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Objective: Prevention of diabetic retinopathy would benefit from availability of drugs that preempt the effects of hyperglycemia on retinal vessels. We aimed to identify candidate drug targets by investigating the molecular effects of drugs that prevent retinal capillary demise in the diabetic rat.Research Design and Methods: We examined the gene expression profile of retinal vessels isolated from rats with 6 months of streptozotocin-induced diabetes and compared it with that of control rats. We then tested whether the aldose reductase inhibitor sorbinil and aspirin, which have different mechanisms of action, prevented common molecular abnormalities induced by diabetes. The Affymetrix GeneChip Rat Genome 230 2.0 array was complemented by real-time RT-PCR, immunoblotting, and immunohistochemistry.Results: The retinal vessels of diabetic rats showed differential expression of 20 genes of the transforming growth factor (TGF)-beta pathway, in addition to genes involved in oxidative stress, inflammation, vascular remodeling, and apoptosis. The complete loop of TGF-beta signaling, including Smad2 phosphorylation, was enhanced in the retinal vessels, but not in the neural retina. Sorbinil normalized the expression of 71% of the genes related to oxidative stress and 62% of those related to inflammation. Aspirin had minimal or no effect on these two categories. The two drugs were instead concordant in reducing the upregulation of genes of the TGF-beta pathway (55% for sorbinil and 40% for aspirin) and apoptosis (74 and 42%, respectively).Conclusions: Oxidative and inflammatory stress is the distinct signature that the polyol pathway leaves on retinal vessels. TGF-beta and apoptosis are, however, the ultimate targets to prevent the capillary demise in diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Microbes have developed their own specific strategies to cope with reactive oxygen species (ROS). Catalase, a heme-containing tetramer expressed in a broad range of aerobic fungi, shows remarkable efficiency in degrading hydrogen peroxide (H 2 O 2 ) for fungal survival and host invasion. Here, it is demonstrated that catalase inactivation by blue light renders fungal cells highly susceptible to ROS attack. To confirm catalase as a major molecular target of blue light, wild type Candida albicans are systematically compared with a catalase-deficient mutant strain regarding their susceptibility to ROS through 410 nm treatment. Upon testing a wide range of fungal species, it is found that intracellular catalase can be effectively and universally inactivated by 410 nm blue light. It is also found that photoinactivation of catalase in combination with ROS-generating agents is highly effective in total eradication of various fungal species, including multiple Candida auris strains, the causative agent of the global fungal epidemic. In addition, photoinactivation of catalase is shown to facilitate macrophage killing of intracellular Candida albicans. The antifungal efficacy of catalase photoinactivation is further validated using a C. albicans-induced mouse model of skin abrasion. Taken together, the findings offer a novel catalase-photoinactivation approach to address multidrug-resistant Candida infections., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background: Solid organ transplant (SOT) and stem cell transplant (SCT) recipients are at increased risk of invasive fungal disease despite normal neutrophil counts. Here, we measure neutrophil anti-Candida activity., Methods: Twenty-one SOT and 19 SCT recipients were enrolled 2-4 months posttransplant and compared to 23 healthy control patients (HC). Neutrophils were coincubated with Candida albicans, and percentage killing and swarming responses were measured., Results: Neutrophils from transplant patients had decreased fungicidal capacity compared to HC (42%, 43%, and 72% for SCT, SOT, and HC, respectively; SCT vs HC: P < .0001; SOT vs HC: P < .0001; SOT vs SCT: P = .8), including diminished ability to control hyphal growth (HC vs SOT: 0.1455 vs 0.3894, P ≤ .001; HC vs SCT: 0.1455 vs 0.6295, P ≤ .0001, respectively). Serum from SCT, but not SOT, recipients, inhibited the ability of HC neutrophils to control C. albicans (37%, 45%, and 55% for SCT, SOT, and HC, respectively). Neutrophils' control of hyphal growth was partially restored with granulocyte colony-stimulating factor or granulocyte macrophage colony-stimulating factor., Conclusions: Despite normal circulating numbers, our data suggest that neutrophils from SOT and SCT recipients mount dysfunctional responses against C. albicans. Intrinsic neutrophil changes and extrinsic serum factors may be responsible for the dysfunction, which is partially reversed with cytokine augmentation., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Ergosterol-targeting amphotericin B (AmB) is the first line of defense for life-threatening fungal infections. Two models have been proposed to illustrate AmB assembly in the cell membrane; one is the classical ion channel model in which AmB