ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.

In vivo transplantation of fetal human gut-derived enteric neural crest cells.

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75NTR were lentivirally labeled with either GFP or calcium-sensitive GCaMP and transplanted into the hindgut of Rag2- /γc- /C5- -immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut-derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.

Lentiviral labeling of mouse and human enteric nervous system stem cells for regenerative medicine studies.

BACKGROUND: Reliable methods of labeling human enteric nervous system (ENS) stem cells for use in novel cell replacement therapies for enteric neuropathies are lacking. Here, we explore the possibility of using lentiviral vectors expressing fluorescent reporter genes to transduce, label, and trace mouse and human ENS stem cells following transplantation into mouse gut. METHODS: Enteric nervous system precursors, including ENS stem cells, were isolated from enzymatically dissociated mouse and human gut tissues. Lentivirus containing eGFP or mCherry fluorescent reporter genes was added to gut cell cultures at a multiplicity of infection of 2-5. After fluorescence activated cell sorting for eGFP and subsequent analysis with markers of proliferation and cell phenotype, transduced mouse and human cells were transplanted into the gut of C57BL/6 and immune deficient Rag2-/gamma chain-/C5 mice, respectively and analyzed up to 60 days later. KEY RESULTS: Mouse and human transduced cells survived in vitro, maintained intense eGFP expression, proliferated as shown by BrdU incorporation, and formed characteristic neurospheres. When transplanted into mouse gut in vivo and analyzed up to 2 months later, transduced mouse and human cells survived, strongly expressed eGFP and integrated into endogenous ENS networks. CONCLUSIONS & INFERENCES: Lentiviral vectors expressing fluorescent reporter genes enable efficient, stable, long-term labeling of ENS stem cells when transplanted into in vivo mouse gut. This lentiviral approach not only addresses the need for a reliable fluorescent marker of human ENS stem cells for preclinical studies, but also raises the possibility of using lentiviruses for other applications, such as gene therapy.

Enteric neural crest-derived cells and neural stem cells: biology and therapeutic potential.

The enteric nervous system arises from two regions of the neural crest; the vagal neural crest which gives rise to the vast majority of enteric neurones throughout the gastrointestinal tract, and the sacral neural crest which contributes a smaller number of cells that are mainly distributed within the hindgut. The migration of vagal neural crest cells into, and along the gut is promoted by GDNF, which is expressed by the gut mesenchyme and is the ligand for the Ret/GFRalpha1 signalling complex present on migrating vagal-derived crest cells. Sacral neural crest cells enter the gut after it has been colonized by vagal neural crest cells, but the molecular control of sacral neural crest cell development has yet to be elucidated. Under the influence of both intrinsic and extrinsic cues, neural crest cells differentiate into glia and different types of enteric neurones at different developmental stages. Recently, the potential for neural stem cells to form an enteric nervous system has been examined, with the ultimate aim of using neural stem cells as a therapeutic strategy for some gut disorders where enteric neurones are reduced or absent.

Total soft-tissue reconstruction of the middle and lower face with multiple simultaneous free flaps in a pediatric patient.

A 2-year-old boy sustained a massive facial soft-tissue wound secondary to a dog attack. Essentially all the soft tissues of the face were absent, including innervation and intraoral lining. We describe the reconstruction of this defect with five simultaneous free tissue transfers. To our knowledge, this is the first report of five simultaneous free flaps in any patient.

Anti-carcinogenicity of probiotics and prebiotics.

Yoghurt, and the lactic acid producing bacteria (LAB; probiotics) that it contains, have received much attention as potential cancer-preventing agents in the diet. It is usually considered that the mechanism of the action is by increasing the numbers of LAB in the colon, which modifies the ability of the microflora to produce carcinogens. Prebiotics such as non-digestible oligosaccharides (NDO) appear to have similar effects on the microflora by selectively stimulating the growth of LAB in the colon. Evidence for cancer-preventing properties of pro- and prebiotics is derived from studies on faecal enzyme activities in animals and humans, inhibition of genotoxicity of known carcinogens in vitro and In vivo, suppression of carcinogen-induced preneoplastic lesions and tumours in laboratory animals. Some of these studies indicate that combinations of pro and prebiotics ('synbiotics') are more effective. Epidemiological and intervention studies provide some, albelt limited, evidence for protective effects of products containing probiotics in humans.

Residual thermal damage resulting from pulsed and scanned resurfacing lasers.

