The peptide antibiotics from the polymyxin–colistin–circulin family (Vogler & Studer, 1966) are active against Gram-negative bacteria; other peptides, such as polypeptins (Sogn, 1976), jolipeptin (Ito & Koyama, 1972), gavaserin and saltavalin

(Pichard et al., 1995), are active against both Gram-negative and Gram-positive bacteria. The second group includes antibiotics such as gatavalin (Nakajima et al., 1972), fusaricidins (Kajimura & Kaneda, 1996, 1997; Beatty & Jensen, 2002) and LI-F complex (Kurusu et al., 1987), which are active against fungi, actinomycetes and Gram-positive bacteria. Bacteriophage infection of starter cultures remains a significant problem in fermentation Trichostatin A supplier industries. Many bacteriophages are active against strains of the genus Paenibacillus. Most frequently reported are the bacteriophages infecting P. polymyxa and Paenibacillus larvae, and only a few bacteriophages from P. SCH727965 in vitro polymyxa strains have been described in detail thus far. Francis & Rippon (1949) were first to report isolation of four bacteriophages infecting the members of this species. They characterized the host specificity, particle

size, heat resistance, citrate sensitivity and serological reactions of these phages. Other bacteriophages active against P. polymyxa strains were isolated later (Seldin et al., 1984; Starosciak et al., 1985; Matseliukh & Burova, 2004). They were examined by electron microscopy and their lytic spectrum was specified. These double stranded DNA phages are members of the

Siphoviridae and Myoviridae families and, similar to the phages described by Francis & Rippon (1949), they were specific only to the strains of P. polymyxa. One of the bacteriophages – designated IPy1 (Seldin et al., 1984) – was recently used for evaluation of the genetic diversity within the species P. polymyxa (dos Santos et al., 2002). Phage IPy1 DNA served as a probe in hybridization studies. In this study, the bacteriophage ΦBP active against P. polymyxa CCM 7400 is described. We characterized its host spectrum, morphology, structural protein profile, genome size and Methamphetamine presence of the phage sequences on the bacterial host genome, and identified a cassette of lytic genes in its genome. Bacteriophage ΦBP appears to be a virulent mutant of the temperate phage and is the first such phage of P. polymyxa described in detail. Paenibacillus polymyxa CCM 7400 (Czech Collection of Microorganisms, Brno, Czech Republic) was used as the primary host for the isolation, propagation and characterization of the bacteriophage ΦBP. The isolates of P. polymyxa CCM 7400 represented clones of the same strain picked from agar plates. The following strains of the genus Paenibacillus were tested for ΦBP sensitivity: P. polymyxa S292 and P. polymyxa N36 (both from DSMZ, Germany), P. polymyxa CCM 1460, P. polymyxa CCM 1465, P. polymyxa CCM 2000, P. polymyxa CCM 2001 (all from Czech Collection of Microorganisms, Brno, Czech Republic).

These features were apparent for up to 28 days post-operatively. During this post-operative period, the nocifensive behaviour and enhanced reflex activity were significantly attenuated by intrathecal application of MSO (5 μL, 10 mM) but not by vehicle application. In electrophysiological recordings of nociceptive neuronal activity in the MDH, central sensitization was also evident in pulp-exposed rats but not in intact rats and could be significantly attenuated by MSO application but not by vehicle

application. These behavioural and neuronal findings suggest that the astroglial glutamate–glutamine shuttle is responsible for the maintenance of inflammation-induced nocifensive behavioural changes and the accompanying central sensitization in MDH nociceptive neurons in this chronic

pulpitis pain model. “
“The correlation AZD1208 of discharges between single neurons can provide information about the computations and network properties of neuronal populations during Fulvestrant the performance of cognitive tasks. In recent years, dynamic modulation of neuronal correlations by attention has been revealed during the execution of behavioral tasks. Much less is known about the influence of learning and performing a task itself. We therefore sought to quantify the correlated

firing of simultaneously recorded pairs of neurons in the prefrontal cortex of naïve monkeys that were only required to fixate, and to examine how this correlation was altered after they had learned to perform a working memory task. We found that the trial-to-trial correlation of discharge rates between pairs of neurons (noise correlation) differed across neurons depending on their responsiveness and selectivity for stimuli, even before training in a working memory task. After monkeys had learned to perform the task, correlated firing decreased overall, although the effects varied according to the functional properties of the neurons. The greatest decreases were observed on comparison of populations MycoClean Mycoplasma Removal Kit of neurons that exhibited elevated firing rates during the trial events and those that had more similar spatial and temporal tuning. Greater decreases in noise correlation were also observed for pairs comprising one fast spiking neuron (putative interneuron) and one regular spiking neuron (putative pyramidal neuron) than pairs comprising regular spiking neurons only. Our results demonstrate that learning and performance of a cognitive task alters the correlation structure of neuronal firing in the prefrontal cortex.

