Generation of 10 patient-specific induced pluripotent stem cells (iPSCs) to model Pitt-Hopkins Syndrome.

Autosomal dominant mutations in transcription factor 4 (TCF4) are associated with a rare syndromic form of Autism Spectrum Disorder (ASD) called Pitt-Hopkins Syndrome (PTHS). Here, we report the generation of a collection of induced pluripotent stem cells (iPSCs) from 5 patients diagnosed with PTHS and 5 familial controls. These patient-derived iPSCs contain a variety of mutations within the TCF4 gene, possess a normal karyotype and express all the appropriate pluripotent stem cell markers. These novel patient lines will be a useful resource for the research community to study PTHS and the function of TCF4.

Extracellular matrix molecules improve periodontal ligament cell adhesion to anorganic bone matrix.

Bone replacement graft (BRG) materials are used in periodontal therapy to encourage new bone formation. Extracellular matrix proteins may improve periodontal ligament fibroblast (PDLF) attachment to these materials. We demonstrate that PDLFs adhere well to the extracellular matrix (ECM) proteins fibronectin, vitronectin, laminin, and collagen types I and IV. PDLFs express numerous ECM-receptor integrin subunit transcripts (alpha1, alpha2, alpha3, alpha4, alpha5, alpha11, beta1, beta5, and beta8) at high levels, while others (alpha6, alpha9, alphaV, beta3, beta6, and beta7) are expressed at reduced levels. Despite the fact that PDLFs adhere well to fibronectin and collagen type IV bound to plastic, and express integrins that recognize these ECM proteins, they do not attach well to anorganic bovine bone matrix (ABM) coated with these same proteins. However, the addition of vitronectin, laminin, or collagen type I to these same ABMs substantially increased PDL cell attachment. Thus, selective use of ECM proteins may be clinically useful in promoting cell attachment to ABM and bone regrowth.

Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion.

Directed cell adhesion remains an important goal of implant and tissue engineering technology. In this study, surface energy and surface roughness were investigated to ascertain which of these properties show more overall influence on biomaterial-cell adhesion and colonization. Jet impingement was used to quantify cellular adhesion strength. Cellular proliferation and extracellular matrix secretion were used to characterize colonization of 3T3MC fibroblasts on: HS25 (a cobalt based implant alloy, ASTM F75), 316L stainless steel, Ti-6Al4V (a titanium implant alloy), commercially pure tantalum (Ta), polytetrafluoroethylene (PTFE), silicone rubber (SR), and high-density polyethylene (HDPE). The metals exhibited a nearly five-fold greater adhesion strength than the polymeric materials tested. Generally, surface energy was proportional to cellular adhesion strength. Only polymeric materials demonstrated significant increased adhesion strength associated with increased surface roughness. Cellular adhesion on metals demonstrated a linear correlation with surface energy. Less than half as much cellular proliferation was detected on polymeric materials compared to the metals. However the polymers tested demonstrated greater than twice the amount of secreted extracellular matrix (ECM) proteins on a per cell basis than the metallic materials. Thus, surface energy may be a more important determinant of cell adhesion and proliferation, and may be more useful than surface roughness for directing cell adhesion and cell colonization onto engineered tissue scaffoldings.

Revision endoscopic sinus surgery: the Thomas Jefferson University experience.

Since its introduction, functional endoscopic sinus surgery (FESS) has demonstrated success rates of 76% to 98%. A small group of the patients in whom initial FESS and optimal medical therapy fail require revision endoscopic sinus surgery (RESS). This group has recently been studied by several authors, and we have evaluated a group of 90 RESS patients selected from 753 consecutive primary FESS patients. Patients were followed for a mean of 22.8 months. Extent of disease, history of polyps, allergy, previous traditional endonasal sinus surgery, male gender, chronic steroid use, and the presence of a deviated septum all appeared to adversely affect RESS outcome. The surgeon's knowledge of the sinus anatomy is critical, especially in revision sinus cases in which landmarks are distorted or absent. In our review, RESS was associated with a 1% major complication rate and was successful in 67% of patients. Computer-assisted endoscopic sinus surgery integrates preoperative imaging with realtime endoscopic visualization, augments the surgeon's knowledge of anatomy, and helps to minimize patient risk.

Diffusion approximation of frequency sensitive competitive learning.

The focus of this paper is a convergence study of the frequency sensitive competitive learning (FSCL) algorithm. We approximate the final phase of FSCL learning by a diffusion process described by the Fokker-Plank equation. Sufficient and necessary conditions are presented for the convergence of the diffusion process to a local equilibrium. The analysis parallels that by Ritter-Schulten (1988) for Kohonen's self-organizing map. We show that the convergence conditions involve only the learning rate and that they are the same as the conditions for weak convergence described previously. Our analysis thus broadens the class of algorithms that have been shown to have these types of convergence characteristics.

Attributed scattering centers for SAR ATR.

