James Mandell

Structural and functional features of central nervous system lymphatic vessels.

One of the characteristics of the central nervous system is the lack of a classical lymphatic drainage system. Although it is now accepted that the central nervous system undergoes constant immune surveillance that takes place within the meningeal compartment, the mechanisms governing the entrance and exit of immune cells from the central nervous system remain poorly understood. In searching for T-cell gateways into and out of the meninges, we discovered functional lymphatic vessels lining the dural sinuses. These structures express all of the molecular hallmarks of lymphatic endothelial cells, are able to carry both fluid and immune cells from the cerebrospinal fluid, and are connected to the deep cervical lymph nodes. The unique location of these vessels may have impeded their discovery to date, thereby contributing to the long-held concept of the absence of lymphatic vasculature in the central nervous system. The discovery of the central nervous system lymphatic system may call for a reassessment of basic assumptions in neuroimmunology and sheds new light on the aetiology of neuroinflammatory and neurodegenerative diseases associated with immune system dysfunction.

BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms. ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment. However, the receptor(s) upstream of the ELMO/Dock180/Rac module are still unknown. Here we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key ‘eat-me’ signal exposed on apoptotic cells, as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac–GEF complex to mediate the uptake of apoptotic cells.

Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro

The use of autologous urothelium would be advantageous for urothelial replacement in many genitourinary reconstructive procedures. Urothelial tissue grafts might be created using isolated populations of transitional epithelium or tissue in concert with an appropriate synthetic substrate. We describe the results of experiments designed to determine the feasibility of using biodegradable polymers as delivery vehicles for the creation of new urothelial structures in vivo from dissociated cells. Primary cultures enriched in uroepithelial cells were obtained from New Zealand white rabbits using a new technique of cell harvest. Cells were seeded onto nonwoven meshes of polyglycolic acid polymers in culture and, after 1 to 4 days in vitro, the cell-polymer scaffolds were implanted into the mesentery, omentum or retroperitoneum of athymic mice. Polymers implanted without cells served as controls. Animals were sacrificed at 5, 10, 20 and 30 days after implantation and 75 implants were examined histologically. Ten days after implantation isolated single cell layers were seen lining the polymer fibers. At 20 and 30 days polymer degradation was evident and urothelial cells lined the polymer in continuous layers of 1 to 3-cell thickness. Anticytokeratin western blots demonstrated the presence of a urothelium-associated cytokeratin in cell-polymer implants recovered after 30 days. These results demonstrate that urothelial cells can be successfully harvested, survive in culture and attach to artificial biodegradable polymers. The urothelial-polymer scaffolds can be implanted into host animals and the implanted cells can achieve spatial orientation as the polymer undergoes biodegradation. These findings suggest that it may be possible to use autologous urothelium, reconfigured on a synthetic substrate, in reconstructive procedures involving the ureter, bladder and urethra.

Variability in Surgical Caseload and Access to Intensive Care Services

Background Variability in the demand for any service is a significant barrier to efficient distribution of limited resources. In health care, demand is often highly variable and access may be limited when peaks cannot be accommodated in a downsized care delivery system. Intensive care units may frequently present bottlenecks to patient flow, and saturation of these services limits a hospitals responsiveness to new emergencies. Methods Over a 1-yr period, information was collected prospectively on all requests for admission to the intensive care unit of a large, urban childrens hospital. Data included the nature of each request, as well as each patients final disposition. The daily variability of requests was then analyzed and related to the units ability to accommodate new admissions. Results Day-to-day demand for intensive care services was extremely variable. This variability was particularly high among patients undergoing scheduled surgical procedures, with variability of scheduled admissions exceeding that of emergencies. Peaks of demand were associated with diversion of patients both within the hospital (to off-service care sites) and to other institutions (ambulance diversions). Although emergency requests for admission outnumbered scheduled requests, diversion from the intensive care unit was better correlated with scheduled caseload (r = 0.542, P < 0.001) than with unscheduled volume (r = 0.255, P < 0.001). During the busiest periods, nearly 70% of all diversions were associated with variability in the scheduled caseload. Conclusions Variability in scheduled surgical caseload represents a potentially reducible source of stress on intensive care units in hospitals and throughout the healthcare delivery system generally. When uncontrolled, variability limits access to care and impairs overall responsiveness to emergencies.

