William H. Peranteau

IL-10 Overexpression Decreases Inflammatory Mediators and Promotes Regenerative Healing in an Adult Model of Scar Formation

Adult wound healing is characterized by an exuberant inflammatory response and scar formation. In contrast, scarless fetal wound healing has diminished inflammation, a lack of fibroplasia, and restoration of normal architecture. We have previously shown that fetal wounds produce less inflammatory cytokines, and the absence of IL-10, an anti-inflammatory cytokine, results in fetal scar formation. We hypothesized that increased IL-10 would decrease inflammation and create an environment conducive for regenerative healing in the adult. To test this hypothesis, a lentiviral vector expressing IL-10 and green fluorescent protein (GFP) (Lenti-IL-10) or GFP alone (Lenti-GFP) was injected at the wound site 48 hours before wounding. We found that both Lenti-IL-10 and Lenti-GFP were expressed in the wounds at 1 and 3 days post wounding. At 3 days, Lenti-IL-10-treated wounds demonstrated decreased inflammation and decreased quantities of all proinflammatory mediators analyzed with statistically different levels of IL-6, monocyte chemoattractant protein-1, and heat-shock protein 47. At 3 weeks, Lenti-GFP wounds demonstrated scar formation. In contrast, wounds injected with Lenti-IL-10 demonstrated decreased inflammation, a lack of abnormal collagen deposition, and restoration of normal dermal architecture. We conclude that lentivirus-mediated overexpression of IL-10 decreases the inflammatory response to injury, creating an environment conducive for regenerative adult wound healing.

Effect of Maternal Betamethasone Administration on Prenatal Congenital Cystic Adenomatoid Malformation Growth and Fetal Survival

Objective: To evaluate the effect of prenatal steroid treatment on the growth of congenital cystic adenomatoid malformations (CCAM) and survival in affected fetuses not amenable to other percutaneous ultrasound-guided prenatal interventions. Methods: A retrospective review of patients with a CCAM or hybrid lesion treated with two maternal prenatal betamethasone injections was performed. Patients receiving cyst aspiration or thoracoamniotic shunting at the time of or after steroid administration were excluded. Growth rates and survival data were compared to historical non-steroid treated controls. Results: Eleven patients were treated with prenatal steroids (10 microcystic and 1 macrocystic). Survival was 100% in fetuses with hydrops (5/5) or a CCAM volume ratio (CVR) >1.6 (7/7) at the time of steroid administration. This compares to a mortality of 100 and 56.2% respectively in historical non-treated controls. Resolution of hydrops was seen in 80% (4/5) of steroid-treated patients. CCAM growth rates were variable after steroid administration. However, when compared to historical data where CVR and CCAM volume have been documented to increase until 28 weeks’ gestation, the CVR and CCAM volume growth rates decreased in 72.73% and 50% of patients respectively from the time of steroid administration to 28 weeks’ gestation. Conclusions: In the fetus with a CCAM, the presence of hydrops fetalis or a CVR >1.6 is indicative of poor fetal outcome without prenatal intervention. The observed effect of antenatal steroid treatment on CCAM growth is variable, but its potential to improve survival in these high-risk groups is encouraging and warrants further controlled evaluations.

Fetal Myelomeningocele Repair: The Post-MOMS Experience at the Children's Hospital of Philadelphia

Background: Fetal myelomeningocele (fMMC) repair has become accepted as a standard of care option in selected circumstances. We reviewed our outcomes for fMMC repair from referral and evaluation through surgery, delivery and neonatal discharge. Material and Methods: All patients referred for potential fMMC repair were reviewed from January 1, 2011 through March 7, 2014. Maternal and neonatal data were collected on the 100 patients who underwent surgery. Results: 29% of those evaluated met the criteria and underwent fMMC repair (100 cases). The average gestational age was 21.9 weeks at evaluation and 23.4 weeks at fMMC repair. Complications included membrane separation (22.9%), preterm premature rupture of membranes (32.3%) and preterm labor (37.5%). Average gestational age at delivery was 34.3 weeks and 54.2% delivered at ≥35 weeks. The perinatal loss rate was 6.1% (2 intrauterine fetal demises and 4 neonatal demises); 90.8% of women delivered at the Childrens Hospital of Philadelphia and 3.4% received transfusions. With regard to the neonates, 2 received ventriculoperitoneal shunts prior to discharge; 71.1% of neonates had no evidence of hindbrain herniation on MRI. Of the 80 neonates evaluated, 55% were assigned a functional level of one or more better than the prenatal anatomic level. Conclusion: In an experienced program, maternal and neonatal outcomes for patients undergoing fMMC repair are comparable to results of the MOMS trial.