High-frequency radar measurements of man-made targets are dominated by returns from isolated scattering centers, such as corners and flat plates. Characterizing the features of these scattering centers provides a parsimonious, physically relevant signal representation for use in automatic target recognition (ATR). In this paper, we present a framework for feature extraction predicated on parametric models for the radar returns. The models are motivated by the scattering behaviour predicted by the geometrical theory of diffraction. For each scattering center, statistically robust estimation of model parameters provides high-resolution attributes including location, geometry, and polarization response. We present statistical analysis of the scattering model to describe feature uncertainty, and we provide a least-squares algorithm for feature estimation. We survey existing algorithms for simplified models, and derive bounds for the error incurred in adopting the simplified models. A model order selection algorithm is given, and an M-ary generalized likelihood ratio test is given for classifying polarimetric responses in spherically invariant random clutter.

Evaluation of the surface integrity of hydroxyapatite-coated threaded dental implants after insertion.

The surface characteristics and changes of hydroxyapatite-coated threaded dental implants after insertion into bone (veal calf ribs) with similar cortical and medullary characteristics to the human maxilla and posterior mandible (type III or IV bone) were studied. Hydroxyapatite-coated threaded implants from six vendors were coded A, B, C, D, E, and F. Four implants from each vendor were subjected to conventional placement following the manufacturer's instructions, placement without tapping of the osteotomy site, or evaluated as controls. The implants were recovered atraumatically and examined by scanning electron microscopy at x 35 and x 100 magnification. Photomicrographs were examined and graded by two independent examiners. Statistically significant differences were found using Kruskal-Wallis ANOVA and Wilcoxon Signed Ranks tests in surface integrity between conventional and nontapped treatment for groups C and D; between conventional placement and controls for groups A, B, and F; between nontapped placement and controls for groups A, B, C, D, and E; and among the manufacturers with respect to conventional placement for groups A, B, and E as compared with the other groups (P < 0.05). These findings suggest that surface changes of hydroxyapatite-coated threaded implants may occur during placement, particularly in undersized and untapped osteotomy sites. The changes could result in differences in integration and performance of some implant systems.

Three-dimensional reconstruction of the feline larynx with serial histologic sections.

This paper reviews a new technique to develop high-resolution three-dimensional (3-D) images of the larynx using histological sections. Three-dimensional computer-reconstructed histological sections of the cat are used in this study to evaluate the recurrent laryngeal nerve (RLN) in its true anatomic course, with emphasis on its relationship to surrounding structures (laryngeal framework). A cat model was used because of specimen availability and technical ease of tissue preparation. Computer-reconstructed serial histologic sections add a new dimension to the study of laryngeal anatomy, laryngeal trauma, and growth patterns of laryngeal cancer. The technical aspects of three-dimensional reconstruction and future applications are discussed.

Cell adhesion to biomaterials: correlations between surface charge, surface roughness, adsorbed protein, and cell morphology.

Adhesion of cells to a biomaterial surface can be a major factor mediating its biocompatibility. In this investigation, jet impingement techniques were used to quantify strength of cellular adhesion to various material surfaces. The metals tested: HS25 (a cobalt-based alloy similar to F75), 316L stainless steel, Ti-6Al-4V, and commercially pure tantalum, exhibited nearly a fivefold increase in adhesion strength above that characteristic of the polymeric materials tested (PTFE, silicone rubber, and HDPE). The present study examines physical and biological factors that might influence fibroblast adhesion to the biomaterial surface. The relation between surface charge and cellular adhesion was investigated in a controlled manner by measuring adhesion strength over a range of charge densities. The cells showed charge and electrical potential-dependent adhesion maxima, suggesting that surface alloying for optimum adherence may be possible. In a preliminary series of experiments adsorbed serum protein layers on a series of materials of differing adherence were investigated using gel electrophoresis to assess protein composition. Analysis of adsorbed proteins revealed little difference in relative abundance or total adsorption quantity. SEM micrographs of cells on Ti-6Al-4V and silicone rubber (high and low adhesion materials, respectively) demonstrated differences in cell morphology and cell density.

Morphology and physiologic responsiveness of cultured rabbit lacrimal acini.

