Physical Activity Differentially Affects the Cecal Microbiota of Ovariectomized Female Rats Selectively Bred for High and Low Aerobic Capacity

The gut microbiota is considered a relevant factor in obesity and associated metabolic diseases, for which postmenopausal women are particularly at risk. Increasing physical activity has been recognized as an efficacious approach to prevent or treat obesity, yet the impact of physical activity on the microbiota remains under-investigated. We examined the impacts of voluntary exercise on host metabolism and gut microbiota in ovariectomized (OVX) high capacity (HCR) and low capacity running (LCR) rats. HCR and LCR rats (age = 27wk) were OVX and fed a high-fat diet (45% kcal fat) ad libitum and housed in cages equipped with (exercise, EX) or without (sedentary, SED) running wheels for 11wk (n = 7-8/group). We hypothesized that increased physical activity would hinder weight gain, increase metabolic health and shift the microbiota of LCR rats, resulting in populations more similar to that of HCR rats. Animals were compared for characteristic metabolic parameters including body composition, lipid profile and energy expenditure; whereas cecal digesta were collected for DNA extraction. 16S rRNA gene-based amplicon Illumina MiSeq sequencing was performed, followed by analysis using QIIME 1.8.0 to assess cecal microbiota. Voluntary exercise decreased body and fat mass, and normalized fasting NEFA concentrations of LCR rats, despite only running one-third the distance of HCR rats. Exercise, however, increased food intake, weight gain and fat mass of HCR rats. Exercise clustered the gut microbial community of LCR rats, which separated them from the other groups. Assessments of specific taxa revealed significant (p<0.05) line by exercise interactions including shifts in the abundances of Firmicutes, Proteobacteria, and Cyanobacteria. Relative abundance of Christensenellaceae family was higher (p = 0.026) in HCR than LCR rats, and positively correlated (p<0.05) with food intake, body weight and running distance. These findings demonstrate that exercise differentially impacts host metabolism and gut microbial communities of female HCR and LCR rats without ovarian function.     

Neonatal Streptococcus pneumoniae Infection May Aggravate Adulthood Allergic Airways Disease in Association with IL-17A

Epidemiologic studies have demonstrated that some bacteria colonization or infections in early-life increased the risk for subsequent asthma development. However, little is known about the mechanisms by which early-life bacterial infection increases this risk. The aim of this study was to investigate the effect of neonatal Streptococcus pneumoniae infection on the development of adulthood asthma, and to explore the possible mechanism. A non-lethal S. pneumoniae lung infection was established by intranasal inoculation of neonatal (1-week-old) female mice with D39. Mice were sensitized and challenged with ovalbumin in adulthood to induce allergic airways disease (AAD). Twenty-four hours later, the lungs and bronchoalveolar lavage fluid (BALF) were collected to assess AAD. Neonatal S. pneumoniae infection exacerbated adulthood hallmark features of AAD, with enhanced airway hyperresponsiveness and increased neutrophil recruitment into the airways, increased Th17 cells and interleukin (IL)-17A productions. Depletion of IL-17A by i.p. injection of a neutralizing monoclonal antibody reduced neutrophil recruitment into the airways, alleviated airway inflammation and decreased airway hyperresponsiveness. Furthermore, IL-17A depletion partially restored levels of inteferon-γ, but had no effect on the release of IL-5 or IL-13. Our data suggest that neonatal S. pneumoniae infection may promote the development of adulthood asthma in association with increased IL-17A production.     

Dysfunction of Inflammation-Resolving Pathways Is Associated with Exaggerated Postoperative Cognitive Decline in a Rat Model of the Metabolic Syndrome

