Early Gestation Fetuses Research Articles

Prenatally Educated Friendly NK Cells regulate Allo-specific T cell Responses through expansion of Foxp3+CD4+Treg Cells and suppression of Type 1 and 17 CD8+T cell responses.

Abstract Prenatal alloantigen exposure during embryonic development permanently tolerizes the early gestational fetus and provides a tremendous advantage over transplantation after birth. However, the influence of prenatally educated NK cells on alloantigen-specific T cell immune responses is not well elucidated. In previous experiments, we found that prenatally-educated friendly NK (fNK) cells exert suppressive effects on allo-specific T cell responses in vitro. We hypothesized that prenatally-educated fNK cells exert regulatory effects on allo-specific T cells in vivo. To challenge this hypothesis, we examined the regulatory potential of prenatally educated fNK cells through gain and loss of function experiments in stable prenatal Balb/c --> B6.Thy1.2+ chimeras (engrafter mice). We depleted both (Ly49AFG+) fNK cells and Thy1.2+T cells or Thy1.2+T cells alone in host engrafter mice and then adoptively transferred naïve responder Thy1.1+ T cells along with donor-specific allogeneic or syngeneic target cells into the engrafter hosts. We found that the fNK cell-replete animals exhibited a higher frequency of foxp3+Tregs and a lower frequency of allospecific T-bet+ and RORγt+ CD8 T cells compared to the fNK cell-depleted controls. Furthermore, the neutralization of TGF-β1 decreased the fNK cell-induced expansion of Treg cells in vitro. From these studies, we conclude that prenatally educated fNK cells exert regulatory effects on allo-specific T-cell responses through the TGF-β1 mediated expansion of foxp3+Treg cells. Prenatal antigen experience drives the emergence of antigen-specific suppressors that regulate T cell responses. National Institutes of Health Grant R01HL103745 and Lurie Children’s Hospital Research Foundation (to A.F.S.)

Microfocus computed tomography for fetal postmortem imaging: an overview

Over the last few years, fetal postmortem microfocus computed tomography (micro-CT) imaging has increased in popularity for both diagnostic and research purposes. Micro-CT imaging could be a substitute for autopsy, particularly in very early gestation fetuses for whom autopsy can be technically challenging and is often unaccepted by parents. This article provides an overview of the latest research in fetal postmortem micro-CT imaging with a focus on diagnostic accuracy, endovascular staining approaches, placental studies and the reversibility of staining. It also discusses new methods that could prove helpful for micro-CT of larger fetuses. While more research is needed, contrast-enhanced micro-CT has the potential to become a suitable alternative to fetal autopsy. Further research using this novel imaging tool could yield wider applications, such as its practise in imaging rare museum specimens.

The Theoretical Basis of In Utero Hematopoietic Stem Cell Transplantation and Its Use in the Treatment of Blood Disorders

Since its conception, prenatal therapy has been successful in correction of mainly anatomical defects, although the range of application has been limited. Research into minimally invasive fetal surgery techniques and prenatal molecular diagnostics has facilitated the development of in utero stem cell transplantation (IUT)-a method of delivering healthy stem cells to the early gestation fetus with the hope of engraftment, proliferation, and migration to the appropriate hematopoietic compartment. An area of application that shows promise is the treatment of hematopoietic disorders like hemoglobinopathies. The therapeutic rationale of IUT with hematopoietic stem cells (HSCs) is based on the proposed advantages the fetal environment offers based on its unique physiology. These advantages include the immature immune system facilitating the development of donor-specific tolerance, the natural migration of endogenous hematopoietic cells providing space for homing and engraftment of donor cells, and the fetal environment providing HSCs with the same opportunity to survive and proliferate regardless of their origin (donor or host). Maternal immune tolerance to the fetus and placenta also implies that the maternal environment could be accepting of donor cells. In theory, the fetus is a perfect recipient for stem cell transplant. Clinically, however, IUT is yet to see widespread success calling into question these assumptions of fetal physiology. This review aims to discuss and evaluate research surrounding these key assumptions and the clinical success of IUT in the treatment of thalassemia.

