46 research outputs found
Original Article
The present paper deals with an investigation on the changes appearing in the mucous membrane of the nose (physiologic atrophy) in normal persons of different age groups, as contrasted with a wasting of the mucous mambrane of the nose in cases of atrophic rhinitis. The investigation has been performed for the purpose of contributing to the studies of the pathology of atrophic rhinitis. 1. Pathologic changes of a considerable degree were. observed in the epithelium in quite a large section of infants and children where it had been considered normal as a results of macroscopic examinations. 2. Metaplasia of the epithelial cells developing in the mucous membrane in the forepart of the respiratory region seems to occur as a result of the stimulus applied from without. The phenomenon was marked in the front and along the lower edge of the inferior turbinal, showing a tendency to increase in magnitude as the age advance. It did not, however, spread over a wide area, nor was there any marked development of cornification. An increase in mucus secretion, as well as in the number of goblet cells, was noticed in the epithelium as the age advance. Mucous degeneration gradually set in at the end of forties, becoming marked in the sixties. 4. In the basal membrane, the hyaline layer, which is its secondary form, grew in size with age, and a substance which stains with Hale\u27s stain was detected in it. This substance seems to have an important share in the mucus secreting function of the epithelium. 5. It seems that the epithelium of the mucous membrane of the upper respiratory tract continues to function even in considerably advanced ages. 6. The lymphoid tissue situated underneath the epithelium attained the largest quantity in persons about 20 years old; it began to diminish and grow less thick in persons over 40. The presence of the elastic fiber was noticed in the subepithelial layer in all age groups, though the number of persons with this phenomenon was small.7. The glands wers under-developed in children of about 10; they grew rapidly after that age until about 40 when they began to show a tendency to atrophy. 8. It seems that the periglandular lymphocytes, which infiltrate without bringing about the disintegration of the glands, take charge of the metabolism of the glands. A large number of them were found in infancy but they showed a marked decrease in number in persons over about 40. It would seem that, in highly advanced ages, non-inflammatory disintegration of the glands could possibly occur as a result of the infiltration of the lymphoid tissue. 9. The formation of the oncocyte, an unusual cell of the epithelium of the gland which characterizes the old age, was noticed in 7 cases. 10. The blood vessels manifested changes of a high degree in persons of advanced ages: they revealed evidences of functional disturbance of a high degree when stained by the stains of H. E, Weigert, PAS and Hale. This would show the measure of the influence that has been exerted on the function of the mucous membrane. 11. Corpora cavernosa was under-developed in infancy but became well-developed in persons of about 20; a decrease in the number of bodies and a diminution in size of the inner lumen became marked in persons over 40, becoming more marked in persons over 50
Trehalose induces autophagy and activates autophagic flux in CHIP-mutant fibroblasts.
<p>The cells were treated for 24 h with o without trehalose 50 mM in the presence or absence of 10 ĀµM chloroquine (CQ), a lysosomotropic agent widely used to block lysosomal degradation. <b>A</b>) Fluorescence microscopy images showing autophagosome marker LC-3 (green), lysosomal marker Lamp-2A (red) and LC3-LAMP-2A colocalization (yellow). (Scale bar ā=ā20 Āµm). <b>B</b>) Integrated optical density (IOD) quantification of LC3 expression. <b>C</b>) The lysosomal degradation was measured by IOD quantification of colocalization LC-3-LAMP-2A (yellow). <b>D</b>) Western blot showing LC3-II accumulation. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean Ā± SEM. In 7B and 7C values are the mean Ā± S.E.M. of six independent coverslips (each value represents the mean of 20 field for coverslip). Three individuals with the mean of 2 independent coverslips (nā=ā3) in the control group and in the CHIP-mutant group 6 independent coverslips of the only patient (pseudo-replicates, nā=ā6). In 7D values represents the mean of 2 independent dishes of cells of 2 controls (nā=ā2) and for the CHIP-mutant group one patient with four independent dishes (pseudo-replicates, nā=ā4). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents without chloroquine; Ī“Ī“p<0.01, Ī“Ī“Ī“p<0.001 trehalose -CQ <i>vs</i> trehalose + CQ treated cultures.</p
ROS production, Glutathione homeostasis and Mitochondrial membrane potential.
