A good example is invariant natural killer T (iNKT) cells, which make up a large proportion of lymphocytes in human and murine adipose tissue. Here, they are unusually poised to produce anti-inflammatory or regulatory cytokines, however in obesity, iNKT click here cells are greatly reduced. As iNKT cells are potent transactivaors of other immune cells, and can act

as a bridge between innate and adaptive immunity, their loss in obesity represents the loss of a major regulatory population. Restoring iNKT cells, or activating them in obese mice leads to improved glucose handling, insulin sensitivity, and even weight loss, and hence represents an exciting therapeutic avenue to be explored for restoring homeostasis in obese adipose tissue. Adipose tissue is a dynamic tissue serving a primary and essential function in lipid storage, but it also Z-IETD-FMK mw acts as an endocrine

organ, producing many adipokines that regulate satiety, storage capacity, insulin sensitivity and glucose handling.[1] In addition, human and murine adipose tissue contains a distinct collection of immune cells in the lean steady state. Immune cells reside in the stromovascular fraction of adipose tissue, along with vascular endothelial cells, mesenchymal stem cells and pre-adipocytes, and appear to be in contact with neighbouring adipocytes. This adipose-resident immune system is unique in terms of enrichment of certain otherwise rare cells, and in the phenotype of these cells compared with elsewhere in the body. The immune system resident in adipose tissue plays a key role in maintaining homeostasis and keeping inflammation at bay. Resident alternatively activated macrophages may phagocytose dead cells, adipocytes and their contents, to prevent triggering an immune response by free fatty acid release. Other resident cells like regulatory T cells and eosinophils also prevent an inflammatory environment by producing

anti-inflammatory cytokines like interleukin-10 (IL-10) and IL-4 at steady state. However in the obese state, adipocytes are overloaded Tenoxicam and stressed, and they release adipokines, which can modulate the immune system. In the state of chronic excess calorie intake and lipid overload in adipose tissue, the resident immune system is aberrantly activated, which has been shown to contribute to the metabolic disorder that ensues in obesity. Hence, the resident immune system in lean adipose tissue is key to maintaining a healthy controlled state of immune tolerance, and at the same time, in obesity, the resident immune system is a key mediator of chronic inflammation at the heart of metabolic disease. We have discovered the enrichment of one such resident immune cell, the invariant natural killer T (iNKT) cell in human and murine adipose tissue.

For the analyses of target gene expression in the CaCo-2 cells with quantitative RT–PCR, total RNA was isolated (Sigma), reverse transcription was performed with added DNAse treatment, and qPCR analyses were performed

as described above for biopsy samples. Markers of apoptosis were bcl-2 (Hs00608023_m1) and BAX (Hs00180269_m1). Ribosomal 18 s RNA was used as an endogenous control (Hs99999901_s1). The data analysis was performed with SPAW statistics version 17·0 for Windows (SPSS Inc., Chigaco, IL, USA) and GraphPad prism software (San Diego, CA, USA). For comparisons between the groups, the non-parametric Kruskal–Wallis test and Mann–Whitney U-test were used. The Spearman’s rank correlation test was applied to analyse correlations between different parameters. P-values < 0·05 were considered significant. The Ethics buy Tamoxifen Committee of the Hospital for Children and Adolescents, Helsinki University Central Hospital, Finland and the Regional Ethics Committee for Human Research at the University Hospital of Linköping, Sweden approved the study plans and written informed consent was obtained from parents and children. The results of the immunohistochemistry and qPCR analyses of the small intestinal biopsies from the Finnish study population consisting of children with untreated CD, children with T1D and reference children are shown in Fig. 1. The expression of IL-17-positive cells and IL-17-specific

mRNA levels differed significantly between the groups (P = 0·029 and P < 0·001, respectively, Kruskal–Wallis test). The density of intestinal IL-17-positive cells was Aldehyde dehydrogenase increased in untreated CD 5-Fluoracil manufacturer compared to the T1D patients (P = 0·039, Mann–Whitney U-test) (Fig. 1a). Additionally, the IL-17 mRNA level was higher in untreated CD than in subjects with T1D or reference children (P < 0·001 for both comparisons, Mann–Whitney U-test) (Fig. 1b). In T1D, no difference in the number of IL-17-positive cells or transcripts was seen in comparison to the reference children. In children with untreated CD the expression of IL-17-positive cells correlated positively with the IL-17 mRNA

