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Biomedical subjects

Kirby L Johnson

Publications and source records attributed to Kirby L Johnson.

At least 19 recordsLinked to original sources
PubMed2006

Circulating cell-free fetal messenger RNA levels after fetoscopic interventions of complicated pregnancies.

OBJECTIVE: The aim of this study was to examine fetal gene expression in maternal plasma after fetoscopic intervention for twin-twin transfusion syndrome or congenital diaphragmatic hernia. STUDY DESIGN: Twelve women with pregnancies that were complicated by twin-twin transfusion syndrome and 10 women carrying fetuses with congenital diaphragmatic hernia were sampled before and sequentially after treatment. Levels of glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, and gamma globin messenger RNA were measured by real-time reverse transcriptase polymerase chain reaction amplification. RESULTS: At all time points, glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels were higher in the congenital diaphragmatic hernia cases than in the twin-twin transfusion syndrome cases (P < .05), but during the immediate postoperative observation period, there were no significant changes in glyceraldehyde-3-phosphate dehydrogenase, human placental lactogen, or gamma globin messenger RNA levels in individual patients or patients who were grouped by procedure. CONCLUSION: Fetoscopic intervention of complicated pregnancies does not affect circulating fetal messenger RNA levels, which is in contrast to earlier observations that circulating fetal DNA levels increase after laser ablation for twin-twin transfusion syndrome. Plasma glyceraldehyde-3-phosphate dehydrogenase messenger RNA levels could be a potential novel biomarker for fetal trauma.

Female↗
PubMed2006

A prospective analysis of cell-free fetal DNA concentration in maternal plasma as an indicator for adverse pregnancy outcome.

OBJECTIVES: To evaluate whether cell-free fetal (cff) DNA in maternal plasma during the second trimester is a marker for developing pregnancy-associated complications. Two PCR techniques for the detection and quantitation of fetal DNA were compared. METHODS: Plasma samples were prospectively collected from 84 pregnant women carrying male fetuses before amniocentesis (14-29 weeks). We later recorded 26 pregnancies with complicated outcomes, including five cases of fetal chromosomal abnormalities. For statistical analysis, two overlapping subgroups A and B were made. Each group was separately compared for total and fetal DNA with a corresponding group considered normal using Wilcoxon rank sum test. Male fetal DNA concentration in maternal plasma was quantified using real-time quantitative polymerase chain reaction (PCR) of SRY sequences. The samples were also analyzed by quantitative fluorescent PCR (QF-PCR) using highly polymorphic short tandem repeat DNA sequences (STRs), and the percentage of relative fetal allele concentration in maternal alleles was calculated and compared to the fetal/total DNA ratio obtained by real-time PCR. RESULTS: Quantities of total and fetal circulating DNA were significantly correlated (r(2) = 0.44, P < 0.0001) with a median total DNA concentration of 522 GE/mL (range 51-3047) and a median fetal DNA concentration of 8 GE/mL (range 0-879). Neither level was correlated with gestational age in pregnancies with normal (r(2) = -0.05; P = 0.66, and r(2) = 0.02; P = 0.88, respectively) and abnormal (r(2) = 0.45; P = 0.17, and r(2) = 0.11; P = 0.76, respectively) outcomes. Although both total and fetal DNA levels were always higher in women carrying pregnancies with chromosomal aberrations or having other pregnancy complications (P-values range from 0.028 to 0.267), these differences reached statistical significance only for total DNA levels between the group A and corresponding normal pregnancies (P = 0.028). The correlation between the fetal/total DNA ratio obtained by real-time PCR and the percentage of relative fetal allele concentration in maternal alleles obtained by QF-PCR was not found to be statistically significant (r(2) = 0.04; P = 0.76). CONCLUSION: Our results confirm the clinical value of fetal DNA measurement in maternal plasma during the second trimester as a supplement for the diagnosis of aneuploidies. Its use as a screening instrument for complications that develop later in pregnancy seems to be limited but needs further investigation. Although the QF-PCR assay has the advantage of being applicable to both female and male fetuses, this approach cannot be used for quantitation of cff DNA in maternal plasma samples.

