Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Transforming growth factor beta 1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane.

Application of TGF beta 1 (10-100 ng) to the chicken chorioallantoic membrane (CAM) for 72 h resulted in a dose-dependent, gross angiogenic response. The vascular effects induced by TGF beta 1 were qualitatively different than those induced by maximal doses of basic FGF (bFGF) (500 ng). While TGF beta 1 induced the formation of large blood vessels by 72 h, bFGF induced primarily small blood vessels. Histologic analysis revealed that TGF beta 1 stimulated pleiotropic cellular responses in the CAM. Increases in fibroblast and epithelial cell density in the area of TGF beta 1 delivery were observed as early as 4 h after TGF beta 1 treatment. By 8 h, these cell types also demonstrated altered morphology and marked inhibition of proliferation as evidenced by 3H-thymidine labeling. Thus, the TGF beta 1-stimulated accumulation of these cell types was the result of cellular chemotaxis from peripheral areas into the area of TGF beta 1 delivery. Microscopic angiogenesis in the form of capillary sprouts and increased endothelial cell density first became evident at 16 h. By 24 h, capillary cords appeared within the mesenchyme of the CAM, extending towards the point of TGF beta 1 delivery. 3H-thymidine labeling revealed that the growth of these capillary cords was due to endothelial cell proliferation. Finally, perivascular mononuclear inflammation did not become evident until 48 h of treatment, and its presence correlated spatially and temporally with the gross and histological remodelling of newly formed capillary cords into larger blood vessels. In summary, these data suggest that, in the chicken CAM, TGF beta 1 initiates a sequence of cellular responses that results in growth inhibition, cellular accumulation through migration, and microvascular angiogenesis.

Engineering an "infectious" T(reg) biomimetic through chemoselective tethering of TGF-ß1 to PEG brush surfaces.

Modulation of immunological responses to allografts following transplantation is of pivotal importance to improving graft outcome and duration. Of the many approaches, harnessing the dominant tolerance induced by regulatory T cells (Treg) holds tremendous promise. Recent studies have highlighted the unique potency of cell surface-bound TGF-ß1 on Treg for promoting infectious tolerance, i.e. to confer suppressive capacity from one cell to another. To mimic this characteristic, TGF-ß1 was chemoselectively tethered to inert and viable polymer grafting platforms using Staudinger ligation. We report the synthesis and functional characterization of these engineered TGF-ß1 surfaces. Inert beads tethered with TGF-ß1 were capable of efficiently converting naïve CD4(+) CD62L(hi) T cells to functional Treg. Concordantly, translation of conjugation scheme from inert surfaces to viable cells also led to efficient generation of functional Treg. Further, the capacity of these platforms to generate antigen-specific Treg was demonstrated. These findings illustrate the unique faculty of tethered TGF-ß1 biomaterial platforms to function as an "infectious" Treg and provide a compelling approach for generating tolerogenic microenvironments for allograft transplantation.

Tyrosine inhibits intestinal-phase gastric-acid secretion.

BACKGROUND: Intestinal infusion of leucine augments the maximal gastric-acid secretory response to pentagastrin and serves as a bioassay for intestinal-phase acid secretion. Another action of leucine is to decrease tyrosine flux into neural tissues. We hypothesized that the mechanism of leucine-stimulated acid secretion involves its ability to alter tyrosine absorption. METHODS: We administered tyrosine intrajejunally and intravenously to anesthetized, vagotomized rats during maximal pentagastrin stimulation and measured acid output in response to intrajejunal infusion of leucine. RESULTS: Intrajejunal tyrosine produced a dose-dependent inhibition of leucine-stimulated acid secretion but only mild effects on acid secretion in response to pentagastrin alone. Intravenous tyrosine infusions also decreased acid secretion in response to intestinal leucine but required much higher doses. CONCLUSIONS: Tyrosine exerts a specific inhibitory effect on leucine-stimulated acid secretion, which is mediated intraluminally. Because tyrosine is an important regulator of adrenergic nervous system activity, we speculate that the mechanism of leucine-stimulated acid secretion may involve modulation of the sympathetic nervous system, thereby affecting acid secretion.

Cystic neoplasms of the pancreas. A clinicopathologic study, including DNA flow cytometry.