Postmortem microfocus computed tomography for noninvasive autopsies: experience in >250 human fetuses

BackgroundNoninvasive imaging autopsy alternatives for fetuses weighing <500 grams are limited. Microfocus computed tomography has been reported as a viable option in small case series with the potential to avoid an invasive autopsy. Implementation of postmortem microfocus computed tomography in a large cohort as part of routine clinical service has yet been unreported, and realistic “autopsy prevention rates” are unknown.ObjectiveThis study aimed to describe the range of abnormalities detectable on fetal microfocus computed tomography in a clinical setting and additional findings identified on the antenatal ultrasound and to estimate the invasive autopsy avoidance rate (ie, cases in which imaging was sufficient to deem autopsy unnecessary).Study DesignA prospective observational case series of all fetuses referred for microfocus computed tomography imaging at a single institution was conducted for 3 years (2016–2019). Imaging was reported by 2 pediatric radiologists before autopsy, with “decision to proceed” based on the specialist perinatal pathologists’ judgment and parental consent. Agreement rates between microfocus computed tomography and antenatal ultrasound were evaluated, and where feasible, diagnostic accuracy for microfocus computed tomography was calculated using autopsy as a reference standard.ResultsA total of 268 fetuses were included (2–350 grams body weight; 11–24 weeks’ gestation), with cause for demise in 122 of 268 (45.5%). Of the 122 fetuses, 64 (52.5%) exhibited fetal anomalies. Although 221 of 268 (82.5%) fetuses had consent for invasive autopsy, only 29 of the 221 (13.1%) underwent this procedure, which implied an autopsy avoidance rate of 192 of 221 (86.9%). Complete agreement was present for all brain, thoracic, and abdominal pathologies, whereas sensitivity and specificity for cardiac anomalies were 66.7% and 91.7%, respectively. Microfocus computed tomography and antenatal ultrasound agreement was found in 219 of 266 cases (81.9%), with partial agreement in 21 of 266 (7.9%) and disagreement in 26 of 266 (10.5%), mostly because of additional cardiac, soft tissue, or genitourinary findings by microfocus computed tomography, which were not seen on the ultrasound.ConclusionFetal microfocus computed tomography imaging is a viable and useful tool for imaging early gestational fetuses and can avoid the need for invasive autopsy. Confirmation of antenatal diagnoses is achieved in most cases, and additional anomalies may also be detected.

Fetoscopic versus Ultrasound-Guided Intravascular Delivery of Maternal Bone Marrow Cells in Fetal Macaques: A Technical Model for Intrauterine Haemopoietic Cell Transplantation

Introduction: Significant limitations with existing treatments for major haemoglobinopathies motivate the development of effective intrauterine therapy. We assessed the feasibility of fetoscopic and ultrasound-guided intrauterine haemopoietic cell transplantation (IUHCT) in macaque fetuses in early gestation when haemopoietic and immunological ontogeny is anticipated to enable long-term donor cell engraftment. Material and Methods: Fluorescent-labelled bone marrow-derived mononuclear cells from 10 pregnant Macaca fascicularis were injected into their fetuses at E71–114 (18.9–170.0E+6 cells/fetus) by fetoscopic intravenous (n = 7) or ultrasound (US)-guided intracardiac injections, with sacrifice at 24 h to examine donor-cell distribution. Results: Operating times ranged from 35 to 118 min. Chorionic membrane tenting and intrachorionic haemorrhage were observed only with fetoscopy (n = 2). Labelled cells were stereoscopically visualised in lung, spleen, liver, and placenta. Donor-cell chimerism was highest in liver, spleen, and heart by flow cytometry, placenta by unique polymorphism qPCR, and was undetected in blood. Chimerism was 2–3 log-fold lower in individual organs by qPCR than by flow cytometry. Discussion: Both fetoscopic and US-guided IUHCT were technically feasible, but fetoscopy caused more intraoperative complications in our pilot series. The discrepancy in chimerism detection predicts the challenges in long-term surveillance of donor-cell chimerism. Further studies of long-term outcomes in the non-human primate are valuable for the development of clinical protocols for IUHCT.

