Publications by authors named "Kirby L Johnson"

72 Publications

Innovative Approaches in Chronic Disease Management: Health Literacy Solutions and Opportunities for Research Validation.

Stud Health Technol Inform 2017 ;240:433-443

Institute for Healthcare Advancement, U.S.A.

This chapter discusses the need for innovative health literacy solutions to combat extensive chronic disease prevalence and costs. The authors explore the intersection of chronic disease management and health literacy. They provide specific examples of successful health literacy interventions for managing several highly prevalent chronic diseases. This is followed by suggestions on pairing research and practice to support effective disease management programs. In addition, the authors discuss strategies for collection and dissemination of knowledge gained from collaborations between researchers and practitioners. They identify current challenges specific to disseminating information from the health literacy field and offer potential solutions. The chapter concludes with a brief look at future directions and organizational opportunities to integrate health literacy practices to address the need for effective chronic disease management.
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April 2018

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

Hum Genet 2014 Sep 22;133(9):1075-82. Epub 2014 May 22.

Mother Infant Research Institute and Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, MA, USA,

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.
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http://dx.doi.org/10.1007/s00439-014-1448-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384642PMC
September 2014

Global gene expression analysis of amniotic fluid cell-free RNA from recipient twins with twin-twin transfusion syndrome.

Prenat Diagn 2013 Sep 4;33(9):873-83. Epub 2013 Jun 4.

Mother Infant Research Institute, Tufts Medical Center, Boston, MA, USA.

Objective: The objective of this study was to understand the biological pathways involved in twin-twin transfusion syndrome (TTTS) by performing global gene expression analysis of amniotic fluid (AF) cell-free RNA.

Methods: A prospective whole transcriptome microarray study analyzing cell-free RNA in AF from TTTS recipient twins and singleton controls was carried out. Significantly differentially regulated genes in TTTS cases (N = 8) versus matched controls (N = 8) were identified and pathways analyses performed. Significant gene expression differences between stage II TTTS recipients (N = 5) and stage III TTTS recipients with abnormal Doppler measurements (N = 5) were also analyzed.

Results: Analysis of paired data from TTTS cases and controls revealed differential expression of 801 genes, which were significantly enriched for neurological disease and cardiovascular system pathways. We also identified cardiovascular genes and pathways associated with the presence of critically abnormal Doppler measurements in stage III TTTS recipients.

Conclusions: This study provides the first transcriptome-wide data on the impact of TTTS on fetal development. Our results show that gene expression involving neurological and cardiovascular pathways are altered in recipient fetuses prior to surgical treatment. This has relevance for the origins of long-term complications seen in survivors and for the development of future fetal biomarkers.
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http://dx.doi.org/10.1002/pd.4150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3773178PMC
September 2013

The natural history of fetal cells in postpartum murine maternal lung and bone marrow: a two-stage phenomenon.

Chimerism 2012 Jul-Dec;3(3):59-64. Epub 2012 Jul 1.

Mother Infant Research Institute, Floating Hospital at Tufts Medical Center, and Genetics Program, Department of Pathology, Sackler School of Graduate Biomedical Sciences, Boston, MA, USA.

During pregnancy, fetal cells cross into the maternal organs where they reside postpartum. Evidence from multiple laboratories suggests that these microchimeric fetal cells contribute to maternal tissue repair after injury. In mouse models, most injury experiments are performed during pregnancy; however, in a clinical setting most injuries or diseases occur postpartum. Therefore, experiments using animal models should be designed to address questions in the time period following delivery. In order to provide a baseline for such experiments, we analyzed the natural history of fetal cells in the postpartum maternal organs. Female C57BL/6J mice were mated to males homozygous for the enhanced green fluorescent protein gene. Fetal cells in the maternal lungs and bone marrow were identified by their green fluorescence using in a high-speed flow cytometer and their counts were compared between the lung and bone marrow. Spearman correlation analysis was used to identify relationships between the duration of time postpartum and the cell counts and ratio of live and dead cells. Our results show that fetal cells persist in these organs until at least three months postpartum in healthy female mice. We show a two-stage decline, with an initial two and a half-week rapid clearance followed by a trend of gradual decrease. Additionally, an increase in the ratio of live to dead cells within the lung over time suggests that these cells may replicate in vivo. The results presented here will inform the design of future experiments and may have implications for women's health.
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http://dx.doi.org/10.4161/chim.22769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545915PMC
June 2013

Performing discovery-driven neonatal research by transcriptomic analysis of routinely discarded biofluids.

J Matern Fetal Neonatal Med 2012 Dec 27;25(12):2507-11. Epub 2012 Aug 27.