OBJECTIVE: To review the classification, clinical behavior, and appropriate therapy for cystic neoplasms of the pancreas. We examined patient demographics, clinical parameters, preoperative imaging modalities, histologic findings, and tumor DNA content to determine which best predict outcome. DESIGN: Case series and survey of pathologic specimens. SETTING: Tertiary care center. PATIENTS: Twenty-two patients with cystic neoplasms of the pancreas treated at affiliates of Northwestern University Medical School, Chicago, Ill. MAIN OUTCOME MEASURES: Predictive value of preoperative testing, tumor DNA content, patient survival. RESULTS: In 20 patients undergoing computed tomographic scan, the tumor was visualized in every case. All other imaging studies evaluated were less likely to demonstrate the lesion. Eight of 10 patients with serous cystadenomas were alive with no evidence of disease at the time of this report; one patient was alive with local recurrence, and a second patient had died of unrelated causes. All patients with mucinous cystadenomas were alive with no evidence of disease. Three of seven patients with cystadenocarcinomas had aneuploid, high S-phase tumors, and one had a diploid, high S-phase tumor; all four died (mean survival, 4.8 months). Two patients with cystadenocarcinomas had diploid, low S-phase tumors; both were long-term survivors but died of their disease at 8.6 and 9.3 years. CONCLUSIONS: (1) Computed tomographic scan is the most valuable diagnostic imaging study for preoperative evaluation of these patients. (2) Precise preoperative determination of tumor type is not possible. (3) DNA flow cytometry may help identify patients with aggressive tumors who may benefit from adjuvant chemoradiation.

The stabilization of ferrous iron by a toxic beta-amyloid fragment and by an aluminum salt.

Aluminum is a recognized neurotoxin in dialysis encephalopathy and may also be implicated in the etiology of neurodegenerative disease, particularly Alzheimer's disease. Alzheimer's disease is suspected to be associated with oxidative stress, possibly due to the pro-oxidant properties of beta-amyloid present in the senile plaques. The underlying mechanism by which this occurs is not well understood although interactions between amyloid and iron have been proposed. The presence of low molecular weight iron compounds can stimulate free radical production in the brain. This study provides a possible explanation whereby both aluminum and beta-amyloid can potentiate free radical formation by stabilizing iron in its more damaging ferrous (Fe2+) form which can promote the Fenton reaction. The velocity, at which Fe2+ is spontaneously oxidized to Fe3+ at 37 degrees C in 20 mM Bis-Tris buffer at pH 5.8, was significantly slowed in the presence of aluminum salts. A parallel effect of prolongation of stability of soluble ferrous ion, was found in the presence of beta-amyloid fragment (25-35). Ascorbic acid, known to potentiate the pro-oxidant properties of iron, was also capable of markedly stabilizing ferrous ions.

Expression of and response to growth regulatory peptides by two human pancreatic carcinoma cell lines.

Two human pancreatic adenocarcinoma cell lines (PANC 1 and MIA PACA 2) were examined for expression of growth factors that could potentially play a role either in growth regulation of the tumor cells, or in cells that comprise the stromal elements of tumors. Both cell lines expressed transforming growth factor-alpha (TGF alpha), basic fibroblast growth factor (bFGF), c-sis (PDGF B chain), TGF beta 1, and TGF beta 3 mRNA by Northern blot analysis. Only the PANC 1 cells, however, expressed the TGF beta 2 transcript. TGF beta-like competing activity was found in medium conditioned by either cell line, but TGF alpha-like [epidermal growth factor (EGF)-competing] activity was not detected in the medium from either cell line by radioreceptor assay. TGF alpha and EGF caused concentration-dependent stimulation of soft agar colony growth of the MIA PACA 2 cells, while only TGF alpha caused a significant but less dramatic stimulation of soft agar growth of the PANC 1 cells. Insulin stimulated the anchorage-independent growth of MIA PACA 2 but not PANC 1 cells. Likewise, bFGF also caused a concentration-dependent stimulation of MIA PACA 2 but not PANC 1 growth in soft agar, and PDGF had no effect on the growth of either cell line. TGF beta had no inhibitory or stimulatory effect on soft agar colony growth of either the PANC 1 or the MIA PACA 2 cells, although both cell lines exhibited high affinity, saturable TGF beta binding sites, and TGF beta 1 was capable of autoinduction of TGF beta 1 mRNA expression in PANC 1 cells. The ability to continue to respond to positive growth regulatory factors coupled with the loss of responsiveness to negative growth factors may be important in the pathogenicity of these aggressive tumors.

