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Circulating tumor cell (CTC) detection in peripheral blood of colon and other epithelial cancer patients is becoming a scientifically recognised indicator for the presence of primary tumors and/or metastasis. The resulting need to further develop CTC detection-based systems for improved diagnosis, prognosis and assessment of therapy efficacy in tumour patients has prompted the application of different approaches, including expression analysis of tissue-specific and epithelial genes. In this context, lack of specificity of the analysed genes remains a fundamental problem for reliable CTC detection. In this study, we have selected a panel of highly specific epithelial genes: cytokeratin 20 (CK20), cytokeratin 19 (CK19), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC), and performed RT-PCR analysis to assess their expression in total blood and in different cell fractions of peripheral blood (PBMC and CD45-negative population) of cancer patients and healthy controls. Our results demonstrate that analysis of a single gene in a CTC-enriched population (CD45− peripheral blood cells) of cancer patients allows detection of a CTC molecular signature in at most 63.3% of cases, while analysis of all four genes performed in all three sample types increases the detection of positive patient samples to 87.7%. Healthy controls did not show positivity for any combination of these genes, although positivity was observed for the CEA marker alone, which was detected in 3 (6.6%) out of 45 donors, and only in the CD45− fraction. Here, we demonstrate that combined analysis of the genes above, in multiple blood fractions, results in a highly specific and sensitive CTC detection system in patients with metastatic solid tumors. Therefore, we believe that validation on a large scale of this approach, which demonstrates higher specificity in patients compared to controls, could become a relevant CTC screening test in patients with established metastatic disease, and furthermore, may also be useful for evaluating the possible presence of CTCs before the onset of clinically manifested metastatic spreading.

Chimerism, defined as the co-existence of cells of different origin within the same organism, has received much attention in hematopoietic cell and organ transplantation because of the strict relationship between its establishment and the induction of specific tolerance. Traditional methods applied for chimerism detection, such as immunohistochemistry, cytogenetics, fluorescent-activated cell sorter analysis, and serological and biochemical testing, are limited by their sensitivity. We have established a highly sensitive molecular approach based on the amplification of the mitochondrial cytochrome B gene and tested its specificity and sensitivity level in six different mammalian species, including human, pig, mouse, rat, sheep and rabbit. Increased sensitivity of detection of specific amplification products was obtained by the non-radioactive Southern blot technique. This novel approach allows the detection of one cell against the background of 1 to 4 x 10(6) xenogenec cells and will be helpful for high-sensitivity analysis of donor cell engraftment after xenotransplantation procedures in these animal models.

The Wiskott-Aldrich syndrome (WAS), X-linked severe combined immunodeficiency (SCIDX1), and X-linked agammaglobulinemia (XLA) are severe congenital immunodeficiencies with X-linked inheritance. Although rare, they are all associated with severe infections from early in life, and high morbidity and mortality. Female carriers of these diseases can be identified by a non-random pattern of X-chromosomal inactivation in cell lineages targeted by each gene defect. For patients with WAS, SCIDX1 or XLA, the demonstration of non random X-Chromosome inactivation in their mothers can be used to confirm clinical diagnosis. Furthermore, analysis of X-Chromosome inactivation in at risk females allows preconceptional carrier detection, thus representing an important aid in genetic counseling. For each disease we established a PCR-based, non radioactive assay at the human androgen receptor (HUMARA) locus, that allows analysis of X-Chromosome inactivation in the affected cell types and in tissue specific controls to exclude the issue of skewed X-chromosomal inactivation. In our study, 50 females with a known family history of XLA [19], WAS [18], and SCIDX1 [13],were examined. A carrier status was established in 19 females (7 XLA, 6 WAS, 6 SCIDX1) and excluded in 29 ( 11 XLA, 11 WAS, 7 SCIDX1). Only in 2 cases (4%) the assay was not informative.