Epidemiology and major subtypes of congenital heart defects in Hunan Province, China.

Congenital heart defects (CHDs) are the most common birth defects (BDs) and account for nearly one-third of all BDs. The aim of this article was to investigate the epidemiology and major subtypes of CHDs in Hunan Province, China in the last 5 years.CHD surveillance data from 2012 to 2016 were collected from 52 registered hospitals in Hunan. The prevalence rates of CHDs, incidence rates of CHDs combined with other BDs, and rates of termination of pregnancy (TOP) for CHDs among different regions, infant sexes, and maternal ages were calculated for both early fetuses (<28 weeks of gestation) and perinatal infants (PIs) (between 28 weeks of gestation and 7 days after birth). Both the constituent ratio and prevalence rates were computed among subtypes.CHDs were found in 6289 out of 673,060 births. The overall prevalence was 93.44 per 10,000 PIs, with 19.27 and 74.17 per 10,000 in early fetuses and PIs, respectively. The risks of CHDs were higher in infants from urban areas than those from rural areas during the whole gestation and were higher in male infants than in female infants during the perinatal period. The total prevalence of CHDs increased significantly with maternal age (χ2 trend = 141.84, P < .05). Among fetuses in early gestation, there were 288 cases (22.21%) of CHDs combined with other BDs and 1292 cases (99.61%) of TOP for CHD. The 3 major subtypes of CHDs were ventricular septal defect (VSD) (22.06%), Tetralogy of Fallot (TOF) (9.43%), and atrioventricular septal defect (AVSD) (6.69%). Among PIs, there were 1541 cases (30.87%) of CHD diagnosed before delivery and 1184 cases (76.83%) were TOP. The 3 major subtypes were atrial septal defect (ASD) (42.81%), patent ductus arteriosus (PDA) (16.07%), and VSDs (15.21%).The total prevalence of CHD in Hunan Province and the rate of TOP for CHD was high, especially for early-gestation fetuses. Pregnancies in urban women, male PIs, and maternal age were the risk factors for CHDs. Among early-gestation fetuses, the most common types were VSD, TOF, and ASD, and among PIs, the most common types were ASD, PDA, and VSD.

Preliminary Evaluation of Fetal Congenital Heart Defects Changes on Fetal-Maternal Heart Rate Coupling Strength.

Monitoring fetal heart rate in an important aspect in evaluating fetal well being. Maternal-fetal interaction has shown evolution during fetal maturation. In this work, we studied maternal-fetal heart rate synchronization in early and late gestation fetuses. We also evaluated variations in the synchronization due to congenital heart defect (CHD). Maternal-fetal heart rate synchronization for 22 early gestation (Age < 32 weeks), $late gestation (Age >32 weeks) and 7 CHD fetuses (5 of them with gestational age < 32 weeks). The synchronization ratio between the mother and the fetus was more localized at certain fetus heart rate in the early gestation group while it was spreading over more fetal heart rate for the late group. For example, for maternal primary cycle of 3 beat- to-beat (m=3), the synchronization ratio of 5 fetus beats (n=5) contributed 60±30% of the whole coupling ratios for the early group while it contributed 30°30% for the late group (p< 0.01). On the other hand, the coupling ratio of m:n=3:7 contributed 4±17% of the early group and 13±24% for the late group (p< 0.05). The standard deviation of the phase coherence index $(\lambda_{-\mathrm{S}\mathrm{D}})$ for both the late and the CHD groups were significantly higher than the early group at different values. For example, $\lambda -\mathrm{S}\mathrm{D} was 0.006\pm 0.004$ for the early group while it was 0.009±0.008 for the late group (p< 0.01) and 0.01± 0.002 for the CHD group (p< 0.01) for m=3. The variation between the early and late normal groups might indicate a healthy development of the autonomic nervous system while the higher variation in the CHD group could be a good marker for impairment of the cardiac autonomic activity. Further coupling analysis with more abnormal cases is needed to verify these findings.

