RESUMO
The flow cytometry method (FCM) is a widely renowned practice increasingly used to assess the microbial viability of probiotic products. Additionally, the measurement of water activity (aw) can be used to confirm the presence of viable cells in probiotic products throughout their shelf lives. The aim of this study was to investigate the correlation between changes in aw and variations in active fluorescent units (AFU), a unit commonly used in flow cytometry method, during the aging of probiotic products containing freeze-dried bacteria. We controlled the stability of probiotic products for bacterial counts (using ISO 19344 method) and aw levels in commercially available capsules containing freeze-dried bacteria such as Lactobacillus sp. or combinations of Lactobacillus sp. and Bifidobacterium sp. in standard conditions (25 ± 2 °C and 60% relative humidity) over a period of 24 months. During this time, the bacterial contents decreased by 0.12 Log10 in the single-strain product, by 0.16 Log10 in the two-strain product and by 0.26 Log10 in the multi-strain product. With the increase in aw, the number of bacteria decreased but the aw at the end point of the stability study did not exceed 0.15 in each of the three tested products. FCM combined with aw is a prospective analysis that can be used to assess the stability of probiotic products, both for its ability to detect bacterial viability and for practical (analysis time) and economic reasons.
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Dehydroepiandrosterone (DHEA) is an abundant steroid and precursor of sex hormones. During aging, the reduction in DHEA synthesis causes a significant depletion of estrogens and androgens in different organs, such as the ovaries, brain, and liver. Primary Biliary Cholangitis (PBC) is a cholestatic liver disease that begins with immune-mediated bile duct damage, and is followed by liver fibrosis, and finally, cirrhosis. PBC primarily affects postmenopausal women, with an average age of diagnosis of 65 years, but younger women are also affected. Here, we analyzed the levels of DHEA, estradiol (E2), and estriol (E3) in the PBC sera of females at an age of diagnosis under 40 (n = 37) and above 65 (n = 29). Our results indicate that in PBC patients at an age of diagnosis under 40, E2 was significantly lower compared to that in healthy women. In contrast, the levels of DHEA and E3 were in a normal range. Furthermore, ELISA assays revealed that in PBC patients at an age of diagnosis above 65, the levels of DHEA, E2, and E3 significantly declined in comparison to those in younger patients. In addition, flow cytometry analysis showed that the level of IL-8 significantly decreased while the level of TNF-α increased in older PBC patients compared to younger ones. Moreover, we showed for the first time that the sulfonated form of DHEA, DHEA-S, reduces the levels of both pro-inflammatory interleukins, IL-8 and TNF-α, in PBC-like cholangiocytes (H69-miR506), while it diminishes the level of the pro-fibrotic interleukin, IL-13, in hepatocytes (Hep-G2). Finally, we demonstrated that the expression of the pro-fibrotic agent TGF-ß significantly increased in both the early (F0-F3) and cirrhotic (F4) stages of PBC, and this elevation was accompanied by higher α-SMA expression.
Assuntos
Cirrose Hepática Biliar , MicroRNAs , Humanos , Feminino , Idoso , Citocinas/metabolismo , Fator de Necrose Tumoral alfa , Interleucina-8 , Fibrose , Cirrose Hepática , Interleucinas , Desidroepiandrosterona , MicroRNAs/metabolismoRESUMO
Evidence suggests a role of the immune system in the pathogenesis of a number of mental conditions, including schizophrenia (SCH). In terms of physiology, aside from its crucial protective function, the complement cascade (CC) is a critical element of the regeneration processes, including neurogenesis. Few studies have attempted to define the function of the CC components in SCH. To shed more light on this topic, we compared the levels of complement activation products (CAP) (C3a, C5a and C5b-9) in the peripheral blood of 62 patients with chronic SCH and disease duration of ≥ 10 years with 25 healthy controls matched for age, sex, BMI and smoking status. Concentrations of all the investigated CAP were elevated in SCH patients. However, after controlling for potential confounding factors, significant correlations were observed between SCH and C3a (M = 724.98 ng/mL) and C5a (M = 6.06 ng/mL) levels. In addition, multivariate logistic regression showed that C3a and C5b-9 were significant predictors of SCH. There were no significant correlations between any CAP and SCH symptom severity or general psychopathology in SCH patients. However, two significant links emerged between C3a and C5b-9 and global functioning. Increased levels of both complement activation products in the patient group as compared to healthy controls raise questions concerning the role of the CC in the etiology of SCH and further demonstrate dysregulation of the immune system in SCH patients.
