In conclusion, increased frequency of CD4+ Tregs and CD8+ Tregs in HCV-infected patients compared with healthy controls and even higher frequencies in HIV/HCV co-infected patients was found. Furthermore, CD4+ Tregs in HCV-infected and this website HIV/HCV co-infected patients display a more active phenotype. Importantly, Tregs in the liver was found to be associated with the degree of inflammation but

not the current stage of fibrosis. Thus, Tregs may have a role in regulation of inflammation during HCV infection. However, larger prospective studies of patients with chronic HCV are warranted to elucidate this. We gratefully acknowledge the patients and healthy subjects who made this study possible. The authors have no conflicts to disclose. This VX-770 supplier study was funded by The Danish Council for Independent Research, Lundbeck Foundation, Novo Nordisk Foundation and Augustinus Foundation. The funders had no impact

on study design, data collection and analysis, or preparation of the manuscript or decision to publish. Part of the data included in this manuscript has been presented at The International Liver Congress™ 2011, by the European Association for the Study of the Liver (EASL), Berlin. “
“Cells that belong to the family of innate lymphoid cells (ILCs) not only form a first line of defense against invading microbes, but also play essential roles in tissue remodeling and immune pathology. Rorγt+ ILCs, producing the cytokines IL-22 and IL-17, include lymphoid tissue inducer (LTi) cells which are critical for the formation of lymphoid structures. Recently another ILC subset has been identified, which is dependent on RORα for its development and is dedicated to the production of the Th2 cytokines Thymidine kinase IL-5 and IL-13. These ILCs have been termed type 2 ILCs. All ILC subets are considered to belong to the same family that also includes natural killer cells because they all rely on the common γ-chain (γc) of the IL-2 receptor for their development and function, share a lymphoid morphology and depend on the transcriptional repressor Id2 for their development.

Other transcription factors, including Notch, and the aryl hydrocarbon receptor (AhR) in RORγt+ ILCs and GATA3 in type 2 ILCs, also play roles in the development, survival, and function of these ILC subpopulations. Here we review the current knowledge with regard to the transcription factors involved in the development and functions of ILCs. Innate lymphoid cells (ILCs), including RORγt+ ILCs and type 2 ILCs, represent a novel group of cells related to NK cells. ILCs lack antigen receptors encoded by rearranged genes, such as the T-cell receptors expressed by T cells (reviewed in [[1, 2]]). Emerging evidence indicates that ILCs not only function as a first line of defense against invading microbes, but also play essential roles in tissue remodeling.

“
“Alzheimer’s disease (AD) is associated with neuronal degeneration, synaptic loss and deficits in multiple neurotransmitter systems. Alterations in the serotonin 1A (5-HT1A) receptor can contribute to impaired cognitive function in AD, and both in vitro binding and Positron emission tomography (PET) imaging studies have demonstrated that 5-HT1A receptors

in the hippocampus/medial temporal cortex are affected early in AD. This neuropathological study examined the localization and immunoreaction intensity of 5-HT1A receptor protein in AD hippocampus with the goal to determine HM781-36B supplier whether neuronal receptor levels are influenced by the severity of NFT severity defined by Braaks’ pathological staging and to provide immunohistochemical confirmation of the binding assays and PET imaging studies. Subjects included AD patients and non-AD controls (NC) stratified into three Braaks’ stages (Braak 0–II, NC; Braak III/IV and V/VI, AD). In the Braak 0–II group, 5-HT1A-immunoreactivity (ir) was prominent in the neuropil of the