Mixed chimerism following in utero hematopoietic stem cell transplantation in murine models of hemoglobinopathy

OBJECTIVE Mixed hematopoietic chimerism after bone marrow transplantation can provide effective treatment for beta-thalassemia because of the selective advantage that exists for donor erythropoiesis. In utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism, particularly when a selective advantage exists for donor cells. To investigate the biology of IUHSCTx in hemoglobinopathies, we performed fully allogeneic IUHSCTx in murine models of beta-thalassemia (Thal) and sickle cell disease (SCD). MATERIALS AND METHODS We serially assessed and compared levels of mononuclear cell (MNC) and erythroid chimerism after IUHSCTx of either adult bone marrow (BM)- or fetal liver (FL)-derived allogeneic donor cells in the two hemoglobinopathy models, which differ significantly in their degree of anemia (Thal>>SCD) and red cell half-life (Thal<<SCD). RESULTS The mean level of donor MNC chimerism was higher for SCD and Thal chimeras receiving FL- compared to adult BM-derived donor cells and tended to increase over time in the FL recipients. Donor hemoglobin (Hb) levels also were higher in all groups receiving FL compared to adult BM. Donor Hb levels in chimeric Thal mice were significantly higher than those in SCD or wild-type mice. Hematologic parameters such as Hb, hematocrit (Hct), mean cell volume (MCV), membrane-associated denatured Hb, and the oxygen equilibration curve were improved in chimeric hemoglobinopathy mice. However, the improvement in Hb, Hct, and MCV was not sustained despite stable levels of donor leukocyte engraftment. CONCLUSION The severity of the hemoglobinopathy being treated and the source of donor cells may be important determinants of success in the treatment of hemoglobinopathy by IUHSCTx.

Murine bone marrow derived stromal progenitor cells fail to prevent or treat acute graft‐versus‐host disease

Human and murine stromal progenitor cells (SPCs) can suppress alloresponse in vitro, suggesting that SPCs may have clinical application toward prevention or treatment of graft‐versus‐host disease (GVHD). However, th eresults of in vivo studies have been conflicting. This study utilized an established murine model of acute GVHD to assess the ability of bone marrow derived murine SPCs (mSPCs) to prevent or treat GVHD. GVHD was established by transplantation of B6 bone marrow and spleen cells into lethally irradiated (900 cGy) B6 × BALB/c F1 recipients. mSPCs were administered using various dose and timing protocols designed to either prevent or treat GVHD. After transplantation, mice were monitored daily for weight and survival. Differences in symptom severity were compared using a clinical GVHD scoring system. All GVHD control mice died of lethal GVHD. All groups treated with mSPCs for the prevention of GVHD went on to develop clinical GVHD with no alteration of the disease course or severity compared to controls. Administration of mSPC after the development of GVHD failed to improve the disease course. We conclude that in this model, the ability of SPCs to suppress alloresponse in vitro does not correlate with in vivo prevention or treatment of acute GVHD.

Long-term transgene expression in cardiac and skeletal muscle following fetal administration of adenoviral or adeno-associated viral vectors in mice

In utero gene transfer may provide advantages for the correction of congenital genetic disorders. In the present study we compare the ability of adenovirus (AdCMVLacZ), and two serotypes of adeno‐associated virus (AAVCMVLacZ serotypes 2 and 2/5), to target cardiac and skeletal muscle after prenatal systemic or intramuscular injection in mice and assess the immune response to the vectors.

