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Biomedical subjects

A W Flake

Publications and source records attributed to A W Flake.

At least 37 records · Page 2Linked to original sources
PubMed2000

Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.

Adipocytes↗
PubMed2000

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Adult↗
PubMed2000

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep.

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Adipocytes↗
PubMed2000

Echocardiographic evaluation of the fetus with congenital cystic adenomatoid malformation.

OBJECTIVES: The development of nonimmune hydrops is a known complication of congenital cystic adenomatoid malformation (CCAM) in the fetus. The aim of this study was to investigate the impact of CCAM on hemodynamics in the fetus and to determine whether cardiac dysfunction contributes to the development of hydrops in this patient population. METHODS: The echocardiographic data from 41 consecutive fetuses diagnosed prenatally with CCAM were reviewed. The presence or absence of hydrops was noted in each patient. Two-dimensional ultrasonographic and Doppler data were compared between the two groups (with or without hydrops). RESULTS: The mean gestational age for the study group at the time of imaging was 23.2 +/- 3.0 weeks. Hydrops was noted in 15 of 41 fetuses (36.5%). The fetuses that developed hydrops had a lower cardiac/thoracic ratio than those which did not develop hydrops (0.18 vs. 0.23, P = 0.001). The fetuses with hydrops also demonstrated an increase in early ventricular filling. The ratio of early ventricular filling to atrial contraction (E/A ratio) at both the tricuspid and mitral valves was significantly higher in the fetuses with hydrops (P = 0.005 and P = 0.03, respectively). Doppler interrogation of the inferior vena cava demonstrated a greater degree of reversal with atrial contraction in the hydrops group (29.7% vs. 15.1%, P = 0.003). The development of significant atrioventricular valve insufficiency was rare in the fetuses with hydrops (one of 15 cases). CONCLUSIONS: Cystic adenomatoid malformation can result in significant cardiac compression in the fetus. This alters hemodynamics and may result in hydrops on the basis of elevated central venous pressure.

Case-Control Studies↗
PubMed2000

Combined pure esophageal atresia, duodenal atresia, biliary atresia, and pancreatic ductal atresia: prenatal diagnostic features and review of the literature.

The authors recently managed a case of combined pure esophageal, duodenal, biliary, and pancreatic ductal atresia in a fetus and newborn with Trisomy 21. The authors present a case report and review of the literature, emphasizing the prenatal radiographic features of the combined lesions and the high incidence of associated anomalies including Down's syndrome. Prenatal suspicion of these anomalies warrants karyotype analysis as well as careful pre- and postnatal screening for other anomalies.

Abnormalities, Multiple↗
PubMed2000

Treatment of severe congenital diaphragmatic hernia by fetal tracheal occlusion: clinical experience with fifteen cases.

OBJECTIVE: Our purpose was to determine whether prenatal tracheal occlusion improves survival in a selected population of fetuses affected by severe congenital diaphragmatic hernia. STUDY DESIGN: Fetuses with isolated congenital diaphragmatic hernia were selected as candidates for fetal intervention by specific criteria designed to predict a 90% mortality rate with conventional postnatal treatment. RESULTS: Fifteen fetuses underwent tracheal occlusion with 5 survivors (33%). Two fetuses were lost to early preterm labor. In 13 mothers, postoperative gestation ranged from 19 to 68 days, with a mean duration of pregnancy after tracheal occlusion of 38 days. The 5 survivors were hospitalized for an average of 76 days. Despite dramatic lung growth in some fetuses after tracheal occlusion, intensive management was required, and most deaths were caused by respiratory insufficiency. CONCLUSION: Prenatal tracheal occlusion can result in impressive lung growth in a subset of fetuses with severe congenital diaphragmatic hernia. However, survival remains compromised by pulmonary functional abnormality and the consequences of prematurity.

Child Development↗
PubMed2000

Assessment of fetal lung volumes and liver herniation with magnetic resonance imaging in congenital diaphragmatic hernia.