CA1 and subiculum, moderate in the dentate gyrus molecular layer (DGml), and low in the CA3 and CA4. No changes in 5-HT1A-ir were observed in the hippocampus of AD subjects in the Braak III/IV group. Hippocampal 5-HT1A-ir intensity was markedly decreased in the CA1 region in 6/11 (54.5%) subjects in the Braak V/VI group. KU-60019 chemical structure Across all three groups combined, there was a statistically significant association between reduced 5HT1A-ir and neuronal loss in the CA1, but not in the CA3. The present data demonstrate that

hippocampal 5-HT1A receptors are mainly preserved until the end-stage of NFT progression in AD. Thus, the utility of PET imaging using a 5-HT1A-specific radiolabeled probe as a marker of hippocampal neuronal loss may be limited to the CA1 field in advanced stage AD cases. “
“This chapter contains sections titled: Introduction Principles of Anatomical Organization in the Developing Nervous System Early Specification of the Nervous System Correlative Neurodevelopment Comparative Neurodevelopment Principles of Vertebrate Oxymatrine Neurodevelopment Mechanisms of Neurodevelopmental Vulnerability Developmental Neurotoxicity: A Lifelong Menace References “
“Deposition of amyloid beta (Aβ) in the brain is one of the defining abnormalities of Alzheimer’s disease (AD). Phosphorylation of Aβ at serine 8 (pAβ) has been implicated in its aggregation in vitro and pAβ level has been shown to be significantly elevated in AD. We aimed to assess the specificity of pAβ for AD and have investigated associations of pAβ with parenchymal and cerebrovascular accumulation of Aβ, disease progression, angiotensin-converting enzyme activity and APOE genotype.

Patients with acute hepatitis B had greater HBcAg-specific interleukin-21-producing CD4+ T cells in blood compared with chronic hepatitis B patients, and there was no statistical significance between immune active chronic hepatitis B patients and inactive healthy carrier patients for these cells, whereas frequencies of these cells negatively correlated with HBV DNA levels but positively correlated

with HBc18-27-specific IFN-γ-producing CD8+ T cells. Moreover, interleukin-21 sustained HBc18-27-specific CD8+ T cells in vitro, and interleukin-21 production by HBcAg-specific Alisertib research buy IL-21-producing CD4+ T cells of acute hepatitis B patients enhanced IFN-γ and perforin expression by CD8+ T cells from chronic hepatitis B patients. Our results demonstrate that HBcAg-specific interleukin-21-producing CD4+ T cell responses might contribute to viral control by sustaining CD8+

T cell antiviral function. The quantity and quality of adaptive antiviral immune response influences clinical outcome of infection by the non-cytopathic, hepatotropic hepatitis B virus (HBV) [1]. The multispecific and vigorous CD4+ T cell and CD8+ T cell reactivity was present in acute HBV-infected patients who succeed in clearing HBV infection. However, in Ulixertinib nmr chronic HBV infection, the immune responses are weak and oligoclonal. The HBV-specific cytotoxic CD8+ T cells, which are believed to play a crucial role in viral clearance, show exhausted antiviral function almost characterized by an inability to produce cytokines such as IFN-γ and TNF-α, low cytotoxic activities or low proliferation in response to cognate antigen [2]. Studies in other persistent virus infection have shown that exhaustion of specific cytotoxic CD8+ T cell response mainly result from the high levels of virus antigen and low levels of CD4 help T cell[3]. Indeed, virus-specific CD4+ T cell responses are required for the efficient development of effector-specific cytotoxic CD8 T cell and B cell antibody production particularly during chronic HBV infection [4, 5]. A recent study showed that early activation

of CD4+ T cells correlates with an influx of HBV-specific CD8+ T cells into the liver in a chimpanzee model of acute HBV infection, and animals depleted of CD4+ T cells become persistently infected when inoculated with a dose of HBV [6, 7]. These data indicate that virus-specific CD4+ T cell subsets play a critical role in determining immune responses to the virus and disease outcome. However, the mechanisms by which CD4 help T cell required to control HBV infection are not well understood. Recently, several studies in animal model of LCMV infection demonstrate that interleukin-21 (IL-21), a common γ-chain cytokine, is essential for sustained specific CD8+ T cell response and control of viraemia in persistent viral infection [8-10].

