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During pregnancy, a bidirectional exchange of cells develops in utero between the mother and her fetus. In addition, the maternal cells can persist in the circulation and tissues of the children long after delivery. This phenomenon is called microchimerism.The transferred maternal cells have been suspected to trigger autoimmune reactions such as juvenile dermatomyositisin the child. However, more recently, maternal cells have been described as able to adopt the host tissue phenotype. In neonatal lupus congenital heart block, myocardiocytes deriving from the mother can be retrieved in the child’s heart.Similarly, in pityriasis lichenoides or in juvenile diabetes, the presence of maternal keratinocytes or pancreatic islet beta-cells have been reported. These studies suggest that persisting maternal stem cells, such as mesenchymal stem cells, can home to the child’s damaged tissue and differentiate in order to participate in the remodelling process.

Leg ulcers occurrence constitutes an invalidating complication of sickle cell anaemia. Treatment is frustrating because all procedures used in other wounds are usually unsuccessful or followed by a quick recurrence. We report the case of a woman who recovered from her leg ulcers during pregnancy followed by an early relapse. This peculiar course strongly evokes a role for foetal stem cell implication in the observed pregnancy associated ulcers healing.

Neurofibromatosis 1 (NF1) is a common genetic disease with an incidence of about 1 in 2,500, an autosomal dominant mode of inheritance and a high rate of new mutations. Two striking aspects of NF1 are its progression with age and its extreme variability. Its diagnosis is based on 7 criteria of which 2 are required for diagnosis: significant cafe-au-lait macules (CLM), neurofibromas of any type or 1 plexiform neurofibroma, freckling in the axillary or inguinal regions, optic glioma, Lisch nodules, a distinctive osseous lesion such as sphenoid dysplasia or thinning of the long bone cortex with or without pseudarthrosis, a first-degree relative with NF1. Although specificity and sensitivity of these criteria are high during adulthood, their sensitivity is low during early childhood: 97% of NF1 patients meet them by the age of 8 years, but only 70% by the age of 1 year. In France, the diagnosis is often made in young adults, and the detection of the early complications needs precocious diagnosis . We conducted a retrospective audit of practice to identify features associated with early diagnosis.

For those who are not used yet to this rather recent concept, microchimerism refers to the presence of very low numbers of allogeneic cells in an individual. Most interest in this phenomenon has been raised after the demonstration by the group of Diana Bianchi, a researcher working previously on prenatal diagnosis, that fetal cells – present in the peripheral blood of women during pregnancy – may persist decades after delivery [1]. Fetal semi-allogeneic cells were indeed largely known to be tolerated during pregnancy, through various pathways. However, the cause of such an escape of foreign cells from the maternal immune surveillance for a very long time remains an unanswered intriguing question. Some have hypothesized that such an escape could be due to a leakage of paternal antigens from fetal cells, while others evoked a homology in the major histocompatibility complex leading to a ‘compatibility from the mother’s view’ [2]. Nevertheless, whatever the mechanism of this tolerance, the evidence that such foreign cells could persist has been the rationale for many authors to evaluate a possible implication of chimeric cells in deleterious consequences for the host. Why these investigators focused on such a harmful role is easy to understand: several diseases classified as auto-immune predominate in females, the gender in whom persisting fetal circulation from a previous pregnancy may occur, and in addition several of these ‘autoimmune diseases’ looked like ‘allo-immune diseases’, namely graft-versus-host reaction (GvHD). Based on this, several groups have shown an association between fetal cell persistence and systemic sclerosis (SSc) [3–6]. In addition, such fetal cells were also found in involved tissues such as the skin [4]. At that point, a fascinating perspective appeared possible: spontaneous SSc could be an antimaternal reaction of the fetus, similar to posttransfusional GvHD. However, many points were quickly raised against this pathway. First, nearly all studies found a significant level of normal females with chimerism. Second, sclerodermoid GvHD displays several clinical, pathological and immunological differences to SSc [7]. Third, no increase in SSc appeared to be reported after red blood cell transfusion [8]. Finally, other studies failed to find more chimerism in females with SSc as compared with controls [9, 10]. Therefore, a real controversy about the implication of chimerism in SSc is still to be debated. Some consider that the presence of chimeric cells would act as one step in a multistep phenomenon in the pathogenesis of SSc, either by direct T cell antihost reactivity or by inhibition of host T regulatory cells with consecutive auto-immunity. Other authors believe by contrast that these fetal cells are recruited into damaged tissue in order to participate in tissue repair as we well see below. Quickly, after investigating SSc, several auto-immune diseases were evaluated. Interestingly, using different techniques, 2 groups showed in 2000 that juvenile dermatomyositis was associated with the presence of maternal cells in the blood and the muscle of affected sons, while controls with non-inflammatory myopathies had no such cells [11, 12]. We were therefore in the same scenario as in SSc: allogeneic cells, coming from the reverse traf

