Den snart Dr-titulerade forskaren Eva Milgbauer från Linköpings Uni har tillsammans med kollegor från 3 andra säten fått en studie godkänd och publicerad. Innehållet i den ser ut att vara en vidareutveckling från hennes doktorsavhandling. Tyvärr är den inlåst, men Mr Google låter oss få tillräcklig info att se att HoloMonitor använts vid studierna.

Faradaic Fenton Pixel – Reactive Oxygen Species Delivery using Au/Cr Electrochemistry

First published: 15 May 2023

Abstract

Reactive oxygen species (ROS) are an integral part of many anticancer therapies. Fenton-like processes involving reactions of peroxides with transition metal ions are a particularly potent and tunable subset of ROS approaches. Precise on-demand dosing of the Fenton reaction is an area of great interest. Herein, we present a concept of an electrochemical faradaic pixel which produces controlled amounts of ROS via a Fenton-like process. The pixel comprises a cathode and anode, where the cathode reduces dissolved oxygen to hydrogen peroxide. The anode is made of chromium, which is electrochemically corroded to yield chromium ions. Peroxide and chromium interact to form a highly oxidizing mixture of hydroxyl radicals and hexavalent Cr-ions. After benchmarking the electrochemical properties of this type of device, we demonstrate how it can be used under in vitro conditions with a cancer cell line. The faradaic Fenton pixel is a general and scalable concept that can be used for on-demand delivery of redox-active products for controlling a physiological outcome.

Vidimering :

Forskningsrapport nr 2 släpptes för publicering idag, 22/5 och är betitlad :

Mast Cell Tryptase Promotes Airway Remodeling by Inducing Anti-Apoptotic and Cell Growth Properties in Human Alveolar and Bronchial Epithelial Cells

Published: 22 May 2023

8 forskare från Sverige och Danmark är författare till studien.Här låter jag google översätta abstract och slutsats.

Abstrakt

Bronkial och alveolär ombyggnad och nedsatt epitelfunktion är egenskaper hos kroniska andningssjukdomar.

Hos dessa patienter infiltrerar ett ökat antal mastceller (MC) som är positiva för serinproteaser, tryptas och chymas, epitel och alveolär parenkym. Emellertid är lite känt om implikationen av intraepitelial MC på den lokala miljön, såsom epitelcellfunktion och egenskaper. I denna studie undersökte vi om MC-tryptas är involverat i bronkial och alveolär remodellering och mekanismerna för reglering under inflammation.

Med hjälp av ny holografisk levande cellavbildning fann vi att MC-tryptas förbättrade human bronkial och alveolär epitelcellstillväxt och förkortade celldelningsintervallen. Den förhöjda celltillväxten inducerad av tryptas förblev i ett proinflammatoriskt tillstånd. Tryptas ökade också uttrycket av det anti-apoptotiska proteinet BIRC3, liksom frisättning av tillväxtfaktor i epitelceller. Således innebär våra data att intraepitelial och alveolär MC-frisättning av tryptas kan spela en kritisk roll för att störa bronkial epitelial och alveolär homeostas genom att förändra celltillväxt-dödsreglering.

2. Materials and Methods

2.3. Cigarette Smoke Extraction

Cigarette smoke extract (CSE) was prepared according to Gellner et al. (2017) [21]. Research cigarettes (University of Kentucky, USA) were kindly provided by Prof. Arne Egesten, Lund University. Briefly, 8 cigarettes were marked with a permanent marker 23 mm from the end and attached to the apparatus containing 40 mL RPMI medium. Cigarette smoke extraction was performed inside a fume hood. The cigarettes were lighted and slow puffs (2–4 s) were made via a vacuum suction and repeated every 30 s until the marking of the cigarette was reached (total of 10–12 puffs per cigarette). A new cigarette was inserted into the holder and this procedure was repeated until all of the cigarettes had been smoked. This solution was defined as 100% CSE. The CSE was aliquoted and stored in a −20 °C freezer until use. To quantitate an appropriate dose for the cell experiments, dose–response experiments using the HoloMonitor and LDH assay analysis of the supernatants were performed.

2.4. Holographic Live Cell Imaging (HoloMonitor M4)

Bronchial and alveolar cell growth and cell division properties were studied using the HoloMonitor M4 live cell imaging system (Phase Holographic Imaging, Lund, Sweden). Cells were cultured in Sarstedt TC 6-well plates (Nümbrecht, Germany) 24 h prior to stimulation, at a cell concentration of 10,000 cells/cm2. A total of 7–10 focus points were taken per well/stimulation and images were captured every 15 min over 72 h, giving >2000 images per well and per repeat. To investigate the effect of tryptase on cell growth and division rate, data were based on three independent repeats and at least 15 focus points/stimulation. Cell growths are presented as a percentage of cell growth (relative to the starting number of cells) or number of cells at an indicated time point.

3. Results

3.1. Mast Cell Tryptase Enhances Cell Growth in Alveolar and Bronchial Epithelial Cells

To better understand the implications of MC infiltration into the airways, this study focused on the role of MC proteases on alveolar and bronchial epithelial cell growth and homeostasis. By using holographic live cell imaging, we were able to perform thorough and detailed cell growth analysis, comparing the cell growth of untreated and tryptase-treated cells. Images (567 µm × 567 µm, 20× magnification) were captured every 15 min over 72 h, generating 288 images per focus point and ≥5 focus points per well, and stimulation was analyzed. In total, this resulted in ≥1440 analyzed images and approximately 375 analyzed cells per every single treatment and per repeat. As shown in the cell growth curves (Figure 1A,C), tryptase enhanced epithelial cell growth, both in AECs and BECs when compared to the non-stimulated (NS) cells. At 72 h, we found significantly elevated cell growth in the tryptase-treated cells compared to the untreated cells (Figure 1B,D) (AEC NS: 82 cells ± 41, AEC T: 120 cells ± 43, p = 0.009, BEC NS: 88 cells ± 42, BEC T: 138 cells ± 63, p = 0.003). Representative images were captured using the HoloMonitor M4 at the starting time (0 h) and 72 h post-treatment. Optimal cell viability and proliferation over time were observed in all groups using the HoloMonitor M4.

