Lung Lavage Fluid Research Articles

Effect of Moringa oleifera Lam. Seed Extract on Ovalbumin-Induced Airway Inflammation in Guinea Pigs

To determine the therapeutic potential of herbal medicine Moringa oleifera Lam. family: Moringaceae in the control of allergic diseases, the efficacy of the ethanolic extract of the seeds of the plant (MOEE) against ovalbumin (OVA)-induced airway inflammation in guinea pigs was examined. During the experimental period, the test drugs (MOEE or dexamethasone) were administered by oral route prior to challenge with aerosolized 0.5% OVA. Bronchoconstriction tests were performed and respiratory parameters (i.e., tidal volume and respiratory rate) were measured. At the end of experiment, blood was collected from each animal to perform total and differential counts and serum was used for assay of IL-4, IL-6, and TNFα. Lung lavage fluid (BAL) was collected for estimation of cellular content and cytokine levels. Lung tissue histamine assays were performed using the homogenate of one lobe from each animal; a separate lobe and the trachea were subjected to histopathology to measure the degree of any airway inflammation. The results suggest that in OVA-sensitized control animals that did not receive either drug, tidal volume (Vt) was decreased, respiration rate (f) was increased, and both the total and differential cell counts in blood and BAL fluid were increased significantly. MOEE-treatment of sensitized hosts resulted in improvement in all parameters except BAL TNFα and IL-4. Moreover, MOEE-treatment also showed protection against acetylcholine-induced broncho-constriction and airway inflammation which was confirmed by histological observations. The results of these studies confirm the traditional claim for the usefulness of this herb in the treatment of allergic disorders like asthma.

In vivo and in vitro effects of salbutamol on alveolar epithelial repair in acute lung injury

Background: Acute lung injury is an important cause of respiratory failure in the critically ill patient. It is caused by damage to the alveolar barrier with subsequent alveolar flooding leading...

The properties and turnover of hyaluronan.

Hyaluronan (HA) was discovered over 50 years ago but its metabolism and cellular interactions have only recently received detailed attention. HA is synthesized in the plasma membrane by addition of monosaccharides to the reducing terminal. In tissues, it occurs bound to plasma membranes, aggregated with other macromolecules, or as free polysaccharide. Tissue HA enters the bloodstream in significant amounts through the lymph and is rapidly absorbed via a receptor into liver endothelial cells, where degradation follows. HA levels in serum are normally 10-100 micrograms/l, but can be elevated in cirrhosis, rheumatoid arthritis and scleroderma, due either to impaired hepatic uptake or to increased production. Studies on aqueous humour, middle ear secretion, amniotic fluid, lung lavage fluid, urine, skin diseases and cancer have identified other causes of deranged HA metabolism. HA can be visualized on some cell surfaces as a coating impermeable to particulate material. Specific HA binding occurs on lymphoma cell lines, lung macrophages and SV-3T3 cells but, except in synthesis or uptake, the significance of membrane-associated HA is incompletely understood. It has been reported to activate macrophages and granulocytes, protect cells, control cell migration, and cooperate with intercellular matrix in cell detachment; it also plays a central role in growth control, cellular differentiation and tissue morphogenesis.

Ambient ozone primes pulmonary innate immunity in mice.

Exposure to ozone in air pollution in urban environments is associated with increases in pulmonary-related hospitalizations and mortality. Because ozone also alters clearance of pulmonary bacterial pathogens, we hypothesized that inhalation of ozone modifies innate immunity in the lung. To address our hypothesis, we exposed C57BL/6J mice to either free air or ozone, and then subsequently challenged with an aerosol of Escherichia coli LPS. Pre-exposure to ozone resulted in [corrected] higher concentrations of both total protein and proinflammatory cytokines in lung lavage fluid, enhanced LPS-mediated signaling in lung tissue, and higher concentrations of serum IL-6 following inhalation of LPS. However, pre-exposure to ozone dramatically reduced inflammatory cell accumulation to the lower airways in response to inhaled LPS. The reduced concentration of cells in the lower airways was associated with enhanced apoptosis of both lung macrophages and systemic circulating monocytes. Moreover, both flow cytometry and confocal microscopy indicate that inhaled ozone causes altered distribution of TLR4 on alveolar macrophages and enhanced functional response to endotoxin by macrophages. These observations indicate that ozone exposure increases both the pulmonary and the systemic biologic response to inhaled LPS by priming the innate immune system.

