The IFN-γ pathway is central for ECM development after blood-stage PbA infection. We first assessed the role of this pathway

in preerythrocytic/intrahepatic stage infection by investigating ECM neurological signs development in IFN-γR1−/− mice. Following the injection of 1000 sporozoites, 60% of the WT control mice developed typical ECM neurological symptoms, such as ataxia, loss of grip strength, progressive paralysis, and coma, and succumbed within 8–9 days, as previously described [22]. In contrast, IFN-γR1−/− mice were fully resistant to the same challenge, surviving 30 days with no ECM neurological signs (Fig. 1A). Therefore, type II IFN-γ pathway is essential for ECM development after PbA sporozoite infection. The role of type I IFN-α/β versus type II IFN-γ pathways in ECM development after infection with hepatic or blood-stage PbA was then assessed in mice deficient for either IFNAR1 or IFN-γR1. HDAC inhibitors list DAPT supplier IFNAR1−/− mice were partially protected against ECM following sporozoite-initiated infection, only 20% dying before day 10, and 40% eventually developing typical ECM neurological symptoms, which reflected a delayed ECM development after infection with sporozoites (Fig. 1A),

as compared with WT control mice, 60% of which developed ECM and died before day 10, and IFN-γR1-deficient mice, which were fully resistant to PbA challenge. After injection of PbA-parasited red blood cells (105 pRBC/mouse), WT mice succumbed within 7–9 days with typical ECM neurological signs,

while IFN-γR1−/− mice were resistant, surviving over 20 days after infection with no ECM neurological signs. IFNAR1−/− mice were partially protected, 41% dying before day 9 postinfection and a further 36% developing delayed ECM from day 9 to 11 (Fig. 1B). Partial protection of IFNAR1−/− mice was also seen in response to higher dose PbA-infected erythrocytes injection (106pRBC/mouse; data not shown). Parasitemia was analyzed by flow cytometry using GFP transfected parasites [23]. There was no delay in parasitemia in IFN-γR1−/− and IFNAR1−/− mice following either sporozoite or blood-stage PbA infection. At 9 days after Reverse transcriptase PbA sporozoite infection, parasitemia was about 2% in all groups with no significant differences between WT, IFN-γR1−/−, and IFNAR1−/− mice (Fig. 1C), while after blood-stage PbA infection parasitemia was 11–12% at 7 days in WT and IFN-γR1−/− mice, and was slightly increased in IFNAR1−/− mice (Fig. 1D). IFN-γR1−/− and IFNAR1−/− mice succumbed at later stages to either sporozoite or blood-stage infection with high parasitemia (Fig. 1E and F) and severe anemia (Fig. 2A and B) in the absence of neurological signs. Thus, our data confirm the essential role of type II IFN-γ pathway in ECM development after either PbA merozoite or sporozoite infection and demonstrate a contribution of type I IFN-α/β pathways in ECM development that was not associated with any direct effect on parasite growth.

Presented

results showed that C. albicans cells opsonization with sera significantly improved the killing efficiency of PMN. The ability of immune sera prepared by immunization with M5-BSA conjugate to induced PMN’s killing activity was comparable to or statistically significantly lower (3rd sc dose) than capacity of placebo control sera. The lower efficiency of immune sera to induce candidacidal activity is probably related with lower capacity of specific antibodies KU-60019 nmr to recognize corresponding antigenic structures in cell wall of C. albicans cells and to activate complement, which leads to limited opsonization and reduced induction of PMN’s candidacidal activity. Sera obtained by immunization with M6-BSA conjugate slightly improved the candidacidal activity of PMN with statistically significantly higher effect than control sera for sera after the 1st and the 3rd sc dose SCH 900776 in vitro of conjugate. Comparison of obtained results revealed different functionality of antibodies induced by these two conjugates containing structurally similar α-1,6-branched oligomannosides. Mannan is also able to contribute to the resistance of C. albicans to complement activation through the alternative pathway in the absence of mannan-specific

antibodies [36]. Han and Cutler described protection by a murine IgM and IgG3 antibodies requiring an intact complement system in a mouse model of disseminated candidiasis [6]. We observed mainly decrease in PMN’s candidacidal activity using complement-inactivated sera in comparison with non-inactivated sera; thus, inactivation of complement in sera obtained by immunization with conjugates mainly reduced effectiveness of sera to induce candidacidal activity (Fig. 6). Upon obtained results, we assume different specificity and different