Prospects for fetal gene therapy.

Advances in prenatal diagnosis and gene transfer technology have allowed consideration of prenatal gene therapy. A compelling argument can be made for this strategy in treating genetic diseases that are fatal in the prenatal or perinatal period. In other diseases, the fetal environment may offer unique biological advantages that favor a prenatal gene therapy strategy over treatment after birth. Although issues of safety and efficacy must be resolved before clinical application, the development of fetal gene therapy may become a new molecular therapeutic arm in the field of prenatal intervention.

Configuration of thiols dictates their ability to promote iron-induced reactive oxygen species generation.

Iron catalyzes the production of reactive oxygen species (ROS) through the Fenton reaction. The modification of this phenomenon in the presence of various thiol compounds that are nominally reducing agents has been studied. Using the synaptosomal/mitochondrial (P2) fraction of rat cerebral cortex as a biological source of reactive oxygen species (ROS) production, we studied the influence of four compounds, glutathione (GSH), cysteine, N-acetyl-cysteine (NAC), and homocysteine on iron-induced ROS production. None of the thiol compounds alone, at the concentrations used, affected the basal rate of ROS production in the P2 fraction. GSH, homocysteine and NAC did not alter Fe-induced ROS generation, while cysteine greatly potentiated ROS formation. Measurement of the rate of ROS production in the presence of varying concentrations of cysteine together with 20 microM ferrous iron revealed a dose-response relationship. The mechanism whereby free cysteine, but not the cysteine-containing peptide GSH, homocysteine or NAC with a blocked amino group, exacerbates the pro-oxidant properties of ferrous iron probably involves formation of a complex between iron, a sulfhydryl and a free carboxyl residue located at a critical distance from the -SH group. Cysteine-iron interactions may, in part, account for the excessive toxicity of free cysteine in contrast to GSH and NAC.

BAPS Prize--1997. Fetal gene therapy: efficacy, toxicity, and immunologic effects of early gestation recombinant adenovirus. British Association of Paediatric Surgeons.

BACKGROUND/PURPOSE: Advancements in gene transfer technology and prenatal diagnosis have allowed investigators to consider an in utero gene therapy approach for fatal genetic diseases. The authors sought to develop fetoscopic techniques for gene delivery and investigate the efficacy and safety of recombinant adenoviral vectors in the fetus. METHODS: Fetal sheep between 60 and 130 days' gestation (dGA) underwent either fetoscopic intratracheal injection or umbilical vein (UV) injection of recombinant adenovirus, AdCMVlacZ. At death, fetal organs were examined for beta-galactosidase expression, histopathology, and CD45 immunostaining. Fetal serum was compared with preimmune serum for transaminase levels and the presence of antiadenoviral neutralizing antibodies. RESULTS: Fetoscopic intratracheal delivery of AdCMVlacZ in late-gestation sheep fetuses resulted in efficient alveolar gene transfer, but, antiadenoviral immunologic reactions limited the longevity of transgene expression to 14 days. This prompted an examination of whether early gestational exposure could induce tolerance in the fetus to adenoviral and transgene antigens. AdCMVlacZ (1 x 10(11) particles) was injected via UV into fetuses at 60 dGA. Within 3 days, beta-galactosidase expression was localized to the fetal liver, adrenal glands, kidneys, and endocardium. Although adrenal expression was nearly constant over 28 days, expression in fetal liver disappeared within 14 to 28 days. Loss of hepatic expression did not appear to be immune mediated because there was no evidence of hepatic inflammation or appearance of antiadenoviral neutralizing antibodies. Fetuses injected with AdCMVlacZ at 60 dGA were reinjected with 1 x 10(13) particles at 125 dGA and antiadenoviral humoral immune responses were recorded. Despite early-gestation adenovirus injection, fetuses still responded to the late-gestation adenoviral exposure, developing antiadenoviral neutralizing antibodies similar to control fetuses. CONCLUSIONS: The authors developed fetoscopic access for pulmonary adenovirus delivery in late-gestation sheep. Although initial alveolar transduction was highly efficient, antiadenoviral immune responses limited the duration of transgene expression. In contrast, early-gestation adenoviral delivery did not elicit antiadenoviral immune responses despite achieving efficient transduction of many fetal tissues. Furthermore, early-gestation adenovirus delivery did not affect late-gestation antiadenoviral immune responses. These findings suggest that the early-gestation sheep fetus is not amenable to adenoviral tolerance induction by UV injection and that it is incompetent of immunologic response to adenovirus. For the purposes of in utero gene therapy, recombinant adenovirus may be applied optimally to genetic diseases requiring transient in utero expression.

