hormone levels mediate inflammatory responses. Studies have shown that ozone exposure contributes to lung injury and impairment of innate immunity with differential effects in men and women. Here, we hypothesized that 17β-estradiol enhances inflammation and airway hyperresponsiveness (AHR), triggered by ozone exposure, in the female lung. We performed gonadectomy and hormone treatment (17β-estradiol, 2 wk) in C57BL/6J female and male mice and exposed animals to 1 ppm of ozone or filtered air for 3 h. Twenty-four hours later, we tested lung function, inflammatory gene expression, and changes in bronchoalveolar lavage fluid (BALF). We found increased AHR and expression of inflammatory genes after ozone exposure. These changes were higher in females and were affected by gonadectomy and 17β-estradiol treatment in a sex-specific manner. Gonadectomized male mice displayed higher AHR and inflammatory gene expression than controls exposed to ozone; 17β-estradiol treatment did not affect this response. In females, ovariectomy reduced ozone-induced AHR, which was restored by 17β-estradiol treatment. Ozone exposure also increased BALF lipocalin-2, which was reduced in both male and female gonadectomized mice. Treatment with 17β-estradiol increased lipocalin-2 levels in females but lowered them in males. Gonadectomy also reduced ozone-induced expression of lung IL-6 and macrophage inflammatory protein-3 in females, which was restored by treatment with 17β-estradiol. Together, these results indicate that 17β-estradiol increases ozone-induced inflammation and AHR in females but not in males. Future studies examining diseases associated with air pollution exposure should consider the patient's sex and hormonal status.

Metabolic Consequences of Chronic Alcohol Abuse in Non-Smokers: A Pilot Study.

An alcohol use disorder (AUD) is associated with an increased susceptibility to respiratory infection and injury and, upon hospitalization, higher mortality rates. Studies in model systems show effects of alcohol on mitochondrial function, lipid metabolism and antioxidant systems. The present study applied high-resolution metabolomics to test for these changes in bronchoalveolar lavage fluid (BALF) of subjects with an AUD. Smokers were excluded to avoid confounding effects and compliance was verified by cotinine measurements. Statistically significant metabolic features, differentially expressed by control and AUD subjects, were identified by statistical and bioinformatic methods. The results show that fatty acid and acylcarnitine concentrations were increased in AUD subjects, consistent with perturbed mitochondrial and lipid metabolism. Decreased concentrations of methyl-donor compounds suggest altered one-carbon metabolism and oxidative stress. An accumulation of peptides suggests proteolytic activity, which could reflect altered epithelial barrier function. Two metabolites of possible microbial origin suggest subclinical bacterial infection. Furthermore, increased diacetylspermine suggests additional metabolic perturbations, which could contribute to dysregulated alveolar macrophage function and vulnerability to infection. Together, the results show an extended metabolic consequence of AUD in the bronchoalveolar space.

Environmental exposures and airway inflammation in young thoroughbred horses.

BackgroundInflammatory airway disease (IAD) in horses is a widespread, performance‐limiting syndrome believed to develop in response to inhaled irritants in the barn environment.ObjectivesTo evaluate changes in bronchoalveolar lavage fluid (BALF) cytology and exposure to particulates, endotoxin, and ammonia during horses' first month in training.AnimalsForty‐nine client‐owned 12‐ to 36‐month‐old Thoroughbred horses entering race training.MethodsIn this prospective cohort study, a convenience sample of horses was assigned to be fed hay from a net (n = 16), whereas the remaining horses were fed hay from the ground (n = 33). BALF was collected at enrollment and after 14 and 28 days in training. Respirable particulate, inhalable particulate, respirable endotoxin, and ammonia concentrations were measured at the breathing zone of each horse weekly.ResultsMedian respirable particulates were significantly higher when horses were fed from hay nets than when fed hay from the ground (hay net 0.28 mg/m3, no hay net 0.055 mg/m3, P < .001). Likewise, inhalable particulate (hay net 8.3 mg/m3, no hay net 3.3 mg/m3, P = .0064) and respirable endotoxin (hay net 173.4 EU/m3, no hay net 59.2 EU/m3, P = .018) exposures were significantly higher when horses were fed from hay nets. Feeding hay from a net resulted in significantly higher BALF eosinophil proportions over time (P < .001). BALF eosinophils were significantly related to respirable particulate exposure (14 days in training r s = 0.37, P = .012, 28 days in training, r s = 0.38, P = .017).Conclusions and Clinical ImportancePulmonary eosinophilic inflammation develops in response to respirable particulate exposure in young Thoroughbreds, indicating a potential hypersensitivity to inhaled particulate allergens.

