Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells

A large number of biologic, technological, and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long- term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures, and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established, long- term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long- term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore, such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion.     

Neuronal Nitric Oxide Synthase is a Key Factor in Doxorubicin-Induced Toxicity to Rat-Isolated Cortical Neurons

Doxorubicin (DOX) is neurotoxic to serum-free cultures of rat cortical neurons in a biphasic concentration manner. For concentrations up to 0.5 μM, cell death follows an apoptotic pattern, while for higher concentrations apoptosis is inhibited and necrosis becomes dominant. Considering the potential toxic effects of DOX resulting from its redox-cycling, in this study we investigated the generation of reactive species and subsequent oxidative stress effects, formation of quinoproteins, activation of NF-kB, depletion of energy levels and consequent cell death in cultures of primary rat cortical cells challenged with this antitumour drug. The influence of neuronal nitric oxide synthase (nNOS) on DOX-induced neuronal cell damage was subsequently evaluated. The exposure of rat cortical primary cell cultures to DOX resulted in a significant generation of ROS/RNS, activation of NF-kB, depletion of GSH levels, depletion of ATP, and cell death, in a concentration biphasic manner. Doxorubicin also significantly increased protein-bound quinone products in neurons in a concentration-dependent manner. Inhibition of nNOS decreased neuronal cell death induced by DOX in a significant way, at the first phase of the biphasic curve. In conclusion, this study shows, for the first time, a clear involvement of nNOS and subsequent ROS/RNS generation as crucial signalling mediators of DOX-induced neurotoxicity on isolated cortical neurons. Inhibition of ROS/RNS formation, modulation of NOS isoforms and modulation of NF-kB activation could be of beneficial in preventing damage in the CNS caused by DOX.     

US-guided percutaneous biopsy: use of a screw biopsy stylet to aid needle detection

Insertion of a screw biopsy stylet into a thin-walled biopsy needle greatly enhances detection of the needle during ultrasound-guided percutaneous biopsy. This technique is helpful when precise needle-tip localization is needed for biopsies of small lesions.     

Percutaneous cholecystography in children

Percutaneous cholecystography was performed on 13 children who had biliary system abnormalities: two had biliary hypoplasia, five had sclerosing cholangitis, three had cirrhosis, two had distal choledochal obstruction, and one had an obstructed portoenterostomy. In 12 patients transcholecystic cholangiography showed, without significant complications, the intra-and extrahepatic bile ducts. In one patient with primary sclerosing cholangitis, the intrahepatic bile ducts were not opacified satisfactorily; dilatation of the gallbladder required surgical drainage. The transcholecystic technique is indicated when the intrahepatic bile ducts are either mildly dilated or not dilated.     

Risk of breast implant‐associated anaplastic large cell lymphoma in patients submitted to breast implantation: A systematic review

Although breast implant‐associated anaplastic large cell lymphoma (BIA‐ALCL) is a rare disease, its incidence has been increasing. The aim of this study was to assess the risk of BIA‐ALCL in women with breast implants. A systematic search was carried out in Pubmed, Scopus, ScienceDirect, LIVIVO, Cochrane Library, Google Scholar, and OpenGrey databases. The risk assessment of bias was based on the Newcastle‐Ottawa Scale. The rarity of BIA‐ALCL was a major limitation. Although we have found evidence of an increased risk of BIA‐ALCL, further studies are needed to understand why some large samples did not present any case of the disease.     

The effect of lysosome permeability changes on DNA synthesis and mitosis of cells growing in vitro

Summary Cultures of sheep embryonic cells, when actively growing, were incubated with quinacrine (a lysosome tagg) at concentrations of 10^–7, 10^–6, 10^–5 g/ml of culture medium, for 24 hours. The mean number of mitosis and 24 hour thymidine labelled cells are an exponential function of the initial dye concentration on the culture medium. The variation of S compartment (on one hour pulse autoradiographs) suggests two different transit times of cell population at 10^–6 and 10^–7 g/ml. The distribution obtained by plotting the percentages of cells with acid phosphatase against mitotic index points to an inverse correlation between these two parameters. Multinucleation in cells obtained from 10^–5 g/ml preparations was six times more frequent as compared to the controls. In such preparations 3.2% of the cells showed nucleus-cytoplasmatic bridging with no thymidine label. The intravital stained bodies (the lysosomes) contain acid phosphatase, a PAS positive substance, ribonucleoprotein and a blue fluorescent material resisting nuclease digestion.

