Enrichment of fetal cells and free fetal DNA from maternal blood: An insight into the Basel experience.

Our laboratory is a leading pioneer in the enrichment of fetal cells from maternal blood with the aim of developing a non-invasive risk free form of prenatal diagnosis. By using the then novel Magnetic Activated Cell Sorting (MACS) we were among the first to detect fetal aneuploidies. The efficacy of this methodology is currently being explored in the large scale so-called, NIFTY study conducted under the auspices of the NIH, in which our group is participating. We have extended the scope of these investigations by analysing single micromanipulated erythroblasts by single cell PCR. These studies have shown that fetal genetic loci such as sex and rhesus D status can be identified with great reliability non-invasively. This analysis also irrevocably demonstrated that a large proportion of the erythroblasts in maternal circulation are of fetal origin. This aspect has now been incorporated into the next phase of the NIH study. Recent research from our laboratory has indicated that the traffic of fetal cells into the maternal periphery is significantly enhanced in preeclamptic pregnancies. We have now investigated whether this perturbation takes place early in pregnancy prior to the onset of disease symptoms, by performing a prospective study in which close to 100 pregnant women were recruited at around 20 weeks of gestation. By correlating the number of enriched erythroblasts with subsequent pregnancy outcome, we were able to show that the traffic of fetal cells was indeed significantly elevated in those pregnancies which developed preeclampsia, but not those which were affected by fetal growth retardation. We have also explored the new finding of free fetal DNA in maternal plasma. By the use of sensitive quantitative PCR we were recently able to show that the levels of this circulatory fetal DNA are elevated in pregnancies with certain aneuploidies, thereby opening the prospect of a new additional screening tool.

Both maternal and fetal cell-free DNA in plasma fluctuate.

Elevations in the concentration of cell-free fetal DNA in maternal plasma have recently been determined in various pregnancy-related disorders, including preeclampsia, preterm labor, and polyhydramnios. In addition, almost 2-fold increments in cell-free fetal DNA levels have been recorded in pregnancies with certain aneuploid fetuses, in particular trisomy 21. These findings have led to the speculation that quantitative assessment of circulatory fetal DNA may be useful in the noninvasive prenatal diagnosis of certain fetal genetic constellations or pregnancy-related disorders. A premise for any quantitative analysis is that the quantity of the analyte being assayed does not vary greatly over time. As this aspect has not been examined for circulatory DNA levels, we examined these in normal healthy individuals as well as in pregnant women. Our data indicate that severalfold alterations in circulatory DNA amounts do occur over short periods of time. Of particular note is that we observed almost 2-fold variations in free fetal DNA levels over a period of 3 days, which are in a similar range to the elevations noted in aneuploid pregnancies. Our results, therefore, imply that caution should be used when using small increments in circulatory fetal DNA concentrations for potential diagnostic applications.

Severely reduced presence of tissue macrophages in the basal plate of pre-eclamptic placentae.

Pre-eclampsia is a disorder of unknown aetiology peculiar to human pregnancy. A well-described pathological feature being shallow trophoblast invasion into the spiral arteries during placenta development. Epidemiological studies have revealed an increased risk in pregnancies of primipaternity, and an association with the maternal-fetal HLA-DR relationship, both suggesting the involvement of an immunological component. We were therefore interested in the distribution of HLA-DR expressing myeloid cells in the decidua of healthy and pre-eclamptic placentae. We have studied the monocytes in maternal and fetal peripheral blood as well as in the placenta and identified the cluster of differentiation (CD) 14(+)myeloid cells in the basal plate as mannose receptor (ManR) positive tissue macrophages. In a comparison between peripheral blood monocytes from healthy pregnant and pre-eclamptic women we found no significant difference in the subpopulation size of CD14(+)/CD16(+)monocytes. The number and location of macrophages in the placental villi was similar. However, while the basal plate of the normal decidua contained numerous CD14(+), HLA-DR(bright), ManR(+)tissue macrophages, this compartment was virtually void of these phagocytic cells in the pre-eclamptic placenta. This novel finding suggests that in pre-eclampsia not only the migration of endovascular cytotrophoblasts is disturbed, but that also maternal macrophage migration is affected.

Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma.

OBJECTIVE: To examine whether concentrations of free extracellular fetal circulatory DNA in maternal plasma are stable or fluctuate. METHODS: Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). RESULTS: In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. CONCLUSION: The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.

Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses.

Current non-invasive screening methods for the prenatal diagnosis of fetal aneuploidies are hampered by low sensitivities and high false positive rates. Attempts to redress this situation include the enrichment of fetal cells from maternal blood, or the use of fetal DNA in the plasma of pregnant women. By the use of real-time quantitative polymerase chain reaction (PCR) it has recently been shown that circulatory male fetal DNA in maternal plasma is elevated in pregnancies with trisomy 21 fetuses. In this independent study we confirm and extend upon these results by showing that the levels of fetal DNA are also elevated in pregnancies with other chromosomal aneuploidies (mean=185.8 genome equivalents/ml; range=62.2-471.7) when compared to pregnancies with normal male fetuses (mean=81.9 genome equivalents/ml; range=28.8-328.9), p=0.005. This elevation was greatest for fetuses with trisomy 21, whereas it was not significant for fetuses with trisomy 18, p=0.356. These data suggest that a quantitative analysis of such fetal DNA levels may serve as an additional marker for certain fetal chromosomal abnormalities, in particular for trisomy 21.

