Impact on postoperative, neonatal and 2-year neurodevelopmental outcomes of UA-AREDF during and after fetal spina bifida repair.

OBJECTIVE: Absent or reversed end-diastolic flow (AREDF) in the umbilical artery (UA) on Doppler is a known phenomenon during fetal interventions, such as fetal open spina bifida (OSB) repair. We aimed to evaluate the clinical importance of these Doppler findings by investigating the impact of UA-AREDF on postoperative, neonatal and 2-year neurodevelopmental outcomes. METHODS: This was a prospective study of pregnancies undergoing fetal OSB repair at the Zurich Center for Fetal Diagnosis and Therapy between 2010 and 2019. The group with UA-AREDF during or immediately after the intervention was compared to the group with normal UA Doppler. Primary endpoint was the FIGO scores of cardiotocography (CTG) 1, 2 and 6 h postoperatively and on day 1 after surgery. Secondary endpoints were the neonatal parameters and 2-year neurodevelopmental outcome assessed using the Bayley Scales of Infant and Toddler Development, Third Edition. RESULTS: Data of 130 patients were analyzed. None of the fetuses had UA-AREDF before OSB repair. Normal UA Doppler was observed in 107 (82%) patients and UA-AREDF was observed in 23 (18%) during or immediately after OSB surgery. UA-AREDF was more often observed after version of the fetus (P = 0.045). Seventeen (13%) cases had absent end-diastolic flow (UA-AEDF) and six (5%) cases had reversed end-diastolic flow (UA-REDF). UA-AREDF disappeared in all 23 cases within the first day after OSB surgery. One-third of all CTGs were restricted in oscillation after surgery, but no significant difference in CTG 1, 2 and 6 h postoperatively or on the first postoperative day was found between the UA-AREDF and normal-Doppler groups (P > 0.05). Gestational age at delivery, UA pH, 5-min Apgar score and birth weight were comparable between the two groups, and there was no difference in the 2-year neurodevelopmental outcome (P > 0.05). The neonatal and 2-year neurodevelopmental outcomes also did not differ significantly between the UA-REDF and UA-AEDF groups. CONCLUSIONS: Postoperative CTG abnormalities occur and recover at a similar rate in fetuses with transitory UA-AREDF and those with normal Doppler during fetal OSB repair. UA-AREDF during fetal OSB repair did not negatively influence postnatal or 2-year neurodevelopmental outcomes. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Systematic classification of maternal and fetal intervention-related complications following open fetal myelomeningocele repair - results from a large prospective cohort.

OBJECTIVE: To systematically categorise all maternal and fetal intervention-related complications after open fetal myelomeningocele (fMMC) repair of the first 124 cases operated at the Zurich Centre for Fetal Diagnosis and Therapy. DESIGN: A prospective cohort study. SETTING: Single centre. POPULATION: Mothers and fetuses after fMMC repair. METHODS: Between 2010 and 2019, we collected and entered all maternal complications following fMMC repair into the Clavien-Dindo classification. For fetal complications, a classification system based on the Medical Dictionary for Regulatory Activities terminology of Adverse Events was used including the preterm definitions of the World Health Organization. MAIN OUTCOME MEASURES: Systematic classification of maternal and fetal complications following fMMC repair. RESULTS: Gestational ages at surgery and birth were 25.0 ± 0.8 and 35.4 ± 2.0 weeks, respectively. In 17% of all cases, no maternal complications occurred. Maternal intervention-related complications were observed as follows: 69% grade 1, 36% grade 2, 25% grade 3, 6% grade 4 and 0% grade 5. In 34%, no fetal complications were noted; however, 43% of the fetuses developed a grade 1, 14% a grade 2, 8% a grade 3, 2% a grade 4 and 2% a grade 5 complication. CONCLUSION: This study raises awareness of complications following open fMMC repair; 6% of mothers and 2% of fetuses experienced a severe complication (grade 4) and perinatal death rate of 2% was observed (grade 5). These data are useful for prenatal counselling, they help to improve the system of fetal surgical care, and they allow benchmarking with other centres as well as comparison with fetoscopic approaches. TWEETABLE ABSTRACT: Systematic classification of all maternal and fetal intervention-related complications following open fMMC repair.

Intussusception in children: lessons learned from intestinal lymphoma as a rare lead-point.