The cholinergic antiinflammatory pathway (CAP), which terminates in the spleen, attenuates postoperative cognitive decline (PCD) in rodents. Surgical patients with metabolic syndrome exhibit exaggerated and persistent PCD that is reproduced in postoperative rats selectively bred for easy fatigability and that contain all features of metabolic syndrome (low-capacity runners [LCRs]). We compared the CAP and lipoxin A₄ (LXA₄), another inflammation-resolving pathway in LCR, with its counterpart high-capacity runner (HCR) rats. Isoflurane-anesthetized LCR and HCR rats either underwent aseptic trauma involving tibial fracture (surgery) or not (sham). At postoperative d 3 (POD3), compared with HCR, LCR rats exhibited significantly exaggerated PCD (trace fear conditioning freezing time 43% versus 57%). Separate cohorts were killed at POD3 to collect plasma for LXA4 and to isolate splenic mononuclear cells (MNCs) to analyze CAP signaling, regulatory T cells (Tregs) and M2 macrophages (M2 Mφ). Under lipopolysaccharide (LPS) stimulation, tumor necrosis factor (TNF)-α produced by splenic MNCs was 117% higher in LCR sham and 52% higher in LCR surgery compared with HCR sham and surgery rats; LPS-stimulated TNF-α production could not be inhibited by an α7 nicotinic acetylcholine receptor agonist, whereas inhibition by the β₂ adrenergic agonist, salmeterol, was significantly less (−35%) than that obtained in HCR rats. Compared to HCR, sham and surgery LCR rats had reduced β₂ adrenergic receptor–expressing T lymphocytes (59%, 44%), Tregs (47%, 54%) and M2 Mφ (45%, 39%); surgical LCR rats’ hippocampal M2 Mφ was 66% reduced, and plasma LXA4 was decreased by 120%. Rats with the metabolic syndrome have ineffective inflammation-resolving mechanisms that represent plausible reasons for the exaggerated and persistent PCD.     

A Multiple Antigenic Peptide Mimicking Peptidoglycan Induced T Cell Responses to Protect Mice from Systemic Infection with Staphylococcus aureus

Due to the enormous capacity of Staphylococcus aureus to acquire antibiotic resistance, it becomes imperative to develop vaccines for decreasing the risk of its life-threatening infections. Peptidoglycan (PGN) is a conserved and major component of S. aureus cell wall. However, it has not been used as a vaccine candidate since it is a thymus-independent antigen. In this study, we synthesized a multiple antigenic peptide, named MAP27, which comprised four copies of a peptide that mimics the epitope of PGN. After immunization with MAP27 five times and boosting with heat-inactivated bacterium one time, anti-MAP27 serum bound directly to S. aureus or PGN. Immunization with MAP27 decreased the bacterial burden in organs of BALB/c mice and significantly prolonged their survival time after S. aureus lethal-challenge. The percentage of IFN-γ⁺CD3⁺ T cells and IL-17⁺CD4⁺ T cells in spleen, as well as the levels of IFN-γ, IL-17A/F and CCL3 in spleen and lung, significantly increased in the MAP27-immunized mice after infection. Moreover, in vitro incubation of heat-inactivated S. aureus with splenocytes isolated from MAP27-immunized mice stimulated the production of IFN-γ and IL-17A/F. Our findings demonstrated that MAP27, as a thymus-dependent antigen, is efficient at eliciting T cell-mediated responses to protect mice from S. aureus infection. This study sheds light on a possible strategy to design vaccines against S. aureus.     

Brain Activation Patterns at Exhaustion in Rats That Differ in Inherent Exercise Capacity

In order to further understand the genetic basis for variation in inherent (untrained) exercise capacity, we examined the brains of 32 male rats selectively bred for high or low running capacity (HCR and LCR, respectively). The aim was to characterize the activation patterns of brain regions potentially involved in differences in inherent running capacity between HCR and LCR. Using quantitative in situ hybridization techniques, we measured messenger ribonuclease (mRNA) levels of c-Fos, a marker of neuronal activation, in the brains of HCR and LCR rats after a single bout of acute treadmill running (7.5–15 minutes, 15° slope, 10 m/min) or after treadmill running to exhaustion (15–51 minutes, 15° slope, initial velocity 10 m/min). During verification of trait differences, HCR rats ran six times farther and three times longer prior to exhaustion than LCR rats. Running to exhaustion significantly increased c-Fos mRNA activation of several brain areas in HCR, but LCR failed to show significant elevations of c-Fos mRNA at exhaustion in the majority of areas examined compared to acutely run controls. Results from these studies suggest that there are differences in central c-Fos mRNA expression, and potential brain activation patterns, between HCR and LCR rats during treadmill running to exhaustion and these differences could be involved in the variation in inherent running capacity between lines.     

