A 51-year-old man with a diagnosis of myelodysplasia and non-Hodgkin's lymphoma underwent an unmatched allogenic bone marrow transplantation and was treated posttransplant with chronic immunosuppressive medication. Eight months following transplantation, he presented with progressive dysarthria, cognitive and visual decline. Evaluation included brain magnetic resonance (MR) imaging demonstrating multifocal areas of increased T2 and FLAIR (fluid attenuated inversion recovery) signals involving the left frontal, parietal, and occipital lobes. The MR lesions demonstrated diffuse increased signal on DWI (diffusion-weighted images) and normal to low signal on ADC (apparent diffusion coefficients). Contrast-enhanced T1 images were unremarkable. Lumbar puncture revealed a mild elevation in cerebrospinal fluid (CSF) protein. CSF PCR assay for viral DNA fragments were negative on two occasions. Serum serology for HIV was negative as well. A brain biopsy was subsequently performed. The clinical and neuroimaging differential diagnoses as well as neuropathologic correlation are presented.,

Blood. 2006 March 1; 107(5): 2170–2179.

Gene array analysisTotal RNA was isolated from passage 1 (young p1) and passage 5 (aged p5) MEFs with a RNeasy mini kit (Qiagen, Venlo, The Netherlands). During cDNA synthesis, samples were labeled with [33P]-deoxycytidine triphosphate (dCTP; MP Biochemicals, Irvine, CA). Mouse filter gene arrays (GF400a; Research Genetics, Invitrogen) were hybridized and analysis was performed as described previously.34 Expression patterns were verified by reverse transcription-polymerase chain reaction (RT-PCR).

Blood. 2006 March 1; 107(5): 2170–2179.

AnimalsTimed pregnant female C57BL/6 mice were used to obtain day 14 postcoitus embryos from which mouse embryonic fibroblasts (MEFs) were isolated. Eight- to 12-week-old female B6.SJL-PtprcaPep3b/BoyJ (CD45.1) mice were used as donors for transplantation and were bred at the Central Animal Facility of the University of Groningen. Eight- to 12-week-old female C57BL/6 (CD45.2) mice were purchased from Harlan (Horst, The Netherlands) and were used as recipients.

Blood. 2006 March 1; 107(5): 2170–2179.

Retroviral overexpression of Ezh2 in HSCsPrimary bone marrow (BM) cells were transduced as previously described with some adjustments.35 Briefly, BM cells were extracted from mice (CD45.1) given intraperitoneal injections of 150 mg/kg 5-fluorouracil (5-FU; Pharmachemie Haarlem, Haarlem, The Netherlands) 4 days earlier. Cells were cultured for 48 hours in StemSpan (StemCell Technologies, Vancouver, BC, Canada) supplemented with 10% FCS, 300 ng/mL PEG-rrSCF (Amgen, Thousand Oaks, CA), 20 ng/mL rmIL-11 (R&D Systems, Minneapolis, MN), 10 ng/mL Flt3 ligand (Amgen), penicillin, and streptomycin. Viral supernatant was harvested 24 and 48 hours after transfection of ecotropic Phoenix cells and inoculated in 6-well plates that were coated with 50 μg retronectin (Takara, Kyoto, Japan). Plates containing the viral supernatant were spun for 1 hour at 800 g at room temperature. Four hours later viral supernatant was removed, and 7.5 × 105 cultured BM cells were inoculated per well together with 4 μg Polybrene (Sigma). At the second transduction 2 μg Polybrene was added. Four days later transduction efficiency was determined by flow cytometry (FACSCalibur; Becton Dickinson, Palo Alto, CA).

Blood. 2006 March 1; 107(5): 2170–2179.

In vitro differentiation of purified HSCsPurified Lin-Sca-1+c-kit+ (LSK) cells were cultured in IMDM (Invitrogen) containing 100 ng/mL PEG-rrSCF (Amgen) and 10 ng/mL GM-CSF (Behringwerke, Marburg, Germany) to stimulate rapid in vitro differentiation. Total cell number was assessed using trypan blue exclusion. At different time points during culturing RNA was isolated for gene expression analysis.

Blood. 2006 March 1; 107(5): 2170–2179.