BACKGROUND: Most cases of intussusception in children are idiopathic. Rarely, a malignant disease such as intestinal lymphoma may cause intussusception. Due to dramatic changes of expected outcome with chemotherapy regime alone, the surgical management of patients with intestinal lymphoma presenting with intussusception has to be reevaluated. METHODS: Retrospective chart review from May 2011 to February 2017. We included all patients with intestinal lymphoma presenting with intussusception. RESULTS: We found five patients with a mean age of 6.4 years (range 3-16). The most common presenting symptom was abdominal pain for several weeks which had acutely worsened. In all but one patient an ultrasound before pneumatic or hydrostatic reduction showed a finding suspicious of a pathological lead-point. Pneumatic or hydrostatic reduction was attempted in all patients, no complications were noted. In one patient reduction was not successful. Recurrence after reduction occurred in two patients. Two patients needed surgery, three had a percutaneous ultrasound-guided biopsy for diagnostic purposes. All patients had aggressive mature B cell non-Hodgkin lymphoma. CONCLUSION: A high index of suspicion for the presence of a pathological lead-point in children older than 4 years and children with recurrent intussusception is necessary in patients presenting with intussusception. Malignant, highly aggressive B cell non-Hodgkin lymphoma, although rare, must actively be searched for. Pneumatic or hydrostatic reduction should remain the first line treatment in most cases.

Intestinal IMINO transporter SIT1 is not expressed in human newborns.

The expression of amino acid transporters in small intestine epithelia of human newborns has not been studied yet. It is further not known whether the maturation of imino acid (proline) transport is delayed as in the kidney proximal tubule. The possibility to obtain small intestinal tissue from patients undergoing surgery for jejunal or ileal atresia during their first days after birth was used to address these questions. As control, adult terminal ileum tissue was sampled during routine endoscopies. Gene expression of luminal imino and amino acid transporter SIT1 (SLC6A20) was approximately threefold lower in newborns versus adults. mRNA levels of all other luminal and basolateral amino acid transporters and accessory proteins tested were similar in newborn mucosa compared with adults. At the protein level, the major luminal neutral amino acid transporter B0AT1 (SLC6A19) and its accessory protein angiotensin-converting enzyme 2 were shown by immunofluorescence to be expressed similarly in newborns and in adults. SIT1 protein was not detectable in the small intestine of human newborns, in contrast to adults. The morphology of newborn intestinal mucosa proximal and distal to the obstruction was generally normal, but a decreased proliferation rate was visualized distally of the atresia by lower levels of the mitosis marker Ki-67. The mRNA level of the 13 tested amino acid transporters and accessory proteins was nonetheless similar, suggesting that the intestinal obstruction and interruption of amniotic fluid passage through the small intestinal lumen did not affect amino acid transporter expression. NEW & NOTEWORTHY System IMINO transporter SIT1 is not expressed in the small intestine of human newborns. This new finding resembles the situation in the proximal kidney tubule leading to iminoglycinuria. Lack of amniotic fluid passage in small intestinal atresia does not affect amino acid transporter expression distal to intestinal occlusion.

Successful grafting of tissue-engineered fetal skin.

PURPOSE: Fetal repair of spina bifida results in improved outcomes and has therefore become a standard clinical procedure in some highly specialized centers. However, optimization of the procedure technique and timing is needed. Both might be achieved by facilitating the procedure using laboratory-grown fetal skin substitutes. The aim of this study was therefore to test in vivo the suitability of such a fetal skin substitute for an in utero application. METHODS: Collagen-based hydrogels containing fetal ovine fibroblasts were seeded with fetal ovine keratinocytes and transplanted on immuno-incompetent nu/nu rats. After 3 weeks, grafts were harvested and analyzed histologically and by immunohistochemistry. RESULTS: Laboratory-grown fetal ovine dermo-epidermal skin substitutes showed successful engraftment at 3 weeks. Histologically, grafts revealed a neo-dermis populated by fibroblasts and with ingrowth of vessels, and an epidermis with an adult-like, mature appearance depicting clearly basal, spinous, granular, and a corneal layer. Immunostaining confirmed a physiologically organized epidermis. CONCLUSION: Fetal dermo-epidermal skin substitutes of ovine origin can successfully be grafted in vivo. In a next step, we will have to test whether favorable results can also be obtained when grafts are used in utero. If so, then human fetal spina bifida repair using laboratory-grown autologous fetal skin for defect closure may be envisaged.

Myelinated and unmyelinated nerve fibers reinnervate tissue-engineered dermo-epidermal human skin analogs in an in vivo model.

