Supplemental Material - Respiratory impairments in patients suffering from Fabry disease – A cross-sectional study

Supplemental Material for Respiratory impairments in patients suffering from Fabry disease – A cross-sectional study by Huma Ahmed, Vibeke Backer, Grigoris Effraimidis, Åse Krogh Rasmussen, Caroline Michaela Kistorp and Ulla Feldt-Rasmussen in Chronic Respiratory Disease</p

The influence of ghrelin on the thyroid-stimulating hormone (TSH)-induced increase in thyroglobulin (Tg) and cAMP production.

<p>The influence of ghrelin on the TSH-induced increase in Tg and cAMP production at three different concentrations of TSH (0.1 IU/L, 0.5 IU/L and 1 IU/L). The basal levels, i.e. the values in the absence of TSH, were subtracted, before the groups were compared. Grey = vehicle, pattern = ghrelin (100 nM). Means (+SEM). *P < 0.05 compared to the control (vehicle). <b>A)</b> Ghrelin inhibited the TSH-induced increase in Tg production measured by enzyme-linked immunosorbent assay (ELISA) in primary cultures of human thyroid cells for the TSH concentration of 0.1 IU/L. n = 8 (0.1 IU/L) and n = 6 (0.5 and 1 IU/L) in triplets. Two patient samples were excluded due to lack of basal TSH-induced Tg production. <b>B)</b> No influence of ghrelin on the TSH-induced increase in cAMP production at three different concentrations of TSH (0.1 IU/L, 0.5 IU/L and 1 IU/L) measured by a competitive protein binding method in primary cultures of human thyroid cells. n = 8 (0.1 IU/L and 1 IU/L) and n = 6 (0.5 IU/L) in triplets.</p

Clinical and genetic features of the Fabry patients.

<p>“+” yes</p><p>“-”no</p><p>*Chaparone-therapy.</p><p>ERT: Enzyme replacement treatment</p><p>Cerebrovascular disease: Infarct or hemorrhage</p><p>Cardiovascular disease: Arrhythmia, congestive heart failure or myocardial infarction</p><p>Renal event: Dialysis (D) or kidney transplantation (KT)</p><p>Unknown: U</p><p>Clinical and genetic features of the Fabry patients.</p

Primer sequences for RT-qPCR.

<p>Primer sequences for RT-qPCR.</p

The influence of ghrelin on the thyroid-stimulating hormone (TSH)-induced (0.1 IU/L) mRNA expression of four thyroid components.

<p>The expression of the TSH receptor (TSH-R), thyroperoxidase (TPO), thyroglobulin (Tg) and sodium iodide symporter (NIS) measured by real-time quantitative polymerase chain reaction (RT-qPCR) in a primary culture of human thyroid cells in presence and absence of ghrelin. Indicated as fold change of mRNA expression compared to basal level (dashed line). IL-6 was used as a negative control. Grey = vehicle, pattern = ghrelin (100 nM). Means (±SEM), n = 6. *P < 0.05 compared to the control (vehicle). Two patients were excluded due to unknown sample material.</p

Ghrelin receptor (GhrR) mRNA expression level.

<p>GhrR mRNA expression level in relation to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression level in human brain, thyroid tissue and cell cultures measured by real-time quantitative polymerase chain reaction (RT-qPCR). n = 2.</p

Quantitative 3-dimensional surface projection-analysis of FDG-uptake in patient no 25.

<p>PET-images of patient no. 25 are normalized to whole brain using a database of normal subjects and scaled to Z values from - 4.0 to 4.0 (Neurostat). Projections of Z values are shown onto, respectively, the right and left lateral hemispheric surfaces, the superior and inferior surfaces, and the anterior and posterior surfaces. The uptake in the left cerebellar hemisphere is reduced in addition to uptake in the right frontal cortex. MRI surface projections are presented for anatomical reference in a standard stereotactic space</p

Influence of Phthalates on Cytokine Production in Monocytes and Macrophages: A Systematic Review of Experimental Trials

<div><p>Background</p><p>Phthalates are a group of endocrine disrupting chemicals suspected to influence the immune system. The aim of this systematic review is to summarise the present knowledge on the influence of phthalates on monocyte and macrophage production and secretion of cytokines, an influence which could affect both pro- and anti-inflammatory abilities of these cells.</p><p>Strategy and Results</p><p>A systematic search was performed in Medline, Embase and Toxline in June 2013, last updated 3rd of August 2014. Criteria used to select studies were described and published beforehand online on Prospero (<a href="http://www.crd.york.ac.uk/NIHR_PROSPERO" target="_blank">http://www.crd.york.ac.uk/NIHR_PROSPERO</a>, registration number CRD42013004236). <i>In vivo</i>, <i>ex vivo</i> and <i>in vitro</i> studies investigating the influence of phthalates on cytokine mRNA expression and cytokine secretion in animals and humans were included. A total of 11 reports, containing 12 studies, were found eligible for inclusion. In these, a total of four different phthalate diesters, six primary metabolites (phthalate monoesters) and seven different cytokines were investigated. Though all studies varied greatly in study design and species sources, four out of five studies that investigated di-2-ethylhexyl phthalate found an increased tumour necrosis factor-α secretion/production from monocytes or macrophages. A summary of cytokine measurements was not possible since few studies were comparable in study design and due to insufficient reporting of raw data for most of the included studies.</p><p>Conclusion</p><p>Results from this review have suggested that at least one phthalate (di-2-ethylhexyl phthalate) has the ability to enhance tumour necrosis factor-α production/secretion from monocytes/macrophages <i>in vitro</i>, but also observed <i>ex vivo</i>. Influence of other phthalates on other cytokines has only been investigated in few studies. Thus, <i>in vitro</i> studies on primary human monocytes/macrophages as well as more <i>in vivo</i> studies are needed to confirm or dispute these findings.</p></div

Primary and secondary outcomes from individual studies.

<p>Footnote: BBzP: Butylbenzyl phthalate, DEHP: Di-2-ethylhexyl phthalate, DiNP: Di-iso-nonyl phthalate, DnBP: Di-n-butyl phthalate, IL: Interleukin, LDH: lactate dehydrogenase, MEHP: Mono-(2-ethylhexyl) phthalate, ND: not done, PI: Propidium Iodide, RAW 264 cell line: mouse leukemic monocyte-macrophage cell line, SD: standard deviation, SEM: standard error of the mean, THP-1 cell line: acute monocytic cell line, TNF: Tumour necrosis factor.</p><p>Primary and secondary outcomes from individual studies.</p

FDG-PET and MRI-features of the Fabry patients.

<p><b><i>n</i>.<i>a</i>.</b><i>not applicable</i></p><p><b><i>-</i></b><i>WML grade 0 or pathology not present</i></p><p><b><i>- / -</i></b><i>no changes in neither PET nor MRI</i></p><p>^<b>a</b> Patient no. 5: symmetrical mildly reduced activity parietotemporally bilaterally</p><p>^<b>b</b> Patient no. 22: symmetrical mildly reduced activity in both thalami <i>Cont</i>.</p><p>^<b>c</b> Progression of pathology on either PET or MRI was detected in the following patients:</p><p>Patient no. 3: PET-study period: seven years</p><p>Patient no. 4: PET-study period: six years.</p><p>Patient no. 8: MRI study period: three years</p><p>Patient no. 9: PET-study period: five years. MRI study period: seven years.</p><p>Patient no. 19: MRI study period: five years</p><p>Patient no. 25: PET/MRI study period: two years.</p><p>FDG-PET and MRI-features of the Fabry patients.</p