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

SEQUENCE ANALYSIS FOR THE GENDER-INDEPENDENT ASSESSMENT OF FETAL CELLS IN MATERNAL BLOOD DONGHYUN CHA, KIARASH KHOSROTEHRANI, DIANA BIANCHI, KIRBY JOHNSON, Pochon University, Obstetrics and Gynecology, Seoul, South Korea, Tufts University, Pediatrics, Boston, Massachusetts, Tufts University, Pediatrics, Obstetrics & Gynecology, Boston, Massachusetts OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (fNRBCs) 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 fNRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. STUDY DESIGN: fNRBCs 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 (4 cases), 18 (1 case), 13 (2 cases), and other genetic abnormalities (2 cases). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR 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 based on morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and non-shared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSION: 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. Women and Infants’ Hospital, Maternal Fetal Medicine, Providence, Rhode Island, University of North Carolina, Department of Obstetrics and Gynecology, Chapel Hill, North Carolina OBJECTIVE: Vaginal bleeding in the first trimester may result in a disruption in the maternal-fetal interface with subsequent transfer of hormones into the maternal circulation. Elevated maternal serum concentrations of AFP have been observed in the first and second trimester in women with a history of first trimester vaginal bleeding. Our goal was to examine the effect of first trimester vaginal bleeding on first trimester serum levels of PAPP-A, free b-hCG, and nuchal translucency (NT) using a prospectively-collected large database. STUDY DESIGN: Women enrolled in the FASTER trial had a NT measurement and PAPP-A and free b-hCG levels drawn at 10 3/7-13 6/7 weeks. Patients with fetal anomalies, a diagnosis of diabetes prior to becoming pregnant or who conceived with invitro fertilization were excluded from this analysis. Patients were divided into three groups: (1) no bleeding, (2) light bleeding, or (3) heavy bleeding. The log transformed multiples of the medians (MoMs) of NT, PAPPA, and free b-hCG for the three groups were compared using analysis of variance for crude effects and analysis of covariance adjusting for possible confounding variables. For presentation, the results were back transformed to medians in MoMs. RESULTS: The study included 34,548 patients: 29,639 patients without bleeding, 4341 patients with light bleeding, and 547 patients with heavy bleeding. Adjusted medians of the MoMs of the markers are listed in the table. CONCLUSION: First trimester vaginal bleeding does not appear to affect PAPP-A or free b-hCG levels nor does bleeding impact NT measurement in this large study.

Highlights • The goal of osteogenesis imperfecta (OI) cell therapy is osteoblast replacement • In utero transplantation (IUT) of bone marrow was conducted in OI mice • Donor-derived osteoblasts were absent following bone marrow IUT • Donor-derived hematopoietic cells included osteoclasts and osteal macrophages, In this article, Millard and colleagues show that intrauterine bone marrow transplantation in the oim/oim mouse model of osteogenesis imperfecta yields hematopoietic microchimerism in the absence of donor osteopoiesis or phenotypic improvement. Bone-associated donor cells were not bone-forming osteoblasts, but osteoclasts (bone resorbing cells of the hematopoietic lineage) and osteal macrophages (bone regulatory cells of the hematopoietic lineage).

