Interleukin-27 (IL-27) is an immunoregulatory cytokine that suppresses inflammation through multiple mechanisms, including induction of IL-10, but the transcriptional network mediating its diverse functions remains unclear. Combining temporal RNA profiling with computational algorithms, we predict 79 transcription factors induced by IL-27 in T cells. We validate 11 known and discover 5 positive (Cebpb, Fosl2, Tbx21, Hlx, and Atf3) and 2 negative (Irf9 and Irf8) ll10 regulators, generating an experimentally refined regulatory network for ll10. We report two central regulators, Prdm1 and Maf, that cooperatively drive the expression of signature genes induced by IL-27 in type 1 regulatory T cells, mediate IL-10 expression in all T helper cells, and determine the regulatory phenotype of colonic Foxp3(+) regulatory T cells. Prdm1/Maf double-knockout mice develop spontaneous colitis, phenocopying ll10-deficient mice. Our work provides insights into IL-27-driven transcriptional networks and identifies two shared ll10 regulators that orchestrate immunoregulatory programs across T helper cell subsets.

The transfer of fetal cells to maternal organs occurs in mouse and human pregnancy. Techniques such as polymerase chain reaction and flow cytometry do not permit study of fetal cell morphology or anatomic location. Using a green fluorescent protein (GFP) transgenic mouse model, our objective was to determine whether GFP+ signal emanates from intact or degraded fetal cells, and whether they have a characteristic appearance and location within maternal lung. Four wild-type female mice were mated to males homozygous for the Gfp transgene and studied at days e16-18. Controls were 2 females mated to wild-type males. Morphologic appearance and anatomic position of each GFP+ object within maternal lung was recorded. GFP signals were sufficiently bright to be visualized without anti-GFP antibody and were confirmed by confocal microscopy to be separate from fluorescent artifact. Of 438 GFP+ objects detected, 375 (85.6%) were from intact cells, and 63 (14.4%) were acellular. Four distinct categories of intact cells were observed. Of these, 23.2% had mononuclear morphology with a relatively large nucleus and GFP+ cytoplasm (Group A). An additional group of cells (10.1%) had mononuclear morphology and podocyte extensions (Group B). The remainder of cells had fragmented nuclei or cytoplasm. Both intact cells and acellular fragments were predominantly localized to the maternal alveolar septum (P<0.0001). This study demonstrates that fetal GFP+ cells are predominantly located in the alveolar septum and have characteristic morphologies, although it remains unclear whether these represent distinct categories of cells or degrading cells. Nevertheless, this naturally acquired population of fetal cells in maternal lung should be considered in studies of lung biology and repair.

The objective of this study was to determine if fetal–maternal cell trafficking is affected by maternal immune competence and/or parental background strain using fluorescence-activated cell sorting (FACS). In our experience the sensitivity of FACS allows for the detection of 5 fetal in 107 maternal cells and assessment of cell surface phenotype. Wild-type C57BL/6J (n=18), FVB/NJ (n=15), and immunodeficient B6129S7-Rag1tm1Mom/J (n=16) female mice were mated to C57BL/6J males homozygous for the green fluorescent protein (GFP) transgene. Single cell suspensions of maternal lung, liver, spleen, bone marrow, and blood were analyzed between late gestation (day e16–18) and 1 day post-partum for the number of GFP-positive fetal cells in relation to 107 maternal cells and the percentage of GFP-positive cells that expressed the surface markers CD11b, CD29, CD34, CD44, or CD105. The highest relative proportions of GFP-positive fetal cells were observed in maternal lungs and livers from immunocompetent allogenic females. Among congenic matings, fetal cell microchimerism was higher in immunodeficient compared with immunocompetent females. Maternal strain and strain differences between the mother and father statistically significantly affected both the numbers of fetal cells and the relative distribution of cell types in maternal organs. The highest relative proportion of fetal cells was observed in allogenic matings with immunocompetent females. Since allogenic matings are more similar to those that occur in humans, future studies using animal models of microchimerism should consider incorporating this type of experimental design.

