Background The difficulty of antifungal substances to penetrate keratin and slow nail growth limit the efficacy of topical therapy in onychomycosis. One promising alternative is photodynamic antimicrobial chemotherapy, or PACT : an irradiated photosensitizer creates singlet oxygen molecules which destroy pathogens without damaging human cells. Objective As PACT has demonstrated strong antifungal capabilities, we wanted to investigate its efficacy in an in vitro model of onychomycosis. Methods PACT was tested in a microdilution assay, in an in vitro onychomycosis model as well as in a patient. Results PACT inhibited fungal growth in the microdilution assay with no colonies of T. rubrum detectable. Fungal growth was also inhibited in an onychomycosis model, after 30 min of LED irradiation. Subsequently, a patient with distolateral onychomycosis was treated on three consecutive days and showed significant and durable improvement of nail morphology 6 months after. Conclusion PACT appears to be an effective treatment of onychomycosis in vitro . The promising results need to be validated by clinical trials.

BackgroundPlatelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood.ObjectivesSerum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far.Methods and ResultsElectron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-)). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3-induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b.ConclusionsThe present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.

OBJECTIVE:Atherosclerosis, an inflammatory disease of arterial vessel walls, requires migration and matrix metalloproteinase (MMP)-9-dependent invasion of monocytes/macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor nuclear factor-κB, which is regulated by inhibitor κB (IκB) and thus IκB kinase. Regulators of nuclear factor-κB include serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. APPROACH AND RESULTS:Gene-targeted apolipoprotein E (ApoE)-deficient mice without (apoe(-/-)sgk1(+/+)) or with (apoe(-/-)sgk1(-/-)) additional SGK1 knockout received 16-week cholesterol-rich diet. According to immunohistochemistry atherosclerotic lesions in aorta and carotid artery, vascular CD45(+) leukocyte infiltration, Mac-3(+) macrophage infiltration, vascular smooth muscle cell content, MMP-2, and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe(-/-)sgk1(-/-)mice than in apoe(-/-)sgk1(+/+)mice. As determined by Boyden chamber, thioglycollate-induced peritonitis and air pouch model, migration of SGK1-deficient CD11b(+)F4/80(+) macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1(-/-) than in sgk1(+/+)macrophages and in control plasmid-transfected or inactive (K127N)SGK1-transfected than in constitutively active (S422D)SGK1-transfected THP-1 cells. Confocal microscopy revealed reduced macrophage number and macrophage MMP-9 content in plaques of apoe(-/-)sgk1(-/-) mice. In THP-1 cells, MMP-inhibitor GM6001 (25 μmol/L) abrogated (S422D)SGK1-induced MMP-9 production and invasion. According to reverse transcription polymerase chain reaction, MMP-9 transcript levels were significantly reduced in sgk1(-/-)macrophages and strongly upregulated in (S422D)SGK1-transfected THP-1 cells compared with control plasmid-transfected or (K127N)SGK1-transfected THP-1 cells. According to immunoblotting and confocal microscopy, phosphorylation of IκB kinase and inhibitor κB and nuclear translocation of p50 were significantly lower in sgk1(-/-)macrophages than in sgk1(+/+)macrophages and significantly higher in (S422D)SGK1-transfected THP-1 cells than in control plasmid-transfected or (K127N)SGK1-transfected THP-1 cells. Treatment of (S422D)SGK1-transfected THP-1 cells with IκB kinase-inhibitor BMS-345541 (10 μmol/L) abolished (S422D)SGK1-induced increase of MMP-9 transcription and gelatinase activity. CONCLUSIONS:SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of monocyte/macrophage migration and MMP-9 transcription via regulation of nuclear factor-κB.

Background: Platelets are activated by increase of cytosolic Ca2+ concentration ([Ca2+]i) following stimulation of store operated Ca2+ entry (SOCE), which is accomplished by the pore forming unit Orai1 and its regulator STIM1. In other cell types Ca2+ transport across the cell membrane is regulated by 1,25(OH)2 vitamin D. Formation of 1,25(OH)2 vitamin D is inhibited by anti-aging protein klotho. Thus, 1,25(OH)2 vitamin D plasma levels are excessive in klotho-deficient mice (kl/kl), which suffer from severe vascular calcification, early appearance of age related disorders and a life span shortened to some 12 weeks. The present study explored whether klotho deficiency impacts on [Ca2+]i regulation and function of platelets. Methods and results: As a result, SOCE and agonist-induced [Ca2+]i increase were significantly blunted in platelets from kl/kl mice. Similarly, degranulation, integrin αIIbβ3 activation as well as aggregation and in vitro thrombus formation were significantly impaired in platelets from kl/kl mice. Low vitamin D diet (LVD) normalized plasma 1,25(OH)2 vitamin D concentrations in klotho-deficient mice and abrogated impaired Ca2+ signaling as well as Ca2+-dependent activation of platelets from kl/kl mice. Platelet and megakaryocyte mRNA levels and protein expression of platelet SOCE-mediating molecules STIM1 and Orai1 were significantly reduced in kl/kl mice, an effect reversed after treatment with LVD. The effect of excessive Treatment with 1,25(OH)2 vitamin D mimicked the phenotype of megakaryocytes from klotho-deficient mice since 1,25(OH)2 vitamin D-treated megakaryocytic MEG-01 cells displayed significantly decreased mRNA levels and protein abundance of STIM1 and Orai1. Nuclear abundance of NF-κB subunits p50 and p65 were significantly lower in megakaryocytes derived from kl/kl mice than in megakaryocytes from klotho wildtype mice, an effect again found reversed in megakaryocytes from kl/kl mice treated with LVD. Transfection of MEG-01 cells with p50/p65 significantly increased STIM1 and Orai1 expression on mRNA as well as on protein level. Conclusions: In conclusion, klotho deficiency leads to blunted SOCE in platelets resulting in impaired thrombus formation at least in part due to impairment of NF-κB-dependent STIM1 and Orai1 expression in megakaryocytes by excessive 1,25(OH)2 vitamin D levels.