Summary Background X-linked severe combined immunodeficiency (SCIDX1) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common γ chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDX1 can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. Methods A male fetus was diagnosed as having SCIDX1 by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E resetting. Chimerism analysis was by HLA-DQα typing and γ-chain staining on cord blood. Findings A healthy 3·6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3·5 months of age the infant is well and his T-cell counts and function are normal. Interpretation In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Fetal membranes (amnion and chorion) have recently raised significant attention as potential sources of stem cells. We have recently demonstrated that cells derived from human term placenta show stem cell phenotype, high plasticity, and display low immunogenicity both in vitro and in vivo. Moreover, placenta-derived cells, after xenotransplantation, are able to engraft in solid organs including the lung. On these bases, we studied the effects of fetal membrane-derived cells on a mouse model of bleomycin-induced lung fibrosis. Fetal membrane-derived cells were infused 15 min after intratracheal bleomycin instillation. Different delivery routes were used: intraperitoneal or intratracheal for both xenogeneic and allogeneic cells, and intravenous for allogeneic cells. The effects of the transplanted cells on bleomycin-induced inflammatory and fibrotic processes were then scored and compared between transplanted and control animals at different time points. By PCR and immunohistochemistry analyses, we demonstrated the presence of transplanted cells 3, 7, 9, and 14 days after transplantation. Concomitantly, we observed a clear decrease in neutrophil infiltration and a significant reduction in the severity of bleomycin-induced lung fibrosis in mice treated with placenta-derived cells, irrespective of the source (allogeneic or xenogeneic) or delivery route. Our findings constitute further evidence in support of the hypothesis that placenta-derived cells could be useful for clinical application, and warrant further studies toward the use of these cells for the repair of tissue damage associated with inflammatory and fibrotic degeneration.

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G0 phase and abolishes the production of inflammatory cytokines such as TNF-α, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Cells derived from the amniotic membrane of human placenta have been receiving particular attention because of their stem cell potentiality and immunomodulatory properties, which make them an attractive candidate source for cell therapy approaches. In this study, we isolated cells from the mesenchymal region of amnion and identified two subpopulations discordant for expression of the HLA-DR, CD45, CD14, and CD86 cellular markers. We therefore refer to the unfractionated cell population derived from this region as amniotic mesenchymal tissue cells (AMTC). We studied the suppressive and stimulatory characteristics of the unfractionated, HLA-DR-positive, and HLA-DR-negative AMTC populations and demonstrated that all three fail to induce an allogeneic T-cell response. However, unfractionated AMTC, which could inhibit T-cell allogeneic proliferation responses, induced proliferation of T cells stimulated via the T-cell receptor (TcR), in a cell-cell contact setting. We have shown that this stimulatory capacity can be attributed to the HLA-DR-positive AMTC subpopulation. Indeed, even though the HLA-DR-positive AMTC fraction surprisingly failed to induce proliferation of resting allogeneic T cells, they could cause strong proliferation of anti-CD3-primed allogeneic T cells. This stimulatory effect was not observed using the HLA-DR-negative AMTC fraction. The revelation that human amniotic mesenchyme possesses cell populations with both suppressive and stimulatory properties sheds additional light on the immunomodulatory functions of this tissue and may contribute to the clarification of some ongoing controversies associated with mesenchymal stromal cells of other sources, such as the presence of HLA-DR-positive cells and the suppressive versus stimulatory properties of these cells.Disclosure of potential conflicts of interest is found at the end of this article.

Bone marrow (BM) multipotent mesenchymal stromal cells (MSCs) present with multipotent differentiation potential and immunomodulatory properties. As an alternative to bone marrow, we have examined fetal membranes, amnion and chorion, of term human placenta as a potential source of multipotent MSCs. Here we show that amnion mesenchymal cells (AMCs) and chorion mesenchymal cells (CMCs), isolated by mechanical separation and subsequent enzymatic digestion, demonstrate plastic adherence and fibroblast-like morphology and are able to form colonies that could be expanded for at least 15 passages. By FACS analysis, AMCs and CMCs were shown to be phenotypically similar to BM-MSCs and, when cultured in differentiation media, they demonstrated high morphogenetic plasticity by differentiating into osteocytes, chondrocytes and adipocytes. In an attempt to isolate cells with MSC characteristics from human fetal membranes, AMCs and CMCs expressing CD271 were enriched by immunomagnetic isolation and were demonstrated to possess higher clonogenic and osteogenic differentiation potential than CD271-depleted fractions. Based on these findings, amnion and chorion can be considered as a novel and convenient source of adult MSCs.

