Comparison of human B cell activation by TLR7 and TLR9 agonists

Research articl

A robust method to quantify low molecular weight contaminants in heparin: detection of tris(2-n-butoxyethyl) phosphate

Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes similar to 120 min against similar to 3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. the procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.Brazilian agency Fundacao AraucariaBrazilian agency FINEP (PRONEX-CARBOIDRATOS, PADCT II/SBIO)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Parana, Dept Bioquim & Biol Mol, BR-81531980 Curitiba, PR, BrazilIst Ric Chim & Biochim G Ronzoni, I-20133 Milan, ItalyUniversidade Federal de São Paulo, Dept Bioquim & Biol Mol, BR-04044020 São Paulo, SP, BrazilUniv Liverpool, Sch Biol Sci, Liverpool L69 7ZB, Merseyside, EnglandUniversidade Federal de São Paulo, Dept Bioquim & Biol Mol, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

Mild hypothermia delays the development of stone heart from untreated sustained ventricular fibrillation - a cardiovascular magnetic resonance study

<p>Abstract</p> <p>Background</p> <p>'Stone heart' resulting from ischemic contracture of the myocardium, precludes successful resuscitation from ventricular fibrillation (VF). We hypothesized that mild hypothermia might slow the progression to stone heart.</p> <p>Methods</p> <p>Fourteen swine (27 ± 1 kg) were randomized to normothermia (group I; n = 6) or hypothermia groups (group II; n = 8). Mild hypothermia (34 ± 2°C) was induced with ice packs prior to VF induction. The LV and right ventricular (RV) cross-sectional areas were followed by cardiovascular magnetic resonance until the development of stone heart. A commercial 1.5T GE Signa NV-CV/i scanner was used. Complete anatomic coverage of the heart was acquired using a steady-state free precession (SSFP) pulse sequence gated at baseline prior to VF onset. Un-gated SSFP images were obtained serially after VF induction. The ventricular endocardium was manually traced and LV and RV volumes were calculated at each time point.</p> <p>Results</p> <p>In group I, the LV was dilated compared to baseline at 5 minutes after VF and this remained for 20 minutes. Stone heart, arbitrarily defined as LV volume <1/3 of baseline at the onset of VF, occurred at 29 ± 3 minutes. In group II, there was less early dilation of the LV (p < 0.05) and the development of stone heart was delayed to 52 ± 4 minutes after onset of VF (P < 0.001).</p> <p>Conclusions</p> <p>In this closed-chest swine model of prolonged untreated VF, hypothermia reduced the early LV dilatation and importantly, delayed the onset of stone heart thereby extending a known, morphologic limit of resuscitability.</p

Purified B cells were stimulated with the indicated IRM or with CpG2006

Gene expression changes were assessed by quantitative real time RT-PCR at 2, 8 or 24 hours after stimulation. The log2 of the maximum fold change over the time course from 2 to 24 hours for 1 representative donor is shown. Hierarchical clustering was performed as described in methods.<p><b>Copyright information:</b></p><p>Taken from "Comparison of human B cell activation by TLR7 and TLR9 agonists"</p><p>http://www.biomedcentral.com/1471-2172/9/39</p><p>BMC Immunology 2008;9():39-39.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2503978.</p><p></p

Purified B cells from 3 different donors were stimulated with the indicated IRM or with CpG2006 for 2, 8 or 24 hours, and then were harvested for mRNA analysis

The fold change in gene expression at each time point, normalized to vehicle control, is shown for CCL4 (MIP1β) (), Conditioned media from the stimulated cells were collected at 8 and 24 hours after stimulation for analysis of protein production. The amount of secreted CCL4 (MIP1β) (in pg/ml) is shown in . The concentrations of the TLR agonists were: 3M-006, 5 μM; 3M-003, 1 μM; 852A, 3 μM; 3M-002, 5 μM; CpG 2006, 3 μM.<p><b>Copyright information:</b></p><p>Taken from "Comparison of human B cell activation by TLR7 and TLR9 agonists"</p><p>http://www.biomedcentral.com/1471-2172/9/39</p><p>BMC Immunology 2008;9():39-39.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2503978.</p><p></p

Purified B cells were cultured for 10 days in the presence of the indicated concentrations of an IRM or CpG2006

Conditioned media from these cultures was analyzed for production of IgM () or IgG (). Data from a representative donor are shown.<p><b>Copyright information:</b></p><p>Taken from "Comparison of human B cell activation by TLR7 and TLR9 agonists"</p><p>http://www.biomedcentral.com/1471-2172/9/39</p><p>BMC Immunology 2008;9():39-39.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2503978.</p><p></p

Highly purified B cells from 3 different donors were examined for expression of the TLRs by RT-PCR

The copy number for TLR2 to TLR10 mRNA was normalized to that for GAPDH to compare expression between donors.<p><b>Copyright information:</b></p><p>Taken from "Comparison of human B cell activation by TLR7 and TLR9 agonists"</p><p>http://www.biomedcentral.com/1471-2172/9/39</p><p>BMC Immunology 2008;9():39-39.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2503978.</p><p></p