FRET was measured by monitoring excitation at 280 nm and emission

FRET was measured by monitoring excitation at 280 nm and emission at 450 nm, and normalized to total AMCA fluorescence (excitation at 350 nm and emission at 450 nm). Complete digestion of HLA-DR1 during the reaction was verified by SDS-PAGE analysis and silver staining of recovered reaction mixtures. Samples were boiled after the addition of 8 × Laemmli SDS-PAGE sample buffer with 5% v/v 2-mercaptoethanol, run on 12% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Freiburg, Germany). Membranes were blocked for 1 hr in blocking buffer (1× PBS, 0·05% Tween 20 and 1× Rotiblock; Roth, Karlsruhe, Germany) and incubated for an

additional hour with the HLA-DR-specific antiserum CHAMP32 diluted in blocking buffer. After washing in PBS with 0·05% Tween 20, horseradish peroxidase (HRP)-conjugated secondary antibody (donkey Ceritinib cost anti-rabbit immunoglobulin; GE Healthcare) was diluted 1 : 5000 in blocking buffer and HM781-36B concentration incubated for 1 hr. Following additional washes, HRP activity was revealed using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare) and visualized using Hyperfilm ECL (GE Healthcare). Cathepsin G−/− (CG−/−) mice on a C57BL/6 background were received from the laboratory of C. Pham (Department of Internal Medicine, Washington University School of Medicine, Saint Louis,

MO). C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were bred and maintained at the Stanford University Research Animal not Facility. The handling of all mice followed guidelines and requirements established by the National Institutes of Health and Stanford University animal research committee. Mice were killed with compressed CO2 gas and by cervical dislocation, and spleens were removed. Single-cell suspensions were prepared by mechanical disruption of the spleen through a 70-μm filter. Spleens were then treated with 1 × red blood cell (RBC) lysis buffer [1·68 m NH4Cl, 0·10 m potassium bicarbonate and 1 mm ethylenediaminetetraacetic acid (EDTA)], washed twice, and used directly for analysis. The following

antibodies were purchased from BD Biosciences (San Jose, CA): anti-mouse I-Ab phycoerythrin (PE), anti-mouse CD11b PE-Cy7, anti-mouse CD11c allophycocyanin (APC), and anti-mouse CD19 APC-Cy7. Anti-mouse CD3 Pacific Blue, anti-mouse CD45R (B220) fluorescein isothiocyanate (FITC), and anti-mouse F4/80 PerCP-Cy5.5 were purchased from eBiosciences (San Diego, CA). Before staining, cell preparations were blocked with 3·3 μg/ml anti-mouse CD16/CD32 (Fc Block; BD Biosciences) in PBS containing 0·5% bovine serum albumin (BSA) and 0·1% NaN3 for 15 min. For intracellular staining, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min on ice, and washed with 1 × Perm/Wash Buffer (BD Biosciences).

Background: Home dialysis provides significant autonomy for most

Background: Home dialysis provides significant autonomy for most people. In Australia 60% of households have 1 or more domestic pets (37% dogs & 26% cats, ABS). Whilst pets provide significant social benefit, little is documented about the potential hazards in the home dialysis setting. Methods: In addition to our local case, the Peritoneal Dialysis Peritonitis registry at ANZDATA was searched for episodes of PD peritonitis

due to P. multicida from 1/1/2011 to 31/12/12. Results: Our local case was a 40yo woman with ESKD due to reflux nephropathy. Dialysis consisted of APD for 2 years following a previous transplant. She worked nightshift as a registered nurse. Her cat slept in the bed with her whilst she was connected to APD and had been noted to lick the Tenckhoff catheter at times. A total of 5 previous episodes of peritonitis in 5 people (4 Caucasian, 3 female), mean age RG7420 cost BI 2536 50 years were identified in the ANZDATA peritonitis registry. All were on APD using glucose-based solutions. Final treatment consisted of Amoxycillin, Gentamicin and Ceftriaxone in 1 case each and Cefazolin in 2 cases. Mean duration of treatment was 16 days (range 14 to 19). Outcome was good in all cases with no deaths, no recurrence, no removal of catheter and no transfer to HD. Conclusions: PD peritonitis

due to Pasteurella multicida is an uncommon but preventable cause of peritonitis. Education of people on PD around the potential hazards of domestic animals should be included in all training for home therapies. 252 JUST A SPOONFUL OF SUGAR – MEDICAL GRADE HONEY FOR PAEDIATRIC PERITONEAL DIALYSIS EXIT-SITE INFECTION, A CASE SERIES TA FORBES1, L SHAW1, Z MILLARD1, J KAUSMAN1,2, Megestrol Acetate AM WALKER1, C QUINLAN1,2 1Royal Children’s Hospital, Melbourne, Victoria; 2Murdoch

