[Clinical analysis of 10 patients with Fabry disease].

We aimed to analyze the clinical data of 10 patients (6 male and 4 female) with Fabry disease (FD). The mean age of the patients was (28.80±9.27) years. Seven patients had classical FD and three had delayed onset FD. Among the 10 patients, six had skin involvement and cutaneous angiokeratoma; five had hypohidrosis or anhidrosis; nine had intermittent neuralgia; and three had supraorbital ridge protrusion, forehead bulge, and lip thickening. Five patients had proteinuria, including one with chronic kidney disease stage 3 and one with chronic kidney disease stage 5. Cardiac involvement occurred in three patients, two had myocardial hypertrophy and one had valvular insufficiency. The activity of galactosidase decreased in seven patients (2.80-1.55 µmol·L-1·h-1). Plasma deacetyl-GL-3 was elevated in all 10 patients(3.12-120.00 ng/ml). Three patients underwent renal biopsy, wherein two cases of focal segmental glomerulosclerosis and one of mesangial proliferative glomerulonephritis was found. A large number of myeloid and zebra bodies were found in the podocytes in three patients, including a small number of myeloid and zebra bodies in the renal tubular epithelial cells in one patient with occasional zebra bodies in the renal interstitium. Nine patients had GLA gene mutations. One patient was c.102T>A, a de novo mutation. Four patients were treated with agalsidase α injection (0.2 mg/kg, intravenous infusion every 2 weeks), and their prognosis was good. FD has various clinical manifestations and multi-system involvement, which requires multidisciplinary cooperation. Detection of galactosidase activity, plasma globotriaosylsphingosine, and GLA gene mutation can help for accurate diagnosis.

Selective photodynamic eradication of senescent cells with a ß-galactosidase-activated photosensitiser.

A ß-galactosidase-responsive photosensitiser has been designed and synthesised. It contains a galactosyl substrate, a boron dipyrromethene-based photosensitising unit and a black hole quencher 2 connected via an AB2-type self-immolative linker. This novel photosensitiser can be selectively activated by the senescence-associated ß-galactosidase in senescent cells, leading to restoration in fluorescence emission and effective killing of the cells via photodynamic action.

Cordycepin Enhances SIRT1 Expression and Maintains Stemness of Human Mesenchymal Stem Cells.

BACKGROUND/AIM: Mesenchymal stem cells (MSCs) have been employed for therapeutic applications of various degenerative diseases. However, the major concern is MSC aging during the in vitro cultivation. Thus, the approach to delay MSC aging was examined in this research by focusing on the expression of Sirtuin 1 (SIRT1), a key anti-aging marker. MATERIALS AND METHODS: Cordycepin, a bioactive compound derived from Cordyceps militaris, was used to up-regulate SIRT1 and maintain stemness of MSCs. Upon treatment with cordycepin, MSCs were investigated for cell viability, doubling time, key gene/protein expression, galactosidase-associated senescence assay, relative telomere length, and telomerase expression. RESULTS: Cordycepin significantly increased the expression of SIRT1 in MSCs by activating the adenosine monophosphate activated protein kinase (AMPK)-SIRT1 signalling pathway. Moreover, cordycepin maintained the stemness of MSCs by deacetylating SRY-box transcription factor 2 (SOX2) via SIRT1, and cordycepin delayed cellular senescence and aging of MSCs by enhancing autophagy, inhibiting the activity of senescence-associated-galactosidase, maintaining proliferation rate, and increasing telomere activity. CONCLUSION: Cordycepin could be used to increase SIRT1 expression in MSCs for anti-aging applications.

Biopharmaceutical applications of α-galactosidases.

α-Galactosidases are exoglycosidases that are active on galactose-containing side chains in oligosaccharides, polysaccharides, glycolipids, and glycoproteins. α-Galactosidases are gaining increased interest in human medicine, especially in the enzyme replacement therapy for Fabry's disease. α-Galactosidases with regioselectivity toward α-1,3-linked galactose find application in xenotransplantation and blood group transformation. The use of α-galactosidases as a therapeutic agent in alleviating the postprandial symptoms of irritable bowel syndrome is much acclaimed. The excellent therapeutic applications of α-galactosidases have led to an upwelling of worldwide research interventions to identify novel α-galactosidases with improved catalytic efficiency. In addition to these therapeutic applications, α-galactosidases also have interesting applications in the industrial sectors like food, feed, probiotics, sugar, and paper pulp. The current review focuses on the diverse therapeutic applications of α-galactosidases and their prospects.

