Meijers J.C.M.,Sanquin |
Schmaier A.H.,Case Western Reserve University
Blood | Year: 2017
In this issue of Blood, Ivanov et al1 demonstrate that factor XII (FXII) does not have to be cleaved into a 2-chain protein to initiate contact activation by creating a form of single-chain FXIIa (scXIIa) with low-level proteolytic activity.
Kooistra K.,Sanquin |
Mesman H.J.,Sanquin |
de Waal M.,Sanquin |
Koppelman M.H.G.M.,National Screening Laboratory |
Vox Sanguinis | Year: 2011
Background and Objectives: Plasma derivatives and blood components with low levels of parvovirus B19 (B19) seem not infectious, but recently infected, highly viraemic donors may transmit B19. We studied the incidence of high-level B19 viraemia (B19 DNA >10 6IU/ml) in 6·5 million Dutch blood donations. Materials and Methods: Between 2003 and 2009, all Dutch blood and plasma donations were screened for the presence of B19 DNA, via pools of 480. Reactive pools were resolved and demographic parameters were obtained for all donors with B19 viraemia >10 6IU/ml. In a subset, IgG and IgM antibodies to B19 were determined. Results: Four hundred and eleven donations (1/15815) were identified with B19 DNA levels above 10 6IU/ml, predominantly (83%) occurring in donors aged 18-47years. Each year infection rates were elevated between December and July, with April accounting for 16% of infections. The years 2004 and 2009 were epidemic, with up to 1/4880 highly viraemic donations in May 2004. In a subset of 67 viraemic donations, 47/67 (70%) tested negative for IgG and IgM antibodies to B19; 16/67 (24%) showed isolated IgM and 4/67 (6%) contained IgG and IgM antibodies. The seasonal pattern of asymptomatic B19 infection in blood donors followed the notification rate of clinical cases. Geographically, B19 infection was randomly spread over the Netherlands. Conclusions: In epidemic seasons, blood donations with high levels of parvovirus, without concurrent antibodies, are common. They may infect immunocompromised and parvovirus-naïve recipients. The feasibility of preventive measures should be studied. © 2010 Sanquin Blood Supply Foundation. Vox Sanguinis © 2010 International Society of Blood Transfusion.
Denys B.,Erasmus Medical Center |
Denys B.,Ghent University |
Van Der Sluijs-Gelling A.J.,Dutch Childhood Oncology Group |
Homburg C.,Sanquin |
And 7 more authors.
Leukemia | Year: 2013
Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics. © 2013 Macmillan Publishers Limited All rights reserved.
News Article | March 2, 2017
NIJMEGEN & AMSTERDAM, the Netherlands--(BUSINESS WIRE)--Synthon Biopharmaceuticals BV, an international biopharmaceutical company that is focused on developing new molecular entities for treating cancer and autoimmune diseases, today announced that it has entered a license and collaboration agreement for the development of novel immuno-oncology antibodies with Sanquin Blood Supply Foundation (‘Sanquin’), Amsterdam. Under the terms of the agreement, Synthon has obtained worldwide exclusive rights to Sanquin’s know-how, lead antibodies and intellectual property regarding the CD47-SIRPα pathway to develop new immuno-oncology treatments. CD47 is a broadly expressed membrane protein that interacts with the myeloid inhibitory immunoreceptor SIRPα (also termed CD172a or SHPS-1). When SIRPα engages with CD47, it provides a downregulatory signal that inhibits host cell phagocytosis, and CD47 therefore acts as a “don't-eat-me” signal. Blocking CD47-SIRPα interactions promotes the destruction of, for example, cancer cells by phagocytes through antibody-dependent mechanisms. Dr. Marco Timmers, chief scientific officer of Synthon Biopharmaceuticals commented: “We are particularly pleased with this license and collaboration agreement, which combines Sanquin’s outstanding research capabilities with Synthon’s biopharmaceutical drug development and manufacturing excellence. This will enable us to accelerate the availability of important new therapeutic treatment options for cancer patients who may benefit from these.” Prof. Dr. René van Lier, director of Research and Member of the Executive Board at Sanquin: "The collaboration agreement with Synthon, with their knowledge of next-generation medicines, is a great example of Sanquin's efforts to provide patients with innovative therapeutic treatment options." Financial details of the agreement were not disclosed. Sanquin is responsible for the blood supply in the Netherlands on a not-for-profit basis. Hundreds of thousands of non-remunerated blood donors make this possible. They are the heart of the organization. The core activity is safe and efficient production and distribution of blood and plasma products