Department of Pediatrics, The Floating Hospital for Children at Tufts Medical Center, Boston, MA 02111, USA.

Objective: To perform discovery-driven research on the neonatal salivary and cord blood transcriptomes.

Methods: Two separate cohorts of infants were enrolled in this study. In one, cord blood (n = 10) and in the other, saliva samples (n = 10) were collected at term gestation. Total RNA was extracted, amplified and hybridized onto Affymetrix HG U133a gene expression microarrays. Following normalization, genes expressed in the highest quintile (≥ 80%) across all subjects in each biofluid were analyzed with Ingenuity Pathway Analysis. Over-represented pathways relating to organ specific development and physiological functions in the newborn were explored.

Results: There were 303 genes in neonatal saliva and 282 genes in umbilical cord blood that met statistical criteria. Of these, 114 were common to both biofluids. Pathway analyses revealed the important roles of redox balance, cellular proliferation, and smooth muscle relaxation. In blood, hematopoiesis and immune response pathways predominated. In saliva, pathways associated with the gastrointestinal system were highlighted.

Conclusions: Neonatal cord blood and saliva provide a wealth of transcriptomic information. These normally discarded biofluids should be considered an important source of real-time gene expression data that may elucidate key pathways in neonatal physiology and pathology.
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http://dx.doi.org/10.3109/14767058.2012.717126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511475PMC
December 2012

Increased death of adipose cells, a path to release cell-free DNA into systemic circulation of obese women.

Obesity (Silver Spring) 2012 Nov 7;20(11):2213-9. Epub 2012 Jun 7.

Department of Obstetrics and Gynecology, MetroHealth Medical Center Case Western Reserve University, Cleveland, Ohio, USA.

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study, we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell-free DNA (cfDNA) into their systemic circulation. Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) positive CD68 macrophages in obese vs. lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a twofold increase in circulating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.
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http://dx.doi.org/10.1038/oby.2012.138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483388PMC
November 2012

Novel neurodevelopmental information revealed in amniotic fluid supernatant transcripts from fetuses with trisomies 18 and 21.

Hum Genet 2012 Nov 3;131(11):1751-9. Epub 2012 Jul 3.

Mother Infant Research Institute and the Division of Genetics, Department of Pediatrics, The Floating Hospital for Children at Tufts Medical Center, 800 Washington St, Boston, MA 02111, USA.

Trisomies 18 and 21 are the two most common live born autosomal aneuploidies in humans. While the anatomic abnormalities in affected fetuses are well documented, the dysregulated biological pathways associated with the development of the aneuploid phenotype are less clear. Amniotic fluid (AF) cell-free RNA is a valuable source of biological information obtainable from live fetuses. In this study, we mined gene expression data previously produced by our group from mid-trimester AF supernatant samples. We identified the euploid, trisomy 18 and trisomy 21 AF transcriptomes, and analyzed them with a particular focus on the nervous system. We used multiple bioinformatics resources, including DAVID, Ingenuity Pathway Analysis, and the BioGPS Gene Expression Atlas. Our analyses confirmed that AF supernatant from aneuploid fetuses is enriched for nervous system gene expression and neurological disease pathways. Tissue analysis showed that fetal brain cortex and Cajal-Retzius cells were significantly enriched for genes contained in the AF transcriptomes. We also examined AF transcripts known to be dysregulated in aneuploid fetuses compared with euploid controls and identified several brain-specific transcripts among them. Many of these genes play critical roles in nervous system development. NEUROD2, which was downregulated in trisomy 18, induces neurogenic differentiation. SOX11, downregulated in trisomy 21, is a transcription factor that is essential for pan-neuronal protein expression and axonal growth of sensory neurons. Our results show that whole transcriptome analysis of cell-free RNA in AF from live pregnancies permits discovery of biomarkers of abnormal human neurodevelopment and advances our understanding of the pathophysiology of aneuploidy.
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http://dx.doi.org/10.1007/s00439-012-1195-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472090PMC
November 2012

Comprehensive analysis of genes expressed by rare microchimeric fetal cells in the maternal mouse lung.

Biol Reprod 2012 Aug 23;87(2):42. Epub 2012 Aug 23.

Mother Infant Research Institute, Floating Hospital at Tufts Medical Center, Boston, Massachusetts, USA.

