There were no serious systemic complications. Although we have described limited cases and supporting data are lacking, we ITF2357 molecular weight feel that this procedure might

be useful for microsurgical reconstruction of the lower limb. © 2010 Wiley-Liss, Inc. Microsurgery 30:376–379, 2010. “
“Venous flow-through flaps (venous flaps) are useful reconstructive options, particularly in the repair of defects with segmental vessel loss. They are relatively easy to harvest and confer several benefits at the donor site. However, given that they are based on a single central vein, their survival is notoriously unreliable and they are susceptible to ischemia and venous congestion. Various designs have been suggested to improve the circulatory physiology, and hence survival, of venous flap. More recent designs involve adaptations to the arrangement and number of efferent veins draining arterialized venous flaps. The most commonly used classification

system for venous flaps, proposed by Chen, Tang, and Noordhoff, does not afford adequate description of these alternate designs. This article offers a classification system that can incorporate all reported modifications to venous flaps. This simple adaptation to the classification system proposed by Chen et al. restores its usefulness in describing modern variations to venous flap design. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“When reconstructing combined defects of the cervical spine and the posterior pharyngeal wall

the goals are bone stability along with continuity of the aerodigestive tract. We present a case of a patient with a cervical spine Raf inhibitor review defect, including C1 to C3, associated with a posterior pharyngeal wall defect after excision of a chordoma and postoperative radiotherapy. The situation was successfully solved with a free fibula osteo-adipofascial flap. The reconstruction with a fibula osteo-adipofascial flap provided several benefits Thiamet G in comparison with a fibula osteo-cutaneous flap in our case, including an easier insetting of the soft tissue component at the pharyngeal level and less bulkiness of the flap allowing our patient to resume normal deglutition. © 2013 Wiley Periodicals, Inc. Microsurgery 34:314–318, 2014. “
“The objective of this preliminary study was to develop a reabsorbable vascular patch that did not require in vitro cell or biochemical preconditioning for vascular wall repair. Patches were composed only of hyaluronic acid (HA). Twenty male Wistar rats weighing 250–350 g were used. The abdominal aorta was exposed and isolated. A rectangular breach (1 mm × 5 mm) was made on vessel wall and arterial defect was repaired with HA made patch. Performance was assessed at 1, 2, 4, 8, and 16 weeks after surgery by histology and immunohistochemistry. Extracellular matrix components were evaluated by molecular biological methods.

Both of these hospitals are major central referral centers to which many patients from other areas of Iran are referred. In all, 183 immunocompromised patients were included in this study. Eligibility criteria Panobinostat solubility dmso were immunosuppression

due to HIV infection (with decreased white cell counts), hematological malignancies and use of immunosuppressive drugs after solid organ transplant or for treatment of chronic or intractable hematologic diseases. The ethics committee of Baqiyatallah University of Medical Sciences approved the study protocol. After informed written consent had been obtained, the study nurse administered a comprehensive questionnaire to each patient. This author-compiled checklist included items on patient variables including age, sex and weight; sociodemographic and intra-familial factors; location of dwelling; occupation; number of household members with diarrhea; zoonotic factors including exposure to pets and farm animals; and environmental factors including source of drinking water and exposure Daporinad datasheet to lake, river or swimming pools. Clinical characteristics including diarrhea, weight loss, vomiting, abdominal pain and nausea, presence of concomitant microbial infections, antiretroviral use and laboratory characteristics including CD4 + T-cell counts were recorded. This checklist was filled out

by a physician who confirmed patient’s symptoms by physical examination and so on. Diarrhea was defined as three or more watery or loose stools in a 24-hour period. Diarrhea that persisted for more than two weeks was considered chronic; otherwise, it was classified as acute. Weight loss was considered significant when referred patients lost more than 10% of their baseline body weight during their hospitalization. Three fecal samples were collected at two days intervals from each patient and placed in a disposable plastic cup. The samples were taken immediately to the laboratory and stored at −20°C until analysis. The fecal specimens were concentrated using a sucrose solution with a specific gravity of 1.200 at a centrifuge speed of 800

