Efficient transduction of neural cells in vitro and in vivo by a baculovirus-derived vector

Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.     

Copy number variants in patients with short stature

Height is a highly heritable and classic polygenic trait. Recent genome-wide association studies (GWAS) have revealed that at least 180 genetic variants influence adult height. However, these variants explain only about 10% of the phenotypic variation in height. Genetic analysis of short individuals can lead to the discovery of novel rare gene defects with a large effect on growth. In an effort to identify novel genes associated with short stature, genome-wide analysis for copy number variants (CNVs), using single-nucleotide polymorphism arrays, in 162 patients (149 families) with short stature was performed. Segregation analysis was performed if possible, and genes in CNVs were compared with information from GWAS, gene expression in rodents'' growth plates and published information. CNVs were detected in 40 families. In six families, a known cause of short stature was found (SHOX deletion or duplication, IGF1R deletion), in two combined with a de novo potentially pathogenic CNV. Thirty-three families had one or more potentially pathogenic CNVs (n=40). In 24 of these families, segregation analysis could be performed, identifying three de novo CNVs and nine CNVs segregating with short stature. Four were located near loci associated with height in GWAS (ADAMTS17, TULP4, PRKG2/BMP3 and PAPPA). Besides six CNVs known to be causative for short stature, 40 CNVs with possible pathogenicity were identified. Segregation studies and bioinformatics analysis suggested various potential candidate genes.     

Color change of some aesthetic dental materials: Effect of immersion solutions and finishing of their surfaces

ObjectivesThe purpose of this research was to evaluate the color change of five aesthetic dental materials, before and after immersion in distilled water and blue food color solution for 7 and 21 days, and to study the effect of finishing the surfaces on any color change.MethodsDisc shaped samples of five types of light curing composite (A2) (n = 10 samples/composite) were prepared and all samples were light-cured with a Plasma Arc light cure unit for ten seconds. One side of each sample disc was finished and polished with a Super-Snap system all samples. After 24 h, color measurements of each sample were conducted using a digital spectrophotometer. Five sample discs from each composite group were immersed in 30 ml of food color solution for 7 and 21 days, while the remaining five sample discs were immersed in 30 ml of distilled water as a control. Color measurements were repeated for all samples at 7 and 21 days after immersion. The color changes were statistically analyzed using t-tests within the same group. A result was considered statistically significant at α = 0.05.ResultsThe color differences (ΔE) ranged from 0.4 to 4.66 and statistically significant differences on the finished and unfinished surfaces were observed after immersion in the food color solution for 7 days. No significant differences were found in any group after immersion in the food color solution for 21 days. The Tetric EvoCeram and Arabesk groups showed less color differences after 7 and 21 days than other composites.ConclusionFinished composite surfaces showed less coloration than unfinished surfaces after 7 days, but all surfaces (finished and unfinished) were highly colored for all composite types after 21 days.     

Ischemic stroke in the setting of chronic immune thrombocytopenia in an elderly patient--a therapeutic dilemma

Chronic immune thrombocytopenia (ITP) carries a poor prognosis in the elderly patients. Increasing evidence proposes that a subgroup of patients with chronic ITP may be more susceptible to ischemic stroke. An 84-year-old Caucasian man with multiple ischemic stroke risk factors presented with acute onset of slurred speech, confusion, and unsteady gait. Physical examination and neurologic imaging were consistent with a new left thalamic infarct. Platelet counts ranged between 40 000 × 10(9)/L and 65 000 × 10(9) /L. Antiplatelet therapy for his newly acquired stroke was not initiated considering his low platelet counts and for mildly symptomatic thrombocytopenia, and the patient was discharged home. Both hematologic and neurologic guidelines for the management of chronic ITP and stroke have contradictory goals. Although anticoagulation is mandated in acute stroke, ITP causes low platelet counts that increase bleeding complications.     