<p><b>A</b>) Representative images of DCF staining in control and CHIP-mutant fibroblasts for the indicated treatment. Scale bar ā=ā10 Āµm. <b>B</b>) Percentage of DCF positive fibroblasts. One hundred fifty to two hundred cells per coverslip cells were counted. <b>C</b>) GSH levels in control and CHIP mutant human fibroblasts with 7 and 12 passes. <b>D</b>) Representative images of MitoTracker TM Ros and nuclei Hoescht staining in control and CHIP-mutant fibroblasts cultured in the presence of epoxomicin (10 nM) and/or trehalose (50 mM). Yellow arrows indicate small depolarized mitochondria (scale bar ā=ā30 Āµm). <b>E</b>) Quantification in IOD of fibroblasts with altered mitochondria morphology by fluorescence imaging analysis in 10 random fields of 6 coverslips from control and CHIP-mutant cultures (Scale bar ā=ā15 Āµm). <b>F</b>) Complex IV staining showed an abundant and granular perinuclear pattern indicating mitochondrial compartmentalization (scale bar ā=ā10 <i>Āµ</i>m). A cell of this micrograph has been magnified in the pictures below. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean Ā± SEM. Values of 3B are the mean of two independent experiments of 4 independent cell dishes ānā=ā4ā (pseudo-replicates) of the patient group and in control group ānā=ā2ā: the mean of 2 different controls with 4 dishes of cells each one. In 3C and 3E values are the mean Ā± SEM from two experiments with 6 independent cells dishes (nā=ā6, pseudo-replicates). Statistical analysis was performed by Student's t-test. *p<0.01, **p<0.01, ***p<0.001 vs solvents; Ī“p<0.05, Ī“Ī“p<0.01, Ī“Ī“Ī“p<0.001 trehalose + epoxomicin vs epoxomicin treated cultures; ā§ā§ā§p<0.001 fibroblasts with 12 <i>vs</i> 7 passages.</p
Proliferation and apoptosis of human fibroblasts with compound heterozygous CHIP mutations.
<p>Trehalose protects from apoptosis and epoxomicin-induced cell death in CHIP-mutant human fibroblasts. <b>A</b>) Photomicrographs of dose-dependent effects of epoxomicin and trehalose pre-treatment 15 min before epoxomicin for 24 h on the expression of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts (scale bar ā=ā30 Āµm). <b>B</b>) Percentage of cleaved caspase-3 positive cells in control and CHIP-mutant fibroblasts treated with 0, 2 and 10 nM epoxomicin for 24 h and trehalose 50 mM pre-treatment after 5 days in vitro. One hundred fifty to two hundred cells per coverslip cells were counted. <b>C</b>) Photomicrographs of dividing BrdU<sup>+</sup> cells and total nuclei stained with bis-benzimide. (Scale bar ā=ā30 Āµm). <b>D</b>) Percentage of BrdU<sup>+</sup> cells with respect to the total number of fibroblasts with 7 and 12 passages, respectively. Sixty to eighty cells were counted per coverslip to obtain the percentages of BrdU<sup>+</sup> cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean Ā± SEM. Values are the mean of two experiments of 6 independent cell dishes (pseudo-replicates, nā=ā6) of the patient group and in control group: the mean of the 3 different controls with 3 dishes of cells each one (nā=ā3). Statistical analysis was performed by Student's t-test. **p<0.01, ***p<0.001 <i>vs</i> solvents; Ī“Ī“Ī“ p<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin; ā§ā§ā§p<0.001 fibroblasts with 12 <i>vs</i> 7 passages.</p
Trehalose increases autophagy activity in CHIP-mutant human fibroblasts.
<p>After 5 DIV, fibroblasts were pre-treated with trehalose (50 or 100 mM) for 15 minutes followed by addition of epoxomicin or solvent for another 24 h. <b>A</b>) LC3 staining (green) and nuclei (Hoescht, blue) immunofluorescence in CHIP-mutant fibroblast treated with trehalose (100 mM) and their quantification <b>B</b>) in percentage of cells with more than 50 autophagic vesicles (Scale bar ā=ā20 Āµm). <b>C</b>) Representative microphotographs of Lysosomal-associated membrane protein, LAMP-2A, present in lysosomes and endosomes, (Scale bar ā=ā20 Āµm) and <b>D</b>) quantification in percentage of cells with LAMP-2A positive vesicle distribution in the perinuclear region, around the nucleus. <b>E</b>) Images of fibroblasts expressing CD63, as a late endo/lysosomal marker (Scale bar ā=ā30 Āµm) and <b>F</b>) quantification in percentage of optical intensity for CD63 inmunoreactivity. The insets show boxed regions at higher magnification (Scale bar ā=ā15 Āµm). <b>G</b>) Immunofluorescence for HSC70 and LAMP-2A in CHIP-mutant cultures treated with trehalose 100 mM for 48 h (Scale bar ā=ā20 Āµm) and <b>H</b>) percentage of co-localization LAMP-2A/HSC70 positive cells. <b>I</b>) Percentage of LAMP-2A positive cells. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean Ā± SEM. Values in 5B, 5D, 5H and 5I are the mean Ā± S.E.M. of two experiments with six cover slips, from 150 to 250 cells of every group were counted. In 5F, in CD63 immunostaining data are the mean of 4 independent dishes of cells for 2 control individuals (nā=ā2), in the CHIP-mutant group 6 independent dishes of cells of the only patient (pseudo-replicates, nā=ā6). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents; Ī“p<0.05, Ī“Ī“Ī“p<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin treated cultures.</p
Expression of p62, Atg5āAtg12 and Beclin-1 in controls and CHIP-mutant fibroblasts.