During pregnancy, cells from each fetus travel into the maternal circulation and organs, resulting in the development of microchimerism. Identification of the cell types in this microchimeric population would permit better understanding of possible mechanisms by which they affect maternal health. However, comprehensive analysis of fetal cells has been hampered by their rarity. In this study, we sought to overcome this obstacle by combining flow cytometry with multidimensional gene expression microarray analysis of fetal cells isolated from the murine maternal lung during late pregnancy. Fetal cells were collected from the lungs of pregnant female mice. cDNA was amplified and hybridized to gene expression microarrays. The resulting fetal cell core transcriptome was interrogated using multiple methods including Ingenuity Pathway Analysis, the BioGPS gene expression database, principal component analysis, the Eurexpress gene expression atlas, and primary literature. Here we report that small numbers of fetal cells can be flow sorted from the maternal lung, facilitating discovery-driven gene expression analysis. We additionally show that gene expression data can provide functional information about fetal cells. Our results suggest that fetal cells in the murine maternal lung are a mixed population, consisting of trophoblasts, mesenchymal stem cells, and cells of the immune system. Detection of trophoblasts and immune cells in the maternal lung may facilitate future mechanistic studies related to the development of immune tolerance and pregnancy-related complications, such as pre-eclampsia. Furthermore, the presence and persistence of mesenchymal stem cells in maternal organs may have implications for long-term postpartum maternal health.
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http://dx.doi.org/10.1095/biolreprod.112.101147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431427PMC
August 2012

Neuropeptide Y2 receptor (NPY2R) expression in saliva predicts feeding immaturity in the premature neonate.

PLoS One 2012 21;7(5):e37870. Epub 2012 May 21.

Division of Newborn Medicine, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, Massachusetts, United States of America.

Background: The current practice in newborn medicine is to subjectively assess when a premature infant is ready to feed by mouth. When the assessment is inaccurate, the resulting feeding morbidities may be significant, resulting in long-term health consequences and millions of health care dollars annually. We hypothesized that the developmental maturation of hypothalamic regulation of feeding behavior is a predictor of successful oral feeding in the premature infant. To test this hypothesis, we analyzed the gene expression of neuropeptide Y2 receptor (NPY2R), a known hypothalamic regulator of feeding behavior, in neonatal saliva to determine its role as a biomarker in predicting oral feeding success in the neonate.

Methodology/principal Findings: Salivary samples (n = 116), were prospectively collected from 63 preterm and 13 term neonates (post-conceptual age (PCA) 26 4/7 to 41 4/7 weeks) from five predefined feeding stages. Expression of NPY2R in neonatal saliva was determined by multiplex RT-qPCR amplification. Expression results were retrospectively correlated with feeding status at time of sample collection. Statistical analysis revealed that expression of NPY2R had a 95% positive predictive value for feeding immaturity. NPY2R expression statistically significantly decreased with advancing PCA (Wilcoxon test p value<0.01), and was associated with feeding status (chi square p value  =  0.013).

Conclusions/significance: Developmental maturation of hypothalamic regulation of feeding behavior is an essential component of oral feeding success in the newborn. NPY2R expression in neonatal saliva is predictive of an immature feeding pattern. It is a clinically relevant biomarker that may be monitored in saliva to improve clinical care and reduce significant feeding-associated morbidities that affect the premature neonate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037870PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357390PMC
December 2012

The amniotic fluid transcriptome: a source of novel information about human fetal development.

Obstet Gynecol 2012 Jan;119(1):111-8

Mother Infant Research Institute and Division of Genetics, Department of Pediatrics, The Floating Hospital for Children, Tufts Medical Center, Boston, Massachusetts, USA.

Objective: Amniotic fluid is a complex biological material that provides a unique window into the developing human. Residual amniotic fluid supernatant contains cell-free fetal RNA. The objective of this study was to develop an understanding of the amniotic fluid core transcriptome by analyzing the transcripts ubiquitously present in the amniotic fluid supernatant of euploid midtrimester fetuses.

Methods: This was an in silico (computational) investigation using publicly available gene expression data previously produced by our group from 12 euploid midtrimester amniotic fluid samples. Functional analyses were performed using a web-based software analysis tool. Organ specificity was examined for each transcript using a gene expression atlas. For fetal organs not represented in the atlas, manual literature searching and the web-based software analysis tool were used to generate fetal organ-associated gene lists.

Results: There were 476 well-annotated genes present in 12 of 12 amniotic fluid samples. Functional analysis identified six physiologic systems represented in the amniotic fluid core transcriptome, including musculoskeletal and nervous system development and function and embryonic and organismal development. Mammalian target of rapamycin signaling was identified as a key canonical pathway. Twenty-three highly organ-specific transcripts were identified; six of these are known to be highly expressed in the fetal brain.