Staphylococcus aureus-induced immunosuppression mediated by IL-10 and IL-27 facilitates nasal colonisation

Staphylococcus aureus persistently colonises the anterior nares of a significant proportion of the healthy population, however the local immune response elicited during S. aureus nasal colonisation remains ill-defined. Local activation of IL-17/IL-22 producing T cells are critical for controlling bacterial clearance from the nasal cavity. However, recurrent and long-term colonisation is commonplace indicating efficient clearance does not invariably occur. Here we identify a central role for the regulatory cytokine IL-10 in facilitating bacterial persistence during S. aureus nasal colonisation in a murine model. IL-10 is produced rapidly within the nasal cavity following S. aureus colonisation, primarily by myeloid cells. Colonised IL-10^-/- mice demonstrate enhanced IL-17+ and IL-22+ T cell responses and more rapidly clear bacteria from the nasal tissues as compared with wild-type mice. S. aureus also induces the regulatory cytokine IL-27 within the nasal tissue, which acts upstream of IL-10 promoting its production. IL-27 blockade reduces IL-10 production within the nasal cavity and improves bacterial clearance. TLR2 signalling was confirmed to be central to controlling the IL-10 response. Our findings conclude that during nasal colonisation S. aureus creates an immunosuppressive microenvironment through the local induction of IL-27 and IL-10, to dampen protective T cell responses and facilitate its persistence.     

Role of Staphylococcus aureus Virulence Factors in Inducing Inflammation and Vascular Permeability in a Mouse Model of Bacterial Endophthalmitis

Staphylococcus (S.) aureus is a common causative agent of bacterial endophthalmitis, a vision threatening complication of eye surgeries. The relative contribution of S. aureus virulence factors in the pathogenesis of endophthalmitis remains unclear. Here, we comprehensively analyzed the development of intraocular inflammation, vascular permeability, and the loss of retinal function in C57BL/6 mouse eyes, challenged with live S. aureus, heat-killed S. aureus (HKSA), peptidoglycan (PGN), lipoteichoic acid (LTA), staphylococcal protein A (SPA), α-toxin, and Toxic-shock syndrome toxin 1 (TSST1). Our data showed a dose-dependent (range 0.01 μg/eye to 1.0 μg/eye) increase in the levels of inflammatory mediators by all virulence factors. The cell wall components, particularly PGN and LTA, seem to induce higher levels of TNF-α, IL-6, KC, and MIP2, whereas the toxins induced IL-1β. Similarly, among the virulence factors, PGN induced higher PMN infiltration. The vascular permeability assay revealed significant leakage in eyes challenged with live SA (12-fold) and HKSA (7.3-fold), in comparison to other virulence factors (~2-fold) and controls. These changes coincided with retinal tissue damage, as evidenced by histological analysis. The electroretinogram (ERG) analysis revealed a significant decline in retinal function in eyes inoculated with live SA, followed by HKSA, SPA, and α-toxin. Together, these findings demonstrate the differential innate responses of the retina to S. aureus virulence factors, which contribute to intraocular inflammation and retinal function loss in endophthalmitis.     

Artificial Selection for Whole Animal Low Intrinsic Aerobic Capacity Co-Segregates with Hypoxia-Induced Cardiac Pump Failure

Oxygen metabolism is a strong predictor of the general health and fitness of an organism. In this study, we hypothesized that a divergence in intrinsic aerobic fitness would co-segregate with susceptibility for cardiovascular dysfunction. To test this hypothesis, cardiac function was assessed in rats specifically selected over nineteen generations for their low (LCR) and high (HCR) intrinsic aerobic running capacity. As an integrative marker of native aerobic capacity, run time to exhaustion between LCR and HCR rats had markedly diverged by 436% at generation nineteen of artificial selection. In vivo assessment of baseline cardiac function by echocardiography and catheter-based conductance micromanometry showed no marked difference in cardiac performance. However, when challenged by exposure to acute hypoxia, cardiac pump failure occurred significantly earlier in LCR rats compared to HCR animals. Acute cardiac decompensation in LCR rats was exclusively due to the development of intractable irregular ventricular contractions. Analysis of isolated cardiac myocytes showed significantly slower sarcomeric relaxation and delayed kinetics of calcium cycling in LCR myocytes compared to HCR myocytes. This study also revealed that artificial selection for low native aerobic capacity is a novel pathologic stimulus that results in myosin heavy chain isoform switching in the heart as shown by increased levels of β-MHC in LCR rats. Together, these results provide evidence that alterations in sub-cellular calcium handling and MHC isoform composition are associated with susceptibility to compensatory cardiac remodeling and hypoxia induced pump failure in animals with low intrinsic aerobic capacity.     

IL-4 increases surfactant and regulates metabolism in vivo

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.     