<p>Trehalose treatment increases the autophagic activity. Fibroblasts were exposed to 50 mM trehalose for 24 hours and the autophagy-related proteins were analyzed by Western blot. <b>A</b>) Western blot showing the autophagic degradation of p62, a major selective substrate for autophagy. <b>B</b>) Beclin-1 and <b>C</b>) Atg5-12, essential factors for autophagosome formation were increased by trehalose. Data of the control and CHIP-mutant groups were analyzed independently, no inter-group statistical analysis was performed. Data are expressed as the mean Ā± SEM. Values are the mean of two experiments of independent dishes of one patient (pseudo-replicates, nā=ā4ā6) and for control group 3 different controls with the mean of different dishes of cells (nā=ā3). Statistical analysis was performed by Student's t-test. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> solvents; Ī“Ī“p<0.01, Ī“Ī“Ī“p<0.001 trehalose + epoxomicin <i>vs</i> epoxomicin treated cultures.</p
Additional file 1: of C9orf72 is differentially expressed in the central nervous system and myeloid cells and consistently reduced in C9orf72, MAPT and GRN mutation carriers
Supplementary Data. Figure S1: Definition of TSSs at the C9orf72 locus; Figure S2: C9orf72 expression changes in challenged CD14+ monocytes. Figure S3: Distinct TSSs expression at C9orf72 locus in CNS, myeloid and lymphoid cells. Figure S4: Sense and antisense C9orf72 transcripts at the C9orf72 locus. Figure S5: Distinct C9orf72 TSSs expression in control brains. Figure S6: C9orf72 expression in adult and fetal cortex. Figure S7: C9orf72 expression level in CD14+ monocytes, brain tissue and microglia. Figure S8: C9orf72 expression in brains of patients with different neurodegenerative diseases. Figure S9: C9orf72 expression in CD14+ monocytes. Suppl. excel File 1: WGCNA results. Suppl. excel File 2: Correlations values between C9orf72 TSS(s) and all the other TSSs in the co-expressed modules. Table S1: Expression of C9orf72 TSSs as defined in CAGEseq dataset 1. Table S2: Expression of C9orf72 TSSs as defined in CAGEseq dataset 2. Table S3: List of primers used in this study. Table S4: Biological functions significant to the three modules related to C9orf72 TSSs as identified by WGCNA. Table S5: Biological functions significant for genes that correlate with C9orf72 TSSs in the WGCNA identified modules. Table S6: Summary of the Mann-Whitney test performed on NRQ values from qPCR experiments on medial frontal gyrus. (ZIP 20675ąø¢ļæ½kb
Mutation Frequency of the Major Frontotemporal Dementia Genes, <i>MAPT</i>, <i>GRN</i> and <i>C9ORF72</i> in a Turkish Cohort of Dementia Patients
<div><p><i>āMicrotubule-associated protein tauā (MAPT)</i>, <i>āgranulinā (GRN)</i> and ā<i>chromosome 9 open reading frame72ā</i> (<i>C9ORF72)</i> gene mutations are the major known genetic causes of frontotemporal dementia (FTD). Recent studies suggest that mutations in these genes may also be associated with other forms of dementia. Therefore we investigated whether <i>MAPT</i>, <i>GRN</i> and <i>C9ORF72</i> gene mutations are major contributors to dementia in a random, unselected Turkish cohort of dementia patients. A combination of whole-exome sequencing, Sanger sequencing and fragment analysis/Southern blot was performed in order to identify pathogenic mutations and novel variants in these genes as well as other FTD-related genes such as the ā<i>charged multivesicular body protein 2Bā (CHMP2B)</i>, the ā<i>FUS RNA binding proteinā (FUS)</i>, the ā<i>TAR DNA binding proteinā (TARDBP)</i>, the ā<i>sequestosome1ā (SQSTM1)</i>, and the ā<i>valosin containing proteinā (VCP)</i>. We determined one pathogenic <i>MAPT</i> mutation (c.1906C>T, p.P636L) and one novel missense variant (c.38A>G, p.D13G). In <i>GRN</i> we identified a probably pathogenic TGAG deletion in the splice donor site of exon 6. Three patients were found to carry the GGGGCC expansions in the non-coding region of the <i>C9ORF72</i> gene. In summary, a complete screening for mutations in <i>MAPT</i>, <i>GRN</i> and <i>C9ORF72</i> genes revealed a frequency of 5.4% of pathogenic mutations in a random cohort of 93 Turkish index patients with dementia.</p></div
LD-plot of the region of interest in PCLO.
<p>The SNPs with the lowest P-values, rs2715147 and rs2715148 are in high LD with eachother and with rs2522833. This supports the hypothesis that either rs2522833 or a SNP in high LD with it is the most likely causal variant in this cohort.</p
Base pairs and percentage of region covered on the Sequence Capture arrays.
<p>Base pairs and percentage of region covered on the Sequence Capture arrays.</p