for the treatment of patients. Sanquin conducts scientific research, adding to our knowledge of blood and transfusion medicine. This enables Sanquin to advise hospitals and anyone treating patients. http://www.sanquin.nl/en/research/ Synthon, with headquarters in Nijmegen, the Netherlands, is an international pharmaceutical company and a leader in the field of generic medicines. The company started its biopharmaceutical franchise in 2007 and is building a promising portfolio of innovative next-generation medicines. Synthon is developing into a specialty pharmaceutical company, focusing on the therapeutic areas of oncology and auto-immune diseases. Synthon products are currently approved by regulatory agencies in around 100 countries worldwide and marketed through strategic partnerships and – in dedicated areas – through direct sales. Synthon employs about 1,900 staff worldwide, and in 2016 it recorded a turnover of EUR 258 million. For more information, go to www.synthonbiopharmaceuticals.com
News Article | December 1, 2016
AIDSmonument in Amsterdam Counts Down Till the end of AIDS Elsevier/ RELX Group on behalf of the NAMES Project Netherlands From December 1st 2016, World Aids Day, onwards the Netherlands have their own HIV/AIDSmonument, officially unveiled by the mayor of Amsterdam, Eberhard van der Laan, together with the artist Jean-Michel Othoniel and Louise van Deth, managing director of AIDS Fonds (AIDS Foundation) the Netherlands. It is located on the south bank of the river IJ on a permanent location between Central Station and the concert hall Muziekgebouw aan 't IJ. The monument "Living by Numbers" was created by the French artist Jean-Michel Othoniel. The striking features of the landmark are the great scarlet beads made of hand-blown glass; the monument has the shape of a giant abacus, which counts down till the end of AIDS. Development of the monument is an initiative of the NAMES Project Netherlands Foundation, and could be realized thanks to the support of main sponsors: Aids Fonds (Aids Foundation), OLVG hospital of Amsterdam, RELX Group and ViiV Healthcare. Other contributing sponsors include among others, Amsterdams Fonds voor de Kunst, The Art of Impact, Zabawas, and the Centrum district of Amsterdam; funding was also raised through a crowdfunding campaign. The monument is a beacon of hope and support to anyone living with HIV. At the same time it is a tribute to all buddies, supporters, medical employees, activists and scientists. Moreover it is also a memorial, dedicated to all loved ones who have died of AIDS. The initiators of developing the monument chose Amsterdam because this city has been and is a sanctuary for many people with HIV who were not welcome in their own countries. "Othoniel demonstrates with his design that an AIDSmonument does not necessarily have to be somber. He combines very delicately a heavy subject with elegance and beauty. The wonderful light at the bank of the river IJ will be freely reflected in the shining beads, made of hand-blown glass. The monument will match its surroundings perfectly. The AIDSmonument will enrich Amsterdam with a significant work of art," the appointed art committee had said upon selecting the design of the monument. See for more information: http://www.hiv-aidsmonument.nl - and further details provided in appendix below. Twitter hashtags: #Aidsmonument #WorldAIDSDay What is the significance of the HIV/AIDSmonument? The aim of this monument is to keep the attention for AIDS and HIV alive and kicking. At this moment there are approximately 22,100 people in the Netherlands with AIDS and HIV. Every week there are still 19 people in this country who are getting the diagnosis that they have HIV. Moreover a quarter of people with HIV don't even know that he or she is carrying the HIV virus. Therefore HIV and AIDS deserve all our attention, and was the AIDSmonument unveiled December 1st 2016 - Worlds Aids Day - in the footsteps of other cities such as New York, San Francisco and Vancouver. The AIDSmonument was designed in the form of an abacus; an instrument to teach people to count. The abacus is significant, because it symbolizes the countdown to the moment that AIDS will have disappeared from this world for good. The beads in the abacus are made of hand blown glass and therefore all slightly different. They catch the light of the river IJ, reflecting the light in bright colors. It puts a spell on the eyes of anyone passing by. Every year the number of beads will be adjusted and this will be clarified during an event. The beads will, for example, show how many people have been cured of HIV. In that way the AIDSmonument will reflect not