Conclusion: 3D CTA with stereotactic fiducials allows surgeons to adequately estimate abdominal flap volume before surgery, potentially giving guidance in the amount of tissue that can be harvested from a patient’s lower abdomen. © 2011 Wiley-Liss, Inc. Microsurgery, 2011 “
“The present study investigates the vascular anatomy of the vastus lateralis motor nerve (VLMN) to be used as a vascularized nerve graft in facial nerve reconstruction. We evaluated the maximum length of the nerve that can be included in the flap and its vascular pedicle. In addition, we discuss its adequacy for use in early reconstruction of the facial Neratinib purchase nerve both as ipsilateral facial nerve reconstruction and

as cross-facial nerve graft. Five fresh cadavers were used in this study. In all specimens, the VLMN and its vascular pedicle were dissected, photodocumented and measured using calipers. In addition, two vascularized

VLMN were injected with a radiopaque Vemurafenib cell line contrast and underwent CT angiography and three dimensional reconstructions were scanned to illustrate the vascular supply of the nerve using OsiriX Software. The VLMN was divided into two divisions, an oblique proximal and a descending distal, in 70% of the dissections with a mean maximal length of 8.4 ± 4.5 cm for the oblique division and 15.03 ± 3.87 cm for the descending division. The length of the oblique division, when present, was shorter than the length of the descending branch in all specimens. The mean length of the pedicle was 2.93 ± 1.69 cm, and 3.27 ± 1.49 cm until crossing the oblique and the descending division of the nerve respectively.

The mean caliber of the nerve was 2.4 ± 0.62 mm. Three-dimensional computed tomography angiography demonstrated perfusion throughout the entire VLMN by branches from the descending branch of the lateral femoral circumflex artery which ran parallel to the descending division of the VLMN. Additionally, we observed that technically it was possible to preserve the this website oblique branch of the VLMN. This study confirms that VLMN presents adequate anatomic features to be used as a vascularized nerve graft for facial nerve reconstruction in terms of length, pedicle, and caliber. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“Introduction: Although, the success of free flaps has increased in the last years, more details about its characteristics might improve the clinical outcome of the flaps. This study examined the thermoregulatory ability as a sign of neural re-innervation of two different types of microsurgical free flaps in the postoperative course. Methods: A total of 22 patients were examined after grafting two different flap types: The latissimus dorsi myocutaneous (LDM) flap (n = 11) and the anterolateral thigh (ALT) flap (n = 11).

Peyer’s patches may also support some IgA production through a TI mechanism [[78]]. In addition to IgA-inducing FDCs, Peyer’s patches include TipDCs, a TNF-inducible nitric oxide synthase (iNOS)-producing DC subset that usually occupies the intestinal lamina propria [[79]]. These TipDCs elicit IgA production Talazoparib in vitro by increasing the expression of the TGF-β receptor on B cells via nitric oxide, thereby rendering B cells more responsive to IgA-inducing signals provided by TGF-β [[79]]. Of note, recent findings

show that IgA-secreting plasma cells acquire TipDC-like phenotypic features in the intestinal microenvironment, including expression of the antimicrobial mediators, TNF and iNOS [[80]]. Thus, some of the functions previously ascribed to intestinal TipDCs also involve IgA-secreting plasma cells. Follicular B cells from Peyer’s patches and mesenteric lymph nodes further undergo IgA CSR and production in response to TI signals from plasmacytoid Selleck LDK378 DCs (pDCs), which release large amounts of BAFF and APRIL upon being “primed” by type I interferon from intestinal stromal cells [[81]]. Together with Peyer’s patches and mesenteric lymph nodes, isolated lymphoid follicles represent another intestinal site for IgA induction. Isolated lymphoid follicles contain lymphoid tissue-inducer cells that

release the TNF family member lymphotoxin-β upon exposure to TLR signals from commensals [[42]]. The interaction of lymphotoxin-β with its cognate receptor stimulates local stromal cells to release TNF and DC-attracting chemokines

