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Biomedical subjects

A W Flake

Publications and source records attributed to A W Flake.

At least 19 recordsLinked to original sources
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↗
PubMed1998

In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses.

We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.

Animals↗
PubMed1998

In utero transplantation for thalassemia.

In utero hematopoietic stem cell transplantation is a promising approach for the treatment of a variety of congenital hematologic diseases. Although the approach has been successful for immunodeficiency syndromes, attempts thus far to treat the hemoglobinopathies have failed. In most of these cases the late gestational age at transplantation, source of donor cells, or procedure-related complications, provide an explanation for failure. Nevertheless the biology of thalassemia, in the context of prenatal transplantation, requires examination. In contrast to postnatal bone marrow transplant regimens, engraftment after in utero transplantation requires donor cells to effectively complete for developing receptive sites in the recipient hematopoietic microenvironment. Effective prenatal treatment of thalassemia will depend on the ability of normal cells to engraft and complete in the thalassemic microenvironment. Clinical observations after bone marrow transplantation of amelioration of anemia in beta-thalassemia by relatively low degrees of mixed chimerism, and the apparent selective advantage observed for donor erythropoiesis, suggest prenatal transplantation could succeed. Prenatal strategies involving multiple transplants, donor-specific tolerance induction, and postnatal same-donor transplants should be considered.

Anemia, Sickle Cell↗
PubMed1998

Microchimerism and tolerance after in utero bone marrow transplantation in mice.

BACKGROUND: Donor-specific tolerance has been induced after both fetal and neonatal hematopoietic stem cell (HSC) transplantation in mice. However, the relationship between hematopoietic microchimerism and tolerance in these models has not been defined due to the insensitivity of donor cell detection methodology. To address this problem we developed a semiquantitative polymerase chain reaction (PCR)-based assay for detection of microchimerism after major histocompatibility (MHC) class I disparate HSC transplantation. This assay was used to examine the relationship between microchimerism and tolerance after fetal and neonatal transplantation of fully allogeneic bone marrow cells. MATERIALS AND METHODS: C57BL/6 mice (H2-Kb) were used as adult bone marrow donors and Balb/c mice (H2-Kd) were used as fetal or newborn recipients. A dose of 10(10) BM cells/kg was injected intraperitoneally into recipient animals. Peripheral blood of animals which survived beyond 3 weeks of age was analyzed by PCR for the presence of donor MHC class I DNA. Tolerance was tested by placement of donor-specific skin grafts after determination of chimerism status. RESULTS: Our assay was found to be specific for H2-Kb donor cells in an H2-Kd background with a sensitivity of <0.0001%. Of 49 animals injected in utero 19 (38%) had donor DNA present in peripheral blood at low levels (<0.1%) whereas only 1 of 18 neonatally injected animals had detectable donor cells (P < 0.01). Tolerance to donor-specific skin grafts was found in 6 of 9 animals which were chimeric after in utero HSC transplantation whereas none of the 18 neonatally injected animals including the chimeric animal were tolerant. CONCLUSIONS: Our results indicate the following. ( 1) Hematopoietic microchimerism can be detected by PCR in peripheral blood after in utero injection of fully allogeneic HSCs. ( 2) Fetal injections yield a higher incidence of microchimerism than newborn injections. ( 3) Tolerance can be induced across a fully allogeneic barrier by in utero HSC transplantation and this is associated with the presence of peripheral blood microchimerism.

Animals↗
PubMed1998

Fetal lung lesions: management and outcome.

