Biomedical subjects

T M Crombleholme

Publications and source records attributed to T M Crombleholme.

At least 19 recordsLinked to original sources

PubMed2001

Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Adenoviridae↗

PubMed2001

Alpha5 and alpha2 integrin gene transfers mimic the PDGF-B-induced transformed phenotype of fibroblasts in human skin.

Platelet-derived growth factor (PDGF)-B is a proto-oncogene capable of transforming fibroblasts. Using adenoviral vectors, we tested whether endogenous PDGF-B expression in human skin xenotransplants leads to changes in the expression of alpha5 and alpha2 integrin subunits and whether integrin overexpression leads to PDGF-related changes in the skin. In vitro, transduction of fibroblasts with PDGF-B or the integrin alpha5 subunit stimulated multilayered growth and spindle-type morphology, both markers of mesenchymal cell transformation. In vivo, PDGF-B transduction of the human dermis was associated with up-regulation of collagen and fibronectin synthesis, increases in alpha5 and alpha2 integrin subunit expression, vessel formation, and proliferation of fibroblasts, keratinocytes, and pericytes. A similar stromal response was induced when alpha5 and alpha2 integrin subunits were overexpressed in the human dermis, suggesting that integrins play a major role in the induction of a transformed phenotype of fibroblasts by PDGF-B.

Antigens, CD↗

PubMed2001

Synthetic hybrid grafts for craniofacial reconstruction: sustained gene delivery using a calcium phosphate bone mineral substitute.

These experiments were performed to evaluate the efficacy of a biocompatible bone cement, Norian CRS, engineered as a hybrid graft for simultaneous bone matrix reconstruction and sustained, site-directed gene transfer using an adenoviral vector. Norian CRS was cured ex vivo by mixing a calcium source powder with a phosphate source solution to form a paste. To 1.0 ml of the cement was added 50 microl of a solution containing 1 x 10(8) plaque-forming units of a replication-deficient adenoviral vector containing a bacterial beta-galactosidase reporter gene (AdLacZ). In vitro, fragments of the hybrid Norian-AdLacZ construct were placed into 12-microm-pore culture plate inserts and cocultured with human fibroblasts. The same insert was transferred to a new well of fibroblasts every 48 hours for 30 days, and, after allowing 72 hours for gene expression, fibroblasts were examined for transgene expression by 5 bromo-4-chloro-3-indoyl-beta-D-galactosidase (X-gal) staining. In vivo, the Norian-AdLacZ hybrid was implanted into 10-mm frontal bone defects in 3-week-old piglets. The implant sites were harvested after 5 days and were examined for transgene expression by X-gal staining. X-gal staining of fibroblasts incubated with the hybrid Norian-AdLacZ construct was observed throughout the 30-day period. Transgene expression was also observed about the periphery of the calvarial defects treated with hybrid Norian-AdLacZ constructs. Thus, adenoviral vectors may be incorporated successfully into a synthetic calcium phosphate bone mineral substitute to provide effective, sustained local gene delivery.

Adenoviridae↗

PubMed2000

Diminished interleukin 6 (IL-6) production during scarless human fetal wound repair.

Fetal wound healing is characterized by minimal inflammation and scarless repair. IL-6 stimulates inflammation in postnatal wound healing. We hypothesized that fetal skin has a diminished IL-6 response and that exogenous IL-6 will result in scar formation. Human adult or fetal skin was placed subcutaneously in SCID mice and incisionally wounded. Wounds were excised after 4, 12, 24 or 72 h for IL-6 mRNA quantification by RT-PCR. In other grafts, 5 microgram of IL-6 was injected at wounding and then harvested at 7 days for analysis of scar formation. IL-6 production was examined in primary cultures of human fetal or adult dermal fibroblasts incubated for 8 h with 0, 0.1, 1 or 10 ng/ml of PDGF-BB. IL-6 mRNA was detected 4 h after wounding in fetal and adult wounds, but by 12 h there was no IL-6 mRNA in the fetal wounds. Adult wounds had IL-6 mRNA persisting to 72 h. IL-6 administration to fetal wounds resulted in scar formation. Fetal fibroblasts produced less IL-6 protein and mRNA at all points examined (P<0.01 vs adult). Diminished production of inflammatory cytokines such as IL-6 may be responsible for the lack of inflammation seen during fetal wound healing. Diminished inflammation may provide a permissive environment for scarless wound healing.

Adult↗

PubMed2000

FGF2-Targeted adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation.