Haploidentical In Utero Hematopoietic Cell Transplantation Improves Phenotype and Can Induce Tolerance for Postnatal Same-Donor Transplants in the Canine Leukocyte Adhesion Deficiency Model

In the murine model, in utero hematopoietic cell transplantation (IUHCT) has been shown to achieve low levels of allogeneic chimerism and associated donor-specific tolerance permitting minimal conditioning postnatal hematopoietic stem cell transplantation (HSCT). In this pilot study, we investigated IUHCT in the canine leukocyte adhesion deficiency (CLAD) model. Haploidentical IUHCT resulted in stable low-level donor cell chimerism in all dogs that could be analyzed by sensitive detection methodology (4 of 10) through 18 months of follow-up. In the 2 CLAD recipients, low-level chimerism resulted in amelioration and complete reversal of the CLAD phenotype, respectively. Six recipients of IUHCT (5 carriers and 1 CLAD) subsequently received postnatal HSCT from the same haploidentical prenatal donor after minimal conditioning with busulfan 10 mg/kg. Chimerism in 2 of 5 CLAD carriers that underwent HSCT increased from < 1% pre-HSCT to sustained levels of 35% to 45%. Control animals undergoing postnatal haploidentical HSCT without IUHCT had no detectable donor chimerism. These results demonstrate that haploidentical IUHCT in the CLAD model can result in low-level donor chimerism that can prevent the lethal phenotype in CLAD dogs, and can result in donor-specific tolerance that can facilitate postnatal minimal conditioning HSCT.

Postnatal Chest Wall Deformities after Fetal Thoracoamniotic Shunting for Congenital Cystic Adenomatoid Malformation

Objectives: Large macrocystic congenital cystic adenomatoid malformations (CCAMs) can be treated with thoracoamniotic (TA) shunting to reduce CCAM volume. Two CCAM fetuses treated with TA shunt had postnatal radiographic rib deformities. Study Design: Retrospective review of prenatal TA shunting for large macrocystic CCAMs evaluated for the presence of rib deformities. Comparison groups not eligible for TA shunting included nonshunted CCAMs resected postnatally (group A) and size-matched nonshunted CCAMs resected postnatally (group B). Results: Chest wall abnormalities were identified in 77% of newborns ranging from severe concavity and fractures (in two fetuses shunted at 18 and 20 weeks of gestation) to rib thinning compared to comparison groups A and B. The severity of chest wall deformity correlated with earlier gestational age at shunting. Conclusions: TA shunting at less than 21 weeks of gestational age may result in postnatal chest wall deformity. This observation should be discussed during counseling for this procedure.

Prenatal course and postnatal management of peripheral bronchial atresia: Association with congenital cystic adenomatoid malformation of the lung

Objective: Peripheral bronchial atresia (PBA), a newly identified fetal lung lesion, is often asymptomatic and managed nonoperatively. However, recent studies suggest that bronchial atresia plays a role in the etiology of microcystic maldevelopment present in congenital cystic adenomatoid malformations (CCAM) which require resection to decrease the risk of infection, pneumothorax and malignant degeneration. The purpose of this study was to evaluate the prenatal radiographic and postnatal computed tomography (CT) scan/pathology findings with attention to the pathologic diagnosis of microcystic maldevelopment in infants with the presumptive diagnosis of PBA. Methods: A retrospective review of prenatal and postnatal records of patients diagnosed with fetal lung lesions was performed. Two groups of patients were identified: (1) patients diagnosed with PBA on postnatal CT scan (n = 16), and (2) patients with the pathologically confirmed diagnosis of PBA independent of postnatal CT findings (n = 23). Results: Prenatal ultrasound diagnosis of these lesions included CCAMs, hybrid lesions, bronchopulmonary sequestrations and bronchial atresia. Eleven of the 16 patients in group 1 with the postnatal radiologic diagnosis of PBA underwent surgical resection, 6 of which were found to have microcystic changes consistent with CCAM. Evaluation of the 23 patients in group 2 with pathologically confirmed PBA identified 65% that had evidence of microcystic maldevelopment consistent with the small cyst type of CCAM. Conclusion: Radiographically diagnosed PBA as well as pathologically confirmed PBA is frequently associated with microcystic changes consistent with the small cyst type of CCAM. Thus, operative management should be considered for PBA to decrease CCAM-associated risks.

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have recently shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this study we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8–10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.