Postmortem microfocus computed tomography for early gestation fetuses: a validation study against conventional autopsy

Perinatal autopsy provides useful clinical information in up to 40% of cases. However, there is a substantial unmet clinical need with regards to postmortem investigation of early gestation fetal loss for parents for whom standard autopsy is either not available or not acceptable. Parents dislike the invasive nature of autopsy, but current clinical imaging techniques do not provide high-enough imaging resolution in small fetuses. We hypothesized that microfocus computed tomography, which is a rapid high-resolution imaging technique, could give accurate diagnostic imaging after early gestation fetal loss. The objective of the study was to evaluate the diagnostic accuracy of microfocus computed tomography for noninvasive human fetal autopsy for early gestation fetuses, with the use of conventional autopsy as the reference standard. We compared iodinated whole body microfocus computed tomography in 20 prospectively recruited fetuses (11-21 weeks gestation from 2 centers) with conventional autopsy in a double-blinded manner for a main diagnosis and findings in specific body organs. Fetuses were prepared with 10% formalin/potassium tri-iodide. Images were acquired with a microfocus computed tomography scanner with size-appropriate parameters. Images were evaluated independently by 2 pediatric radiologists, who were blinded to formal perinatal autopsy results, across 40 individual indices to reach consensus. The primary outcome was agreement between microfocus computed tomography and conventional autopsy for overall diagnosis. Postmortem whole body fetal microfocus computed tomography gave noninvasive autopsy in minutes, at a mean resolution of 27μm, with high diagnostic accuracy in fetuses at <22 weeks gestation. Autopsy demonstrated that 13 of 20 fetuses had structural abnormalities, 12 of which were also identified by microfocus computed tomography (92.3%). Overall, microfocus computed tomography agreed with overall autopsy findings in 35 of 38 diagnoses (15 true positive, 18 true negative; sensitivity 93.8% [95% confidence interval, 71.7-98.9%], specificity 100% [95% confidence interval, 82.4-100%]), with 100% agreement for body imaging diagnoses. Furthermore, after removal of nondiagnostic indices, there was agreement for 700 of 718 individual body organ indices that were assessed on microfocus computed tomography andautopsy (agreement, 97.5%; 95% confidence interval, 96.1-98.4%), with no overall differences between fetuses at ≤14 or>14 weeks gestation (agreement, 97.2% and 97.9%, respectively). Within first-trimester fetal loss cases (<14 weeks gestation), microfocus computed tomography analysis yielded significantly fewer nondiagnostic indices than autopsy examination (22/440 vs 48/348, respectively; P<.001). Postmortem whole-body fetal microfocus computed tomography gives noninvasive, detailed anatomic examinations that are achieved in minutes at high resolution. Microfocus computed tomography may be preferable to magnetic resonance imaging in early gestation fetuses and may offer an acceptable method of examination after fetal loss for parents who decline invasive autopsy. This will facilitate autopsy and subsequent discussions between medical professionals who are involved in patient care and counselling for future pregnancies.

Analysis of maternal–fetal heart rate coupling directions with partial directed coherence

Maternal psycho-physiological activities affect the fetal heart rate and heart rate variability. However, directions and patterns of maternal and fetal heartbeat coupling are still poorly understood. The aim of this study was to quantify the direction of short-term maternal–fetal cardiac coupling in early, mid and late gestation fetuses by using partial directed coherence (PDC) analysis approach. The analysis was based on fetal electrocardiograms (fECGs) of 66 healthy fetuses; 22 from early gestation 16–25 weeks, 22 from mid gestation 26–30 weeks and 22 from late gestation 32–41 weeks. Results of analyzing PDC demonstrated a causal influence of fetal on maternal heart rate in the early gestation, while it significantly decreased from early to mid gestation along with a significant increase of maternal to fetal coupling strength. The causal influence of maternal on fetal heart rate was the strongest in the mid gestation and remained dominant in the late gestation. In conclusion, the application of PDC revealed detailed information about short-term maternal–fetal cardiac couplings and regulatory mechanisms (patterns) of developing autonomic nervous system function.