OBJECTIVE: We evaluated the use of fetal magnetic resonance imaging in predicting outcomes after ultrasonographic diagnosis of left-sided congenital diaphragmatic hernia. STUDY DESIGN: Forty-one pregnant women carrying fetuses with congenital diaphragmatic hernia underwent 43 magnetic resonance imaging scans. Lung volumes were calculated by summing the areas on 6-mm axial sections. The presence or absence of liver herniation was noted. A liver/diaphragm ratio was obtained by using the distances from the superior aspect of the liver and the diaphragmatic remnant to the apex of the chest. RESULTS: Mean gestational age was 26 weeks and overall survival was 59%. Neither right, left, nor total lung volume measurements were predictive of survival. Liver herniation into the left side of the chest was predictive of outcome at P<.05. The liver/diaphragm ratio was predictive of outcome at P = .03. CONCLUSION: Fetal magnetic resonance imaging permits calculation of lung volumes, but these volumes are not predictive of outcome. However, both the presence of liver herniation and the volume of liver within the chest, as reflected by the liver/diaphragm ratio, help predict outcome in left-sided congenital diaphragmatic hernia.

Fetus↗
PubMed1999

In utero stem cell transplantation for the treatment of genetic diseases.

In utero haematopoietic cell transplantation is currently in its embryonic stage of development, but holds considerable developmental promise as a therapeutic approach for the treatment of a large number of congenital haematological diseases. Despite, until recently, limited evidence of clinical efficacy, interest in the field has been gaining momentum, and clinical application is likely to increase. Parallel advances in prenatal diagnosis, fetal intervention, and haematopoietic stem cell technology have removed many of the practical, technical, and ethical obstacles to clinical application. With this progress, there has been a significant increase in the number of centers around the world with both the stated interest and perceived expertise to develop clinical programs. At this point in the evolution of in utero haematopoietic stem cell transplantation there are more questions than answers. Widespread clinical applications are premature based on the extremely limited clinical success, which has been achieved. The biology of each disease is unique and expectations of success or failure can only be based upon sound clinical investigation guided by an understanding of the relevant issues and careful selection and evaluation of patients. Clinical centers should be associated with an active research effort to solve the remaining problems with this potentially valuable therapeutic approach. In the near future, advances in our understanding of stem cell biology, developmental ontogeny, and gene therapy may allow prenatal stem cell and gene therapy to achieve their full potential.

Animals↗
PubMed1999

Improvement in hindbrain herniation demonstrated by serial fetal magnetic resonance imaging following fetal surgery for myelomeningocele.

CONTEXT: Hindbrain herniation occurs in a large percentage of children with myelomeningocele and is the leading cause of death in this population. The effect of early fetal closure of myelomeningocele on hindbrain herniation is unknown. OBJECTIVE: To determine whether early fetal closure of myelomeningocele affects hindbrain herniation. DESIGN: Case series of patients undergoing fetal myelomeningocele closure with serial measurements of hindbrain herniation and a mean follow-up of 182 days. SETTING: Tertiary care medical center. PARTICIPANTS: Ten patients undergoing fetal myelomeningocele closure at 22 to 25 weeks' gestation between March 1998 and February 1999. MAIN OUTCOME MEASURES: Need for shunt placement; degree of hindbrain herniation (grades 0-3) found on magnetic resonance imaging (MRI) performed prior to surgery and 3 and 6 weeks after fetal surgery, as well as shortly after birth; gestational age at delivery. RESULTS: All initial fetal MRI scans performed at 19 to 24 weeks' gestation showed significant (grade 3) cerebellar herniation and absence of spinal fluid spaces around the cerebellum. Six fetuses were delivered electively at 36 weeks' gestation after lung maturity was established. The other 4 were delivered prematurely, at 25, 30, 30, and 31 weeks of gestation, and the 25-week gestation neonate died. All 9 surviving neonates showed improvement in the hindbrain hernia at the 3-week postoperative fetal scan (grade 2, n = 4; grade 1, n = 5). On the postnatal scan, all patients showed grade 1 hindbrain herniation. Only 1 patient required placement of a ventriculoperitoneal shunt. CONCLUSION: In this series of patients, fetal myelomeningocele closure resulted in improvement in hindbrain herniation as demonstrated by serial MRI scans.

