An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10 Expression across T Helper Cell Subsets.

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) Il10 regulators, generating an experimentally refined regulatory network for Il10. 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 Il10 regulators that orchestrate immunoregulatory programs across T helper cell subsets.

Effects of testosterone replacement in men with opioid-induced androgen deficiency: a randomized controlled trial.

Symptomatic androgen deficiency is common in patients taking opioid analgesics, as these drugs potently suppress the hypothalamic-pituitary-gonadal axis. However, the efficacy of testosterone replacement in this setting remains unclear. The objective of this trial was to evaluate the efficacy of testosterone replacement on pain perception and other androgen-dependent outcomes in men with opioid-induced androgen deficiency. We conducted a randomized, double-blind, parallel placebo-controlled trial at an outpatient academic research center. Participants were men aged 18 to 64 years on opioid analgesics for chronic noncancer pain, and total testosterone levels were <350 ng/dL. Participants were randomly assigned to 14 weeks of daily transdermal gel that contained 5 g of testosterone or placebo. Primary outcomes were changes in self-reported clinical pain and objectively assessed pain sensitivity. Sexual function, quality of life, and body composition were also assessed. The mean age was 49 years. The median total and free testosterone levels at baseline were 243 ng/dL and 47 pg/mL and 251 ng/dL and 43 pg/mL in the testosterone and placebo arm, respectively. Of the 84 randomized participants, 65 had follow-up data on efficacy outcomes. Compared with men assigned to the placebo arm, those assigned to testosterone replacement experienced greater improvements in pressure and mechanical hyperalgesia, sexual desire, and role limitation due to emotional problems. Testosterone administration was also associated with an improvement in body composition. There were no between-group differences in changes in self-reported pain. In conclusion, in men with opioid-induced androgen deficiency, testosterone administration improved pain sensitivity, sexual desire, body composition, and aspects of quality of life.

Testosterone dose-response relationships with cardiovascular risk markers in androgen-deficient women: a randomized, placebo-controlled trial.

OBJECTIVE: To determine dose-dependent effects of T administration on cardiovascular risk markers in women with low T levels. METHODS: Seventy-one hysterectomized women with or without oophorectomy with total T < 31 ng/dL and/or free T < 3.5 pg/mL received a standardized transdermal estradiol regimen during the 12-week run-in period and were then randomized to receive weekly im injections of placebo or 3-, 6.25-, 12.5-, or 25-mg T enanthate for 24 weeks. Total and free T levels were measured by liquid chromatography-tandem mass spectrometry and equilibrium dialysis, respectively. Insulin resistance and inflammatory markers were measured at baseline and 24 weeks. In a subset of women, magnetic resonance imaging of the abdomen was performed to quantify abdominal fat volume. RESULTS: Fifty-nine women who completed the 24-week intervention were included in the final analysis. The five groups were similar at baseline. Mean on-treatment nadir total T concentrations were 14, 79, 105, 130, and 232 ng/dL in the placebo group and the 3-, 6.25-, 12.5-, and 25-mg groups, respectively. No significant changes in fasting glucose, fasting insulin, homeostatic model assessment of insulin resistance, high sensitivity C-reactive protein, adiponectin, blood pressure, and heart rate were observed at any T dose when compared to placebo. Similarly, no dose- or concentration-dependent changes were observed in abdominal fat on magnetic resonance imaging. CONCLUSION: Short-term T administration over a wide range of doses for 24 weeks in women with low T levels was not associated with worsening of cardiovascular risk markers.

Testosterone dose-response relationships in hysterectomized women with or without oophorectomy: effects on sexual function, body composition, muscle performance and physical function in a randomized trial.

OBJECTIVE: This study aims to determine the dose-dependent effects of testosterone on sexual function, body composition, muscle performance, and physical function in hysterectomized women with or without oophorectomy. METHODS: Seventy-one postmenopausal women who previously underwent hysterectomy with or without oophorectomy and had total testosterone levels less than 31 ng/dL or free testosterone levels less than 3.5 pg/mL received a standardized transdermal estradiol regimen during the 12-week run-in period and were randomized to receive weekly intramuscular injections of placebo or 3, 6.25, 12.5, or 25 mg of testosterone enanthate for 24 weeks. Total and free testosterone levels were measured by liquid chromatography-tandem mass spectrometry and equilibrium dialysis, respectively. The primary outcome was change in sexual function measured by the Brief Index of Sexual Functioning for Women. Secondary outcomes included changes in sexual activity, sexual distress, Derogatis Interview for Sexual Functioning, lean body mass, fat mass, muscle strength and power, and physical function. RESULTS: Seventy-one women were randomized; five groups were similar at baseline. Sixty-two women with analyzable data for the primary outcome were included in the final analysis. The mean on-treatment total testosterone concentrations were 19, 78, 102, 128, and 210 ng/dL in the placebo, 3-mg, 6.25-mg, 12.5-mg, and 25-mg groups, respectively. Changes in composite Brief Index of Sexual Functioning for Women scores, thoughts/desire, arousal, frequency of sexual activity, lean body mass, chest-press power, and loaded stair-climb power were significantly related to increases in free testosterone concentrations; compared with placebo, changes were significantly greater in women assigned to the 25-mg group, but not in women in the lower-dose groups. Sexual activity increased by 2.7 encounters per week in the 25-mg group. The frequency of androgenic adverse events was low. CONCLUSIONS: Testosterone administration in hysterectomized women with or without oophorectomy for 24 weeks was associated with dose and concentration-dependent gains in several domains of sexual function, lean body mass, chest-press power, and loaded stair-climb power. Long-term trials are needed to weigh improvements in these outcomes against potential long-term adverse effects.

Fetal cells in the murine maternal lung have well-defined characteristics and are preferentially located in alveolar septum.

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.

Maternal background strain influences fetal-maternal trafficking more than maternal immune competence in mice.

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 10(7) maternal cells and assessment of cell surface phenotype. Wild-type C57BL/6J (n=18), FVB/NJ (n=15), and immunodeficient B6129S7-Rag1(tm1Mom)/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 10(7) 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.

Increased fetal cell trafficking in murine lung following complete pregnancy loss from exposure to lipopolysaccharide.

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.

Array-CGH analysis of cell-free fetal DNA in 10 mL of amniotic fluid supernatant.

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.

Spot counting to locate fetal cells in maternal blood and tissue: a comparison of manual and automated microscopy.

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 (400x magnification), the number of cells located and duration of scoring were recorded. For automated scanning using the Metasystems Metafer3/Metafer4 Scanning System (200x 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).

Cell-free fetal DNA in amniotic fluid: unique fragmentation signatures in euploid and aneuploid fetuses.

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.

Natural history of fetal cell microchimerism during and following murine pregnancy.

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.

Cervical cancer and microchimerism.

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-positive 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.

Fetal cell isolation from maternal blood cultures by flow cytometric hemoglobin profiles. Results of a preliminary clinical trial.

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 post-termination 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.