Effects of COX-2 inhibitor on ventilator-induced lung injury in rats

Mechanical ventilation especially with large tidal volume has been demonstrated to activate inflammatory response inducing lung injury, which could be attenuated by cyclooxygenase (COX)-2 inhibitors. As the main small integral membrane proteins that selectively conduct water molecules' transportation, aquaporin (AQP)-1 downregulation significantly related to lung edema and inflammation. This study aims to investigate the role of AQP1 in ventilator-induced lung injury in rats and evaluates the effects of COX-2 inhibition. Forty rats were allocated into four groups, where rats in Groups LD (low volume+DMSO) and LN (low volume+NS-398) were given intravenously 2ml DMSO and 8mg/kg NS-398 (a specific COX-2 inhibitor, dissolved in 2ml DMSO) before 4-hour lower tidal volume ventilation (8ml/kg), respectively, while DMSO and NS-398 were administrated in the same manner before 4-hour injurious ventilation (40ml/kg) in Groups HD (high volume+DMSO) and HN (high volume+NS-398). The arachidonic acid metabolites (6-keto prostaglandin F1α, thromboxane B2), inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, 6, 8) and total protein levels in bronchoalveolar lavage (BAL) fluid and COX-2 mRNA and AQP1 protein expression in lung tissue were detected; water content and lung morphology were also evaluated. Compared to Groups LD and LN, the rats in Groups HD and HN suffered obvious lung morphological changes with higher wet-to-dry weight ratio and lung injury score, and the levels of arachidonic acid metabolites, inflammatory cytokines and total protein in BAL fluid were increased, the expression of COX-2 mRNA was significantly upregulated and AQP1 protein was downregulated in lung tissue (p<0.05). The changes in BAL fluid and the severity of lung injury were attenuated, and AQP1 expression was upregulated in Group HN as compared to HD (p<0.05). Ventilation with large tidal volume causes inflammatory mediator production and AQP1 downregulation, which could be attenuated by COX-2 inhibition.

Proteomic Analysis of Bronchoalveolar Lavage Fluid Proteins from Mice Infected with Francisella tularensis ssp. novicida

Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA), and Pseudomonas aeruginosa. The composition of bronchoalveolar lavage fluid (BALF) proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress, and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system; however, the timing of their induction varied. F. tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection; however, within 24 h, they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response; however, this response is diminished by 24 h. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.

Comparison of mouse strains and exposure conditions in acute cigarette smoke inhalation studies

Cigarette smoke exposures in mice have been conducted under various exposure conditions using different strains as animal models of smoke-related diseases. We exposed cigarette smoke to two strains of mice [C57BL/6J (C57) and AKR/J (AKR)] under two different exposure regimens (1 h or 4 h/day) at equivalent daily exposure amount (concentration × time). After 2 weeks exposure, mice were evaluated using exposure markers and biological responses. Smoke exposure suppressed respiratory parameters dependent on exposure concentration. The 1-h regimen groups generally showed a greater degree of respiratory suppression and relatively lower exposure markers of urinary nicotine metabolites than the corresponding 4-h regimen groups. Tidal volume was more suppressed in AKR compared to C57, while respiratory rate was more suppressed in C57. Plasma exposure markers and respiratory parameters suggested that C57 inhaled more volume of smoke than AKR. Changes in bronchoalveolar lavage fluid (BALF) cytology and enzyme parameters were most noticeable in the 1 h AKR groups. In BALF cytokine concentration, TARC concentration in C57 was higher than AKR, while KC and MCP-1 in AKR were higher than C57. Relative lung/body weight ratio in smoke-exposed C57 was generally higher, as well as the incidence and severity of lesions in respiratory organs compared to AKR. In summary, C57 appeared to inhale relatively more smoke and displayed greater inflammatory changes in respiratory tract than AKR. Comparison of exposure regimens suggests that a longer exposure duration at lower WTPM concentration might deliver a larger dose of smoke than a shorter exposure duration at higher WTPM concentration.