PURPOSE: The clinical application of autologous tissue-engineered skin analogs is an important strategy to cover large skin defects. Investigating biological dynamics, such as reinnervation after transplantation, is essential to improve the quality of such skin analogs. Previously, we have examined that our skin substitutes are reinnervated by host peripheral nerve fibers as early as 8 weeks after transplantation. Here, we wanted to investigate the presence and possible differences regarding myelinated and unmyelinated host nerve fibers 15 weeks after the transplantation of light and dark human tissue-engineered skin analogs. METHODS: Human epidermal keratinocytes, melanocytes, and dermal fibroblasts were isolated from human light and dark skin biopsies. Keratinocytes and melanocytes were seeded on fibroblast-containing collagen type I hydrogels after expansion in culture. After additional culturing, the tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds created on the back of immuno-incompetent rats. Skin substitutes were excised and analyzed 15 weeks after transplantation. Histological sections were examined with regard to the ingrowth pattern of myelinated and unmyelinated nerve fibers into the skin analogs using markers, such as Substance P, NF200, and S100-Beta. RESULTS: We found myelinated and unmyelinated peripheral host nerve fibers 15 weeks after transplantation in the dermal part of our human skin substitutes. In particular, we identified large-diameter-myelinated Aß- and Aδ-fibers, and small-diameter C-fibers. Furthermore, we observed myelinated nerves in close proximity to CD31-positive blood capillaries. In the long run, both types of ingrown host fibers showed an identical pattern in both light and dark skin analogs. CONCLUSION: Our data suggest that myelinated and unmyelinated peripheral nerves reinnervate human skin substitutes in a long-term in vivo transplantation assay. Our tissue-engineered skin analogs attract A- and C-fibers to supply both light and dark skin analogs. Potentially, this process restores skin sensitivity and has, therefore, a significant relevance with regard to future application of autologous pigmented dermo-epidermal skin substitutes onto patients.

Experimental tissue engineering of fetal skin.

PURPOSE: In some human fetuses undergoing prenatal spina bifida repair, the skin defect is too large for primary closure. The aim of this study was to engineer an autologous fetal skin analogue suitable for in utero skin reconstruction during spina bifida repair. METHODS: Keratinocytes (KC) and fibroblasts (FB) isolated from skin biopsies of 90-day-old sheep fetuses were cultured. Thereafter, plastically compressed collagen hydrogels and fibrin gels containing FB were prepared. KC were seeded onto these dermal constructs and allowed to proliferate using different culture media. Constructs were analyzed histologically and by immunohistochemistry and compared to normal ovine fetal skin. RESULTS: Development of a stratified epidermis covering the entire surface of the collagen gel was observed. The number of KC layers and degree of organization was dependent on the cell culture media used. The collagen hydrogels exhibited a strong tendency to shrink after eight to ten days of culture in vitro. On fibrin gels, we did not observe the formation of a physiologically organized epidermis. CONCLUSION: Collagen-gel-based ovine fetal cell-derived skin analogues with near normal anatomy can be engineered in vitro and may be suitable for autologous fetal transplantation.

In utero surgery rescues neurological function at birth in sheep with spina bifida.

We hypothesize that the neurologic deficit associated with open spina bifida is not directly caused by the primary defect but rather is due to chronic mechanical and chemical trauma since the unprotected neural tissue is exposed to the intrauterine environment. We report here that exposure of the normal spinal cord to the amniotic cavity in midgestational sheep fetuses leads to a human-like open spina bifida with paraplegia at birth, indicating that the exposed neural tissue is progressively destroyed during pregnancy. When open spina bifida was repaired in utero at an intermediate stage, the animals had near-normal neurologic function. The spinal cord was deformed but largely preserved. These findings suggest that secondary neural tissue destruction during pregnancy is primarily responsible for the functional loss and that timely in utero repair of open spina bifida might rescue neurologic function.

Association of uterine activity and maternal volatile anesthetic exposure during open fetal surgery for spina bifida: a retrospective analysis.