Human fetal wound healing: a review of molecular and cellular aspects

The physiological answer to after birth skin lesions is scarring, which compromises the function and the aesthetics of the injured area. However, fetuses in early gestation (24 weeks or less) respond to this damage with skin regeneration. To explain this difference, several factors are considered, such as increased production of collagen III in fetal fibroblasts and increased presence of this collagen in the skins of these fetuses. Increased hyaluronic acid in fetal matrix correlates with greater capacity for migration of fibroblasts in scarless repair. The fact that myofibroblasts in the wound appear only after the fetal stage of pregnancy which forms scars can also be correlated. Additionally, there is an increase in the amount of adhesion molecules in repair without scarring, which would multiply cell adhesion and migration. Lower levels of bTGF1 in fetal wound are correlated with reduced amounts of collagen I and may be the result of higher relative expression of bTGF3, which downregulates bTGF1. Amniotic fluid itself might be a stimulating factor to human skin’s fibroblasts proliferation through cytokines such as bFGF and PDGF. A hypoxic environment in the fetal wound, associated with increased presence of Dot cells in blood, is also observed, and both facts can be related to a difference in the repair of the skin. Distinct gene expression guides those different responses and may also help to elucidate fetal skin regeneration. When the mechanisms responsible for the absence of scars in wounded fetuses are enlightened, it will be a significant mark in the studies of wound cicatrization and its therapeutic applications shall be extremely valuable.

The Male Fetal Biomarker INSL3 Reveals Substantial Hormone Exchange between Fetuses in Early Pig Gestation.

The peptide hormone INSL3 is uniquely produced by the fetal testis to promote the transabdominal phase of testicular descent. Because it is fetal sex specific, and is present in only very low amounts in the maternal circulation, INSL3 acts as an ideal biomarker with which to monitor the movement of fetal hormones within the pregnant uterus of a polytocous species, the pig. INSL3 production by the fetal testis begins at around GD30. At GD45 of the ca. 114 day gestation, a time at which testicular descent is promoted, INSL3 evidently moves from male to female allantoic compartments, presumably impacting also on the female fetal circulation. At later time-points (GD63, GD92) there is less inter-fetal transfer, although there still appears to be significant INSL3, presumably of male origin, in the plasma of female fetuses. This study thus provides evidence for substantial transfer of a peptide hormone between fetuses, and probably also across the placenta, emphasizing the vulnerability of the fetus to extrinsic hormonal influences within the uterus.

Characterization of equine hyalocytes: their immunohistochemical properties, morphologies and distribution.

In horse, the characterizations of hyalocytes under the steady state are still unclear. Therefore, we investigated characterizations of hyalocytes in normal equine eyes by their immunohistochemical phenotype, histomorphology and distribution. Thirty-one eyes from 18 horses, divided into 4 groups (G) by age, were used: early (G1) and late gestation (G2) fetuses, 1- to 3-year-old (G3) and 8- to 24-year-old (G4) horses. Equine hyalocytes were histologically classified into 4 types, and they immunohistochemically expressed MHC II and CD163. Hyalocytes were detected on and/or around ciliary processes and pars plana in G2, G3 and G4, but were not located on retina and optic papilla. A significant increase in distribution was found between G2 and both G3 and G4, and the largest distribution was found at ciliary processes in these groups. Equine hyalocytes were characterized as residential ocular macrophage and MHC II antigen-bearing cell, accompanied by a pleomorphic appearance and located in the contiguous ciliary body. Our data provided characterizations of hyalocytes in normal equine eyes and may well contribute to improving the understanding of pathogenesis of equine ocular disease.