Multiplex and real-time quantitative PCR on fetal DNA in maternal plasma. A comparison with fetal cells isolated from maternal blood.

Fetal DNA has recently been detected in maternal plasma by PCR and has shown promise for the prenatal determination of fetal sex or rhesus D. In order to obtain the maximum amount of information from this fetal genetic material, we have devised a sensitive multiplex PCR method to permit simultaneous analysis for both the SRY locus and the rhesus D gene. Our studies show that this technique is very sensitive and specific. In the 22 cases from rhesus D negative women examined, we were able to determine both fetal genotypes correctly. In the parallel enrichment for fetal cells, fetal erythroblasts were only detected in 14 of the 19 cases. Our data also indicate that fetal DNA from rhesus D positive fetuses is present in maternal plasma even after prophylactic anti-D treatment. Furthermore, since fetal cells have been reported to be elevated in pregnancies with aneuploid fetuses, we have quantified the amount of fetal DNA present in the maternal plasma of 10 such affected pregnancies by real-time PCR. Our results indicate that fetal DNA is elevated under such circumstances when compared to gestationally matched normal pregnancies (mean of 7% in aneuploid samples versus 3.5% in normal pregnancies). These results indicate that the quantification of fetal DNA in maternal plasma may be an additional screening tool for pregnancies at risk of bearing an aneuploid fetus.

Functional inactivation in the whole population of human V gamma 9/V delta 2 T lymphocytes induced by a nonpeptidic antagonist.

Nonpeptidic compounds stimulate human T cells bearing the TCR-gamma delta in the absence of major histocompatibility complex restriction. We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of V gamma 9/V delta T cells ex vivo, antagonizes the same cell population after repetitive activation. Stimulation with DPG results in partial early protein tyrosine phosphorylation and a prolonged, but reversible, state of unresponsiveness to agonist ligands in V gamma 9/V delta 2, but not in other T cells. These findings show that TCR antagonism is a general phenomenon of T cells. However, in contrast to the clonal specificity of altered peptides antagonizing alpha beta T cells, all the tested V gamma 9/V delta 2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.

Interferon beta-1b inhibits gelatinase secretion and in vitro migration of human T cells: a possible mechanism for treatment efficacy in multiple sclerosis.

Treatment with interferon beta-1b has substantial clinical benefit in the demyelinating disease multiple sclerosis, yet the mechanism of action in the disease remains largely unknown. Gelatinase A (matrix metalloproteinase-2, 72-kd gelatinase) and B (matrix metalloproteinase-9, 92-kd gelatinase) are matrix metalloproteinases capable of enzymatic digestion of subendothelial basement membrane constituents. In human T cells, interleukin-2 induces gelatinase secretion and enhances gelatinase-dependent migration across an artificial basement membrane-like layer in vitro. Pretreatment of T cells with interferon beta-1b for 48 hours decreased interleukin-2-induced gelatinase production and secretion as determined by zymography. In parallel to the downregulation of gelatinase secretion, pretreatment with interferon beta-1b inhibited T-cell migration across the basement membrane in vitro by up to 90%, but had only a minor impact on cell locomotion per se. For both gelatinase secretion and T-cell migration, the inhibitory effect mediated by exposure to interferon beta-1b was dose dependent. Fluorescence-activated cell sorter analysis also showed that interferon beta-1b downregulates the interleukin-2 receptor alpha-chain and lowered the affinity of interleukin-2 to the cell surface by 30%, which may represent an additional mechanism for the observed effects of interferon beta-1b. The dramatic effects of interferon beta-1b on gelatinase expression and migration raise the possibility that its beneficial effects in multiple sclerosis may result from interference with the capacity of activated T cells to traverse the basement membrane and migrate to the central nervous system.

Human V gamma 9-V delta 2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites.

Many different pathogens stimulate cells bearing the V gamma 9-V delta 2 T cell receptor (TCR), which represent the most abundant population of human gamma delta cells. The antigens responsible for the stimulation of these gamma delta cells are not well characterized. Here, we describe six non-peptidic molecules which share this property: isopentenylpyrophosphate, dimethylallylpyrophosphate, 2,3-diphosphoglyceric acid, glycerol-3-phosphoric acid, xylose-1-phosphate, and ribose-1-phosphate. All these molecules are naturally occurring metabolites in prokaryotic and eukaryotic cells, and stimulate freshly isolated gamma delta cells from peripheral blood of different donors as well as established gamma delta clones. Comparison of their structure with that of similar but inactive molecules showed that both the number and position of the phosphate groups, as well as the residues connected with the carbon backbone are required for stimulation. The CD3-TCR complex is involved in cell triggering as shown by inhibition with anti-CD3 Fab fragments. However, all gamma delta clones were broadly cross-reactive and we could not isolate cells specific for only one ligand. The capacity of this frequent subset of gamma delta cells to recognize common bacterial metabolites confers the advantage to react rapidly to different invading pathogens.