vertically forms transmembrane tunnel and the other is a recently proposed sterol sponge model where AmB is laterally adsorbed onto the membrane surface. To address this controversy, we use polarization-sensitive stimulated Raman scattering from fingerprint C═C stretching vibration to visualize AmB, ergosterol, and lipid in single fungal cells. Intracellular lipid droplet accumulation in response to AmB treatment is found. AmB is located in membrane and intracellular droplets. In the 16 strains studied, AmB residing inside cell membrane was highly ordered, and its orientation is primarily parallel to phospholipid acyl chains, supporting the ion channel model. Label-free imaging of AmB and chemical contents offers an analytical platform for developing low-toxicity, resistance-refractory antifungal agents., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Neutrophils are the most abundant white blood cell in the body and are key participants in the defense against fungal infections. Fungal infections occur often in patients with cirrhosis and are associated with increased 30-day and 90-day mortality. Previous studies have shown that specific neutrophil functions are abnormal in patients with cirrhosis, although the extent of neutrophil dysfunction is not well understood. We tested the ability of neutrophils from 21 hospitalized patients with cirrhosis and 23 healthy control patients to kill Candida albicans , a common fungal pathogen in patients with cirrhosis. Using an assay, we also measured the ability of neutrophils to coordinate multicellular, synchronized control of C. albicans hyphae through a process known as swarming. We found that neutrophils from patients with cirrhosis have significantly decreased fungicidal capacity compared with healthy control neutrophils (53% vs. 74%, P  < 0.0001) and diminished ability to control hyphal growth normalized as a ratio to healthy control (0.22 vs. 0.65, P  < 0.0001). Moreover, serum from patients with cirrhosis decreases the ability of healthy control neutrophils to kill C. albicans (from 60% to 41%, P  < 0.003). Circulating concentration of the inflammatory cytokines tumor necrosis factor α, interleukin-6, and interleukin-8 were found to be significantly elevated in patients with cirrhosis compared to healthy controls. Following pretreatment with granulocyte-colony stimulating factor and granulocyte-macrophage colony-stimulating factor, neutrophil function was restored to almost that of healthy controls. Conclusion: Our data establish profound neutrophil dysfunction against, and altered swarming to, C. albicans in patients with cirrhosis. This dysfunction can be partially reversed with cytokine augmentation ex vivo ., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Background: The efficacy of interleukin-6 receptor blockade in hospitalized patients with coronavirus disease 2019 (Covid-19) who are not receiving mechanical ventilation is unclear., Methods: We performed a randomized, double-blind, placebo-controlled trial involving patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, hyperinflammatory states, and at least two of the following signs: fever (body temperature >38°C), pulmonary infiltrates, or the need for supplemental oxygen in order to maintain an oxygen saturation greater than 92%. Patients were randomly assigned in a 2:1 ratio to receive standard care plus a single dose of either tocilizumab (8 mg per kilogram of body weight) or placebo. The primary outcome was intubation or death, assessed in a time-to-event analysis. The secondary efficacy outcomes were clinical worsening and discontinuation of supplemental oxygen among patients who had been receiving it at baseline, both assessed in time-to-event analyses., Results: We enrolled 243 patients; 141 (58%) were men, and 102 (42%) were women. The median age was 59.8 years (range, 21.7 to 85.4), and 45% of the patients were Hispanic or Latino. The hazard ratio for intubation or death in the tocilizumab group as compared with the placebo group was 0.83 (95% confidence interval [CI], 0.38 to 1.81; P = 0.64), and the hazard ratio for disease worsening was 1.11 (95% CI, 0.59 to 2.10; P = 0.73). At 14 days, 18.0% of the patients in the tocilizumab group and 14.9% of the patients in the placebo group had had worsening of disease. The median time to discontinuation of supplemental oxygen was 5.0 days (95% CI, 3.8 to 7.6) in the tocilizumab group and 4.9 days (95% CI, 3.8 to 7.8) in the placebo group (P = 0.69). At 14 days, 24.6% of the patients in the tocilizumab group and 21.2% of the patients in the placebo group were still receiving supplemental oxygen. Patients who received tocilizumab had fewer serious infections than patients who received placebo., Conclusions: Tocilizumab was not effective for preventing intubation or death in moderately ill hospitalized patients with Covid-19. Some benefit or harm cannot be ruled out, however, because the confidence intervals for efficacy comparisons were wide. (Funded by Genentech; ClinicalTrials.gov number, NCT04356937.)., (Copyright © 2020 Massachusetts Medical Society.)

