The synergistic effects of microfracture, perforated decalcified cortical bone matrix and adenovirus-bone morphogenetic protein-4 in cartilage defect repair

We reported a technique for articular cartilage repair, consisting of microfracture, a biomaterial scaffold of perforated decalcified cortical bone matrix (DCBM) and adenovirus-bone morphogenetic protein-4 (Ad-BMP4) gene therapy. In the present study, we evaluated its effects on the quality and quantity for induction of articular cartilage regeneration. Full-thickness defects were created in the articular cartilage of the trochlear groove of rabbits. Four groups were assigned: Ad-BMP4/perforated DCBM composite (group I); perforated DCBM alone without Ad-BMP4 (group II); DCBM without perforated (group III) and microfracture alone (group IV). Animals were sacrificed 6, 12 and 24 weeks postoperation. The harvested tissues were analyzed by magnetic resonance image, scanning electron microscope, histological examination and immunohistochemistry. Group I showed vigorous and rapid repair leading to regeneration of hyaline articular cartilage at 6 weeks and to complete repair of articular cartilage and subchondral bone at 12 weeks. Groups II and III completely repaired the defect with hyaline cartilage at 24 weeks, but group II was more rapid than group III in the regeneration of repair tissue. In group IV the defects were concave and filled with fibrous tissue at 24 weeks. These findings demonstrated that this composite biotechnology can rapidly repair large areas of cartilage defect with regeneration of native hyaline articular cartilage.     

Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three-stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore, we have provided evidence that the hESC-derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen, and generation and secretion of plasma proteins. More importantly, the hESC-derived hepatocytes express several members of cytochrome P450 isozymes, and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes, which may be useful as an in vitro system for toxicity screening in drug discovery.     

Determination of Red Blood Cell Velocity by Video Shuttering and Image Analysis

A novel modification of conventional video imaging techniques has been developed to determine the velocity of red blood cells (RBCs), which offers compatibility with existing video-based methods for determining blood oxygenation and hemoglobin concentration. Traditional frame-by-frame analysis of video recordings limits the maximum velocity that can be measured for individual cells in vivo to about 2 mm/s. We have extended this range to about 20 mm/s, by electronic shuttering of an intensified charge-coupled device camera to produce multiple images of a single RBC in the same video frame. RBCs were labeled with fluorescein isothiocyanate and the labeled cells (FRBCs) were used as probes to determine RBC velocities in microvessels of the hamster retractor muscle. Velocity was computed as the product of the distance between centroids of two consecutive image positions of a FRBC and the shuttering frequency of the camera intensifier. In vitro calibrations of the system using FRBC and Sephadex beads coated onto a rotating disk yielded an average coefficient of variation of about 6%. Flow conservation studies at bifurcations indicated that the maximum diameter of microvessels below which all the FRBCs in the lumen could be detected was 50 m. The technique was used to estimate mean-FRBC velocity distributions in vessels with diameters ranging from 8 to 50 m. The mean-FRBC velocity profiles were found to be blunter than would be expected for Poiseuille flow. Single FRBCs tracked along an unbranched arteriole exhibited significant temporal variations in velocity. © 1999 Biomedical Engineering Society. PAC99: 8719Tt, 8717Jj, 4279Pw, 8780Tq, 8719Ff, 4230Va, 0705Pj     

Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones

Hypercapnia as well as lowered intracellular pH (pH_i) increase the bioelectric activity of CO₂/H⁺-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na⁺/H⁺ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5–1 μmol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO₂: 60–100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05–0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO₂/H⁺-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 μmol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO₂/H⁺-sensitive neurones within the ventrolateral medulla oblongata. Received: 12 February 1999 / Received after revision and accepted: 15 April 1999     

N-Terminal deletions of rKv1.4 channels affect the voltage dependence of channel availability