BACKGROUND: Recent warnings postulate a possible damaging effect of volatile anesthetics on the fetus. In our archive of fetal surgeries, we found wide variation in dosing of volatile anesthetics during spina bifida surgeries. We hypothesized that there was an association between volatile anesthetic exposure and uterine activity. METHODS: Sixty anesthesia records from spina bifida operations were assessed. We analyzed the course of the administered volatile anesthetic during surgery and calculated from each patient's anesthesia record the volatile anesthetic exposure expressed in vol%h. We divided the records into two post hoc groups of the 20 lowest exposure (Group L) versus the 20 highest exposure (Group H), and compared them for uterine activity and fetal heart rate. RESULTS: The number of contractions per hour was significantly greater in Group H (mean 1.3, SD ±â€¯1.2) compared with Group L (mean 0.5, SD ±â€¯0.6, P=0.049). There was no difference between the groups for the administration of the tocolytic drug atosiban (P=0.29). The course of the mean arterial pressure did not significantly differ but group H needed significantly more vasoactive medication (P <0.05). CONCLUSIONS: We found that a lower intra-operative volatile anesthetic exposure than recommended in the MOMS-trial (i.e. <2.0 minimum alveolar concentration [MAC]) was not associated with an increase in intra-operative uterine activity. This is an indication that during spina bifida surgery, 2.0 MAC may not be necessary to avoid potentially harmful uterine activity.

Cross-linking of the dermo-epidermal junction of skin regenerating from keratinocyte autografts. Anchoring fibrils are a target for tissue transglutaminase.

Since transglutaminases create covalent gamma-glutamyl-epsilon-lysine cross-links between extracellular matrix proteins they are prime candidates for stabilizing tissue during wound healing. Therefore, we studied the temporo-spatial expression of transglutaminase activity in skin regenerating from cultured epithelial autografts in severely burned children by the specific incorporation of monodansylcadaverine into cryostat sections from skin biopsies obtained between 5 d to 17 mo after grafting. The dansyl label was subsequently immunolocalized in the epidermis, dermal connective tissue, and along the basement membrane. Incubation of cryosections of normal and regenerating skin with purified tissue transglutaminase confirmed the dermo-epidermal junction and the papillary dermis as targets for this enzyme and revealed that in regenerating skin transamidation of the basement membrane zone was completed only 4-5 mo after grafting. Immunoelectron microscopy revealed that three distinct regions on the central portion of anchoring fibrils were positive for monodansylcadaverine in normal skin which were negative during the initial phase of de novo formation of anchoring fibrils in regenerating skin. Biochemically, we identified collagen VII as potential substrate for tissue transglutaminase. Thus, tissue transglutaminase appears to play an important role not only in cross-linking of the papillary dermis but also of the dermo-epidermal junction in particular.

Impact of carbon dioxide versus air pneumoperitoneum on peritoneal cell migration and cell fate.

BACKGROUND: Postoperative systemic immune function is suppressed after open abdominal surgery, as compared with that after minimally invasive abdominal surgery. As a first line of defense, peritoneal macrophages (PMo) and polymorphonuclear neutrophil granulocytes (PMNs) are of primary importance in protecting the body from microorganisms. Previous studies have shown changes in these cell populations over time after open versus laparoscopic surgery. This study aimed to investigate the dynamics of cell recruitment and clearance of peritoneal cells. METHODS: Female NMRI mice (33 +/- 2 g) were randomly assigned to carbon dioxide (CO2) or air insufflation. Intravasal cells with phagocytic capabilities were selectively stained by intravenous injection of the fluorescent dye PKH26 24 h before surgery. Gas was insufflated into the peritoneal cavity through a catheter, and the pneumoperitoneum was maintained for 30 min. Peritoneal lavage was performed 1, 3, 8, or 24 h after surgery. Apoptotic cells were assessed by flow cytometry using a general caspase substrate. RESULTS: The total peritoneal cell count did not differ between groups. The PKH26-positive PMo level was significantly increased after CO2, as compared with air, at 1 h and 24 h. The ratio of apoptotic PMo did not differ between the groups. In the peritoneal lavage, polymorphonuclear leukocytes (PMNs) were tripled in the air group, as compared with the CO2 group, whereas the ratio of apoptotic PMNs was significantly decreased. There was a higher fraction of PKH26-positive PMNs after air exposure, as compared with that after CO2. CONCLUSIONS: Air exposure triggered a higher transmigration rate of PMNs from the blood compartment into the peritoneal cavity and decreased PMN apoptosis, as compared with CO2. The lower proportion of PKH26-positive peritoneal macrophages in the air group might have been attributable to a higher inflammatory stimulation than in the CO2 group, leading to increased emigration of PMo to draining lymph nodes. All the findings underscore a complex cell-specific regulation of cell recruitment and clearance in the peritoneal compartment.

Gene expression along the cerebral-spinal axis after regional gene delivery.