In diabetes there is a long latency between the onset of hyperglycemia and the appearance of structural microangiopathy. Because Ly6C low patrolling monocytes (PMo) behave as housekeepers of the vasculature, we tested whether PMo protect microvessels against diabetes. We found that in wild-type mice, diabetes reduced PMo in the general circulation but increased by fourfold the absolute number of PMo adherent to retinal vessels (leukostasis). Conversely, in diabetic NR4A1 -/- mice, a model of absence of PMo, there was no increase in leukostasis, and at 6 months of diabetes, the number of retinal acellular capillaries almost doubled compared with diabetic wild-type mice. Circulating PMo showed gene expression changes indicative of enhanced migratory, vasculoprotective, and housekeeping activities, as well as profound suppression of genes related to inflammation and apoptosis. Promigratory CXCR4 was no longer upregulated at longer duration when retinal acellular capillaries begin to increase. Thus, after a short diabetes duration, PMo are the cells preferentially recruited to the retinal vessels and protect vessels from diabetic damage. These observations support the need for reinterpretation of the functional meaning of leukostasis in diabetes and document within the natural history of diabetic retinopathy processes of protection and repair that can provide novel paradigms for prevention., (© 2020 by the American Diabetes Association.)

Electrophilic compounds present in humans, originating from endogenous processes or pollutant exposures, pose a risk to health though their reaction with nucleophilic sites in protein and DNA. Among this chemical class, aldehydes are mainly present in indoor air and they can also be produced by endogenous lipid peroxidation arising from oxidative stress. Known to be very reactive, aldehydes have the ability to form exocyclic adducts to DNA that, for the most if not repaired correctly, are mutagenic and by consequence potential agents involved in carcinogenesis. The aim of this work was to establish profiles of exocyclic DNA adducts induced by aldehyde mixtures, which could ultimately be considered as a genotoxic marker of endogenous and environmental aldehyde exposure. Adducts were quantified by an accurate, sensitive and validated ultra high performance liquid chromatography-electrospray ionization analytical method coupled to mass spectrometry in the tandem mode (UHPLC-ESI-MS/MS). We simultaneously measured nine exocyclic DNA adducts generated during the exposure in vitro of calf thymus DNA to different concentrations of each aldehyde along, as well as, to an equimolar mixture of these aldehydes. This approach has enabled us to establish dose-response relationships that allowed displaying the specific reactivity of aldehydes towards corresponding adducts formation. Profiles of these adducts determined in DNA of current smokers and non-smokers blood samples supported these findings. These first results are encouraging to explore genotoxicity induced by aldehyde mixtures and can furthermore be used as future reference for adductomic approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to Candida species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens Candida albicans , Candida glabrata , and Candida auris Neutrophil responses to Candida species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to Candida species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics. IMPORTANCE Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen Candida The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic Candida species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders., (Copyright © 2020 Negoro et al.)

Necrotizing fasciitis is a potentially fatal soft tissue infection that requires prompt clinical suspicion, pharmacological and surgical interventions. Bacterial pathogens, such as beta-hemolytic streptococcus and Staphylococcus aureus , are the main etiology of necrotizing fasciitis, however, rare cases caused by fungal pathogens, such as Candida albicans , have been reported following trauma. Here, we present the first case of C. albicans necrotizing fasciitis following an elective surgical procedure in an immunocompetent adult., (© 2020 The Authors.)

