Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells

Cytokines secreted by cells of the immune system can alter the behavior and properties of immune or other cells. At a site of inflammation, sets of cytokines interact with immune cells, and their combined effect is often more important than the function of one isolated component. Conventional techniques, such as enzyme-linked immunosorbent assays, generally require large quantities of cells to characterize a complete cytokine profile of activated lymphocytes. The Bio-Plex system from Bio-Rad Laboratories combines the principle of a sandwich immunoassay with the Luminex fluorescent-bead-based technology. We developed a multiplex cytokine assay to detect different cytokines simultaneously in culture supernatant of human peripheral blood mononuclear cells stimulated with antigen and with mitogen. Fifteen human cytokines (interleukin 1α [IL-1α], IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-18, gamma interferon, and tumor necrosis factor alpha) were validated with a panel of healthy individuals, rheumatoid arthritis patients, and juvenile idiopathic arthritis patients. Comparing the multiplex assay with a regular enzyme-linked immunosorbent assay technique with this donor panel resulted in correlation coefficients for all cytokines ranging from 0.75 to 0.99. Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.     

Interleukin-2 activated NK cells do not use the CD95L- and TRAIL-pathways in the rapid induction of apoptosis of rat colon carcinoma CC531s cells

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-α pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-α pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Timing-Invariant CT Angiography Derived from CT Perfusion Imaging in Acute Stroke: A Diagnostic Performance Study

BACKGROUND AND PURPOSE:Timing-invariant (or delay-insensitive) CT angiography derived from CT perfusion data may obviate a separate cranial CTA in acute stroke, thus enhancing patient safety by reducing total examination time, radiation dose, and volume of contrast material. We assessed the diagnostic accuracy of timing-invariant CTA for detecting intracranial artery occlusion in acute ischemic stroke, to examine whether standard CTA can be omitted.MATERIALS AND METHODS:Patients with suspected ischemic stroke were prospectively enrolled and underwent CTA and CTP imaging at admission. Timing-invariant CTA was derived from the CTP data. Five neuroradiologic observers assessed all images for the presence and location of intracranial artery occlusion in a blinded and randomized manner. Sensitivity and specificity of timing-invariant CTA and standard CTA were calculated by using an independent expert panel as the reference standard. Interrater agreement was determined by using κ statistics.RESULTS:We included 108 patients with 47 vessel occlusions. Overall, standard CTA and timing-invariant CTA provided similar high diagnostic accuracy for occlusion detection with a sensitivity of 96% (95% CI, 90%–100%) and a specificity of 100% (99%–100%) for standard CTA and a sensitivity of 98% (95% CI, 94%–100%) and a specificity of 100% (95% CI, 100%–100%) for timing-invariant CTA. For proximal large-vessel occlusions, defined as occlusions of the ICA, basilar artery, and M1, the sensitivity and specificity were 100% (95% CI, 100%–100%) for both techniques. Interrater agreement was good for both techniques (mean κ value, 0.75 and 0.76).CONCLUSIONS:Timing-invariant CTA derived from CTP data provides diagnostic accuracy similar to that of standard CTA for the detection of artery occlusions in acute stroke.

Stroke imaging research currently focuses on prediction of patient outcome and identifying patients who are suitable for neurointerventional treatment.^1,2 For these purposes, advanced stroke imaging protocols typically add CT perfusion imaging or diffusion-weighted MR imaging to the traditional work-up, consisting of noncontrast CT and CT angiography.^2,3 Noncontrast CT is used to differentiate hemorrhagic stroke from ischemic stroke and to assess early signs of ischemia. CTA is used to localize arterial occlusions and to identify proximal large-vessel occlusions that may be suitable for endovascular treatment. CT perfusion imaging and DWI are used to assess the extent and severity of hypoperfusion and particularly increase the sensitivity of imaging in the early stages of ischemic stroke.⁴ The practical advantages of CT perfusion imaging are that it is widely available and does not delay treatment decisions because it is fast and most patients already undergo CT scanning.³Currently, CTA can be derived from CT perfusion data. Such an approach allows the enhancement of patient safety by reducing the total scanning time, radiation dose, and amount of contrast material needed.⁵ In CT perfusion imaging, multiple scans after intravenous injection of contrast material are obtained with time, generating a 4D dataset, which is used to derive cerebral perfusion maps such as the cerebral blood flow, cerebral blood volume, and arrival times. When imaging is performed on a CT scanner with large spatial coverage, however, this 4D data can also be used to provide CT angiographic information, referred to as 4D-CTA or dynamic CTA. Previous studies have assessed whether 4D-CTA can be used for detection of vascular occlusion in a stroke setting but found that image quality was moderate and diagnostic performance for stroke assessment was limited because large-vessel occlusions may be missed.^5–8 Recently, a different approach to obtain CTA from CT perfusion source data was presented that combines the whole 4D-CTA dataset into 1 high-quality 3D-CTA dataset by displaying maximum contrast enhancement with time.⁵ This technique is referred to as “timing-invariant CTA” because it is insensitive to delayed contrast arrival and was shown to provide similar-to-superior image quality compared with standard CTA.⁵The aim of our study was to test the diagnostic performance of timing-invariant CTA for stroke evaluation, to assess whether standard CTA can be omitted when CT perfusion imaging has been performed.     