potential protective efficacy of antibodies induced by immunization with M5-BSA and M6-BSA conjugates. The importance of antibodies specificity seems to be critical for induction of candidacidal activity and obtained result confirmed low correlation between protection and mannan or whole cell–specific antibodies levels alone [13, 14]. In addition, results Fossariinae obtained with M5-BSA and M6-BSA conjugates revealed lower ability of α-1,6-branched oligomannoside – BSA conjugates in comparison with linear oligomannoside – BSA conjugates to induce production of antibodies with strong reactivity to corresponding antigenic determinants in natural cell wall mannan and lower capacity to induce antibodies significantly enhancing candidacidal activity of PMN in comparison with previously published results obtained with linear oligomannoside – BSA conjugates [13, 14].

Finally, the release of the constrictive status of the AVA during CIVD may be the direct result of cold acting on the contractile elements in the smooth muscle [43]. It is undisputed that CIVD magnitude and onset time is also strongly dependent on central factors and sympathetic activity, which is clearly visible in the strong effect of manipulations in core temperature on the CIVD response [16,25,26,28]. Supporting evidence was

found by Mekjavic et al. [55] in their finding that, after 15 days of immersing one hand in 8°C water, both the acclimated and contralateral (nonacclimated) hand demonstrated decreased CIVD frequency and finger temperatures. Such observations have resulted in an additional central Trichostatin A model explaining

CIVD, wherein the release of peripheral vasoconstriction serves to release excess heat from the body assuming sufficient body heat content in the core [25–27]. The most likely explanation of CIVD is probably a combination of vasodilators released in cold tissue, a neuromuscular blockade at the sympathetic nerve/AVA junction and direct effect of cold on the contractile mechanism of the AVA. Overall, this lack of consensus makes it difficult to speculate on the potential mechanisms that may be responsible for an enhanced CIVD response with repeated cold exposure. However, initial work is starting to explore the effects of repeated cold exposure on sympathetic drive and selleck products also blood-borne dilatory substances. Changes in sympathetic Selleck Venetoclax outflow over time may contribute to CIVD adaptation, as the repeated immersions should result in a reduced sympathetic outflow over time [46,66]. Many authors reported a decrease in pain or subjective thermal discomfort with repeated local cold exposure [18,22,36,67]. In turn, the reduced pain sensation amplifies the decrease in sympathetic outflow as pain activates the sympathetic system. The reduction in pain sensation may be caused by less sensory input, but is more likely caused by central nervous inhibition

of the afferent sensory input. However, others have suggested that the stress of cold exposure causes an elevation in sympathetic activity, resulting in enhanced vasoconstrictory tone and negative adaptations to local cold acclimation [55]. Only one study measured blood values related to sympathetic outflow [35]. They found no changes in catecholamines over the acclimation protocol. However, as they also observed no changes in CIVD response, the potential role of these factors in any changes in finger thermal responses to repeated cold exposure remains inconclusive. The relative change in sympathetic/parasympathetic drive may be estimated using heart-rate variability measurements during repeated cold immersions of the hands but, to our knowledge, heart rate variability has not been employed in any CIVD study.

89 [95%CI 1.45–2.46, P < 0.001] with an increase in RI of 0.1 (Fig. 2). Conclusion: Higher RI resulted in an increase in the risk for CKD progression. LIM SOO KUN1, THEVARAJAH MALATHI2, CHEW YEE YEAN2, NG KOK PENG1, TAN LI PING1, WONG CHEW MING1, KENG TEE CHAU1, CHONG YIP BOON1, KONG WAI YEW1 1Renal Division, Department of Medicine, University of Malaya; 2Department of Pathology, University of Malaya Introduction: Quantification

of proteinuria is an essential part of chronic LY2157299 datasheet kidney disease management. Proteinuria predicts progression of kidney disease and long term cardiovascular risk. Twenty-four-hour urine protein is the gold standard method of proteinuria quantification but have major limitations. A spot urine sample for protein-creatinine see more ratio (uPCR) and albumin-creatinine ratio (uACR) have been commonly used in routine practices but there is no consensus on the optimal test to estimate proteinuria in kidney diseases. Objectives: 1. To examine the relationship between uPCR and uACR and 24-hour urine protein. 2. To study the diagnostic

performance of uPCR and uACR in estimating proteinuria. Methodology: This is a prospective cross-sectional study that recruited patients who attended renal clinic and had urine dipstick positive for protein. Twenty-four-hour urine samples were collected as per standard protocol. Spot urine samples were collected in the morning upon completion of 24-hour urine collection and uPCR and uACR were performed. Demographic details, clinical data and laboratory test results were captured from patient medical records. The correlation between uPCR and uACR to 24-hour urine protein excretion was assessed. The diagnostic value of uPCR and uACR was expressed in sensitivity and specificity. Results: 187 patients were recruited with mean age of 58.3 ± 14.6 years and 51% were male. Diabetes mellitus