×g for 10 mins. All samples were stained by the modified Ziehl-Neelsen method and examined under Staurosporine bright field microscopy. A sample was considered Cryptosporidium positive if typical oocysts 4–6 μm in diameter were visible. Fecal samples were subjected to six cycles of freeze–thaw in liquid nitrogen and a 95°C water bath to rupture the oocysts. DNA was isolated from aliquots of frozen stool using the QIAamp DNA stool minikit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. A two-step nested PCR protocol was used to amplify the 18S rRNA gene (830 bp). The fragment of the 18S rRNA gene was amplified by PCR using the following primers: 5′-TTCTAGAGCTAATACATGCG-3′ and 5′-CCCATTTCCTTCGAAACAGGA-3′ for primary PCR and 5′-GGAAGGGTTGTATTTATTAGATAAAG-3′ and 5′-AAGGAGTAAGGAACAACCTCCA-3′ for secondary PCR.

Conclusion: The fructuation of CH50 after the transition to on-line HDF was

correlated with nutrition status such as TP, Alb, UA, K or cholesterol, and might be one of the early indicators for permanence of on-line HDF. KIMURA KEIKO1, KASUGA HIROTAKE1, TAKAHASHI RYO1, MATSUBARA CHIEKO1, KAWASHIMA KIYOHITO1, KAWAHARA HIROHISA1, DMXAA concentration MIZUNO MASASHI2, SUZUKI YASUHIRO2, MARUYAMA SHOUICHI2, ITO YASUHIKO2, MATSUO SEIICHI2 1Nagoya Kyoritu Hospital; 2Nephrology, Nagoya University Nagoya University Introduction: Ankle brachial index (ABI) has been widely recognized as a marker of systemic atherosclerosis in various population including hemodialysis (HD) patients. Protein-energy wasting (PEW), currently considered to be due to inflammatory process rather than poor nutritional intake, is highly prevalent in HD patients, and is also associated with increasing risk of mortality. We investigated the association of ABI and PEW with mortality in HD patients. Methods: A total of 1036 HD patients were divided into three groups according to ABI PD98059 chemical structure levels; normal group: 0.9–1.4 (n = 682), high group: >1.4 (n = 150) and low group: <0.9 (n = 204) and were also divided into tertiles according to geriatric nutritional risk index (GNRI) levels as a simplified marker of PEW state; tertile 1 (T1): <90.8, T2: 90.8–97.3 and T3: >97.3 (Table 2). GNRI was calculated as follows; GNRI = (14.89 × albumin) + [41.7 × (body

weight Lck / body weight at BMI of 22)]. They were followed up for 8 years. Results: Declined GNRI levels were independently associated with abnormal ABI (<0.9 or >1.4) (odds ratio 0.97, 95%CI 0.96–0.99, p = 0.0009). By Kaplan-Meier analysis, 8-year event-free survival rates from mortality were 62.8%, 46.2% and 27.3% among normal,

high and low ABI group (p < 0.0001), and were 34.3%, 59.7% and 68.0% among T1, T2 and T3 of GNRI, respectively (p < 0.0001). After adjusting for other confounders, both ABI and GNRI were independent predictors for mortality. In the combined setting of ABI and GNRI, the risk of mortality was 4.26-fold (95%CI 2.63–6.90) higher in the low ABI group with T1 of GNRI and 3.69-fold (95%CI 2.30–5.91) higher in the high ABI group with T1 of GNRI compared to the normal ABI group with T3 of GNRI, respectively Similar results were also obtained from cardiovascular mortality. Conclusion: Abnormal ABI and lower GNRI, might reflect PEW state, were closely linked, and were additively associated with increasing risk of mortality in HD patients. ZHAO LIJUN1, HUANG SONGMIN1, LIANG TING2, TANG HONG2 1Department of Nephrology, West China Hospital of Sichuan University; 2Department of Cardiology, West China Hospital of Sichuan University Introduction: While chronic dialysis therapy has been exhibited a high prevalence of pulmonary hypertension, occurrence of right heart failure during dialysis treatment is associated with high mortality in patients with pulmonary hypertension.