Childrens Research Institute, Melbourne, Victoria, Australia Aim: A photographic case series and literature review presenting Medihoney as an effective treatment for peritoneal exit-site infections and over-granulation. Background: International guidelines in peritoneal dialysis (PD) advocate for regular application of topical mupirocin in chronic PD exit-site care. A strong evidence base links this treatment to reduced rates of exit-site infections and peritonitis (ESIP), however emerging reports of increasing mupirocin resistance and gram negative exit-site floral replacement and ESIP are threatening the long-term viability of topical antibiotic ointments as a prophylactic treatment. Honey has multiple, proven, antibacterial and wound healing properties. Cochrane review of topical honey for wound healing found some benefit for superficial and partial thickness burns. Recent randomised controlled trials have not proven honey to be superior to mupirocin in ESIP prophylaxis.

Taken together, these results show that B melitensis exopolysacc

Taken together, these results show that B. melitensis exopolysaccharide is a new mannose-rich polymeric structure. Besides exopolysaccharide, extracellular matrices often contain DNA, which may contribute to the structural integrity of biofilms (Whitchurch et al., 2002; Steinberger & Holden, 2005). To test whether Brucella’s clumps include DNA, culture samples were incubated in

the presence of DNAseI and the enzyme effect was observed under a microscope. Two hours after DNAseI incubation (Fig. 5b), clumps appeared to be digested by the nuclease while culture samples incubated with the enzyme buffer did not (Fig. 5a). This effect was increased after 24 h of incubation (Fig. 5c). Brucella melitensis wild-type strain or bearing a control vector (MG200 strain), used as negative aggregation controls, showed no effect of DNAseI treatment. These results Cobimetinib clinical trial demonstrate that DNA is a component of the

extracellular matrix of B. melitensis aggregates CP-673451 and contributes significantly to their structure. Because a recent study showed that OMVs are classical components of biofilm matrices (Schooling & Beveridge, 2006), we wondered whether our MG210 clumping strain could overproduce OMVs. We tested this hypothesis using transmission electron microscopy (TEM). We analyzed the abundance of OMVs’ structure in culture samples from MG210 and the wild-type strain collected in the stationary growth phase. Compared with the wild-type strain, we observed that the production of OMV-like structures was strongly increased in the clumping strain (Fig. 6a and b). Moreover, we took a set of minimum 20 TEM pictures for each strain on which we counted both the number of OMVs-like structures and the amount of bacteria to obtain quantitative data. Counting was performed in triplicate for each strain. As shown in Fig. 6c, we counted a mean of 73 OMVs per 100 bacteria in the

aggregative strain, but only four OMVs per 100 bacteria in the wild-type strain. These data indicate that OMVs could be a component of the matrix of the clumps formed by B. melitensis as described for other biofilm matrices. To confirm this hypothesis, we compared the abundance of two major OMPs of the OMVs formed by Brucella (Omp25 and Omp31) (Gamazo & Moriyon, 1987; Boigegrain et al., 2004) in B. melitensis wild-type and MG210 strains by dot-blot analysis using specific MAbs (Cloeckaert et al., 1990). Omp16 (PAL lipoprotein) was used as an internal loading control. Dot blotting was carried out with B. melitensis culture supernatants (containing the OMVs fraction) (Fig. 7) from stationary-phase cultures. OD600 nm were used to normalize all samples. As shown in Fig. 7, the abundance of both tested OMPs of B. melitensis’ OMVs is strongly increased in MG210 supernatants compared with the control strain. Omp16 presented almost the same relative abundance in the two strains tested.

2A, panel III compared with Fig 1A panel VI) Based on the resul

2A, panel III compared with Fig. 1A panel VI). Based on the results in our 3D collagen culture experiments, we cannot conclude that enhanced neutrophil accumulation into tumour colonies also led to enhanced tumour destruction.