(49%) is the main aetiology of kidney disease, followed by lupus nephritis (16%), IgA nephropathy (14%) and hypertension (11%). The Chlormezanone mean serum creatinine was 181 μmol/L with estimated glomerular filtration rate of 46 ml/min/1.73 m2. There is good correlation between uPCR and uACR with 24-hour urine protein, with r value of 0.84 and 0.89 respectively (p < 0.01). For proteinuriavs 16%). For significant proteinuria (≥1 g per day), both uPCR and uACR have almost similar sensitivity and specificity (98 to 100%). Conclusion: Spot uPCR and uACR correlate well with 24-hour urine protein excretion. uPCR is a better test to estimate proteinuria, with better specificity, in case of positive urine dipstick for protein.

[24, 70] Given that M1 macrophages do not express legumain, this legumain-based FDA-approved Drug Library DNA vaccine may be particularly useful for destroying M2-like TAMs. Another membrane protein involved in T-cell-mediated TAM depletion is CD1d, a strict target of Vα24-invariant natural killer T (NKT) cells. NKT cells are an independent factor

for favourable outcome in various human cancers.[71] The earlier explanation for the tumoricidal role of NKT cells emphasized the expression of CD1d on tumour cells, such as leukaemia and lymphoma cells.[71] However, this explanation faced a great challenge because the majority of human tumour cells are actually CD1d-negative. How do NKT cells reject CD1d-negative tumours? Song et al.[72, 73] provided an alternative answer to this question. They stated that TAMs were the major CD1d-positive cells co-localizing with NKT cells in primary human neuroblastomas and in AZD1208 mouse xenografts of neuroblastoma, and that TAMs were the major targets of NKT cells in CD1d-negative tumours. This discovery is important because it may guide the designs of NKT-mediated immunotherapy, alone or

in combination with other standard therapies. According to this notion, the agents that can promote the expression of CD1d in TAMs may improve the tumoricidal function of NKT cells. One such agent is retinoic acid, which can strongly up-regulate the CD1d expression in macrophages[74] and is now used as a standard therapeutic drug for high-risk neuroblastoma Teicoplanin in clinic.[71] However, the contribution of the NKT–TAM axis to the effects of retinoic acid on tumour suppression needs to be further explored. Although most TAMs exhibit immunosuppressive M2-like properties, they remain the plasticity for polarization,[75] which provides a potential for TAMs to re-polarize from tumour-promoting M2-type to tumoricidal M1-type. It is known that the polarization of macrophages largely depends on the local cytokine profiles. In detail, when high levels of Th1 cytokines, such as tumour necrosis factor (TNF), IL-12 and interferons (IFNs), are present, the pro-inflammatory M1 macrophages

will be established; whereas when exposed to Th2 cytokines, such as IL-4, IL-10, IL-13 and transforming growth factor-β (TGF-β), macrophages will polarize to M2 status.[4] Until now, several signalling pathways, especially the nuclear factor-κB (NF-κB) and the signalling transducer and activator of transcription (STAT) pathways are known to play pivotal roles in the transcriptional profile of macrophages.[6] Among those transcriptional factors, STAT1 and canonical NF-κB (p50p65 heterodimer) are essential for the M1 tumoricidal functions and trigger the expression of pro-inflammatory cytokines.[6] In contrast, TAMs harbouring activated STAT3 and STAT6 are not tumoricidal; instead, they exhibit M2 properties and facilitate cancer development.