only loss, but will also emphasize that there is hope for the future. The monument is an initiative of the foundation NAMENproject Nederland, an organization that works with volunteers who want to keep the attention for HIV and AIDS alive and kicking. In search of a suitable design the foundation was advised by an independent commission of arts. Moreover there was a web-election among the public. This resulted in a longlist of forty artists. Eventually the foundation chose a work of art by the internationally well-known French artist Jean-Michel Othoniel. The river IJ seems a perfect site for the AIDSmonument due to its history. This river symbolizes trade overseas. Sailors did not only exchange goods; they also exchanged diseases. Such as syphilis in the fifteenth century and plague in the seventeenth century. In that way the river IJ reflects almost tangibly the international dimension of AIDS. But there is more: only a few decades ago the De Ruyterkade was the place for hookers and junkies, with just around the corner social workers and the bus that was providing methadone. For gay men the eastern side of the quay was a popular car-cruising-area and Café West-Indië - no longer existing - at De Ruyterkade 110 was the meeting point of Motor Sportclub Amsterdam, a motorclub for gay people. Nowadays the 'back of Amsterdam' shows the dynamics of the capital of the Netherlands. The site, where the AIDSmonument is located, is now a beautiful spot at the river IJ, among exceptional architecture and international splendor. Nowadays the south bank of the river IJ offers a 'coming and going' of many people due to the Central Station, the river-ferries, rivercruise-ships and the Passenger Terminal Amsterdam (PTA), which is the harbor for international cruise-ships. Every year hundreds of thousands residents and visitors will see the AIDSmonument: from the street, from passing busses, trams and trains, from bigger and smaller ships and boats. The AIDSmonument will also be seen by pedestrians and by people driving their cars and riding their bikes. Amongst them there will be many, many tourists. The AIDSmonument will not only be a place for official ceremonies, but also for intimate memorials by individuals. Therefore Othoniel has designed a bench right underneath the abacus with a beautiful view of the river. Underneath the huge abacus people will be able to daydream and reflect, while they look at the horizon, pondering on the future. In this way the AIDSmonument is to give people the courage to go on. How is the artist involved? Jean-Michel Othoniel (Saint-Étienne, 1964) was eighteen, when a mysterious disease got a name: AIDS. Since the eighties AIDS plays an important part in Othoniel's work. The absence of bodies is a significant recurrent theme. For this Living by Numbers Othoniel was inspired by figures: the number of people with HIV and AIDS. Counting is a recurrent theme in this design: the number of victims, the number of people who live with HIV, the number of CD4-cells. All this led to the idea of the abacus; a calculation tool from the past. The hand blown beads symbolize hope, especially our hope that one day people will be able to stop counting. Colored balls have been the principal material that Jean-Michel Othoniel has been using in his work since the late 1990's. Therefore his work looks like monumental jewels. Othoniel says: "Beauty in art seems kind of taboo in the Western world, whereas beauty, compassion and hope are absolutely necessary in our world." The foundation NAMENproject Nederland could only realize the HIV/AIDSmonument thanks to many companies and organisations that supported this monument ever since 2014. The main sponsors are the Aids Fonds (Aids Foundation), the Amsterdam hospital OLVG, RELX Group (formerly Reed Elsevier) and ViiV Healthcare. Founding sponsors are DoubleTree by Hilton and Gilead. Many individuals donated money due to crowdfundation through http://www.voordekunst.nl And there was support by major (art)foundations: the Amsterdams Fonds voor de Kunst (Amsterdam Foundation for the Arts), Art of Impact and Zabawas. Other sponsors are the hospital Academisch Medisch Centrum (AMC), het Amsterdamsche Fonds, Amsterdam Lowlanders Rugby Club, Michael Bakish & Peter M. Hirsch, Canal Company, COC Nederland, Cordaan, dance4life, Drugspastoraat Amsterdam, Eelco & Frank, Elsevier Women's Network & Elsevier Pride, Les Enfants Terribles, Firma Netjes, Stichting GALA, Gebr. Silvestri, GGD Amsterdam, hello gorgeous, Stichting HIV Monitoring, Hiv Vereniging Nederland, i.m. Kees Rümke, Stichting Homomonument, IHLIA LGBT Heritage, Joep Lange Institute, Lloyd Hotel & Culturele Ambassade, Mr. B, De