To determine whether chemically induced miscarriage affects fetomaternal trafficking in a mouse model, we measured the amount of fetal DNA present in various maternal organs by polymerase chain reaction amplification following exposure to lipopolysaccharide (LPS). As the frequency of fetal cells and the number of animals with detectable microchimerism following LPS injection were significantly increased, particularly in lung tissue compared to controls, with no signs of an inflammatory response, we conclude that LPS-induced miscarriage results in increased murine fetomaternal cell trafficking, supporting a relationship between fetal loss and the establishment of fetal cell microchimerism.

BACKGROUND:Circulating cell-free fetal deoxyribonucleic acids (cffDNA) are novel biomarkers with many clinical applications. Amniotic fluid (AF) is a rich source of cffDNA. We investigated the biophysical characteristics of cffDNA in AF, hypothesizing that they would differ from cffDNA in maternal plasma.METHODS:We obtained 10 mL of fresh AF supernatant from women carrying euploid fetuses (n = 39) and aneuploid fetuses (n = 4). To test the effects of storage and karyotype, samples from euploid fetuses (n = 19) and aneuploid fetuses with trisomies 21 (n = 16), 18 (n = 9), or 13 (n = 3); triploidy (n = 4); or monosomy X (n = 2) were frozen at -80 degrees C. AF cffDNA was characterized by real-time quantitative PCR amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and analysis of the DNA fragmentation signature.RESULTS:We observed a significant correlation of concentration with gestational age for fresh AF cffDNA from euploid fetuses (R(2) = 0.77, P <0.0001) but not for frozen cffDNA (P = 0.63). The median amount of cffDNA in frozen euploid samples was significantly lower than in fresh samples (P <0.0001). After adjustment for gestational age, there was a statistically significant decrease in the median amount of cffDNA in frozen aneuploidy samples compared with frozen euploid samples (P = 0.0005). Analysis of the cffDNA size distribution showed different and qualitatively unique patterns for each karyotype.CONCLUSIONS:Gestational age, karyotype, and sample storage time affect concentrations and fragment size of AF cff DNA. These effects may be attributable to fundamental differences in tissue sources, excretion modes, or kinetic pathways. Characteristic signature patterns for each common aneuploidy offer the possibility of using DNA fragmentation analysis as a means of triaging AF samples.

Background: Fetal cell detection in maternal tissue requires an accurate, efficient, and reproducible microscopy method. Our objective was to compare manual scoring to a commercially available automated scanning system for the detection of chromosome signals by fluorescence in situ hybridization (FISH). Methods: X and Y chromosome FISH signals were detected on slides of calibrated mixtures of blood, paraffin‐embedded liver sections, and post‐termination blood. For manual scoring (400× magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200× magnification), duration of scanning, number of gallery images generated, duration of manual review of gallery images, and number of confirmed fetal cells were recorded. Results: From all slides the number of target fetal cells located by manual and automated microscopy was highly correlated ( r = 0.90). However, automated scanning required on average 4‐fold more time than manual scoring ( P < 0.0001), with an average automated scanning time of 9.7 h per slide compared with 2.4 h per slide when scored manually. Conclusions: In general, the accuracy of automated and manual microscopy is comparable, although manual scoring is more efficient because of the level of magnification necessary for automated scanning of cells, and a large number of gallery images generated by automated scanning that must then be reviewed manually. This suggests that when rapid analysis is required (i.e., clinical situations), manual microscopy is preferable. In contrast, automated scanning may have advantages over manual microscopy when time constraints are less imposed (i.e., research situations). Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.

BACKGROUND: In humans, fetal microchimeric cells transferred to maternal tissues during pregnancy can adopt a hepatocyte phenotype. Our objective was to determine whether fetal cells participate in the response to specific murine post-partum hepatic injuries. METHODS: Wild-type female mice were bred to males transgenic for the enhanced green fluorescent protein (GFP) (n = 42). Following delivery, we created models of chemical or surgical injury with carbon tetrachloride (CCl4) injection or by performing partial hepatectomy. Liver injury was assessed histologically. Fetal cells in maternal liver were detected and measured by real-time PCR amplification of the gfp transgene and by immunofluorescence using anti-GFP antibodies. RESULTS: PCR results showed that in chemical but not surgical injury, fetal GFP+ cells were detectable in maternal liver and spleen and that fetal cell presence was significantly increased over time following injury (4 versus 8 weeks, P = 0.006 for liver and P = 0.0006 for spleen). In some animals, following chemical injury, GFP+ cells were detected by immunofluorescence. CONCLUSIONS: The results of this preliminary study suggest that specific types of injury may elicit different fetal cell responses in maternal organs. There is a significant effect of time on fetal cell presence in liver and spleen. Furthermore, real-time PCR amplification is more sensitive than immunofluorescence for the detection of microchimeric fetal cells.