haematobium also suggests that

co-infection may favour immune regulation via IL-10. However, it is also possible that compared to S. mansoni, infection with S. haematobium is more favourable to IL-10 production, rather than being just a result of co-infection see more with the two species. Inclusion of a group of patients infected with S. haematobium alone would clarify the relative role of the two species. Should co-infected individuals exhibit a more regulated early immune response, this may predispose the host to developing down-regulated response to later stages of parasite development. Indeed, a recent study in the same region of Senegal suggests that Selleck Alectinib co-infection with S. mansoni may reduce the risk of S. haematobium-associated bladder morbidity [23], and it is possible that IL-10 induced by cercarial E/S material may contribute to this phenomenon. Repeated exposure to cercarial E/S in a schistosome-endemic setting may favour down-regulation of egg-associated pathology in a manner akin to that seen in a murine model of repeated infections [10]. Another possible factor to explain the greater

IL-10: TNFα cytokine ratios in co-infected patients might be infection intensity as it has been shown that systemic IL-10 levels are higher in individuals with a greater worm burden [29-31]. It might be concluded that co-infected individuals had greater water contact (i.e. increased incidences of exposure leading to infection with both species and/or exposure to a greater number of cercariae) and therefore have higher worm burdens. Indeed, it has previously been shown that S. mansoni egg output is greater in co-infected subjects than those infected only with S. mansoni in the Diokhor Tack community [22]. However, this was not observed in the subcohort of participants in the current study. There was also no correlation between either S. mansoni or S. haematobium egg output

and the production of any of the 0–3 h RP-specific cytokines tested (data not shown). The composition of various leucocyte subsets in WB Cobimetinib nmr may also affect the cytokine profile of cultured WB. Although we found no difference in the proportions of neutrophils, monocytes, lymphocytes or basophils, there was a significant increase in the proportion of eosinophils in the WB from both schistosome-infected groups compared with the uninfected control group. Eosinophilia is a common feature of human schistosome infections [32], and eosinophils are a potential source of IL-10 [33, 34] but a correlation between elevated eosinophil counts and IL-10 production was not observed. Due to its small size, our study may have lacked statistical power to detect significant correlation between egg output and cytokine production, or leucocyte composition, of WB.

The recombinant histidine-tagged protein TcSPR was purified by Ni2+ chelation chromatography selleck screening library following the Gibco BRL procedure for the Protein Expression System, pROEX-1 vector (cat. No. 10197-010, Gaithersburg, MD, USA), from Escherichia coli transformed with the plasmid pRSETTcSPR. The recombinant proteins TcSP, His-TcSPA and His-TcSPC were obtained as inclusion bodies from E. coli transformed with the plasmids pRSETTcSP, pRSETTcSPA and pRSETTcSPC,

respectively. Sodium deoxycholate-washed inclusion bodies (2% in 50 mM Tris-HCl pH 7·5, 50 mM EDTA) were resuspended in 100 mM Tris-HCl, pH 12·5 and solubilized by gradually adding 2 M urea. After centrifugation, supernatants containing solubilized

