Timothy M. Crombleholme

Fetal intervention in obstructive uropathy: Prognosticindicators and efficacy of intervention

Management of the fetus with bilateral hydronephrosis is controversial; ability to predict outcome and efficacy of prenatal intervention are unknown. We studied 40 fetuses referred for ultrasonography, examination of fetal urine, and possible therapy. We retrospectively assigned fetuses to a good prognosis group if fetal urine was hypotonic (sodium less than 100 mEq/L, chloride less than 90 mEq/L, osmolarity less than 210 mOsm/L) and there was no ultrasonographic evidence of dysplasia; we assigned fetuses to a poor prognosis group if even one criterion was abnormal. Survival was greater in the good prognosis group than in the poor prognosis group (81% vs 12.5%; 87% vs 30%, excluding abortions) (p less than 0.005). We then attempted to assess the efficacy of prenatal urinary decompression by comparing outcome within the good and poor prognosis groups. Survival with intervention was greater in both the good prognosis group and the poor prognosis group (89% vs 70% and 30% vs 0%). In 6 of the 8 survivors in the good prognosis group, severe oligohydramnios was reversed by decompression. We conclude the fetal urine electrolyte levels and ultrasonographic appear helpful in predicting residual fetal renal function and neonatal outcome and that prenatal decompression may prevent the development of fatal pulmonary hypoplasia.

Congenital cystic lung disease: contemporary antenatal and postnatal management

Congenital cystic lung disease comprises a broad spectrum of rare but clinically significant developmental abnormalities, including congenital pulmonary adenomatoid malformations, bronchopulmonary sequestrations, bronchogenic cysts, and congenital lobar emphysema that result from perturbations in lung and airway embryogenesis. As congenital lung lesions are now more commonly recognized antenatally, mothers require accurate prenatal counseling and appropriate perinatal management. In light of long-term complications of infection and malignancy, there is growing consensus that infants with asymptomatic lesions should undergo elective excision of congenital pulmonary adenomatoid malformation (CPAM) or bronchopulmonary sequestration (BPS). This review will focus on advancements and current practice in the diagnosis and management of CPAM and BPS, identifying aspects of the literature that are confusing or controversial. Although our knowledge and pre- and postnatal management of lung lesions will continue to evolve and improve, there is a compelling need for a unified clinical and pathological classification system that creates a common platform for discussion, clinical management, and research.

Adenoviral mediated gene transfer of PDGF-B enhances wound healing in type I and type II diabetic wounds

We have shown that the genetically diabetic mouse (C57BLKS/J‐m+/+Leprdb) has a wound healing and neovascularization deficit associated with an inability to recruit endothelial precursor cells (EPCs) to the wound. This may account for a fundamental mechanism in impaired diabetic wound healing. We hypothesized that the adenoviral mediated overexpression of platelet‐derived growth factor‐B (PDGF‐B) would enhance wound healing, improve neovascularization, and recruit EPCs to the epithelial wound in three diabetic mouse models. Eight‐mm full‐thickness flank wounds were made in db/db, nonobese NOD/Ltj, streptozotocin, and C57BLKS/J mice. Wounds were treated with either 1 × 108 PFU Ad‐PDGF‐B or Ad LacZ or phosphate buffered saline solution. Wounds harvested at seven days were analyzed for epithelial gap, blood vessel density, granulation tissue area, and EPCs per high powered field. All three diabetic models have a significant wound healing and neovascularization defect compared to C57BLKS/J controls. Adenoviral‐PDGF‐B treatment significantly enhanced epithelial gap closure in db/db, streptozotocin, and nonobese NOD/Ltj mice as compared to diabetic phosphate buffered saline solution or Ad LacZ controls. A similar increase in the formation of granulation tissue and vessel density was also observed. All three models had reduced levels of GATA‐2 positive EPCs in the wound bed that was corrected by the adenoviral mediated gene transfer of PDGF. EPC recruitment was positively correlated with neovascularization and wound healing. Three different diabetic models have a wound healing impairment and a decreased ability to recruit EPCs. The vulnerary effect of adenoviral mediated gene therapy with PDGF‐B significantly enhanced wound healing and neovascularization in diabetic wounds. The PDGF‐B mediated augmentation of EPC recruitment to the wound bed may be a fundamental mechanism of these results.