Human exposure to aldehydes is implicated in several diseases including cancer. These strong electrophilic compounds can react with nucleophilic sites in DNA to form reversible and irreversible modifications. These modifications, if not repaired, can contribute to pathogenesis. The aim of our study was to provide a mass spectrometry (MS)-based profiling method for identifying potential biomarkers of aldehydes exposure. We have developed and validated a highly sensitive method using ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) for the simultaneous quantitation of 9 exocyclic DNA adducts derived from 8 main exogenous and endogenous aldehydes, namely formaldehyde, acetaldehyde, acrolein, crotonaldehyde, malondialdehyde, 4-hydroxy-2-nonenal, glyoxal and methylglyoxal. Finally, we applied the established method to quantify adducts in genomic DNA isolated from the blood of a smoker and a non-smoker blood samples in order to demonstrate its applicability., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections., Competing Interests: The authors have declared that no competing interests exist.

Macrophages play a critical role in the elimination of fungal pathogens. They are sensed via cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live Candida glabrata in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of C. glabrata did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled C. glabrata were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry. C. glabrata remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of C. glabrata with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage-fungal interaction. In conclusion, we demonstrate a new method of tracking division of C. glabrata using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed C. glabrata .

We hypothesized that third-party fecal microbiota transplantation (FMT) may restore intestinal microbiome diversity after allogeneic hematopoietic cell transplantation (allo-HCT). In this open-label single-group pilot study, 18 subjects were enrolled before allo-HCT and planned to receive third-party FMT capsules. FMT capsules were administered no later than 4 weeks after neutrophil engraftment, and antibiotics were not allowed within 48 hours before FMT. Five patients did not receive FMT because of the development of early acute gastrointestinal (GI) graft-versus-host disease (GVHD) before FMT (n = 3), persistent HCT-associated GI toxicity (n = 1), or patient decision (n = 1). Thirteen patients received FMT at a median of 27 days (range, 19-45 days) after HCT. Participants were able to swallow and tolerate all FMT capsules, meeting the primary study endpoint of feasibility. FMT was tolerated well, with 1 treatment-related significant adverse event (abdominal pain). Two patients subsequently developed acute GI GVHD, with 1 patient also having concurrent bacteremia. No additional cases of bacteremia occurred. Median follow-up for survivors is 15 months (range, 13-20 months). The Kaplan-Meier estimates for 12-month overall survival and progression-free survival after FMT were 85% (95% confidence interval, 51%-96%) and 85% (95% confidence interval, 51%-96%), respectively. There was 1 nonrelapse death resulting from acute GI GVHD (12-month nonrelapse mortality, 8%; 95% confidence interval, 0%-30%). Analysis of stool composition and urine 3-indoxyl sulfate concentration indicated improvement in intestinal microbiome diversity after FMT that was associated with expansion of stool-donor taxa. These results indicate that empiric third-party FMT after allo-HCT appears to be feasible, safe, and associated with expansion of recipient microbiome diversity. This trial was registered at www.clinicaltrials.gov as #NCT02733744., (© 2018 by The American Society of Hematology.)

Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC 50 of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC 50 in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections.

The roles of transforming growth factor (TGF)-β in extracellular matrix production and vascular remodeling, coupled with increased TGF-β expression and signaling in diabetes, suggest TGF-β as an important contributor to the microangiopathy of diabetic retinopathy and nephropathy. To investigate whether increased TGF-β signaling could be a therapeutic target for preventing retinopathy, we used a pharmacologic approach (SM16, a selective inhibitor of the type 1 TGF-β receptor activin receptor-like kinase 5, orally active) to inhibit the increased, but not the basal, Tgf-β signaling in retinal vessels of diabetic rats. At the level of vascular gene expression, 3.5 months' diabetes induced minimal changes. Diabetes + SM16 for 3 weeks caused widespread changes in gene expression poised to enhance vascular inflammation, thrombosis, leakage, and wall instability; these changes were not observed in control rats given SM16. The synergy of diabetes and SM16 in altering gene expression was not observed in the lung. At the level of vascular network morphology, 7 months' diabetes induced no detectable changes. Diabetes + SM16 for 3 weeks caused instead distorted morphology and decreased density. Thus, in diabetes, retinal vessels become dependent on a small increase in TGF-β signaling via activin receptor-like kinase 5 to maintain early integrity. The increased TGF-β signaling may protect against rapid retinopathy progression and should not be a target of inhibitory interventions., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