Induction of interferon-gamma and downstream pathways during establishment of fetal persistent infection with bovine viral diarrhea virus

Development of transplacental infection depends on the ability of the virus to cross the placenta and replicate within the fetus while counteracting maternal and fetal immune responses. Unfortunately, little is known about this complex process. Non-cytopathic (ncp) strains of bovine viral diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, cause persistent infection in early gestational fetuses (<150 days; persistently infected, PI), but are cleared by immunocompetent animals and late gestational fetuses (>150 days; transiently infected, TI). Evasion of innate immune response and development of immunotolerance to ncp BVDV have been suggested as possible mechanisms for the establishment of the persistent infection. Previously we have observed a robust temporal induction of interferon (IFN) type I (innate immune response) and upregulation of IFN stimulated genes (ISGs) in BVDV TI fetuses. Modest chronic upregulation of ISGs in PI fetuses and calves reflects a stimulated innate immune response during persistent BVDV infection. We hypothesized that establishing persistent fetal BVDV infection is also accompanied by the induction of IFN-gamma (IFN-γ). The aims of the present study were to determine IFN-γ concentration in blood and amniotic fluid from control, TI and PI fetuses during BVDV infection and analyze induction of the IFN-γ downstream pathways in fetal lymphoid tissues. Two experiments with in vivo BVDV infections were completed. In Experiment 1, pregnant heifers were infected with ncp BVDV type 2 on day 75 or 175 of gestation or kept naïve to generate PI, TI and control fetuses, respectively. Fetuses were collected by Cesarean section on day 190. In Experiment 2, fetuses were collected on days 82, 89, 97, 192 and 245 following infection of pregnant heifers on day 75 of gestation. The results were consistent with the hypothesis that ncp BVDV infection induces IFN-γ secretion during acute infection in both TI and PI fetuses and that lymphoid tissues such as spleen, liver and thymus, serve both as possible sources of IFN-γ and target organs for its effects. Notably, induction of IFN-γ coincides with a decrease in BVDV RNA concentrations in PI fetal blood and tissues. This is the first report indicating the possible presence of an adaptive immune response in persistent BVDV infections, which may be contributing to the observed reduction of viremia in PI fetuses.

Fetal Mouse Skin Heals Scarlessly in a Chick Chorioallantoic Membrane Model System

In mammals, the early-gestation fetus has the regenerative ability to heal skin wounds without scar formation. This observation was first reported more than 3 decades ago, and has been confirmed in a number of in vivo animal models. Although an intensive research effort has focused on unraveling the mechanisms underlying scarless fetal wound repair, no suitable model of in vitro fetal skin healing has been developed. In this article, we report a novel model for the study of fetal wound healing. Fetal skin from gestational day 16.5 Balb/c mice (total gestation, 20 days) was grafted onto the chorioallantoic membrane of 12-day-old chicken embryos and cultured for up to 7 days. At 48 hours postengraftment, circular wounds (diameter = 1 mm) were made in the fetal skin using a rotating titanium sapphire laser (N = 45). The tissue was examined daily by visual inspection to look for signs of infection and ischemia. The grafts and the surrounding host tissue were examined histologically. In all fetal skin grafts, the wounds completely reepithelialized by postinjury day 7, with regeneration of the dermis. Fetal mouse skin xenografts transplanted onto the chorioallantoic membrane of fertilized chicken eggs provides a useful model for the study of fetal wound healing. This model can be used as an adjunct to traditional in vivo mammalian models of fetal repair.

Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice

Transplantation of allogeneic stem cells into the early gestational fetus, a treatment termed in utero hematopoietic cell transplantation (IUHCTx), could potentially overcome the limitations of bone marrow transplants, including graft rejection and the chronic immunosuppression required to prevent rejection. However, clinical use of IUHCTx has been hampered by poor engraftment, possibly due to a host immune response against the graft. Since the fetal immune system is relatively immature, we hypothesized that maternal cells trafficking into the fetus may pose the true barrier to effective IUHCTx. Here, we have demonstrated that there is macrochimerism of maternal leukocytes in the blood of unmanipulated mouse fetuses, with substantial increases in T cell trafficking after IUHCTx. To determine the contribution of these maternal lymphocytes to rejection after IUHCTx, we bred T and/or B cell-deficient mothers to wild-type fathers and performed allogeneic IUHCTx into the immunocompetent fetuses. There was a marked improvement in engraftment if the mother lacked T cells but not B cells, indicating that maternal T cells are the main barrier to engraftment. Furthermore, when the graft was matched to the mother, there was no difference in engraftment between syngeneic and allogeneic fetal recipients. Our study suggests that the clinical success of IUHCTx may be improved by transplanting cells matched to the mother.

