Hematopoietic chimerism achieved by in utero hematopoietic stem cell injection does not induce donor-specific tolerance for renal allografts in sheep.
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Animals↗
Biomedical subjects
A W Flake
Publications and source records attributed to A W Flake.
Massive thymic hyperplasia: characterization of a rare mediastinal mass.
Thymic hyperplasia is a rare cause of an anterior mediastinal mass in children. True thymic hyperplasia is characterized by massive thymic hypertrophy with retention of normal thymic architecture, and must be distinguished from more commonly seen tumors of the anterior mediastinum. Previous reports of thymic hyperplasia primarily have been descriptive, with minimal analysis of the cellular characteristics of the tumor. To better describe the cellular characteristics of thymic hyperplasia, the authors report on a 10-year-old boy found to have an asymptomatic left paracardiac mass during cardiac evaluation. The mass enlarged rapidly during the following 2 weeks and filled the entire left side of the chest. An open biopsy specimen showed normal thymic architecture. Because of the size and rapid growth of the mass, the authors decided to resect it. Cellular analysis of the mass did not show differences from normal thymic elements. There was conserved thymic architecture. The expression of cell surface markers and the proliferative response of thymocytes to cytokines appeared to be normal. Similarly, a long-term cell culture of the thymocytes did not result in clonal proliferation. Immunohistologic staining of stromal cells showed no striking differences from normal thymic elements. Moreover, immunologic analysis of the patient by standard hematological parameters, lymphocyte subsets, quantitative immunoglobins, and immunoelectrophoresis showed no abnormalities except for lymphocytosis, which resolved after tumor resection. The authors conclude that thymic hyperplasia occurs in immunologically normal children and may exhibit (1) rapid growth with minimal associated symptoms, (2) normal thymic architecture, (3) normal thymocyte and stromal growth characteristics in vitro, and (4) a normal thymocyte response to cytokine stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Child↗
Amniotic fluid phospholipid analysis in the fetus with congenital diaphragmatic hernia.
The substantial morbidity and mortality of congenital diaphragmatic hernia (CDH) is attributed to the pulmonary hypoplasia caused by the presence of abdominal viscera in the chest during intrauterine life. Recent experimental studies suggest that surfactant deficiency may also contribute to CDH pathophysiology. Clinically, the amniocentesis-derived lecithin to sphingomyelin (L/S) ratio and phosphatidylglycerol (PG) data are used to assess fetal lung maturity. We have performed amniotic fluid phospholipid analyses at 33 to 38 weeks' gestation in 18 fetuses with prenatally diagnosed CDH to assess fetal lung maturity, plan optimal timing for delivery, and selectively employ prenatal glucocorticoid or postnatal surfactant therapy. Compared with published control values from uncomplicated pregnancies, there was no difference in the L/S ratio or PG in the CDH fetus. Based on amniotic fluid phospholipid data, the human CDH fetus is not surfactant-deficient.
Amniotic Fluid↗
Effect of nitric oxide on the colonic smooth muscle of patients with Hirschsprung's disease.
Hirschsprung's disease results in bowel obstruction because of a failure of smooth muscle relaxation in both the aganglionic segment of bowel and the internal anal sphincter (IAS). Nonadrenergic noncholinergic (NANC) nerves, which use nitric oxide (NO) as their chemical messenger, are responsible for relaxing smooth muscle in normal bowel and the IAS. Previous work indicates that the cause of the aganglionic colon's inability to relax may be a lack of NANC nerves. To test this hypothesis, the authors compared the effect of an exogenous source of NO, S-nitroso-N-acetylpenicillamine (SNAP), on the isometric tension of smooth muscle strips taken from the ganglionic colon, aganglionic colon, and IAS of patients with Hirschsprung's disease. Exposure of ganglionic and aganglionic colon specimens to SNAP (10(-3) to 10(-5) mol/L) resulted in up to 70% reduction of resting tension. This relaxation occurred in a dose-dependent fashion and could be promptly reversed by the addition of the NO antagonist methylene blue. However, SNAP had no demonstrable effect on the smooth muscle strips taken from the IAS of patients with Hirschsprung's disease. This finding suggests that, in the aganglionic colon, a deficiency of NANC nerves contributes to the development of bowel obstruction. However, the failure of the IAS to relax in Hirschsprung's disease appears to be unrelated to NO and the NANC nervous system.
Colon↗
Congenital high airway obstruction syndrome (CHAOS): a potential for perinatal intervention.