proteins were passed through a DEAE-cellulose column (DE-52; Amersham Pharmacia, Piscataway, NJ, USA), and bound proteins were eluted with a linear gradient of NaCl 0·0–0·5 M in 100 mM Tris-HCl, pH 12·5. Purified recombinant proteins were dialysed against phosphate-buffered saline (PBS) and analysed by SDS-polyacrylamide gel electrophoresis. Plasmid DNA was purified by anion-exchange chromatography using Qiagen maxi kits. DNA used for immunizations was sterilized by ethanol precipitation and resuspended cancer metabolism inhibitor in lipopolysaccharide-free PBS (Gibco). Details on the coding region of the TcSP gene and the transcribed amino acids are shown in Table 1. Groups of 4–10 BALB/c mice were used in this study. The mice were immunized by intraperitoneal (i.p.) injection of 10 μg of the recombinant proteins emulsified in Freund’s complete adjuvant (Sigma) and boosted twice with 10 μg of the recombinant proteins in Freund’s incomplete adjuvant every 2 weeks. For DNA-based immunizations, 100 μg of recombinant plasmids or vector DNA was dissolved in 50 μL of PBS, injected intramuscularly (i.m.) in the tibialis anterior muscle and boosted twice Oxalosuccinic acid every 2 weeks [25]. Mice immunized with DNA or recombinant proteins were challenged

i.p. 2 weeks after the last boost with 80 × 103 blood trypomastigotes. The selected dose of the parasite has previously been shown to be high enough to produce acute parasitemia and mortality in infected mice [25]. Eight days after challenge, parasitemia was monitored by counting peripheral parasites every 3 days in 40 μL of blood diluted 1/25 in PBS by direct microscopy examination in a Neubauer chamber. Obtained data are presented as parasites/mL (104) of blood. The mice died naturally, and mortality was recorded daily. Proteins were resolved on SDS-PAGE [26] and either visualized by staining with Coomassie blue or electrophoretically transferred onto nitrocellulose membranes for immunoblotting [27].

(7). Freshly prepared MRS broth was supplemented with 0, 1, 2, and 3 mg/ml concentration selleck screening library of oxgall as a bile source (Sigma, St Louis, MO, USA). A filter-sterilized cholesterol solution

(10 mg/ml in ethanol) was added to the broth to a final concentration of 100 μg/ml, inoculated with each strain (at 2%), and incubated at 42°C for 19 and 48 hr. After the incubation period, cells were removed from the broth by centrifugation for 20 min at 10 000 ×g and 1°C. A modified colorimetric method as described by Rudel and Morris (15) was used to determine the amount of cholesterol in the resuspended cells and spent broth. The amount of cholesterol removed was estimated by subtracting the cholesterol amount in the spent broth from that in the uninoculated control broth. Cholesterol uptake was determined according to a modified method of Kimoto et al. (16). Overnight cultures

of the strains were inoculated into 10 ml of MRS broth and incubated at 42°C for 19 hr. After incubation, the cells were harvested by centrifugation for 15 min at 1800 ×g, washed twice with sterile distilled water, and resuspended in 10 ml of distilled water. The suspension was divided into two portions. The first portion was autoclaved for 15 min at 121°C to prepare heat-killed AP24534 cells whereas the other portion was not processed (i.e. resting cells). The heat-killed cells were suspended in MRS broth containing oxgall (3 mg/ml) and cholesterol (100 μg/ml), which was previously adjusted at pH 6.8. In the case of the resting cells, they were suspended with 0.05 mol/l PBS buffer (pH 6.8) containing oxgall (3 mg/ml) and

cholesterol (100 μg/ml). The process of incubation and centrifugation was the same as above. The spent broth was assayed for cholesterol. EPS production in MRS broth supplemented with 0 μg/ml and 100 μg/ml cholesterol was determined according to modified methods of Valerie and Rawson (17) and Dubois et al. (18). Overnight cultures of the strains were inoculated with 5 ml of MRS broth supplemented with 100 μg/ml and without cholesterol. After incubation at 42°C for 19 hr, 1 ml aliquots of the samples were taken to small test tubes and Thymidine kinase tested for EPS production. For the immobilization procedure, modified methods of Sheu and Marshall (19) and Sultana et al. (20) were used. Overnight cultures of the strains were inoculated with 500 ml of MRS broth and incubated at 42°C for 19 hr. Tubes were centrifuged for 15 min at 5000 ×g and 1°C and washed with PBS (pH 6.2) three times. The pellet was suspended with 50 ml NaCl solution (9 g/l) and cell density was determined according to Mac Farland 6 (Bio Mérieux, Marcy l’Etoile, France) and equalized for all samples. This suspension was mixed with a sterile Na–Alginate mixture (2 mg/100 ml; Sigma-Aldrich GmbH, Steinheim, Germany) and homogenized with a magnetic mixer (Heidolph, EKT 3001, Kelheim, Germany). The cell pellet solution–alginate mixture was dropped into a sterile 0.4 mol/l CaCl2 solution with a peristaltic pump.