Conclusion: Amniotic fluid cell-free fetal RNA can provide biological information on multiple fetal organ systems. The presence of fetal-brain specific transcripts in amniotic fluid suggests novel approaches to the study of developmental disorders that involve the central nervous system. The finding that the mammalian target of rapamycin signaling is enriched in midtrimester fetuses may have future applications in the study of fetal growth disorders.
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http://dx.doi.org/10.1097/AOG.0b013e31823d4150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273331PMC
January 2012

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

Stem Cells Dev 2012 Jan 14;21(1):158-65. Epub 2011 Oct 14.

Mother Infant Research Institute at Tufts Medical Center, Boston, Massachusetts 02111, USA.

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.
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http://dx.doi.org/10.1089/scd.2010.0518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245672PMC
January 2012

Proof of concept study to assess fetal gene expression in amniotic fluid by nanoarray PCR.

J Mol Diagn 2011 Sep 9;13(5):565-70. Epub 2011 Aug 9.

Division of Genetics, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, Massachusetts, USA.

Microarray analysis of cell-free RNA in amniotic fluid (AF) supernatant has revealed differential fetal gene expression as a function of gestational age and karyotype. Once informative genes are identified, research moves to a more focused platform such as quantitative reverse transcriptase-PCR. Standardized NanoArray PCR (SNAP) is a recently developed gene profiling technology that enables the measurement of transcripts from samples containing reduced quantities or degraded nucleic acids. We used a previously developed SNAP gene panel as proof of concept to determine whether fetal functional gene expression could be ascertained from AF supernatant. RNA was extracted and converted to cDNA from 19 AF supernatant samples of euploid fetuses between 15 to 20 weeks of gestation, and transcript abundance of 21 genes was measured. Statistically significant differences in expression, as a function of advancing gestational age, were observed for 5 of 21 genes. ANXA5, GUSB, and PPIA showed decreasing gene expression over time, whereas CASC3 and ZNF264 showed increasing gene expression over time. Statistically significantly increased expression of MTOR and STAT2 was seen in female compared with male fetuses. This study demonstrates the feasibility of focused fetal gene expression analysis using SNAP technology. In the future, this technique could be optimized to examine specific genes instrumental in fetal organ system function, which could be a useful addition to prenatal care.
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http://dx.doi.org/10.1016/j.jmoldx.2011.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157615PMC
September 2011

Optimal techniques for mRNA extraction from neonatal salivary supernatant.

Neonatology 2012 26;101(1):55-60. Epub 2011 Jul 26.

Division of Newborn Medicine, Department of Pediatrics, The Floating Hospital for Children at Tufts Medical Center, Medford, MA, USA.

Background: Gene expression profiling of the salivary supernatant is emerging as a new and important source of real-time, systemic, biological information. However, existing technologies prevent RNA extraction of small quantities found in neonatal salivary supernatant.

Objective: The aim of this study was to develop techniques to enhance extraction of cell-free RNA from neonatal salivary supernatant.

Methods: Two saliva samples (10-100 μl) were serially collected from newborns (36-41 weeks' gestation) (n = 13) and stabilized. Total RNA was extracted from salivary supernatant with the use of two modified extraction techniques: Qiagen RNAprotect® Saliva Mini Kit (method 1) and the QIAamp Viral RNA Mini Kit (method 2). Quantitative RT-PCR amplification for GAPDH was performed on extracted salivary samples. Statistical analyses were performed on mean threshold cycle (Ct) levels to compare RNA yield from each protocol. Paired microarray analyses were made between neonatal whole saliva and supernatant (n = 3) to discern gene expression differences between these biolayers.

Results: mRNA was successfully extracted and amplified from all salivary supernatant samples. Extraction with method 2 yielded more RNA than with method 1 (p = 0.008). There was a 7.5% discordance between paired gene expression analyses for whole saliva and supernatant. Genes that were statistically significantly upregulated in supernatant highlighted 16 distinct biological functions not seen in whole saliva. Conversely, only two biological functions were unique to whole saliva.

Conclusion: Neonatal cell-free salivary supernatant mRNA may be readily extracted and utilized on downstream applications. These technical enhancements allow for further exploration of the diagnostic potential of the neonatal salivary transcriptome.
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http://dx.doi.org/10.1159/000328026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3151004PMC
July 2012

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

J Reprod Immunol 2011 Aug 5;90(2):188-94. Epub 2011 Jul 5.

Division of Hematology and Oncology, Department of Medicine, Tufts Medical Center, Boston, MA 02111, USA.

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.
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http://dx.doi.org/10.1016/j.jri.2011.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143711PMC
August 2011

Fetal cell microchimerism and cancer: a nexus of reproduction, immunology, and tumor biology.

