Molecular mechanisms of pancreas development and insulin regulation in beta cells.

2006/2007Type 1 or “juvenile” Diabetes is an autoimmune disease in which the insulin secreting beta-cells, essential for glucose homeostasis, are destroyed by a target immune attack. To date, patients with type 1 Diabetes can rely only on exogenous insulin injection to control the glucose levels, but, unfortunately, the related chronic and devastating complications cannot be prevented. Identification of beta-cell-specific genes playing critical roles during the development will help to create new sources of beta-cells from stem/progenitor cells. Interestingly, endocrine cell fate appears to be governed by the “peaked” expression of the transcription factor Ngn3. However, Ngn3’s targets are still not well identified. As for pancreas development also insulin gene regulation lacks important information. Therefore the aims of this Thesis work have been to test an set-up an efficient system for the transient expression of Ngn3, mimicking its physiological behavior; identify the major factors that lie directly downstream the Ngn3 expression and characterize a novel beta-cell specific insulin gene regulator. As result from this work, an innovative system mimicking pancreatic differentiation and Ngn3 pulsed expression has been developed. Furthermore, a novel Ngn3’s downstream target, the zinc-finger protein “OVO_like 1, has been identified opening a new, and interesting, scenario of Ngn3 gene’s regulation. Last, but not least, the beta-cell-insulin-gene regulator A2.2 has been characterized and a reproducible purification system has been developed.197

β-MSCs: Successful fusion of MSCs with β-cells results in a β-cell like phenotype

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, antiapoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow-derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes. Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherryexpressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment. With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP. After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of betacell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes

Enhanced-Precision Measurement of Glutathionyl Hemoglobin by MALDI-ToF MS

Glutathionyl-hemoglobin (HbSSG) is used as a human biomarker to pinpoint systemic oxidative stress caused by various pathological conditions, noxious lifestyles, and exposure to drugs and environmental or workplace toxicants. Measurement by MALDI mass spectrometry is most frequently used, however, the method suffers from excessive uncontrolled variability. This article describes the improvement of a MALDI-ToF mass spectrometry method for HbSSG measurement through enhanced precision, based on strict control of sample preparation steps and spreadsheet-based data analysis. This improved method displays enhanced precision in the analysis of several hundred samples deriving from studies in different classes of healthy and diseased human subjects. Levels span from 0.5% (lower limit of detection) up to 30%, measured with a precision (as SE%) < 0.5%. We optimized this global procedure to improve data quality and to enable the Operator to work with a reduced physical and psychological strain. Application of this method, for which full instruction and the data analysis spreadsheet are supplied, can encourage the exploitation of HbSSG to study human oxidative stress in a variety of pathological and living conditions and to rationally test the efficacy of antioxidant measures and treatments in the frame of health promotion

Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

AIMS: Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. METHODS: The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. RESULTS: Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. CONCLUSIONS: This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels

Differential Redox State and Iron Regulation in Chronic Obstructive Pulmonary Disease, Acute Respiratory Distress Syndrome and Coronavirus Disease 2019

In patients affected by Acute Respiratory Distress Syndrome (ARDS), Chronic Obstructive Pulmonary Disease (COPD) and Coronavirus Disease 2019 (COVID-19), unclear mechanisms negatively interfere with the hematopoietic response to hypoxia. Although stimulated by physiological hypoxia, pulmonary hypoxic patients usually develop anemia, which may ultimately complicate the outcome. To characterize this non-adaptive response, we dissected the interplay among the redox state, iron regulation, and inflammation in patients challenged by either acute (ARDS and COVID-19) or chronic (COPD) hypoxia. To this purpose, we evaluated a panel of redox state biomarkers that may integrate the routine iron metabolism assays to monitor the patients’ inflammatory and oxidative state. We measured redox and hematopoietic regulators in 20 ARDS patients, 20 ambulatory COPD patients, 9 COVID-19 ARDS-like patients, and 10 age-matched non-hypoxic healthy volunteers (controls). All the examined pathological conditions induced hypoxia, with ARDS and COVID-19 depressing the hematopoietic response without remarkable effects on erythropoietin. Free iron was higher than the controls in all patients, with higher levels of hepcidin and soluble transferrin receptor in ARDS and COVID-19. All markers of the redox state and antioxidant barrier were overexpressed in ARDS and COVID-19. However, glutathionyl hemoglobin, a candidate marker for the redox imbalance, was especially low in ARDS, despite depressed levels of glutathione being present in all patients. Although iron regulation was dysfunctional in all groups, the depressed antioxidant barrier in ARDS, and to a lesser extent in COVID-19, might induce greater inflammatory responses with consequent anemia

Comparison of the C_T-values generated by Series A&B.

<p>Real-Time PCR quantification of blood samples collected with Tempus^TM and PAXgene^TM collection tubes and RNA extracted by Norgen. <b>(A)</b> Series A: Norgen RNA extraction with 500 ng RNA input (n = 12 each for Tempus and for PAXgene blood collection), <b>(B)</b> Series B: Norgen RNA extraction and a second DNase1 digestion with 250 ng input RNA (n = 12 each for Tempus^TM and PAXgene^TM collection tubes, experimental triplicates from DNA digestion onwards). Data are means of all donors ± SE *p<0.05 vs. PAXgene collection tubes.</p

Study overview and RNA yields.

<p><b>(A)</b> Study workflow of sample collection and RNA extraction. In the first sample set (n = 12 donors, 2 samples from each), all samples were extracted with the corresponding commercial Norgen Biotek kit, specified for both tested collection systems (series A&B). Series B was additionally DNase1 digested. A second sample set (series C; n = 5 donors, with 8 separately collected tubes from each donor for PAX and Tempus^TM) was tested with the recommended PAXgene^TM blood RNA isolation kit. <b>(B)</b> RNA yield obtained with spectrophotometry (Nanodrop photometer) and <b>(C)</b> intact RNA yield measured with the Ribogreen method. ¹⁾In 5 random samples no RNA was detectable. Therefore, those samples were excluded from calculations. *p<0.05 vs. PAXgene collection tubes/ Norgen RNA extraction, **p<0.05 vs. Tempus collection tubes/ Norgen RNA extraction and PAXgene collection tubes/ Norgen RNA extraction.</p

Coupling of PAX collection tubes with PAXgene RNA extraction kit improved RNA stability, quantity and quality and enhanced reproducibility.

<p>Plotted dots of donors 1–12 from different reference C_T values from the analyses in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161778#pone.0161778.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161778#pone.0161778.g004" target="_blank">4</a> (Series A,B,C). Results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161778#pone.0161778.g002" target="_blank">Fig 2</a> were used and C_T values from house keeping genes were compared from the 12 different donors. Data are obtained <b>(A)</b> from series A with 500 ng RNA (n = 12; both sampling systems), <b>(B)</b> from series B with 250 ng RNA with additional DNase1 digestion and (<b>C)</b> 5 donors with 4 samples each from Tempus^TM and PAXgene^TM collection (100 ng each measured in experimental duplicates).</p