Western blottingCells were resuspended in PBS, lysed by addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and sonicated. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. After blocking in 3% skim milk, membranes were probed with a 1:100 dilution of mouse anti-Ezh2 mAb (M18EZH2, kindly provided by Prof Dr A. P. Otte, Swammerdam Institute for Life Science, University of Amsterdam, The Netherlands37,38), washed, and incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Buckinghamshire, United Kingdom). Membranes were developed using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences). Equal loading of membranes was verified with rabbit anti-γ-tubulin (Sigma).

Blood. 2006 March 1; 107(5): 2170–2179.

Quantitative PCRTotal RNA was isolated using the RNeasy Mini kit (Qiagen) and standard cDNA synthesis was performed. Quantitative PCRs (qPCRs) were performed in triplicate. PCR amplification, using SYBR Green, was performed in 96-well microtiter plates in an iCycler thermal cycler (Bio-Rad, Hercules, CA). Sample cDNAs were compared with expression of house-keeping genes Gapdh and actin using the relative quantification ΔΔCT technique.39 Relative expression levels in the different LSK populations were estimated by first calculating the number of molecules formed at reaching the CT. By correcting this value for the initial number of cells used, relative expression was determined.

Blood. 2005 September 1; 106(5): 1574–1580.

MiceSingle-cell suspensions for FACS analysis and RNA extraction were prepared from 8- to 10-week-old female C57BL/6j mice. Transplantation studies used 8-week-old male donor C57BL/6-Ly5.1 (CD45.1; B6.SJL-Ptprca Pep3b/BoyJ) or C57BL/6-GFP mice that constitutively and ubiquitously express the enhanced green fluorescent protein (GFP) reporter gene under regulatory control of the chicken beta-actin promoter with cytomegalovirus enhancer (C57BL/6j-Tg(ACTB-EGFP)1Osb/J). Recipient mice were congenic 8- to 10-week-old female C57BL/6-Ly5.2 (CD45.2). All mice were obtained from Jackson Labs (Bar Harbor, ME) and maintained in a pathogen-free animal facility. Animal studies were approved by the Standing Committee on Animals of Harvard Medical School.

Blood. 2005 September 1; 106(5): 1574–1580.

Flow cytometryCell sorting was performed on a MoFlo triple laser instrument (DakoCytomation, Fort Collins, CO) using Summit 3.1 software (Dako Cytomation). Analysis of raw data was completed with FlowJo software (Treestar, Ashland, OR). The laser emissions were 488, 350, and 647 nm. Fluorescence was detected with the following bandpass filters: 530/40 for FITC and GFP, 580/30 for PE, 670/30 for PI, 405/30 for Hoechst blue, 570/20 for Hoechst red, and 670/20 for APC (Omega Optical, Brattleboro, VT). Hoechst blue and red fluorescence were detected in linear-scale acquisition. First, a live cell gate was created excluding cell fragments (low forward scatter) or events that contained high PI fluorescence. Cells within this live cell gate were displayed on a Hoechst-red–Hoechst-blue histogram, and side population (SP) cells were identified after collecting at least 1 × 105 events (gating algorithm illustrated in Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).

Blood. 2005 September 1; 106(5): 1574–1580.

Reverse-transcription–polymerase chain reaction (RT-PCR)Total RNA was extracted from 8500 cells sorted from each bone marrow or liver population (Absolutely RNA Nanoprep Kit; Stratagene, La Jolla, CA). DNase treatment of all RNA extracts was included according to the manufacturer's instructions. cDNA was prepared by reverse transcription under standard conditions using random hexamer priming (First-Strand cDNA Synthesis Kit; Amersham Biosciences). PCR amplification of c-kit and glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA was performed using FastStart Taq DNA Polymerase (Roche) and the following primer pairs: c-kit forward, 5′-GCACTTGAGTGCTACACTCTTGCACCT-3′; c-kit reverse, 5′-TCTTCAGAACTGTCAACAGTTGGACAACA-3′; GAPDH forward, 5′-TTCCAGTATGACTCCACTCACG-3′; and GAPDH reverse, 5′-GTTCACACCCATCACAAACATG-3′. Conventional PCR conditions using one primer pair per reaction tube were as follows: 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, 40 cycles. Multiplex PCR to amplify both genes in the same reaction tube was performed under identical conditions with both primer pairs added to the same PCR reaction tube.

Ann Gen Psychiatry. 2007; 6: 25.

