In vitro  survival of  Listeria monocytogenes  in human amniotic fluid

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

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G-CSF and Erythropoietin Stability in Amniotic Fluid during Simulated in vitro Digestion Conditions

Journal of Pharmacy Technology ◽

10.1177/875512250201800603 ◽

2002 ◽

Vol 18 (6) ◽

pp. 310-315 ◽

Cited By ~ 11

Author(s):

Darlene A Calhoun ◽

Brooke E Richards ◽

Jason A Gersting ◽

Sandra E Sullivan ◽

Robert D Christensen

Keyword(s):

Amniotic Fluid ◽

Preterm Neonate ◽

In Vitro Digestion ◽

Human Amniotic Fluid ◽

Time Zero ◽

Simulated Digestion ◽

The Stability ◽

Stimulating Factor ◽

Degree Of Degradation

Objective: To determine the stability of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo) in human amniotic fluid and recombinant G-CSF (Neupogen) and Epo (Epogen) in simulated amniotic fluid to digestions at pH concentrations of 3.2, 4.5, and 5.8 to assess their bioavailability to the neonate. Design: A simulated amniotic fluid containing Neupogen and Epogen was subjected to in vitro conditions that mimicked preprandial and postprandial neonatal intestinal digestion. Human amniotic fluid was tested using identical digestion conditions as well as human amniotic fluid to which Epogen and Neupogen had been added. Main Outcome Measures: The percentages of G-CSF/Epo and Neupogen/Epogen remaining after 1 and 2 hours of simulated digestions were compared with those at time zero, and concentrations at 2 hours were compared with those at 1 hour and time zero. Results: In simulated amniotic fluid at pH 3.2, significant degradation of G-CSF was observed at 1 hour (p = 0.03). No differences were observed at 1 or 2 hours for either pH 4.5 (p = 0.30 and 0.11, respectively) or pH 5.8 (p = 0.20 and 0.49, respectively). Human amniotic fluid exhibited significant degradation pH 3.2 (p = 0.04) and pH 4.5 (p < 0.05) at 1 hour; no difference was noted at pH 5.8 at 1 hour (p = 0.34). When additional Neupogen was added to human amniotic fluid, significant degradation was observed at pH 3.2 (p < 0.05) and pH 4.5 (p = 0.03) at 1 hour; no difference was noted at 1 hour at pH 5.8 (p = 0.11). In simulated amniotic fluid at pH 3.2, significant degradation of Epo occurred at 1 hour (p < 0.05). There were no differences at 1 hour for pH 4.5 (p = 0.50) or pH 5.8 (p = 0.17). Human amniotic fluid exhibited significant degradation at pH 3.2 (p < 0.05) and pH 4.5 (p < 0.05) at 1 hour; no difference was noted at 1 hour at pH 5.8 (p = 0.34). When additional Epogen was added to human amniotic fluid, significant degradation was observed at pH 3.2 (p = 0.001) and pH 4.5 (p = 0.003); no difference was noted at 1 hour at pH 5.8 (p = 0.31). Conclusions: G-CSF/Epo in human amniotic fluid and Neupogen/Epogen in simulated amniotic fluid are preserved to varying degrees during simulated digestion conditions. The degree of degradation of both cytokines was time- and pH-dependent. Measurable quantities of G-CSF and Epo are biologically available when swallowed by the fetus or a preterm neonate.

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In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp

Digestive and Liver Disease ◽

10.1016/j.dld.2013.02.007 ◽

2013 ◽

Vol 45 (8) ◽

pp. 669-676 ◽

Cited By ~ 44

Author(s):

Gianluca Carnevale ◽

Massimo Riccio ◽

Alessandra Pisciotta ◽

Francesca Beretti ◽

Tullia Maraldi ◽

...

Keyword(s):

Stem Cells ◽

Amniotic Fluid ◽

Dental Pulp ◽

In Vitro Differentiation ◽

Β Cells ◽

Human Amniotic Fluid

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Nuclear Nox4 Role in Stemness Power of Human Amniotic Fluid Stem Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2015/101304 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Tullia Maraldi ◽

Marianna Guida ◽

Manuela Zavatti ◽

Elisa Resca ◽

Laura Bertoni ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Amniotic Fluid ◽

Human Stem Cells ◽

Human Amniotic Fluid ◽

Differentiation Potential ◽

Amniotic Fluid Stem Cells ◽

Target Molecules ◽

Cellular Compartments

Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expandin vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

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