To determine whether rSj16 could induce regulatory T cells in vitro, spleen mononuclear cells were isolated from the naïve mice and cultured in the presence of rSj16, SEA or OVA, respectively. Four days later, cells were analysed by flow cytometry (FCM) for the expression of CD4, CD25 and Foxp3, a regulatory function-related marker that is known to be expressed in regulatory T cells and not in activated T cells (24). The results showed that the proportion of CD4+CD25+Foxp3+ T cells in rSj16-treated groups significantly increased compared with SEA, OVA or medium-treated groups (Figure 1a). We then examined whether CD4+CD25+Foxp3+ T cells could be induced by rSj16 in vivo. CD4+ T cells were isolated from the

spleens of mice injected with rSj16, SEA, OVA, incomplete Freund’s adjuvant (IFA) or PBS, respectively. Mitomycin C order The number of CD4+CD25+Foxp3+ T cells was detected by FCM. The proportion of CD4+CD25+Foxp3+ T cells in rSj16-injected group significantly increased compared to SEA, OVA or PBS-injected groups (Figure 1b). Taken together, these results indicated that rSj16 treatment increased CD4+CD25+Foxp3+ T-cell populations both in vivo and in vitro. To further test whether CD4+CD25− T cells can be differentiated into CD4+CD25+Foxp3+ T cells by rSj16; CD4+CD25− T cells were purified and stimulated in vitro with rSj16 in presence of APCs. The number of CD4+CD25+Foxp3+ T cells was also detected by FCM. The results

showed that the proportion of CD4+CD25+Foxp3+ T cells in rSj16-treated groups significantly increased compared with SEA, OVA or medium-treated groups (Figure 1c). The results suggested that the increase of CD4+CD25+Foxp3+ T cells was selleck chemical from the conversion of CD4+CD25− T cells. To determine whether the suppressive activity of CD4+CD25+ T cells could be enhanced by rSj16 in vitro,

CD4+CD25+ T cells from naïve mice were pretreated in vitro with rSj16, OVA or PBS, respectively, then cocultured with responder naïve murine CD4+CD25− T cells in presence of anti-CD3 and APCs (25,26). It is showed that all OVA-, PBS- and rSj16-pretreated Tregs were able to inhibit proliferation of CD4+CD25− T cells, but the degree of inhibition was enhanced in rSj16-treated cells compared with PBS- or OVA-pretreated cells (Figure 2a). We then tested whether Tregs generated by injection with rSj16 could exhibit inhibitory activity in vivo. CD4+CD25+ T cells purified from Nintedanib (BIBF 1120) rSj16-, SEA-, OVA- or PBS-injected mice were cocultured with responder cells, and the degree of suppression was assessed as described above. The results showed that CD4+CD25+ T cells from SEA-, OVA- or PBS-injected mice were effective in suppressing CD4+CD25− T-cell proliferation, but the degree of inhibition was even higher for CD4+CD25+ T cells purified from rSj16-injected mice (Figure 2b). To study the types of suppression of rSj16-induced regulatory T cells, we measured the concentration of the cytokines in supernatants of naïve mouse splenocytes cocultured with different antigens.

m. did induce Gag-specific gut-homing T cells [20]. Thus, in the BALB/c mice, triple regimens of DCM and TGF-beta inhibitor DMC elicited robust CD8+ TEM cells early after vaccination, which converted into TCM and in the spleen maintained the markers for GALT homing. In the first part of this work, we rederived ChAdV-68 by inserting its whole genome into a BAC in a single step, which simultaneously generated a deletion at the E1 locus, for easy further manipulation using recombineering. This simplified cloning strategy paves a way for future derivation and exploration of other human and animal adenoviruses so far untested

for vaccine delivery [40]. The application of BAC recombineering also facilitates modulation of adenovirus immunogenic properties by rational activation of various innate pathways, which will in turn lead to functionally distinct properties of vaccine-elicited

adaptive responses. Clearly, not all the HIV-1-host interactions and workings of the immune system are yet completely understood to, on one hand, identify desired protective properties of vaccine-elicited T cells and, on the other hand, manipulate the intra- and intercellular signaling so as to bias the actively induced responses toward a desired type. Nevertheless, BAC-facilitated www.selleckchem.com/products/gsk1120212-jtp-74057.html genetic manipulation prepares the grounds for such future molecular manipulations. The AMQ epitope-mediated protection of BALB/c mice against EcoHIV/NDK infection [35] best approximates the clearance of HIV-1-infected cells during primary HIV-1 infection. For human vaccines, Gag is a suitable first-generation immunogen, to which broad and robust T-cell responses correlate with good control of chronic HIV-1 infection

[43]. For more efficient early protection particularly in humans, responses to conserved regions of HIV-1 [44, 45] that the virus cannot easily change without Tacrolimus (FK506) a likely significant fitness cost and/or mosaic protein design [46] might be even more beneficial due to the increase coverage of HIV-1 variants and escape mutants. However, these are theoretical arguments: which vaccine design will induce the most effective T-cell responses can be only determined by protection of humans against acquisition of HIV-1 and/or decrease of virus load at set point, which in turn delays development of AIDS possibly without the need of antiretroviral drugs. While the HIV-1-derived Tg determines specificity of the vaccine-elicited T cells, route of immunization and choice of vaccine vector(s) determine the T-cell quality, tissue localization and longevity [47, 48]. In this respect, ChAdVs are gaining center stage as vectors for subunit vaccines against a number of challenging infections such are malaria, HCV, pandemic influenza virus, and HIV-1 [7].