expression levels (R = 0·444; P = 0·111, Spearman), whereas no such correlation was seen in the reference group (R = −0·247; P = 0·555, Spearman) or in children with T1D (R = −0·104; P = 0·775, Spearman). The number of FoxP3-positive cells and FoxP3-specific mRNA differed significantly between the groups (P = 0·003 and P = 0·008, respectively, Kruskal–Wallis test) (Fig. 1c,d). Increased numbers of FoxP3-positive cells were found in untreated CD when compared to T1D and reference children (P = 0·003 and P = 0·006, respectively, Mann–Whitney U-test) (Fig. 1c). Additionally, untreated CD had higher FoxP3 mRNA levels than subjects with T1D and reference children (P = 0·007 and P = 0·015, respectively, Mann–Whitney U-test) (Fig. 1d).

Results are

expressed as means ± standard deviation (SD) and were compared using an unpaired Student’s t test. To determine the effectiveness of the sublingual immunization, mice were immunized with 25k-hagA, 25k-hagA-MBP, or PBS. Sublingual immunization with 25k-hagA-MBP induced significant serum IgG and IgA 7 days after the final immunization (Fig. 1a). In contrast, 25k-hagA-immunized and nonimmunized mice induced low or no detectable titers, respectively, after sublingual immunization. In addition, the serum IgG and IgA Ab responses KPT 330 induced by 25k-hagA-MBP persisted for almost 1 year (Fig. 1b). When the subclasses of antigen-specific IgG antibodies induced by sublingual 25k-hagA or 25k-hagA-MBP

www.selleckchem.com/products/iwr-1-endo.html challenge were determined, all IgG subclasses were significantly enhanced in 25k-hagA-MBP group. On the other hand, 25k-hagA-immunized group showed a low level of IgG1 (and sparse IgG2b) (Fig. 1c). Sublingual immunization of 25k-hagA-MBP induced high levels of 25k-hagA-MBP-specific IgA Ab responses in saliva (Fig. 2a). In contrast, essentially no IgA was detected in the saliva of mice sublingually treated with 25k-hagA or PBS. The most 25k-hagA-MBP-specific IgA AFCs were detected in the salivary glands suspensions (Fig. 2b). As sublingual immunization with 25k-hagA-MBP elicited 25k-hagA-MBP-specific Ab responses in both mucosal and systemic compartments, establishing the nature of the T cell help supporting the responses was important. When mononuclear cells from the SMLs of immunized mice were restimulated with 25k-hagA-MBP in vitro, significant levels of proliferative responses were induced (Fig. 3a). In contrast, no significant proliferation or cytokine production was observed in hagA-immunized mice (data not shown). Furthermore, mononuclear cells isolated from SMLs immunized with 25k-hagA-MBP showed higher production

of IL-4, IFN-γ, and TGF-β (Fig. 3b). These data find more indicate that sublingually immunized 25k-hagA-MBP-specific Th1-type and Th2-type responses are induced in SMLs. Given that sublingual immunization with 25k-hagA-MBP elicited long-term antigen-specific Ab responses in sera, we sought to determine whether these antibodies were capable of suppressing the alveolar bone absorption caused by P. gingivalis infection. Thus, mice given 25k-hagA, 25k-hagA-MBP, and PBS were infected orally with P. gingivalis 7 days after the last immunization. Mice immunized with 25k-hagA-MBP showed a significant protection and reduced bone loss caused by P. gingivalis infection (Fig. 4). In contrast, mice immunized with 25k-hagA alone did not show the reduced level of bone loss by P. gingivalis infection. These findings indicate that sublingual immunization with 25k-hagA-MBP is protective against oral infection by P. gingivalis.