Nederlandsche Bank, Nederlandse Vereniging van Hemofilie Patiënten (NVHP), Nederlandse Vereniging van HIV Behandelaren (NVHB), Nieuwezijds Gay Sauna, Pascal van den Noort, Mark Noyons & Partners, PlanetRomeo Foundation, De Regenboog Groep, RutgersWPF, Sanquin Bloedvoorziening, Thalys, Thermos Sauna, Tijgertje, TrutFonds, VU Medisch Centrum and Zorggroep Amsterdam Oost (Flevohuis | De Open Hof). Other supporters were the Municipality of Amsterdam and the central district of Amsterdam. The Netherlands have always been a runner-up in the international battle against AIDS. Scientists and experts from our country have been important in the development of the combination therapy. Due to this therapy of HIV-inhibitors dying of AIDS became living with HIV. This meant a huge breakthrough, but meanwhile the danger remains that HIV has become invisible. People tend to forget that even this year and even in The Netherlands there are still people dying of AIDS. See for more information: http://www.hiv-aidsmonument.nl
News Article | December 14, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Human skin tissue was obtained from healthy donors undergoing corrective breast or abdominal surgery after informed consent in accordance with our institutional guidelines. This study was approved by the Medical Ethics Review Committee of the Academic Medical Center. Split-skin grafts of 0.3 mm in thickness were obtained using a dermatome (Zimmer). After incubation with Dispase II (1 U ml−1, Roche Diagnostics), epidermal sheets were separated from the dermis and cultured in in Iscoves Modified Dulbeccos’s Medium (IMDM, Thermo Fischer Scientific) supplemented with 10% FCS, gentamycine (20 μg ml−1, Centrafarm), pencilline/streptomycin (10 U ml−1 and 10 μg ml−1, respectively; Invitrogen). Further LC purification was performed using a Ficoll gradient (Axis-shield) and CD1a microbeads (Miltenyl Biotec) as described before4, 10. Isolated LCs were routinely 90% pure and expressed high levels of Langerin and CD1a. MUTZ-LCs were differentiated from CD34+ human AML cell line MUTZ3 progenitors in the presence of GM-CSF (100 ng ml−1, Invitrogen), TGF-β (10 ng ml−1, R&D) and TNF-α (2.5 ng ml−1, R&D) and cultured as described before14. Immature DCs were differentiated from monocytes, isolated from buffy coats of healthy volunteer blood donors (Sanquin, The Netherlands), in the presence of IL-4 (500 U ml−1, Invitrogen) and GM-CSF (800 U ml−1, Invitrogen) and used at day 6 or 7 as previously described20. CD4+ T cells were obtained from peripheral blood mononuclear cells (PBMCs) activated with phytohaemagglutinin (1 mg ml−1; L2769, Sigma Aldrich) for 3 days, enriched for CD4+ T cells by negative selection using MACS beads (130-096-533, Miltenyi) and cultured overnight with IL-2 (20 U ml−1; 130-097-745, Miltenyi) as described before5. The following inhibitors were used: rapamycin (mTOR inhibitor, tlrl-rap, Invivogen), bafilomycin A1 (V-ATPase inhibitor; tlrl-baf1; Invivogen) and MG-132 (proteasome inhibitor; 474790; Calbiochem). All cell lines were obtained from ATCC and tested negative for mycoplasma contamination, determined in 3-day-old cell cultures by PCR. Langerin and Langerin mutant W264R expression plasmid pcDNA3.1 were obtained from Life Technologies and subcloned into lentiviral construct pWPXLd (Addgene). HIV-1-based lentiviruses were produced by co-transfection of 293T cells with the lentiviral vector construct, the packaging construct (psPAX2, Addgene) and vesicular stomatitis virus glycoprotein envelope (pMD2.G, Addgene) as described previously31. U87 cell lines stably expressing CD4 and wild-type CCR5 co-receptor (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: U87 CD4+CCR5+ cells from H. K. Deng and D. R. Littman32) were transduced with HIV-1-based lentiviruses expressing sequences coding human TRIM5α33, rhesus TRIM5α33, wild-type Langerin or Langerin(W264R). NL4.3, NL4.3-BaL, SF162, NL4.3eGFP-BaL, NL4.3-BlaM-Vpr and VSV-G-pseudotyped NL4.3(ΔEnv) HIV-1 were generated as described10. All produced viruses were quantified by p24 ELISA (Perkin Elmer Life Sciences) and titrated using the indicator cells TZM-Bl. Primary LCs and MUTZ-LCs were infected with a multiplicity of infection of 0.2–0.4 and HIV-1 infection was assessed by flow cytometry at day 7 after infection by intracellular p24 staining. Double staining with CD1a (LCs marker; HI149-APC; BD Pharmigen) and p24 (KC57-RD1-PE; Beckman Coulter) was used to discriminate the percentage of CD1a+p24+ infected LCs. CD4+CCR5+ U87 parental or transduced cells were infected at a multiplicity of infection of 0.1–0.2 and HIV-1 infection was assessed at day 3 after infection by