During the last few years, the induction of toxicological mechanisms by atmospheric ultrafine particles (UFP) has become one of the most studied topics in toxicology and a subject of huge debates. Fine particles (FP) and UFP collected at urban and rural sites in Lebanon were studied for their chemical composition and toxicological effects. UFP were found more enriched in trace elements, secondary inorganic ions, total carbon and organic compounds than FP. For toxicological analysis, BEAS-2B cells were exposed for 24, 48 and 72 h to increasing concentrations of FP, water-UFP suspension (UFPw) and UFP organic extract (UFPorg). Our findings showed that UFP caused earlier alterations of mitochondrial metabolism and membrane integrity from the lowest concentrations. Moreover, a significant induction of CYP1A1, CYP1B1 and AhRR genes expression was showed after cells exposure to UFPorg and to a lesser extent to UFPw and FP samples., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Assessment of air pollution by particulate matter (PM) is strongly required in Lebanon in the absence of an air quality law including updated air quality standards. Using two different PM2.5-0.3 samples collected at an urban and a rural site, we examined genotoxic/epigenotoxic effects of PM exposure within a human bronchial epithelial cell line (BEAS-2B). Inorganic and organic contents evidence the major contribution of traffic and generating sets in the PM2.5-0.3 composition. Urban PM2.5-0.3 sample increased the phosphorylation of H2AX, the telomerase activity and the miR-21 up-regulation in BEAS-2B cells in a dose-dependent manner. Furthermore, urban PM2.5-0.3 induced a significant increase in CYP1A1, CYP1B1 and AhRR genes expression. The variable concentrations of transition metals and organic compounds detected in the collected PM2.5-0.3 samples might be the active agents leading to a cumulative DNA damage, critical for carcinogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Traffic-related volatile organic compounds (VOCs) pollution has frequently been demonstrated to be a serious problem in the developing countries. Benzene and 1,3-butadiene (BD) have been classified as a human carcinogen based on evidence for an increased genotoxic and epigenotoxic effects in both occupational exposure assessment and in vivo/in vitro studies. We have undertaken a biomonitoring of 25 traffic policemen and 23 office policemen in Beirut, through personal air monitoring, assessed by diffusive samplers, as well as through the use of biomarkers of exposure to benzene and BD. Personal benzene, toluene, ethylbenzene, and xylene (BTEX) exposure were quantified by GC-MS/MS, urinary trans, trans-muconic acid (t,t-MA) by HPLC/UV, S-phenyl mercapturic acid (S-PMA), monohydroxy-butenyl mercapturic acid (MHBMA) and dihydroxybutyl mercapturic acid (DHBMA) by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC/ESI(-)-MS/MS) in MRM (Multiple Reaction Monitoring) mode. We found that individual exposure to benzene in the traffic policemen was higher than that measured in traffic policemen in Prague, in Bologna, in Ioannina and in Bangkok. t,t-MA levels could distinguish between office and traffic policemen. However, median MHBMA levels in traffic policemen were slightly elevated, though not significantly higher than in office policemen. Alternatively, DHBMA concentrations could significantly distinguish between office and traffic policemen and showed a better correlation with personal total BTEX exposure. DHMBA, measured in the post-shift urine samples, correlated with both pre-shift MHMBA and pre-shift DHMBA. Moreover, there was not a marked effect of smoking habits on DHBMA. Taken together, these findings suggested that DHBMA is more suitable than MHBMA as biomarker of exposure to BD in humans. Traffic policemen, who are exposed to benzene and BD at the roadside in central Beirut, are potentially at a higher risk for development of diseases such as cancer than office policemen., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.