Alleles↗
PubMed2006

Fetal nucleic acids in maternal body fluids: an update.

Our laboratory continues to be actively involved in the development of new biomarkers for prenatal diagnosis using maternal blood and amniotic fluid. We have also developed a mouse model that demonstrates that cell-free fetal (cff) DNA is detectable in the pregnant maternal mouse. In human maternal plasma and serum we have analyzed factors that are important in the clinical interpretation of cff DNA levels. Maternal race, parity, and type of conception (natural or assisted) do not affect cff DNA levels, but maternal weight does. We have also analyzed the relationship between placental volume, using a three-dimensionsal ultrasound examination, and cff DNA levels. Surprisingly, there is no association between these values. Finally, we are using specific disease models (such as congenital diaphragmatic hernia and twin-to-twin transfusion) to understand the effects of gestational age and specific pathology on fetal gene expression by analyzing cell-free mRNA levels in maternal plasma. In the amniotic fluid we have focused on improvements in recovery of cff DNA and mRNA. By optimizing recovery we have made some interesting observations about differences in fetal DNA between blood and amniotic fluid. In addition, we have successfully hybridized cff DNA in amniotic fluid to DNA microarrays, permitting assessment of fetal molecular karyotype. We also have preliminary data on fetal gene expression in amniotic fluid. Finally, we remain actively involved in promoting noninvasive prenatal testing in the United States, such as encouraging the use of fetal DNA for fetal rhesus D assessment. On the other hand, we are cautious and concerned about the accuracy of "at-home" kits for fetal gender detection.

Amniotic Fluid↗
PubMed2005

Cell-free fetal DNA levels in pregnancies conceived by IVF.

BACKGROUND: Increased second-trimester levels of maternal serum HCG in IVF conceptions lead to an increased false-positive rate in Down syndrome screening. Increased levels of cell-free fetal DNA (cffDNA) in maternal plasma have been correlated with increased HCG levels. Our aim was to determine whether cffDNA levels are elevated in IVF pregnancies compared with natural pregnancies. METHODS: Sixteen archived second-trimester serum samples from IVF pregnancies were matched with five control samples from naturally conceived pregnancies per case, all carrying a singleton male fetus. cffDNA concentrations were measured by real-time PCR amplification of a Y chromosome sequence and compared with four standard second trimester serum screening markers (alpha-fetoprotein, estriol, HCG and inhibin A). RESULTS: Mean cffDNA levels for cases and controls were 57.9 and 57.1 genome equivalents/ml, respectively (P = 0.95). Mean observed rank (from 1 to 6) of cffDNA was 3.625 in the IVF conceived group, compared with an expected value of 3.5 (P = 0.53). No significant correlations were observed between cffDNA and serum markers. CONCLUSIONS: IVF does not affect levels of cffDNA, which appears to be independent of traditional screening markers (e.g. HCG). Therefore, cffDNA can be used as an additional serum marker (e.g. Down syndrome screening) without adjustment for IVF pregnancies.

Biomarkers↗
PubMed2005

Global gene expression analysis of the living human fetus using cell-free messenger RNA in amniotic fluid.