Cancer Res 2011 Jan;71(1):8-12

Division of Hematology and Oncology, Department of Medicine, Tufts Medical Center, Boston, Massachusetts 02111, USA.

Fetal cell microchimerism (FCM) is the persistence of fetal cells in the maternal circulation and organs following pregnancy. Proposed hypotheses about the function of fetal cells in the pathogenesis of maternal cancer include promotion of tumorigenesis, protection by providing immunosurveillance, and participation in tissue repair. To date, studies of FCM and cancer have been primarily descriptive and quantitative. More research is needed to understand the cellular phenotype of the microchimeric cells in maternal tumors and whether they have a functional role. This research will require further study using a multidisciplinary approach, incorporating knowledge of the fetomaternal relationship, tumor biology, immunology, and clinical oncology.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059599PMC
January 2011

Transcriptomic analysis of cell-free fetal RNA suggests a specific molecular phenotype in trisomy 18.

Hum Genet 2011 Mar 9;129(3):295-305. Epub 2010 Dec 9.

Division of Genetics, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, 800 Washington Street, Box 394, Boston, MA 02111, USA.

Trisomy 18 is a common human aneuploidy that is associated with significant perinatal mortality. Unlike the well-characterized "critical region" in trisomy 21 (21q22), there is no corresponding region on chromosome 18 associated with its pathogenesis. The high morbidity and mortality of affected individuals has limited extensive investigations. In order to better understand the molecular mechanisms underlying the congenital anomalies observed in this condition, we investigated the in utero gene expression profile of second trimester fetuses affected with trisomy 18. Total RNA was extracted from cell-free amniotic fluid supernatant from aneuploid fetuses and euploid controls matched for gestational age and hybridized to Affymetrix U133 Plus 2.0 arrays. Individual differentially expressed transcripts were obtained by two-tailed t tests. Over-represented functional pathways among these genes were identified with DAVID and Ingenuity(®) Pathways Analysis. Results show that three hundred and fifty-two probe sets representing 251 annotated genes were statistically significantly differentially expressed between trisomy 18 and controls. Only 7 genes (2.8% of the annotated total) were located on chromosome 18, including ROCK1, an up-regulated gene involved in valvuloseptal and endocardial cushion formation. Pathway analysis indicated disrupted function in ion transport, MHCII/T cell mediated immunity, DNA repair, G-protein mediated signaling, kinases, and glycosylation. Significant down-regulation of genes involved in adrenal development was identified, which may explain both the abnormal maternal serum estriols and the pre and postnatal growth restriction in trisomy 18. Comparison of this gene set to one previously generated for trisomy 21 fetuses revealed only six overlapping differentially regulated genes. This study contributes novel information regarding functional developmental gene expression differences in fetuses with trisomy 18.
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http://dx.doi.org/10.1007/s00439-010-0923-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206603PMC
March 2011

Insights into fetal and neonatal development through analysis of cell-free RNA in body fluids.

Early Hum Dev 2010 Nov 18;86(11):747-52. Epub 2010 Sep 18.

Mother Infant Research Institute at Tufts Medical Center, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, MA 02111, USA.

The use of cell-free nucleic acids in the circulation of pregnant women for noninvasive prenatal diagnosis is arguably one of the hottest current topics in prenatal medicine. Between 1997 and the present era this field has gone from basic research to clinical application for diagnosis of fetal gender and Rhesus D status. Over the next few years it is likely that noninvasive prenatal diagnosis for Down syndrome will also be possible. Here we summarize current and future clinical applications of analyzing cell-free fetal DNA and RNA in both maternal and neonatal body fluids, including maternal plasma, serum, whole blood, amniotic fluid, and neonatal saliva. We describe methods to evaluate normal and abnormal fetal and neonatal development using gene expression microarrays. We also discuss the ways in which differentially-regulated gene lists can advance knowledge of both fetal and neonatal biology, as well as suggest novel possibilities for fetal and neonatal treatment.
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http://dx.doi.org/10.1016/j.earlhumdev.2010.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967661PMC
November 2010

Relationships between cell-free DNA and serum analytes in the first and second trimesters of pregnancy.

Obstet Gynecol 2010 Sep;116(3):673-678

From the Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Tufts Medical Center, Boston, Massachusetts; the Division of Genetics, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, Massachusetts; the Department of Pathology and Laboratory Medicine, Women and Infants Hospital and the Alpert School of Medicine at Brown University, Providence, Rhode Island; the Institute of Clinical Research and Health Policy Studies, Tufts Medical Center, Boston, Massachusetts; and the Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York.