Insulin-like Growth Factor-binding Protein-3 (IGFBP-3) Blocks the Effects of Asthma by Negatively Regulating NF-κB Signaling through IGFBP-3R-mediated Activation of Caspases

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein known for modulating mitogenic and metabolic actions of IGFs as well as exerting a variety of biological actions not involving IGFs. Here, we show that IGFBP-3 blocks specific physiological consequences of asthma in an IGF-independent manner in vitro and in vivo. IGFBP-3 treatment effectively reduced all physiological manifestations of asthma examined in vivo (airway hyper-responsiveness, cellular and pathological changes in bronchoalveolar lavage fluid and lung tissue, and expression of numerous proinflammatory molecules). These unique IGFBP-3 effects were further confirmed in IGFBP-3-transgenic mice, thus strengthening the notion of IGFBP-3 actions within the respiratory system. Using human epithelial cells, we demonstrated the following: 1) IGFBP-3 blocks TNF-α-induced expression of proinflammatory molecules; 2) IGFBP-3 attenuates the TNF-α-induced migratory response of eosinophils; and 3) IGFBP-3 negatively regulates TNF-α-induced expression of the key NF-κB regulatory molecules IκBα and p65-NF-κB at the post-translational level. We identified that IGFBP-3 degrades IκBα and p65-NF-κB proteins through IGFBP-3 receptor (IGFBP-3R)-mediated activation of caspases thereby inhibiting TNF-α-induced activation of NF-κB signaling cascades. This unique IGFBP-3/IGFBP-3R action was further confirmed by demonstrating complete inhibition of IGFBP-3 action in the presence of caspase inhibitors as well as IGFBP-3R siRNAs. Non-IGF-binding IGFBP-3 mutants further proved the IGF-independent action of IGFBP-3. Our findings indicate that IGFBP-3 inhibits airway inflammation and hyper-responsiveness via an IGF-independent mechanism that involves activation of IGFBP-3R signaling and cross-talk with NF-κB signaling. The IGFBP-3/IGFBP-3R system therefore plays a pivotal role in the pathogenesis of asthma and can serve as a newly identified potential therapeutic target for this debilitating disease.

Ultraviolet B light–exposed human platelets mediate acute lung injury in a two‐event mouse model of transfusion

Ultraviolet B (UVB) light has been used alone on platelet (PLT) transfusion products to prevent alloimmunization or with chemical sensitizers to reduce pathogens. Such processing can damage PLTs and potentiate their storage lesion. Transfusion-related acute lung injury (ALI) has occurred in patients whose underlying condition led to an inflamed endothelium and who were transfused with products that contained either HLA or HNA antibodies or biologic modifiers such as lipids or antigens from stored cells. Clinical trials of UV-treated PLTs in patients with thrombocytopenia generated controversy regarding association of these cells with respiratory distress. We evaluated whether UVB PLTs could mediate ALI in an animal model of ALI. We used a two-event animal model where the sensitizing event was lipopolysaccharide (LPS) and the second event was infusion of human PLTs or UVB human PLTs (2.4 J/cm(2) ). Infused human PLTs were followed with whole animal imaging, lung histology, confocal microscopy, lung water, and changes in bronchoalveolar lavage fluid (BALF) related to ALI. In LPS-treated mice UVB human PLTs accumulated in the lungs and were associated with ALI manifested by increased protein and white blood cells (WBCs) in BALF. Untreated human PLTs did not accumulate in the lungs or increase BALF protein or WBC counts. We provide a proof of principle that UVB human PLTs can accumulate in lungs of LPS-primed animals and mediate ALI. PLTs exposed to high doses of UVB could potentially mediate similar effects in patients predisposed with sepsis or other causes of endothelial cell inflammation.