Studies in fetal wound healing. IV. Hyaluronic acid-stimulating activity distinguishes fetal wound fluid from adult wound fluid.

Recent clinical and experimental evidence suggests that the fetus responds to injury in a fashion fundamentally different from that of the adult. Our initial experience with human open fetal surgery reinforces experimental observations that the fetal wounds heal without the scarring, inflammation, and contraction that often accompany adult wounds. In this study we examine fetal wound fluid in an attempt to elucidate the control mechanisms that endow the fetus with unique healing properties. The extracellular matrix of fetal wounds is rich in hyaluronic acid, a glycosaminoglycan found in high concentrations whenever there is tissue proliferation, regeneration, and repair. We establish that wound fluid from the fetus contains high levels of hyaluronic acid-stimulating activity that may underlie the elevated deposition of hyaluronic acid in the fetal wound matrix. In contrast there was no hyaluronic acid-stimulating activity present in adult wound fluid. Hyaluronic acid, in turn, fosters an extracellular environment permissive for cell motility and proliferation that may account for the unique properties observed in fetal wound healing.

Studies in fetal wound healing. VII, Fetal wound healing may be modulated by hyaluronic acid stimulating activity in amniotic fluid

Fetal wound healing occurs rapidly and without inflammation, fibrosis, or scar formation. It is a process fundamentally different from adult wound healing. The mechanisms that underlie such unique healing properties are unknown. However, hyaluronic acid, a glycosaminoglycan component of the extracellular matrix, is prominent throughout the course of fetal wound healing, and is thought to play a major role in the healing process. Amniotic fluid contains high levels of hyaluronic acid. Amniotic fluid also contains a number of potent growth factors that are critical for fetal development. In this report, a new factor in amniotic fluid that stimulates deposition of hyaluronic acid is described. This activity is measured in an in vitro assay system in which cultured fibrosarcoma cells are used as indicator cells. Amniotic fluid thus provides two separate mechanisms for the deposition of hyaluronic acid. One is by exogenous application directly onto fetal skin wounds; the second is by providing a factor to increase the production of hyaluronic acid endogenously, by stimulating cells around the wound site. The resulting hyaluronic acid-rich area may support the ability of the fetal wound to heal with its unique properties.

Regenerative versus reparative healing in tendon: a study of biomechanical and histological properties in fetal sheep.

AbstractPrevious studies have shown fetal tissues heal in a regenerative fashion without scar formation. The objective of this study is to compare the healing properties of adult and fetal tendons. Time-mated pregnant ewes at 80–85 days of gestation were utilized. A partial, midsubstance tenotomy was performed in the lateral extensor fetal tendons, and analogous tenotomies were created in the maternal limbs. One week after injury, the fetal and adult animals were sacrificed, and tendons were histologically and mechanically evaluated. Immunohistochemical staining for transforming growth factor beta isoform 1 (TGF-β1) was performed. Histologically, a gap with granulation tissue and inflammatory cells was visible in the site of wounding in the adult tendons. In the fetal tendons, no abnormalities were noted in the wound, with reconstitution of collagen architecture. TGF-β1 expression was low in fetal but upregulated in the adult wounds. No significant differences were found in the biomechanical properties between groups. We identified regenerative healing properties in injured fetal tendon, while adult tendon tissue healed reparatively with scar formation. Fetal tendons demonstrated a limited recovery of mechanical properties after injury that was no better than that of the adult tendons at seven days. A better understanding of the mechanisms of fetal healing may lead to novel therapeutic strategies in the clinical setting.