Fetoscopic gene therapy for congenital lung disease.

Fetal gene therapy offers the promise of cure for certain genetic diseases, like cystic fibrosis and surfactant protein B deficiency. The authors hypothesized that a fetoscopic approach could attain a high level of organ-specific gene transfer to the fetal lung late in gestation. To test this hypothesis the authors examined the efficacy, specificity, and toxicity of recombinant adenovirus-mediated transfer of the beta-galactosidase marker gene to the lung of late gestation fetal sheep using a fetoscopic technique. Twelve fetal sheep of 125 to 135 days' gestation (term, 145 days) underwent fetoscopic bronchoscopy and intratracheal administration of a replication-deficient adenoviral vector that transduces the beta-galactosidase marker gene. Escape of administered virus was prevented by the fetoscopic deployment of a detachable silicone balloon in the fetal trachea. All fetuses survived until being killed at 2 days after vector delivery for the histopathologic assessment of vector efficacy and specificity. Optimal beta-galactosidase transgene expression was observed at a viral titer of 2 x 10(12) particles per milliliter of administered volume. Expression was greatest in the distal pulmonary parenchyma, particularly in type II pneumocytes, and extended out to the pleura. There was no evidence of gene transfer in either the large conducting airways or in any other fetal organ. The authors have developed a minimally invasive technique for the specific pulmonary delivery of gene therapy vectors to the fetus with no associated acute toxicity. Gene transfer to the late gestation fetus for the treatment of congenital pulmonary disease may be feasible through fetoscopy.

Myofibroblast persistence in fetal sheep wounds is associated with scar formation.

The scarless repair capabilities of the fetus are influenced by the size of the wound and the gestational age of the fetus. Whereas small wounds heal scarlessly, large wounds in the same fetus heal with scar. Myofibroblasts are specialized fibroblasts that express alpha-smooth muscle actin (alpha-SMA), a contractile cytoskeletal protein. The authors hypothesized that small fetal wounds that heal scarlessly will have a relative absence of myofibroblasts, whereas large wounds that heal with scar will have abundant myofibroblasts. In this study, an incisional wound and four punch biopsy wounds of 2, 4, 6, and 10 mm diameter were placed on the backs of 60- to 90-day-gestation fetal sheep (term, 145 days). Fourteen days after wounding, the healed fetal wounds were harvested, the repair morphology was determined (scarless, transitional repair, or scar), and the expression of alpha-SMA was analyzed by immunohistochemistry. In the second part of this study the authors analyzed the temporal expression of alpha-SMA in fetal wounds at 1, 2, 3, and 7 days after wounding in 70-day-gestation fetal sheep. In the 14-day wounds, the authors found that alpha-SMA was not expressed in any incisional or 2-mm wound that healed scarlessly, but it was expressed in all wounds that healed with scar. Overall, alpha-SMA expression strongly correlated with increasing wound size (P < .005). Myofibroblasts were seen as early as 24 hours after wounding, and at 3 and 7 days all wounds showed strong expression of alpha-SMA. These results demonstrate that although myofibroblasts are induced early in fetal wound repair, by 14 days there is a notable lack of myofibroblasts in wounds that heal scarlessly and an abundance of myofibroblasts in those wounds that scar. By determining the factors that regulate the disappearance of the myofibroblast in scarless fetal wounds, the authors hope to gain new insights into the mechanisms of scarless fetal repair.

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.