Congenital high airway obstruction syndrome (CHAOS) results in a predictable constellation of findings: large echogenic lungs, flattened or inverted diaphragms, dilated airways distal to the obstruction, and fetal ascites and/or hydrops. The authors report on four fetuses referred for evaluation. None of them survived. Postmortem evaluation showed that three fetuses had laryngeal atresia, and one had tracheal stenosis. Coexistent fetal anomalies were accurately diagnosed by ultrasound in three of the four patients. The finding of CHAOS on prenatal ultrasound examination is diagnostic of complete or near-complete obstruction of the fetal upper airway, most likely caused by laryngeal atresia. A greater understanding of the natural history of CHAOS may permit improved prenatal and perinatal management.
Adult↗
Plug the lung until it grows (PLUG): a new method to treat congenital diaphragmatic hernia in utero.
Fetal lungs normally produce fluid that flows through the upper airway into the amniotic fluid. In fetuses with congenital diaphragmatic hernia (CDH), obstructing the flow of lung fluid may expand the lungs and propel the viscera from the chest, alleviating the pulmonary hypoplasia associated with CDH. To test this hypothesis, left-sided diaphragmatic hernias were created in sixteen 75-day-gestation fetal lambs (full-term, 145 days). At 120 days, the trachea was ligated in eight lambs; it was left unligated in the other eight. At 135 to 140 days, the fetuses were delivered, and a tracheostomy performed. Newborns were ventilated for 1 hour and then killed. Blood gas analysis was performed at 0,5,20,40, and 60 minutes. Lung dry weight, DNA, protein, and lipid analyses, as well as plasma cortisol measurements were performed. At autopsy, in the ligated lambs, the abdominal viscera was reduced from the thorax; however, the unligated lambs had viscera completely occupying the left chest. The lungs of the ligated lambs had a higher dry weight (4.22 +/- 1.37 g/kg v 1.95 +/- 0.59 g/kg; P =.001), DNA (193.8 +/- 90.5 mg/kg v 91.5 +/- 66.4 mg/kg; P = .02), and protein (1798 +/- 691.6 mg/kg v 766.6 +/- 201 mg/kg; P = .004). Lung saturated phosphatidyl choline (SPC) levels, DNA:protein ratio, and plasma cortisol were not different between the groups. Neonatal Po2 at 60 minutes was higher in the ligated group (179.4 +/- 127.0 mm Hg v 60.9 +/- 62.4 mm Hg; P < .05), and Pco2 was lower (44.1 +/- 21.4 v 83.9 +/- 23.5; P < .05) in the ligated group.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Stromal microenvironment of human fetal hematopoiesis: in vitro morphologic studies.
The stromal microenvironment is essential for the proliferation and differentiation of hematopoietic progenitors. The features of stroma which contribute to normal hematopoietic stem cell ontogeny or to observed differences in hematopoiesis between fetal and adult hematopoietic organs remain to be fully characterized. In this study, we used long-term culture conditions to grow human fetal liver, fetal bone marrow and adult bone marrow-derived stroma. The stromal layers in all cultures were observed to support multilineage hematopoiesis. Routine and electron microscopic evaluation of the stromal layers reveal the presence of two distinct cell types: a large cell with extensive cytoplasmic projections, and a smaller cell resembling a macrophage. In contrast to some previous reports from in vitro stromal studies, there were no adipocytes, endothelial cells, or cells which could conclusively be identified as fibroblasts in the stromal layer. These findings were further substantiated by negative findings on sections stained with oil-red-O for fat, trichrome for collagen, and factor VIII-related antigen for endothelial elements. There were no morphological differences in the stromal layers from fetal liver, fetal bone marrow, and adult bone marrow sources. This finding is important because it suggests that differences in the behavior of hematopoietic stem cells, when supported in these various hematopoietic microenvironments, are less likely to be explained by obvious differences in the cytologic architecture of stroma than by differences in stem cell biology or growth factor interactions.
Adult↗
Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep.
We previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human fetal liver hematopoietic stem cells in sheep after transplantation in utero. That the engraftment of long-term repopulating pluripotent stem cells occurred in these animals was shown here by the fact that transplantation of human CD45+ cells isolated from bone marrow of these chimeric animals into preimmune fetal sheep resulted in engraftment and expression of human cells. Marrow cells were obtained from three chimeric sheep at 3.2-3.6 yr after transplant. The relative percentage of human CD45+ cells present in these marrows was 3.3 +/- 0.32%. A total of 29 x 10(6) CD45+ cells were isolated by panning, pooled, and transplanted into six preimmune sheep fetuses (4.8 x 10(6) cells/fetus). All six recipients were born alive. Hematopoietic progenitors exhibiting human karyotype were detected in marrows of two lambs soon after birth. Cells expressing human CD45 antigen were also detected in blood and marrow of both lambs. Human cell expression has been multilineage and has persisted for > 1 yr. These results demonstrate that the expression of human cells in this large animal model resulted from engraftment of long-term repopulating pluripotent human stem cells.