He continued to have fever despite 2 weeks of broad-spectrum antibiotics, and was transferred to Barnes-Jewish Hospital/Washington University in St. Louis, MO, USA, for further evaluation. Our differential diagnosis included malaria, typhoid, typhus, leptospirosis, relapsing fever, and tuberculosis. On examination, the patient complained of chills, and thick and thin blood smears were immediately obtained. Both revealed 1% parasitemia with gametocytes of selleck chemical Plasmodium vivax. The patient was treated with mefloquine 1,200 mg PO once and primaquine 15 mg PO daily for 2 weeks. At follow-up, his symptoms had completely resolved. Vector-borne

and environmentally acquired infections are a threat to all travelers to endemic locations, but military personnel are at

elevated risk because of the duration and intensity of environmental exposure. An analysis of 17,353 travelers revealed that the majority, around 64%, present with symptoms of infection within the first month of travel.1 However, this analysis did not PLX3397 include military personnel. When evaluating fever in military personnel, a careful history should include country and terrain of any deployments, both recent and distant. Malaria represents one of the most important infectious disease threats to deployed military forces; 15 of the last 17 major or minor military deployments were to malarious locations. Afghanistan has large endemic areas of malaria, especially below 2,000 m above sea-level.2 This disease has reemerged in the north-eastern river valleys used for

growing rice because of lapsed control measures, intensified agricultural activity, and returning refugees,3 with an annual incidence of 240 per 1,000 people around Jalalabad, where our patient was exposed.4 In 2004, the attack rate of troops deployed to Afghanistan was 52.4 cases per 1,000 soldiers.5 Malaria has also been reported in both British and German soldiers returning from Afghanistan.6,7 In 2004, P. vivax infection acquired in Afghanistan accounted for 25% of the 56 cases of malaria diagnosed among US Army soldiers. Soldiers presented for care weeks to 20 months after return to the United States.8 Thalidomide The median time to diagnosis of malaria in returning Army Rangers was 233 days.5 Vivax malaria presented in German soldiers as late as 9 months after return from Afghanistan.7 This report highlights the importance of a high index of suspicion for tropical infections in returning military personnel; it also underscores an important feature of malaria infection, the possibility of delayed presentation. Low index of suspicion in patients presenting long after exposure is further complicated by the poor sensitivity of malaria smears when trained and experienced microscopists are not readily available; malaria was suspected at the regional hospital but smears were read as negative, delaying the initiation of appropriate treatment.

This noncognitive-based algorithm should prove useful to identify HIV-infected patients with advanced disease at high risk of HAND who require more formal assessment. We propose staged guidelines, using the algorithm, for improved HAND therapeutic management. Future larger, international studies are planned to test the predictive effect of nadir CD4 cell count, hepatitis C virus infection, gender, ethnicity and HIV viral clade. We recommend the use of this first version for HIV-infected Caucasian men with advanced disease. The clinical management of HIV-positive

persons is increasingly complicated in the era of combination antiretroviral therapy (CART). One aspect of management that requires extensive training relates to the early identification of neurocognitive complications Ion Channel Ligand Library chemical structure of HIV infection. In many countries the general practitioner or the HIV physician is often the primary patient’s interlocutor

[1]. Without specific screening using procedures that are still relatively lengthy Protease Inhibitor Library clinical trial or require specific training, especially for interpretation [2], physicians may sometimes overlook patients in need of further neurological evaluation. In the CART era, the prevalence of neurocognitive impairment remains high (up to 50% [3]) and HIV-associated neurocognitive disorder (HAND) has shifted towards a milder clinical presentation [4]. Such a mild clinical presentation can escape detection without formal neurological assessment and neuropsychological testing [5]. HAND, even in its mild form, is independently predictive of death [6] as well as HIV-associated dementia [7]. Short-term outcomes include significant impact on independence in daily activities including employment [8], and perhaps most importantly medication adherence [9]. The availability of a tool that can easily be used to predict HAND is therefore necessary.

