RESUMEN
The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.
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Médula Ósea , Células Madre Mesenquimatosas , Humanos , Medios de Cultivo/farmacología , Suplementos Dietéticos , Ácido LácticoRESUMEN
BACKGROUND: Multiple organ dysfunction syndrome (MODS) and the consecutive multiple organ failure (MOF) are severe and dreaded complications with a high mortality in multiple trauma patients. The aim of this study was to investigate the potential of the adipokines leptin, resistin, interleukin-17A and interleukin-33 as possible biomarkers in the early posttraumatic inflammatory response and for identifying severely traumatized patients at risk of developing MODS. METHODS: In total, 14 multiple trauma patients with an injury severity score (ISS) ≥ 16 as well as a control group of 14 non-multiple trauma patients were included in this study and blood samples were taken at the time points 0, 6, 24, 48 and 72 h after admission. For the trauma patients, the SIRS and Denver MOF score were determined daily. The quantitative measurement of the plasma concentrations of the adipokines was performed using ELISA. RESULTS: In the statistical analysis, the multiple trauma patients showed statistically significant higher plasma concentrations of leptin, resistin, IL-17A and IL-33 compared to the control group. In addition, there was a statistically significant positive correlation between the concentrations of resistin, IL-17A and IL-33 and the corresponding SIRS scores and between the concentrations of resistin, IL-17A and IL-33 and the corresponding Denver MOF scores. Finally, ROC curve analysis revealed that the adipokines leptin and IL-17A are suitable diagnostic markers for the discrimination between multiple trauma patients with and without MOF. CONCLUSIONS: Leptin and IL-17A could be suitable diagnostic markers to identify severely injured patients with a developing SIRS and MOF earlier, to adjust surgical therapy planning and intensive care.
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Adipoquinas/sangre , Biomarcadores/sangre , Insuficiencia Multiorgánica/diagnóstico , Traumatismo Múltiple/complicaciones , Síndrome de Respuesta Inflamatoria Sistémica/diagnóstico , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Insuficiencia Multiorgánica/sangre , Insuficiencia Multiorgánica/etiología , Pronóstico , Curva ROC , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Adulto JovenRESUMEN
ABSTRACT: A suite of software tools has been developed for dose estimation (BAT, WinFRAT) and prediction of acute health effects (WinFRAT, H-Module) using clinical symptoms and/or changes in blood cell counts. We constructed a database of 191 ARS cases using the METREPOL (n = 167) and the SEARCH-database (n = 24). The cases ranged from unexposed (RC0), to mild (RC1), moderate (RC2), severe (RC3), and lethal ARS (RC4). From 2015-2019, radiobiology students and participants of two NATO meetings predicted clinical outcomes (RC, H-ARS, and hospitalization) based on clinical symptoms. We evaluated the prediction outcomes using the same input datasets with a total of 32 teams and 94 participants. We found that: (1) unexposed (RC0) and mildly exposed individuals (RC1) could not be discriminated; (2) the severity of RC2 and RC3 were systematically overestimated, but almost all lethal cases (RC4) were correctly predicted; (3) introducing a prior education component for non-physicians significantly increased the correct predictions of RC, ARS, and hospitalization by around 10% (p<0.005) with a threefold reduction in variance and a halving of the evaluation time per case; (4) correct outcome prediction was independent of the software tools used; and (5) comparing the dose estimates generated by the teams with H-ARS severity reflected known limitations of dose alone as a surrogate for H-ARS severity. We found inexperienced personnel can use software tools to make accurate diagnostic and treatment recommendations with up to 98% accuracy. Educational training improved the quality of decision making and enabled participants lacking a medical background to perform comparably to experts.
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Síndrome de Radiación Aguda , Síndrome de Radiación Aguda/diagnóstico , Síndrome de Radiación Aguda/etiología , Síndrome de Radiación Aguda/terapia , Bases de Datos Factuales , Hospitalización , Humanos , Radiobiología/educación , Programas InformáticosRESUMEN
HemoDose is a software tool, which estimates absorbed doses based on blood cell counts (BCC). The aim of our study was to validate HemoDose for early dose estimates. Dose estimates generated by HemoDose were compared with dose estimates stored in SEARCH from radiation victims. Moreover, BCC from unirradiated donors and corresponding HemoDose dose estimates were analysed. We employed linear or logistic regression analysis. There was a significant correlation between calculated doses by HemoDose based on single and multiple lymphocyte counts when omitting lowest and highest dose estimates. Furthermore, there was a significant correlation between calculated doses by HemoDose based on lymphocyte counts and the estimated doses based on DIC. The dose estimates by HemoDose based on lymphocyte counts and DIC showed a comparable correlation with HARS degrees 0 and 4. In conclusion, HemoDose dose estimation based on early lymphocyte counts appears to be a promising biodosimetry tool under certain considerations.