UVB Light Exposed Human Platelets Mediate Acute Lung Injury In a Two-Event Mouse Model of Transfusion

AbstractAbstract 3354Transfusion related acute lung injury (TRALI) has occurred in patients whose underlying condition led to an inflamed endothelium, and who were transfused with a transfusion product that contained either HLA or HNA antibodies or biologic modifiers such as lipids or antigens from stored cells. Similar two-event reactions can be modeled in animals pretreated with lipopolysaccharide (LPS) and infused with similar types of antibodies or media from stored transfusion products. The first event induces activation of the endothelium and sequestration of neutrophils in the lungs while the second event activates neutrophils to cause local tissue damage. In some animal models of acute lung damage platelet depletion reduces the lung damage while in other models infusion of activated platelets potentiates it. Ultraviolet B (UVB) light has been used on platelet transfusion products to prevent alloimmunization or with chemical sensitizers to reduce pathogens. Such processing may damage platelets and potentiate their storage lesion. UVB exposed human platelets (HPs) were evaluated in a two-event animal model of acute lung injury (ALI) where the sensitizing event was LPS and the second event was infusion of HPs or UVB HPs (2.4 J/cm2). Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). UVB exposure of HPs increased their p-selectin expression (control 17.8±0.3% vs. UVB 35.9±3.2%) and reduced their JC-1 dye ratio indicating mitochondrial damage (8.9±0.7 control vs 1.9±1.2 UVB). Internal organ distribution of intravenous (IV) infused HPs was followed with whole animal imaging, confocal microscopy and with pathophysiological changes in bronchoalveolar lavage fluid (BALF). In LPS-treated mice, UVB HPs labeled with fluorescent dye had more accumulation in lungs compared to untreated HPs (29±12% vs 15±5% respectively; % of total fluorescence recovered), while the accumulation in lungs of healthy animals was equivalent for both UVB treated and untreated HPs. In separate experiments, LPS pretreated mice were infused with UVB exposed HPs or control HPs, and lungs were examined by histology and with confocal microscopy for fluorescent staining for CD41 and CD62 expression. Histology sections revealed extensive changes in lungs, such as thickening of the alveolar septa and obliteration of lung architecture in LPS animals infused with UVB HPs as compared to healthy animals infused with control HPs or with HPs treated with UVB. Confocal microscopy with specific antibodies identified HP accumulation in lungs of LPS treated animals infused with UVB exposed HPs. However, HP accumulation in lungs did not occur with control HPs or in healthy mice. Accumulation of UVB HPs in lungs of LPS pretreated mice was associated with increased (3-4 fold compared to control HPs) protein concentration and leukocyte accumulation in BALF. Increased alveolar permeability to protein and leukocyte accumulation in alveoli is associated with acute lung damage. To examine whether lung damage occurred due to presence of UVB HPs in the lungs or due to biologic mediators released into plasma from UVB exposed HPs we separated plasma from HPs post UVB treatment and infused the UVB plasma alone. Plasma isolated from UVB HPs did not alter BALF protein levels or leukocyte counts even though the UVB HPs did. In conclusion, UVB HPs can accumulate in lungs of LPS primed animals and are associated with lung damage as indicated by histological changes and increased protein and WBCs in BALF fluid. The ALI is associated with direct platelet accumulation in the lungs but not with biologic modifiers released into plasma. Our animal model of ALI suggests that HPs exposed to high doses of UVB could mediate similar effects in patients predisposed to TRALI with sepsis or other causes of endothelial cell inflammation. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Cellular changes in bronchoalveolar lavage fluid in hyperoxia-induced lung injury

It is well known that high concentration oxygen exposure is a model of acute lung injury (ALI). However, controversy exists over the mechanism. This study was designed to clarify the cellular characteristics in bronchoalveolar lavage fluid (BALF) and body weight loss of rats exposed to oxygen(> 90%). Young male Wistar rats, aged 6 weeks, were divided into three groups: (1) room air group (exposed to room air, n = 22); (2) hyperoxia < 48 h group (exposed to over 90% oxygen for less than 48 h, n = 18); (3) hyperoxia 66–72 h group (exposed to over 90% oxygen for 66–72 h group, n = 7). Compared to the room air group, the total cell counts in the hyperoxia 66–72 h group decreased, whereas the neutrophils increased significantly. The body weights of the rats exposed to room air continued to increase. However, the body weights of oxygen-exposed rats increased slightly on the first day and weight loss was seen from the second day. All rats were noted to have bilateral pleural effusion in the hyperoxia 66–72 h group. The data suggests that (1) an increase in neutrophil count is an evident feature of hyperoxia-induced lung injury; (2) high concentration oxygen exposure can give rise to anorexia and malnutrition, which may play a role in hyperoxia-induced lung injury. Blocking neutrophil influx into lung tissue in the early phase and improving malnutrition are two effective methods to reduce hyperoxic lung injury.