Losartan Attenuates Ventilator-Induced Lung Injury

Accumulating evidence shows that angiotensin II (ANG II) can be generated locally in the lung tissue and may have autocrine and/or paracrine actions on the cellular level. In addition, ANG II precursor, angiotensinogen, as well as ANG II type 1 receptor (AT(1)), are also expressed in the lung tissue. Recent studies revealed that ANG II promoted acute lung injury induced by acid aspiration or sepsis, and that ANG II receptor blockade had a protective effect against acute lung injury. Therefore, the authors hypothesized that ventilator-induced lung injury might also be exacerbated by local ANG II action, and that ANG II receptor blockade would protect the lung from ventilator-induced lung injury. Forty Sprague Dawley rats weighing 300-350 g were randomly divided into the following experimental groups (10 rats in each group): (1) control group: rats were unventilated; (2) LVT (low volume ventilation) group: rats were ventilated with 8 mL/kg tidal volume room air for 2 h; (3) HVT (high volume ventilation) group: rats were ventilated with 40 mL/kg tidal volume room air for 2 h; (4) HVT + Losartan group: rats were pretreated with Losartan (30 mg/kg, i.p.) prior to high volume ventilation. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. The expression of angiotensinogen and AT(1) receptor mRNA in lung tissue was measured by reverse transcriptase-polymerase chain reaction. Apoptosis of the lung cells was assayed with terminal deoxynucleodityl transferase-mediated nick-end labeling method. Lung pathological changes were examined with optical microscopy. Total protein, wet/dry ratios (W/D), myeloperoxidase (MPO) activity, and neutrophil counts of the lung tissue or lavage fluid were measured with corresponding methods. Compared with control or LVT, HVT caused significant ventilator-induced lung injury and increased the expression of angiotensinogen and AT(1) receptor mRNA in the lung. Total protein, the number of apoptotic cells, W/D ratio, MPO activity, and neutrophil counts were significantly higher in the HVT group than in the LVT or control group. Pretreatment with Losartan attenuated ventilator-induced lung injury and prevented the increase in total protein, the number of apoptotic cells, W/D ratio, MPO, and neutrophil counts caused by high volume ventilation. Our study indicates that HVT causes remarkable lung injury and up-regulates angiotensinogen and AT(1) receptor expression of in the lung, and that Losartan, a selective inhibitor of subtype AT(1) receptors for angiotensin II, can relieve acute lung injury caused by high volume ventilation.

Developmental changes in rat surfactant lipidomics in the context of species variability

Lung surfactant comprises mainly phosphatidylcholine (PC) species together with phosphatidylglycerols and surfactant proteins (SP) SP-A to -D. Changes in the concentrations of its principal components dipalmitoyl-PC, palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC relative to developmental, structural and physiological differences are only partially understood. Particularly, their attribution to differences in air-liquid interface curvature, compared with dynamic parameters, such as respiratory rate, are controversial. We postulated that during alveolarization the changes in these principal PC components of surfactant differ from those in other phospholipid parameters, and that across endothermic vertebrates their concentrations are related to lung physiology rather than structure. We therefore investigated in rats from postnatal day (d)1 to d42 the pattern of surfactant phospholipids relative to alveolarization (d4-d14), and we discuss these changes in terms of molecular adaptation to pulmonary structure or physiology. Contrary to mammals with advanced alveolarization and increased respiratory rate (RR) at term, concentrations of dipalmitoyl-PC (49-52%) and palmitoylmyristoyl-PC (7-9%) in lung lavage fluid were identical at d1 and d42. At d7-d14, when in rats RR is increased, palmitoyl-myristoyl-PC transiently increased by 2.5- to 3.9-fold at the expense of dipalmitoyl-PC (-32% to 34%) and palmitoyl-palmitoleoyl-PC (-16%). Other lipidomic changes followed essentially different patterns of increase or decrease. Palmitoyl-myristoyl-PC was increased in large aggregates suggesting that it is an integral component of active surfactant. In the overall context of vertebrates, irrespective of age and lung structure, fractions of palmitoyl-myristoyl-PC, dipalmitoyl-PC and palmitoyl-palmitoleoyl-PC correlate with differences in RR rather than alveolar curvature. In adult mammals, however, only concentrations of palmitoyl-palmitoleoyl-PC correlate with RR.