Clinical protocol: Phase I trial to evaluate the safety of H5.020CMV.PDGF-B for the treatment of a diabetic insensate foot ulcer.

Most patients with chronic wounds fail to heal in a reasonable period of time. Despite considerable advances in elucidating the molecular basis of wound repair, attempts at developing new therapies have been disappointing. In fact, in the few studies where cytokine growth factors have been efficacious, their effect has been dramatically less than would have been predicted from animal studies. We hypothesize that platelet‐derived growth factor‐BB, a growth factor associated with wound healing, when produced in large quantities within the wound bed due to adenovirus mediated gene overexpression by the cells of the wound bed will dramatically enhance wound healing. Simply stated, we plan to insure the delivery of the growth factor by using gene therapy techniques so that cells locally involved in the wound healing process will temporarily increase their production of platelet‐derived growth factor‐BB. We present the first step in the series of human investigations to test this hypothesis which is a phase I clinical trial. Our proposed study is designed to assess local and systemic toxicity, and the feasibility of using the maximum tolerated dose of H5.020CMV.PDGF‐b associated with in vivo platelet‐derived growth factor‐BB gene transduction via an intraulcer injection of H5.020CMV.PDGF‐b in patients with a diabetic insensate foot ulcer.

Salvage of a Fetus with Congenital High Airway Obstruction Syndrome by ex utero Intrapartum Treatment (EXIT) Procedure

A fetus with congenital high airway obstruction syndrome (CHAOS) due to complete tracheal atresia was referred at 31 weeks of gestation after 12 weeks of massive hydrops. The fetus was delivered by the ex utero intrapartum treatment procedure allowing sufficient time while on placental support for bronchoscopy to confirm tracheal atresia and tracheostomy to secure the airway. His postnatal course was complicated by severe capillary leak syndrome secondary to hydrops, diaphragmatic paralysis, tracheobronchial malacia, and the need for chronic ventilatory support. The infant’s tracheobronchial malacia resolved by 5 months of age and normal diaphragmatic function was restored at 9 months allowing him to be weaned from mechanical ventilation. He underwent tracheal reconstruction at 17 months of age. At follow up at 32 months of age he has a patent airway and is the first long-term survivor with CHAOS.

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.

Adenoviral-mediated gene transfer in wound healing

The application of gene transfer strategies to wound healing is not an obvious use of this technology until one considers the important role of cytokines and growth factors in the normal wound healing response. Several gene transfer strategies have been proposed, from in vitro retroviral‐mediated gene transfer with autologous transplantation, to in vivo plasmid based gene transfer as retroviral gene transfer. The limitations of these approaches have been efficiency of gene transfer, transgene expression and biologic response. Adenoviral‐mediated gene transfer in wound healing is a relatively new application of this vector. The advantage of the adenovirus as a gene transfer vector lies in its ability to transduce nondividing cells of all types at very high efficiency without integration into the host cells genome. The disadvantage of adenovirus as a vector is the relatively short duration of transgene expression and the inflammatory response it elicits. In the setting of wound healing brief duration of high levels of transgene may be all that is necessary to favorably influence wound healing. Secondly, as wound healing is fundamentally an inflammatory response, the inflammation elicited by the adenovirus may not be detrimental as long as the transgene is a growth factor with significant vulnerary effects such as platelet‐derived growth factor‐B. This review summarizes the current state of adenoviral‐mediated gene transfer in experimental models of impaired wound healing which have laid the groundwork for proposed phase I clinical trials of adenoviral‐mediated gene transfer of platelet‐derived growth factor‐B in chronic venous leg ulcers and chronic nonhealing diabetic foot ulcers. Adenoviral‐mediated gene transfer is a useful tool in the study of the role of specific cytokines and growth factors in normal and impaired wound healing. Adenoviral‐mediated gene transfer may hold significant promise for clinical application as a means of efficient growth factor delivery in correcting impaired wound healing.