Here we propose a screening algorithm for HAND that was developed in a sample of 97 HIV-positive individuals with advanced disease. This algorithm was based on risk factors that have been documented for HAND: age [10], educational achievement [11], plasma viral load [12], previous central nervous system (CNS) HIV-related insult [13], haemoglobin levels [14], HIV infection duration [15], tetracosactide CART CNS penetration characteristics [16] and duration of current CART [17]. The development of the screening algorithm was based on support vector machine (SVM) methodology. Because the aim of our study was to provide a simplified algorithm from a complex set of predictors, SVM was the most appropriate procedure [18]. The SVM has been shown to be extremely robust in solving prediction problems while handling large sets of predictors [18]. It is an alternative to more standard statistical techniques such as logistic regression and in certain situations has been found to be superior to logistic regression for finding a robust fit with fewer predictors [18–21].

Control larvae were injected with 0.9% NaCl. To determine whether DHA confers a protective effect to Burkholderia-infected larvae, a single dose of DHA (50 mM) was administrated 6 h postinfection. To determine intracellular bacterial numbers, hemolymph was obtained from three infected

larvae by puncturing the larval abdomen with a sterile needle. The out-flowing hemolymph was immediately transferred into a sterile Eppendorf tube containing a few crystals of phenylthiourea to prevent melanization. Then, hemolymph was serially diluted in 0.9% AZD8055 mw NaCl and plated on LB agar. Colonies were counted after incubation at 37 °C for 24 h. Three larvae per treatment for each time point (10 and 21 h postinfection) were cryopreserved, sliced and homogenized in 1 mL of Trizol reagent (Sigma–Aldrich). Whole animal RNA was extracted according to the manufacturer’s protocol. RNA was treated with Turbo DNase (Ambio, Applied Biosystems) according to manufacturer’s recommendations and quantified spectrophotometrically (NanoDrop ND-1000). Quantitative real-time reverse transcription PCR (RT–PCR) was performed check details with the 7500 real-time PCR system (Applied Biosystems), according to the protocols of the manufacturer. Briefly, cDNA was synthesized from 200 ng of total RNA using Taqman kit (Roche, Applied Biosystems) and then analyzed with Power SYBR Green

master mix (Applied Biosystems), using primers to amplify the genes encoding antimicrobial peptides: gallerimycin (Altincicek & Vilcinskas, 2006), galliomycin (Wojda et al., 2009), inducible metalloproteinase inhibitor (IMPI) (Altincicek & Vilcinskas, 2006), lysozyme (Altincicek & Vilcinskas, 2006) and the housekeeping gene actin (Altincicek & Vilcinskas, 2006). All samples were analyzed in triplicate, and the amount of mRNA detected normalized to control actin mRNA values. Relative quantification of genes expression was calculated by using the ∆∆CT method (Livak & Schmittgen, 2001). All experiments were performed a minimum of three times. Relative comparisons were done between corrected values with anova test for significance.

A P-value Baricitinib < 0.05 was considered statistically significant. The antibacterial activity of eight LCUFAs with various carboxyl lengths (18 carbons or more) was quantitatively evaluated against B. cenocepacia K56-2. The growth was monitored for 24 h at 37 °C in the presence of 20 mM of each LCUFA by measuring the OD640 nm. Of the eight lipids tested, only 3 [Petroselinic acid 18:1 (n-6), DHA 22:6 (n-3) and nervonic acid 24:1 (n-9)] showed growth inhibition effects. DHA caused the greatest growth inhibition (50–60%) compared with the control (Fig. 1), so it was selected for further studies. A control assay with 2.7% ethanol had no effect on the growth of B. cenocepacia K56-2 (Fig. 1). DHA against B. cenocepacia K56-2 recorded a MIC range of 40–50 mM, determined after 24 h by the reference broth microdilution method.