Splenocytes from experimental animals (7 weeks post-cGVHD) were enriched for CD4+ T cells (as above) and rested for 24 h in complete media prior to re-stimulation. A total of 2 × 106 cells were labelled with 5 μM CFSE (Molecular Probes, USA) and re-stimulated with 2 × 106 irradiated APCs isolated from B6Kd, CBA or BALB/c mice. CD3+CD28+-coated beads (Dynal Invitrogen, UK) were used as positive controls. Mixed lymphocyte reactions were incubated over 4 days after which cells were stained with anti-H-2Kd PE, anti-CD4

and live-dead exclusion dye (Invitrogen) and analysed by flow cytometry to examine the percentage of proliferating T cells (CFSE dim), relative to unstimulated cells, after gating on live CD4+ Osimertinib donor H-2Kd− or recipient H-2Kd+ T cells. Cytokines produced by 5 × 106 splenocytes isolated from experimental cGVHD and PBS control groups was detected by analysis of cell supernatants harvested 5 days after in vitro culture. Screening for IL-6, IL-12, IL-1β, IFN-γ, TNF-α and IL-10 was performed using the MSD mouse pro-inflammatory multiplex cytokine kit and platform (Mesoscale, Maryland, USA). Data shown is mean ± SD, or mean ± SEM, where indicated. Statistical comparisons between experimental groups were made using two-tailed unpaired-Student’s t-tests. Statistical comparisons of percentage of proliferating cells following in vitro re-stimulation selleck chemicals between

treatment groups was made using two-way ANOVA (α-significance level 99.9%) Bonferroni post tests. Statistical significance is denoted as follows, p < 0.0001***, p < 0.001**, p < 0.05* throughout. This research was supported by the National Institute for Health Research (NIHR) Biomedical Resveratrol Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department

of Health. This work was also supported by the British Heart Foundation and Guy’s & St. Thomas’s Charity. Authors declare no financial or commercial conflict of interest. “
“Granulysin and interferon-gamma (IFN-γ) have broad antimicrobial activity which controls Mycobacterium tuberculosis (M. tuberculosis) infection. Circulating granulysin and IFN-γ concentrations were measured and correlated with clinical disease in Thai patients with newly diagnosed, relapsed and chronic tuberculosis (TB). Compared to controls, patients with newly diagnosed, relapsed and chronic TB had lower circulating granulysin concentrations, these differences being significant only in newly diagnosed and relapsed TB (P < 0.001 and 0.004, respectively). Granulysin concentrations in patients with newly diagnosed and relapsed TB were significantly lower than in those with chronic TB (P= 0.003 and P= 0.022, respectively).

The

glomerular basement membrane and the podocytes are typically not affected as seen in electron microscopic images. However, despite the lack of microscopic evidence of podocyte damage, there are data that podocytes have a role in preeclampsia. Podocyturia has been RG7420 purchase demonstrated in patients with glomerular diseases.55 More recently women with clinically established preeclampsia have been shown to excrete viable podocytes in their urine56,57 called ‘footprints in the urine’.58 Women with normotensive pregnancies and women with gestational hypertension did not have podocyturia. The significance of these results remains to be confirmed in larger clinical studies. Molecular and cellular studies by Garovic and others have shown marked downregulation of podocyte expression of nephrin and synaptopodin

and this combined with the endothelial cell injury is likely to explain the proteinuria.47 The possible mechanism of the proteinuria is that the decrease in nephrin is due to its release from the slit diaphragm by proteolytic cleavage.59,60 Nephrin shedding could be due to increased endothelin61 and decreased VEGF62 both of which are implicated in the endothelial injury. The recent finding of the reduced availability of podocyte-produced VEGF indicates a mechanism whereby the endothelium loses its fenestrations and this alteration contributes to protein loss in the urine.63–66 In this instance, there is reduction in endothelial signalling and subsequent endothelial swelling, and thus a reduction in both

the size and density Wnt inhibitor Resveratrol of the fenestrations on the endothelial cells.65 The hypothesis therefore is that the endothelial injury is the primary insult and that podocyte damage directly results from these events. An increase in circulating sFLT-1 (soluble VEGF receptor) reduces the available free VEGF, resulting in an increase in endothelin-1 production and secretion by the glomerular endothelial cell61 (Fig. 2). The animal model of proteinuria in which antibodies to VEGF are infused into rats66 confirms podocyte damage as well as endothelial dysfunction.62,65 The importance of the endothelial cell/podocyte interrelationship is further evidenced by the effect of circulating sFLT-1 binding podocyte-produced VEGF resulting in endothelial thickening, which may be responsible for the reduction in both the size and the density of endothelial fenestrations.66 Other potential mechanisms include CD2AP, epithelial protein 1, GLEPP, Twerk and cytokines,34,67 but their roles are not fully elucidated. The proteinuria per se may be damaging to podocyte function.68,69 Given the profound haemodynamic renal adaptation required for normal pregnancy, it is no wonder that underlying renal disease poses a particular risk in pregnancy.