Gene therapy has yet to achieve reproducible clinical efficacy, due to inadequate gene delivery, inadequate gene expression, or dose-limiting toxicity. We have developed a gene therapy technology for tissue repair and regeneration that employs a structural matrix for DNA delivery. The matrix holds the DNA vector at the treatment site and provides a scaffolding for in-growth and accumulation of repair cells and efficient DNA transfection. We now report, for the first time, matrix-mediated delivery of targeted DNA vectors for soft tissue repair. A collagen matrix was used to deliver an adenoviral vector encoding platelet-derived growth factor-B (AdPDGF-B), resulting in efficient transgene expression in vitro and in vivo. Increases in the overall levels of expression and in the relative amounts of secreted PDGF-BB were achieved when AdPDGF-B was conjugated to fibroblast growth factor (FGF2) such that the virus was targeted for cellular uptake via FGF receptors. Matrix-mediated delivery of AdPDGF-B enhanced wound healing responses in vivo, and FGF2 targeting generated effects comparable to nontargeted vectors at significantly lower doses. Therefore, matrix-mediated delivery in combination with FGF2 targeting overcomes some of the safety and efficacy limitations of current gene therapy strategies and is an attractive therapeutic approach for tissue repair and regeneration.

Animals↗

PubMed2000

Human melanoma progression in skin reconstructs : biological significance of bFGF.

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

Basement Membrane↗

PubMed2000

Echocardiographic evaluation of the fetus with congenital cystic adenomatoid malformation.

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

Case-Control Studies↗

PubMed2000

Superior mesenteric venous thrombosis in malrotation with chronic volvulus.

Malrotation can be difficult to diagnose after the newborn period because of intermittent symptoms and vague clinical findings, but malrotation with midgut volvulus is usually quite striking in its presentation. Early diagnosis and surgical treatment are essential to prevent acute ischemic infarction of the bowel, although chronic complications are rare. The authors present an unusual case of mesenteric venous thrombosis secondary to chronic midgut volvulus. A 13-year-old girl presented with an 11-year history of recurrent bouts of abdominal pain evaluated at 3 other institutions without a diagnosis. At the referring hospital, an episode of bilious emesis associated with abdominal pain prompted a computerized tomography scan of the abdomen. This showed a calcified thrombus within the superior mesenteric vein (SMV). At laparotomy, malrotation with chronic 270 degree volvulus was found with evidence of mesenteric venous hypertension. Segmental occlusion was documented on magnetic resonance angiography. SMV thrombosis is an unusual complication of malrotation with chronic midgut volvulus.

Adolescent↗

PubMed2000

Fetal wound repair results in scar formation in interleukin-10-deficient mice in a syngeneic murine model of scarless fetal wound repair.

BACKGROUND: Fetal dermal wound healing is characterized by minimal inflammation, restoration of normal dermal architecture, and scarless repair. The authors have shown that proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) are diminished during fetal wound repair. Interleukin-10 (IL-10) is an antiinflammatory cytokine that decreases production of IL-6 and IL-8. The authors hypothesized that diminished IL-6 and IL-8 and minimal inflammation may be caused by IL-10. METHODS: To test this hypothesis, the authors developed a new syngeneic murine model of fetal wound repair in which 15-day-gestation skin from either normal C57BL/6 or transgenic C57BL/6 IL-10 knockout mice was grafted to the back of the same strain adult mice. The grafts were incisionally wounded after 5 days, harvested at 1 week, and analyzed for inflammatory response and scar formation. RESULTS: Wounds in normal fetal skin grafts showed minimal inflammation and normal dermal reticular collagen pattern at the site of the wound, consistent with scarless repair. In contrast, wounds in IL-10 knockout fetal skin grafts showed significant inflammation and scar formation. CONCLUSIONS: Fetal skin grafts on adult syngeneic mice heal without inflammation or scar formation. The absence of IL-10 in fetal skin results in scar formation. Intrinsic lack of IL-10 may result in continued amplification of the inflammatory cytokine cascade, continued stimulation of fibroblasts, and abnormal collagen deposition. IL-10 is necessary for scarless wound repair to occur.

Animals↗

PubMed2000

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

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

Child Development↗

PubMed2000

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

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

Fetus↗

PubMed2000

Anesthesia for fetoscopic fetal surgery: twin reverse arterial perfusion sequence and twin-twin transfusions syndrome.

IMPLICATIONS: Twin reversed arterial perfusion sequence and twin-twin transfusion syndrome can be managed by fetoscopic fetal surgery. It is important to consider the fetal, uteroplacental, and maternal issues when choosing an anesthetic technique. We report on three patients with differing anesthetic issues using fetoscopic surgery for umbilical cord coagulation.

Adult↗

PubMed1999

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

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

Female↗

PubMed1999

Elevated platelet-derived growth factor-B in congenital cystic adenomatoid malformations requiring fetal resection.