Background Previously, we showed that analysis of amniotic fluid (AF) supernatant cell-free fetal (cff) DNA using DNA microarrays (array-CGH) allows for detection of whole chromosome differences between test and reference DNA. Subsequent technical advances have increased both the yield and quality of extracted cffDNA. Here we determined whether array-CGH using smaller volumes of both fresh and frozen AF cffDNA could identify fetal aneuploidy.Methods CffDNA was extracted from 10 mL of residual AF supernatant. The test AF samples (n = 10) included one with a normal karyotype, and nine with the following fetal aneuploidies: trisomies 13 (n = 1), 18 (n = 3), 21 (n = 2), trisomy 9 mosaicism (47,XX,+9[18]/46,XX[2]), triploidy (69,XXY) and Turner syndrome (45,X).Results Array-CGH using AF cffDNA from aneuploid fetuses, compared to euploid reference AF cffDNA, detected whole chromosome aneuploidy in 8 of 9 cases tested, including the case of trisomy 9 mosaicism. The case of triploidy was not detected.Conclusions CffDNA extracted from 10 mL AF supernatant can be analyzed using array-CGH to correctly identify human chromosome abnormalities. This technology allows for rapid screening of AF samples for whole chromosomal changes by using routinely discarded supernatant, and may augment standard prenatal karyotyping techniques by providing additional molecular information. Copyright (c) 2007 John Wiley & Sons, Ltd.

Cell-free fetal nucleic acids (cffDNA) are present in maternal plasma and serum (1), but amniotic fluid (AF) also provides an attractive source of cffDNA. The concentration of cffDNA is 100- to 200-fold higher in AF than in maternal plasma/serum (2), but low yields of cffDNA compromise testing by techniques such as genomic microarrays, which require a minimum of 100 ng of DNA (3). For protocol optimization, we used 5 large-volume AF supernatant samples from patients who had undergone therapeutic amnioreduction for twin-twin transfusion syndrome. After optimization, we compared the DNA yield of the old and new protocols for freshly discarded AF supernatant samples from 29 euploid singleton pregnancies. Approval for this study was obtained from the Institutional Review Boards of Tufts-New England Medical Center and Women and Infant’s Hospital. The median gestational age at amniocentesis …

In humans, fetal cells enter the maternal circulation during all pregnancies and can persist for decades. Human studies, however, are often limited by the number of subjects and the availability of healthy and diseased tissues for analysis. We sought to develop a murine model to establish the natural history of fetal cell microchimerism in various maternal tissues during and after healthy pregnancies resulting from congenic and allogenic matings. We bred C57BL/6J and DBA/2J virgin female mice to C57BL/6J males transgenic for the enhanced green fluorescent protein (GFP), which shows autosomal dominant inheritance with complete penetrance and is under the control of a ubiquitous chicken beta-actin promoter and a cytomegalovirus enhancer. During pregnancy and at different times after delivery, female mice were sacrificed. Tissues were collected and the presence of the gfp transgene and GFP+ cells was assessed by real-time quantitative PCR and by immunofluorescence. During pregnancy, microchimerism was detected in all tissues from mice carrying GFP+ fetuses. Fetal cells were often mononuclear. The frequency of fetal cells in the lungs was significantly higher compared to other tissues. The level of microchimerism was also significantly higher in congenic compared to allogenic matings. After delivery, the frequency of fetal cells decreased and fetal cells were undetectable at 2 and 3 weeks after the first delivery. However, some mice that had three gestations had detectable fetal cells 3 weeks after their last delivery. Using sensitive methods of detection, we demonstrate that fetal cell microchimerism occurs during all murine pregnancies. We describe a useful model for the study of the consequences of this phenomenon.

