Bloggen har fått mejl (1/2)

Titt som tätt mejlar phi,are bloggen frågor men även kanske användbar information.Mycket av det som man vill jag ska kika närmare på är inte riktigt av den kalibern att jag tycker det är värt att uppmärksamma på bloggen.Till de som skickar sådan information återkopplar jag med anledning till icke publicerat material,det om man lämnar mejladress, Men nu har jag fått in 2 tips att kika närmare på och det har jag alltså gjort då dessa håller bra nivå.Först en forskningsrapport från Ungern,närmare bestämt från phi-kända universitetet Semmelweis.

Tyvärr hittar jag ingen engelsk version av studien så vi får hålla till godo med ungersk text.Eftersom studien är 108 sidor lång finns det ingen chans att sitta och översätta sida efter sida.Istället har jag valt ut stycken som berättar vad denna studie handlar om.Det är alltså en doktorsavhandling som då naturligtvis är tämligen gedigen och väl genomgången med referenser till 160 tidigare studier som bas.

Forskare från Semmelweis har sen tidigare specialiserat sig på åkommor som uppstår vid en graviditet.Åkommor i den bemärkelsen att graviditeten kan utlösa och resultera i allvarliga sjukdomsfall som ex cancertumörer och i värsta fall dödsfall.Söker man på Semmelweis universitet hos ställen forskare får sina studier publicerade hittar man volymer just med studier inom detta område.Exempelvis denna studie som Nature publicerade 2018. https://www.nature.com/articles/s41598-018-23706-7

Innehållet i denna doktorsavhandling handlar om immunsystemets funktion hos den gravida kvinnan och det foster hon bär på.Att dessa 2 immunsystem kan "krocka" med varandra och resultera i ex avstötning av fostret.

Men till själva studien,som i översättning blir The mechanism of pregnancy immune tolerance.

A terhességi immuntolerancia mechanizmusának vizsgálatai

The birth of a new life depends on the cooperation of two different genomes. The developing embryo and continuous lively communication between the maternal immune system is outstanding important.

 One of the most dynamic forms of intercellular communication is mediated by cell membrane-surrounded blisters, i.e., extracellular vesicles (EVs) communication. EVs are evolutionarily conserved, active, unidirectional, or bidirectional create a horizontal system of connections between cells, playing a role in autocrine,in both paracrine and endocrine signaling. It is subpicomolar in the blood plasma are on average between 100 and 1 000 nm and 1 have a typical mass per gigadalt. 

Their presence has been studied so far, reproductive relevant biological fluids (eg follicular fluid, amniotic fluid,breast milk, embryo culture medium, semen, blood plasma, serum). The EVs play an active role in the maturation processes, implantation and presumably in the development and maintenance of immune tolerance in relation to pregnancy is. Fetal trophoblast cells and trophoblast-derived cells direct contact of fragments / particles with maternal circulation as early as 1893 assumed.

It was later verified that it originated from the maternal-fetal interface trophoblast-derived EVs continuously enter the maternal circulation and their presence a in peripheral blood as early as the 6th week of pregnancy. From a methodological point of view,The presence of EVs can be confirmed and characterized by a flow cytometer, confocal microscopy, mass spectrometry, next generation sequencing (NGS) and by transmission electron microscopy5–9. In terms of their biochemical composition, EVs transport key regulatory proteins, lipids, and nucleic acids a cells, thereby affecting reproductive performance

Ur studien klipper jag in relevant HoloMonitorinfo.Översatt + ungerskt original.

CD3 + / CD25high + / CD127low + Treg cells were sorted for further holomicroscopy under sterile conditions (SONY SH800S) (Figure 5).

5. ábra. In vitro differenciáltatott Treg-sejtek szortolásához alkalmazott kapuzási stratégia, FSC-H – előreszórás, SSC-H - oldalszórás

 

CD25 + generated in vitro/ CD127lo Treg cells with cell sorter under sterile conditions sorted and then characterized by differentiation by holomicroscopy for 3 h cell viability. To assess viability, cell migration and motility parameters (cell pathway, motility rate) were examined. Cell migration is between 30 and 90 minutes showed the greatest heterogeneity in the study interval. Cell migration is relative showed a steady rate of cell migration (Fig. 27). By sorted Treg cells examination of the path taken, i.e., monitoring of cell migration activity is appropriate.

27. ábra. In vitro differenciáltatott Treg-sejtek holomikroszkópos sejtmigrációs és motilitási paramétereinek regisztrálása a sejtviabilitás igazolására. A vizsgált Treg-sejtek növekvő tendenciájú sejtmigrációs aktivitást (kék vonal), illetve kvázi szabályos sejtmotilitást (piros vonal) és sejtmotilitási sebességet (zöld vonal) mutatnak. 

 

 

IEV-induced THP-1 cell migration was examined by holomicroscopy compared to untreated THP-1 cells in both preeclampsia,both healthy iEVs stimulated migration of THP-1 cells but preeclampsia
and healthy iEV effect was not significantly different (preeclampsia iEV: 0.142 ± 0.003 slope, healthy iEV: 0.139 ± 0.006 slope). However the preeclampsia iEVs induced significantly lower cell motility in the healthy compared to iEVs (preeclampsia iEV: 3.22 ± 0.008 slope, healthy iEV: 4.82 ± 0.06 slope) (Fig. 40). Pregnancy-associated EVs affect THP-1 cells.