The patient was a 55-year-old married female, mother of two healthy grown-up children, with a history of type-1 DM since the age of 11. For her diabetes, she was initially treated with insulin (Novolente) once and then twice daily for 35 years. Subsequently, she was administered an intensified insulin regimen comprising insulin (NPH) twice daily and rapid-acting regular insulin three times daily pre-prandially, followed by a regimen of NPH and very-rapid acting insulin analogue (lispro) pre-prandially. During the last 3 years the patient has been treated with continuous subcutaneous insulin infusion through a pump, requiring 18–20 units/day as a basal regimen and a total of around 18–20 units/day as bolus injections. However, during the last year, due to the patient's poor compliance with the antidiabetic regimen and the required dietary pattern, her glucemic control deteriorated markedly, with frequent hypoglycemic episodes (and even comas) 2–3 times a week, followed by hyperglycemia events. In fact, the patient often felt hungry and, after the extra meals and fearing that her glucose level had risen excessively, she self-administered more insulin than required, thus causing the hypoglycemic episodes. Over the years she had also developed severe diabetic retinopathy, requiring laser treatments, as well as renal vascular complications of DM.

Ann Gen Psychiatry. 2007; 6: 25.

Case ReportA 73-year-old white man presented complaining of recent episodes of spontaneous bleeding. He had noticed 3 nose bleeds in the prior month, ecchymosis on his hands and arms after very minor trauma, frequent hemorrhoidal bleeding, and bleeding from his ear after striking it with a blunt comb. The patient was a healthy, retired pediatrician. He had a remote history of bleeding after using aspirin, occasional, mild hemorrhoidal bleeding, and only 1 or 2 previous nosebleeds. The patient had no excessive bleeding after previous surgical procedures. He was taking no prescription medications, but did take vitamins A, C, D, E, and folic acid for preventive care and had used ginkgo, 75 mg per day, for the previous 6 months as an aid to his “failing memory.” The ginkgo supplement, which was produced by a large U.S. manufacturer and distributed by a large supermarket chain, was standardized to 27% ginkgo flavone glycosides and 10% terpene lactones. He had never been diagnosed with cognitive dysfunction, but felt that ginkgo improved both his memory and clarity of thought.

PLoS Med. 2006 October; 3(10): e420.

Tumor SamplesTumors for this study were randomly selected from 700 freshly frozen lung cancer tissues from patients who had undergone complete, curative resection of their disease from 1993 to 2001 at Johns Hopkins Hospital, Baltimore, Maryland, United States. Tumors were macrodissected and only those specimens with greater than 50% neoplastic cells were used. A total of 56 cases of lung tumor, including 40 paired lung tumors and adjacent normal tissues (frozen tissue) and 16 pleural fluid (PF) samples, were chosen in accordance with the Institutional Review Board protocol, and DNA was isolated using DNeasy Kit (Qiagen, Valencia, California, United States). Details of the lung tumor samples are listed in Table S1.

PLoS Med. 2006 October; 3(10): e420.

Western Blot AnalysisTo obtain total protein lysates, cancer cells or tissues were lysed in 50 mM Tris (pH 7.2), 1% Triton X-100 containing Halt Protease Inhibitor cocktail (Pierce, Rockford, Illinois, United States) and centrifuged at 12,000g for 15 min at 4 °C. To obtain nuclear extracts, NE-PER Nuclear Extraction Reagents (Pierce) were used. Protein concentrations were estimated by the BCA method (Pierce). For immunoblot analysis, 30 μg and 100 μg of protein from nuclear extracts and total protein lysates, respectively, were used and resolved on 10% SDS-PAGE gels. Proteins were transferred onto PVDF membranes, and the following antibodies were used for immunoblotting: anti-KEAP1 (gift from M. Velichkova, University of California San Diego, La Jolla, California, United States), anti-NRF2 (H-300; Santa Cruz Biotechnology, Santa Cruz, California, United States), anti–Lamin B1 (Santa Cruz Biotechnology), and anti-GAPDH (Imgenex, Sorrento Valley, California, United States). All primary antibodies were diluted in PBS-T/5% nonfat dry milk and incubated overnight at 4 °C.

PLoS Med. 2006 October; 3(10): e420.

Enzyme AssayEnzyme activities of GST, GSR, and NQO1 were determined in the total protein lysates by following methods previously described [8]. Total glutathione (oxidized and reduced) was determined using a modified Tietze method [25] by measuring reduction of 5,5′-dithiobis-2-nitrobenzoic acid in a GSR-coupled assay.