[1] Donor-derived T cells are considered the main effector cells mediating acute GVHD because they recognize MHC disparities (allo-antigen) between the donor and recipient, which are presented by antigen-presenting cells (APC). T-cell activation in response to allo-antigen STA-9090 datasheet requires two stimulatory signals.[1] The primary signal is delivered through the T-cell receptor (TCR), which recognizes antigens on MHC molecules. This signal is necessary but not sufficient to induce full T-cell activation, which also requires co-stimulation that drives T cells to proliferate and produce cytokines. The co-stimulation signal is mediated by a number of ligand–receptor pairs expressed

on APC and T cells, and is a composite or net effect of stimulatory and inhibitory signals mediated between these two

cells. The inhibitory TCR include cytotoxic T-lymphocyte antigen-4 (CTLA-4),[2] programmed cell death-1 (PD-1)[3] and B- and T-lymphocyte attenuator BAY 80-6946 price (BTLA).[4] Studies using experimental models of acute GVHD have shown that co-stimulatory molecules play a pivotal role in initiating acute GVHD.[5] By contrast, much less is known about co-inhibitory pathways in this process, better understanding of which would make them useful therapeutic targets. Recently, we discovered a new co-inhibitory pathway composed of DC-HIL on APC and syndecan-4 (SD-4) on activated T cells.[6, 7] DC-HIL is a highly-glycosylated type I transmembrane receptor (95 000–120 000 molecular weight) expressed constitutively by many APC sub-sets including

macrophages, monocytes, epidermal Langerhans cells, CD11c+ CD4+ lymphoid dendritic cells (DC), CD11c+ CD8+ myeloid DC and CD11c+ PDCA-1+ plasmacytoid DC.[8] It is also known as glycoprotein nmb (Gpnmb),[9] osteoactivin[10] and haematopoietic growth factor-inducible neurokinin-1 type (HGFIN).[11] DC-HIL binds to heparan sulphate-like structures on SD-4 expressed by activated (but not resting) T cells, isothipendyl and their binding inhibits strongly the anti-CD3 response of T cells, resulting in cessation of interleukin-2 (IL-2) production and prevention of T-cell entry into the cell cycle.[6, 12] Consistent with a previous finding that SD-4 is expressed primarily by effector/memory (but not recently activated) T cells,[13] infusion of DC-HIL or SD-4 soluble receptor during the elicitation (but not sensitization) phase of contact hypersensitivity effectively blocked the inhibitory function of the endogenous DC-HIL/SD-4 pathway, thereby enhancing ear-swelling responses in this experimental model.[7] Conversely, depletion of SD-4+ T cells by infusion of toxin-conjugated DC-HIL inhibited elicitation (but not sensitization) of contact hypersensitivity.[13] These findings support the concept that binding of DC-HIL to SD-4 inhibits pre-primed T-cell responses. To determine whether SD-4 is the sole T-cell ligand of DC-HIL and whether its negative regulatory role applies to acute GVHD, we took advantage of SD-4 knockout (KO) mice.