Reduction of endotoxin may protect lung from injury in rats with hepatopulmonary syndrome

To study the protective effects of ofloxacin by reduction of endotoxin on lung in rats with hepatopulmonary syndrome (HPS). Rat models of HPS were induced by ligating the bile duct to result in the biliary cirrhosis. Thirty male SD rats were randomly divided into three groups with 10 animals in each one: sham operation group (the bile ducts were isolated and 2 ml NS was injected i.p), HPS model group (the bile ducts were ligated and 2 ml NS was injected i.p) and levofloxacin group (the bile ducts were did as the former group and 20 mg/kg ofloxacin injected i.p). After 6 weeks the portal pressure, ratios of dry weight to wet weight of lung (D/W), weight of spleen, plasma levels of endotoxin, MPO active and MDA contents in the lung. Plasma and lavage fluid of lung were examined. The results of the blood-gas analysis, the bacterial culture of blood, bile and ascites and the evaluation of pathologic change of lung be also investigated. In levofloxacin group, portal pressure [(19.28 +/- 2.3) compare with (17.80 +/- 2.18)cm H2O, P<0.01], D/W [(5.1 +/- 0.7) compare with (4.9 +/- 0.7), P <0.01], plasma endotoxin levels [(59 +/- 6.2) compare with (268 +/- 35.6)ng/L, P<0.01], MPO active [(0.40 +/- 0.10) compare with (0.24 +/- 0.03)U, P<0.01] and MDA [(0.32 +/- 0.05) compare with (0.22 +/- 0.03) micromol/L, P<0.01] contents in the lung were significantly decreased compared with those in HPS group, and the inflammation of lung was also obviously alleviated. Reduction of endotoxin can attenuate the injury of lung in HPS rats.

Distribution of intracellular and secreted surfactant during postnatal rat lung development

Pulmonary surfactant prevents alveolar collapse via reduction of surface tension. In contrast to human neonates, rats are born with saccular lungs. Therefore, rat lungs serve as a model for investigation of the surfactant system during postnatal alveolar formation. We hypothesized that this process is associated with characteristic structural and biochemical surfactant alterations. We aimed to discriminate changes related to alveolarization from those being either invariable or follow continuous patterns of postnatal changes. Secreted active (mainly tubular myelin (tm)) and inactive (unilamellar vesicles (ulv)) surfactant subtypes as well as intracellular surfactant (lamellar bodies (lb)) in type II pneumocytes (PNII) were quantified before (day (d) 1), during (d 7), at the end of alveolarization (d 14), and after completion of lung maturation (d 42) using electron microscopic methods supplemented by biochemical analyses (phospholipid quantification, immunoblotting for SP-A). Immunoelectron microscopy determined the localization of surfactant protein A (SP-A). (1) At d 1 secreted surfactant was increased relative to d 7-42 and then decreased significantly. (2) Air spaces of neonatal lungs comprised lower fractions of tm and increased ulv, which correlated with low SP-A concentrations in lung lavage fluid (LLF) and increased respiratory rates, respectively. (3) Alveolarization (d 7-14) was associated with decreasing PNII size although volume and sizes of Lb continuously increased. (4) The volume fractions of Lb correlated well with the pool sizes of phospholipids in lavaged lungs. Our study emphasizes differential patterns of developmental changes of the surfactant system relative to postnatal alveolarization.

High‐frequency oscillation (HFO) prevents activation of NF‐κB found with conventional mechanical ventilation (CMV) in surfactant‐depleted rabbit lung

High-frequency oscillation (HFO) has been recognized as an effective ventilatory strategy to minimize lung injury during respiratory support. Conventional mechanical ventilation (CMV) compared with HFO was shown to result in an increased number of PMNs and inflammatory cytokines in the lung lavage fluid. However how mechanical forces can be sensed by cells and converted into biochemical signals for intracellular signal transduction is still unknown. In this current study, we sought to determine whether the activation of Nuclear factor-kappa B (NF-kappaB) might be involved in the lung injury caused by CMV. Surfactant-depleted Japanese white rabbits received 1- or 4-hr CMV or 1- or 4-hr HFO. Then, activation of NF-kappaB in the lungs was assessed by conducting electrophoretic mobility shift assays (EMSA). In the experiment with whole lungs, NF-kappaB activity was much higher in the 4-hr CMV lungs than in the 4-hr HFO lungs. To clarify the origin of the cells in which NF-kappaB was activated, we did a second lung lavage at the end of ventilation and washed out the cells that had infiltrated the alveoli. The levels of NF-kappaB activity were the similar in the lungs of 4-hr HFO rabbits and in those of 4-hr CMV ones. On the other hand, NF-kappaB activity was much higher in the 4-hr CMV lungs than in the 4-hr HFO lungs in the experiment with the lung lavage fluid cells. These results show that the increase in NF-kappaB activity in the lungs of 4-hr CMV rabbits was due mainly to the cells that had infiltrated the alveoli.