BACKGROUND: Laser resurfacing with rapidly scanned or pulsed carbon dioxide (CO2) lasers has evolved rapidly in recent years. These lasers vaporize small amounts of tissue, while leaving minimal residual thermal damage. OBJECTIVE: To compare the depth of residual thermal damage of two of the most commonly used CO2 laser systems. A rapidly scanned laser was compared to a short-pulse laser system. METHODS: Laser treatment was performed on abdominoplasty specimens prior to removal in four subjects. One, two, or three passes of the two most commonly used energies were administered using each laser system. RESULTS: The depth of thermal damage increased with a greater number of passes with each laser system. Higher energies resulted in greater residual thermal damage with each system after the first pass up to three passes, which was the maximum number of passes administered. Combining the second and third passes, residual thermal damage was remarkably similar when comparing the pulsed and scanned lasers. CONCLUSIONS: The most commonly used energy settings of two lasers with very different modes of action resulted in remarkably similar depths of thermal damage, suggesting that the zone of thermal damage may correlate with clinical outcome. In addition, the zone of thermal damage enlarges as the number of passes increases from one to three.

Long-term assessment of CO2 facial laser resurfacing: aesthetic results and complications.

Several series have documented the ability of the carbon dioxide laser to smooth facial rhytids; however, follow-up has been limited to several months. Since 1995, more than 600 full or partial facial resurfacings were performed with the pulsed CO2 laser. To assess the long-term efficacy and safety of this procedure, the results of 211 resurfacings were retrospectively reviewed using a custom-designed database. Variables that were input included patient demographics, Fitzpatrick skin type, smoking history, prior and concurrent facial procedures, laser pass data, and postoperative complications. Short and long-term aesthetic results were graded by a blinded panel of plastic surgery reviewers (none of whom performed the laser resurfacing) using a standardized photographic rhytid scale. For each facial region, this scale consisted of eight high-resolution photographs depicting increasingly severe wrinkling. Facial rhytids were almost completely ablated at the 3 and 6 month follow-up. Some relapse was seen at 1 year, but the overall aesthetic result remained very good. Regions with dynamic rhytids (e.g., the perioral region) showed more recurrence. The best and most durable results were seen in the cheeks. Infection and scleral show each occurred in 13 patients (6 percent). Forty-five patients (21 percent) developed postprocedure hyperpigmentation, but the overwhelming majority of this group were treated before our postoperative antipigment regimen. Hypopigmentation was noted in 17 patients (8 percent) in this early follow-up group. Two patients (1 percent) developed postoperative scarring. It is hoped that these data will serve to provide additional information on the long-term results of laserbrasion.

CO2 laser safety considerations in facial skin resurfacing.

Carbon dioxide lasers have been used increasingly in the field of aesthetic plastic surgery, specifically for facial resurfacing procedures. As many plastic surgeons are now venturing into the arena of laser surgery for the first time, it is paramount to understand basic laser safety principles to protect our patients, the operating room personnel, and the laser surgeon. This article reviews basic laser principles and practices and delineates the safety requirements needed to perform laser resurfacing using the CO2 laser system. We subjected several common objects present in the operative field during resurfacing procedures to multiple passes of both the Coherent 5000 C laser and the Laser Industries (Sharplan) model 150XJ laser Silktouch to assess flammability and margins of safety. We tested endotracheal tubes, wet and dry towels, wet and dry gauze sponges, cottonoids, eye protectors, and ophthalmic ointments. Neither flame nor burn was incited in the moistened preparations. The dry objects tested produced flame. The plastic corneal protectors began to melt by the third pass and produced significant heat. Lastly, both the Lacrilube and Bacitracin ophthalmic ointments began to vaporize after three laser passes. On the basis of our findings in this study, we recommend guidelines for prudent and safe CO2 laser usage in facial skin resurfacing.

Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry.

Interstitial cells of Cajal (ICC) of various morphologies have been described in the gastrointestinal (GI) tracts of mammals. Different classes of ICC are likely to have different functional roles. ICC of the mouse GI tract have been shown to express c-kit, a proto-oncogene that codes for a receptor tyrosine kinase. We have studied the distribution of ICC within the guinea pig GI tract using antibodies to c-Kit protein and immunohistochemical techniques. c-Kit-like immunoreactivity revealed at least 6 types of ICC: (1) intramuscular ICC (IC-IM1) that lie within the muscle layers of the esophagus, stomach, and cecum, (2) ICC within the myenteric plexus region (IC-MY1) in the corpus, antrum, small intestine, and colon, (3) ICC that populate the deep muscular plexus of the small intestine (IC-DMP), (4) ICC at the submucosal surface of the circular muscle layer in the colon (IC-SM), (5) stellate ICC that are closely associated with the myenteric plexus (IC-MY2) and orientated toward the longitudinal muscle layer in the colon, and (6) branching intramuscular ICC (IC-IM2) in the proximal colon within the circular and longitudinal muscle layers. c-Kit immunohistochemistry appears to be an excellent and selective technique for labeling ICC of the guinea-pig GI tract. Labeling of these cells at the light-microscopic level provides an opportunity for characterizing the distribution, density, organization, and relationship between ICC and other cell types.