We demonstrate here that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome: DNA complexes (CLDCs) produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Gene expression was identified both within the spinal cord and the brain after intracerebroventricular or intrathecal injection of either CLDCs or plasmid DNA alone. Intracerebroventricular or intrathecal injection of CLDCs containing the beta-galactosidase (beta-Gal) gene produced patchy, widely scattered areas of beta-Gal expression. The chloramphenicol acetyltransferase (CAT) reporter gene product reached peak levels between 24 hr and 1 week postinjection, and was still present at significant levels 3 weeks after a single intracerebroventricular or intrathecal injection. Intrathecal injection of the human granulocyte colony-stimulating factor (G-CSF) gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine nerve growth factor (NGF) gene increased mNGF levels in the hippocampus, a target region for cholinergic neurons in the medial septum, and increased cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) activity within the brain, a well-characterized effect of both purified and recombinant NGF protein. These findings indicate that intracerebroventricular or intrathecal injection of CLDCs can produce significant levels of expression of biologically and therapeutically relevant genes within the CNS. Efficient gene transfer into the CNS will facilitate the evaluation of gene function and regulation within the brain and spinal cord. We attempted to transfer and express genes within the brain and spinal cord by direct CNS injection of either DNA alone or CLDCs into the intraventricular and subarachnoid compartments. We show that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or CLDCs produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Intrathecal injection of the hG-CSF gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine NGF gene increased mNGF levels in the hippocampus, and increased cholinergic neurotransmitter synthetic enzyme ChAT activity within the brain. Locoregional diffusion of gene products expressed by transfected meningeal lining cells into brain and spinal cord parenchyma could potentially target secreted proteins within brain and spinal cord regions relevant to neuropathological states while limiting peripheral side effects.

Confocal laser scanning analysis of the association of fibulin-2 with fibrillin-1 and fibronectin define different stages of skin regeneration.

The fibulins represent a novel family of extracellular matrix proteins. We report the temporo-spatial expression of fibulin-2 in skin regenerating from keratinocyte autografts. In normal dermis, fibulin-2 was associated with the fibrillin-containing microfibrillar apparatus, except for the portion immediately adjacent to the dermo-epidermal junction. In contrast, early regenerating dermis showed numerous fusiform fibrillin-microfibrils along the basement membrane, whereas fibulin-2 was present in a distinct and separate layer below. Both proteins formed independent fibrillar systems also in the reticular dermis without significant colocalization; however, over time both fibril systems became congruent: after 4 mo there was extensive colocalization of fibulin-2/fibrillin in the reticular dermis, after 17 and 24 mo this also occurred in the papillary dermis. Simultaneous visualization of fibulin-2 and fibronectin revealed an inverse pattern: complete colocalization at 7 d and discordant distribution 17-24 mo after grafting. In particular, the fibrillar fibronectin pattern at early time points changed into a faint granular distribution throughout the dermis and along the subbasement membrane region as in normal skin. Dermal fibroblast cultures showed that fibrillin and fibronectin participated in distinct fibrillar systems; however, fibulin-2 colocalized with either protein. We propose that, in regenerating skin, fibulin-2 is a late component of the cutaneous microfibrillar apparatus with an earlier existence in a fibrillar matrix mediated by fibronectin. This suggests interaction of fibulin-2 with both fibronectin fibrils and fibrillin microfibrils, and is consistent with in vitro binding data.

The cutaneous microfibrillar apparatus contains latent transforming growth factor-beta binding protein-1 (LTBP-1) and is a repository for latent TGF-beta1.

The transforming growth factors-beta1 and beta2 (TGF-beta) stimulate synthesis of extracellular matrix proteins in vitro and appear upregulated in fibrotic conditions, in scar formation, and in wound healing. The extracellular matrix in turn might also act as a scavenger or repository for TGF-beta. We therefore studied the in situ distribution of latent TGF binding protein-1 (LTBP-1) and latent TGF-beta1 on extracellular matrix elements of normal human skin and skin regenerating from cultured keratinocyte autografts. We localized both LTBP-1 and latent TGF-beta1 to fibrillin-containing (elastic) microfibrils. Both LTBP-1 and latent TGF-beta1 were already present during the earliest stages of the de novo formation of the microfibrillar apparatus, i.e., on fusiform, randomly oriented microfibrils that later coalesced to form the typical candelabra-like structures in the papillary dermis. We show herewith that LTBP-1 exerts a dual role as a component of fibrillin-microfibrils of the skin and in targeting latent TGF-beta1 to the cutaneous microfibrillar apparatus. Thus, this major connective tissue structure does not only serve as a force bearing element and scaffold for elastin deposition in the dermis, but also as an important repository for latent TGF-beta in the skin.

Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.

The temporo-spatial expression of fibrillin and elastin in skin regenerating from autologous keratinocyte grafts was studied in three burned children. Skin biopsies taken between 5 days and 17 months after grafting were investigated by conventional immunofluorescence, confocal laser scanning, and electron microscopy. Fibrillin, the major component of 10-12nm microfibrils, appeared 5 days after grafting in a band-like fashion similar to collagen VII at the prospective basement membrane, and the formed the characteristic microfibrillar candelabra at the dermo-epidermal junction by fusion of several fine microfibrils to communicating microfibrils projecting downward into the reticular layer of the neodermis. Four to five months after grafting, several communicating microfibrils were connected to a web of horizontally undulating microfibrils of the neodermis which had developed independently. Elastin was first identified in the deeper neodermis 1 month after grafting as granular aggregates and 4 months after grafting on fibrillar structures and surrounding capillaries of the upper neodermis. Association of elastin with microfibrils in the papillary dermis was not detectable before month 17. Our findings suggest that the cutaneous microfibrillar apparatus develops simultaneously at both the dermo-epidermal junction and the reticular dermis and is a prerequisite for elastic fiber formation. In addition, it might be a driving force for the formation of the papilla-rete ridge pattern.

Efficient gene expression in skin wound sites following local plasmid injection.

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

Experimental fetal transesophageal and intracardiac echocardiography utilizing intravascular ultrasound technology.

Fetal transesophageal and intracardiac echocardiography by utilizing ultrasound technology permits accurate definition of cardiac anatomy in fetal sheep. Because fetal transesophageal echocardiography is less invasive than intracardiac echocardiography, it has the potential to serve as a monitoring tool for currently developed open and fetoscopic fetal cardiac interventions.

Oxygen-regulated expression of TGF-beta 3, a growth factor involved in trophoblast differentiation.

The transforming growth factor-beta 3 (TGF-beta 3) is involved in oxygen-dependent differentiation processes during placental development and pregnancy disorders. However, the importance of oxygen partial pressure for the regulation of TGF-beta 3 expression is presently unclear. We and others presented preliminary evidence that the hypoxia-inducible factor-1 (HIF-1) confers TGF-beta 3 transcription but it was unknown whether this occurred directly or indirectly. To analyze how HIF-1 regulates TGF-beta 3 gene transcription, we cloned and sequenced the mouse TGF-beta 3 promoter region. Multiple putative HIF-1 binding sites (HBSs) were identified, many of which co-localized with two G+C rich CpG islands 5' to the TGF-beta 3 transcription start site. A 6.8 kb fragment of the TGF-beta 3 promoter induced reporter gene expression under hypoxic conditions or when treated with an iron chelator known to stabilize and activate the HIF-1 alpha subunit. Deletion of a 2.4 kb fragment upstream of the distal CpG island abolished inducibility of reporter gene expression. Two HBSs (HBS1 and HBS6) that bound the HIF-1 protein could be identified within this 2.4 kb fragment. These results suggest that TGF-beta 3 gene expression is directly regulated by HIF-1.

Dexamethasone therapy and endogenous cortisol production in severe pediatric head injury.

A prospective randomised study was performed on 25 children aged 1.4 to 15.8 years with severe head injury (Glasgow Coma Scale less than or equal to 7) to determine the clinical effectiveness and the impact on endogenous cortisol production of high-dose steroid therapy. Thirteen patients (group 1) received dexamethasone 1 mg/kg/day during the first 3 days and 12 (group 2) not. All patients were treated with a standardized regimen. Urinary free cortisol was measured by radioimmunoassay, and the clinical data were recorded at hourly intervals. Outcome was assessed 6 months later using the Glasgow Outcome Scale. We found a higher frequency of bacterial pneumonias in the dexamethasone-treated patients (7/13 versus 2/12). Group 1 showed a suppression of endogenous cortisol production from day 1 to day 6. In group 2, mean free cortisol was up to 5-fold higher than under basal conditions. The results in group 2 showed that the endogenous steroid production reacts adequately to the stress of severe head injury. It probably is sufficient to elicit maximum glucocorticoid effects. There was no other statistically significant difference in the clinical and laboratory data between the two groups. We conclude that dexamethasone in high doses suppresses endogenous cortisol production up to 6 days and may increase the risk of bacterial infection without affecting the outcome or the clinical and laboratory data.