CONTEXT: No molecular biological tests are available to monitor the ongoing development of human fetuses in vivo. OBJECTIVE: To determine whether cell-free fetal messenger RNA (mRNA) in amniotic fluid can be detected using oligonucleotide microarrays to study large-scale gene expression in living human fetuses, with analysis of sex, gestational age, and fetal pathology as variables. DESIGN, SETTING, AND PATIENTS: Four samples of cell-free amniotic fluid were analyzed from pregnant women between 20 and 32 weeks' gestation and undergoing amnioreduction for polyhydramnios associated with twin-twin transfusion syndrome or hydrops fetalis (cases). The control consisted of 6 pooled amniotic fluid samples from women at 17 weeks' gestation and undergoing genetic amniocentesis. After extraction from the normally discarded fraction of amniotic fluid, RNA was amplified twice, labeled, and analyzed using gene expression microarrays. MAIN OUTCOME MEASURE: Relative mRNA expression in cell-free samples of amniotic fluid from fetuses with polyhydramnios at different gestational ages vs cell-free amniotic fluid from a pooled control. RESULTS: Thirty-six percent of 22,283 probe sets represented on the arrays were present in the cell-free amniotic fluid, and a median of 20% of all probe sets differed between cases and the pooled control. Only male samples expressed 1 Y chromosome transcript. The expression of some developmental transcripts, such as surfactant proteins, mucins, and keratins, changed with gestational age by up to 64-fold. A water transporter gene transcript was increased up to 18-fold in both twin-twin transfusion samples. Placental gene transcripts were not present in any samples. CONCLUSIONS: This pilot study demonstrates that cell-free fetal mRNA can be extracted from amniotic fluid and successfully hybridized to gene expression microarrays. Preliminary analysis suggests that gene expression changes can be detected in fetuses of different sexes, gestational age, and disease status. Cell-free mRNA in amniotic fluid appears to originate from the fetus and not the placenta.

Amniocentesis↗
PubMed2005

The utility of an erythroblast scoring system and gender-independent short tandem repeat (STR) analysis for the detection of aneuploid fetal cells in maternal blood.

OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (NRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system based on the unique morphological and hemoglobin staining characteristics of this cell type. Presumptive fetal NRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. METHODS: NRBCs were isolated by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection from peripheral blood of nine women following termination of pregnancy for trisomy 21 (n=4), 18 (n=1), 13 (n=2), and other genetic abnormalities (n=2). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR (polymerase chain reaction) amplification of chromosome 21 (D21S1411, D21S11) and chromosome 18 (D18S535) short tandem repeat (STR) DNA polymorphisms. RESULTS: In all cases, candidate fetal NRBCs were accurately identified on the basis of morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and nonshared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSIONS: Using the erythroblast scoring system and subsequent analysis of inherited DNA polymorphisms, we were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation and genetic analysis is a promising method for noninvasive prenatal diagnostic applications.

Aneuploidy↗
PubMed2005

Placental volume, as measured by 3-dimensional sonography and levels of maternal plasma cell-free fetal DNA.

OBJECTIVE: Measurement of cell-free fetal (cff) DNA in maternal plasma may have clinical application in prenatal screening for fetal Down syndrome and preeclampsia. Little is known regarding the tissue of origin of these fetal-derived sequences. We tested the hypothesis that if the placenta is the major contributor of cff DNA, then an increased placental volume should be associated with higher maternal plasma cff DNA levels. STUDY DESIGN: We enrolled 143 pregnant women who underwent first-trimester placental volume measurement using 3-dimensional ultrasonography. Cff DNA in maternal plasma on the day of the scan was quantified by real-time polymerase chain reaction (PCR) amplification of a Y-chromosome sequence. The association between measured placental volume and maternal plasma cff DNA levels was analyzed along with relevant clinical variables. RESULTS: The median (25th, 75th percentiles) maternal plasma cff DNA level was 16.9 genome equivalents (GE)/mL (10.8, 28.7). Raw values were adjusted for gestational age and maternal body mass index. The median (25th, 75th percentiles) placental volume was 53.2 mL (43.0, 64.7), and median placental quotient (ratio of placental volume to fetal crown-rump length) was 1 mm2 (0.8, 1.1). Based on multivariate linear regression analyses, neither of the above placental measurements showed a significant association with maternal plasma cff DNA levels (P = .43 and .43, respectively). A modest association was found between plasma cff DNA levels and gravidity (P = .03). CONCLUSION: Our data did not show a significant association between either the placental volume or placental quotient, and maternal plasma cff DNA levels. We speculate that it is the extent of placental apoptosis that primarily affects the amount of cff DNA released into the maternal circulation.

Cross-Sectional Studies↗
PubMed2005

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.