Figure 1. Holographic live cell imaging was used to investigate alveolar and bronchial cell growth. The growth curves show that MC tryptase increased the cell growth over time in both AECs (A) and BECs (C), compared to the NS cells. At 72 h, tryptase significantly enhanced the cell growth in alveolar and bronchial cells (B,D), in comparison to the NS cells. Data were based on three independent experiments and >15 focus points per group (≥4320 images/group across 72 h). Representative images (E) of alveolar (left) and bronchial (right) epithelial cells taken using the HoloMonitor M4 demonstrating cell numbers for NS and tryptase-treated cells at 0 h and 72 h.
A PAR-2 inhibitor, I-191, was used to test whether the elevated cell growth induced by tryptase was a result of PAR-2 activation in the AECs. The results obtained using HoloMonitorM4 showed no statistical difference in cell growth when comparing NS alveolar epithelial cells with cells pre-stimulated with I-191 and then stimulated with tryptase (NS: 82 cells ± 41, T + I-191: 97 cells ± 40) (Figure 1F,G). As a control, we also analyzed cell growth in AECs treated with the inhibitor alone (100 cells ± 34), which did not induce a statistical difference in cell growth in comparison to NS.

Figure 3. Tryptase altered cell division rates in alveolar and bronchial epithelial cells. Two-dimensional and three-dimensional images captured using HoloMonitor M4 illustrating cells’ pre- and post-cell division with a 15 min time difference (A). Tryptase induced significant shorter cell division intervals in both early and late phases of the experiment in AECs, compared to NS cells (B), whereas tryptase effect on BECs was not seen in the early phase but the later phase of the experiment (C). Values are shown as mean of three independent experiments and a minimum of 10 cells/group, and images were manually tracked throughout the experiment (144 pictures across 36 h). The analysis of individual cells showed a reduction in the time interval between cell divisions in tryptase-treated cells, both for AECs (D) and BECs (E). (F) (AECs) and (G) (BECs) demonstrate the obtained data from the HoloMonitorM4 in a schematic illustration of cell division properties of NS and tryptase-stimulated cells. (D–G) are based on tracking a minimum of 8 cells per image and stimulation across 72 h (in total more than 288 images/stimulation analyzed). Statistical significance between early and late responses was tested using the Mann–Whitney test. Data represent the mean (±SD). ** p < 0.01, **** p < 0.0001.

5. Slutsatser

I denna studie avslöjar våra resultat att tryptas utövar förmågan att inducera celltillväxt och förkortning av celldelningsintervall och förändra anti-apoptotisk reglering och tillväxtfaktorfrisättning i både humana alveolära och bronkiala epitelceller. Dessutom visar vi att tryptasbehandlade primära bronkialepitelceller från astmapatienter förbättrade proteinuttrycket av det anti-apoptotiska proteinet BIRC3, förändrade frisättningen av tillväxtfaktor och förhöjde uttrycket av den proliferativa markören Ki67, även om ingen funktionell skillnad observerades. Sammantaget innebär våra resultat att intraepitelial MC och deras frisättning av tryptas kan vara nyckelaktörer i luftvägsremodellering genom att störa normal celltillväxt-dödshomeostas och därmed främja epitelcelltillväxt och överlevnad och eventuellt andra strukturella celler i lungvävnaden också. Förutom att utveckla begreppet MCs roll vid kroniska luftvägssjukdomar, kan dessa observationer ge viktiga insikter om hur man kan förbättra behandlingsstrategier genom att rikta tryptas.

Acknowledgments

We want to thank Phase Holographic Imaging, PHI, and especially Kersti Alm for their kind and invaluable support with the HoloMonitorM4.

Min kommentar
Lite ovanligt numera att se HoloMonitor användas utanför cancerforskningen, men en bra påminnelse att konstatera instrumentets versatilitet. Forskarnas krav på att studera ickeinfärgade levande celler vinner mark.

Mvh the99

Tillägg

Alliance for Regenerativ Medicine och deras lobbande i Washington Capitol Hill börjar ge resultat.

This week, the House Health Subcommittee markup included the Medicaid VBPs for Patients (MVP) Act.

We applaud the subcommittee for advancing this important legislation, which will promote value-based payments of cell and gene therapies for patients.

This bipartisan legislation is a step in the right direction to promoting access to cell and gene therapies for Medicaid patients who need them.

We look forward to continuing our work with the bill sponsors and hope to see it advance out of the Energy and Commerce Committee.

Översättning :

Den här veckan inkluderade House Health Subcommittee-markeringen Medicaid VBP for Patients (MVP) Act.

Vi applåderar underutskottet för att ha främjat denna viktiga lagstiftning, som kommer att främja värdebaserade betalningar av cell- och genterapier för patienter.

Denna tvåpartslagstiftning är ett steg i rätt riktning för att främja tillgången till cell- och genterapier för Medicaid-patienter som behöver dem.

Vi ser fram emot att fortsätta vårt arbete med lagförslagssponsorerna och hoppas att det går vidare från energi- och handelsutskottet.

Som service till alla ev HoloMonitornyfikna forskare : HoloMonitor Demo

THE99

måndag 22 maj 2023

2 nya forskningsrapporter