Female↗
PubMed1999

A kinetic model for the homing and migration of prenatally transplanted marrow.

Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (10(6) LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

Animals↗
PubMed1999

Homing of human cells in the fetal sheep model: modulation by antibodies activating or inhibiting very late activation antigen-4-dependent function.

The mechanisms by which intravenously (IV)-administered hematopoietic cells home to the bone marrow (BM) are poorly defined. Although insightful information has been obtained in mice, our knowledge about homing of human cells is very limited. In the present study, we investigated the importance of very late activation antigen (VLA)-4 in the early phases of lodgment of human CD34(+) progenitors into the sheep hematopoietic compartment after in utero transplantation. We have found that preincubation of donor cells with anti-VLA-4 blocking antibodies resulted in a profound reduction of human cell lodgment in the fetal BM at 24 and 48 hours after transplantation, with a corresponding increase of human cells in the peripheral circulation. Furthermore, IV infusion of the anti-VLA-4 antibody at later times (posttransplantation days 21 to 24) resulted in redistribution or mobilization of human progenitors from the BM to the peripheral blood. In an attempt to positively modulate homing, we also pretreated human donor cells with an activating antibody to beta1 integrins. This treatment resulted in increased lodgment of donor cells in the fetal liver, presumably for hemodynamic reasons, at the expense of the BM. Given previous involvement of the VLA-4/vascular cell adhesion molecule (VCAM)-1 adhesion pathway in homing and mobilization in the murine system, our present data suggest that cross-reacting ligands (likely VCAM-1) for human VLA-4 exist in sheep BM, thereby implicating conservation of molecular mechanisms of homing and mobilization across disparate species barriers. Thus, information from xenogeneic models of human hematopoiesis and specifically, the human/sheep model of in utero transplantation, may provide valuable insights into human hematopoietic transplantation biology.

Animals↗
PubMed1999

Postnatal booster injections increase engraftment after in utero stem cell transplantation.

BACKGROUND: The primary barrier to clinical application of in utero hematopoietic stem cell (HSC) transplantation is limited donor cell engraftment. We hypothesized that limited engraftment was due to competition between host and donor cells for available niches. We reasoned that increased engraftment might be achieved by performing multiple transplants separated by brief intervals to allow time for formation of new niches. To test this we performed multiple transplants in a congenic combination to avoid confounding immunologic effects. MATERIALS AND METHODS: C57Pep3B (H2Kb, CD45.1) mice were used as donors of adult bone marrow and C57Bl/6 (H2Kb, CD45.2) mice were used as 14-day-gestation fetal recipients. All fetuses were injected intraperitoneally with 1 x 10(6) mononuclear cells. Boosted neonates were injected at Days 2, 4, and 7 of life with 5 x 10(6) cells. All animals were analyzed for donor cell engraftment by dual-color flow cytometry using CD45 and CD45.1 antigens. Results are reported as the mean +/- SD. Statistical analysis was performed using the two-tailed Student t test with P < 0.05 considered significant. RESULTS: Postnatally boosted animals demonstrated significantly elevated levels of donor cell engraftment (3.30 +/- 0.8%; n = 8; P < 0.00001) when compared to the control animals (0.69 +/- 0.5%; n = 9) as determined by peripheral blood analysis at 6 weeks of age. This elevated level of engraftment was stable long term. CONCLUSIONS: Our results demonstrate a significant increase in donor cell engraftment with postnatal booster injections after in utero transplantation. This supports the hypothesis that a limited number of niches may be a major component of the barrier to engraftment. It also suggests that postnatal booster injections may be a viable therapeutic strategy for improving donor cell engraftment after in utero HSC transplantation.