Recent basic studies regarding pathogenesis, the new agents concerning inflammation, mitochondria biogenesis may possible new therapeutic targets for AKI.

Novel biomarker-guided early diagnosis of AKI may facilitate exploration of novel anti-inflammatory and antioxidant therapies in specific AKI syndromes, such as sepsis-induced AKI, and open new avenues to facilitate renal recovery and prevent short and long-term complications. Recently, survivors of episodes of AKI who are at risk for the development or worsening of CKD need greater attention. The burden of CKD on the global health care system is well documented, so the importance of preventing or minimizing CKD progression in survivors of AKI episodes cannot be overstated. To this end, the recent KDIGO clinical practice guideline proposed a new conceptual model, called acute kidney disease, to selleck inhibitor highlight the need to follow survivors of AKI episodes in the near term and monitor development of signs and symptoms of CKD, with a focus on screening for markers of kidney damage (i.e., proteinuria) and/or reduced GFR. Major risk factors for CKD progression after AKI include PD-0332991 purchase advanced age, diabetes mellitus, hypertension, heart failure, preexisting CKD (as defined by proteinuria or educed GFR), and low levels of serum albumin, a dual marker of nutrition and inflammation. The presence of these risk factors should alert

practitioners to be especially vigilant for CKD development after an episode of AKI. The survive episodes of AKI be followed regularly to assess for early evidence of CKD (i.e., development of hypertension, proteinuria, or reduced GFR) to slow progression of diagnosed CKD to ESRD. In this section, many drug therapy including renin-angiotensin system blocker is available. In summary besides renal replacement therapy, no other supportive drugs are available for patients with AKI. The best therapy for patients with AKI seems to be the avoidance of further injury to the kidney. Selleckchem MK-3475 AKI to CKD transition should be cared by

monitoring by change of GFR, presence of proteinuria or hypertension. ZHANG HONG Renal Division, Department of Medicine, Peking University First Hospital & Peking University Institute of Nephrology, China IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and widely considered to be a polygenic disease. Although exact pathogenesis is still unclear, multi-hit mechanism was proposed for this disease. To further clarify genetic factors involved in IgAN and draw clues to the underlying pathogenesis, three groups of scientists from England, US and China, independently performed genome-wide association studies (GWAS) in IgAN and identified several IgAN susceptible loci. One of the identified genomic region 1q32 contains complement factor H (CFH) and the related CFHR3, CFHR1, CFHR4, CHFR2, CFHR5 genes.

Briefly, a mouse was placed into the main chamber of the plethysmograph. The mouse was exposed to nebulized PBS and methacholine (Sigma-Aldrich) in PBS using an ultrasonic nebulizer. As an index of in vivo airway obstruction, buy BYL719 enhanced pause (Penh) values were measured and expressed as relative values compared to baseline Penh values following PBS exposure for each methacholine concentration (1–25 mg/ml). Levels of plasma OVA-specific IgE

(OVA-IgE) in challenged mice were measured by enzyme-linked immunosorbent assay (ELISA), as described previously [16]. Th1 and Th2 cytokine levels (IL-4, IL-5, IL-13, IFN-γ) were measured in BALF by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. To estimate OVA-specific T cell proliferation in vivo, we used OTII CD4+ cells labelled with CFSE; Molecular Probes, Eugene, OR, USA). Single-cell spleen suspensions from OTII mice were depleted of dendritic cells (DCs) using CD11c microbead and automatic magnetic-activated cell sorting (autoMACS) system