Parabens are among the most frequently used preservatives to inhibit microbial growth and extend the shelf life of a range of consumer products. The objective of the present study was to gain insight into the metabolism of parabens in breast cancer cells (MCF7) since they have demonstrated estrogenic activity towards these cells and have been detected in breast cancer tissues. The toxicity of parabens to MCF7 cells was determined using MTT assays. Hydrolysis of methyl-, butyl and benzyl-paraben to p-hydroxybenzoic acid was analyzed in cultured MCF7 cells and in cellular homogenates. Glucuronidation and sulfoconjugation were studied in MCF7 homogenates, and parabens were analyzed by HPLC. Methyl-paraben was shown to be far less toxic than butyl and benzyl-paraben. Parabens were completely stable in MCF7 homogenates whereas p-nitrophenyl acetate, a substrate type, underwent hydrolysis. MCF7 cell homogenates did not express glucuronidation and sulfoconjugation activities toward parabens. The higher stability of parabens may explain their accumulation in breast cancer tissue as previously reported in the literature., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 mum min(-1). Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte-hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed., (Copyright 2009 Elsevier Ltd. All rights reserved.)

To contribute to complete the knowledge of the underlying mechanisms of action involved in air pollution particulate matter (PM)-induced cytotoxicity, an aerosol was collected in Dunkerque, a French seaside City heavily industrialized. In this work, we focused our attention on its physical and chemical characteristics, its cytotoxicity, and its role in the induction of the volatile organic compound (VOC) and/or polycyclic aromatic hydrocarbon (PAH)-metabolizing enzymes in human lung epithelial cells (A549). Size distribution showed that 92.15% of the collected PM were PM2.5 and the specific surface area was 1 m2/g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. VOC, PAH, etc.) chemicals were found in collected PM, revealing that much of them derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. The thermal desorption study indicated that organic chemicals were not only adsorbed onto the surface but also highly incrusted in the structure of PM. The lethal concentrations at 10% and 50% of collected PM were 23.72 microg/mL (or 6.33microg/cm2) and 118.60 microg/mL (or 31.63 microg/cm2), respectively. The VOC and/or PAH-coated onto PM induced significant increases in mRNA expressions of cytochrome P450 (cyp) 1a1, cyp2e1, cyp2f1, nadph quinone oxydo-reductase-1, and glutathione s-transferase-pi 1, versus controls. Hence, we concluded that the metabolic activation of the very low doses of VOC and/or PAH-coated onto the inorganic condensation nuclei from Dunkerque City's PM is one of the underlying mechanisms of action closely involved in its cytotoxicity in human lung epithelial cells.

To contribute to improving knowledge on the adverse health effects induced by particulate matter (PM) air pollution, an extensive investigation was undertaken of the underlying mechanisms of action activated by PM(2.5) air pollution collected in Dunkerque, a strongly industrialized French seaside city. Their chemical and physical characteristics have been previously determined, and earlier in vitro short-term studies have shown them to cause dose-dependent and time-dependent oxidative damage, gene expression and protein secretion of inflammatory mediators, and apoptotic events in human lung epithelial cells (L132) in culture. Hence, this work studied the activation of nuclear factor-kappa B (NF-kappaB)/inhibitory kappa B (IkappaB) by Dunkerque city PM(2.5) in these target cells, by determination of phosphorylated p65 and phosphorylated IkappaBalpha protein levels in cytoplasmic extracts, and p65 and p50 DNA binding in nuclear extracts. In PM-exposed L132 cells, there were concentration- and/or time-dependent increases in nuclear p65 and cytoplasmic IkB-alpha phosphorylation, and nuclear p65 and p50 DNA binding. Taken together, these results showed that Dunkerque city PM(2.5) were involved in the activation of the NF-kappaB/IkappaB complex, notably through the occurrence of oxidative stress conditions, and, therefore, in the gene expression and protein secretion of inflammatory mediators in target L132 cells. Hence, these findings suggested that the activation of the NF-kappaB/IkappaB complex preceded cytotoxicity in Dunkerque city PM-exposed L132 cells., ((c) 2007 John Wiley & Sons, Ltd.)