Risk factors for complications in pediatric tissue expansion.

Tissue expansion in children has been associated with complication rates of 20 to 40 percent. A critical analysis of 6 years' (1988-1993) experience with 180 expanders placed in 82 consecutive children was performed to identify those factors which predispose to complications. Major and minor complications each occurred in 9 percent of patients. The factors associated with a statistically significant increase in complications were burns and soft-tissue loss, patient age under 7 years, use of internal expander ports, and a history of two or more prior expansions. In addition, complications were significantly more likely to occur within the first 90 days than during any subsequent expansion. Factors that did not influence complication rate included patient gender, wound drainage upon expander insertion or removal, intraoperative use of antibiotic irrigation, number of expanders placed, use of customized expanders, and operating surgeon.

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.

Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants.

Electrical rhythmicity in the gastrointestinal tract may originate in interstitial cells of Cajal (IC). Development of IC in the small intestine is linked to signaling via the tyrosine kinase receptor, c-kit. IC express c-kit protein, and disruption of c-kit signaling causes breakdown in IC networks and loss of slow waves. We tested whether mutations in steel factor, the ligand for c-kit, affect the development of IC networks. IC were found in the region of the myenteric plexus (IC-MY) in mice with steel mutations (i.e., Sl/Sld) at 5-10 days postpartum, but these cells formed an abnormal network. IC-MY were not observed in adult Sl/Sld animals. IC in the deep muscular plexus (IC-DMP) appeared normal in Sl/Sld animals. Electrical slow waves, normally present in the small intestine, were absent in Sl/Sld animals (10-30 days postpartum). Neural inputs were intact in Sl/Sld animals. Steel factor appears important for the development of certain classes of IC, and IC-MY appear to be involved in the generation of electrical rhythmicity in the small intestine.

Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.

1. Interstitial cells of Cajal (ICs) have been proposed as pacemakers in the gastrointestinal tract. We studied the characteristics and distribution of ICs and electrical activity of small intestinal muscles from mice with mutations at the dominant-white spotting/c-kit (W) locus because the tyrosine kinase function of c-kit may be important in the development of the IC network. 2. W/WV mutants (days 3-30 postpartum) had few ICs in the myenteric plexus region compared with wild type (+/+) siblings. The few ICs present were associated with neural elements and lay between myenteric ganglia and the longitudinal muscle layer. 3. Electrical recordings from intestinal muscle strips showed that electrical slow waves were always present in muscles of +/+ siblings, but were absent in W/WV mice. 4. Muscles from W/WV mice responded to stimulation of intrinsic nerves. Neural responses, attributed to the release of acetylcholine, nitric oxide and other unidentified transmitters, were recorded. 5. These findings are consistent with the hypothesis that ICs are a critical element in the generation of electrical rhythmicity in intestinal muscles. The data also show that neural regulation of gastrointestinal muscles can develop independently of the IC network. 6. W locus mutants provide a powerful new model for studies of the physiological role of ICs and the significance of electrical rhythmicity to normal gastrointestinal motility.

Is there an association between hemangioma and syndromes with dysmorphic features?

Most vascular birthmarks can be categorized, based on clinical and cellular criteria, as either (1) a hemangioma, or (2) a malformation, or (3) a macular stain. Macular stains are commonly seen in newborns, and they consist of faint vascular stains of the glabella, eyelids, and nuchal region called "nevus flammeus," "stork bite," "salmon patch," etc. Unfortunately, the term "hemangioma" is frequently applied to all three types of cutaneous vascular lesions. Usually, these disparate vascular anomalies are listed in association with various malformative syndromes and are generically labeled "hemangioma." This study attempts to define accurately the specific vascular anomalies seen in children born with syndromes with dysmorphic features. This review of five standard textbooks of genetics showed that the majority of vascular anomalies reported in syndromic newborns are not hemangiomas. Rather, they are macular stains, and the vast majority of these fade with time. Congenital telangiectasias and other vascular malformations (capillary, lymphatic, venous, arterial, and combinations thereof) also occur in association with dysmorphic syndromes. In contrast, hemangioma, the most common neonatal tumor, is seen only incidentally with rare dysmorphic conditions. Specifically, hemangioma was found to occur only in association with midline (sternal, abdominal) clefting, right-sided aortic arch coarctation, and with a constellation of sacral and genitourinary defects.