In vitro studies on hirudin elimination by haemofiltration: comparison of three high-flux membranes.

BACKGROUND: Recombinant hirudin (r-hirudin) is a highly selective thrombin inhibitor used for anticoagulation in heparin-induced thrombocytopenia type II. R-hirudin is increasingly applied to patients with renal failure and on renal replacement therapy. Since kidney function impairment strongly prolongs r-hirudin elimination half-life, severe accumulation and bleeding complications may occur. Data on the r-hirudin permeability and elimination capacity of different haemofilters are limited. METHODS: Three haemofilter types were investigated: high-flux polysulphone (Fresenius), AN69 (Hospal), and polyamide (Gambro). We used two in vitro haemofiltration models: (i) an open post-dilution haemofiltration model with ultrafiltration and fluid substitution (model 1) simulating hirudin intoxication, and (ii) a closed model with ultrafiltrate reinfusion (model 2) to determine steady-state sieving coefficients (SC). Fresh human heparinized blood (2 IU unfractionated heparin/ml blood) was used. In model 2, SC obtained with human whole blood were compared with isotonic saline. RESULTS: In model 1, r-hirudin levels decreased significantly faster with polysulphone than with AN69 or polyamide (P<0.05). In accordance with this, in model 2 the observed SC in whole blood were 1.11+/-0.28 (polysulphone), 0.61+/-0.15 (AN69) and 0.33+/-0.13 (polyamide), and clearances were 28+/-7 (polysulphone), 15+/-4 (AN69) and 8+/-3 ml/min (polyamide) (P<0.001 for all comparisons). The SC in saline were slightly but significantly lower for polysulphone (0.88+/-0.12), similar for AN69 (0.59+/-0.1), and significantly improved for polyamide (0.83+/-0.1). CONCLUSIONS: Elimination of r-hirudin by haemofiltration strongly depended on the membrane material. Using human blood, we observed large differences between the three high-flux membranes. The saline experiments suggest a membrane-dependent impact of plasma proteins and pH on hirudin sieving. Our findings have implications for r-hirudin dosage in haemofiltration, for treatment of overdosage, and for future in vitro haemofiltration studies.     

Deep neural models for color classification and color constancy

Color constancy is our ability to perceive constant colors across varying illuminations. Here, we trained deep neural networks to be color constant and evaluated their performance with varying cues. Inputs to the networks consisted of two-dimensional images of simulated cone excitations derived from three-dimensional (3D) rendered scenes of 2,115 different 3D shapes, with spectral reflectances of 1,600 different Munsell chips, illuminated under 278 different natural illuminations. The models were trained to classify the reflectance of the objects. Testing was done with four new illuminations with equally spaced CIEL*a*b* chromaticities, two along the daylight locus and two orthogonal to it. High levels of color constancy were achieved with different deep neural networks, and constancy was higher along the daylight locus. When gradually removing cues from the scene, constancy decreased. Both ResNets and classical ConvNets of varying degrees of complexity performed well. However, DeepCC, our simplest sequential convolutional network, represented colors along the three color dimensions of human color vision, while ResNets showed a more complex representation.     

Variable sensitivity to noxious heat is mediated by differential expression of the CGRP gene

Heat sensitivity shows considerable functional variability in humans and laboratory animals, and is fundamental to inflammatory and possibly neuropathic pain. In the mouse, at least, much of this variability is genetic because inbred strains differ robustly in their behavioral sensitivity to noxious heat. These strain differences are shown here to reflect differential responsiveness of primary afferent thermal nociceptors to heat stimuli. We further present convergent behavioral and electrophysiological evidence that the variable responses to noxious heat are due to strain-dependence of CGRP expression and sensitivity. Strain differences in behavioral response to noxious heat could be abolished by peripheral injection of CGRP, blockade of cutaneous and spinal CGRP receptors, or long-term inactivation of CGRP with a CGRP-binding Spiegelmer. Linkage mapping supports the contention that the genetic variant determining variable heat pain sensitivity across mouse strains affects the expression of the Calca gene that codes for CGRPalpha.     

Loss of CD103~+ intestinal dendritic cells during colonic inflammation

AIM:To investigate possible differences in dendritic cells(DC)within intestinal tissue of mice before and after induction of colitis. METHODS:Mucosal DC derived from intestinal tissue,as well as from mesenteric lymph nodes and spleen,were analyzed by fluorescence activated cell sorting(FACS) analysis.Supernatants of these cells were analyzed for secretion of different pro-and anti-inflammatory cytokines. Immunohistochemistry and immunofluorescence were performed on cryosections of mucosal tissue derived fro...     