OBJECTIVE: Our purpose was to review our experience with fetal congenital cystic adenomatoid malformation and extralobar pulmonary sequestration emphasizing natural history, management, and outcome. STUDY DESIGN: We conducted a retrospective review of 175 fetal lung lesions diagnosed by antenatal ultrasonography at 2 fetal treatment centers. RESULTS: There were 134 congenital cystic adenomatoid malformation cases. Fourteen women underwent elective abortion, 101 women were managed expectantly, 13 women had fetal surgery, and 6 women had placement of a thoracoamniotic shunt. For the congenital cystic adenomatoid malformation lesions that were not associated with nonimmune hydrops, all babies survived. Of 25 large congenital cystic adenomatoid malformations that had associated hydrops that were followed expectantly, all fetuses died before or shortly after birth. Fetal surgical resection of the tumor (fetal lobectomy) was performed at 21 to 29 weeks' gestation in 13 hydropic fetuses with 8 fetuses continuing gestation with subsequent hydrops resolution, impressive in utero lung growth, and neonatal survival. Six fetuses with a very large solitary cyst underwent thoracoamniotic shunting and 5 survived. There were 41 extralobar pulmonary sequestration cases. Twenty-eight extralobar pulmonary sequestrations dramatically regressed on serial prenatal sonography, were asymptomatic after birth, and were only detectable by imaging studies postnatally (no resection required). Of the remaining 13 extralobar pulmonary sequestration cases, 2 underwent elective abortion, 7 symptomatic lesions were resected after birth with survival, 1 hydropic fetus died, and 3 fetuses had an associated tension hydrothorax with secondary hydrops that was successfully treated by either fetal thoracenteses or thoracoamniotic shunting followed by postnatal resection. CONCLUSIONS: The natural history of prenatally diagnosed lung masses is variable, and associated anomalies are rare. Most congenital cystic adenomatoid malformation lesions can be managed with maternal transport, planned term delivery, and postnatal resection. Many extralobar pulmonary sequestrations dramatically decrease in size before birth and may not need treatment after birth. Fetal therapy is now an option for lung lesions associated with nonimmune hydrops.

Amnion↗
PubMed1998

Tracheal occlusion in the fetal rat: a new experimental model for the study of accelerated lung growth.

BACKGROUND: Prenatal tracheal occlusion accelerates fetal lung growth, but the mechanism of this phenomenon is unknown. Previous animal models have been limited by expense, lack of species-specific molecular probes, or the stage of lung development when studies could be performed. To provide a model that is more amenable to systematic analysis, we have developed an in vivo rat model of prenatal tracheal occlusion. METHODS: Time-dated pregnant rats underwent laparotomy at 19 days' gestational age (term, 22 days). The fetal head and neck were exteriorized through a hysterotomy, and the trachea was ligated under a dissecting microscope. The fetus was returned to the amniotic cavity, and the uterine and maternal abdominal incisions were closed. The dam and the fetuses were killed at 21.5 days' gestational age, and the fetal lungs were assessed for lung growth and compared with nonoperated littermate controls. RESULTS: Thirty-two of 50 manipulated fetuses survived. Of the 32 survivors, successful tracheal ligation was confirmed in 20, and these 20 fetuses were compared with 33 littermate controls. Fetal body weight (4.81+/-0.26 g v 4.87+/-0.41 g) and heart weight (0.05+/-0.01 g v 0.05+/-0.01 g) were not significantly different between ligated fetuses and littermate controls, whereas the wet lung weight (0.30+/-0.06 g v 0.13+/-0.02 g, P<.01), lung-to-body-weight ratio (6.34+/-1.16% v 2.64+/-0.41%, P<.01), dry lung weight (17.4+/-2.45 mg v 12.1+/-1.87 mg, P<.01), total lung DNA (1210+/-371 microg v 828+/-208 microg, P<.01), and total lung protein (14.3+/-5.3 mg v 8.7+/-1.7 mg, P<.01) were increased significantly in the ligated fetuses. The enlarged lung demonstrated normal histology findings after inflation fixation. CONCLUSIONS: Prenatal tracheal occlusion during the canalicular stage of lung development accelerates lung growth in the rat. In comparison with other large animal models, this relatively inexpensive small animal model has the distinct advantages of a short gestation, a large number of fetuses per litter, the availability of a developmental model of congenital diaphragmatic hernia, and the availability of well-defined molecular probes to investigate the mechanism of tracheal occlusion-induced lung growth.

Animals↗
PubMed1998

The association of complete laryngotracheoesophageal cleft with left lung agenesis: pathophysiological clues provided by an experiment of nature.

The authors present a patient with complete laryngotracheoesophageal cleft and concurrent left lung agenesis and microgastria. Prenatal ultrasound scan showed polyhydramnios and a hypertrophic right lung. The authors propose that the combination of right lung hypertrophy, polyhydramnios, and microgastria in the absence of a competent laryngeal mechanism may suggest that the preferential path for swallowed amniotic fluid was into the lung, rather than the normal route through the stomach. This case illustrates the prenatal findings suggestive of complete laryngotracheoesophageal cleft and lung agenesis, and suggests a potential causal relationship between shunting of swallowed amniotic fluid into the bronchial tree and prenatal lung hypertrophy and microgastria.