such as CCL19 and CCL21 [[42]]. By inducing DC production of matrix metalloproteases 9 and 13, TNF stimulates DCs to process active TGF-β from a latent precursor protein [[42]]. In the presence of TLR signals, DCs further release BAFF and APRIL, which activate 4-Aminobutyrate aminotransferase a TI pathway for IgA production by cooperating with TGF-β [[42]]. In addition to isolated lymphoid follicles, the intestinal lamina propria contains a diffuse lymphoid tissue comprised of scattered B cells that can undergo IgA class switching and production, although less efficiently and at a lower frequency than follicular B cells (reviewed in [[82, 83]]). This IgA production is supported by multiple subsets of lamina propria DCs that can activate B cells in a TI manner. When exposed to microbial TLR signals, lamina propria TipDCs release nitric oxide, which in turn enhances the production of BAFF and APRIL [[79]]. Another lamina propria DC subset with IgA-licensing function is represented by DCs constitutively expressing the flagellin receptor TLR5 [[84]]. These DCs express little or no TLR4 and induce TI IgA class switching and production by releasing retinoic acid and IL-6 upon sensing flagellin from commensal bacteria [[84]]. Also, epithelial cells deliver IgA-inducing signals to lamina propria B cells by releasing BAFF and APRIL after recognizing bacteria via multiple TLRs [[38, 85]].

Therefore, we hypothesized that reduced Th1-cell responses in the il17ra−/− BCG-vaccinated mice was due to decreased induction of IL-17-dependent IL-12 production. Consistent with this hypothesis, significantly reduced IL-12p35 Proteases inhibitor and IL-12p40 mRNA levels were detected in DLN cells of BCG-vaccinated il17ra−/− mice (Fig. 1D) and correlated with decreased mRNA expression of the Th1-cell transcription factor, Tbet 21. As expected 12, there was increased induction of IL-17 mRNA in the il17ra−/− BCG-vaccinated DLN cells compared with DLN cells isolated from B6 BCG-vaccinated mice (Fig. 1D). Also, the increased levels

of IL-17 mRNA correlated with increased expression of the Th17-cell transcription factor, RORγt 22 in DLN cells from il17ra−/− BCG-vaccinated mice (Fig. 1D). These data suggest that IL-17 is required for the induction of vaccine-induced Th1-cell responses following BCG vaccination. IL-23 is critical for Th17-cell responses in vivo following mycobacterial exposure 23–25 and therefore, we vaccinated B6 mice and IL-23 gene-deficient mice (il23p19−/−) and evaluated the generation of Ag85B-specific Th17- and Th1-cell responses in the DLNs. The generation of Ag85B-specific

Th17-cell responses (Fig. 2A) and Th1-cell responses (Fig. 2B) were significantly decreased in il23p19−/− mice when compared with B6 BCG-vaccinated mice. Induction of an effective Th1-cell vaccine response is crucial for vaccine-induced protection AZD1208 concentration against M. tuberculosis challenge 25. Therefore,

we tested whether reduced Th17- and Th1-cell vaccine-induced responses resulted in decreased protection Chlormezanone in the M. tuberculosis-challenged il23p19−/− BCG-vaccinated mice. il23p19−/− mice were vaccinated with BCG, rested for 30 days, following which they were challenged with aerosolized M. tuberculosis and the lung bacterial burdens determined in BCG-vaccinated and unvaccinated mice. As previously described, no differences in bacterial burden in the lungs of B6 and il23p19−/− unvaccinated mice were detected 23 (Fig. 2C). However, we found significantly higher lung bacterial burden in il23p19−/− M. tuberculosis challenged BCG-vaccinated mice when compared with B6 BCG-vaccinated mice (Fig. 2C). These data demonstrate the importance of the IL-23/Th17 pathway in mediating Th1-cell responses and protective BCG vaccine-induced immunity in response to pulmonary M. tuberculosis challenge. Since IL-17 appeared to be a prerequisite for effective generation of BCG-induced Th1-cell responses (Fig. 1), we determined the kinetics of Ag85B-specific Th1- and Th17-cell responses in B6 and il17ra−/− BCG-vaccinated mice. We found that significant Ag85B-specific Th17-cell responses occurred between days 4 and 8 in the DLNs of BCG-vaccinated B6 mice, which was prior to the detection of Ag85B-specific Th1-cell responses on day 14 postvaccination (Fig. 2D).