Long-Term Urological Response of Neonates With Myelodysplasia Treated Proactively With Intermittent Catheterization and Anticholinergic Therapy

PURPOSE Urinary tract management in children with myelodysplasia is controversial. Some advocate observation alone, while others believe that the prophylactic institution of intermittent catheterization and anticholinergic therapy may help to prevent deterioration. MATERIALS AND METHODS A nonrandomized prospective study was instituted to compare the urological outcomes of a cohort of children who were at risk for urological deterioration on the basis of bladder-sphincter dyssynergia and/or high filling or voiding pressures. Those at risk were observed until deterioration occurred, or were placed on prophylactic intermittent catheterization with or without anticholinergic medication. RESULTS Of 44 children at risk 35 followed by observation alone had urinary tract deterioration, whereas only 3 of 20 at risk treated with prophylactic intermittent catheterization had deterioration with time. CONCLUSIONS Proactive bladder treatment significantly reduced the incidence of upper urinary tract deterioration and need for surgical intervention.

Laparoscopic Evaluation of the Nonpalpable Testis: A Prospective Assessment of Accuracy

To assess diagnostic accuracy, laparoscopy and surgical exploration were prospectively performed in 104 children with 126 nonpalpable testes. Laparoscopic localization of the testis was correct in 90% (114 of 126 testes) and was nondiagnostic in 8% largely due to preperitoneal insufflation. No surgical complications occurred. Using the criteria of blind-ending vas deferens and spermatic vessels as diagnostic of an intra-abdominal vanishing testis, the accuracy of diagnosis was 100% but the inability to identify either vas or vessels was associated with intra-abdominal testes in 2 of 3 cases. Identification of canalicular vas deferens and spermatic vessels was associated with testes in 36 of 75 cases (48%). Bilateral nonpalpable testes were significantly less likely to have an absent testes (5%) than a unilateral nonpalpable testis (59%), suggesting the possibility of different pathophysiological mechanisms in those entities. Diagnosis and surgical management of nonpalpable testes were directly impacted by laparoscopy in 42 of 117 testes (36%) by identifying intra-abdominal vanishing testis, the location of an intra-abdominal testes or the need for retroperitoneal exploration when vas deferens and spermatic vessels were not found. Accurate knowledge of testis location in 97% of the testes facilitated development of an appropriate surgical strategy (that is laparoscopic/laparoscopic assisted versus open procedure).

The Response of the Fetal Kidney to Obstruction

In a fetal ovine model the renal effects of different anatomic levels of fetal urinary obstruction were studied. Parameters of prenatal renal growth and differentiation were characterized and correlated with the patterns of renal response to in utero obstruction. Complete ureteral or urethral obstruction was produced in the sheep fetus at 55 to 60 days of gestation. Animals were delivered and sacrificed at near term (140 days), and the kidneys were removed and prepared for analysis. Parameters examined included weight, histology, glomerular number and total surface area, as well as urinary sodium, creatinine, osmolarity and N-acetyl glucosaminidase. Three patterns of response were identified, producing hydronephrotic, cystic or dysgenetic kidneys. Hydronephrotic kidneys were usually the result of bladder outlet obstruction or ureteral obstruction with spontaneous urinary decompression. These kidneys were large (20.7 gm. versus normal 10.8 gm., p less than 0.0001), with thinning of cortical parenchyma that was structurally intact. Glomerular number and surface area were normal. Cystic kidneys were large (14.2 gm., p less than 0.05) with grossly visible cysts and an effaced medulla. Cortical structure was distorted by cysts but basic elements were intact. Glomerular number and surface area were not reduced. Dysgenetic kidneys were small (3.9 gm., p less than 0.0001) with markedly abnormal cortical structure and little recognizable medulla. Histological elements similar to fetal structures were present, including cuboidal/columnar tubular epithelium and peritubular mesenchymal collars. Glomerular number and surface area were significantly less than normal (p less than 0.001). The kidneys contralateral to unilaterally obstructed kidneys were significantly larger than normal (16.2 gm., p less than 0.0001), with normal histology, glomerular number and surface area, indicating in utero contralateral renal hypertrophy. Urinary sodium was variably affected in the hydronephrotic kidneys and was identical to plasma in the dysgenetic kidneys. These results indicate the technical feasibility of in utero models of urinary obstruction. Renal growth and patterns of differentiation were markedly affected by in utero obstruction. They should be a major focus in the investigation of congenital obstructive uropathy, since normal processes of renal growth and differentiation form the basis for postnatal function.