Expression of Urokinase-Type Plasminogen Activator in Platelets Decreases Oxygen-Induced Lung Injury in Mice.

We have previously shown that urokinase (uPA) can be ectopically expressed and stored in platelets in a transgenic mouse model and that such platelets can effectively prevent untoward thrombosis. One major advantage of this uPA delivery system is that there is no systemic fibrinolysis and the uPA is selectively released at sites of vascular injury. We have now tested whether such a delivery model may be of use in the care of patients with lung injury where inhibition of fibrinolytic activity and intra-alveolar fibrin deposition are of pathogenic importance. In this study, we evaluated (O2)-induced lung injury in transgenic mice expressing platelet uPA compared to wildtype (WT) littermate controls. Given that pulmonary sequestration of activated inflammatory cells, including neutrophils (PMN) and platelets, occurs relatively early in response to O2, and given the ability of the transgenic uPA platelets to enhance fibrin lysis, we hypothesized that hyperoxic-induced lung injury would be lessened in the transgenic animals. Lung injury was assessed by changes in bronchoalveolar lavage fluid (BAL) protein concentration. In this model, a significant increase in BAL protein concentration, indicative of injury to the alveolar capillary barrier, occurred in WT mice after 72 hr of >95% O2 exposure compared to controls [6.9 ± 0.9 vs. 0.14 ± 0.01 mg/ml (mean ± SEM)], respectively. BAL protein was significantly decreased in platelet uPA+ mice [1.5 ± 0.4 vs. 6.9 ± 0.9 mg/ml, n=6–8, p<0.05]. Immunohistochemical detection of fibrin in control and platelet uPA+ mice lungs using anti-murine fibrin(ogen) antibody showed that very little fibrin(ogen) was detected in control (0 hr) mice or WT mice exposed to >95% O2 for 24 hr. By 72 hr of O2 in WT mice, however, fibrin(ogen) immunoreactivity was seen within the alveolar space and diffusely throughout the interstitium. Fibrin clots within small blood vessels were also seen. In contrast, fibrin(ogen) immunoreactivity was greatly reduced in uPA+ mice. Moreover survival time of uPA+ mice was significantly longer than WT animals (127 ± 28 hr vs. 97 ± 14 hrs, respectively, n=12, p<0.003). Total white blood cell counts and % PMN in the BAL were significantly higher after 72 hr exposure to O2, suggesting that increased pulmonary inflammation is not necessarily associated with increased lung injury, a finding consistent with other studies showing that neutrophils do not play a significant role in the genesis of this lung injury. These data support the hypothesis that a decrease in lung fibrinolytic activity is a primary factor in the development of O2-induced lung injury and that platelet delivery of uPA may be an especially effective and safe way to prevent the development of such lung injury.

Time course of bleomycin-induced lung fibrosis.

Intratracheal instillation (IT) of bleomycin is a widely used experimental model for lung fibrosis. In this study we describe the time-course of bleomycin-induced lung fibrosis in mice using computer-assisted morphometry. C57Bl/6J mice were treated with a single IT dose of bleomycin or control saline. Animals were killed 3, 6, 14 and 21 days post-IT. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, hydroxyproline concentration in the lung, routine light microscopic examination resulting in a semiquantitative morphological index (SMI) of lung injury, and quantitative morphological measurements (fibrosis fraction and alveolar wall area fraction) aided by optimas image analysis software. Changes in BAL fluid attributed to bleomycin treatment include increased total cell count (days 14 and 21), and increased percentage of neutrophils (days 3 and 6) followed by a sustained increase in lymphocytes (days 6, 14 and 21). Hydroxyproline levels increased in bleomycin-treated mice on days 14 and 21. Median SMI grades were significantly elevated on days 3, 14 and 21. Computer-assisted morphometry demonstrated a 3-fold increase in fibrosis fraction and a 1.3-fold increase in wall area fraction in bleomycin-treated mice on day 14, with no further increase on day 21. These data also demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days after IT instillation of bleomycin, based on the observation that at 14 days the animals developed extensive fibrosis, but had less variability in the fibrotic response and lower mortality than later at 21 days. Computer-assisted morphometry provides objective and quantitative measurements that are a useful tool for the evaluation of bleomycin-induced lung injury.