intracellular p24 staining or GFP expression. For analysis of transmission of HIV-1 to T cells, LCs were stringently washed 3 days after infection followed by co-culture with activated allogeneic CD4+ T cells for 3 days. Triple staining with CD1a (LCs marker), CD3 (T cells marker; 552851-PercP, BD Pharmigen) and p24 was used to discriminate the percentage of CD3+CD1a−p24+ infected T cells. HIV-1 infection and transmission was assessed by FACSCanto II flow cytometer (BD Biosciences) and data analysis was carried out with FlowJo software (Treestar). HIV-1 production was determined by a p24 antigen ELISA in culture supernatants (ZeptoMetrix). mRNA was isolated with an mRNA Capture kit (Roche) and cDNA was synthesized with a reverse-transcriptase kit (Promega). For real-time PCR analysis, PCR amplification was performed in the presence of SYBR green in a 7500 Fast Realtime PCR System (ABI). Specific primers were designed with Primer Express 2.0 (Applied Biosystems; Extended Data Table 1). The cycling threshold (C ) value is defined as the number of PCR cycles in which the fluorescence signal exceeds the detection threshold value. For each sample, the normalized amount of target mRNA (N ) was calculated from the C values obtained for both target and household (GAPDH, primary LCs, DCs and U87 cells lines; β-actin, MUTZ-LCs) mRNA with the equation N = 2Ct(control) − Ct(target). For relative mRNA expression, control siRNA sample was set at 1 within the experiment and for each donor. A two-step Alu-long terminal repeat (LTR) PCR was used to quantify the integrated HIV-1 DNA in infected cells as previously described20. Total cell DNA was isolated at 16 h after infection (multiplicity of infection of 0.4) with a QIAamp blood isolation kit (Qiagen). In the first round of PCR, the DNA sequence between HIV-1 LTR (LTR R region, extended with a marker region at the 5′ end) and the nearest Alu repeat was amplified (primer sequences, Extended Data Table 1). The second round was nested quantitative real-time PCR of the first-round PCR products using primers annealing to the aforementioned marker region in combination with another HIV-1-specific primer (LTR U5 region) by real-time quantitative PCR. Two different dilutions of the PCR products from the first-round of PCR were assayed to ensure that PCR inhibitors were absent. For monitoring the signal contributed by unintegrated HIV-1 DNA, the first-round PCR was also performed using the HIV-1-specific primer (LTR R region) only. HIV-1 integration was normalized relative to GAPDH DNA levels. For relative HIV-1 integration, control siRNA-infected cells (total signal; Supplementary Table 1) was set as 1 for one experiment or for each donor. A BlaM-Vpr-based assay was used to quantify fusion of HIV-1 to the host membrane in infected LCs as previously described10. LCs were infected with NL4.3-BlaM-Vpr for 2 h and then loaded with CCF2/AM (1 mM, LiveBLAzer FRET-B/G Loading Kit, Life technologies) in serum-free IMDM medium for 1 h at 25 °C. After washing, BlaM reaction was allowed to develop for 16 h at 22 °C in IMDM supplemented with 10% FCS and 2.5 mM anion transport inhibitor probenecid (Sigma Pharmaceuticals). HIV-1 fusion was determined by monitoring the changes in fluorescence of CCF2/AM dye, which reflect the presence of BlaM-Vpr into the cytoplasm of target cells upon viral fusion. The shift from green emission fluorescence (500 nm) to blue emission fluorescence (450 nm) of CCF2/AM dye was assessed by flow cytometer LSRFortessa (BD Biosciences) and data analysis was carried out with FlowJo software. Percentages of blue fluorescent CCF2/AM+ cells are depicted as percentage of HIV-1 fusion. A fluorescent bead adhesion assay was used to examine the ability of HIV-1 gp120-coated fluorescent beads to bind Langerin in CD4+CCR5+ U87 transfectants as previously described5. Binding was measured by FACSCanto II flow cytometer and data analysis was carried out with FlowJo software. Skin LCs and DCs were transfected with 50 nm siRNA with the transfection reagent DF4 (Dharmacon) whereas MUTZ-LCs, CD4+CCR5+ U87 parental or transduced cells were transfected with transfection reagent DF1 (Dharmacon) and were used for experiments 48–72 h after transfection. The siRNA (SMARTpool; Dharmacon) were specific for Atg5, (M-004374-04), Atg16L1 (M-021033), LSP-1 (M-012640-00), TRIM5α (M-007100-00) and non-targeting siRNA (D-001206-13) served as control. Langerin was silenced in MUTZ-LCs by electroporation with Neon Transfection System (ThermoFischer Scientific) using siRNA Langerin (10 μM siRNA, M-013059-01, SMARTpool; Dharmacon). Silencing of the aforementioned targets was verified by real-time PCR, flow