tuberculosis infection in humans, and that responsiveness to the triggering molecules (TNFR1/2

and Fas) is decreased in macrophage/monocytes, potentially to the advantage of the pathogen. There is a substantial body of evidence that suggests that apoptosis may be an important factor in the control of TB. Attention has been focused in particular on apoptosis of the macrophage, since the alveolar macrophage is the cell most likely to first come in contact with M. tuberculosis after inhalation of the bacteria and depending on activation, can either eliminate the pathogen or become a host cell for it 46, Dinaciclib in vitro 47. Apoptosis appears to be a critical link in this process, but precisely how this is controlled remains unclear. The literature is complex – as are the interacting and overlapping apoptosis pathways themselves: apoptosis does not result from a simple activation, but is the outcome of the balance of multiple factors

that promote or inhibit the development of the cascade. These apoptotic modulating factors are themselves controlled by other factors. As a result the literature contains evidence – even down to the causative genes in the pathogen – that M. tuberculosis promotes apoptosis in infected cells 29 or inhibits it 30. Depending on conditions, either or both of these activities is likely to occur and whether cell death results is likely to be CB-839 cost due to the balance of multiple factors. The issue is further complicated by the fact that many studies focus on in vitro models where infection is examined in isolation. While a reductive approach of this sort is necessary to mapping out the pathways involved and identifying factors that could be involved (and more or less required, given the number of modulating proteins that could be involved), it is not necessarily predictive of the situation in human disease. We have Adenosine triphosphate therefore addressed

only the question of the activation of the extrinsic pathway of cell death, which has been suggested as an important method of removing infected cells. We took blood from three cohorts in a TB endemic country – Ethiopia – and examined gene activation of the earliest triggering and regulating factors on the extrinsic pathway of apoptosis. The high incidence of M. tuberculosis infection in Ethiopia means that everyone in the three cohorts has potentially been exposed to infection, and prior work makes it plain that a substantial proportion of even the CC group is most likely latently infected 18, 48. Thus, the three clinical cohorts can be thought of as representing a spectrum. The assumptions we have made, based on prior studies, are that TB patients represent the disease in its active and most pathological state.

Multiparity induces transferable-specific hypo-responsiveness or even true tolerance to either HY or paternal alloantigens.53,54

Placental products, be them placental PLX-4720 price extracts or water-soluble material obtained from these, co-injected with alloantigenic cells, induce systemic antigen-specific LyT2+ Ts.81 These were traced in the first pregnancy in mice and in rats by Baines and Liburd. Similarly, antigen-specific MHC-restricted Ts were found in humans.82 Controversies about in vitro assays can still be traced in proceedings of the Gusberg meeting.83 In the 1980s, we studied, in detail, the in vitro properties and mode of action of these suppressor cells (specificity, mediation by a soluble factor). A part of these studies was carried out with anti I–J antisera, as many other labs working on suppression did at the time. Lee Hood’s demonstration that the I–J region does not exist while properties of the suppressor BIBW2992 ic50 factor(s) of

Gershon and Cantor were more and more improbable doomed Ts. For an excellent revision of the history of Ts, see references.84,85 We nevertheless still tested/published the role of Ts in CBA × DBA/2 matings.51 As reviewed, in,86 the CD25 and Foxp3 markers again boosted Ts on the forefront. Yet the I–J trauma lead to a more benign denomination of ‘regulatory T cells’ (Tregs), rather than ‘CD4+ Ts’, which we first saw in 1981, but termed ‘inducers’ .87 CD8+ cells are still important partners, as shown in studies by Arck, Clark and coworkers.88 Aluvihare and Darasse convincingly demonstrated that CD4+ CD25+ elimination causes foetal deaths in allopregnancy by transfer or direct in vivo experiments.89,90 Saito traced/ quantified Foxp3 cells Tenofovir molecular weight (T regs) in human decidua as well as regulatory NK/T cells.91 Robertson and coworkers92 showed that the Foxp3