Rat Kv1.4 potassium channels undergo rapid inactivation, which is mediated by the N-terminal structure of the polypeptide. This inactivation can be removed by N-terminal deletion of about 20 residues. However, more substantial deletion (e.g. 37 residues) restores inactivation suggesting a second inactivating domain [Kondoh et al. J Biol Chem 272:19333–19338, 1997]. Here we provide evidence that this inactivation shares all properties with N-type inactivation. Pore mutations, which are supposed to affect C-type inactivation, have no effect. In addition, the redox regulation of inactivation, which is typical for Kv1.4 channels, can be conferred to the inactivation of the deleted constructs by incorporation of an N-terminal cysteine residue. The most remarkable feature of this secondary inactivation is the existence of two components in the steady-state voltage dependence of inactivation. For mutant rKv1.4Δ2–37 about 90% of the channels only activate when the holding membrane potential is more negative than about –120 mV; the remaining 10% show the typical threshold at –60 mV. Mutagenesis of the truncated channel affected the relative amplitudes of these two components, but not the voltage dependence. The results suggest that the secondary ball structures of rKv1.4 channels interact with the protein structures responsible for activation. Received: 3 February 1999 / Accepted: 15 March 1999     

Cytologic features of lymphoepithelial cyst of the pancreas: two preoperatively diagnosed cases based on fine-needle aspiration.

We describe the cytologic features seen in fine-needle aspiration (FNA) specimens from two cases of preoperatively diagnosed lymphoepithelial cyst (LEC) of the pancreas. Pancreatic LEC is a rare, true cyst of uncertain histogenesis that may clinically and radiologically mimic a pseudocyst or cystic neoplasm. Both our patients were middle-aged men who presented with vague abdominal pain. Computed tomography (CT) of the abdomen revealed a mass in or around the pancreas, and CT-guided percutaneous FNA (patient 1) and endoscopic ultrasound-guided FNA (patient 2) yielded paste-like yellow-gray material. Cytologic smears showed numerous anucleated squamous cells in a background of keratinous and amorphous debris. A few benign nucleated squamous cells and plate-like cholesterol crystals were also seen. Unlike LEC of the head and neck region, only rare lymphocytes and histiocytes were present. Pancreatic LEC was diagnosed based on these cytologic findings and was histologically confirmed following cyst enucleation (patient 1) and partial pancreatectomy (patient 2). We conclude that preoperative FNA and recognition of the characteristic cytologic pattern will enable conservative surgical management of pancreatic LEC. Diagn. Cytopathol. 1999;21:346-350.     

Roles of PilC and PilE Proteins in Pilus-Mediated Adherence of Neisseria gonorrhoeae and Neisseria meningitidis to Human Erythrocytes and Endothelial and Epithelial Cells

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coli interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.     

FTDP‐17 with Pick body‐like inclusions associated with a novel tau mutation,p.E372G

Mutations in microtubule‐associated protein tau gene (MAPT) cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP‐17). Here, we describe a patient with FTDP‐17 and a novel missense mutation in exon 13 of MAPT, p.E372G. We compare clinicopathologic features of this patient to two previously unreported patients with another exon 13 mutation, p.G389R. The patient with the p.E372G mutation was a 40‐year‐old man with behavioral variant frontotemporal dementia (bvFTD), who subsequently developed agrammatic speech and parkinsonism. One of the FTDP‐17 patients with p.G389R mutation presented at age 24 with agrammatic variant of primary progressive aphasia, and subsequently behavioral dysfunction. The other presented at age 53 with bvFTD, followed by agrammatic speech and corticobasal syndrome. Neuropathologic features of FTDP‐17 due to p.E372G were similar to those of p.G389R, including tau‐immunoreactive Pick body‐like neuronal inclusions and swollen, tapering thread‐like processes in white matter immunoreactive for 3‐repeat and 4‐repeat tau. Biochemical analysis of insoluble tau showed similar isoform compositions in p.E372G and p.G389R. Functional studies of the p.E372G mutation showed marked increase in tau filament formation and its reduced ability to promote microtubule assembly. Together these findings indicate that p.E372G is a pathogenic MAPT mutation that causes FTDP‐17 similar to p.G389R.     

Epidemiology of anaphylaxis at a tertiary care center: A report of 730 cases

Background

Recent data reveal that the rate of anaphylaxis is increasing and suggest that idiopathic anaphylaxis may account for most of these cases.