Aging↗
PubMed1999

Maintenance of CD34 expression during proliferation of CD34+ cord blood cells on glycosaminoglycan surfaces.

Recent studies have indicated that glycosaminoglycan (GAG) interactions with hematopoietic progenitors play a significant role in the regulation of hematopoiesis. However, the details of these interactions are not clear. In this study, we examined the role of soluble and immobilized GAGs in the proliferation of CD34+ cells. Chitosan, a cationic polysaccharide, was used to immobilize GAGs in ionic complex membranes. The GAGs studied were heparin, hyaluronate, and chondroitin sulfates A, B, and C. CD34-enriched umbilical cord blood cells were seeded onto tissue culture plates coated with the GAG-chitosan complex membranes. Cultures were maintained in medium supplemented with stem cell factor and interleukin 3 for up to six weeks, during which total and CD34+ cell numbers were determined by flow cytometry. Total cell number expansion ranged from 25-fold to 40-fold after six weeks. However, only heparin and chondroitin sulfate B (CSB) surfaces retained a significant CD34+ fraction. All other surfaces exhibited declines in CD34 expression, with negligible CD34+ percentages remaining after four weeks. In contrast, heparin and CSB surfaces exhibited CD34+ fractions as high as 90% after four weeks. GAG desorption studies indicated that the observed effects were partly mediated by desorbed GAGs in a concentration dependent manner. Subsequent studies showed that sustained high (160 microg/ml) heparin levels had toxic effects, while the same concentration of CSB exhibited more rapid early proliferation of CD34+ cells. In conclusion, this culture system has demonstrated the ability to produce simultaneous proliferation and CD34+ cell enrichment of a partially purified cord blood population by controlling the nature and levels of GAG moieties to which the cells are exposed. The results indicate that specific GAGs can significantly influence the growth and differentiation characteristics of cultured CD34+ cells.

Animals↗
PubMed1999

TGF-beta2 is increased after fetal tracheal occlusion.

BACKGROUND/PURPOSE: Fetal tracheal occlusion (TO) accelerates lung growth in normal and hypoplastic fetal lung. The mechanism of accelerated lung growth remains unknown but may be a result of growth factor induction. Previous studies of growth factors induced by tracheal ligation have characterized mRNA rather than protein expression. Although the transforming growth factor-beta (TGF-beta) family participates in normal lung morphogenesis, its role in lung growth after TO is unclear. The authors hypothesize that TGF-beta expression is increased with TO and may contribute to the accelerated lung growth seen after TO. METHODS: Diaphragmatic hernia (DH) was created in 80-day-gestation sheep (n = 6; term, 145) by excising the left diaphragm. At 110 days, the trachea was occluded (n = 4) with a clip. DH controls (n = 2) were not occluded. Fetuses were killed at 139 days, and lung samples were snap frozen for tissue analysis. Non-DH control lungs were harvested from full-term animals (n = 2). TGF-beta mRNA was analyzed by semiquantitative reverse transcriptionase-polymerase chain reaction (RT-PCR). TGF-beta protein was assessed by Western blot analysis. RESULTS: TGF-beta1 mRNA and protein were not increased with tracheal ligation compared with either non-DH or DH controls. TGF-beta2, however, was markedly increased, at both the mRNA and protein level, in ligated lungs compared with nonligated controls. CONCLUSIONS: TGF-beta2 protein, but not TGF-beta1, is increased in the hypoplastic lungs of fetal sheep after tracheal occlusion. Increased TGF-beta2 expression appears to result from increased or prolonged expression of mRNA transcripts. This is the first study to document a change in growth factor protein levels after TO. Increased TGF-beta2 expression may contribute to accelerated lung growth and decreased surfactant production observed after tracheal occlusion.

Animals↗
PubMed1999

In utero bone marrow transplantation induces donor-specific tolerance by a combination of clonal deletion and clonal anergy.