Animals↗
Nitric oxide synthase is deficient in the aganglionic colon of patients with Hirschsprung's disease.
OBJECTIVES: The cause of Hirschsprung's disease is unknown but defects in nonadrenergic, non-cholinergic innervation could prevent relaxation of aganglionic colon in patients with this disease. Nonadrenergic, noncholinergic nerves induce relaxation by using nitric oxide synthase to produce the smooth muscle relaxant nitric oxide (NO). In this study we asked whether aganglionic colon in patients with Hirschsprung's disease is deficient in NO synthase-containing nerves. METHODOLOGY: Using the tetrazolium blue dye method of demonstrating nicotinamide adenine dinucleotide phosphate-diaphorase enzymes, we examined eight colon specimens (four aganglionic and four ganglionic) from patients with Hirschsprung's disease for the presence of NO synthase. We further quantified NO synthase enzyme activity in these eight specimens by using the [3H]arginine-to-[3H]citrulline conversion assay. RESULTS: The nicotinamide adenine dinucleotide phosphate-diaphorase staining showed that aganglionic colon contained less NO synthase than ganglionic colon. This NO synthase deficiency was located primarily in the nerves of the circular muscle layer of the colon. In addition, there was a striking difference in the NO synthase enzyme activity between aganglionic and ganglionic colon as measured by the [3H]arginine-to-[3H]citrulline conversion assay. Total NO synthase activity, as measured by this assay, was found to be less in aganglionic than in ganglionic colon. When the total activity was divided into its four known isoforms, aganglionic colon was noted to be striking deficient in the isoform derived primarily from nerves. CONCLUSION: We conclude that aganglionic colon is deficient in NO synthase-containing nerves. This deficiency could prevent smooth muscle relaxation in the aganglionic colon of patients with Hirschsprung's disease.
Amino Acid Oxidoreductases↗
An effective strategy for decontamination, ex vivo expansion, and storage of human fetal liver hematopoietic stem cells.
The transplantation of human fetal tissue has the potential to cure a variety of life-threatening diseases. The strategy for procurement, quality control, and functional assessment of human fetal liver HSC may prove useful for the transplantation of other fetal tissues. In addition to technical limitations, there are ethical and legal issues which need to be resolved before widespread use of fetal tissue. Further development of regulatory standards for the acquisition and distribution of fetal tissues will foster the application of this novel technology.
Abortion, Induced↗
Retention and multilineage expression of human hematopoietic stem cells in human-sheep chimeras.
We have taken advantage of the permissive environment of the preimmune fetus to achieve engraftment of hematopoietic stem cells (HSC) from fetal and postnatal human sources in sheep. The resulting chimeric lambs exhibited long-term multilineage (erythroid, myeloid, lymphoid) expression of donor cells that remained responsive to human-specific cytokines. The small size of the fetus allowed the assessment of the engraftment potential of relatively small numbers of highly characterized human HSC populations, while the large size of the chimeric sheep permitted the long-term evaluation of the HSC activity and response in vivo. The human/sheep xenograft may offer a useful large animal model for the assay and long-term study of the human HSC subsets.
Animals↗
In utero transplantation of rat hematopoietic stem cells induces xenogeneic chimerism in mice.
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Animals↗
Human fetal liver hematopoietic cell expansion with a novel bioreactor system.
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Animals↗
Fetal therapy--1994.
Treatment of fetal anomalies has allowed the intact survival of increasing numbers of fetuses with previously fatal or very likely fatal disorders. Percutaneous in-utero surgery for obstructed bladders and thoracic effusions, and open surgery for diaphragmatic hernias and lung malformations has become increasingly successful. Medical and gene therapy approaches are likewise expanding and will continue to become more available.
Congenital Abnormalities↗
Persistence of human multilineage, self-renewing lymphohematopoietic stem cells in chimeric sheep.