cytometer and immunoblotting (Extended Data Figs 1d, e, 2a–k). Cells were pre-treated with bafilomycin A1 for 2 h or left untreated followed by incubation with HIV-1 for 16 h. Quantification of intracellular LC3 II levels by saponin extraction was performed as described before34, 35. LCs were washed in PBS and permeabilized with 0.05% saponin in PBS. Cells were incubated at 4 °C for 30 min with mouse anti-LC3 primary antibody (M152-3; MBL International) or with mouse anti-IgG1 isotype control (MOPC-21; BD Pharmingen) followed by incubation with Alexa Fluor 488-conjugated goat-anti mouse IgG antibody (A-21121, Life Technologies) in saponin buffer. Intracellular LC3 II levels were assessed by FACSScan or FACSCanto II flow cytometers (BD Biosciences) and data analysis was carried out with FlowJo. Cells were pre-treated with bafilomycin for 2 h or left untreated followed by incubation with HIV-1 for 4 h. Quantification of intracellular LC3 II levels by saponin extraction was performed as described before35. Whole-cell extracts were prepared using RIPA lysis buffer supplemented with protease inhibitors (9806; Cell Signalling). 20–30 μg of extract were resolved by SDS–PAGE (15%) and immunoblotted with LC3 (2G6; Nanotools) and β-actin (sc-81178; Santa Cruz) antibodies, followed by incubation with HRP-conjugated secondary rabbit-anti-mouse antibody (P0161; Dako) and luminol-based enhanced chemiluminescence (ECL) detection (34075; Thermo Scientific). For gel source data, see Supplementary Fig. 1. MUTZ-LCs (2 × 106) were incubated for 16 h with HIV-1 NL4.3 (multiplicity of infection, 0.5) or left untreated as a control, fixed in 4% paraformaldehyde and 1% glutaraldehyde in sodium cacodylate buffer for 10 min at room temperature followed by 24 h at 4 °C. After fixation, cells were collected by centrifugation and the pellet was washed in sodium cacodylate buffer. Cells were post-fixed for 1 h at 4 °C (1% osmium tetroxide, 0.8% potassium ferrocyanide in the same buffer), contrasted in 0.5% uranyl acetate, dehydrated in a graded ethanol series and embedded in epon LX112. Ultrathin sections were stained with uranylacetate/lead citrate and examined with a FEI Tecnai-12 transmission electron microscope. Numbers of autophagosomes per cell was determined in 50 cells for each condition counted by two independent researchers. LCs were left to adhere onto poly-l-lysine coated slides. Cells were fixed in 4% paraformaldehyde and permeabilized with PBS/0.1% saponin/1% BSA/1 mM Hepes. Cells were stained with anti-Langerin (AF2088; R&D Systems) and TRIM5α (ab109709; Abcam) antibodies followed by Alexa Fluor 647-conjugated anti-goat (A-21447; Life Technologies) and Alexa Fluor 488-conjugated anti-rabbit (A-21206; Life Technologies). For detection of autophagic vesicles, LCs were pre-loaded with the Cyto-ID Green detection autophagy reagent (ENZ-51031; Enzo Life Sciences), which was previously shown to specifically stain autophagic vesicles36 before adherence to microscope slides and stained with p24 (KC57-RD1-PE; Beckman Coulter) followed by Alexa-Fluor-546-conjugated anti-mouse (A-11003; Life Technologies). Nuclei were counterstained with Hoechst (10 μg ml−1; Molecular Probes). Single plane images were obtained by Leica TCS SP-8 X confocal microscope and data analysis was carried out with Leica LAS AF Lite (Leica Microsystems). Whole-cell extracts were prepared using RIPA lysis buffer supplemented with protease inhibitors. Atg16L1, DC-SIGN, Langerin, p62 and TRIM5α were immunoprecipitated from 40 μg of extract with anti- Atg16L1 (PM040; MBL International), DC-SIGN (AZN-D1)19, Langerin (10E2)5, p62 (ab56416; Abcam), TRIM5α (ab109709; Abcam), mouse IgG1 isotype control (MOPC-21; BD Pharmingen), mouse IgG2a isotype control (IC003A; R&D systems) and rabbit IgG control (sc-2077; Santa Cruz) coated on protein A/G PLUS agarose beads (sc-2003; Santa Cruz), washed twice with ice-cold RIPA lysis buffer and resuspended in Laemmli sample buffer (161-0747, Bio-Rad). Immunoprecipitated samples were resolved by SDS–PAGE (12.5%), and detected by immunoblotting with Atg5 (PM050; MBL), Atg16L1 (MBL), DC-SIGN (551186; BD Biosciences), Langerin (AF2088; R&D Systems), LSP-1 (3812S; Cell Signalling), TRIM5α (Abcam) and HIV-p24 (KC57-RD1-PE; Beckman Coulter) antibodies, followed by incubation with Clean-Blot IP Detection Kit-HRP (21232; Thermo Scientific) and ECL detection (34075; Thermo Scientific). Data acquisition was carried out with ImageQuant LAS 4000 (GE Healthcare). Immunoprecipitation with TRIM5α, Langerin, DC-SIGN, Atg16L1 and p62 pulls-down mostly the TRIM5α (approximately 56 kDa) form. Relative intensity of the bands was quantified using Image Studio Lite 5.2 software by normalizing β-actin and set at 1 in untreated cells. For gel source data, see Supplementary Fig. 1. Two-tailed Student’s t-test for paired observations (differences of stimulations within the same donor or cell-type) or unpaired observation (differences between U87 transfectants). Statistical analyses were performed using GraphPad 6.0 software and significance was set at P < 0.05 (*P < 0.05; **P < 0.01). The data that support the findings of this study are available from the corresponding author upon reasonable request.