Actins↗
PubMed2005

Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction.

We have identified a subpopulation of stem cells within adult human BM, isolated at the single-cell level, that self-renew without loss of multipotency for more than 140 population doublings and exhibit the capacity for differentiation into cells of all 3 germ layers. Based on surface marker expression, these clonally expanded human BM-derived multipotent stem cells (hBMSCs) do not appear to belong to any previously described BM-derived stem cell population. Intramyocardial transplantation of hBMSCs after myocardial infarction resulted in robust engraftment of transplanted cells, which exhibited colocalization with markers of cardiomyocyte (CMC), EC, and smooth muscle cell (SMC) identity, consistent with differentiation of hBMSCs into multiple lineages in vivo. Furthermore, upregulation of paracrine factors including angiogenic cytokines and antiapoptotic factors, and proliferation of host ECs and CMCs, were observed in the hBMSC-transplanted hearts. Coculture of hBMSCs with CMCs, ECs, or SMCs revealed that phenotypic changes of hBMSCs result from both differentiation and fusion. Collectively, the favorable effect of hBMSC transplantation after myocardial infarction appears to be due to augmentation of proliferation and preservation of host myocardial tissues as well as differentiation of hBMSCs for tissue regeneration and repair. To our knowledge, this is the first demonstration that a specific population of multipotent human BM-derived stem cells can induce both therapeutic neovascularization and endogenous and exogenous cardiomyogenesis.

Adult↗
PubMed2004

Fetal cells in maternal tissue following pregnancy: what are the consequences?

The presence and persistence of fetal cells in murine maternal tissue was first reported over 20 years ago, although it is only more recently that the occurrence and potential consequences of fetomaternal cell trafficking in humans have been fully appreciated. Fetal cell microchimerism is a growing field of investigation, although the data are contradictory relative to the health consequences of persistent fetal cells in maternal tissues. Understanding of the types of cells being transferred from fetus to mother, the location of these fetal cells within the various maternal tissue types, and the functionality of these cells may ultimately lead to measures to minimize or eliminate the deleterious effects of the cells, or to efforts to take advantage of the presence of these cells for therapeutic purposes. This review focuses on the origins of fetal cell microchimerism research and the different hypotheses regarding the consequences of persistent fetal cells in the mother, the various diseases that have been evaluated with respect to fetomaternal cell trafficking, the potential variables associated with the frequency, persistence and tissue distribution of fetal cells in maternal tissue, and an assessment of future direction in this innovative field of inquiry.

Animals↗
PubMed2004

Fetal cell-free DNA circulates in the plasma of pregnant mice: relevance for animal models of fetomaternal trafficking.

BACKGROUND: Cell-free fetal DNA (fDNA) can be detected in maternal plasma throughout human pregnancy and is rapidly cleared after delivery. fDNA measurement has clinical application in many complications of pregnancy. Our aim was to determine if fDNA could be detected in maternal plasma during pregnancy in a mouse model system. We then compared the levels of fDNA during pregnancies in which the mother and fetus were either congenic or allogenic. METHODS: C57BL/6J (H-2b) or DBA/2J (H-2d) wild-type female mice were mated to C57BL/6J mice transgenic for the enhanced green fluorescent protein (gfp) and sacrificed while pregnant. C57BL/6J female mice that had previously given birth to three to six litters after mating with transgenic males were sacrificed after delivery. We used real-time quantitative PCR amplification to detect and measure gfp sequences in maternal plasma. RESULTS: fDNA was consistently detected in maternal plasma during pregnancy and was always absent after delivery [median 211 genome equivalents (GE)/ml vs 0 GE/ml, respectively, P=0.0001]. The level of fDNA was higher in allogenic matings compared to congenic matings (median 167 GE/ml/GFP + fetus vs 81 GE/ml/GFP + fetus, respectively). CONCLUSIONS: fDNA sequences can be reliably detected in maternal plasma during murine pregnancy. Our data lends further support to the use of nonhuman species to investigate the mechanisms involved in fetomaternal trafficking.