Objective: To assess the relationship between first- and second-trimester cell-free DNA levels and maternal serum screening markers.

Methods: First- and second-trimester residual maternal serum samples from 50 women were obtained. First-trimester (pregnancy-associated plasma protein A and beta-hCG) and second-trimester serum analytes (beta-hCG, alpha-fetoprotein, unconjugated estriol, and inhibin A) had been measured at the time of sample receipt. All fetuses were male as confirmed by birth records. Cell-free DNA was extracted and measured by real-time quantitative polymerase chain reaction amplification using glyceraldehyde phosphate dehydrogenase and DYS1 as markers of total DNA and fetal DNA, respectively. Determination of linear associations between first- and second-trimester serum markers and cell-free DNA levels using Pearson correlations was performed.

Results: Statistically significant correlations between first-trimester pregnancy-associated plasma protein A multiples of the median and both total (r=0.36, P=.016) and fetal (r=0.41, P=.006) DNA in the first trimester were observed. There were no significant correlations between first-trimester serum human chorionic gonadotropin or any second-trimester serum marker with DNA levels.

Conclusion: Correlation between serum pregnancy-associated plasma protein A and first-trimester circulating cell-free fetal and total DNA levels is a novel finding. Pregnancy-associated plasma protein A is a glycoprotein of placental origin, and its correlation to cell-free fetal DNA in maternal serum suggests a common tissue origin through apoptosis of placental cells. However, because pregnancy-associated plasma protein A and cell-free DNA were only marginally correlated and cell-free DNA can be reliably detected in the first trimester, the addition of cell-free DNA to serum screening strategies may be helpful in predicting adverse pregnancy outcome.

Level Of Evidence: II.
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http://dx.doi.org/10.1097/AOG.0b013e3181ea2dd1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2975594PMC
September 2010

Cord blood genomic analysis highlights the role of redox balance.

Free Radic Biol Med 2010 Sep 8;49(6):992-6. Epub 2010 Jun 8.

Division of Newborn Medicine, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, MA 02111, USA.

Neonates are exposed to elevated levels of reactive oxygen species as they transition from a hypoxic intrauterine to a normoxic extrauterine environment at birth. This increased oxidative stress is associated with neonatal morbidity. Current antioxidant supplementation treatment strategies have yet to translate into improved neonatal outcomes. Our understanding of a newborn's intricate redox balance, particularly at the genomic level, remains limited. Here, we performed genomic microarray analyses (approximately 14,500 genes) on extracted mRNA from umbilical cord whole blood at term gestation (n=10). Bioinformatic analyses identified 282 genes (2.0%) that were consistently present within the highest quintile of expressed genes. These genes were highly associated with oxidant stress and included superoxide dismutase 1, catalase, peroxiredoxins, and uncoupling proteins. Pathway analyses identified statistically significantly overrepresented functional pathways including "oxidative stress," "oxidative stress response mediated by nuclear factor-erythroid 2-related factor," "hypoxia-inducible factor signaling," and "mitochondrial dysfunction" (p<0.05). These results suggest that neonates require high levels of antioxidants and an intricate cellular redox balance to ensure a successful transition to the extrauterine environment. Understanding the genes necessary to maintain this delicate redox balance may lead to the development of alternative treatment strategies.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921475PMC
September 2010

High-throughput discovery and characterization of fetal protein trafficking in the blood of pregnant women.

Proteomics Clin Appl 2009 Dec;3(12):1389-96

Floating Hospital for Children at Tufts Medical Center, Department of Pediatrics, Division of Newborn Medicine 800 Washington Street, Box 44 Boston, MA 02111.

Although the measurement of fetal proteins in maternal serum is part of standard prenatal screening for aneuploidy and neural tube defects, attempts to better understand the extent of feto-maternal protein trafficking and its clinical and biological significance have been hindered by the presence of abundant maternal proteins. The objective of this study was to circumvent maternal protein interference by using a computational predictive approach for the development of a noninvasive, comprehensive, protein network analysis of the developing fetus in maternal whole blood. From a set of 157 previously identified fetal gene transcripts, 46 were classified into known protein networks, and 222 downstream proteins were predicted. Statistically significantly over-represented pathways were diverse and included T-cell biology, neurodevelopment and cancer biology. Western blot analyses validated the computational predictive model and confirmed the presence of specific downstream fetal proteins in the whole blood of pregnant women and their newborns, with absence or reduced detection of the protein in the maternal postpartum samples. This work demonstrates that extensive feto-maternal protein trafficking occurs during pregnancy, and can be predicted and verified to develop novel noninvasive biomarkers. This study raises important questions regarding the biological effects of fetal proteins on the pregnant woman.
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http://dx.doi.org/10.1002/prca.200900109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825712PMC
December 2009

Circulating cell-free DNA levels increase variably following chorionic villus sampling.