Filamentous Influenza A Virus Infection Predisposes Mice to Fatal Septicemia following Superinfection with Streptococcus pneumoniae Serotype 3

Previous studies have demonstrated that animals exposed to Streptococcus pneumoniae while recovering from influenza A virus infection exhibit exacerbated disease symptoms. However, many of the current animal models exploring dual viral and bacterial synergistic exacerbations of respiratory disease have utilized mouse-adapted influenza virus and strains of Streptococcus pneumoniae that in themselves are highly lethal to mice. Here we describe a mouse model of bacterial superinfection in which a mild, self-limiting influenza virus infection is followed by mild, self-limiting superinfection with S. pneumoniae serotype 3. S. pneumoniae superinfection results in rapid dissemination of the bacterium from the respiratory tract and systemic spread to all major organs of the mice, resulting in fatal septicemia. This phenomenon in mice was observed in superinfected animals undergoing an active viral infection as well as in mice that had completely cleared the virus 7 to 8 days prior to superinfection. Neutrophils were the predominant cellular inflammatory infiltrate in the lungs of superinfected mice compared to singly infected animals. Among other cytokines and chemokines, the neutrophil activator granulocyte colony-stimulating factor (G-CSF) was found to be significantly overexpressed in the spleens, lungs, and brains of superinfected animals. High G-CSF protein levels were observed in sera and lung lavage fluid from superinfected animals, suggesting that G-CSF is a major contributor to synergistic exacerbation of disease leading to fatal septicemia.

CD14 is an essential mediator of LPS-induced airway disease

Chronic lipopolysaccharide (LPS) inhalation in rodents recapitulates many classic features of chronic obstructive pulmonary disease seen in humans, including airways hyperresponsiveness, neutrophilic inflammation, cytokine production in the lung, and small airways remodeling. CD14-deficient mice (C57BL/6(CD14-/-)) have an altered response to systemic LPS, and yet the role of CD14 in the response to inhaled LPS has not been defined. We observed that C57BL/6(CD14-/-) mice demonstrate no discernable physiological or inflammatory response to a single LPS inhalation challenge. However, the physiological (airways hyperresponsiveness) and inflammatory (presence of neutrophils and TNF-alpha in whole lung lavage fluid) responsiveness to inhaled LPS in C57BL/6(CD14-/-) mice was restored by instilling soluble CD14 intratracheally. Intratracheal instillation of wild-type macrophages into C57BL/6(CD14-/-) mice restored neutrophilic inflammation only and failed to restore airways hyperresponsiveness or TNF-alpha protein in whole lung lavage. These findings demonstrate that CD14 is critical to LPS-induced airway disease and that macrophage CD14 is sufficient to initiate neutrophil recruitment into the airways but that CD14 may need to interact with other cell types as well for the development of airways hyperresponsiveness and for cytokine production.

Detection of respiratory pathogens in porcine lung tissue and lavage fluid

The objective of this study was to compare the detection rate of bacterial agents in bronchoalveolar lavage fluid (BALF), taken without visual control, to that in affected lung tissue obtained from the same pig at necropsy. BALF and affected lung tissue were examined for Mycoplasma hyopneumoniae using PCR, and standard cultural methods were used for Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida and Streptococcus suis. All pigs with a history of respiratory symptoms were submitted as live animals for routine diagnostic examination. In each animal the site of lavage, marked by injecting methylene blue, differed from the site of pneumonic lesions. M. hyopneumoniae was detected more frequently in lung tissue than in BALF in cases with moderate or severe lung lesions. The detection rates of M. hyopneumoniae were higher in the BALF of pigs with mild lesions. Cultural examination of BALF was at least as satisfactory as affected lung tissue for detecting B. bronchiseptica, H. parasuis and P. multocida.