Animals↗
PubMed2004

Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype.

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.

Amniocentesis↗
PubMed2004

Transfer of fetal cells with multilineage potential to maternal tissue.

CONTEXT: During pregnancy, fetal CD34+ cells enter the maternal circulation, persist for decades, and create a state of physiologic microchimerism. Many studies have confirmed the residual presence of fetal cells in maternal blood and tissues following pregnancy. Fetal cells may respond to maternal injury by developing multilineage capacity in maternal organs. OBJECTIVE: To verify that fetal microchimeric cells express markers of epithelial, leukocyte, and hepatocyte differentiation within maternal organs. DESIGN, SETTING, AND PATIENTS: Archived paraffin-embedded tissue section specimens from 10 women who had male offspring and were previously found to have high numbers of microchimeric cells, and 11 control women who had no prior male pregnancies. Male cells were identified by fluorescence in situ hybridization, using X and Y chromosome-specific probes, followed by histologic and immunochemical studies using anticytokeratin (AE1/AE3) as a marker of epithelial cells, anti-CD45 as a leukocyte marker, and heppar-1 as a hepatocyte marker. MAIN OUTCOME MEASURE: Percentage of microchimeric cells expressing nonhematopoietic markers. RESULTS: A total of 701 male (XY+) microchimeric cells were identified (mean [SD], 227 [128] XY+ cells per million maternal cells). In maternal epithelial tissues (thyroid, cervix, intestine, and gallbladder), 14% to 60% of XY+ cells expressed cytokeratin. Conversely, in hematopoietic tissues, such as lymph nodes and spleen, 90% of XY+ cells expressed CD45. In 1 liver sample, 4% of XY+ cells expressed heppar-1. Histologic and immunochemical evidence of differentiation, as assessed by independent observers, was highly concordant (kappa = 0.72). CONCLUSION: The detection of microchimeric male cells, bearing epithelial, leukocyte, or hepatocyte markers, in a variety of maternal tissue specimens suggests the presence of fetal cells that may have multilineage capacity.

Adult↗
PubMed2004

Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR.

BACKGROUND: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. METHODS: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. RESULTS: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93-100%) with only four false-positive results obtained across the entire study. CONCLUSIONS: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.

Clinical Laboratory Techniques↗
PubMed2004

ROC analysis of an erythroblast morphologic scoring system to improve identification of fetal cells in maternal blood.

OBJECTIVES: Nucleated red blood cells are used in research settings as a target cell type for investigations of noninvasive prenatal diagnosis, and these cells have a characteristic nuclear morphology and hemoglobin staining pattern that makes them distinguishable from maternal cells. Recently, we developed an erythroblast scoring system based on these characteristics. Here, we employ statistical analyses to further characterize the utility of this scoring system. METHODS: A total of 170 nucleated red blood cells isolated from peripheral blood of four women undergoing elective termination of a trisomy 21 male fetus were analyzed. Each of the four scoring system parameters, whose values range from 0 to 3 points, served as an independent variable to determine its significance for the correct identification of a cell as fetal or maternal. A logistic regression was used as a multivariable statistical tool. RESULTS: Forty-four patterns of the four parameters were found in the overall series. Some patterns were exclusively associated with maternal cells (e.g. 1-1-1-1 and 1-1-2-2), and others were exclusively associated with fetal cells (e.g. 3-2-2-2 and 3-2-3-2). A detection rate of 73.9% at a false-positive rate of 5% resulted from a random simulation model performed with a 1:5 case:control matched set. The variables most predictive of a cell being fetal were gamma hemoglobin staining intensity of cytoplasm and nuclear roundness. CONCLUSIONS: The modified scoring system presented here improves upon the previously reported, unmodified erythroblast scoring system. These statistical analyses suggest that the scoring system is a promising method that aids in distinguishing fetal and maternal NRBCs for prenatal diagnostic applications and that it may be amenable to automated microscopy by applying the discrete morphological parameters as computational classifiers.

Cell Nucleus↗