6C). Nevertheless, splenocytes from mice injected with DCs matured with the VSGs significantly downregulated IL-17 production comparable to the T-cell cytokine profile of TNF-DC-treated animals. Mice treated with MiTat-matured DCs, however, HIF-1 pathway were not able to block the nonprotective IFN-γ production as TNF-DC-treated animals, but in addition, retained high production of the disease-preventing cytokines IL-13 and IL-10 (Fig. 6C). Moreover, repetitive injections of differentially

matured DCs did not alter the frequencies of FoxP3-expressing Treg cells in spleens of EAE-diseased mice (Supporting Information Fig. 5D). This suggests that semi-mature DCs regulate EAE by protective mechanisms other than CD25+ FoxP3+ Treg-cell induction. In sum, the partial DC maturation stages were all equally effective in creating a protective Th2/Tr1-cell environment, which was able to block the Th1/Th17-cell mediated EAE. In this study, we showed that similar partial maturation stages of DCs can be achieved with the proinflammatory cytokine TNF and the T. brucei antigens check details mfVSG and MiTat1.5 sVSG. Our data further indicate that low concentrations of pathogen-derived

TLR-mediated stimuli program DCs similarly to the inflammatory cytokine TNF for the differentiation toward an inflammatory, semi-mature DC phenotype. These partial DC maturation stages were able to induce Th2-cell priming in vitro and in vivo and induced only quantitative differences in the extent of Th2-cell differentiation. Moreover, these Th2-cell signatures did not differ in their intrinsic quality to heal autoimmune diseases such as EAE and had no influence on allergic asthma. These data have important implications for the understanding of parasitic immune

evasion, the design of vaccines and provide further insights how DC maturation signatures critically contribute to the differentiation of defined Th-cell subsets. The stimulus LPS triggers DC maturation through TLR4 ligation and directs Th-cell differentiation toward Th1-cells. Less is known which PRRs drive Th2-cell associated immune responses. Recent reports suggest that house dust mite allergens initiate asthmatic Cyclin-dependent kinase 3 inflammation by signaling through the TLR4 receptor complex in part by LPS contamination 45, 46. Our data show that the T. brucei antigen MiTat1.5 sVSG-conditioned DCs to produce IL-6 and IL-1β, which is dependent on TLR4 and the adaptor molecule MyD88. A novel TLR4-mediated signaling pathway was identified in which TLR4 stimuli trigger a rapid increase in intracellular cAMP followed by translocation of the transcription factor CREB and IL-6 production 47. Further investigation is needed to address whether MiTat1.5 sVSG activation of DCs is accompanied with an intracellular cAMP rise and CREB transcription factor translocation. The T. brucei AnTat1.

The defects in IL-17 responses to S. aureus in cells isolated from this family were milder compared to the ‘classical’ HIES patients, as they were still able to release approximately 30% of the normal IL-17 production. In line with the presence of candidiasis as a clinical symptom in the family, IL-17 production after C. albicans stimulation was equally defective compared to the other patients. In addition to IL-17, other defects in the cytokine response of HIES patients have also been reported, such as a defective IFN-γ production [17,22], and increased granulocyte–macrophage

colony-stimulating Selinexor ic50 factor (GM-CSF) [23]. In line with these previous studies, in our study IFN-γ production was decreased in HIES patients, while IL-10 release

was significantly higher compared to controls. Production of IFN-γ was defective in response to both C. albicans and S. aureus. IFN-γ is the prototype of Th1 cytokines and plays a crucial role in activation of the innate and adaptive host response against these pathogens [24]. Therefore, the defective IFN-γ response could be at least as relevant as the defect found in IL-17. Furthermore, it should be kept in mind that IFN-γ therapy is a relatively safe therapeutic ��-catenin signaling option [25] and it has been reported that recombinant IFN-γ can enhance neutrophil chemotactic responses in patients with HIES [26]. Together, these data argue strongly for a dysbalance of Th subsets in patients with HIES, with defective responses of the proinflammatory subsets Th1 and Th17, and increased function of the anti-inflammatory