Retinoid-Related Orphan Receptor γ Controls Immunoglobulin Production and Th1/Th2 Cytokine Balance in the Adaptive Immune Response to Allergen

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. RORgamma, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of RORgamma-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORgamma, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-gamma and IL-10 production was markedly greater in splenocytes from RORgamma-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged RORgamma-deficient animals in vivo. These data reveal a critical role for RORgamma in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.

Distribution of Clara cell secretory protein expression in the tracheobronchial airways of rhesus monkeys

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties; to be present in all adult mammals; and to be well conserved in rodents, humans, and nonhuman primates. The rationale for this study is to define the distribution and abundance of CCSP in the airway epithelium and lavage fluid of the adult rhesus monkey and to provide information for evaluating CCSP as a marker of Clara cells and as a biomarker of lung health. Lung tissue and lavage fluid from 3-yr-old rhesus monkeys were examined using histopathology and immunohistochemistry. Proximal bronchi, midlevel bronchi, and terminal/respiratory bronchioles were compared for immunohistochemical localization of CCSP in three-dimensional whole mounts as well as in paraffin and Araldite sections. Immunoreactive CCSP was found in nonciliated cells throughout the airway epithelium. Proximal and midlevel airways had the highest labeling. CCSP decreased in distal airways, and respiratory bronchioles had little to no CCSP. CCSP in the most distal airways was in tall cuboidal cells adjacent to the pulmonary artery. Although a large number of cells were present in the terminal bronchioles that would be classified as Clara cells based on morphology (nonciliated cells with apical protrusions), only a small number stained positively for immunoreactive CCSP. Semiquantitative analysis of Western blots indicated that changes in lavage CCSP are consistent with, and may be predictive of, overall CCSP levels in the airway epithelium in this primate species that is phylogenetically similar to humans.

Effects of Corticosteroids on Expression of Interleukin-18 in the Airway Mucosa of a Mouse Model of Allergic Rhinitis

In this study, we aimed to investigate the release and response of interleukin (IL)-18 to steroid treatment in a mouse model of allergic rhinitis. BALB/c mice were sensitized systemically by intraperitoneal injection of ovalbumin and locally by ovalbumin inhalation. Dexamethasone sodium phosphate was given by intraperitoneal injection in the steroid treatment group. Symptom scores, eosinophil counts, and IL-18 concentrations in the nasal and lung lavage fluids were analyzed. The symptom scores and eosinophil counts of the negative control and steroid treatment groups were significantly lower than those of the positive control group (p < .01). The mean IL-18 concentrations in the nasal lavage fluid were not significantly different among the three groups (56.68 +/- 9.57,63.39 +/- 8.93, and 64.47 +/- 6.83 pg/mL, respectively). The IL-18 concentrations in the lung lavage fluid were significantly different between the positive control group and the steroid treatment group (430.75 +/- 154.54 and 69.94 +/- 14.26 pg/mL, respectively, p = .028). The IL-18 concentration was found to be increased in the lung lavage fluid, but not in the nasal lavage fluid, in a mouse model of allergic rhinitis. Increased IL-18 concentrations returned toward the previous concentrations after steroid treatment. These results suggest that the roles of IL-18 may be different in the pathogenesis of allergic rhinitis and the pathogenesis of asthma.

Interaction of Pneumocystis carinii With Dendritic Cells and Resulting Host Responses to P. carinii

To assess the interaction of Pneumocystis carinii with dendritic cells (DCs), and the consequences of the response of the host immune system to P. carinii antigens when DC are pulsed with P. carinii, murine DC were pulsed with P. carinii, and the resultant P. carinii host responses assessed in vitro and in vivo. P. carinii interacted with murine bone marrow-derived DC in vitro in part via mannose receptors. DC pulsed with P. carinii did not demonstrate increased expression of the cell surface markers MHC II, CD40, CD54, CD80 (B7.1), and CD86 (B7.2). The release of interleukin (IL)-4 was increased, but there was no increase in the release of interleukin (IL)-12p40, IL-10, tumor necrosis factor-alpha, IL-6, and nitrite compared with naive DC. In vivo administration of DC pulsed with P. carinii induced a P. carinii-specific response, generating CD4+ cells that proliferated and released IL-4, but not interferon-gamma, in response to P. carinii-pulsed DC in vitro. In vivo administration of DC pulsed with P. carinii also induced P. carinii-specific immunoglobulin (Ig)G1, IgG2a, and IgG2b, but not IgG3, antibodies in serum, and lung lavage fluid. Finally, CD4+ depleted mice immunized with DC pulsed with P. carinii demonstrated suppression of lung growth of P. carinii after intratracheal challenge with P. carinii at 3 and 16 weeks after immunization. These observations provide insight into DC-P. carinii interactions, and support the concept that a vaccine that includes DC pulsed with P. carinii can mount a humoral and T helper 2-type cellular response to P. carinii sufficient to suppress the growth of P. carinii in the lung.