BioTechniquesVol. 34, No. 2 BenchmarksOpen AccessCombined FISH and Immunolabeling on Paraffin-Embedded Tissue Sections for the Study of MicrochimerismKiarash Khosrotehrani, Helene Stroh, Diana W. Bianchi & Kirby L. JohnsonKiarash KhosrotehraniTufts-New England Medical Center, Boston, MA, USA, Helene StrohTufts-New England Medical Center, Boston, MA, USA, Diana W. BianchiTufts-New England Medical Center, Boston, MA, USA & Kirby L. Johnson*Address correspondence to Dr. Kirby L. Johnson, Division of Genetics, Department of Pediatrics, Tufts-New England Medical Center, Box 394, 750 Washington Street, Boston, MA 02111, USA. e-mail: E-mail Address: kjohnson@lifespan.orgTufts-New England Medical Center, Boston, MA, USAPublished Online:24 Sep 2018https://doi.org/10.2144/03342bm01AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit FiguresReferencesRelatedDetailsCited ByFetal Cell Microchimerism; Normal and Immunocompromised Gestations in Mice22 August 2019 | Fetal and Pediatric Pathology, Vol. 39, No. 4Cancer22 June 2018Microchimeric fetal cells play a role in maternal wound healing after pregnancy9 April 2014 | Chimerism, Vol. 5, No. 2Recycling and Long-Term Storage of Fluorescence In Situ Hybridization Slides1 March 2014 | American Journal of Clinical Pathology, Vol. 141, No. 3Identifying the Origin and Phenotype of Cells in Tumor Xenografts21 August 2013Microchimerism in Mouse PregnancyOrigin of Langerhans cells in normal skin and chronic GVHD after hematopoietic stem-cell transplantation30 December 2013 | Experimental Dermatology, Vol. 23, No. 1Pregnancy-Acquired Fetal Progenitors as Natural Cell Therapy19 February 2013Fetal Cells in the Murine Maternal Lung Have Well-Defined Characteristics and Are Preferentially Located in Alveolar SeptumStem Cells and Development, Vol. 21, No. 1Automated analysis of protein expression and gene amplification within the same cells of paraffin-embedded tumour tissue9 June 2011 | Cellular Oncology, Vol. 34, No. 4Fetal microchimerism as an explanation of disease21 December 2010 | Nature Reviews Endocrinology, Vol. 7, No. 2Placenta as a Source of Stem Cells and as a Key Organ for Fetomaternal Tolerance29 November 2010Combined detection of Her2/neu gene amplification and protein overexpression in effusions from patients with breast and ovarian cancer9 March 2010 | Journal of Cancer Research and Clinical Oncology, Vol. 136, No. 9CD34+ cells in maternal placental blood are mainly fetal in origin and express endothelial markersLaboratory Investigation, Vol. 89, No. 8Microchimerism in type 1 diabetes21 March 2009 | Current Diabetes Reports, Vol. 9, No. 2Increased fetal cell microchimerism in high grade breast carcinomas occurring during pregnancyInternational Journal of Cancer, Vol. 124, No. 5Fetal Microchimeric Cells Participate in Tumour Angiogenesis in Melanomas Occurring during PregnancyThe American Journal of Pathology, Vol. 174, No. 2Fetal Cell Microchimerism in Papillary Thyroid Cancer: A Possible Role in Tumor Damage and Tissue Repair15 October 2008 | Cancer Research, Vol. 68, No. 20Breast cancer stroma frequently recruits fetal derived cells during pregnancy20 December 2008 | Breast Cancer Research, Vol. 10, No. 1Cord blood in regenerative medicine: do we need immune suppression?30 January 2007 | Journal of Translational Medicine, Vol. 5, No. 1Donor‐Derived Human Bone Marrow Cells Contribute to Solid Organ Cancers Developing After Bone Marrow Transplantation2 January 2009 | STEM CELLS, Vol. 25, No. 11Spot Counting to Locate Fetal Cells in Maternal Blood and Tissue: A Comparison of Manual and Automated Microscopy1 January 2007 | Microscopy Research and Technique, Vol. 70, No. 7Microchimerism and Stem Cell Transplantation in Multiple SclerosisImpact of fetal–maternal microchimerism on women's health—a review7 July 2009 | The Journal of Maternal-Fetal & Neonatal Medicine, Vol. 20, No. 1Feto-maternal cell traffickingStem Cell Reviews, Vol. 2, No. 2Presence of Chimeric Maternally Derived Keratinocytes in Cutaneous Inflammatory Diseases of Children: The Example of Pityriasis LichenoidesJournal of Investigative Dermatology, Vol. 126, No. 2Maternal origin of inflammatory leukocytes in preterm fetal membranes, shown by fluorescence in situ hybridisationPlacenta, Vol. 26, No. 8-9Multi-lineage potential of fetal cells in maternal tissue: a legacy in reverseJournal of Cell Science, Vol. 118, No. 8Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer24 February 2005 | The Journal of Pathology, Vol. 205, No. 5Skin Carcinoma Arising From Donor Cells in a Kidney Transplant Recipient7 March 2005 | Cancer Research, Vol. 65, No. 5Cervical Cancer and MicrochimerismObstetrics & Gynecology, Vol. 102, No. 4 Vol. 34, No. 2 Follow us on social media for the latest updates Metrics Downloaded 233 times History Published online 24 September 2018 Published in print February 2003 Information© 2018 Author(s)PDF download