Epidemiological studies have associated the increase of respiratory and cardiovascular mortality and morbidity with high levels of air pollution particulate matter (PM). However, the underlying mechanisms of actions by which PM induce adverse health effects are still unclear. We have recently undertaken an extensive investigation of the adverse health effects of air pollution PM(2.5), and shown that in vitro short-term exposure to PM(2.5) induced oxidative stress and inflammation in human lung epithelial cells (L132). Hence, it was convenient to complete the physical and chemical characterization of PM and to investigate whether in vitro short-term exposure to PM could be imply in the activation of apoptosis. Accordingly, we found that 92.15% of PM were equal or smaller than 2.5 microm and their specific surface area was 1m(2)/g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. polycyclic aromatic hydrocarbons) chemicals were found in PM, suggesting that much of them derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. In other respects, we showed that PM exposure induced apoptosis by activating not only the tumor necrosis factor-alpha (TNF-alpha)-induced pathway (i.e. TNF-alpha secretion, caspase-8 and -3 activation), but also the mitochondrial pathway (i.e. 8-hydroxy-2'-desoxyguanosine formation, cytochrome c release from mitochondria, caspase-9 and -3 activation). Moreover, changes in the transcription rates of p53, bcl-2, and bax genes, on the one hand, and DNA fragmentation, on the other hand, were reported in PM-exposed proliferating L132 cells, revealing the occurrence of apoptotic events. Taken together, these findings suggested that in vitro short-term exposure to PM(2.5) induced apoptosis in L132 cells.

Exposure to urban airborne particulate matter (PM) has been associated with adverse health effects. In this work, we focused our attention on the capacity of air pollution PM to induce cytotoxic, oxidative stress, and inflammatory responses in human epithelial lung cells (L132) in culture. PM were collected in Dunkerque, a French seaside city, and their physical and chemical characteristics were carried out. Their size distribution showed that 92.15% of the PM were equal or smaller than 2.5 and their specific surface area was 1 m2/g. Inorganic (i.e. Fe, Al, Ca, Na, K, Mg, Pb, etc.) and organic (i.e. VOC, PAH, etc.) chemicals were found in PM. Physical and chemical properties of Dunkerque City's PM suggested that much of the collected PM derived from wind-borne dust from the industrial complex and the heavy motor vehicle traffic. Their cytotoxicity, as evaluated by survival rate determination, lactate dehydrogenase activity, and mitochondrial dehydrogenase activity showed concentration and time-dependent effects in L132 cells (LC10 = 18.84 microg PM/ml; LC50 = 75.36 microg PM/ml). Moreover, in PM-exposed L132 cells, there were concentration- and time-dependent changes in lipid peroxidation, superoxide dismutase activity, 8-hydroxy-2'-deoxyguanosine formation, and poly(ADP-ribosyl)ation, on the one hand, and in tumor necrosis factor-alpha secretion, inducible nitric oxide synthase activity, and nitric oxide release, on the other hand. Taken together, these findings suggested that oxidative stress and inflammatory responses proceeded cytotoxicity in PM-exposed L132 cells.

The prevention of diabetic retinopathy requires drugs that leverage the benefits of glycemic control without adding the burden of side effects. Aspirin at dosages of 1-1.5 g/day has prevented manifestations of diabetic retinal microangiopathy in a clinical trial as well as in studies with dogs. Because lower and safer doses of aspirin could be used if its beneficial effects on retinopathy were due to antithrombotic effects, we compared the effects of a selective antiplatelet drug (clopidogrel) to those of aspirin in streptozotocin-induced diabetic rats. Clopidogrel did not prevent neuronal apoptosis, glial reactivity, capillary cell apoptosis, or acellular capillaries in the retina of diabetic rats. Aspirin, at doses yielding serum levels (<0.6 mmol/l) well below the anti-inflammatory range for humans, prevented apoptosis of capillary cells and the development of acellular capillaries but did not prevent neuroglial abnormalities. The aldose reductase inhibitor sorbinil, used as the benchmark for the effect of the other drugs, prevented all abnormalities. The diabetic rat retina showed increased expression of the transcription factor CCAAT/enhancer-binding protein-beta, one of the known targets of low-intermediate concentrations of aspirin. Thus we found a spectrum of drug efficacy on the prevention of experimental diabetic retinopathy, ranging from the absent effect of a selective antiplatelet drug to the prevention of all abnormalities by an aldose reductase inhibitor. Aspirin at low-intermediate concentrations selectively prevented microangiopathy. The minimal effective dose of aspirin should now be sought.