PURPOSE: To assess the morphology and physiologic response of intact rabbit lacrimal acini maintained in culture for as long as 14 days and to compare the characteristics of the cultured acini with untreated fresh lacrimal gland, incubated lacrimal tissue slices, and freshly isolated acini. METHODS: Acini were obtained by enzymatic digestion of lacrimal glands of adults male New Zealand white rabbits and cultured for as long as 14 days on Matrigel-coated inserts in DMEM-F12. Untreated fresh lacrimal gland, incubated lacrimal tissue slices, freshly isolated acini, and intact cultured acini were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Maintenance of physiologic responsiveness of the cultured acini was assessed by the comparison of the release of protein in response to muscarinic cholinergic, alpha 1- and beta-adrenergic, and peptidergic stimulation of the cultured acini with the secretory response of incubated tissue slices. RESULTS: As examined by TEM, the ultrastructure of intact acini cultured for 3 and 7 days was notably similar to fresh tissue and incubated tissue slices. Recovery of the cultured acini from perturbations induced by the digestion procedure resulted in acini that were firmly attached to the filter supports and that exhibited histotypic acinar morphology, including lumenal microvilli, apically situated secretory granules and junctional complexes, lateral desmosomes, and appropriate secretory organelles. Secretory granules and associated organelles also were prominent in 14-day cultures; however, by day 14, as visualized by SEM, the acini had flattened. TEM of acini at this time showed that they had lost much of their acinar organization and cellular polarization. Outgrowths composed of acinar cells could be observed by day 7 by both TEM and SEM, and SEM demonstrated non-acinar cells growing on the filter surface in 14-day cultures. The morphologic and physiologic response of the acinar cells to autonomic agonists was similar in incubated tissue slices and cultured acini. Morphologically, acinar cells in both preparations responded to cholinergic stimulation by releasing secretory granules, resulting in honeycombed regions in the cell. Assay of secreted protein in response to cholinergic, alpha 1- and beta-adrenergic, and peptidergic stimulation indicated that 3-day cultures of acini retain the response to carbachol, phenylephrine, and vasoactive intestinal peptide. However, the response to isoproterenol is diminished when compared with incubated tissue slices. CONCLUSIONS: Under the conditions described, cultures of intact lacrimal acini from New Zealand white rabbits maintain histotypic morphology and physiologic responsiveness similar to fresh lacrimal gland and incubated tissue slices. Culture of the acini affords a useful alternative for the study of chronic, as well as acute, effects of neurotransmitters, neuropeptides, hormones, and cytokines on the structure and function of the gland.

Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures.

Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.

Culture of rat renal medullary tissue in media made hyperosmotic with NaCl and urea.

During antidiuresis, the rat kidney maintains a variable and steep osmotic gradient from the cortex (300 mOsm) to the inner medulla (at least 2,600 mOsm). Therefore, cells in the renal medulla must be able to adapt to a variably hyperosmotic environment. We have examined the ability of tissue fragments taken from various points on the cortical-medullary axis to survive and grow when cultured in media made hyperosmotic with urea and NaCl. Survival and growth were measured by the explants' ability to produce epithelial outgrowths. At osmotic concentrations of 1,100 and 1,200 mOsm, only explants from the inner medulla produced epithelial outgrowths. At 700 mOsm, all explants produced outgrowths but outgrowth size was a function of position on the cortical-medullary axis, with inner medullary fragments producing the largest outgrowths. Growth was most rapid at all osmolalities when the Na+:urea ratio was 1:1. These results are consistent with the hypothesis that renal medullary cells are adapted to elevated concentrations of Na+ and urea. Both explants and epithelial outgrowths were examined using light and electron microscopy. Physical continuities between the epithelial outgrowths and collecting duct epithelium in the explants, as well as the ultrastructural characteristics of the outgrowths at 700 mOsm, indicated that the outgrowths may have originated from collecting duct epithelium.

Effects of chronic nicotine on cerebral glucose utilization in the rat.

Effects of chronic nicotine on glucose utilization in 45 CNS regions were examined using the 2-deoxy-D-[1-14C]glucose technique in rats. Either L-nicotine (1.0 mg/kg) or saline was injected subcutaneously twice daily for 10 days and once the morning of day 11. On the following afternoon, rats received either nicotine challenge (0.3 mg/kg) or saline subcutaneously 2 min before an intravenous pulse of 2-deoxy-D-[1-14C]glucose. Drug-naive rats given nicotine challenge showed increases of glucose utilization in thalamic nuclei and components of the visual system. Tolerance to nicotine challenge in rats given nicotine chronically was seen in the ventral tegmental area, some components of visual pathways, the cerebellum, and vestibular nuclei; no regions showed sensitization. The percent of rats manifesting most nicotine-induced behaviors either increased (Straub tail, locomotor stimulation, tremors) or did not change (ataxia) over the 10 days of chronic treatment. Plasma from rats given nicotine chronically showed low concentrations of nicotine (12 +/- 1 ng/ml) and higher concentrations of cotinine (124 +/- 6 ng/ml) at the time of assay, with no apparent effect of chronic treatment on nicotine levels after the challenge dose. Changes in regional brain activity, as measured by the 2-deoxy-D-[1-14C]glucose technique, generally do not explain the observed sensitization to nicotine in behavioral assays.

Phagocytosis of Mycobacterium leprae by cardiac muscle cells--a preliminary report.

Fetal cardiac muscle cells were shown to ingest M. leprae easily within 20 minutes of exposure in vitro. This phagocytosis is considered nonspecific and facilitated by the lipid coat of the mycobacteria. The presence of M. leprae free in the cytoplasm of the muscle cells did not seriously affect the morphology or rhythmic contractions of the cells. The significance of the presence of M. leprae in somatic cells needs further study.