BAPS prize-1999: Lung growth induced by prenatal tracheal occlusion and its modifying factors: a study in the rat model of congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Prenatal tracheal occlusion (TO) has been shown to accelerate lung growth in animal models and models of pulmonary hypoplasia. However, these models may not mimic early events in human congenital diaphragmatic hernia (CDH). The authors previously have developed a model of TO in the rat. The purpose of this study was to apply this technique to characterize TO-induced lung growth in the early onset nitrofen-induced model of CDH, and to address the clinically important questions of the effect of timing of TO and maternal infusion of terbutaline on TO-induced lung growth. METHODS: Left-sided CDH was induced in the fetuses of time-dated pregnant Sprague-Dawley rats by feeding 100 mg of nitrofen on day 9 of gestation. TO was performed via maternal laparotomy and hysterotomy at 19 days' gestation. At harvest (21.5 days' gestation), lungs from nitrofen-exposed fetuses without CDH (non-CDH), with CDH (CDH), and with CDH and TO (CDH-TO) were compared by analysis of wet and dry weight, DNA and protein content, and stereologic morphometry. A second study was performed to assess relative lung growth achieved by equal intervals of TO after "early" (19 days) versus "late" (20 days) gestational TO. Finally, the effect of maternal infusion of terbutaline, a commonly used tocolytic for fetal surgery, on TO-induced lung growth was analyzed. RESULTS: Analysis of lung growth showed consistent and significant lung growth in CDH-TO lungs. Lung growth after TO was proliferative and characterized by an increase in parenchymal volume as manifest by increased total saccular number and surface area and radial saccular count. Although visceral reduction was partially achieved, herniated liver was reduced incompletely. The majority of lung growth occurred during the latter half of the TO period. Early gestational age at TO and maternal terbutaline administration adversely influenced lung growth in CDH-TO fetuses. CONCLUSIONS: Prenatal TO induces dramatic lung growth in the early onset, nitrofen-induced rat model of CDH. TO is more effective later in gestation presumably because of the advanced stage of lung development and lung fluid production. This effect could be counterbalanced by the use of beta-mimetic tocolytic, which inhibits fetal lung fluid production late in gestation. Multiple factors including fetal lung fluid production and absorption, pharmacologic agents, space-occupying herniated viscera, and timing and duration of TO may be important clinical variables. The development of the rat model should facilitate further studies into the cellular and molecular mechanisms responsible for TO-induced lung growth.

Epidermal integrin expression is upregulated rapidly in human fetal wound repair.

BACKGROUND/PURPOSE: The fetus heals skin wounds rapidly and scarlessly. The mechanisms that mediate the rapid reepithelialization that is seen in this process are unknown. Integrins are a family of cell surface receptors that bind fibronectin, tenascin, collagen, and other extracellular matrix proteins that are deposited rapidly in fetal wounds. The authors hypothesized that epidermal integrin receptors specific for fibronectin and other wound matrix proteins are upregulated rapidly during human fetal repair. METHODS: To investigate the spatial and temporal expression of integrins in scarless fetal repair, fetal skin from six human abortuses (16 to 23 weeks' gestation) was transplanted subcutaneously into severe combined immunodeficient mice. After graft take, full-thickness incisional wounds were made in the grafts, and grafts were harvested at various time-points from 4 hours to 28 days after wounding. Integrin receptor protein expression was analyzed at each time-point using immunohistochemistry with monoclonal antibodies specific for the receptors that bind fibronectin, tenascin, collagen, and laminin (alpha5, alpha(v), beta6, alpha2, alpha3, alpha6, and beta4). RESULTS: In this model, wounded human fetal skin grafts reepithelialized rapidly (within 24 to 36 hours) and healed scarlessly. Within 4 hours of wounding, the grafts showed increased, suprabasal expression (alpha2, alpha3, alpha6, beta4) or neoexpression (alpha5, alpha(b), beta6) of integrins at the epidermal wound edge. This increased expression persisted until reepithelialization was complete. CONCLUSIONS: Early upregulation of integrins in fetal wounds may permit rapid keratinocyte migration and reepithelialization, and may be important in limiting the induction of inflammatory mediators and scar.

Wound size and gestational age modulate scar formation in fetal wound repair.