Ectopic expression of a grapevine alkaline α-galactosidase seed imbibition protein VvSIP enhanced salinity tolerance in transgenic tobacco plants.

Alpha-galactosidase seed imbibition protein (VvSIP) isolated from Vitis vinifera is up-regulated upon salt stress and mediates osmotic stress responses in a tolerant grapevine cultivar. So far, little is known about the putative role of this stress-responsive gene. In the present study, VvSIP function was investigated in model tobacco plants via Agrobacterium-mediated genetic transformation. Our results showed that overexpression of VvSIP exhibited increased tolerance to salinity at germination and late vegetative stage in transgenic Nicotiana benthamiana compared to the nontransgenic plants based on the measurement of the germination rate and biomass production. High salt concentrations of 200 and 400 mM NaCl in greenhouse-grown pot assay resulted in better relative water content, higher leaf osmotic potential, and leaf water potential in transgenic lines when compared to the wild-type (WT) plants. These physiological changes attributed to efficient osmotic adjustment improved plant performance and tolerance to salinity compared to the WT. Moreover, the VvSIP-expressing lines SIP1 and SIP2 showed elevated amounts of chlorophyll with lower malondialdehyde content indicating a reduced lipid peroxidation required to maintain membrane stability. When subjected to high salinity conditions, the transgenic tobacco VvSIP exhibited higher soluble sugar content, which may suggest an enhancement of the carbohydrate metabolism. Our findings indicate that the VvSIP is involved in plant salt tolerance by functioning as a positive regulator of osmotic adjustment and sugar metabolism, both of which are responsible for stress mitigation. Such a candidate gene is highly suitable to alleviate environmental stresses and thus could be a promising candidate for crop improvement.

[Isolation, culture and inflammatory characteristics of astrocytes in aged rats nervous tissue].

Objective: To establish an optimized method for the isolation and purification of astrocytes from the neural tissues of young and aged rats. Then, the morphological and functional differences of astrocytes between young and aged rats were compared to explore the functional changes of astrocytes after aging and its possible mechanism in the aging process. Methods: Young (2 months old) and aged (20 months old) SD rats were used. Astrocytes in brain and spinal cord tissue were purified by 50% - 35% percoll density gradient centrifugation. Each group of cells was set up with three duplicate wells. After 72 h of culture, Glial fibrillary acidic protein (GFAP) which was astrocyte specific marker were detected by immunofluorescence to evaluate the morphological characteristics. Cell senescence markers (p16 and p21) and ß- Galactosidase were detected by qPCR and staining respectively. The expressions of pro-inflammatory cytokines (IL-1ß, TNF-α) and anti-inflammatory cytokines were detected by qPCR. Results: Using 50%-35% percoll gradient separation, astrocytes were obtained with large number, good activity and purity of more than 95%, which could be used in subsequent experiments. Compared with the astrocytes in the nerve tissue of young rats, the astrocytes in the nervous tissue of the aged rats had fewer protrusions and tended to be activated in cell morphology; the positive rate of ß -galactosidase staining was increased significantly and the expressions of p16 and p21 were increased (P<0.01). The expressions of pro-inflammatory cytokines (IL-1ß, TNF-α) were increased (P<0.05), and the expression of anti-inflammatory cytokine (IL-10) was decreased (P<0.05) in astrocytes of the aged rats nervous tissue. Conclusion: The percoll gradient of 50% - 35% could be used as a method for separation, purification and primary culture of astrocytes. With the increase of age, astrocytes undergo cellular senescence, showing a pro-inflammatory phenotype, promoting inflammaging of the nervous system, which may be one of the mechanisms of nervous system aging and neurodegenerative diseases.

Late-onset Fabry disease due to a new (p.Pro380Leu) pathogenic variant of GLA Gene.