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Very small embryonic-like stem cells (VSELs) are a dormant population of development early stem cells deposited in adult tissues that as demonstrated contribute to tissue/organ repair and regeneration. We postulated developmental relationship of these cells to migrating primordial germ cells (PGCs) and explained the quiescent state of these cells by the erasure of differently methylated regions (DMRs) at some of the paternally imprinted genes involved in embryogenesis. Recently, we reported that VSELs began to proliferate and expand in vivo in murine bone marrow (BM) after exposure to nicotinamide (NAM) and selected pituitary and gonadal sex hormones. In the current report, we performed proteomic analysis of VSELs purified from murine bone marrow (BM) after repeated injections of NAM + Follicle-Stimulating Hormone (FSH) that in our previous studies turned out to be an effective combination to expand these cells. By employing the Gene Ontology (GO) resources, we have performed a combination of standard GO annotations (GO-CAM) to produce a network between BM steady-state conditions VSELs (SSC-VSELS) and FSH + NAM expanded VSELs (FSH + NAM VSELs). We have identified several GO biological processes regulating development, organogenesis, gene expression, signal transduction, Wnt signaling, insulin signaling, cytoskeleton organization, cell adhesion, inhibiting apoptosis, responses to extra- and intracellular stimuli, protein transport and stabilization, protein phosphorylation and ubiquitination, DNA repair, immune response, and regulation of circadian rhythm. We report that VSELs express a unique panel of proteins that only partially overlapped with the proteome of BM - derived hematopoietic stem cells (HSCs) and hematopoietic mononuclear cells (MNCs) and respond to FSH + NAM stimulation by expressing proteins involved in the development of all three germ layers. Thus, our current data supports further germ-lineage origin and multi germ layer differentiation potential of these cells.
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Medula Óssea , Proteômica , Animais , Camundongos , Diferenciação Celular , Células-Tronco Hematopoéticas , Hormônio Foliculoestimulante/metabolismo , Camadas GerminativasRESUMO
Finasteride (Fin) causes androgen imbalance by inhibiting the conversion of testosterone (T) to its more active metabolite, dihydrotestosterone (DHT). Androgen receptors (AR) are present (e.g., in hepatocytes), which have well-developed endoplasmic reticulum (ERet). Cellular protein quality control is carried out by ERet in two paths: (i) unfolded protein response (UPR) and/or (ii) endoplasmic reticulum associated degradation (ERAD). ERet under continuous stress can generate changes in the UPR and can direct the cell on the pathway of life or death. It has been demonstrated that genes involved in ERet stress are among the genes controlled by androgens in some tissues. Oxidative stress is also one of the factors affecting the functions of ERet and androgens are one of the regulators of antioxidant enzyme activity. In this paper, we discuss/analyze a possible relationship between androgen imbalance in paternal generation with ERet stress and liver disorders in both paternal and filial generation. In our rat model, hyperglycemia and subsequent higher accumulation of hepatic glycogen were observed in all filial generation obtained from females fertilized by Fin-treated males (F1:Fin). Importantly, genes encoding enzymes involved in glucose and glycogen metabolism have been previously recognized among UPR targets.
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Although regenerative and inflammatory processes are involved in the etiopathogenesis of many psychiatric disorders, their roles are poorly understood. We investigate the potential role of stem cells (SC) and factors influencing the trafficking thereof, such as complement cascade (CC) components, phospholipid substrates, and chemokines, in the etiology of schizophrenia. We measured sphingosine-1-phosphate (S1P), stromal-derived factor 1 (SDF-1), and CC cleavage fragments (C3a, C5a, and C5b-C9; also known as the membrane attack complex) in the peripheral blood of 49 unrelated patients: 9 patients with ultra-high risk of psychosis (UHR), 22 patients with first-episode psychosis (FEP), and 18 healthy controls (HC). When compared with the HC group, the UHR and FEP groups had higher levels of C3a. We found no significant differences in hematopoietic SC, very small embryonic-like stem cell (VSEL), C5a, S1P, or SDF-1 levels in the UHR and FEP groups. However, among FEP patients, there was a significant positive correlation between VSELs (CD133+) and negative symptoms. These preliminary findings support the role of the immune system and regenerative processes in the etiology of schizophrenia. To establish the relevance of SC and other factors affecting the trafficking thereof as potential biomarkers of schizophrenia, more studies on larger groups of individuals from across the disease spectrum are needed.