An Animal Model for Assessing Cardiovascular Cell Therapy with Human Bone Marrow Stem Cells

AbstractAbstract 4693 Introduction:Ischemic cardiovascular diseases are still the leading cause of morbidity and mortality in the western world. Incomplete surgical myocardial revascularization is a determining factor for early flare of subsequent angina which results in lower patient survival. Cell-based cardiac therapy in the treatment of ischemic heart disease is a new concept that has been studied using numerous cell types, delivery systems, and strategies. Bone-marrow derived stem/progenitor cells (BMDC) have been used in a number of different studies in both animal and human models. Adult BMDC is composed of a multitud of cell types: hematopoietic, mesenchimal, stromal stem cells and other cell types yet to be characterized. More studies are needed to address the timing, relative concentrations, source and route of delivery of each cellular populations in animals models of myocardial ischemia. In vivo studies are clear prerequisites for clinical translation and appropriate animal models are needed. Objective:The aim of this study was to produce a xenogenic large animal model of myocardical infartation and investigate whether this model allows evaluation of angiogenic cell therapy with human BMDC. Methods:The proposed strategy was to develop myocardial ischemia in a human cell graft tolerant sheep model. Chimeric tolerant sheeps were created by human BMDC transplant into early gestational sheep fetuses during the “preimmune” stage. Human CD34+ cells (x = 315×103) were implanted in 45+3 days sheep fetuses (n = 10) by echocardiographic-guided injection (modified Zanjani model).Myocardial infarction (MI) was produced by a combined technique of surgical ligature of the first marginal of the circumflex artery and cryoablation with liquid nitrogen achieving an infarction area with total fibrotic replacement (n=8). MI was created in 1 year chimeric sheep and infarcted areas were treated with saline (control group, n=3) or 260×103 human CD34+cells (treated group, n=7). Animals were euthanazied 1 month after MI and the heart were excised for pathology study. The infarct region was divided for study into three areas respect to the center of the infarct. Blood vessels were identified by BS-1 lectin-histochemistry and capillary density determined. Results:Our results showed no differences in capillary density in the three areas studied but a slightly increase in arteriolar component in the treated group compared with the control group (not significant). Neither significant differences were observed when comparing epicardial, medium and endocardial areas. No immune rejection or graft vs. host disease were observed in all treated animals. Conclusion:This study shows that this model is a useful tool for preclinic in vivo evaluation of human cell therapy strategies in ischemic cardiovascular disease. Using human CD34+ cells at doses mentioned above our results did not showed significantly more angiogenesis and arteriogenesis than in the control group. To improve this model, further studies are needed to fully charaterize the role of dose, cell type and time for the angiogenic effect of cell-based therapies. Understanding the potentential of defined lineages of stem cells or undifferentiated progenitors, will lead to better and more focussed clinical trial designs using each cell type indenpendently or in combination, depending on which particular clinical indication is being targeted. Disclosures:No relevant conflicts of interest to declare.

Expression of Regulatory T cell (Treg) Activation Markers in Endometrial Tissues from Early and Late Pregnancy in the Feline Immunodeficiency Virus (FIV)-Infected Cat

Regulatory T cells (Tregs) support pregnancy maintenance by suppressing placental inflammation, while diminished Treg function may accompany reproductive failure. Experimental FIV infection frequently results in vertical transmission and increased pregnancy failure in the cat. The mechanism of reproductive compromise is unknown. We hypothesized that FIV infection alters endometrial Treg population dynamics and function, potentiating vertical transmission and reproductive failure. RNA collected from early and late gestation reproductive tissue and fetuses from FIV infected and control cats was probed for expression of FIV gag and Treg markers CD25, FOXP3, and CTLA4, using real time reverse-transcriptase (RT)-PCR. Frequent placental and fetal infection and reproductive failure were detected at early and late pregnancy. Expression of FOXP3 and CTLA4 was higher in early gestation tissues from control cats. FIV infection significantly reduced expression of FOXP3 and CTLA4 at early, but not late pregnancy. At late pregnancy, CTLA4 was expressed to higher levels in infected tissues. The number of tissues with decreased co-expression of FOXP3 and CTLA4 was significant in infected cats at early pregnancy. No significant changes in CD25 expression occurred between FIV-infected and control animals at early or late pregnancy. Differences in Treg marker expression were not significant between viable and non-viable pregnancies in infected cats. The detection of Treg markers in these feline tissues provides the first evidence of feline endometrial Tregs and suggests that such cells diminish as pregnancy progresses. These cells may be depleted or rendered less functional by viral infection, but understanding their role in pregnancy requires further study.