The early-gestation fetus heals incisional skin wounds rapidly and scarlessly. The morphology with which the fetus heals excisional skin wounds remains unclear. To characterize excisional fetal wound repair, and to determine whether there is a developmentally regulated wound-size threshold beyond which fetal skin heals with scar, the authors created excisional wounds in fetal lambs of varying gestational age. Time-mated pregnant ewes carrying 22 fetuses at 60 to 90 days' gestation (term, 145 days) underwent laparotomy and hysterotomy. An incisional wound and four circular, punch biopsy wounds of 2, 4, 6, and 10 mm in diameter were placed on the back of each fetal lamb and marked with India ink. The wounds were harvested at 14 days' postwounding and examined grossly and microscopically after serial sectioning and histological staining. Morphological features of all wounds were graded. By 14 days' postwounding all fetal wounds had healed completely. for lambs at each gestational age, increasing wound size was strongly associated with an increase in the frequency of scar. Also, as gestational age increased from 60 to 90 days' gestation the frequency of scarless repair decreased. By understanding the cellular and molecular processes that mediate scar formation with increasing wound size and advancing gestational age, the authors hope to gain further insight into the mechanisms of scarless fetal wound repair.

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.

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.

Hyaluronan receptor expression increases in fetal excisional skin wounds and correlates with fibroplasia.

BACKGROUND/PURPOSE: The midgestation fetus heals incisional skin wounds scarlessly, whereas large excisional wounds scar. High concentrations of hyaluronan (HA) are associated with scarless fetal as opposed to scar-forming adult wound repair. Because expression of the HA receptors, CD44 and RHAMM (Receptor for HA-Mediated Motility), has been associated with adult wound fibroplasia, the authors postulated that fetal excisional wounds would show increased expression of CD44 and RHAMM as compared with incisional wounds. METHODS: Two models of fetal wound healing were examined. Fetal skin from human abortuses was heterotransplanted subcutaneously into severe combined immunodeficient (SCID) mice. Fourteen days after grafting, incisional or 2-mm excisional wounds were created (n = 6 per time-point). In addition, incisional and excisional (6 to 10 mm) wounds (n = 5 per time-point) were created on the backs of 70- to 75-day fetal lambs (term, 145 days). Tissue from both models was harvested at sequential time-points after injury. Wounds were studied histologically for fibroplasia and assayed for their HA content. CD44 and RHAMM expression were analyzed by immunohistochemistry and immunoblotting. RESULTS: As expected, in both models, incisional wounds healed scarlessly, whereas excisional wounds showed fibroplasia. Incisional wounds of fetal lambs maintained a significantly higher HA content than excisional wounds 3 days after injury. Between 1 and 7 days in either human or sheep fetal wounds, immunostaining for CD44 and RHAMM markedly increased along the margins of excisional wounds as compared with incisional wounds and unwounded skin. Immunoblot analysis confirmed this increased HA receptor expression in both models. CONCLUSIONS: HA receptor expression increased in both human and sheep fetal excisional wounds and correlated with fibroplasia and a reduced HA content. The authors speculate that strategies to limit the expression or function of HA receptors during postnatal wound repair may modify the development of scar.

Neonatal auditory brainstem response thresholds to air- and bone-conducted clicks: 0 to 96 hours postpartum.

Auditory brainstem response (ABR) thresholds to air- and bone-conducted clicks were investigated in 40 full-term neonates. Subjects were divided into two groups of 20 according to postpartum age: less than 48 hours and between 49 and 96 hours. Mean ABR thresholds to air- and bone-conducted clicks for neonates less than 48 hours postpartum were 14.5 dB nHL (51.5 dB peak SPL) and 1.8 dB nHL (36.8 peak re: 1 microN), respectively, while those for neonates between 49 and 96 hours were 3.8 dB nHL (40.8 dB peak SPL) and 1.5 dB nHL (36.5 dB peak re: 1 microN), respectively. A significant difference was found between the two group mean ABR thresholds to air-conducted stimuli (p < .0001) but not for the bone-conducted stimuli (p < .8959). A statistically significant within-group difference was found between the ABR thresholds to air- and bone-conducted stimuli for only the neonates less than 48 hours of age (p < .0001). When the data was collapsed across groups, simple linear regression analyses revealed a statistically significant relation between postpartum age and ABR threshold to air-conducted stimuli (p < .0001) and a nonsignificant relation between postpartum age and ABR threshold to bone-conducted stimuli (p < .9744). These findings support the notion that some resolution of fluids and residuals in the middle ear occurs during the first 48 hours postpartum and that air-conducted stimuli are attenuated during that period. As such, a physiologic conductive deficit among the younger neonates is suggested.