The Urodynamic Consequences of Posterior Urethral Valves

We evaluated urodynamically 41 patients with posterior urethral valves because of signs or symptoms of incontinence (35), frequency (3), hydronephrosis (2) and infection (1). Findings included normal urodynamic evaluations in 3 patients, 2 had high voiding pressures secondary to outlet resistance and 1 had incontinence on the basis of external urethral sphincter damage. In the remainder 3 patterns of bladder dysfunction were identified. Myogenic failure with overflow incontinence occurred in 14 patients. In this group clean intermittent catheterization or Valsalvas voiding was used for emptying. Hyperreflexic bladders were seen in 10 patients. Pharmacological suppression of instability was effective in 5 of 7 patients treated; 1 required bladder augmentation. Eleven children had a small capacity bladder and poor compliance. Post-void residuals were low and these bladders were generally but not always stable. Pharmacological bladder relaxation was successful in 3 patients, 3 underwent augmentation, 1 did well with alpha-agonists and followup is unavailable on the other 4. These 3 patterns of bladder dysfunction represent an overlapping constellation of residual urodynamic abnormalities due to previous bladder outlet obstruction. Individual patients may show facets of several types of dysfunction associated with 1 predominant pattern.

Phosphorylation State-Specific Antibodies: Applications in Investigative and Diagnostic Pathology

Until recently, the investigation of protein phosphorylation was limited to biochemical studies of enzyme activities in homogenized tissues. The availability of hundreds of phosphorylation state-specific antibodies (PSSAs) now makes possible the study of protein phosphorylation in situ, and is opening many exciting opportunities in investigative and diagnostic pathology. This review illustrates the power of PSSAs, especially in immunohistochemical applications to human disease and animal models. Technical considerations, including antibody specificity and lability of phosphoepitopes, are covered, along with potential pitfalls, illustrated by a case study. In the arena of oncology, PSSAs may prove especially valuable in directly demonstrating the efficacy of chemotherapies targeted at protein kinase cascades. Novel applications of PSSAs are also beginning to reveal molecular mechanisms of inflammatory, degenerative, and toxin-induced diseases.

Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion

Skeletal muscle arises from the fusion of precursor myoblasts into multinucleated myofibres. Although conserved transcription factors and signalling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion by means of signalling through ELMO/Dock180/Rac1 proteins. During myoblast fusion, a fraction of myoblasts within the population underwent apoptosis and exposed phosphatidylserine, an established ligand for BAI1 (ref. 3). Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic myoblasts restored fusion. Furthermore, primary human myoblasts could be induced to form myotubes by adding apoptotic myoblasts, even under normal growth conditions. Mechanistically, apoptotic cells did not directly fuse with the healthy myoblasts, rather the apoptotic cells induced a contact-dependent signalling with neighbours to promote fusion among the healthy myoblasts. In vivo, myofibres from Bai1−/− mice are smaller than those from wild-type littermates. Muscle regeneration after injury was also impaired in Bai1−/−mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify apoptotic cells as a new type of cue that induces signalling via the phosphatidylserine receptor BAI1 to promote fusion of healthy myoblasts, with important implications for muscle development and repair.