We have previously reported the ability of uncharacterized human bone marrow (BM) cells to engraft into preimmune fetal sheep, thereby creating sheep-human chimera suitable for in vivo examination of the properties of human hematopoietic stem cells (HSC). Adult human bone marrow CD34+ HLA-DR- cells have been extensively characterized in vitro and have been demonstrated to contain a number of primitive hematopoietic progenitor cells (PHPC). However, the capacity of such highly purified populations of human marrow CD34+ HLA-DR- cells to undergo in vivo self-renewal and multipotential lymphohematopoietic differentiation has not been previously demonstrated. To achieve that, human CD34+ HLA-DR- cells were transplanted in utero into immunoincompetent fetal sheep to investigate the BM-populating potential of these cells. Long-term chimerism, sustained human hematopoiesis, and expression of human cells belonging to all human blood cell lineages were demonstrated in two animals for more than 7 months' posttransplantation. Chimeric BM contained erythroid, granulocytic/monocytic, and megakaryocytic hematopoietic progenitor cells, as well as the primitive high proliferative potential colony-forming cell (HPP-CFC). Under a variety of in vitro experimental conditions, chimeric BM cells gave rise to human T cells expressing T-lymphocyte-specific markers, human natural killer (NK) cells, and human IgG-producing B cells. In vivo expansion and possibly self-renewal of transplanted PHPC was confirmed by the detection in chimeric BM 130 days' posttransplantation of CD34+ HLA-DR- cells, the phenotype of human cells constituting the stem-cell graft. These studies demonstrate not only the BM-populating capacity, multipotential differentiation, and most likely self-renewal capabilities of human CD34+ HLA-DR- cells, but also that this BM population contains human HSC. Furthermore, it appears that this animal model of xenogeneic stem-cell transplantation is extremely useful for in vivo examination of human hematopoiesis and the behavioral and functional characteristics of human HSC.
Animals↗
Chronic fetal vascular access.
Intensive management of the fetus is limited by our inability to achieve access to the fetal circulation. Using laparoscopic surgery, we can maintain long-term access to the fetal circulation by extra-amniotic catheterisation of chorionic vessels in third-trimester monkeys and early-gestation sheep. We can sample fetal blood, continuously measure blood pressure, and infuse drugs without complications. Safe vascular access will permit assessment of fetal status during fetal surgical procedures and improve postoperative monitoring.
Animals↗
Effect of xenogeneic chimerism in a human/sheep model on natural antibody.
Xenogeneic transplantation is a potential solution to the severe shortage of donor organs for clinical transplantation. The primary limitation to xenogeneic transplantation between widely disparate species is hyperacute rejection, which is triggered by the recipient's natural antibodies reacting against the donor's endothelial cells. Natural antibodies react between widely disparate species but do not react between closely related species. Specific tolerance for xenogeneic transplantation between closely related species can be induced by creating hematopoietic chimerism between donor and recipient. However, whether specific xenograft tolerance can be induced by the creation of chimerism between widely disparate species, where natural antibody reacts, is unknown. We previously have established a model of hematopoietic chimerism between widely disparate species by the in utero transplantation of human fetal hematopoietic stem cells into early gestation fetal lambs. In the present study, we determined whether long-standing hematopoietic chimerism in this human/sheep model reduces the level of natural antibody directed against human endothelial cells. To answer this question, we measured the reactivity of serum from five chimeric sheep, five sheep controls, and five human controls in an in vitro enzyme-linked immunoabsorbance assay directed against human umbilical vein endothelial cells. Unexpectedly, we found that long-standing hematopoietic chimerism in the human/sheep model did not reduce the reactivity of serum against human endothelial cells compared to age-matched sheep controls. These results suggest that the induction of hematopoietic chimerism between widely disparate species will not control the problem of natural antibody and hyperacute rejection.
Animals↗
Fetal surgery for cystic adenomatoid malformation of the lung.
We reviewed our experience with fetal therapy for congenital cystic adenomatoid malformation of the lung (CCAM) at the University of California, San Francisco Fetal Treatment Center. Fetuses with life-threatening CCAM were selected for prenatal treatment according to predetermined guidelines, including the gestational age of the fetus, the size of the intrathoracic lesion, maternal health, and the development of fetal hydrops. The knowledge that fetuses with hydrops are at high risk for fetal or neonatal death led to fetal surgical resection of the massively enlarged pulmonary lobe (fetal lobectomy) in six cases. In the first case, resection was too late, since preoperative labor and maternal preeclampsia could not be reversed, leading to premature delivery of a nonviable infant. In the next four cases, CCAM resection led to resolution of the hydrops, impressive in utero lung growth, and neonatal survival. Right middle and lower lobe resection in the sixth fetus at 21 weeks was successful, but subsequent inexplicable fetal death highlights the need for better postoperative fetal monitoring and treatment. Three other fetuses with a single predominant cyst underwent thoracoamniotic shunt placement alone; two survived after delivery and prompt neonatal surgery with the assistance of high-frequency ventilation or extracorporeal membrane oxygenation. Fetal therapy can now be considered for otherwise fatal space-occupying intrathoracic lesions in the fetus.
Adult↗