Conflict of interest: signaling pathway A. V. S. H. and H. M. are named inventors on a composition of matter patent for MVA85A filed by the University of Oxford, and are shareholders in a Joint Venture formed for the

further development of this vaccine. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“The development of clinical therapeutics that interfere with the migration of leukocytes has revolutionized the treatment of multiple sclerosis and holds great promise for the treatment of a wide range of inflammatory diseases. As the molecules essential for the multi-step adhesion cascade that mediates cellular migration have been elucidated, the number of potential targets available to modulate leukocyte trafficking

has increased exponentially. In this Viewpoint, we briefly review our current understanding of these mole-cular targets and how these targets vary by tissue and leukocyte subset with emphasis on T cells. We then describe the two currently approved therapeutics that target cell migration, natalizumab and fingolimod, and discuss how an improved understanding of their function could pave the way for the development of safer and more efficacious therapies for inflammatory and autoimmune diseases. Nearly 50 years ago, Gowans and Knight published a seminal study demonstrating that labeled lymphocytes injected into rats migrated learn more from the blood into secondary lymphoid organs (SLOs) and then returned to the circulation via the thoracic duct [1]. In MycoClean Mycoplasma Removal Kit an accompanying paper by Marchesi and Gowans, lymphocytes were observed to adhere to what are now called high endothelial venules and pass through the endothelial layer in a directed migration into the lymph node [2]. This process was hypothesized to be selective, as only small lymphocytes emigrated from the venules while larger lymphocytes were excluded. In the time since these first observations were made, knowledge of the molecular mechanisms

that underpin lymphocyte trafficking has exploded. The selective migration observed by Marchesi and Gowans is now understood to be a tightly orchestrated multistep adhesion cascade, regulated by selectins, integrins, chemokines, and chemoattractant lipids, that specifically directs the trafficking of leukocytes into sites essential for their function. Such an improved understanding of the underlying mechanisms involved has resulted in the identification of an array of potential drug targets aimed at modulating cell migration in order to treat a broad range of autoimmune and inflammatory diseases. Today, two drugs targeting cell migration are approved for clinical use in multiple sclerosis, one of which is also approved for Crohn’s disease; and many more are currently in clinical trial for these and other inflammatory diseases.

43 RFM also enhances the production of NO, which might be responsible for parasite killing.44 This was a comprehensive study carried out to investigate the abundance of localized and circulating cytokines in patients with CL caused by L. tropica. Furthermore, the study carried out a comparative assessment of different treatment regimens on the

host immune response, which will help to explore the action of chemotherapy. The higher production of IL-8 in CL patients, leading to excessive inflammatory cell activation, predominantly PMNs that provide shelter to parasites, may allow the parasite to survive and multiply, leading to the development of disease. The observation of high levels of NO and MCP-1 following treatment suggests that MCP-1 orchestrates the induction of leishmanicidal activities in human macrophages HKI-272 mw via the generation

of NO. Financial assistance by the Indian Council of Medical Research is gratefully acknowledged. None of the authors of this paper have conflict of interest to disclose. “
“Natural killer (NK) cells are important components of the innate immune system that mediate effector and regulatory functions. As effector cells, NK cells help control virus-infected cells through cell-mediated antibody-dependent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). Although macaques are an important and reliable