Prenat Diagn 2010 Apr;30(4):325-8

Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Tufts Medical Center, Boston, MA 02111, USA.

Objective: Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA.

Methods: Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 min after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender.

Results: Sensitivity of DYS1 detection in male fetuses was 100% (n = 30); specificity in female fetuses was 100% (n = 22). While a majority of women had > 50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels.

Conclusions: While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women.
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http://dx.doi.org/10.1002/pd.2456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966017PMC
April 2010

Neonatal salivary analysis reveals global developmental gene expression changes in the premature infant.

Clin Chem 2010 Mar 3;56(3):409-16. Epub 2009 Dec 3.

Floating Hospital for Children at Tufts Medical Center, Department of Pediatrics, Division of Newborn Medicine, 800 Washington St., Box 44, Boston, MA 02111, USA.

Background: There is an important need to develop noninvasive biomarkers to detect disease in premature neonates. Our objective was to determine if salivary genomic analysis provides novel information about neonatal expression of developmental genes.

Methods: Saliva (50-200 microL) was prospectively collected from 5 premature infants at 5 time points: before, starting, and advancing enteral nutrition; at the introduction of oral feeds; and at advanced oral feeds. Salivary RNA was extracted, amplified, and hybridized onto whole-genomic microarrays.

Results: Bioinformatics analyses identified 9286 gene transcripts with statistically significant gene expression changes across individuals over time. Of these genes, 3522 (37.9%) were downregulated, and 5764 (62.1%) were upregulated. Gene expression changes were highly associated with developmental pathways. Significantly downregulated expression was seen in embryonic development, connective tissue development and function, hematologic system development and function, and survival of the organism (10(-14) < P < 10(-3)). Conversely, genes associated with behavior, nervous system development, tissue development, organ development, and digestive system development were significantly upregulated (10(-11) < P < 10(-2)).

Conclusions: Comparative genomic salivary analyses provide robust, comprehensive, real-time information regarding nearly all organs and tissues in the developing preterm infant. This innovative and noninvasive technique represents a new approach for monitoring health, disease, and development in this vulnerable patient population. By comparing these data in healthy infants with data from infants who develop medical complications, we expect to identify new biomarkers that will ultimately improve newborn care.
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http://dx.doi.org/10.1373/clinchem.2009.136234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853178PMC
March 2010

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

Fertil Steril 2010 Mar 7;93(5):1718-1721.e2. Epub 2009 Oct 7.

Division of Genetics, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, 800 Washington Street, Box 394, Boston, MA 02111, USA.

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.
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http://dx.doi.org/10.1016/j.fertnstert.2009.08.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842077PMC
March 2010

Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses.

Proc Natl Acad Sci U S A 2009 Jun 27;106(23):9425-9. Epub 2009 May 27.

Department of Computer Science, Tufts University, Medford, MA 02155, USA.

To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS.
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http://dx.doi.org/10.1073/pnas.0903909106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2687148PMC
June 2009

Fetal cells in the pregnant mouse are diverse and express a variety of progenitor and differentiated cell markers.

Biol Reprod 2009 Jul 11;81(1):26-32. Epub 2009 Mar 11.

Division of Genetics, Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, Massachusetts 02111, USA.

To better understand fetomaternal cell trafficking during pregnancy, we used a mouse model to determine the cell surface markers expressed on fetal cells, based on the hypothesis that fetal progenitor cells have the capacity to repair maternal organs, whereas more differentiated cells might initiate graft versus host disease. Wild-type females were mated to either homozygous or hemizygous transgenic males and euthanized in the peripartum period. Using dual color flow cytometry, we analyzed fetal transgene positive cells for the presence of nine markers (ITGAM, ITGB1, PECAM, CD34, CD44, PTPRC, ENG, SLAMF1, and CXCR4) to begin to identify the phenotype and degree of differentiation of fetal cells in nine maternal organs (lung, liver, spleen, blood, bone marrow, kidney, heart, thymus, and brain). Fetal cells were found in all maternal organs following either type of mating, albeit always at a higher frequency following mating with homozygous males. Some organs (e.g., lung and liver) had a wide variety of fetal cell markers present, while other organs (e.g., bone marrow and spleen) had a skewed distribution of fetal cell markers. Fetal cells in the murine pregnant female are diverse. Our results suggest that the fetal cells comprise a mixed population of progenitor and differentiated cells, with different relative proportions in different maternal organs. Future studies will address whether fetal cells cross the placental barrier in a differentiated state or as a homogenous population and subsequently differentiate in target maternal organs.
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http://dx.doi.org/10.1095/biolreprod.108.074468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093984PMC
July 2009

Fetomaternal trafficking in the mouse increases as delivery approaches and is highest in the maternal lung.