News Article | November 16, 2016
— Factors such as rising geriatric population and increasing number of hemophilic patients are boosting the market growth. However, strict government rules, huge cost and limited reimbursements are some of the factors hampering the market. Rising digit of Hemophilic Patients is creating growth opportunities for vendors in the market. By product, immunoglobulin segment dominated the global market with largest share in 2013 owing to growing geriatric population with neurological and autoimmune disease. By region, North America accounted for largest market share followed by Europe. However, Asia-Pacific is anticipated to grow at the highest CAGR owing to increasing digit of hemophilic patients and rising awareness of technologically advanced products in this region. Some of the key players in Plasma Fractionation market include Octapharma AG, Chinese Biological Products, Inc., Baxter International, Inc., Bio Products Laboratory, Grifols International SA, Shanghai Raas Blood Products Co., Ltd, Csl, Ltd., China Biologic Products, Inc., Kedrion S.P.A., Biotest, Sanquin and LFB SA. Regions Covered: • North America o US o Canada o Mexico • Europe o Germany o France o Italy o UK o Spain o Rest of Europe • Asia Pacific o Japan o China o India o Australia o New Zealand o Rest of Asia Pacific • Rest of the World o Middle East o Brazil o Argentina o South Africa o Egypt What our report offers: - Market share assessments for the regional and country level segments - Market share analysis of the top industry players - Strategic recommendations for the new entrants - Market forecasts for a minimum of 7 years of all the mentioned segments, sub segments and the regional markets - Market Trends (Drivers, Constraints, Opportunities, Threats, Challenges, Investment Opportunities, and recommendations) - Strategic recommendations in key business segments based on the market estimations - Competitive landscaping mapping the key common trends - Company profiling with detailed strategies, financials, and recent developments - Supply chain trends mapping the latest technological advancements About Stratistics MRC We offer wide spectrum of research and consulting services with in-depth knowledge of different industries. We are known for customized research services, consulting services and Full Time Equivalent (FTE) services in the research world. We explore the market trends and draw our insights with valid assessments and analytical views. We use advanced techniques and tools among the quantitative and qualitative methodologies to identify the market trends. Our research reports and publications are routed to help our clients to design their business models and enhance their business growth in the competitive market scenario. We have a strong team with hand-picked consultants including project managers, implementers, industry experts, researchers, research evaluators and analysts with years of experience in delivering the complex projects. For more information, please visit http://www.strategymrc.com/
News Article | December 16, 2016
— The report "Plasma Fractionation Market by Product (Albumin, Immunoglobulin, Factor VIII, Protease Inhibitors), Application (Neurology, Hematology, Rheumatology, Immunology) End User (Hospitals, Clinical Research Laboratories) - Global Forecast to 2021", The plasma fractionation market witnessed healthy growth during the last decade and is expected to grow at a CAGR of 6.7% between 2016 and 2021 to reach USD 26.07 Billion by 2021. Growth in this market can be attributed to factors such as rise in aging population worldwide, the increasing on and off-label use of immunoglobulin for various chronic diseases, increased diagnosis rate, and rise in the use of prophylactic treatment for bleeding and immune diseases. The global plasma fractionation market is segmented based on products, applications, end users, and regions. Based on products, the market is further segmented into immunoglobulin, coagulation factor concentrates, albumin, protease inhibitors, and other plasma products. Based on application types, the market is further segmented