However, previous in vitro studies demonstrated that increased effector to target ratios resulted in increased tumour cell killing by neutrophils [8, 10]. It was demonstrated that TNF-α acts not only as a chemo-attractant for neutrophils, but also induces IL-8 production by endothelial cells, which is the prototypic neutrophil chemokine [5]. We therefore tested IL-8 concentrations in supernatants of the collagen cultures. In the presence of FcαRIxHer-2/neu BsAb, low amounts of IL-8 were detected in the absence of HUVECs (Fig. 2C). However, the IL-8 concentration was profoundly amplified in the presence of HUVECs and an FcαRIxHER-2/neu BsAb, supporting the selleck idea that HUVECs produced IL-8 after activation by neutrophils. No IL-8 was detected in the supernatant of collagen cultures in which an anti-Her-2/neu IgG mAb had been added (data not shown). To confirm IL-8 production by HUVECs in resp-onse

to factors that had been secreted by activated neutrophils, we cultured Erlotinib cost HUVEC monolayers in the presence of supernatant that had been harvested from collagen cultures in which SK-BR-3 colonies had been incubated with neutrophils and an FcαRIxHer-2/neu BsAb (in the absence of HUVECs). Although minimal IL-8 levels were detected in the harvested supernatant, the IL-8 concentration increased when this supernatant was added to HUVEC monolayers, indicating IL-8 production by HUVECs (Fig. 2D). Interestingly, the peak of neutrophil migration was observed after 4 h, at which time hardly any IL-8 release was found (Fig. 2B and C). IL-8 therefore does not appear to play a major Chorioepithelioma role in our in vitro experiments, but migration is likely due to release of LTB4 after targeting FcαRI (Fig. 1D and [21]). LTB4 not only acts as chemoattractant, but also

affects the vascular permeability of endothelial cells and transendothelial neutrophil migration [30, 31]. Furthermore, IL-1β and TNF-α (which are also released after FcαRI triggering) are also known to up-regulate BLT receptors on HUVECs with concomitantly enhanced LTB4-mediated responses, such as vascular permeability and transendothelial neutrophil migration [32]. Taken together, targeting FcαRI on neutrophils resulted in release of LTB4, which acted as the major chemoattractant for neutrophil migration. Additionally, release of lactoferrin was observed, reflecting neutrophil degranulation, which resulted in tumour cell killing. IL-8 production was furthermore significantly increased in the presence of endothelial cells, which was due to endothelial cell activation by inflammatory mediators that had been released by neutrophils after activation.

These findings suggest encouraging possibilities for targeting an

These findings suggest encouraging possibilities for targeting angiogenesis (for instance with anti-VEGF) see more as a therapeutic strategy in pilocytic astrocytoma. “
“Adult-onset GM2 gangliosidosis is very rare and only three autopsy cases have been reported up to now. We report herein an autopsy case of adult-onset GM2 gangliosidosis. The patient developed slowly progressive motor neuron disease-like symptoms after longstanding mood disorder and cognitive dysfunction. He developed

gait disturbance and weakness of lower limbs at age 52 years. Because of progressive muscle weakness and atrophy, he became bed-ridden at age 65. At age of 68, he died. His neurological findings presented slight cognitive disturbance, slight manic state, severe muscle weakness, atrophy of four limbs and no extrapyramidal signs and symptoms, and cerebellar ataxia. Neuropathologically, mild neuronal loss and abundant lipid deposits were noted in the neuronal C59 wnt datasheet cytoplasm throughout the nervous system, including peripheral autonomic neurons. The most outstanding findings were marked neuronal loss and distended neurons in the anterior horn of the spinal cord, which supports

his clinical symptomatology of lower motor neuron disease in this case. The presence of lipofuscin, zebra bodies and membranous cytoplasmic bodies (MCB) and the increase of GM2 ganglioside

