Inhibition of AMP-Activated Protein Kinase Accentuates Lipopolysaccharide-Induced Lung Endothelial Barrier Dysfunction and Lung Injury in Vivo

The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction and lung injury in vivo. Both cultured human pulmonary artery endothelial cells (HPAECs) and experimental animals [AMPK subunit α–deficient mice and wild-type (WT) control mice (C57BL/6J)] were used. In cultured HPAECs, LPS increased endothelial permeability in parallel with a decrease in AMPK activity. Consistent with this observation, AMPK activation with the potent AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) attenuated LPS-induced endothelial hyperpermeability in vitro. Intratracheal administration of LPS (1 mg/kg) in WT mice reduced AMPK phosphorylation at Thr172 in lung tissue extracts, increased protein content and cell count in bronchial alveolar lavage fluid, and increased Evans Blue dye infiltration into the lung. These same attributes were similarly enhanced in AMPKα-knockout mice, compared with WT mice. Pretreatment with AICAR reduced these lung injury indicators in LPS-treated WT mice. AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeability by activating the Rac/Cdc42/PAK pathway, with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr658. We conclude that AMPK activity supports normal endothelial barrier function and that LPS exposure inhibits AMPK, thereby contributing to endothelial barrier dysfunction and lung injury.Vascular endothelial permeability plays a pivotal role in regulating many physiological and pathological processes, including angiogenesis, immunity, and inflammation.¹ In lungs, endothelial cells form a semipermeable barrier between the vessel lumen and underlying alveoli, thereby mediating the transmigration of blood cells and maintaining fluid homeostasis. The integrity of the endothelial cell (EC) monolayer therefore directly determines lung vascular permeability. For example, EC barrier dysfunction can lead to an increase in permeation of fluid and macromolecules into the interstitium and alveolar space, resulting in pulmonary edema, a major characteristic of acute lung injury.RhoA, Rac1, and Cdc42 are key members of the Rho GTPase family and are activated on binding GTP at the membrane.² These proteins are intimately involved in regulating cell adhesion and cytoskeletal dynamics, both of which play an important role in endothelial barrier function.^3–5 For example, Rac1 and Cdc42 are important in maintaining, stabilizing, and restoring the endothelial barrier.³ More specifically, Baumer et al⁶ demonstrated that Rac1 is involved in mitigating endothelial hyperpermeability by a subset of agonists, including thrombin and lipopolysaccharide (LPS). In addition, activation of the Rho GTPases Cdc42 and Rac1 restores endothelium integrity after lung injury.⁷ Although the contributions of Rho GTPases in maintaining endothelial barrier function are well established, how Rho GTPase is regulated in endothelial cells is largely unknown.AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase; a catalytic α subunit and regulatory β and γ subunits are important in maintaining the stability of the complex. AMPK belongs to a family of energy-sensing enzymes that function as fuel gauges, monitoring changes in the energy status of the cell.⁸ AMPK is activated in response to a variety of stressors that increase the intracellular ratio of AMP to ATP. On activation, AMPK phosphorylates a number of downstream targets, thereby affecting glucose metabolism, fatty acid oxidation, hepatic lipogenesis, and cholesterol synthesis.⁹ In addition to its regulatory role in metabolism, recent studies have demonstrated a role for AMPK in maintaining normal endothelial function.¹⁰ For example, AMPK subunit α2 exerts protective effects against atherosclerosis by inhibiting the endoplasmic reticulum stress response in ECs.¹¹ Thus, agents that enhance EC barrier function are of potential therapeutic value in a variety of pathological settings, including inflammatory disease, atherosclerosis, and tumor angiogenesis. The effects of AMPK on endothelial barrier function and vascular permeability have not been investigated previously. Thus, the aim of the present study was to investigate whether AMPK protects lung endothelial barrier function and mitigates acute lung injury in response to LPS.     

Lagooning of wastewaters favors dissemination of clinically relevant Pseudomonas aeruginosa

The significance of wastewater treatment lagoons (WWTLs) as point sources of clinically relevant Pseudomonas aeruginosa that can disseminate through rural and peri-urban catchments was investigated. A panel of P. aeruginosa strains collected over three years from WWTLs and community-acquired infections was compared by pulsed field gel electrophoresis (PFGE) DNA fingerprinting and multilocus sequence typing (MLST). Forty-four distantly related PFGE profiles and four clonal complexes were found among the WWTL strains analyzed. Some genotypes were repeatedly detected from different parts of WWTLs, including the influent, suggesting an ability to migrate and persist over time. MLST showed all investigated lineages to match sequence types described in other countries and strains from major clinical clones such as PA14 of ST253 and “C” of ST17 were observed. Some of these genotypes matched isolates from community-acquired infections recorded in the WWTL geographic area. Most WWTL strains harbored the main P. aeruginosa virulence genes; 13% harbored exoU-encoded cytoxins, but on at least six different genomic islands, with some of these showing signs of genomic instability. P. aeruginosa appeared to be highly successful opportunistic colonizers of WWTLs. Lagooning of wastewaters was found to favor dissemination of clinically relevant P. aeruginosa among peri-urban watersheds.     