Abnormalities, Multiple↗
PubMed1998

Increased cell proliferation and decreased apoptosis characterize congenital cystic adenomatoid malformation of the lung.

BACKGROUND/PURPOSE: Congenital cystic adenomatoid malformations (CCAM) are lung lesions that demonstrate abnormalities of both mesenchymal and epithelial tissues. The pathogenesis of these tumors remains unknown. Because normal organogenesis requires a balance between cell proliferation and programmed cell death (apoptosis), the authors hypothesized that CCAM results from an increase in cell proliferation or a decrease in apoptosis within the developing lung, possibly mediated by keratinocyte growth factor (KGF). METHODS: To examine cell cycle control in CCAM, we measured indices of cell proliferation and apoptosis in lesions requiring fetal (n = 4) or neonatal (n = 8) resection compared with those of normal fetal (14 to 28 weeks' gestation; n = 14) and neonatal (n = 3) human lung. Cell proliferation was analyzed by immunostaining for a proliferation marker (Ki-67). Apoptosis was examined using an in situ digoxigenin end-labeling technique to localize apoptotic bodies. The expression of KGF protein and KGF mRNA in CCAM and normal lung was examined using immunohistochemistry and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: CCAM lesions in general showed a twofold increase in cell proliferation index (19.2% +/- 1.4% v 9.6% +/- 0.7%, P < .00005) and a fivefold decrease in apoptotic bodies (0.9 +/- 0.2 v 4.5 +/- 0.5, P < .0005) compared with age-matched normal lung. CCAMs that required resection before birth had the highest cell proliferation index. There were no differences in the expression of KGF protein or KGF mRNA in CCAM and normal lung. CONCLUSIONS: These results demonstrate that CCAM differs from normal lung by increased cell proliferation and decreased apoptosis. The increased proliferation does not appear to be mediated by the pneumocyte mitogen KGF. An examination of factors that control cell proliferation and apoptosis in CCAM may provide further insight into the pathogenesis of this tumor.

Apoptosis↗
PubMed1998

Tracheal occlusion reverses the high impedance to flow in the fetal pulmonary circulation and normalizes its physiological response to oxygen at full term.

PURPOSE: The authors hypothesized that in utero tracheal occlusion would reverse the high impedance to pulmonary blood flow associated with congenital diaphragmatic hernia (CDH) and normalize the fetal physiological response to oxygen at term. METHODS: Three experimental groups were studied. Six fetal lambs (CDH group) underwent creation of a left CDH at 80 days' gestation, an additional six fetal lambs underwent left CDH creation at 80 days' gestation followed by fetal tracheal occlusion performed at 108 days' gestation (CDH + TO group), and four control fetal lambs (control group) underwent a sham procedure at 80 days gestation. All lambs were followed up at 2-week intervals by pulse wave Doppler echocardiography. At each time-point the pulsatility index (PI) was calculated for the left branch pulmonary artery from the Doppler blood velocity waveform. Near term (term, 145 days gestation) at 136 days gestation, measurements were repeated under maternal normoxia and hyperoxia. The fetal lungs were harvested and processed for morphometric analysis by radial alveolar counts (RAC) and lung-to-body-weight ratios (LBWR) as measures of lung growth. RESULTS: At 136 days' gestation the PI of the CDH + TO group (2.88 +/- 0.29) and control group (3.97 +/- 0.37) were significantly lower compared with the PI of the CDH group (9.02 +/- 0.50). There was a significant decrease in the PI of both the CDH + TO group and the control group with maternal hyperoxia at term, whereas the CDH group showed no change. The lungs of the CDH group fetuses were significantly smaller by LBWR and RAC than both CDH + TO and control fetuses. CONCLUSIONS: An elevated PI is associated with pulmonary hypoplasia, fetal tracheal occlusion reverses this finding, and results in a normal fetal physiological response to changes in oxygen tension at term.