BACKGROUND/PURPOSE: In utero bone marrow transplantation can induce donor-specific tolerance to postnatal solid organ transplantation, although the mechanisms remain poorly defined. In this study, we investigated the role of clonal deletion and clonal anergy in the maintenance of tolerance in a murine model of in utero bone marrow transplantation. METHODS: DBA/2 mice (MIs(a+)) were used as donors of adult bone marrow, and 14-day-gestation fetal Balb/c mice (MIs(a-)) were used as recipients. Tolerance was defined by donor-specific skin graft survival for more than 8 weeks. Clonal deletion was assessed by flow cytometry for Vbeta6 T cell receptor usage. A tolerant animal demonstrating partial deletion of CD4+/Vbeta6+ T cells and a nontolerant animal were selected for analysis of clonal anergy by a proliferation assay using plate-bound anti-Vbeta6 antibody for stimulation with or without exogenous interleukin-2 (IL2). RESULTS: Vbeta6+ splenocytes constituted 6.32% of CD4+ T cells in the tolerant animal compared with 9.19% in the nontolerant animal, demonstrating incomplete clonal deletion in the tolerant animal. Stimulation with plate-bound anti-Vbeta6 induced a good proliferative response in the nontolerant animal but a significantly attenuated response in the tolerant animal (P< .001), which was abrogated by the addition of IL2. CONCLUSIONS: In this murine model of in utero bone marrow transplantation, the tolerant state is characterized by partial clonal deletion of donor reactive T cells and clonal anergy of nondeleted donor reactive T cells. The anergic state can be abrogated by exogenous IL2, suggesting that the mechanism of anergy is a deficiency of IL2 production.

Animals↗
PubMed1999

Persistent postnatal transgene expression in both muscle and liver after fetal injection of recombinant adenovirus.

BACKGROUND/PURPOSE: Immune responses to both vector and transgene antigens have limited the efficacy of postnatal gene therapy. We hypothesize that the fetal period may offer immunologic and developmental advantages for successful gene therapy. In this study we examined the efficacy, persistence, and immunologic effects of recombinant adenovirus after intramuscular delivery into fetal mice. METHODS: E1-deleted adenovirus (AdCMVlacZ) containing the beta-galactosidase marker gene was used for injection. Fetal Balb/c mice (14 to 15 days' gestation) were injected with AdCMVlacZ in 10-microL volume in either the shoulder or hindlimb musculature. Animals were killed at 18 to 20 days' gestation and up to 4 months postnatally for analysis of transgene expression and adenoviral genome persistence. RESULTS: Fetuses were injected with doses of AdCMVlacZ from 1 x 10(8) to 2 x 10(10) viral particles (n = 80). Optimal survival rate was 83% at 18 to 20 days' gestation and 55% at 4 weeks of age using a dose of 1 x 10(9) particles. Expression of beta-galactosidasae at 18 to 20 days localized to multiple muscle groups surrounding the site of injection, as well as bone marrow stroma, liver, lung, and dorsal root ganglia. Persistent muscle and liver transgene expression was observed for as long as 16 and 8 weeks, respectively, after injection. The pattern of liver expression was confined to discrete foci of hepatocytes, which appeared to increase in size in older animals. No histological evidence of muscle or liver inflammation was observed at any time after injection. No neutralizing antibodies were observed postnatally. CONCLUSIONS: Our results confirm that gene therapy in the fetus may be advantageous. Distribution of vector in the fetus at the site of injection is clearly broader than in the adult setting. Furthermore, the absence of immune response and persistence of transgene expression suggests that fetal exposure to foreign transgene and vector antigens may induce tolerance. Although we have not proven genomic integration, the histological appearance of transgene expression in the liver supports this conclusion. By understanding the mechanisms that underlie persistent transgene expression, fetal gene therapy may become a feasible strategy for the treatment of fatal genetic diseases.

Adenoviridae↗