Louis, MO, USA). All other chemicals used were obtained from standard commercial suppliers. The Crizotinib manufacturer stain used for the blood smear was the quick panoptic (Laborclin Produtos para Laboratório Ltda, Pinhais, PR, Brazil). ZEA was prepared in olive oil, immediately before administration. Mice were weighed and randomly divided in two groups which received one administration of ZEA (40 mg/kg – 8% of LD50) or olive

oil by gavage (10 ml/kg). Forty eight hours after ZEA or vehicle administration the animals received a dose of pentobarbital (180 mg/kg, i.p.), and blood was collected by cardiac puncture into tubes containing heparin (1 UI/μl). The liver, kidneys and testes were removed, weighed and homogenized in Tris–HCl 50 mM, pH 7.4 for the determination of enzymatic and non-enzymatic indicators of oxidative stress. The epididymis were weighed and used for determining the number and motility of spermatozoa. The open field task is a simple assessment used to determine check details general activity levels, gross locomotor activity and exploration habits in rodents. Two days (48 h) after the treatment with ZEA or vehicle, mice were submitted to the open field test. Mice were placed in a wooden box (20 × 30 cm) with the floor divided in twenty-one identical squares, and the number

of squares crossed with all paws, the number of rearings and the time of cleaning were counted during 10 min. In order to evaluate any possible toxic action of acute ZEA administration, the body and vital organs relative weight were determined. Mice were weighted before, and two days (48 h) after the treatment with ZEA and some vital and reproductive organs (lungs, liver, spleen, kidneys, testes and epydidymis) were weighted relatively to the body weight. Total leucocyte count was performed using 25 ul of blood and 500 ul of solution Turkey in a Neubauer chamber with

the aid of optical microscope with a 40× objective (Nikon Eclipse 50i). Subsequently, Glycogen branching enzyme we applied the technique of blood smears for differential counts of neutrophils (segmented and sticks), eosinophils, lymphocytes and monocytes with 5 ul blood. After performing the same, the slides were stained (panotico fast) and viewed under a microscope according to the method described by (Failace et al., 2009). Assessment of spermatozoa count and motility was performed according to Freund and Carol (1964). The two cauda epididymides from each mouse were homogenized in 2 mL of warmed (37 °C) saline solution (0.9% NaCl). Briefly, 10 μL of the diluted spermatozoa suspension was transferred to each counting chamber of the hemocytometer and was allowed to stand for 5 min. The cells settled during this time were counted with the help of light microscope at 200× magnification (Nikon Eclipse 50i).

The publishing was done under a contractual agreement between Elsevier and GSK. For further information regarding GSK’s contributions, please see the Acknowledgments

section on page XX. Some product-related information contained in this book may be outside the approved labelling for the mentioned products. The information is not intended to offer recommendations for administering the products in a manner inconsistent with the approved labelling. Before using any such products, healthcare practitioners should refer to the approved labelling for the product in their own country. Some information in this book may also relate to candidate vaccines that are still in development and have not yet been licensed. No conclusions should be drawn regarding the safety or efficacy of these unlicensed candidate vaccines. The authors and the editors had complete authority over the content, and received no financial remuneration for learn more the book development. GSK funded the travel expenses and accommodations

related to authors’ meetings. All final text was approved by the authors and independently peer-reviewed before publication. Medical writing and editorial support were provided to the named authors by Markus Voges (employee of GSK) and Slavka Baronikova (employee of GSK). Additional medical writing services, including the preparation of illustrations, were provided by ApotheCom ScopeMedical in accordance with a contract between GSK and ApotheCom ScopeMedical. Elsevier would like to thank Maarten Postma NL Selleckchem GSK2118436 and Ray Spier UK for the critical reviewing of the chapters. Professor Myron Levin, who served as a consultant to the authors of this book, provided his services under a contractual agreement with GSK and was compensated for his services. Paolo Bonanni: has received support for Travel, Board membership, Honoraria for consultancy from

different vaccine-producing companies (GlaxoSmithKline, Sanofi Pasteur MSD, Pfizer, Novartis Vaccines). Wen-Fang Cheng: has received support for Travel, Accommodation and Consultancy (GlaxoSmithKline). Anthony Cunningham: has received support for Travel (Merck, Novartis); Consultancy (GlaxoSmithKline, Merck, Novartis); Honoraria (GlaxoSmithKline, Merck, Novartis). Nathalie Garçon: is an employee of GlaxoSmithKline, Patents, Stock Options, (GlaxoSmithKline). Phospholipase D1 Oberdan Leo: has received support for Travel, Consultancy, Grant, and Lectureships (GlaxoSmithKline). Geert Leroux-Roels: has received support for Institutional Grants (Novartis, GSK, Sanofi-Pasteur, Baxter); Consultancy (GlaxoSmithKline, Novartis). José Ignacio Santos: has received support for Travel (GlaxoSmithKline); Lectureships (GlaxoSmithKline). Lawrence R Stanberry: has received support for Travel, Consultancy (GlaxoSmithKline); Board Membership (Nanobio); Employment (Columbia University); Grants (NIH); Royalties (Elsevier, University Press of Mississippi).