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Zusammenfassung Wachsende Kulturen von Embryonalzellen des Schafes wurden mit Quinacrin, einem die Lysosomen markierenden Farbstoff, in verschiedener Konzentration (10^–5, 10^–6 und 10^–7 g/ml des Kulturmediums) für 24 Std. inkubiert. Die durchschnittliche Zahl der Mitosen und der 24stündlich mit Thymidin markierten Zellen entsprechen einer Exponentialfunktion der anfänglichen Farbstoffkonzentration im Kulturmedium. Die Variationen der S-Phase (gemessen an autoradiographischen Impulsen in einer Stunde) legt das Vorhandensein zweier verschiedener Zeiten für Zellpopulationen bei 10^–6 und 10^–7 g/ml nahe. Der Vergleich des Prozentsatzes der Zellen mit sauerer Phosphatase mit dem Mitoseindex spricht für ein gegensätzliches Verhalten beider Parameter. Vielkernigkeit war bei den Zellen von den 10^–5 g/ml-Präparaten sechsmal häufiger als in den Kontrollen. In den Präparaten zeigten 3,2% der Zellen Kern-Cytoplasma-Brücken ohne Thymidinmarkierung. Die intravital gefärbten Lysosomen enthielten sauere Phosphatase, eine PAS-positive Substanz, Ribonucleoprotein und einen blau fluorescierenden Stoff, der resistent war gegen Nucleaseverdauung.

     

Identification of periodontal pathogens and severity of periodontitis in patients with and without chronic kidney disease

Objective

In this study of patients with chronic periodontitis (CP), the severity of the disease and the main periodontal pathogens identified in patients with chronic kidney disease (CKD) were compared with those detected in individuals without systemic disease.

Design

Nineteen patients with CP without evidence of systemic disease (control group), 25 patients with CP and CKD who were in the pre-dialysis stages (pre-dialysis group), and 22 patients with CP and CKD who were on renal replacement therapy (RRT group) were examined. The severity of CP was based on the investigation of probing depth (PD) and clinical attachment level (CAL). The definition and stage of CKD were based on the criteria proposed by the Kidney Disease Outcomes Quality Initiative of the National Kidney Foundation. Glomerular filtration rate (GFR) was estimated using the equation of Modification of Diet in Renal Disease and the identification of microorganisms in subgingival plaque was performed using polymerase chain reaction (PCR).

Results

Candida albicans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were more common in patients who were on RRT and pre-dialysis than in control subjects. CP was more severe in patients with CKD. A strong association was observed between the frequency of C. albicans (P = 0.056), P.gingivalis (P = 0.008), T. denticola (P = 0.013) and CAL, when CKD patients were compared with the control group.

Conclusion

CP is more severe and is associated with increased frequency of C. albicans, P. gingivalis, T. forsythia, and T. denticola in patients with CKD.     

Simple measurement of gluconeogenesis by direct 2H NMR analysis of menthol glucuronide enrichment from 2H2O.

The contribution of gluconeogenesis to fasting glucose production was determined by a simple measurement of urinary menthol glucuronide (MG) 2H enrichment from 2H2O. Following ingestion of 2H2O (0.5% body water) during an overnight fast and a pharmacological dose (400 mg) of a commercial peppermint oil preparation the next morning, 364 micromol MG was quantitatively recovered from a 2-h urine collection by ether extraction and a 125 micromol portion was directly analyzed by 2H NMR. The glucuronide 2H-signals were fully resolved and their relative intensities matched those of the monoacetone glucose derivative. The pharmacokinetics and yields of urinary MG after ingestion of 400 mg peppermint oil as either gelatin or enteric-coated capsules 1 h before breakfast were quantified in five healthy subjects. Gelatin capsules yielded 197 +/- 81 micromol of MG from the initial 2-h urine collection while enteric-coated capsules gave 238 +/- 84 micromol MG from the 2- to 4-h urine collection.     

Rupture of the posterior tibial tendon: CT and surgical findings

Computed tomography (CT) was performed in 42 patients with 49 clinically suspected tears of the posterior tibial tendon. Twenty-eight of the 49 suspected tears were subsequently surgically explored and repaired. Three patterns of tendon abnormalities were recognized on CT scans: type I-intact, hypertrophied, heterogeneous tendon; type II-attenuated tendon; and type III-absence of a portion of a tendon. Types I and II correlated with partial rupture seen during surgery, and type III correlated with complete rupture of the tendon. CT findings were accurate in 96% of the patients who underwent surgery. In four cases (14%), tendon rupture was seen on CT scans, but the extent of the injury was underestimated and the rupture was misclassified. Reactive periostitis of the distal tibia was seen in 71% of diseased tendons and may represent an important factor in the diagnosis of tendon rupture.