Antithrombotic Therapy for Stroke Prevention in Patients With Heart Failure

OPINION STATEMENT: Congestive heart failure (CHF) is associated with an increased risk of stroke mainly due stasis leading to increased risk of thrombus formation in the left ventricle and subsequent cerebral embolism. CHF patients are also at increased risk of atrial fibrillation (AF) that also leads to cerebral embolism. Aggressive medical management to prevent cardiac decompensation and maintain sinus rhythm is indicated in CHF patients. All patients with CHF and AF should be anticoagulated with warfarin or one of the newer oral anticoagulants. There is no clear indication for anticoagulation in CHF patients due to ischemic cardiomyopathy who are in sinus rhythm. Based on data from the WARCEF study (see below), those patients with CHF due to non-ischemic etiologies who are in sinus rhythm and have a left ventricular ejection fraction (LVEF) less than 30?% to 35?% may benefit from warfarin for the reduction of ischemic stroke risk, but warfarin does not increase survival. Whether warfarin is particularly beneficial for CHF patients who have a prior history of stroke or transient ischemic attack (TIA) is unknown. If, however, there is high enough suspicion that the stroke was of cardioembolic origin in patients with low LVEF, then anticoagulation would possibly be a reasonable option for prevention of recurrent stroke or TIA. Warfarin is indicated for stroke prophylaxis among those CHF patients who have an implanted mechanical device. The role of newer anticoagulants in patients with CHF who do not have AF is unknown at this time. Theoretically, there should be no reason against using these agents in place of warfarin in selected patients, particularly those with highly variable International Normalized Ratios (INR) in the context of warfarin therapy, but the newer anticoagulants have not yet been studied among CHF patients without concomitant AF.     

CD44 enhances tumor formation and lung metastasis in experimental osteosarcoma and is an additional predictor for poor patient outcome

Formation of metastases in the lungs is the major cause of death in patients suffering from osteosarcoma (OS). Metastases at presentation and poor response to preoperative chemotherapy are strong predictors for poor patient outcome. The elucidation of molecular markers that promote metastasis formation and/or chemoresistance is therefore of importance. CD44 is a plasma membrane glycoprotein that binds to the extracellular matrix component hyaluronan (HA) and has been shown to be involved in metastasis formation in a variety of other tumors. Here we investigated the role of CD44 expression on OS tumor formation and metastasis. High CD44 expression, evaluated with a tissue microarray including samples from 53 OS patients and stained with a pan‐CD44 antibody (Hermes3), showed a tendency (p < 0.08) to shortened overall survival. However, nonresponders and patients with lung metastases and high CD44 expression had significantly poorer prognosis than patients with low CD44 expression. Overexpression of the standard CD44 isoform (CD44s) and its HA‐binding defective mutant R41A in osteoblastic SaOS‐2 cells resulted in HA‐independent higher migration rates and increased chemoresistance, partially dependent on HA. In an orthotopic mouse model of OS, overexpression of CD44s in SaOS‐2 cells resulted in an HA‐dependent increased primary tumor formation and increased numbers of micrometastases and macrometastases in the lungs. In conclusion, although CD44 failed to be an independent predictor for patient outcome in this limited cohort of OS patients, increased CD44 expression was associated with even worse survival in patients with chemoresistance and with lung metastases. CD44‐associated chemoresistance was also observed in vitro, and increased formation of lung metastases was found in vivo in SCID mice. © 2013 American Society for Bone and Mineral Research.     

Impact of Preoperative Breast MRI on Surgical Decision Making and Clinical Outcomes: A Systematic Review

Background

Preoperative magnetic resonance imaging (MRI) is increasingly used in the workup of breast cancer patients and could lead to changes in surgical management. It is unclear how the information gained from MRI studies affects surgical decision making and influences clinical outcomes. These issues are addressed in this review.

Methods

PubMed database searches were performed to retrieve and analyze respective original research and review articles on preoperative MRI in the evaluation of breast cancer patients.