Immunosuppression, myeloablation, and the use of immunologically immature tissue can overcome major histocompatibility complex barriers by inducing tolerance. With the goal of inducing tolerance to BALB/c-derived murine hybridoma cells producing the 4C6 monoclonal antibody (mAb), we transplanted BALB/c fetal tissue into neonatal pigs during a regimen of low-dose conditioning with busulfan and cyclophosphamide. After the tolerance induction phase, animals received intraperitoneal injections of 4C6 mAb hybridoma cells. Evidence of persistence of injected cells over time was sought by molecular analysis of peripheral blood for the presence of mouse genomic sequences and circulating 4C6 mAb. Persistence of donor polymerase chain reaction signal during the entire duration of the study, detectable mAb titer for 6 weeks, and a twofold increase of mAb concentration after a boost hybridoma infusion was observed in one animal receiving six consecutive administrations of the conditioning regimen. Our model has the distinctive advantage of allowing functional monitoring of engrafted cells for studying tolerance induction strategies. In addition, this model could be the basis for approaches aimed at producing mAbs in tolerized large animals.

BACKGROUND: Fetal membranes are tissues of particular interest for several reasons, including their role in preventing rejection of the fetus and their early embryologic origin. which may entail progenitor potential. The immunologic reactivity and the transplantation potential of amnion and chorion cells, however, remain to be elucidated. METHODS: Amnion and chorion cells were isolated from human term placenta and characterized by immunohistochemistry, flow cytometric analysis, and expression profile of relevant genes. The immunomodulatory characteristics of these cells were studied in allogeneic and xenogeneic mixed lymphocyte reactions and their engraftment potential analyzed by transplantation into neonatal swine and rats. Posttransplant chimerism was determined by polymerase chain reaction analysis with probes specific for human DNA. RESULTS: Phenotypic and gene expression studies indicated mesenchymal stem cell-like profiles in both amnion and chorion cells that were positive for neuronal, pulmonary, adhesion, and migration markers. In addition, cells isolated both from amnion and chorion did not induce allogeneic nor xenogeneic lymphocyte proliferation responses and were able to actively suppress lymphocyte responsiveness. Transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues. CONCLUSIONS: Human amnion and chorion cells from term placenta can successfully engraft neonatal swine and rats. These results may be explained by the peculiar immunologic characteristics and mesenchymal stem cell-like phenotype of these cells. These findings suggest that amnion and chorion cells may represent an advantageous source of progenitor cells with potential applications in a variety of cell therapy and transplantation procedures.

We have performed prenatal diagnosis for Wiskott-Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation. Copyright © 1999 John Wiley & Sons, Ltd.

X-linked severe combined immunodeficiency (SCIDX1) is an inherited disease characterized by profound abnormalities of cell-mediated and humoral immunity. Patients with SCIDX1 have defects in the common cytokine receptor gamma chain gene (IL2RG) that encodes a shared, essential component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9 and IL-15. We have characterized nine SCIDX1 families by using a DNA-based, non-radioactive screening method and DNA sequencing. Nine different mutations were found, scattered from exon 1 to exon 5 of the IL2RG gene. Two of these mutations have been previously identified in other unrelated patients; the other seven are novel mutations that differ from all of the 95 already reported in the IL2RG mutation data base. In addition to describing novel mutations in the IL2RG gene, this study shows that the knowledge of the genetic defect and the use of an efficient, non-radioactive, and rapid screening approach have important implications for prenatal and postnatal diagnosis, carrier female identification, and possibly prenatal therapy.

X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis.A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood.A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal.In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Wiskott-Aldrich syndrome (WAS) is a fully penetrant X-linked recessive disorder characterized by immunodeficiency, thrombocytopenia, and severe eczema. WAS is a life-threatening disease, with a poor quality of life and high mortality rate in childhood. The gene responsible for the disease has been localized to the proximal short arm of the X- chromosome and recently isolated through positional cloning and named WAS protein (WASP). We have characterized 17 WAS families. We have developed a rapid, nonradioactive screening protocol for identifying WASP gene alterations in genomic DNA. Our method allows simultaneous evaluation of single strand confirmation polymorphism and heteroduplex formation. We have identified 15 novel mutations that involve single basepair changes, or small insertions or deletions, all of which result in premature stop cordon, frame shift with secondary premature stop codon, or splice site defect. These studies document the considerable heterogeneity of the location of mutations in the WASP gene causing full-blown WAS and show the efficiency and rapidity of a screening approach for mutation identification in WAS that will be useful for carrier detection and prenatal diagnosis.