Objective: We conducted a trial to test if the blood of pregnant women contains fetal clonogenic erythroid cells the progeny of which can be identified and isolated by a newly developed flow-sorting procedure. Methods: We have previously demonstrated the identification of fetal nucleated red cells in cocultures of fetal and adult blood. The procedure is based on profiles of the correlated contents of fetal and adult hemoglobin (HbF and HbA, respectively), using antibodies specific for the different hemoglobin chains. In such profiles, fetal cells contain only HbF, while the vast majority of adult cells contain either only HbA or a combination of HbA and HbF. HbF+ HbA– cells are flow sorted and fetal cells identified by fluorescence in situ hybridization, using chromosome-specific probes. This technique provides a yield that approaches 100%, meaning that fetal cells will be found even if the culture contains only a single fetal erythroid colony among thousands of maternal colonies. Peripheral blood samples were obtained from 11 women carrying chromosomally normal male fetuses, from 5 women carrying trisomy 21 fetuses, and from 2 women carrying trisomy 18 fetuses. A further six samples came from women with an unknown fetal karyotype. As positive controls, we used blood samples drawn after termination procedures that tended to induce some fetomaternal hemorrhage. In parallel to the method being tested, we employed alternative techniques of fetal cell detection: one third of the mononuclear cell preparations from each maternal blood sample was not cultured but labeled with anti-HbF antibodies for flow sorting of F+ cells. Ten percent of the total harvested cell population of each culture was subjected to quantitative polymerase chain reaction analysis targeting a Y-chromosome-specific sequence. Results: Most posttermination blood samples yielded fetal cells with high purity which demonstrates the validity of the method. However, no fetal cells were found in any of the maternal blood samples with normal or abnormal pregnancies, neither before nor after culture. Conclusion: We conclude that a cell culture approach targeting clonogenic erythroid cells offers no advantage over established methods of direct isolation.

OBJECTIVE: To determine whether microchimerism is involved in the pathogenesis or progression of cervical cancer.METHODS: Cervical tissue was obtained from eight women who had at least one live-born son and who underwent radical hysterectomy after a diagnosis of cervical cancer. Control tissue was obtained from four women without cervical cancer who had at least one live-born son and from three women with cervical cancer and no male births. Tissue sections were analyzed with fluorescence in situ hybridization for the presence of fetal cells, defined by an X and Y chromosome. Immunolabeling was used to determine the phenotype of the presumed fetal cells.RESULTS: Male cells were found in cervical tissue from all four patients for whom large sections (approximately 1.5 X 2 cm) were analyzed. Only one male cell was found in two of the four patients for whom small biopsy specimens (approximately 0.1 X 0.5 cm) were analyzed. No male cells were found in tissue specimens from controls, whether they were small or large sections. In immunolabeling studies, eight of 18 male cells from one patient were CD45-posiltive and nine of 37 male cells from two patients were cytokeratin-positive. No cells were positive for both markers.CONCLUSION: Cervical cancer might be associated with microchimerism, possibly from fetomaternal cell trafficking. These results further expand the potential relationship between microchimerism and disease in women. (C) 2003 by The American College of Obstetricians and Gynecologists.