BACKGROUND: During lung development, platelet-derived growth factor-BB (PDGF-BB) is maximal during the canalicular stage and decreases by the saccular stage. PDGF-BB stimulates lung growth by increasing cell proliferation. Fetal CCAMs have been shown to have an elevated proliferative index, but it is not known why some CCAMs rapidly enlarge in utero and cause fetal hydrops. The authors hypothesized that the high proliferative index and rapid enlargement of some fetal CCAMs may be caused by persistently elevated PDGF-BB production compared with normal fetal lung. METHODS: To test this hypothesis, tissue was obtained at the time of resection from two fetal CCAMs (22 weeks), three full-term CCAMs, and three normal fetal lungs (21 to 22 weeks). PDGF-BB production by fetal CCAMs was compared with normal age-matched fetal lung using immunohistochemistry, reverse transcriptionase-polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: CCAMs resulting in fetal hydrops and requiring fetal resection had strong mesenchymal immunostaining for PDGF-BB next to epithelial lined cysts, increased PDGF-B gene expression by RT-PCR, and elevated PDGF-BB protein by Western blot, compared with normal age-matched fetal lung. Term CCAMs had minimal PDGF-BB staining, PDGF-B gene expression, and PDGF-BB protein production. CONCLUSIONS: CCAMs that grew rapidly and progressed to hydrops, requiring in utero resection, demonstrated increased mesenchymal PDGF-B gene expression and PDGF-BB protein production compared with age-matched normal fetal lung, which may, in part, be responsible for the autonomous growth and proliferation seen in hydropic fetal CCAMs.

Blotting, Western↗

PubMed1999

The ex utero intrapartum treatment procedure for a large fetal neck mass in a twin gestation.

BACKGROUND: Large fetal neck masses can make it difficult or impossible to secure airways at birth, with associated risks of hypoxia, brain injury, and death. Based on a MEDLINE search from 1966 to June 1998, using the keywords EXIT procedure, placental support, twins, and neck mass, we report the first ex utero intrapartum treatment procedure performed in a twin gestation complicated by a large fetal neck mass. CASE: A giant fetal cervical mass was diagnosed in one fetus of a 20-week twin gestation by sonography and magnetic resonance imaging. At 35 weeks' gestation, the ex utero intrapartum treatment procedure was performed successfully for delivery of the normal twin, followed by intrapartum airway access of the twin with the neck mass. CONCLUSION: Even in twin gestations, the ex utero intrapartum treatment procedure is the delivery method of choice for fetuses with giant neck masses.

Adult↗

PubMed1999

Open fetal surgery for life-threatening fetal malformations.

After more than two decades of experimental and clinical work, fetal surgery has become an accepted treatment modality for selected fetuses with life-threatening anomalies. Color Doppler ultrasound and ultrafast fetal magnetic resonance imaging have enhanced the accuracy of prenatal evaluation traditionally made by ultrasound alone. Fetal lung masses associated with hydrops are nearly 100% fatal. These lesions can be resected in utero if they are predominantly solid or multicystic. Thoracoamniotic shunting may be effective in the setting of a single large predominant cyst. Fetuses diagnosed with left congenital diaphragmatic hernia before 26 weeks' gestation with liver herniation and a sonographic right lung to head circumference ratio (LHR) of less than one may benefit from fetal tracheal occlusion. Fetal sacrococcygeal teratoma complicated with placentomegaly, hydrops, or progressive high output heart failure may benefit from in utero resection of the tumor. Although preterm labor still remains the Achilles heel of open fetal surgery, effective tocolysis may, in the future, expand the scope of fetal surgery.

Bronchopulmonary Sequestration↗

PubMed1999

Troubles with twins: fetoscopic therapy.

Monochorionic twin gestations present challenges in perinatal management not seen in dichorionic twins or singleton pregnancies. Monochorionic twin gestations may be complicated by (1) a monoamniotic presentation, (2) a structurally anomalous cotwin, (3) twin reversed arterial perfusion sequence, (4) and the twin-twin transfusion syndrome. Each of these conditions is associated with increased morbidity and mortality rates. Although a variety of therapeutic interventions are available, fetoscopic surgery is a promising new approach to these conditions. Advances in microinstrumentation have allowed for the wider application of fetoscopy for a variety of prenatally diagnosed conditions, including "troubles with twins." This article examines the natural history, pathophysiology, diagnosis, and indications for fetoscopic surgery in complicated monochorionic twin gestations.

Chorion↗

PubMed1999

Management of fetal airway obstruction.

Fetal airway obstruction can make it difficult if not impossible to secure the airway at birth, before hypoxia, brain injury, or death results. Fetal airway obstruction can result from an intrinsic defect in the airway, such as the congenital high airway obstruction syndrome or extrinsic compression of the airway caused by a cervical mass, most commonly a cervical teratoma or lymphangioma. As fetuses with fetal airway obstruction reach viability, they should be monitored closely for the development or progression of hydrops in intrinsic obstruction cases or polyhydramnios in extrinsic obstruction cases. The fetus should be delivered by using the ex utero intrapartum treatment procedure, with maintenance of uteroplacental circulation and gas exchange. This approach provides time to perform procedures such as direct laryngoscopy, bronchoscopy, or tracheostomy to secure the fetal airway, thereby converting an emergent airway crisis into a controlled situation.

Airway Obstruction↗