40. ábra. Egészséges és preeklampsziás iEV-k THP-1 sejtek migrációs aktivitására kifejtett hatásának vizsgálata. A sejtmotilitás magasabb volt az iEV kezelés hatására (n=6, p<0,05). HP-egészséges, PE- preeklampsziás iEV

 

Materials and Methods

3.5.3 Holomikroszkópia

The HoloMonitor M4 (Phase Holographic Imaging, Lund, Sweden) is a living cell a method suitable for quantitative, unlabeled testing, which allows for more between time-lapse monitoring of cell motility and cell migration. Experimental the chemokinetic effects of circulating EVs on THP-1 cells in our systems (1),and (2) assayed in vitro differentiated Treg cells. The following parameters were determined: (1) cell migration: done between the start point and the end point shortest direct route length (µm); (2) motility: between the starting point and the end point the length of the current route traveled (µm). For THP-1 cell assays, 4 x 104 THP-1 cells.
It was placed in a Petri dish (d = 35 mm) and cultured for 24 h, then 8 x 104 healthy ill. treated with iEVs isolated from the plasma of preeclampsia pregnant women. Induced by iEV migration and motility changes over 2 hours with 30-second sampling detected using the interval. Untreated THP-1 cells as a control we used. For differentiated Treg cells, 60 seconds for 3 hours measurements were performed using sampling intervals.To evaluate the images, use the automatic background threshold method (“Minimal error sets”).algorithm, cut-off = 128) was used. 50 cells were evaluated as our field of view. The 42 the number of fields of view evaluated was 240. During the assessment in the marginal zones located cells were not considered. 

To analyze the data HoloStudio M4 2.5 program was used.

 

Summary

The birth of a new life depends on the collaboration between two different genomes. 

Continuous dynamic communication between the developing embryo and the maternal immune system, in which EVs can play an important role. Our aim was to investigate the role of EVs in immune tolerance. The major target cells of trophoblast derived EVs are CD4+ T cells, which are the precursors of regulatory T cells. The BeWo trophoblastic cell line was used as an in vitro system. A miRNA network favouring immune tolerance was identified in EVs, which plays a role in regulatory T cell differentiation. We found tolerogenic proteins in the protein composition of EVs, and identified the HSPE1 protein, which we demonstrated first to induce regulatory T cell differentiation in human CD4+ T cells. Next, we proved the association of HSPE1 with BeWo-derived EVs. We have shown that BeWo-derived EVs induce regulatory T cell differentiation. We used single cell transcriptomic analysis to examine the heterogeneity of regulatory T cells and revealed that in the memory regulatory T cell show high HSPE1 expression, as well the CAPG is a potential marker gene. To explore ex vivo the immunotolerance, we choose preeclampsia, one of the most common pregnancy-specific disorder. 

In preeclampsia the immunotolerance is altered and exaggerated systemic inflammation occurs. Monocyte cells are important players in immune homeostasis. We examined the circulating EVs in 25 preeclamptic and 20 healthy pregnant women. Preeclamptic EVs pattern contains significantly less platelets and more tissue factor+ and monocyte derived EVs. We identified monocyte cells as target cells of circulating EVs. The THP-1 monocytic cell line was used in our experiments. Preeclamptic EVs were less phagocytosed by THP-1 cells. Furthermore, preeclamptic EVs induced decreased cell migration and enhanced cell adhesion, and inflammatory cytokine (TNF, IL-6) expression of THP-1 cells. Altogether our data suggest that placental extracellular vesicles at the maternofetal interface and the heterogenous circulating extracellular vesicles play a role in the development and maintenance of immune tolerance by influencing maternal regulatory T cell polarization and monocyte cell cytokine secretion and migration patterns. 

Min kommentar

I denna studie har forskaren med kollegor grottat ner sig till DNA-nivå,alltså arvsmassan hos gravid kvinna och det  foster hon bär på. Genome = arvsmassa. Hur sen forskaren kommit till sina slutsatser är jag inte kompetent nog att förstå även om jag kör en svensk översättning. Menar, dessa rader är helt glasklara för gemene man eller hur? Yehh right.

"Sammantaget tyder våra data på att extracellulära vesiklar i placenta vid maternofetalt gränssnitt och de heterogena cirkulerande extracellulära vesiklarna spelar en roll i utvecklingen och upprätthållandet av immuntoleransen genom att påverka moderns regulatoriska T-cellpolarisering och monocytcellcytokinutsöndring och migrationsmönster."

Det undertecknad kan konstatera är istället att doktoranden m kollegor haft stor nytta och användning av PHI`s excellenta HoloMonitor för att nå dessa nya insikter och den kunskap det även ger andra forskare inom fertilitetsområdet.Inte bara cancerforskningen ser instrumentets användbarhet.