C5a negatively regulates toll-like receptor 4-induced immune responses

The complement system and the Toll-like receptors (TLRs) are two central arms of innate immunity that are critical to host defense as well as the development of adaptive immunity. Most pathogens activate both complement and TLRs, suggesting the potential for crosstalk between the two systems. We show here that the complement-derived C5a anaphylatoxin negatively regulates TLR4- and CD40-induced synthesis of IL-12 family cytokines (IL-12, IL-23, and IL-27) from inflammatory macrophages (M phi s) by extracellular signal-regulated kinase- and phosphoinositide 3 kinase-dependent pathways. This decreased cytokine response translates into a decreased T helper type 1 (Th1) response in vitro and in vivo. Accordingly, we found enhanced Th1 immunity in C5a receptor-deficient mice, something that conferred protection from Leishmania major infection. Our findings identify the negative impact of C5a on IL-12 family cytokines as an important mechanism for regulating Th1 polarization in response to innate and adaptive immune network activation.     

Preventive Effects of Escherichia coli Strain Nissle 1917 on Acute and Chronic Intestinal Inflammation in Two Different Murine Models of Colitis

Escherichia coli strain Nissle 1917 (EcN) is as effective in maintaining remission in ulcerative colitis as is treatment with mesalazine. This study aims to evaluate murine models of acute and chronic intestinal inflammation to study the antiinflammatory effect of EcN in vivo. Acute colitis was induced in mice with 2% dextran-sodium sulfate (DSS) in drinking water. EcN was administered from day −2 to day +7. Chronic colitis was induced by transfer of CD4⁺ CD62L⁺ T lymphocytes from BALB/c mice in SCID mice. EcN was administered three times/week from week 1 to week 8 after cell transfer. Mesenteric lymph node (MLN) cytokine secretion (of gamma interferon [IFN-γ], interleukin 5 [IL-5], IL-6, and IL-10) was measured by enzyme-linked immunosorbent assay. Histologic sections of the colon were analyzed by using a score system ranging from 0 to 4. Intestinal contents and homogenized MLN were cultured, and the number of E. coli-like colonies was determined. EcN was identified by repetitive extragenic palindromic (REP) PCR. EcN administration to DSS-treated mice reduced the secretion of proinflammatory cytokines (IFN-γ, 32,477 ± 6,377 versus 9,734 ± 1,717 [P = 0.004]; IL-6, 231 ± 35 versus 121 ± 17 [P = 0.02]) but had no effect on the mucosal inflammation. In the chronic experimental colitis of the transfer model, EcN ameliorated the intestinal inflammation (histology score, 2.7 ± 0.2 versus 1.9 ± 0.3 [P = 0.02]) and reduced the secretion of proinflammatory cytokines. Translocation of EcN and resident E. coli into MLN was observed in the chronic colitis model but not in healthy controls. Administration of EcN ameliorated acute and chronic experimental colitis by modifying proinflammatory cytokine secretion but had no influence on the acute DSS-induced colitis. In this model, preexisting colitis was necessary for translocation of EcN and resident E. coli into MLN.     

Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMC during long-term storage

Analysis of cryopreserved peripheral mononuclear cells (PBMC) is important for evaluating new vaccines in immune based therapies and in pathogenesis studies. To ensure comparable assay results from different laboratories and points of time, collaborative research in multicenter trials needs reliable and reproducible cryopreservation protocols that maintain cell viability and functionality. Current cryomedia consist largely of fetal bovine serum (FBS), a natural mix of growth factors, cytokines, and undefined compounds. Standardized procedures are not possible, as FBS can affect the antigen-specific T-cell response, the most important parameter in functionality assays. Also, worldwide sample exchange is complicated by the strict import restrictions on FBS, because of transfection risk. After establishing a serum-free cryopreservation protocol that maintains cell viability, recovery and antigen-specific T-cell response of PBMC comparably to FBS-based cryomedia (Germann et al., 2011), the aim of this study was the complete avoidance of animal proteins and products in combination with efficient cryopreservation. As long-term stability of the cryopreservation process is crucial for retrospective evaluation of samples at different points of time, PBMC were analyzed after storage for maximal four weeks and again after approximately six months. The cryopreservation efficiency of the protein-free and fully chemically defined cryomedium was comparable to FBS-medium after storage for few weeks and several months. Directly after thawing, this medium yielded viabilities over 97% and recovery values over 84%. Also, the specific T-cell functionality was preserved. Additionally, short-term and six month cryopreservation gave comparable results. The fully chemically defined medium presented here will increase standardization and reproducibility of analysis in multicenter-studies or in retrospective evaluation.