Interfollicular epidermal (IFE) homeostasis is a major physiological process allowing maintenance of the skin barrier function. Despite progress in our understanding of stem cell populations in different hair follicle compartments, cellular mechanisms of IFE maintenance, in particular, whether a hierarchy of progenitors exists within this compartment, have remained controversial. We here used multicolour lineage tracing with Brainbow transgenic labels activated in the epidermis to track individual keratinocyte clones. Two modes of clonal progression could be observed in the adult murine dorsal skin. Clones attached to hair follicles showed rapid increase in size during the growth phase of the hair cycle. On the other hand, clones distant from hair follicles were slow cycling, but could be mobilized by a proliferative stimulus. Reinforced by mathematical modelling, these data support a model where progenitor cycling characteristics are differentially regulated in areas surrounding or away from growing hair follicles. Thus, while IFE progenitors follow a non-hierarchical mode of development, spatiotemporal control by their environment can change their potentialities, with far-reaching implications for epidermal homeostasis, wound repair and cancer development.

Fetal cells enter the maternal circulation during all human and murine pregnancy. The presence of fetal microchimeric cells has been demonstrated in lesional skin of pregnant women affected with polymorphic eruptions of pregnancy. We aimed at determining in a mouse model the role of maternal inflammation in attracting microchimeric cells. Wild type female mice were mated to transgenic males for various reporter genes or wild type male controls. We used males transgenic for the luciferase gene under the control of an ubiquitous (CMV)(C-Luc) or the VEGFR2 (V-Luc) promoter. Alternatively we used males transgenic for the EGFP under the control of the beta-actin promoter. Female pregnant mice were sensitized on day 10 of pregnancy with 2% Oxazolone. On day 16 we revealed a contact hypersensitivity reaction (CHR) by painting the right ear with oxazolone while the left ear received only the vehicle. The presence of fetal cells was then monitored using one of the following: in vivo imaging to detect luciferase expressing fetal cell real time quantitative amplification of the egfp transgene or Y chromosome in situ hybridization to detect male microchimeric cells. On femalemice bearing V-Luc fetuses, a fetal signal could be detected only on the right ear that was significantly higher when compared to controls (3362 versus 1507 photons/s/cm2, p=0.02). Wealso detected fetal cells by real time quantitative PCR in the right as well as the left ear at similar frequencies. Finally, we demonstrated the presence of fetal cells in maternal inflamed ears. Fetal cells were either among the inflammatory cells or were part of the vascular wall. We demonstrate that maternal inflamed skin can recruit fetal microchimeric cells. The fetal cells adopt an endothelial phenotype in the inflamed maternal skin. Fetal cells may therefore participate in maternal angiogenesis and inflammation.

T lymphocytes of fetal origin found in maternal circulation after gestation have been reported as a possible cause for autoimmune diseases. During gestation, mothers acquire CD34+CD38+ cells of fetal origin that persist decades. In this study, we asked whether fetal T and B cells could develop from these progenitors in the maternal thymus and bone marrow during and after gestation. RAG−/−-deficient female mice (Ly5.2) were mated to congenic wild-type Ly5.1 mice (RAG+/+). Fetal double-positive T cells (CD4+CD8+) with characteristic TCR and IL-7R expression patterns could be recovered in maternal thymus during the resulting pregnancies. We made similar observations in the thymus of immunocompetent mothers. Such phenomenon was observed overall in 12 of 68 tested mice compared with 0 of 51 controls (p = 0.001). T cells could also be found in maternal spleen and produced IFN-γ in the presence of an allogenic or an Ag-specific stimulus. Similarly, CD19+IgM+ fetal B cells as well as plasma Igs could be found in maternal RAG−/− bone marrow and spleen after similar matings. Our results suggest that during gestation mothers acquire fetal lymphoid progenitors that develop into functional T cells. This fetal cell microchimerism may have a direct impact on maternal health.