To eliminate this possibility, we examined the time course of the fluorescence of SNAP-Kir2.1 with a SDS-PAGE analysis, in which cell division does not affect the decrease. The fluorescence decreased in a similar time course (Fig. 4). The half-life of SNAP-Kir2.1 with the CMV promoter was 19.6±2.4 h (n=3), which is comparable to that on microscopic estimation (18.2 h), suggesting a minor contribution of cell division. These findings raise a question about the current or the amount of Kir2.1, which accelerates Kir2.1 degradation. To test this, we added 0.3 mM BaCl2 to the medium after wash out of SNAP-Cell-TMR-Star and examined the effect on the decrease

in fluorescence (Fig. 5A). Such low concentrations of Ba2+ are known to suppress K+ currents, especially currents flowing through Kir2.1 channels (Sakmann and Trube, 1984). The addition of Ba2+ significantly slowed EPZ015666 solubility dmso the decrease in fluorescence (Fig. 5A), and prolonged the half-life to 38.8±3.8 h (Fig. 5B and E). As a negative control, we expressed SNAP-β2-Adrenoceptors and found no effects of Ba2+ on their degradation (Fig. 5C). To further examine the dependency of the degradation on current, we constructed a SNAP-tagged mutant of Kir2.1, Ruxolitinib cell line E224G. E224G is less sensitive to physiological intracellular blockers (Mg2+ and polyamines) than wild-type Kir2.1: larger outward currents flow

under physiological conditions (Yang et al., 1995). The expression level of E224G was 30% higher than that of wild-type. Kir2.1-E224G was degraded faster than the wild type (Fig. 5A), and the half-life was shortened to 9.6±0.7 h (n=4)( Fig. 5D and F). To further test the current-dependency, we mutated the K+ ion selective filter sequence GYG to AAA. This mutation results in a dominant-negative form of Kir2.1 ( Fig. 1A). The half-life of dominant-negative

form of Kir2.1 was elongated to 34.9±3.2 h ( Fig. 5F), which is comparable to that of Ba2+ treated wild-type channel. These results suggest that the degradation is regulated by the current through Kir2.1 rather than the amount of the channel proteins. To exclude the possibility that the membrane potential is the determinant for the protein degradation rate, we measured the resting membrane potentials of CMV- and SV40-promoter plasmids transfected cells. There are no significant difference in the CMV- and SV40-promoter plasmids transfected cells (−89.5+2.6 aminophylline and −88.2±1.3 mV (24 h), −88.8+4.0 and −88.5±3.9 mV (48 h), respectively, n=4), suggesting that membrane potential is not the determinant of the degradation rate. To confirm the current dependency in a different way, we added CHX (10 μg/ml) to block the de novo synthesis of proteins. Blockade of protein synthesis should have similar effect to the current blockade. As described above, the SNAP-Kir2.1 proteins were internalized from the plasma membrane in the absence of CHX, whereas they still stayed at the plasma membrane 24 h after the addition of CHX (Fig. 6A).

These spiked

samples were serially diluted 1 in 4 in assay buffer and measured on the mAb assay. Serial dilutions DNA Damage inhibitor were replicated five times within the same plate, and the limit of detection for each mAb was then assessed. The limit of detection for each mAb was determined by selecting the lowest concentration detected by the mAb assay above the blank well containing only assay buffer (no BJ protein). Assay linearity was assessed by serially diluting three serum samples containing elevated levels of monoclonal κ FLC and three samples containing elevated levels of monoclonal λ FLC, two-fold in assay buffer. These six samples were serially diluted nine times with three replicates of each dilution conducted within the same plate. Linearity of the mAb assay was then assessed on the ten sample dilutions. Because competitive inhibition assays are inherently non-linear, a strategy for demonstrating linearity was conducted by comparing the expected results against the acquired results from the serial dilutions. Assay batch-to-batch variability was assessed by analysing fifty serum samples with varying FLC levels once, on separate assay days, using