Stimulation of Mucosal and Systemic Antibody Responses against Recombinant Transferrin-binding Protein B of Actinobacillus pleuropneumoniae with Chitosan after Tracheal Administration in Piglets

This study evaluated the suitability of using a chitosan formulation as an adjuvant to enhance both the mucosal and systemic immune responses against recombinant transferrin-binding protein B (rTbp B) of Actinobacillus pleuropneumoniae via direct tracheal administration. The chitosan formulation was found to enhance mucosal immune response, as measured by the secretory IgA level in lung lavage fluid and lung homogenate extracts, and systemic immune response, as measured by the serum IgG level.

Mucosal Delivery of a Pneumococcal Vaccine UsingLactococcus lactisAffords Protection against Respiratory Infection

Economical and effective vaccines against Streptococcus pneumoniae (pneumococcus) are needed for implementation in poorer countries where the disease burden is highest. Here, we evaluated Lactococcus lactis intracellularly producing the pneumococcal surface protein A (PspA) as a mucosal vaccine in conferring protection against pneumococcal disease. Mice were intranasally (inl) immunized with the lactococcal vaccine. Control groups were also immunized with similar amounts of recombinant PspA administered inl or subcutaneously with alum. PspA-specific antibodies in serum samples and lung lavage fluids were measured before challenge in intraperitoneal sepsis and inl respiratory-infection models of pneumococcal disease. The lactococcal vaccine afforded better protection against respiratory challenge with pneumococcus than did vaccination with purified antigen given inl or by injection with alum. This finding was associated with a shift toward a Th1-mediated immune response characterized by reduced antibody titers to the PspA antigen. In the sepsis model, the lactococcal vaccine afforded resistance to disease on a par with that obtained with the injected vaccine, demonstrating its efficacy against different forms of pneumococcal disease. Given the safety profile of L. lactis, there is considerable potential to develop a pneumococcal vaccine for use in humans and to broaden this approach to combat other major pathogens.

Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method

The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.

Protective effect of curcumin on endotoxin-induced acute lung injury in rats

To investigate the protective effect of curcumin on endotoxin-induced acute lung injury in rats, and explore the underlying mechanisms, 24 male Wistar rats were randomly divided into 4 experimental groups: sham-vehicle (S), sham-curcumin (C), lipopolysaccharide (LPS)-vehicle (L), and curcumin-lipopolysaccharide (C-L) groups. The wet/dry (W/D) weight ratio of the lung and bronchoalveolar lavage (BAL) fluid protein content were used as measures of lung injury. Neutrophil recruitment and activation were evaluated by BAL fluid cellularity and myeloperoxidase (MPO) activity in cell-free BAL and lung tissue. The levels of cytokine-induced neutrophil chemoattractant-I (CINC-1) in lung tissues were measured by ELISA. The histopathological changes of lung tissues were observed by using the HE staining. Our results showed that lung injury parameters, including the wet/dry weight ratio and protein content in BALF, were significantly higher in the L group than in the S group (P<0.01). In the L group, higher numbers of neutrophils and greater MPO activity in cell-free BAL and lung homogenates were observed when compared with the S group (P<0.01). There was a marked increase in CINC-1 levels in lung tissues in response to LPS challenge (P<0.01, L group vs S group). Curcumin pretreatment significantly attenuated LPS-induced changes in these indices. LPS caused extensive morphological lung damage, which was also lessened after curcumin pretreatment. All the above-mentioned parameters in the C group were not significantly different from those of the S group. It is concluded that curcumin pretreatment attenuates LPS-induced lung injury in rats. This beneficial effect of curcumin may involves, in part, inhibition of neutrophilic recruitment and activity, possibly through inhibition of lung CINC-1 expression.