Fabry disease is a rare X-linked lysosomal storage disorder due to pathogenic variants of the galactosidase alpha (GLA) gene, leading to a deficiency of alpha-galactosidase A. The inadequate enzymatic activity leads to progressive glycosphingolipids accumulation within tissues and subsequent multi-systemic dysfunction, with predominant involvement of heart, kidney, and nervous system. Two subtypes are recognized: the classic type and the late-onset type. We here describe the clinical characteristics of a patient with late-onset Fabry disease carrying a not previously identified GLA gene variant. This 50-year-old man came to hospital because of an acute ischemic stroke. He also complained of acroparesthesia and had angiokeratomas in the nape and the back. Blood alpha-galactosidase A activity was low, plasmatic lyso-Gb3 level was borderline, cardiac MRI showed cardiac fibrosis, brain MRI documented cerebrovascular disease, and skin biopsy revealed small fiber neuropathy without globotriaosylceramide-3 skin deposits. Genetic study by means of targeted next-generation sequencing analysis disclosed a missense substitution c.1139C>T (p.Pro380Leu) in the GLA gene. We suggest that this novel variant should be considered as pathogenic and associated with a late-onset variant of Fabry disease with a predominant neurological phenotype.

Generation of the induced pluripotent stem cell line UKWNLi005-A derived from a patient with the GLA mutation c.376A > G of unknown pathogenicity in Fabry disease.

Human dermal fibroblasts (HDF) were obtained by skin punch biopsy from a 51-year old man with suspected Fabry disease (FD), carrying the hemizygous c.376A > G variant in the α-galactosidase A gene (GLA). Cultured HDF were reprogrammed to induced pluripotent stem cells (iPSC) using a non-modified RNA-based transfection protocol. GLA-S126G-iPSC exhibit typical embryonic stem cell-like morphology, normal karyotype, expression of all tested pluripotency markers, and three germ layer differentiation potential. We provide a novel patient-specific cell line that can be used to investigate a genetic variation of yet unknown significance.

Circular RNA-based biomarkers in blood of patients with Fabry disease and related phenotypes.

BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disease caused by mutations in the galactosidase α gene. Deficient activity of α-galactosidase A leads to glycosphingolipid accumulations in multiple organs. Circular RNAs represent strong regulators of gene expression. Their circular structure ensures high stability in blood. We hypothesised that blood-based circular RNA profiles improve phenotypic assignment and therapeutic monitoring of Fabry disease. METHODS: A genome-wide circular RNA expression analysis was performed in blood of genetically diagnosed patients with Fabry disease (n=58), age-matched and sex-matched healthy volunteers (n=14) and disease control patients with acute kidney injury (n=109). Most highly dysregulated circular RNAs were validated by quantitative real-time PCR. Circular RNA biomarker sensitivity, specificity, predictive values and area under the curve (AUC) were determined. Linear regression analyses were conducted for validated circular RNA biomarkers and clinical patient characteristics. RESULTS: A distinct circular RNA transcriptome signature identified patients with Fabry disease. Level of circular RNAs hsa_circ_0006853 (AUC=0.73), hsa_circ_0083766 (AUC=0.8) and hsa_circ_0002397 (AUC=0.8) distinguished patients with Fabry disease from both healthy controls and patients with acute kidney injury. Hsa_circ_0002397 was, furthermore, female-specifically expressed. Circular RNA level were significantly related to galactosidase α gene mutations, early symptoms, phenotypes, disease severities, specific therapies and long-term complications of Fabry disease. CONCLUSION: The discovery of circular RNA-based and Fabry disease-specific biomarkers may advance future diagnosis of Fabry disease and help to distinguish related phenotypes.

A late-onset male Fabry disease patient with somatic mosaicism of a classical GLA mutation: a case report.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA). Male patients of FD develop early sign and symptoms in childhood or adolescence. However, "de novo somatic mosaicism" is rare and might be developed a relatively mild phenotype despite carrying a classic type. A 34-year-old male patient visited with foamy urine. Renal biopsy findings were consistent with FD. Leukocyte α-galactosidase activity was markedly reduced at 5.3 nmol/hr/mg (normal range, 25-126). Sequence analysis of the patient's GLA gene identified mosaicism for the mutation GLA[NM_000169.2] c.820=/G>C. This mutation results in a substitution of the amino acid in position 274 from glycine to arginine. However, no family members showed FD-related symptoms, and the daughter of the patient was also tested for paternity and was identified as a real biological daughter, but DNA sequence analysis for FD showed no mutations. Based on these results, we diagnosed the patients as de novo mutation with somatic mosaicism. Next generation sequencing turned out that 58% of the readings had the mutated allele in buccal cells, 84% in blood, and 85% in urine, when 100% should be expected in a hemizygous affected male confirming the presence of somatic mosaicisms. The patient has been on treatment for enzyme replacement therapy (agalsidase-ß, 1.0 mg/kg biweekly) for past 9 years and has maintained normal renal function (serum creatinine 1.0 mg/dL) with mild albuminuria (123 mg/g Cr). Therefore, this case suggests somatic mosaicism is one of important phenotype modifiers.