Cutaneous Scarring: A Clinical Review

Cutaneous scarring can cause patients symptoms ranging from the psychological to physical pain. Although the process of normal scarring is well described the ultimate cause of pathological scarring remains unknown. Similarly, exactly how early gestation fetuses can heal scarlessly remains unsolved. These questions are crucial in the search for a preventative or curative antiscarring agent. Such a discovery would be of enormous medical and commercial importance, not least because it may have application in other tissues. In the clinical context the assessment of scars is becoming more sophisticated and new physical, medical and surgical therapies are being introduced. This review aims to summarise some of the recent developments in scarring research for non-specialists and specialists alike.

Reciprocal Expression and Signaling of TLR4 and TLR9 in the Pathogenesis and Treatment of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a common and often fatal inflammatory disorder affecting preterm infants that develops upon interaction of indigenous bacteria with the premature intestine. We now demonstrate that the developing mouse intestine shows reciprocal patterns of expression of TLR4 and TLR9, the receptor for bacterial DNA (CpG-DNA). Using a novel ultrasound-guided in utero injection system, we administered LPS directly into the stomachs of early and late gestation fetuses to induce TLR4 signaling and demonstrated that TLR4-mediated signaling within the developing intestine follows its expression pattern. Murine and human NEC were associated with increased intestinal TLR4 and decreased TLR9 expression, suggesting that reciprocal TLR4 and TLR9 signaling may occur in the pathogenesis of NEC. Enteral administration of adenovirus expressing mutant TLR4 to neonatal mice reduced the severity of NEC and increased TLR9 expression within the intestine. Activation of TLR9 with CpG-DNA inhibited LPS-mediated TLR4 signaling in enterocytes in a mechanism dependent upon the inhibitory molecule IRAK-M. Strikingly, TLR9 activation with CpG-DNA significantly reduced NEC severity, whereas TLR9-deficient mice exhibited increased NEC severity. Thus, the reciprocal nature of TLR4 and TLR9 signaling within the neonatal intestine plays a role in the development of NEC and provides novel therapeutic approaches to this disease.

Analysis of lymphocyte phenotypes in cord blood from early gestation fetuses.

Using cord blood samples obtained from fetuses between 16 and 40 weeks gestation, we have used a lysed whole blood flow cytometric technique to study the natural history of lymphocyte phenotypes known to be highly represented in cord blood at birth. The majority (51.0 +/- 14.7%) of lymphocytes expressed CD45RA, a marker of 'virgin' cells and there was a correlation between increasing percentages of CD45RA+lymphocytes and gestational age (r = 0.44, P < 0.01). Few cells (8.5 +/- 4.2%) expressed the CD45RO marker of primed lymphocytes and very few (1.0 +/- 0.7%) co-expressed CD45RA and RO, indicating little traffic between the two maturation markers. The percentage of B lymphocytes co-expressing CD5 was high in the fetal circulation (55.5 +/- 10.5%) compared with healthy adults (23.2 +/- 14.3%; P < 0.00001) and the level of CD5+ B cells declined with gestational age in an exponential manner (r = -0.45, P < 0.05). Similarly, levels of T lymphocytes expressing the gamma delta T cell receptor (TCR) declined exponentially (r = -0.59, P < 0.005). These results demonstrate that lymphocytes remain almost entirely unprimed before birth. In addition, CD5+ B lymphocytes and TCR-gamma delta+ T lymphocytes decline exponentially towards birth, in a manner suggesting that they may be seeding peripheral sites such as the spleen, skin and mucosae.