Hemodynamic Differences in Intracranial Aneurysms before and after Rupture

BACKGROUND AND PURPOSE:Rupture risk of intracranial aneurysms may depend on hemodynamic characteristics. This has been assessed by comparing hemodynamic data of ruptured and unruptured aneurysms. However, aneurysm geometry may change before, during, or just after rupture; this difference causes potential changes in hemodynamics. We assessed changes in hemodynamics in a series of intracranial aneurysms, by using 3D imaging before and after rupture.MATERIALS AND METHODS:For 9 aneurysms in 9 patients, we used MRA, CTA, and 3D rotational angiography before and after rupture to generate geometric models of the aneurysm and perianeurysmal vasculature. Intra-aneurysmal hemodynamics were simulated by using computational fluid dynamics. Two neuroradiologists qualitatively assessed flow complexity, flow stability, inflow concentration, and flow impingement in consensus, by using flow-velocity streamlines and wall shear stress distributions.RESULTS:Hemodynamics changed in 6 of the 9 aneurysms. The median time between imaging before and after rupture was 678 days (range, 14–1461 days) in these 6 cases, compared with 151 days (range, 34–183 days) in the 3 cases with unaltered hemodynamics. Changes were observed for flow complexity (n = 3), flow stability (n = 3), inflow concentration (n = 2), and region of flow impingement (n = 3). These changes were in all instances associated with aneurysm displacement due to rupture-related hematomas, growth, or newly formed lobulations.CONCLUSIONS:Hemodynamic characteristics of intracranial aneurysms can be altered by geometric changes before, during, or just after rupture. Associations of hemodynamic characteristics with aneurysm rupture obtained from case-control studies comparing ruptured with unruptured aneurysms should therefore be interpreted with caution.

Intracranial aneurysms are found in 1%–5% of the adult population.^1,2 For ruptured intracranial aneurysms, case morbidity and fatality rates are high.^1,3 However, 50%–80% of all intracranial aneurysms do not rupture during an individual''s lifetime.¹ More commonly, unruptured aneurysms are incidentally found due to increasing use of imaging.^4,5 The risk of rupture should be balanced against the risk of treatment when deciding whether an aneurysm should be treated. In clinical practice, the location and size of the aneurysm are the most important parameters for determining the risk of rupture.^1,6 However, these geometric predictors are insufficient for optimal treatment selection. Therefore, the search for better predictors for rupture continues.^7–9 Previous studies have associated intra-aneurysmal flow patterns and wall shear stress (WSS) distributions with aneurysm rupture status.^7,8,10 However, these results are still controversial. For example, both high and low aneurysmal WSS were separately associated with aneurysm growth and rupture.^11,12 In these risk-assessment studies, potential changes in hemodynamics due to the rupture itself were systematically neglected. Recently, 2 studies have shown changes in aneurysm geometry after rupture.^13,14 These rupture-associated geometric changes may result in differences in hemodynamic characteristics as well.In this study, we had the opportunity to use high-quality 3D imaging data of 9 patients with intracranial aneurysms, obtained before and after rupture, to assess potential differences in hemodynamic characteristics associated with rupture.     

ATG16L1 and NOD2 polymorphisms enhance phagocytosis in monocytes of Crohn’s disease patients

AIM:To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohn’s disease(CD).METHODS:In this study,we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls.As both autophagy related like 1(ATG16L1)and immunityrelated guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleo-tide-binding ligomerization domain-containing protein2(NOD2)has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction.The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo?Escherichia coli Bioparticles Phagocytosis kit for flowcytometry.RESULTS:In this study,we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity(ratio of mean fluorescence intensity)between the patient groups and the healthy controls.CD patients show a significantly higher phagocytic capacity(ratio mean percentage of phagocytic cells)compared to healthy controls(51.91%±2.85%vs 37.67%±7.06%,P=0.05).The extend of disease was not of influence.However,variants of ATG16L1(WT:2.03±0.19 vs homozygoot variant:4.38±0.37,P<0.009)as well as NOD2(C-ins)(heterozygous variant:42.08±2.94 vs homozygous variant:75.58±4.34(P=0.05)are associated with the phagocytic activity in patients with CD.CONCLUSION:Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants.This could be part of the pathophysiological mechanism resulting in the disease.