three consecutive PF-562271 mouse batches of anti-FLC mAbs, calibrators and other appropriate assay reagents. Assay imprecision was estimated by calculating the intra-assay coefficient of variation percentage (CV%) and the inter-assay CV%. For these tests, pools of samples with low, medium and high levels of κ and λ FLCs were used. All

samples were analysed in duplicate, every morning, for ten working consecutive days, and all tests were conducted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline EP5-A2 (Tholen et al., 2004). The susceptibility of the MTMR9 mAb assay to interference was measured by adding known quantities of interference agents to a pool of National Health Service Blood and Transplant Service (NHSBT) plasma samples containing normal κ (11.12 mg/L) and λ (7.62 mg/L) FLC levels. Individual aliquots of the plasma pool were spiked with purified IgG-κ (3.5 g/L), IgG-λ (3.6 g/L), IgA-κ (1.5 g/L), IgA-λ (3.2 g/L), IgM-κ (6.5 g/L), IgM-λ (3.7 g/L), haemoglobin (4 g/L), bilirubin (0.2 g/L), cholesterol (2 g/L), triglyceride (5 g/L), as well as κ FLC (2 g/L) or λ FLC (2 g/L). Interference testing was conducted in accordance with CLSI guidelines EP7-A2 (McEnroe et al., 2005). For all tests on assay dynamics, except mAb limit of detection, the maximal value obtained from each anti-κ FLC mAb (BUCIS 01 or BUCIS 04) was used as the final κ result, and the same approach was used for each anti-λ FLC mAb (BUCIS 03 and BUCIS 09) for λ FLC results. 250 plasma samples obtained from healthy random donors (NHSBT UK) were measured using the Freelite™ and mAb assays; results obtained by all four anti-FLC mAbs were used for these analyses.

The fractions that contained ptaquiloside were combined and separated a final time using reverse phase HPLC (10 mm × 300 mm C18 column; gradient elution with H2O/MeOH; 30% MeOH – 95% MeOH for 20 min; UV detection at 220 nm). The purified ptaquiloside was assayed to be >98% using HPLC-apci-MS and NMR analysis. Ptaquiloside was used at a dose of 5.3 mg/kg for the in vivo experiments, as previously described

( Latorre et al., 2011). For the in vitro studies, a concentration of 4.4 μg/ml of ptaquiloside was used. This concentration was determined by preliminary tests that demonstrated a reduction in NK cell cytotoxicity in vitro. Sodium selenite (Na2SeO3) (Labsynth, Brazil) was used as the source of selenium and will be described throughout this article as selenium. Importantly, selleckchem none of the mice in this study were selenium deficient because they received standard diet (Nuvilab-CR1®, Nuvital Nutrientes LTDA) containing 0.05 ppm selenium. As in our previous work (Latorre et al., 2011),

we used a dose of 1.3 mg/kg selenium for the in vivo experiments, based on the results of Albers et al. (2003), and a concentration of 0.1 mM for the in vitro studies. This concentration was determined by preliminary tests that demonstrated an increase in NK cell cytotoxicity in vitro. Mice were separated into four groups, with five mice per group, as follows: control (Co), ptaquiloside (Pt), ptaquiloside and selenium (PtSe), and selenium (Se). In general, experimental www.selleckchem.com/products/ABT-737.html mice were treated by daily gavage for 14 days with ptaquiloside (5.3 mg/kg) and/or selenium (1.3 mg/kg). The Co mice received only water and were treated at the same time as the experimental mice. The body weight of each mouse was measured every 3 days for dose adjustment. On day 15 of the experiment, mice from all groups were killed with Immune system an overdose of CO2 and splenic

cell suspensions were then prepared to isolate NK cells (see below). Spleens were removed aseptically and made into a single-cell suspension. Briefly, for each mouse, the isolated spleen was gently squeezed by the distal end of a syringe into a plate of cold RPMI medium (Gibco). The erythrocytes present in the suspension were then lysed using sterile 0.4% ammonium chloride solution. Splenocytes were centrifuged at 1200 rpm (4 °C, 8 min), and the pelleted cells were then re-suspended in RPMI-complete medium (supplemented with 10% FBS, Gibco). To separate non-adherent from adherent cells, the samples were incubated on 6-well plates for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. Next, non-adherent cells were harvested and filtered through a 70 μm cell strainer. Untouched NK cells were isolated according to the manufacturer’s protocol using an NK cell isolation kit, LS columns and a QuadroMACS cell separator system (Miltenyi Biotec, Inc.).