Atrial Myocyte Function and Ca2+ Handling Is Associated with Inborn Aerobic Capacity

Background

Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO_{2 max}) had impaired atrial myocyte contractile function when compared to rats with high inborn VO_{2 max}.

Methods and Results

Atrial myocyte function was depressed in Low Capacity Runners (LCR) relative to High Capacity Runners (HCR) which was associated with impaired Ca²⁺ handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca²⁺ amplitude and diastolic Ca²⁺ level were observed at 5 Hz stimulation where Ca²⁺ amplitude was 70% lower and diastolic Ca²⁺ level was 11% higher in LCR rats. Prolonged time to 50% Ca²⁺ decay was associated with reduced sarcoplasmic reticulum (SR) Ca²⁺ ATPase function in LCR (39%). Na⁺/Ca²⁺ exchanger activity was comparable between the groups. Diastolic SR Ca²⁺ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca²⁺-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca²⁺ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca²⁺-signal onset.

Conclusion

This data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats.     

Lower oxidative DNA damage despite greater ROS production in muscles from rats selectively bred for high running capacity

Artificial selection in rat has yielded high-capacity runners (HCR) and low-capacity runners (LCR) that differ in intrinsic (untrained) aerobic exercise ability and metabolic disease risk. To gain insight into how oxygen metabolism may have been affected by selection, we compared mitochondrial function, oxidative DNA damage (8-dihydroxy-guanosine; 8dOHG), and antioxidant enzyme activities in soleus muscle (Sol) and gastrocnemius muscle (Gas) of adult and aged LCR vs. HCR rats. In Sol of adult HCR rats, maximal ADP-stimulated respiration was 37% greater, whereas in Gas of adult HCR rats, there was a 23% greater complex IV-driven respiratory capacity and 54% greater leak as a fraction of electron transport capacity (suggesting looser mitochondrial coupling) vs. LCR rats. H(2)O(2) emission per gram of muscle was 24-26% greater for both muscles in adult HCR rats vs. LCR, although H(2)O(2) emission in Gas was 17% lower in HCR, after normalizing for citrate synthase activity (marker of mitochondrial content). Despite greater H(2)O(2) emission, 8dOHG levels were 62-78% lower in HCR rats due to 62-96% higher superoxide dismutase activity in both muscles and 47% higher catalase activity in Sol muscle in adult HCR rats, with no evidence for higher 8 oxoguanine glycosylase (OGG1; DNA repair enzyme) protein expression. We conclude that genetic segregation for high running capacity has generated a molecular network of cellular adaptations, facilitating a superior response to oxidative stress.     

Transcriptional Events during the Recovery from MRSA Lung Infection: A Mouse Pneumonia Model

Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging threat to human health throughout the world. Rodent MRSA pneumonia models mainly focus on the early innate immune responses to MRSA lung infection. However, the molecular pattern and mechanisms of recovery from MRSA lung infection are largely unknown. In this study, a sublethal mouse MRSA pneumonia model was employed to investigate late events during the recovery from MRSA lung infection. We compared lung bacterial clearance, bronchoalveolar lavage fluid (BALF) characterization, lung histology, lung cell proliferation, lung vascular permeability and lung gene expression profiling between days 1 and 3 post MRSA lung infection. Compared to day 1 post infection, bacterial colony counts, BALF total cell number and BALF protein concentration significantly decreased at day 3 post infection. Lung cDNA microarray analysis identified 47 significantly up-regulated and 35 down-regulated genes (p<0.01, 1.5 fold change [up and down]). The pattern of gene expression suggests that lung recovery is characterized by enhanced cell division, vascularization, wound healing and adjustment of host adaptive immune responses. Proliferation assay by PCNA staining further confirmed that at day 3 lungs have significantly higher cell proliferation than at day 1. Furthermore, at day 3 lungs displayed significantly lower levels of vascular permeability to albumin, compared to day 1. Collectively, this data helps us elucidate the molecular mechanisms of the recovery after MRSA lung infection.     

Toxin-Induced Necroptosis Is a Major Mechanism of Staphylococcus aureus Lung Damage

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1β production, suggesting a link to the inflammasome. Rip3 ^-/- mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1β in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.     

Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

Background

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response.

Methods

Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation.

Results

MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury.

Conclusions

Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.     

Vinculin and Rab5 Complex Is Requited for Uptake of Staphyrococcus aureus and Interleukin-6 Expression

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.     