Digest the Sugar, Kill the Parasite: A New Experimental Concept in Treating Alveolar Echinococcosis.

INTRODUCTION: The E. multilocularis laminated layer (LL) is a heavily glycosylated parasitic structure that plays an important role in protecting the larval stage (metacestode) of this parasite from physiological and immunological host reactions. We elaborated an experimental design with the idea to modify the (glycan) surface of the LL by a targeted digestion. This should allow the host defense to more easily recognize and attack (or kill) the parasite by immune-mediated effects. METHODS: Experimentally, E. multilocularis (clone H95) metacestodes were cultured in vitro with or without addition of α1-3,4,6-galactosidase or ß1-3-galactosidase in the medium. Morphological changes were subsequently measured by microscopy at different time points. Parasites were then recovered at day 5 and reinjected into mice for assessing their viability and infectious status. For finally recovered parasites, the respective load was assessed ex vivo by wet weight measurement, and host-related PD1 and IL-10 levels were determined as the key immunoregulators by using flow cytometry. RESULTS: Our experiments demonstrated that the parasite vesicular structure can be directly destroyed by adding galactosidases into the in vitro culture system, resulting in the fact that the parasite metacestode vesicles could not anymore infect and develop in mice after this glycan digestion. Moreover, when compared to the mice inoculated with E. multilocularis metacestode without galactosidases, PD1 expression was upregulated in CD4+ Teffs from mice inoculated with E. multilocularis metacestode pretreated with ß1-3-galactosidase, with a lower IL-10 secretion from CD4+ Teffs; there was no difference of PD1 and IL-10 expression levels regarding CD4+ Teff from mice inoculated with E. multilocularis metacestode pretreated with α1-3,4,6-galac-tosidase. DISCUSSION: We raised our hypothesis that this "aborting" effect may be linked to an altered PD1 and IL-10 response fine-tuning between immunopathology and immune protection. These findings justify a continuation of these experiments upon therapeutical in vivo administration of the enzymes.

A Pathogenic Galactosidase A Mutation Coexisting With an MYBPC3 Mutation in a Female Patient With Hypertrophic Cardiomyopathy.

The coexistence of GLA (Pro259Ser, c.775C>T) and MYBPC3 (c.1351+2T>C) mutations was found in a female patient with hypertrophic cardiomyopathy. Histology documented abundant vacuolisation with osmiophilic lamellar bodies and positive Gb3 immunohistochemistry. In the presence of a hypertrophic cardiomyopathy phenotype, the systematic search for unusual findings is mandatory to rule out a phenocopy.

Nanoparticles decorated carbon nanotubes as novel matrix: A comparative study of influences of immobilization on the catalytic properties of Lensculinarisß-galactosidase (Lcß-gal).

In the present study, Multiwalled carbon nanotubes (MWCNT) decorated with two different nanoparticles namely tungsten disulfide (WS2) and tin oxide (SnO2), nanocomposites (NCs) were synthesized via hydrothermal method. Spectroscopic studies showed that both synthesized NCs possess nearly same functional groups but MWCNT-SnO2 NCs are rich in O-functional group. Microscopic studies revealed that both NCs have different morphological microstructure. Lens culinaris ß-galactosidase (Lcß-gal) was immobilized using glutaraldehyde cross-linker resulted in immobilization efficiency of 91.5% and 88% with MWCNT-WS2 and MWCNT-SnO2 NCs, respectively. Remarkable increase in rate of hydrolysis of whey lactose has been observed with both NCs i.e. Lcß-gal immobilized MWCNT-WS2 hydrolyzes the 97% whey lactose in 1.5 h while MWCNT-SnO2 showed maximum 92% of whey hydrolysis in 2 h at optimum conditions. Both nanobiocatalyst could serve as a promising candidates for dairy industries and would offer a potential platform for enzyme based biosensor fabrication.