by biochemistry led to diagnosis of GM2 gangliosidosis. “
“S. Sharma, R. Bandopadhyay, T. Lashley, A. E. M. Renton, A. E. Kingsbury, R. Kumaran, C. Kallis, C. Vilariño-Güell, S. S. O’Sullivan, A. J. Lees, T. Revesz, out N. W. Wood and J. L. Holton (2011) Neuropathology and Applied Neurobiology37, 777–790 LRRK2 expression in idiopathic and G2019S positive Parkinson’s disease subjects: a morphological and quantitative study Aims: Mutations in the gene encoding leucine-rich repeat kinase-2 (LRRK2) have been established as a common genetic cause of Parkinson’s disease (PD). The distribution of LRRK2 mRNA and protein in the human brain has previously been described, although it has not been reported in PD cases with the common LRRK2 G2019S mutation. Methods: To further elucidate the role of LRRK2 in PD, we determined the localization of LRRK2 mRNA and protein in post-mortem brain tissue from control, idiopathic PD (IPD) and G2019S positive PD cases. Results: Widespread neuronal expression of LRRK2 mRNA and protein was recorded and no difference was observed in the morphological localization of LRRK2 mRNA or protein between control, IPD and G2019S positive PD cases.

In the absence of ARA, if an APC presents a total of 105 peptide-

In the absence of ARA, if an APC presents a total of 105 peptide-Class II MHC epitopes and even if as little as 10% of its total presented epitopes are self, then a response to at least 104 S-epitopes would be at risk of breaking tolerance compared to the one S-epitope expressed on >95% of the cross-reactive NS-antigens. The probability HM781-36B that an eTh anti-NS will break tolerance by signalling an iT anti-S in ARA is very low compared to what it would be in its absence. The APC would have to express <10−4 of its processed epitopes as S, before ARA becomes irrelevant to Module

2. It is possible to envisage a situation in which the APC cannot present exogeneous S-antigen by assuming that uptake is dependent on the formation of an antigen-antibody complex. This, in and of itself, would significantly reduce the proportion of S-epitopes presented. If, in addition, the uptake of an NS-antigen-antibody complex shuts off endogeneous presentation of S for a period sufficiently long for T-T interactions to occur, then activation approaching the specificity of ARA might be possible [6]. Bretscher [32–34], who has pioneered a good deal of the thinking in this field, has given us a food-for-thought

suggestion to solve the problem of ARA for T-T interactions [35]. If the B cell acted as the sole APC for T-T interactions, the fact that the B cell presents a single NS-antigen KU-60019 solubility dmso would

ipso facto solve ARA for that antigen. The assumption that the B cell is the APC used for T-T interactions appears to solve the problem of ARA. The proposal is so seductive that one wonders why so many reasons to question it arise. 1  Mutant animals without B cells have T-responses that are normal [36–39]. In sum, this proposal is tenuous in spite of the fact that a B cell is known to be able to act as an APC. One competing assumption is that the professional APC can process an antigen into a signalling patch that maintains the derived peptides together, and across which a T-T signalling interaction occurs [6, 8]. This suggestion has its difficulties with mechanism, as does the assumption that the APC can present peptides Oxymatrine from only one antigen at any moment in time. This latter idea is an analogue of the B-cell/APC model with the advantage that it might be able to solve the problem of rare cells interacting. The almost universally popular assumption lacks rationale, namely that Signal 2 is ‘costimulation’ delivered by an APC to any iT-cell receiving Signal 1. Given that peripheral tolerance exists, a solution to the mechanism of ARA in eTh-APC-iT Signal 2 transmission is mandated [7, 35]. The postulated obligatory role for ARA in Module 3 will be analysed next. The mechanism of ARA by T cells interacting on a ‘professional’ APC (dendritic cell) will eventually have to be faced.

Individuals with values above these

were identified as po

Individuals with values above these

were identified as positive responders. Hence, 50% of healthy controls demonstrated positive IFN-γ responses compared to only 11% of individuals with latent infection and 0% for individuals with active TB infection (P = 0·02). Similar results were observed for IL-17- and IL-22-producing CD4+ T cells with P-values of 0·03 for both groups. One Epigenetics inhibitor individual with active TB had a very high proportion of IL-17-producing CD4+ T cells (83·2%), which was excluded from analysis due to suspected systematic error. Four out of 10 latent TB individuals co-expressed elevated proportions of IL-17+ CD4 T cells and IL-22+ CD4 T cells. Because Th17 cells produce IL-17 and IL-22 and recruit neutrophils to the site of inflammation [18,31], we determined if circulating neutrophils also produce IL-17 and IL-22. As neutrophils comprise approximately 90% of granulocytes, we measured the expression of IL-17 and IL-22 in total granulocytes. The granulocytes were gated according to size and granularity using forward-scatter and side-scatter by flow cytometry (Fig. 2a, left panel). CD4-CD8- cells were then gated from