Th2 subset. In contrast to Th-derived cytokines, the release of IL-1β was normal in HIES patients. aminophylline As IL-1β is important for the generation of Th17 cells [27], this result suggests that it is not a defective IL-1β/IL-1RI axis that is responsible for the defects of IL-17 production in HIES patients. This hypothesis is sustained by the normal generation of Th17 responses in individuals with MyD88 or IRAK4 mutations that are defective in the IL-1RI signalling [as well as Toll-like receptor (TLR) and IL-18R pathways][11]. The defective generation of Th17 responses in HIES must therefore be located at the level of another immunological pathway, the most obvious being the IL-6/STAT3 axis [6]. To test this hypothesis, we investigated the effect of IL-17 co-stimulation with microbial stimuli in combination with IL-6. While IL-6 potentiated the production of IL-17 induced by C. albicans or S. aureus in healthy individuals, no such effect was observed in either the ‘classical’ HIES or the family with the variant HIES.

A careful preventive monitoring as well as an optimal blood pressure control may reduce the risk of AD and improve the outcome of these patients. “
“Background:  Both the presence of peripheral arterial disease and chronic kidney disease has been reported to be independent

risk factors associating with poor prognosis. However, the impact of combination of peripheral arterial disease and chronic kidney disease remains unknown. Methods:  The long-term outcome in 715 consecutive patients who had undergone coronary angiogram for the evaluation of chest pain was analyzed. Patients on haemodialysis were excluded from this analysis. Cohort patients were divided into four groups according to the https://www.selleckchem.com/products/VX-809.html Ankle Brachial Index (ABI <0.9) and glomerular Decitabine ic50 filtration rate (GFR <60 mL/min per m2): group A (n= 498; ABI >0.9, GFR >60); B (n = 65, ABI <0.9, GFR >60); C (n = 99; ABI >0.9, GFR <60); and D (n = 53; ABI <0.9, GFR <60). The mean follow-up period was 620 ± 270 days and evaluated the major cardiac adverse events included survival, stroke, acute coronary syndrome and heart failure. Results:  The mean follow-up period was 620 ± 270 days. Total long-term event was present in 89 patients (groups A–D were 9.4%, 18.5%, 15.2% and 28.3%, respectively). Long-term event rate was 28.3% for patients with the presence of peripheral arterial disease and chronic kidney disease,

compared to 9.4% for those PAK6 without peripheral arterial disease and chronic kidney disease (P < 0.0001). Kaplan–Meier event-free survival curves also showed that the combination of peripheral arterial disease and chronic kidney disease predicted long-term event rate. Conclusion:  The combination of chronic kidney disease and ABI of less than 0.9 undergoing coronary angiogram is strongly associated with long-term event rate. "
“Sepsis has been shown to induce the expansion of CD4+CD25+ regulatory T cells (Tregs), and this paradoxical immune suppression has been suggested to

be closely associated with the development of sepsis-induced organ dysfunction. In the present study, we aimed to investigate the possible link between immune suppression and the development of septic acute kidney injury (AKI). We prospectively enrolled patients with a diagnosis of sepsis, with or without AKI and as well as patients with AKI but without sepsis. Serum and urine samples at the time of the diagnosis were collected to measure neutrophil gelatinase-associated lipocalin (NGAL), cytokines, and soluble CD25 (sCD25). Of the 82 patients enrolled, 44, 18, and 20 patients were classified into septic-AKI, sepsis-non AKI and non-septic AKI groups. There were no differences in the baseline characteristics in all three groups and the severity of infection in the two sepsis groups. Serum levels of interleukin (IL)-10 were significantly elevated in patients with septic-AKI compared to the other two groups.

0 mm; 2-h postprandial glucose of 11.1 mm). Selleckchem FDA approved Drug Library 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 selleck (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, MYO10 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.