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Transtornos Psicóticos , Esquizofrenia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Psicopatologia , Transtornos Psicóticos/diagnósticoRESUMO
BACKGROUND: Alveolar type 2 (AT2) cells and bronchioalveolar stem cells (BASC) perform critical regenerative functions in response to lung damage. Published data show that nonhematopoietic, bone marrow-derived "very small embryonic-like stem cells" (VSELs) can differentiate in vivo into surfactant protein C (SPC)-producing AT2 cells in the lung. Here, we test directly whether VSEL-derived BASC and AT2 cells function to produce differentiated progeny. METHODS: using a reporter mouse in which the H2B-GFP fusion protein is driven from the murine SPC promoter, we tested whether bone marrow-derived VSELs or non-VSEL/nonhematopoietic stem cells (non-VSEL/non-HSCs) can differentiate into AT2 and BASC cells that function as progenitor cells. Immediately following bleomycin administration, WT recipient mice underwent intravenous administration of VSELs or non-VSEL/non-HSCs from SPC H2B-GFP mice. GFP+ AT2 and BASC were isolated and tested for progenitor activity using in vitro organoid assays. RESULTS: after 21 days in vivo, we observed differentiation of VSELs but not non-VSEL/non-HSCs into phenotypic AT2 and BASC consistent with previous data in irradiated recipients. Subsequent in vitro organoid assays revealed that VSEL-derived AT2 and BASC maintained physiological potential for differentiation and self-renewal. CONCLUSION: these findings prove that VSELs produce functional BASC and AT2 cells, and this may open new avenues using VSELs to develop effective cell therapy approaches for patients with lung injury.
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Células da Medula Óssea/citologia , Células-Tronco Embrionárias/transplante , Células Epiteliais/citologia , Lesão Pulmonar/patologia , Células Epiteliais Alveolares/metabolismo , Animais , Bleomicina , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Organoides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The effect of a sucrose diet and repeated one-day starvation on oxidative status in the ovary and uterus is still unknown. Our analysis focused on carbohydrate-lipid metabolism parameters and the changes in red blood cells, ovary and uterus superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities and malonylodialdehyde (MDA) concentration in rats fed with a diet containing 16% of sucrose and subjected to systematic one-day starvation when using such a diet. It was found that a diet with 16% sucrose contributed to the increase of antioxidant enzyme activity in the blood (GPx and CAT) and uterus (SOD), without changes in MDA concentrations, which indicates an increase in reactive oxygen species (ROS) concentration in these tissues, being balanced by an increase in antioxidant enzyme activity. The introduction of a regular one-day starvation period into the diet intensified oxidative stress and led to a redox imbalance in the reproductive tissues of female rats. This was manifested by higher GPx activity, lower CAT activity and higher MDA concentration in the uterus and lower GPx and CAT activities and lower MDA concentration in the ovaries. The observed changes may be the cause of fertility disorders and possible problems with fertilised egg cell implantation into the uterine tissue.
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Sacarose Alimentar , Ovário , Estresse Oxidativo , Inanição/metabolismo , Útero , Animais , Sacarose Alimentar/administração & dosagem , Sacarose Alimentar/farmacologia , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Ovário/química , Ovário/efeitos dos fármacos , Ovário/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Útero/química , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Umbilical cord blood (UCB) is a rich source of stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitors cells (EPCs), and very small embryonic-like stem cells (VSELs). These cells most likely are mobilized into UCB in response to hypoxia and delivery stress. We have hypothesized that they may play a role in repairing certain tissue/organ injuries that occur in the newborn child after delivery. Here we asked whether delivery also mobilizes stem cells into maternal blood, as the mother also experiences hypoxia and several types of internal tissue injuries, particularly in the reproductive tract. We observed that the number of HSCs, MSCs, EPCs, and VSELs increases in maternal blood at 24 h after physiological delivery (n = 17). Based on this observation, we propose that delivery stress is associated with an increase in the number of circulating stem cells, not only on the fetal side but also on the maternal side of the fetal-maternal circulatory barrier.
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Diferenciação Celular/genética , Células Progenitoras Endoteliais/citologia , Sangue Fetal/citologia , Células-Tronco de Sangue Periférico , Adulto , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Sangue Fetal/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Relações Materno-Fetais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismoRESUMO
Bone marrow (BM) residing stem cells are mobilized from their BM niches into peripheral blood (PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells (SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblastlike stem cells (VSELs) and mesenchymal stem cells (MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate (S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer (CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas (BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting (FACS) we evaluated the number of BM-derived SCs circulating in PB: i) CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii) CD45-/CD105+/CD90+/CD29+ MSCs; iii) CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells (EPCs); and iv) CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells (HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor 1 (SDF1), vascular endothelial growth factor (VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.