the granulocyte-enriched cell population (Fig. 2a, middle panel) and analysed for IL-17 and IL-22 expression (Fig. 2a, right panel). The intracellular IL-22 Birinapant purchase was detected in a significant proportion of granulocytes from healthy individuals (20–90%). However, intracellular IL-17 was not detected in granulocytes from normal controls and individuals nearly with latent and active TB

infection (data not shown). The proportion of IL-22-expressing granulocytes was significantly lower in individuals with latent and active TB infection compared to healthy controls (P = 0·02; Fig. 2b). IL-22 expression in pure granulocytes isolated from blood was confirmed by counterstaining with another granulocyte marker CD15 (data not shown). To confirm whether IL-22 is transcribed in granulocytes, IL-22 mRNA expression was evaluated at the mRNA level by quantitative real-time PCR (qPCR) in granulocytes isolated from three healthy individuals. Granulocytes were either unstimulated or were stimulated with PMA for 4, 24 and 48 h. Surprisingly, IL-22 mRNA was not detected in unstimulated granulocytes after isolation. However, IL-22 was induced in granulocytes stimulated with PMA and ionomycin (Fig. 2c) with the peak expression at 24 h post-stimulation. To determine whether antigen-specific CD4+ T cells in latent and active TB subjects produce IL-17, IL-22 and IFN-γ in response to mycobacterial antigens, PBMC were stimulated with mycobacterial culture filtrate for 7 days prior to analysis of intracellular cytokines. The induction of cytokine expressing cells was calculated as a percentage increase following stimulation with mycobacterial antigens compared to the unstimulated cells.

We show that AMPKα1 activates rapidly in response to the metaboli

We show that AMPKα1 activates rapidly in response to the metabolic stress caused by glucose deprivation of CD8 cytotoxic T lymphocytes (CTLs). Moreover, AMPKα1 restrains mammalian target of rapamycin complex 1 activity under conditions of glucose stress. AMPKα1 activity is dispensable for proliferation and differentiation of CTLs. However, AMPKα1 is required for in vivo survival of CTLs following withdrawal

of immune stimulation. AMPKα1null T cells also show a striking defect in their ability to generate memory CD8 T-cell responses during Listeria monocytogenes infection. These results show that AMPKα1 monitors energy stress in CTLs and controls CD8 T-cell memory. “
“Dendritic cells 5-Fluoracil orchestrate innate and adaptive immune responses, which are central to establishing efficient responses to vaccination. Wall-associated protein A (WapA) of Streptococcus mutans was previously used as a vaccine in animal studies for immunization Opaganib nmr against dental caries. However, as a cell surface protein, whether WapA activates innate immune responses and the effects of WapA on DCs remain unclear. In this study, WapA was cloned into the GST fusion vector pEBG, which can be expressed efficiently in mammalian cells. We found that when added before stimulation with LPS, purified WapA-GST protein increased TLR4-induced

NF-κB and MAPK signalling pathway activation. Pretreatment with WapA-GST also increased LPS-induced proinflammatory cytokine production

by DCs, including IL-12, IL-6 and TNF-α. Furthermore, expression of the DC DCLK1 maturation markers CD80/86, CD40 and MHC II was also increased by WapA pretreatment. These data indicate that WapA is recognized by DCs and promotes DC maturation. “
“Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls.

In the same group, 66·2% of

physicians had patients treat

In the same group, 66·2% of

physicians had patients treated at home by a home infusion service. About 20% of these practitioners permitted self-infused IVIG in the home. In the United States, as elsewhere, the increasing use of s.c.-delivered Ig has also proved satisfactory, providing similar doses of Ig with similar efficacy rates Selleckchem INCB024360 as for intravenous delivery. This appears to approach 33% use for immune-deficient patients in the United States at this time. In the early phases of treatment, the objective is to make the therapy as easy as possible. This includes starting with doses that are not likely to lead to reactions, and that will introduce the patient to this form of therapy in a way is both reassuring and efficient. It is our practice to use half the intended dose given i.v. for the first time, to achieve both objectives. Premedication for the i.v. route can be given, Selleck CH5424802 but is usually not required. The choice of treatment location is best decided based on convenience to the patient, as is the choice of the i.v. or s.c. route. Both