THE NONCLASSICAL major histocompatibility complex molecule, HLA-G, is mainly expressed on cytotrophoblast cells during pregnancy, where it is likely involved in the protection of the semiallogeneic fetus from maternal rejection. The HLA-G gene contains eight exons that encode the peptide signal (exon 1), three extracellular domains (exons 2, 3, and 4), a transmembrane region (exon 5), and a reduced cytoplasmic tail due to the presence of a premature stop codon in exon 6. The primary transcript of the HLA-G gene is alternatively spliced, resulting in at least six different HLA-G mRNAs that potentially encode four membrane-bound (HLA-G1, -G2, -G3, and -G4) and two soluble (HLA-G5 and -G6) protein isoforms. While the full-length HLA-G1, which presents similar classical HLA class I structure, was widely investigated over the past few years at both biochemical and functional levels, HLAG2, -G3, and -G4 remain to be well defined. Previous experiments have demonstrated that HLA-G1 transfected into HLA class I-negative cells lines, such as LCL 721.221 and K562, reaches the cell surface and is responsible for NK cytolysis inhibition. This inhibition may occur through interaction with at least three inhibitory receptors, namely ILT-2, p49, and KIR2DL4. However, besides the HLA-A and -B-negative placental tissue, HLA-G expression was also reported in HLA class I-positive cells such as chorionic fetal vessels, thymic epithelial cells, and cytokine activated monocytes, as well as in some melanoma biopsies. In this latter situation, the expression of HLA-G2, -G3, and -G4 isoforms was strongly suggested. Accordingly, to characterize these isoforms, the cDNA encoding each HLA-G isoform was transfected into an HLA-class I-positive cell line, allowing us to mimick the situation in which HLA-G is coexpressed with HLA class I molecules. Each isoform was characterized at both transcriptional and protein levels. Results showed that (1) HLA-G1, -G2, -G3, and -G4 are transcribed in the corresponding transfectant; (2) HLA-G1, -G2, -G3, and -G4 are translated in protein that could be detected by using the 4H84 monoclonal antibody (mAb), which reacts with all denatured isoforms, and (3) only the HLA-G1 protein could be cell-surface detected by the use of the specific anti-HLA-G1 mAb currently available, namely 87G. MATERIALS AND METHODS Cell Lines and HLA-G Vector Constructions

Introduction Breast carcinomas associated with pregnancy display a high frequency of inflammatory types, multifocal lesions and lymph node metastasis. Because pregnancy results in transfer to mothers of foetal stem cells that can migrate and differentiate into various tissues, we addressed the issue of whether such cells are present in breast carcinoma associated with pregnancy. Methods We analyzed women presenting with such tumours who were pregnant with male foetuses using fluorescence in situ hybridization (FISH), targeting X and Y chromosomes. The foetal cell phenotype was then determined by combining FISH and immunohistochemistry with various antibodies. Statistical analysis was performed using t-test or nonparametric Wilcoxon's test. Results We found that foetal cells were present in nine out of 10 carcinomas, in contrast with none of four benign mammary lesions (P < 0.05). Counting foetal and maternal cells showed that the mean number of foetal cells per million maternal cells was 36 in breast cancers and 0 in control samples (P < 0.01). By combining FISH and immunolabelling, we found that foetal cells expressed mainly mesenchymal or, to a lesser degree, epithelial or endothelial markers, but never leucocytes. Conclusion These findings demonstrate the frequent presence of foetal derived cells essentially in tumour stroma. Given the role played by stroma in tumour proliferation, these findings raise the issue of whether foetal cell can be targeted to influence tumour behaviour.