Failure of ticks to transmit Scopulariopsis brevicaulis (Deuteromycota), a common filamentous fungal commensal of ticks

The capacity of ticks to transmit a fungus was examined by analyzing tick saliva, host tissue from feeding sites, and host blood for presence of Scopulariopsis brevicaulis (Sacc.) Bainier, an internal mycosymbiont of the American dog tick, Dermacentor variabilis (Say), and lone star tick, Amnblyomma americanum (L.). Although >85% of ticks were infected with S. brevicaulis, conidia presence was low (0-5% of samples) in microscopic observations and mycological culturing of saliva expressed from larvae, nymphs, and adults. Additionally, the recovery of S. brevicaulis from blood and tissue feeding sites from a rabbit where S. brevicaulis-positive adult ticks had attached and fed was not increased compared with control tissue where no feeding occurred, indicating that transmission does not occur by the blood-feeding route. Tick mouthparts were found to contain S. brevicaulis in addition to Penicillium glabrum (Wehmer) Westling, but these agents were sparse in isolations from the feeding sites, which makes it unlikely that ticks act frequently as a mechanical fungal vector.     

Nitration of tau protein is linked to neurodegeneration in tauopathies

Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimer's disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and alpha-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Pick's disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that tau-positive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions.     

Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4+CD25highFOXP3+ regulatory T cells

Adult bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells that are the subject of intense investigation in regenerative medicine. In addition, MSCs possess immunomodulatory properties with therapeutic potential to prevent graft-versus-host disease (GvHD) in allogeneic hematopoietic cell transplantation. Indeed, MSCs can inhibit natural killer (NK) function, modulate dendritic cell maturation, and suppress allogeneic T-cell response. Here, we report that the nonclassic human leukocyte antigen (HLA) class I molecule HLA-G is responsible for the immunomodulatory properties of MSCs. Our data show that MSCs secrete the soluble isoform HLA-G5 and that such secretion is interleukin-10-dependent. Moreover, cell contact between MSCs and allostimulated T cells is required to obtain a full HLA-G5 secretion and, as consequence, a full immunomodulation from MSCs. Blocking experiments using neutralizing anti-HLA-G antibody demonstrate that HLA-G5 contributes first to the suppression of allogeneic T-cell proliferation and then to the expansion of CD4(+)CD25(high)FOXP3(+) regulatory T cells. Furthermore, we demonstrate that in addition to their action on the adaptive immune system, MSCs, through HLA-G5, affect innate immunity by inhibiting both NK cell-mediated cytolysis and interferon-gamma secretion. Our results provide evidence that HLA-G5 secreted by MSCs is critical to the suppressive functions of MSCs and should contribute to improving clinical therapeutic trials that use MSCs to prevent GvHD.     

Synthesis and characterization of a fluvastatin-releasing hydrogel delivery system to modulate hMSC differentiation and function for bone regeneration

Increases in bone formation have been demonstrated in mice and rats treated with statins, a group of molecules that increase the production of bone morphogenetic proteins-2 (BMP2) by stimulating its promoter. However, clinical use of statins (e.g., fluvastatin) is limited by the lack of a suitable delivery system to localize and sustain release. To harness the therapeutic effect of statins in orthopedic applications, a fluvastatin-releasing macromer was synthesized. When copolymerized with a dimethacrylated poly(ethylene glycol) solution, this fluvastatin-containing molecule was covalently incorporated into hydrogel networks, and hydrolysis of lactic acid ester bonds resulted in the release of the pendantly tethered fluvastatin from the hydrogel into the surrounding solution. The rate of fluvastatin release was controlled by the length of lactic acid spacer (2–6 repeats), and the dose was controlled by the initial comonomer composition (5–500 μg fluvastatin/gel). Released fluvastatin increased human mesenchymal stem cell (hMSC) gene expression of CBFA1, ALP, and COL I by 34-fold, 2.6-fold, and 1.8-fold, respectively, after 14 days of in vitro culture. In addition, treating hMSCs with the released fluvastatin resulted in an average of 2.0- and 1.5-fold greater BMP2 production whereas mineralization increased an average of 3.0-fold and 2.5-fold for 0.01 and 0.1 μM fluvastatin, respectively, over the 2 week culture period. Therefore, fluvastatin-releasing hydrogels may be useful in bone tissue engineering applications, not only for triggering osteogenic differentiation of hMSCs, but also by modulating their function.     

Convergence of heat shock protein 90 with ubiquitin in filamentous alpha-synuclein inclusions of alpha-synucleinopathies

Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are alpha-synucleinopathies characterized by filamentous alpha-synuclein (alpha-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with alpha-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with alpha-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble alpha-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like alpha-syn pathologies. Cell culture studies also revealed that alpha-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of alpha-syn inclusions in PD and related alpha-synucleinopathies.     

Risk reduction counseling is associated with decreased HIV transmission-associated behaviors in high-risk Indian heterosexuals

OBJECTIVE: To estimate the incidence of HIV and study the impact of risk-reduction counseling (RRC) in a cohort of people with high-risk behavior for HIV transmission in Chennai, India. DESIGN: Prospective cohort follow-up of 500 HIV-negative people (250 men and 250 women) at increased risk for HIV acquisition in Chennai, India for a maximum of 1 year was conducted. They received RRC at 0, 6, and 12 months. Generalized estimating equation methodology was used to determine the statistical significance of differences reported in behavior between baseline, 6 months, and 12 months. RESULTS: The overall HIV incidence in this cohort was 0.44 per 100 person-years (95% confidence interval: 0.05-1.60). In the course of the study, both male and female participants reported statistically significant decreases in the number of different sexual partners, the number of new partners, and the proportion of sexual encounters with nonprimary partners. Participants who had more than 3 different partners at baseline and/or exchanged money for sex in the 6 months before enrollment demonstrated the greatest reductions in the number of different sexual partners. CONCLUSIONS: Individualized sexual RRC seems to be a useful intervention to reduce risk-taking behavior among at-risk heterosexuals in India.