Biol Reprod 2008 Nov 16;79(5):841-8. Epub 2008 Jul 16.

Department of Pediatrics, Floating Hospital for Children at Tufts Medical Center, Boston, Massachusetts 02111, USA.

The purpose of the study was to understand in more detail the natural history of fetomaternal cell trafficking in healthy pregnant mice. Our goal was to identify the best target organs and days during pregnancy for further mechanistic studies of the role of fetal cells in maternal disease and injury. C57BL/6J wild-type virgin females (n = 54) were mated with congenic enhanced green fluorescent protein (EGFP) transgenic males. During pregnancy and after delivery, female mice were euthanized, and eight organs and blood were analyzed for the presence of fetal GFP+ cells with flow cytometry and real-time quantitative PCR. Maternal lungs, liver, and spleen were also analyzed by fluorescent stereomicroscopy. Fetal GFP+ cells were first found at low frequency at Embryonic Day 11, increased to a maximum at Embryonic Day 19, and decreased rapidly postpartum. These fetal cell dynamics were significantly reproducible among all mice studied. In addition, there was a consistent distribution of fetal cells within maternal organs, with lung, liver, blood, and spleen having the greatest concentrations; these were highly correlated at all time points (P < 0.0001). Maternal lung contained 10- to 100-fold more fetal cells than any other organ, and using all three techniques, the number of fetal cells detected was the most consistent and reproducible in this organ. Stereomicroscopy showed that within the lung, fetal cells were widely and apparently randomly distributed. Using a murine model, our data demonstrate that fetomaternal cellular trafficking occurs in reproducible patterns, is maximal near term delivery, and has predilection for the maternal lung.
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http://dx.doi.org/10.1095/biolreprod.108.068973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714997PMC
November 2008

pH but not hypoxia affects neonatal gene expression: relevance for housekeeping gene selection.

J Matern Fetal Neonatal Med 2008 Jul;21(7):443-7

Department of Pediatrics, Tufts Medical Center, Boston, MA 02111, USA.

Objective: To identify a candidate neonatal housekeeping gene and to determine the effects of pH and PaO(2) on the stability of newborn gene expression in physiologically hypoxic and acidotic newborn blood.

Methods: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) amplification was performed for four commonly used housekeeping genes (GAPDH, beta-actin, cyclophilin, 28S rRNA) on extracted RNA. Blood gas analyses determined pH and PaO(2) levels.

Results And Conclusions: Beta-Actin was the least variable and GAPDH the most variable housekeeping gene studied. pH negatively correlated with gene expression levels. PaO(2) levels did not significantly affect gene expression. These results inform selection of housekeeping genes for neonatal mRNA research.
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http://dx.doi.org/10.1080/14767050802104843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855685PMC
July 2008

Method for extraction of high-quantity and -quality cell-free DNA from amniotic fluid.

Methods Mol Biol 2008 ;444:303-9

Division of Genetics, Department of Pediatrics, Tufts-New England Medical Center and Floating Hospital for Children, Tufts University School of Medicine, Boston, MA, USA.

Circulating cell-free fetal deoxyribonucleic acids (cffDNAs) are promising biomarkers with various potential clinical applications. Second and third trimester amniotic fluid (AF) is a rich source of cffDNAs. Further improvements to the original protocol for the extraction of cffDNAs from AF supernatant resulted in statistically significant higher yields of high-quality cffDNAs, allowing for a substantial majority of samples to be analyzed with subsequent molecular methods (e.g., comparative genomic hybridization microarrays) to further assess for genetic abnormalities. Several advantages have been realized with the optimized protocol. In addition to an improved yield from a greater proportion of samples compared with the original protocol, the current method, using large silico-membranes, allows for the extraction of cffDNAs from up to 10 samples in <3 h. The replacement of the original lysis buffer eliminates the need for a heating bath during the lysis step, and fewer overall steps are involved in the protocol (e.g., to reduce potential contamination). The improvements in the yield with the current protocol make it possible to augment current standard of care through the analysis of this previously unappreciated source of genetic material. Furthermore, the improvements allow for exploration of widely unknown genetic, pathophysiological, and kinetic issues of cell-free fetal DNA in AF.
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http://dx.doi.org/10.1007/978-1-59745-066-9_24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679523PMC
June 2008