into neurology, immunology, hematology, pulmonology, hemato-oncology, rheumatology, critical care, and other applications. On the basis of end users, the global plasma fractionation market is segmented into hospitals and clinics, clinical research laboratories, and academic institutes. Based on regions, the plasma fractionation market is segmented into North America, Europe, Asia-Pacific, and the Rest of the World. In 2015, immunoglobulin accounted for the largest share of the global plasma fractionation market. However, the protease inhibitors segment is expected to witness the highest growth in the next five years. The growth of protease inhibitors is mainly attributed to its increasing use for respiratory diseases and growing application in various diseases. On the basis of applications, the neurology segment accounted for the largest share of the global plasma fractionation market. The large share of this market segment is mainly due to factors such as increasing use of immunoglobulin in neurological diseases. In the end-user segment, hospitals and clinics accounted for the largest share of the plasma fractionation market and are expected to grow at the highest CAGR in next five years. This large share is due to the increasing use of plasma products for labeled and off-labeled use for the treatment of various diseases in hospitals and clinics. North America is the largest regional segment for plasma fractionation. The large share of this regional segment can be attributed increasing use of immunoglobulins in neurological diseases and increasing use of prophylaxis treatments for diagnosed patients, and the rising number of registered hemophilic patients in this region. However, the market in the Asia-Pacific region is expected to grow at the highest CAGR, owing to the rapid growth in the aging population, increasing use of albumin and immunoglobulin, and increasing number of hemophilic patients. CSL Ltd. (Australia), Grifols S.A (Spain), Baxalta Incorporated (U.S.), Octapharma AG (Switzerland), and Kedrion S.p.A (Italy) held the major share of the plasma fractionation market and will continue to dominate the market between 2016 and 2021. Other major players operating in this market are Bio Products Laboratory (U.K.), Sanquin (Netherland), China Biologic Products, Inc. (China), Biotest AG (Germany), and Laboratoire Français du Fractionnement et des Biotechnologies (France). For more information, please visit http://www.marketsandmarkets.com/Market-Reports/plasma-fractionation-market-93798284.html
Vogelzang E.H.,Jan Van Breemen Research Institute Reade |
Kneepkens E.L.,Jan Van Breemen Research Institute Reade |
Nurmohamed M.T.,Jan Van Breemen Research Institute Reade |
Van Kuijk A.W.R.,Jan Van Breemen Research Institute Reade |
And 3 more authors.
Annals of the Rheumatic Diseases | Year: 2014
Objectives: To investigate the relationship between antidrug antibodies (ADA), adalimumab concentrations and clinical response in patients with psoriatic arthritis (PsA) during 52 weeks of follow-up. Methods: This prospective cohort study included 103 consecutive patients with PsA. Disease Activity Score of 28 joints (DAS28), Erythrocyte Sedimentation Rate, C reactive protein and Psoriasis Area and Severity Index were assessed. Adalimumab concentrations and ADA were measured in serum trough samples, using an ELISA and a radio immunoassay, respectively. Results: Adalimumab concentrations were significantly lower at 28 and 52 weeks in patients with detectable ADA compared with patients without detectable ADA (at week 28: 1.3 mg/L (IQR 0.0-3.2) versus 8.7 mg/L (IQR 5.7-11.5), p<0.001; at week 52: 0.9 mg/L (IQR 0.0-2.9) vs 9.4 mg/L (IQR 5.7-12.1), p=0.0001). DAS28 at 28 weeks (2.16 vs 2.95, p=0.023) and 52 weeks (2.19 vs 2.95, p=0.024) showed a significant difference; patients with detectable ADA had a poorer clinical outcome than patients without. Conclusions: Patients with detectable ADA had lower adalimumab concentrations and a significantly poorer clinical outcome compared with patients in whom ADA were not detected. © 2014, BMJ Publishing Group. All rights reserved.
Guhr T.,University of Amsterdam |
Bloem J.,Sanquin |
Derksen N.I.L.,University of Amsterdam |
Wuhrer M.,Leiden University |
And 3 more authors.
PLoS ONE | Year: 2011
Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA (+)) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA (+) using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA (+) 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA (+) had no effect on the platelet count. Serum levels of IVIg and IVIg-SA (+) were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model. © 2011 Guhr et al.