SHORT-TERM INHALATION TOXICITY OF POLYISOCYANATE AEROSOLS IN RATS: COMPARATIVE ASSESSMENT OF IRRITANT-THRESHOLD CONCENTRATIONS BY BRONCHOALVEOLAR LAVAGE

The object of this study was to compare the relative potency of respirable aerosols of the aliphatic hexamethylene 1,6-diisocyanate homopolymer of the isocyanurate type (HDI-IC) and the aromatic polymeric methylenediphenyl-4,4'-diisocyanate (pMDI) to elicit early changes in bronchoalveolar lavage fluid (BALF). The validity of the concentration × time (C × t) concept was addressed in rats exposed to concentrations from 3.4 to 58.1 mg pMDI/m 3 and exposure durations of 6 h to 23 min, respectively (C × t ≈ 1200 mg/m 3 -min). One additional group of rats was exposed to 2.7 mg MDA/m 3 for 1 × 6 h, a putative product of hydrolysis of pMDI. In rats repeatedly exposed to 12.9 mg pMDI/m 3 (6 h/day, 5 days/wk for 14 days), cumulative exposure-related changes were examined. Results show that total protein and angiotensin-converting enzyme (ACE) in BALF were among the most sensitive endpoints to probe early effects caused by exposure to irritant polyisocyanate aerosols. In the repeated-exposure study, BALF protein was maximal after the first exposure day. Based on these most sensitive endpoints in BALF, a benchmark no-effect threshold concentration of 0.5 and 3 mg/m 3 was estimated for the pMDI and HDI-IC aerosol, respectively. The slope of the concentration-effect curve was steeper following exposure to HDI-IC than to pMDI. These estimated acute no-observed-effect levels (NOELs) were almost identical to those observed in longer term inhalation studies using conventional endpoints. It is concluded that pulmonary irritation caused by polyisocyanate aerosols can readily be quantified in an acute rat bioassay by the analysis of total protein in BALF.

Cytological and immunological changes in bronchoalveolar lavage fluid and histological observation of lung lesions in pigs immunized with Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate

We have compared the cytology of bronchoalveolar lavage fluid (BALF) and the pathology of lung lesions in pigs immunized with/without Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate on experimental infection. Numbers of total cells, macrophages, neutrophils and lymphocytes have decreased in BALF of vaccinated pigs following infection. The mean percentage of lung lesions, inflammatory cell infiltration into the airways and T cells accumulation around the bronchi were reduced in vaccinated pigs. The levels of tumor necrosis factor (TNF)-α also decreased in vaccinated pigs. These results suggest that the vaccination may contribute to decrease TNF-α production, and therefore, inflammatory cell responses in the lung due to M. hyopneumoniae infection were suppressed, resulting in fewer lung lesions.

DIFFERENCES IN THE EXTENT OF INFLAMMATION CAUSED BY INTRATRACHEAL EXPOSURE TO THREE ULTRAFINE METALS: ROLE OF FREE RADICALS

Nickel and cobalt, which belong to the same elemental group, are known to cause interstitial lung disease and bronchial asthma. The ability of these metals to injure lung cells and cause inflammation is likely to be important in their pathogenicity but comparative studies are rare. Additionally, ultrafine (uf) forms of these metals are used increasingly and their is little available information on their toxicity. Thus the inflammatory response following intratracheal instillation of ultrafine particles of Co, Ni, and TiO2 was compared. Physiological saline (PS) was used as a vehicle control and DQ quartz 12 as a positive control. Male Wistar rats were intratracheally instilled with the 3 particle types at a dose of 1 mg suspended in physiological saline. At 1, 3, 7, 15, and 30 d after the injection, lung weight and the cellular and biochemical changes in bronchoalveolar lavage fluid (BALF) were determined. By all of the indices, Uf-Ni appeared to be the most injurious to the lung, causing severe and sustained inflammation, cytotoxicity and increased epithelial permeability. The next most toxic material was DQ quartz, with 12 Uf-Co being closely similar in ability to cause inflammation. Uf-TiO2 was more active than the saline control in all of the indices, but was the least toxic of the particles studied. The present study reveals that three ultrafine particles of the same diameter are dramatically different in their ability to cause inflammation. The three ultrafines were compared as to their ability to cause free-radical damage to supercoiled plasmid DNA, and the result of free-radical activity was found to be Uf-TiO2 << Uf-Co = Uf-Ni. Difference in free-radical-generation activity therefore could underlie the difference in inflammation of these three ultrafine particle types.