Results

Preoperative MRI is a highly sensitive but nonspecific method that leads to changes in surgical management with increased numbers of more extended surgical interventions. It appears that a relatively large proportion of MRI-driven changes in surgical management result in overtreatment without conclusively proven beneficial effects on such clinical outcomes as decrease in reoperation rates or improved patient survival.

Conclusions

Thus, routine use of supplementary preoperative breast MRI should be discouraged until compelling evidence of its effectiveness is available.     

Hair Mercury and Fish Consumption in Residents of O‘ahu,Hawai‘i

Recent studies have established that men are susceptible to cardiotoxicity from methylmercury exposure, which also poses risks to the pregnant woman. Hair samples were obtained and questionnaires for methylmercury exposure assessment were administered to 110 adults (57 men, 53 women) throughout O‘ahu, Hawai‘i during December 2010 to January 2011. Hair samples were analyzed for total mercury with a direct mercury analyzer. Men ≥ 46 years had a median of 2.0 µg/g, which was above the reference dose of 1 µg/g, as compared to younger men with a median 1.0 µg/g (P < 0.05). Hair concentrations from older women had a median of 1.2 µg/g of mercury compared to 0.6 µg/g for younger women. Additionally, 38% of women of childbearing age had a Hazard Index > 1.0. This indicates that both men and women were at risk for excessive methylmercury exposure. In the final regression model, male gender, age > 45 years, length of residency > 10 years in Hawai‘i, and fish consumption frequency > 1 meal per week were significant factors in increased hair mercury levels. Following safe fish consumption practices allows residents to reap health benefits of fish consumption without excessive toxicant exposure.     

Optimizing dose and scheduling of filgrastim (granulocyte colony- stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers [see comments]

To define an optimal regimen for mobilizing and collecting peripheral blood progenitor cells (PBPC) for use in allogeneic transplantation, we evaluated the kinetics of mobilization by filgrastim (recombinant met- human granulocyte colony-stimulating factor [r-metHuG-CSF]) in normal volunteers. Filgrastim was injected subcutaneously for up to 10 days at a dose of 3 (n = 10), 5 (n = 5), or 10 micrograms/kg/d (n = 15). A subset of volunteers from each dose cohort underwent a 7L leukapheresis on study day 6 (after 5 days of filgrastim). Granulocyte-macrophage colony-forming cell (GM-CFC) numbers in the blood were maximal after 5 days of filgrastim; a broader peak was evident for CD34+ cells between days 4 and 6. The 95% confidence intervals (CI) for mean number of PBPC per milliliter of blood in the three dose cohorts overlapped on each study day. However, on the peak day, CD34+ cells were significantly higher in the 10 micrograms/kg/d cohort than in a pool of the 3 and 5 micrograms/kg/d cohorts. Mobilization was not significantly influenced by volunteer age or sex. Leukapheresis products obtained at the 10 micrograms/kg/d dose level contained a median GM-CFC number of 93 x 10(4)/kg (range, 50 x 10(4)/kg to 172 x 10(4)/kg). Collections from volunteers receiving lower doses of filgrastim contained a median GM- CFC number of 36 x 10(4)/kg (range, 5 x 10(4)/kg to 204 x 10(4)/kg). The measurement of CD34+ cells per milliliter of blood on the day of leukapheresis predicted the total yield of PBPC in the leukapheresis product (r = .87, P < .0001). Assuming a minimum GM-CFC requirement of 50 x 10(4)/kg (based on our experience with autologous PBPC transplantation), all seven leukapheresis products obtained at the 10 micrograms/kg/d dose level were potentially sufficient for allogeneic transplantation purposes. We conclude that in normal donors, filgrastim 10 micrograms/kg/d for 5 days with a single leukapheresis on the following day is a highly effective regimen for PBPC mobilization and collection. Further studies are required to determine whether PBPC collected with this regimen reliably produce rapid and sustained engraftment in allogeneic recipients.