supply excellent protection against infections. Having chosen one method does not exclude the other; for example, for those who travel or are away at school, the s.c. route might be used on a temporarily basis, even if the i.v. route is their main method when at home. For patients, the main expectation is that they will not have serious infections, be in the hospital, miss work or school due to illness. Thalidomide For the most part, data from trials on all licensed products will satisfy these expectations. Patients sometimes expect that Ig therapy will stop all infections immediately, but for many reasons this is not a realistic expectation. For those with structural lung damage such as bronchiectasis or those with bronchospasm, the risk of respiratory tract infections will continue, although these episodes are likely to be milder and not lead to hospitalizations. Viral infections as noted above or infections with current influenza strains will still occur. Most

subjects with loss of IgG antibodies will also lack IgA, leaving mucosal surfaces less protected. In most studies of efficacy, episodes of sinusitis and nasopharyngitis continue to occur in a significant proportion, suggesting that this area is less well treated by increasing serum IgG levels [8,14,15]. Potentially for the same reasons, replacing Ig in the serum also does not seem to ameliorate gastrointestinal complaints such as diarrhoea or inflammatory bowel disease. With growing confidence in the benefits of Ig therapy among physicians of all specialities, the increasing use of home therapy and the general mobility of patients, there is a tendency in some cases to allow long lapses between physician visits. In the United States there does not seem to be a consensus about how often a patient should see the physician who is ordering the Ig therapy.

Acquisition and data analysis were performed by FACS To test whe

Acquisition and data analysis were performed by FACS. To test whether HCV core

protein could have any effect on NK cells as previously observed with T cells [19, 20], the YTS NK cell line was transduced with a lentivirus construct expressing HCV core protein and GFP (coreGFP+ YTS NK cells). Initially coreGFP+ YTS NK cells were set up to study the effect of core in the proliferation rate and apoptosis Alvelestat of the cells. The expression of annexin-V was evaluated as an early marker of programmed cell death (Fig. 1 and data not shown). Annexin-V staining was performed every 24 h for a total of 7 days, starting 24 h after transduction. Untransduced cells were also set up in parallel as an additional control. After 24 h, HCV core induced a significant increase in the percentage of apoptotic cells (65 ± 5% annexin-V-positive YTS cells) compared with GFP-expressing YTS cells (41 ± 6%). Those cells expressing the highest level of core were more susceptible, suggesting that high levels of core protein induced the apoptosis in the NK cell line. After the first 24 h, there were no significant differences in the percentage of annexin-V-positive cells between core- and GFP-transduced YTS cells at any time point. The

level of apoptotic cells in untransduced YTS cell cultures ranged between 8% and 15% during the experiment (data not shown). Previous studies examining NK cells from patients infected with HCV have noted alterations in the NK cell phenotypes [21, 22]. While YTS cell line does not express inhibitory receptors, we examined the expression of activating receptors in PF-01367338 research buy the coreGFP+ YTS NK cells at 24 and 120 h

post-transduction (Fig. 2). After 24 h of core protein expression in YTS, only NKp46 showed a significant decrease in expression compared with GFP+ YTS control cells (MFI 16 ± 1 in coreGFP+ YTS NK cells compared with Cyclooxygenase (COX) 20 ± 2 in GFP+ YTS NK cells). At 120 h, coreGFP+ YTS NK cells continued to show altered NKp46 receptor expression (25 ± 3 in coreGFP+ YTS NK cells compared with 35 ± 3 in GFP+ YTS NK cells). None of the other receptors examined seemed to be altered in their expression (Fig. 2). Natural killer cells from HCV-infected patients have been observed to have reduced cytotoxic capabilities [6, 8, 23]. Natural cytotoxic activity of coreGFP+ YTS NK cells was measured against the K562 cell line in a standard 51Cr release assay, at 24 and 120 h after transduction (Fig. 3). At 24 h, no significant differences were found between coreGFP+ YTS NK cells and GFP+ YTS NK cells. However, at 120 h, coreGFP+ YTS cells exhibited a significant decrease in the cytotoxic ability (28.9 ± 6.1% by coreGFP+ YTS NK cells compared with 40.4 ± 2.1% lysis by GFP+ YTS NK cells at 30:1 effector/target ratio; 20.5 ± 3.4% lysis by coreGFP+ YTS NK cells compared with 29.7 ± 1.5% by GFP+ YTS NK cells at 10:1 ratio).