Inflammatory determinants of asthma severity: Mediator and cellular changes in bronchoalveolar lavage fluid of patients with severe asthma

Cellular and mediator profiles in bronchoalveolar lavage have not been compared systematically between patients with asthma of different severities, mainly because the patients with more severe asthma have an increased need for antiinflammatory medication. Information is limited to comparisons of allergic and intrinsic asthma, which can be distinguished clinically. When patients from these two groups with similar degrees of bronchial hyperresponsiveness were compared, both groups showed increased numbers of activated T-helper lymphocytes; those in the allergic group expressed the IL-2 receptor (CD25+), whereas in patients with intrinsic asthma there was also an increased number of T-suppressor cells with the activation markers CD25, class II histocompatibility antigen, and very late activation antigen-1, as well as T-helper cells class II histocompatibility antigen and very late activation antigen-1. This pattern is compatible with a more chronic T-cell activation in patients with intrinsic asthma. In patients with allergic asthma the cytokine pattern is compatible with a pure T H2 response (elevated IL-4 and IL-5); however, intrinsic asthma is characterized by elevated IL-5 and IL-2 but not IL-4. Our own findings show similar concentrations of IL-1, IL-8, and granulocyte-macrophage colony-stimulating factor in bronchoalveolar lavage fluid of patients with allergic and intrinsic asthma, whereas IL-6 and interferon-γ tended to be higher in patients with intrinsic asthma. There are probably fundamental differences in the pathogenesis of allergic and intrinsic asthma. These findings suggest that asthma does not depend on the presence of IgE or IL-4, although both may contribute to the pathogenesis of atopic asthma. The only common pathway in the different presentations of asthma that has been related to clinical symptoms appears to be IL-5-mediated activation of eosinophils; therapies aimed at this mechanism may be promising. (J A LLERGY C LIN I MMUNOL 1996;98:27-40.)

Inflammatory determinants of asthma severity: Mediator and cellular changes in bronchoalveolar lavage fluid of patients with severe asthma

Cellular and mediator profiles in bronchoalveolar lavage have not been compared systematically between patients with asthma of different severities, mainly because the patients with more severe asthma have an increased need for antiinflammatory medication. Information is limited to comparisons of allergic and intrinsic asthma, which can be distinguished clinically. When patients from these two groups with similar degrees of bronchial hyperresponsiveness were compared, both groups showed increased numbers of activated T-helper lymphocytes; those in the allergic group expressed the IL-2 receptor (CD25+), whereas in patients with intrinsic asthma there was also an increased number of T-suppressor cells with the activation markers CD25, class II histocompatibility antigen, and very late activation antigen-1, as well as T-helper cells class II histocompatibility antigen and very late activation antigen-1. This pattern is compatible with a more chronic T-cell activation in patients with intrinsic asthma. In patients with allergic asthma the cytokine pattern is compatible with a pure TH2 response (elevated IL-4 and IL-5); however, intrinsic asthma is characterized by elevated IL-5 and IL-2 but not IL-4. Our own findings show similar concentrations of IL-1, IL-8, and granulocyte-macrophage colony-stimulating factor in bronchoalveolar lavage fluid of patients with allergic and intrinsic asthma, whereas IL-6 and interferon-γ tended to be higher in patients with intrinsic asthma. There are probably fundamental differences in the pathogenesis of allergic and intrinsic asthma. These findings suggest that asthma does not depend on the presence of IgE or IL-4, although both may contribute to the pathogenesis of atopic asthma. The only common pathway in the different presentations of asthma that has been related to clinical symptoms appears to be IL-5-mediated activation of eosinophils; therapies aimed at this mechanism may be promising. (J ALLERGY CLIN IMMUNOL 1996;98:27-40.)