Analysis of Variance↗
PubMed1998

Successful use of extracorporeal membrane oxygenation (ECMO) during BMT for SCID.

An 8-month-old girl with SCID presented with severe bronchiolitis. She received an HLA-identical sibling BMT without conditioning or GVHD prophylaxis. She deteriorated despite mechanical ventilation but had normal cardiac, hepatic and renal function. ECMO was instituted on day +3 and subsequent improvement was seen concurrently with emergence of CD4+ cells on day +11. She was taken off ECMO on day +18 and suffered a left-sided stroke evidenced by a dense left hemiplegia. She was extubated on day +25 and weaned from supplemental oxygen on day +36 and at day +100 has recovered strength in her extremities. This is the first successful use of ECMO as a bridge to engraftment in a BMT patient.

Bone Marrow Transplantation↗
PubMed1998

Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells.

We used the human/sheep competitive engraft model to investigate the in vivo engraftment potential of human CD34-, Lin- cells. In three separate studies, transplantation of CD34+, Lin- and CD34-, Lin- cells from the same normal human donors into preimmune fetal sheep resulted in long-term engraftment and multilineage hematopoietic cell/progenitor expression. Human cell/progenitor activities of the CD34+ group were CD3, 0.52%; CD13, 0.36%; CD45, 1.02%; glyA, 1.78%; HLA-DR, 0.99%; colony-forming units (CFU)-Mix, 7.1%; and granulocyte-macrophage CFU (CFU-GM), 13.7%; those of the CD34- group were CD3, 0.24%; CD45, 2.49%; glyA, 0.75%; HLA-DR, 0.3%; CFU-Mix, 7.1%; and CFU-GM, 4.3%. Of special interest was the detection of highly significant numbers (1 x 10[9] cells) of human CD34+ cells in animals transplanted with CD34- cells. Secondary transplantation and limiting dilution studies confirmed the presence of cells with long-term engraftment potential in CD34- populations. The results presented here demonstrate that the CD34- fraction of normal human bone marrow contains cells capable of engraftment and differentiation into CD34+ progenitors and multiple lymphohematopoietic lineages in primary and secondary hosts.

Adult↗
PubMed1997

In utero hematopoietic stem cell transplantation. A status report.

In utero hematopoietic stem cell transplantation is currently in its early stage of development, but it holds considerable promise as a therapeutic approach for the treatment of a large number of congenital hematologic diseases. Experimental evidence supports the concept of the early gestational fetus as a favorable recipient for cellular therapy. Unique aspects of normal hematologic and immunologic ontogeny allow engraftment and long-term persistence of transplanted hematopoietic stem cells without the requirement for myeloablation or immunosuppression. To date, 21 in utero transplants have been reported. Success has been limited to 4 fetuses, all with immunodeficiency disorders. Despite this limited evidence of clinical efficacy, interest in stem cell transplantation has been gaining momentum, and clinical application is likely to increase. Parallel advances in prenatal diagnosis, fetal intervention, and hematopoietic stem cell technology have removed many of the practical, technical, and ethical obstacles to clinical application. This progress has been accompanied by an increase in the number of centers with both the stated interest and perceived expertise to develop clinical programs. However, there is currently limited consensus among investigators on many important issues, such as the mode or timing of in utero transplantation, the ideal source or dose of donor cells, estimation of maternal and fetal risks, appropriate candidate diseases for treatment, and important ethical considerations in counseling and therapy.

Aborted Fetus↗
PubMed1997

Transplantation of hematopoietic stem cells in utero.