Overweight female rats selectively breed for low aerobic capacity exhibit increased myocardial fibrosis and diastolic dysfunction

The statistical association between endurance exercise capacity and cardiovascular disease suggests that impaired aerobic metabolism underlies the cardiovascular disease risk in men and women. To explore this connection, we applied divergent artificial selection in rats to develop low-capacity runner (LCR) and high-capacity runner (HCR) rats and found that disease risks segregated strongly with low running capacity. Here, we tested if inborn low aerobic capacity promotes differential sex-related cardiovascular effects. Compared with HCR males (HCR-M), LCR males (LCR-M) were overweight by 34% and had heavier retroperitoneal, epididymal, and omental fat pads; LCR females (LCR-F) were 20% heavier than HCR females (HCR-F), and their retroperitoneal, but not perireproductive or omental, fat pads were heavier as well. Unlike HCR-M, blood pressure was elevated in LCR-M, and this was accompanied by left ventricular (LV) hypertrophy. Like HCR-F, LCR-F exhibited normal blood pressure and LV weight as well as increased spontaneous cage activity compared with males. Despite normal blood pressures, LCR-F exhibited increased myocardial interstitial fibrosis and diastolic dysfunction, as indicated by increased LV stiffness, a decrease in the initial filling rate, and an increase in diastolic relaxation time. Although females exhibited increased arterial stiffness, ejection fraction was normal. Increased interstitial fibrosis and diastolic dysfunction in LCR-F was accompanied by the lowest protein levels of phosphorylated AMP-actived protein kinase [phospho-AMPK (Thr(172))] and silent information regulator 1. Thus, the combination of risk factors, including female sex, intrinsic low aerobic capacity, and overweightness, promote myocardial stiffness/fibrosis sufficient to induce diastolic dysfunction in the absence of hypertension and LV hypertrophy.     

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff,Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1intratracheally. J. Appl. Physiol.82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lunglavages from patients with the acute respiratory distress syndrome, andadministering IL-1 intratracheally causes neutrophil accumulation and aneutrophil-dependent oxidative leak in lungs of rats. In the presentstudy, we found that rats pretreated intraperitoneally with lisofylline[(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species,did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1.However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbersof lung lavage neutrophils as rats given IL-1 intratracheally. Lungs ofrats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lungleak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

     

Serine protease inhibitor attenuates ovalbumin induced inflammation in mouse model of allergic airway disease

Background

Serine proteases promote inflammation and tissue remodeling by activating proteinase-activated receptors, urokinase, metalloproteinases and angiotensin. In the present study, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) a serine protease inhibitor was evaluated for prophylactic and therapeutic treatment in mouse model of airway allergy.

Methods

BALB/c mice were sensitized by i.p route and challenged with ovalbumin. They were treated i.n. with 2, 10 and 50 µg of AEBSF, one hour before or after challenge and euthanized to collect BALF (bronchoalveolar lavage fluid), blood and lungs. Proteolytic activity, total cell/eosinophil/neutrophil count eosinophil peroxidase activity (EPO), IL-4, IL-5, IL-10, IL-13, cysteinyl leukotrienes and 8-isoprostane were determined in BALF and immunoglobulins were measured in serum. H&E and PAS stained lung sections were examined for cellular infiltration and airway inflammation.

Results

Mice exposed to ovalbumin and treated with PBS showed increased cellular infiltration in lungs and higher serum IgE, IgG1 and IgG2a levels as compared to sham mice. Treatment with AEBSF reduced total cells/eosinophil/neutrophil infiltration. Both prophylactic and therapeutic AEBSF treatment of 10 or 50 µg reduced serum IgE and IgG1 significantly (p<0.05) than control. AEBSF treatment reduced the proteolytic activity in BALF. IL-4 IL-5 and IL-13 levels decreased significantly (p<0.05) after AEBSF treatment while IL-10 levels increased significantly (p<0.05) in BALF. Airway inflammation and goblet cell hyperplasia reduced as demonstrated by lung histopathology, EPO activity and cysteinyl leukotrienes in BALF after treatment. AEBSF treatment also suppressed oxidative stress in terms of 8-isoprostane in BALF. Among the treatment doses, 10 or 50 µg of AEBSF were most effective in reducing the inflammatory parameters.

Conclusions

Prophylactic and therapeutic treatment with serine protease inhibitor attenuates the airway inflammation in mouse model of airway allergy and have potential for adjunct therapy.