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Radiometría , Programas Informáticos , Recuento de Células Sanguíneas , Aberraciones Cromosómicas , Relación Dosis-Respuesta en la Radiación , Humanos , Dosis de RadiaciónRESUMEN
HDFa (human dermal fibroblasts) are used as cellular models for EMF exposure. To ensure reproducible in vitro experiments, comparable proliferation and differentiation cell conditions must exist, and different donors, passage numbers, culture time, and growth media must be considered. In this study, the authors cultured fibroblasts in DMEM or 106 medium. Growth curves, vitality, morphology, and gene expression of genes coding for proliferation (PCNA, CDKN2A, CDKN1A, SFN), differentiation (PDGFRA, TGM2, ACTA2, PDPN, NTN1, MGP, PPP1R14), and SFN target genes (TP63, MMP1, MMP3) were examined in both media and passage numbers 3-4, 5-6 and >6. At passages 3-4, proliferating cells can be observed in both media. While cells cultured in DMEM proliferate over the passages, from passage 5, cells in 106 medium persisted around the seeded number. TGM2 down-regulation over all passages in both media and cells morphology suggest papillary-type fibroblasts. Downregulation of SFN (negative regulator of mitotic translation and cell differentiation) coincided with proliferating fibroblasts over all examined conditions. Downstream SFN target genes in proliferating cells appeared upregulated (TP63) and downregulated (MMP1/MMP3), suggestive for a status characterized by increased stemnesses (upregulated TP63) and wound healing capacity (downregulated MMP1, MMP3). Resting cells (SFN control values) were associated with control values of TP63 and MMP1/MMP3 expression, suggesting a reduced stemness and wound healing capacity. In conclusion, a set of markers related to proliferation (SFN), differentiation (TGM2), stemnesses (TP63), and wound healing (MMP1/MMP3) allow a culture characterization so that cells under two different conditions can be exposed, thus enabling reproducible EMF experiments or experiments with other exposures.
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Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Fibroblastos/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Piel/metabolismo , Línea Celular , Células Cultivadas , Femenino , Fibroblastos/citología , Humanos , Proteínas/genética , Proteínas/efectos de la radiación , Exposición a la Radiación , Transducción Genética , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
Radiological exposure scenarios involving large numbers of people require a rapid and high-throughput method to identify the unexposed, and those exposed to low- and high-dose radiation. Those with high-dose exposure, e.g., >2 Gy and depending on host characteristics, may develop severe hematological acute radiation syndrome (HARS), requiring hospitalization and treatment. Previously, we identified a set of genes that discriminated these clinically relevant groups. In the current work, we examined the utility of gene expression changes to classify 1,000 split blood samples into HARS severity scores of H0, H1 and H2-4, with the latter indicating likely hospitalization. In several previous radiation dose experiments, we determined that these HARS categories corresponded, respectively, to doses of 0 Gy (unexposed), 0.5 Gy and 5 Gy. The main purpose of this work was to assess the rapidity of blood sample processing using targeted next-generation sequencing (NGS). Peripheral blood samples from two healthy donors were X-ray irradiated in vitro and incubated at 37°C for 24 h. A total of 1,000 samples were evaluated by laboratory personnel blinded to the radiation dose. Changes in gene expression of FDXR, DDB2, POU2AF1 and WNT3 were examined with qRT-PCR as positive controls. Targeted NGS (TREX) was used on all samples for the same four genes. Agreement using both methods was almost 78%. Using NGS, all 1,000 samples were processed within 30 h. Classification of the HARS severity categories corresponding to radiation dose had an overall agreement ranging between 90-97%. Depending on the end point, either a combination of all genes or FDXR alone (H0 HARS or unexposed) provided the best classification. Using this optimized automated methodology, we assessed 100× more samples approximately three times faster compared to standard cytogenetic studies. We showed that a small set of genes, rather than a complex constellation of genes, provided robust positive (97%) and negative (97%) predictive values for HARS categories and radiation doses of 0, 0.5 and 5 Gy. The findings of this study support the potential utility of early radiation-induced gene expression changes for high-throughput biodosimetry and rapid identification of irradiated persons in need of hospitalization.