Hematopoietic stem cell (HSC) transplantation in children and adults with congenital lymphohematopoietic disorders is limited by donor availability, graft failure, graft-versus-host disease (GVHD) and delayed immunological reconstitution. These problems may be circumvented by transplanting the patient before birth. Prenatal cellular therapy for the treatment of congenital diseases has tremendous theoretical appeal. Potential advantages of prenatal transplantation include: A) fetal immunologic immaturity and the potential for induction of donor-specific tolerance; B) available space in the developing bone marrow for engraftment of donor cells; C) the sterile, protective, fetal environment which provides isolation from environmental pathogens, and D) prevention of clinical manifestations of the disease. Normal hematopoietic and immunologic development during ontogeny creates a "window of opportunity" during which events favor the engraftment of transplanted allogeneic (and xenogeneic) HSC and their proliferation. This is a period in which the fetus is immunologically naive and thus incapable of rejecting the foreign HSC, and the expanding bone marrow spaces allow homing and engraftment of HSC without the need for myeloablation. Experiments in sheep have established the optimal age of the recipient, route of donor cell administration, sources of HSC, and other parameters necessary for the successful engraftment and long-term expression of donor HSC. In preclinical studies, transplantation of CD34-enriched or highly purified populations of human adult bone marrow cells in utero resulted in the long-term engraftment and expression of donor HSC without graft failure and GVHD. The strategies developed in allogeneic and xenogeneic fetal sheep models were used to successfully treat human fetuses with X-linked recessive severe combined immunodeficiency.

Animals↗
PubMed1997

Intrapartum airway management for giant fetal neck masses: the EXIT (ex utero intrapartum treatment) procedure.

OBJECTIVE: Our goal was to review our experience with the EXIT (ex utero intrapartum treatment) procedure in the management of five cases with life-threatening fetal neck masses. STUDY DESIGN: We present a retrospective review of prenatal presentation and course, diagnostic accuracy of imaging studies, intraoperative management, complications, and outcomes. RESULTS: Polyhydramnios was the initial presenting symptom in three of five fetuses with a mean gestational age of 25 +/- 6 weeks. Preterm labor occurred in two patients. Fetal magnetic resonance imaging provided accurate diagnosis in all four cases whereas conventional ultrasonography led to the diagnosis in four of five cases. The mean duration of EXIT was 28 +/- 22 minutes. The mean venous cord blood gas values were pH 7.22 +/- 0.05, PCO2 61 +/- 11 mm Hg, and PO2 42 +/- 8 mm Hg. In four of five cases an airway was successfully secured. CONCLUSIONS: The EXIT procedure provides up to 1 hour of good uteroplacental support and is the procedure of choice to secure an airway in the fetus with a giant neck mass.

Airway Obstruction↗
PubMed1997

A new intraluminal antigastroesophageal reflux procedure in baboons.

BACKGROUND: A new endoscopic intraluminal procedure (valvuloplasty) was designed to provide a simple, easy approach to the cardia and to correct and augment any mechanical deficiencies present. The feasibility, durability, and efficacy of this procedure was tested in 13 baboons. METHODS: The valvuloplasty consisted of an intussusception of the gastroesophageal junction into the stomach to create a nipple-type valve. The configuration of the valve was maintained with eight staples and stability was aided by an intramural injection of sodium morrhuate. Gastrointestinal endoscopy and esophageal manometry were performed before and after the procedure. Competency was determined as the intragastric pressure (yield pressure) and volume (yield volume) needed to result in equalization of gastric and esophageal pressure while distending the stomach with water. Comparisons were made with a group of normal baboons (n = 10). RESULTS: All baboons had a normal eating pattern with none showing any evidence of vomiting or regurgitation. Endoscopic circumferential integrity of the valves was 86% at 6 months. The median lower esophageal sphincter length in the valvuloplasty group was significantly (p < 0.01) increased after the procedure from 21 mm to 30 mm. The median yield pressure (22.1 mm Hg) and yield volume (1,525 ml) of the valvuloplasty group was significantly (p < 0.01) greater than the controls (14 mm Hg and 859 ml). CONCLUSIONS: The valvuloplasty is simple, safe, and durable. It augments mechanical function of the cardia and improves competency.

Animals↗
PubMed1997

Cellular therapy.

From a clinical perspective, prenatal transplantation has tremendous potential to broaden the current indications for reconstitution therapy and to offer a safe, efficacious, and cost-effective alternative to conventional postnatal BMT for many congenital hematopoietic diseases. Experimental work and limited clinical experience offer hope for the future. The primary experimental challenges will be to manipulate the biology of in utero HSC transplantation so that the approach will be safe and broadly applicable. This will require strategies to improve engraftment; use alternative sources of cells safely and effectively; and develop techniques for procurement, ex vivo expansion, and tissue banking of safe donor cells. The clinical challenges in the future will be to identify recipients most likely to benefit from the approach, and to define further the clinical and ethical guidelines for its application.

Female↗