Exposure to urban airborne particulate matter (PM) has been associated with adverse health effects. The majority of research articles published on air pollution PM relate to PM10. However, increasing emphasis and stringent regulations have been placed on PM2.5. The mechanisms for PM-induced adverse health effects are not well understood, but inflammation seems to be of importance. We focused our attention also on the capacity of air pollution PM2.5 to induce cytotoxic and inflammatory responses in human epithelial lung cells (L132) in culture. Particulate matter was collected in Dunkerque, a French seaside city characterized by the proximity of industrial activity and heavy motor vehicle traffic. Size distribution results showed that the cumulative frequency of PM2.5 was 92.15% and their specific surface area was 1 m2 g(-1). Inorganic and organic chemicals usually associated with the natural environment but also so-called anthropogenic elements were found in PM, suggesting that much of the PM was derived from wind-borne dust from the industrial complex and the heavy diesel motor vehicle. We observed PM concentration-dependent cytotoxic effects in L132 cells (LC10 = 18.84 microg PM ml(-1); LC50 = 75.36 microg PM ml(-1)). We showed that exposure to Dunkerque City's PM2.5 induced significant increases (in a concentration- and time-dependent manner) in protein secretion and/or gene expression of inflammatory cytokines (i.e. TNF-alpha, IL-1beta, IL-8, GM-CSF, IL-6, TGF-beta1). We hypothesized also that the occurrence of the acute inflammatory response might rely on the capacity of such air pollutants to generate oxidative species, which have been implicated in the stringent regulation of the cytokine network. Hence, we suggest that the development of inflammatory effects that worsen over time stems from the cytotoxicity in Dunkerque City's PM2.5-exposed L132 cells in culture., (Copyright 2005 John Wiley & Sons, Ltd.)

The polyol (sorbitol) pathway of glucose metabolism is activated in many cell types when intracellular glucose concentrations are high, and it can generate cellular stress through several mechanisms. The role of the polyol pathway in the pathogenesis of diabetic retinopathy has remained uncertain, in part because it has been examined preferentially in galactose-induced retinopathy and in part because inhibition studies may not have achieved full blockade of the pathway. Having observed that the streptozotocin-induced diabetic rat accurately models many cellular processes characteristic of human diabetic retinopathy, we tested in the diabetic rat if documented inhibition of the polyol pathway prevents a sequence of retinal vascular abnormalities also present in human diabetes. An inhibitor of aldose reductase, the rate-limiting enzyme in the pathway, prevented the early activation of complement in the wall of retinal vessels and the decreased levels of complement inhibitors in diabetic rats, as well as the later apoptosis of vascular pericytes and endothelial cells and the development of acellular capillaries. Both rat and human retinal endothelial cells showed aldose reductase immunoreactivity, and human retinas exposed to high glucose in organ culture increased the production of sorbitol by a degree similar to that observed in the rat. Excess aldose reductase activity can be a mechanism for human diabetic retinopathy.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2-4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3-kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C(2)-ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia-induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP-activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.

It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of (3)H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with (3)H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.

In several non-vascular tissues in which it has been studied, AMP-activated protein kinase (AMPK) appears to modulate the cellular response to stresses such as ischemia. In liver and muscle, it phosphorylates and inhibits acetyl CoA carboxylase (ACC), leading to an increase in fatty acid oxidation; and in muscle, its activation is associated with an increase in glucose transport. Here we report the presence of both AMPK and ACC in human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with 2 mM AICAR, an AMPK activator, caused a 5-fold activation of AMPK, which was accompanied by a 70% decrease in ACC activity and a 2-fold increase in fatty acid oxidation. Surprisingly, glucose uptake and glycolysis, the dominant energy-producing pathway in HUVEC, were diminished by 40-60%. Despite this, cellular ATP levels were increased by 35%. Thus activation of AMPK by AICAR is associated with major alterations in endothelial cell energy balance. Whether these alterations protect the endothelium during ischemia or other stresses remains to be determined., (Copyright 1999 Academic Press.)