(Miltenyi Biotech, Auburn, CA, USA). The purity of CD4+ cells was estimated to be over 90% using a flow cytometer. Cells were incubated with 5 µM CFSE, according to the manufacturer’s instructions. CFSE-labelled OTII cells (5 × 106 cells) were transferred intravenously into each IgG or PBS-administered wild-type mouse. After injection, mice were challenged with OVA for 30 min a day for 2 days. Seventy-two hours after the OTII cell transfer, mononuclear cells from the thoracic lymph nodes were stained with anti-CD4-magnetic-activated PDK4 cell sorting Selleckchem Stem Cell Compound Library (BD Biosciences, Franklin Lakes, NJ, USA) to analyse transferred CD4+ OTII cell proliferation using a flow cytometer. Data were analysed using Cellquest (BD Biosciences) and FlowJo

software (Treestar, Ashland, OR, USA). To analyse the function of lung CD11c+ antigen-presenting cells (APCs), they were collected 24 h after the mice were administered with 1 mg of IgG or PBS, as described previously [17]. Briefly, mouse lungs were minced and then incubated in the digestion medium consisting of RPMI-1640 (Sigma-Aldrich), 5% fetal bovine serum (Sigma-Aldrich), 1 mg/ml collagenase type 4 (Roche Diagnostics, Indianapolis, IN, USA) and deoxyribonuclease I (bovine pancreas; Wako). Lung CD11c+ APCs were isolated using the CD11c microbeads and autoMACS system according to the manufacturer’s instructions. The purity of CD11+ cells was estimated to be over 80% using a flow cytometer. OTII CD4+ cells were isolated from OTII mouse spleens using the MACS system. OTII CD4+ cells (2·5 × 105 cells/well) were co-cultured in a 96-well plate in complete medium with lung CD11c+ APCs (2·5 × 104 cells/well) from naive WT mice after PBS or IgG administration. Cultures were stimulated in vitro with an OVA323–339 peptide (5 µg/ml; GenWay Biotech, San Diego, CA, USA) or medium for 6 h.

0 mm; 2-h postprandial glucose of 11.1 mm). Forskolin After screening, the following groups of healthy subjects were selected: group A: 78 who were aged 80–102 years (43 men and 35 women; mean age = 85.7 ± 4.6 years);

group B: 128 who were aged 60-79 years (78 men and 50 women; mean age = 69.3 ± 5.2 years); and group C: 60 who were aged 20–59 years (35 men and 25 women; mean age = 34.6 ± 9.5 years). There were no significant differences in gender among the three groups (P > 0.05). Reagents and instruments.  Antibodies for three-colour immunofluorescence studies (CD4-FITC/CD8-PE/CD3-PerCP) and four-colour immunofluorescence studies Buparlisib cost (CD3-FITC/CD16+ CD56-PE/CD45-PerCP/CD19-APC), and RBC haemolysin (FACS Lysing Solution) were purchased from BD Biosciences (San Jose, CA, USA); Ficoll lymphocyte isolation medium was from Shanghai Second Biochemical Reagent Factory (Shanghai, China); RPMI-1640 medium, foetal bovine serum (FBS), phytohemagglutinin (PHA), dimethyl sulfoxide (DMSO) and methyl thiazolyl tetrazolium (MTT) were from Sigma (St. Louis, MO, USA); recombinant human

IL-2, IL-1, γ-INF and anti-human CD3 monoclonal antibody (CD3 mAb) were from Beijing Bangding Biomedical Technology Co., Ltd. (Beijing, check details China) COBE Spectra blood cell separator (Beijing,

China) and Nylon Fiber column T were from WAKO (Tokyo, Japan); BIOCELL HT microplate reader was from Anthos Labtec Instruments (Ges.m.b.H, Salzburg, Austria); FACS Calibur flow cytometer was from BD Biosciences; and K562 cells (NK-sensitive cells) were generated in our laboratory. Measurements.  Peripheral blood T cells and T cell subsets, natural killer (NK) cells and B cells, such as CD4-FITC/CD8-PE/CD3-PerCP (20 μl) and CD3-FITC/CD16+ CD56-PE/CD45-PerCP/CD19-APC (20 μl), were added to a tube (Bead count 52187, Lot 89813) followed by adding EDTA-anticoagulated venous blood (50 μl). This mixture was kept at room temperature for 15 min in the dark. Then, 2 ml of FACS Lysing Solution was added, and the mixture was vortexed and incubated in the dark at room temperature for 15 min. This mixture was then analysed by flow cytometry within 2 h. Gates were set based on CD3-PerCP and CD45-PerCP to separate WBC populations and from which lymphocyte subsets were selected. BD Tritest and Multi TEST software (San Jose, CA, USA) were used for data analysis.