Development of 11-DGA-3-O-Gal-Modified Cantharidin Liposomes for Treatment of Hepatocellular Carcinoma.

BACKGROUND: Liver cancer is a common malignant tumor worldwide, and its morbidity and mortality increase each year. The disease has a short course and high mortality, making it a serious threat to human health. PURPOSE: The objective of this study was to create novel liver-targeting nanoliposomes to encapsulate cantharidin (CTD) as a potential treatment for hepatic carcinoma. METHODS: 3-Galactosidase-30-stearyl deoxyglycyrrhetinic acid (11-DGA-3-O-Gal)-modified liposomes (11-DGA-3-O-Gal-CTD-lip) for the liver-targeted delivery of CTD were prepared via the film-dispersion method and characterized. In vitro analyses of the effects on cellular cytotoxicity, cell migration, cell cycle, and cell apoptosis were carried out and an in vivo pharmacokinetics study and tissue distribution analysis were performed. RESULTS: Compared with unmodified liposomes (CTD-lip), 11-DGA-3-O-Gal-CTD-lip showed higher cytotoxicity and increased the inhibition of HepG2 cell migration, but they did not increase the apoptotic rate of cells. The inhibition mechanism of 11-DGA-3-O-Gal-CTD-lip on hepatocellular carcinoma was partly through cell cycle arrest at the S phase. Analysis of pharmacokinetic parameters indicated that 11-DGA-3-O-Gal-CTD-lip were eliminated more rapidly than CTD-lip. Regarding tissue distribution, the targeting efficiency of 11-DGA-3-O-Gal-CTD-lip to the liver was (41.15 ± 3.28)%, relative targeting efficiency was (1.53 ± 0.31)%, relative uptake rate was( 1.69 ± 0.37)%, and peak concentration ratio was (2.68 ± 0.12)%. CONCLUSION: 11-DGA-3-O-Gal-CTD-lip represent a promising nanocarrier for the liver-targeted delivery of antitumor drugs to treat hepatocellular carcinoma.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2'-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2'-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2'-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2'-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis.

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.

Fabry Nephropathy: An Evidence-Based Narrative Review.

Fabry disease (FD) is a rare, X-linked disorder caused by mutations in the GLA gene encoding the enzyme α-galactosidase A. Complete or partial deficiency in this enzyme leads to intracellular accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in many cell types throughout the body, including the kidney. Progressive accumulation of Gb3 in podocytes, endothelial cells, epithelial cells, and tubular cells contribute to the renal symptoms of FD, which manifest as proteinuria and reduced glomerular filtration rate leading to renal insufficiency. A correct diagnosis of FD, although challenging, has considerable implications regarding treatment, management, and counseling. The diagnosis may be confirmed by demonstrating the enzyme deficiency in males and by identifying the specific GLA gene mutation in male and female patients. Treatment with enzyme replacement therapy, as part of the therapeutic strategy to prevent complications of the disease, may be beneficial in stabilizing renal function or slowing its decline, particularly in the early stages of the disease. Emergent treatments for FD include the recently approved chaperone molecule migalastat for patients with amenable mutations. The objective of this report is to provide an updated overview on Fabry nephropathy, with a focus on the most relevant aspects of its epidemiology, diagnosis, pathophysiology, and treatment options.

Mycophenolic acid induces senescence of vascular precursor cells.

OBJECTIVE: Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function. METHODS: We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects. RESULTS: Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated ß-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA. CONCLUSION: Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.

Oxygen concentration modulates cellular senescence and autophagy in human trophoblast cells.

PROBLEM: We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. METHOD OF STUDY: Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O2 concentrations for 24 hours. We examined the extent of senescence caused using senescence-associated ß-galactosidase (SA-ß-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). RESULTS: Physiological normoxia (5% O2 ) decreased SA-ß-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O2 . Pathophysiological hypoxia (1% O2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-ß-Gal-positive cells and SASP secretion) in O2 -independent manner. CONCLUSION: We conclude that O2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence.

Elastin receptor (S-gal) occupancy by elastin peptides modulates T-cell response during murine emphysema.

Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4+ and CD8+ T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response.