marker decreases in unexplained infertility endometrial biopsies. These, and Fainbolm, detected periodic T reg modulation during the menstrual cycle, peaking in the late follicular phase.93,94 For Fainbolm, T regs from patients with RSA are ‘functionally deficient’,93 and T reg decidual recruitment correlates with expression levels of CCL3, CCL4, CCL5, CCL22, and CX3CL1.90 Finally, placenta-dependent CD8+ T regs have been demonstrated by Shao et al.,95 and this is reminiscent of earlier data in mice about LyT2 Ts.81 Could the placenta escape immune attack by resisting effector cell lysis? We have discussed the Fas/Fas ligand interaction. Membrane and soluble HLA-G (sHLA-G) also play a role, including sHLA-G secretion by the MHC-syncytiotrophoblast. Moreover, trophoblasts (and choriocarcinomas) are resistant intrinsically to cell-mediated lysis.96–98 This resistance is independent of HLA-G.99,100 The once debated soluble factors96,101,102 had properties which fits with what is now known of soluble HLA-G, be it sHLAG1/ G2 characteristics.

In conclusion, we show that receptor repertoire of circulating NK cells is not altered by previous infection with CMV. After exposure to CMV in vitro, however, an HLA class I ligand dependent expansion of KIR2DL1+ and KIR2DL3+ cells occurs, along with expansion of cells expressing NKG2A and KIR3DS1. Changes to the NK-cell receptor repertoire were confined to CMV-IgG positive patients.

Healthy donor buffy coats and sera were collected under an ethical committee approved protocol after written informed consent from SCH727965 supplier all study participants. PBMCs were extracted by using Ficoll. IgG antibodies as a sign of previous infection with CMV were detected using a commercially available assay (Architect CMV IgG, Abbott). Ku 0059436 DNA was extracted from an aliquot of cells by NucleoSpin DNA Extraction Kit (Macherey-Nagel, Düren, Germany), and stored at −20°C until use. The remaining mononuclear cells were cryopreserved until use as described below. mAbs used to stain cell-surface and intracellular Ags were: CD3 (OKT3, eBioscience), CD56 (HCD56, BioLegend), KIR2DL1 (143211, R&D), KIR3DL1 (DX9, Miltenyi), KIR2DL3 (180701, R&D), KIR2DL1/DS1 (HP-MA4, BioLegend), KIR3DL1/S1 (Z27.3.7, Beckman Coulter),

NKG2A (Z199.1, Beckman Coulter), NKG2C (134591, R&D Systems), KIR2DS4 (JJC11.6, Miltenyi), KIR2DL5 (UP-R, BioLegend), KIR2DL2/S2/L3 (DX27, Miltenyi), Ki-67 (20Raj1, eBioscience), CD107a (H4A3, BD-Pharmingen), and IFN-γ (B27,

BD Pharmingen). Samples were acquired on a DAKO CyAn ADP nine-color flow cytometer (Beckman Coulter). For all analyses of NK-cell subsets, we gated on the CD56+/CD3− subset. FACS plots were analyzed with FlowJo software version 9.2. Propidium iodide (BD Pharmingen) was used to exclude dead cells from the analysis. Healthy donor PBMCs (0.2 × 106) were cultured in the presence of 5000 MRC-5 fetal human lung fibroblast cells (kindly provided by H. Hirsch, Basel) on 96-well plates in 200 μL of DMEM plus Vorinostat solubility dmso L-glutamine, 1 mg/mL d-glucose and pyruvate (GIBCO), 10% FCS (Sigma-Aldrich), and 1000 U penicillin/streptomycin (GIBCO). Cells were cultured at 37°C for 14–21 days, and half of the co-culture medium was replaced weekly. At indicated days, cells were harvested and analyzed by FACS for analysis of KIR and NKG2A expression. The MRC-5 cell line was infected with a WT strain of CMV (kindly provided by H. H. Hirsch, Basel) the day before culture and also weekly during the changing of culture medium. Co-culture with uninfected MRC-5 was used as a negative control. Successful infection of MRC-5 cells by CMV was assessed in control cultures demonstrating cytopathic effects. KIR genotype was assessed using sequence-specific primer PCR [25].