animal model for the study of retrovirus-induced human diseases, and despite the crucial role played by NK cells in innate ITF2357 in vivo and adaptive immune responses against simian immunodeficiency virus (SIV), only a few studies have attempted to characterize different macaque NK cell subpopulations. In the present study, we identified a subpopulation of circulatory CD8α− macaque NK cells that express NK lineage markers and exhibit cytotoxic potential. CD8α− NK cells were phenotypically characterized as CD3− CD14− CD20− CD8α− cells Aspartate that express NK cell markers including CD16, CD56, granzyme B, perforin, NKG2D and KIR2D. Based on their CD56/CD16 expression patterns, cells within the CD8α− gate can be divided into four subpopulations: CD56dim CD16bright, CD56dim CD16−, CD56bright CD16−, and CD56− CD16− cells. In contrast, CD8α+ NK cells are 95% CD56dim CD16bright, which correlates with their high cytotoxic potential. Upon interleukin-15 activation, CD8α− cells up-regulated CD69 expression and produced low levels of interferon-γ and tumour necrosis factor-α. Sorted CD8α− NK cells were capable of killing MHC-I-devoid target cells and mediated ADCC responses against SIV gp120-coated target cells in the presence of macaque anti-gp120 antibodies.

Of particular interest in this context are recent studies on human endothelial cell cultures which documented that above a threshold of 135 mmol/L a stepwise increase in the sodium concentration of the incubation medium progressively increases endothelial cell stiffness, causes inhibition of endothelial NO synthase and decreases release of nitric oxide; this effect was abrogated by the mineralocorticoid receptor spironolactone.30 In addition to aldosterone, digitalis-like endogenous

inhibitors of Na+, K+-ATPase have recently been recognized as one class of agents raising blood pressure in response to sodium loads.31 Recent studies clearly documented minor increases in plasma sodium concentration in hypertensive individuals.32 Changes in plasma sodium concentration are transmitted into the cerebrospinal fluid33 triggering the release of cardiotonic steroids, Selleckchem ABT 263 namely, analogues buy CYC202 of digitalis such as ouabain and marinobufagenin.31 In the Dahl salt-sensitive rat, a standard hypertensive animal model with an underlying mutation of the α-1 Na+, K+-ATPase, chronic salt loading increases the excretion of marinobufagenin in the urine.34 Marinobufagenin causes vasoconstriction35 and is increased

in pathological states of sodium overload, for example uraemia and preeclampsia.35,36 The most convincing proof of a key role of sodium and specifically renal sodium handling in the genesis of hypertension has been provided by studies in which heterozygous carriers of mutations of renal sodium transporters were compared with corresponding normotensive control individuals. For instance, in the study of Fava37 in the Framingham population, heterozygous carriers of the Gitelman mutation failed to have phenomena relating to the Gitelman syndrome, but had significantly Tangeritin lower systolic and diastolic pressures compared to matched controls, obviously as the result of higher renal sodium excretion with a shift in the pressure/natriuresis relationship. In summary, the evidence is overwhelming that current intakes of salt contribute in

a major fashion to the current ‘epidemic’ of hypertension. This justifies public health efforts to reduce salt intakes, particularly in commercial food items,38 since it had been shown that only 15% of current salt intakes can be controlled by the patient, whilst 85% of salt is already contained in commercial food items.39 The Author states that there is no conflict of interest regarding the material discussed in the manuscript. “
“Aim:  Podocytes provide a slit diaphragm to inhibit proteinuria, and nephrin between podocytes functions as a barrier during glomerular filtration. Hepatocyte growth factor (HGF) can improve proteinuria in rodents with various renal injuries, but little is known about the role of HGF in podocyte-based events during glomerulonephritis.

50 L, p<0.00001 versus Prosecco, by ANOVA). Furthermore, participants could thoroughly analyze, in a non-blind manner, three independent but very big pieces of 50.00 kg pork-shaped “mortadella” (that some erroneously still call “Bologna”, and was kindly provided by SIICA member Luca Cicchetti), a total of 150.00 kg, compared with 48.00 kg of 24-month-old home-made original parmesan (p<0.001 versus mortadella), and an adequate, but impossible to calculate, amount of “focaccia” and “piadina” (i.e. type of breads you can find only in the Romagna region). The second

day of the meeting saw a strong scientific program dealing with topics related to NK cells and innate immunity, immunodeficiencies, immunoregulation, mucosal immunity and veterinary immunology. The role of radical oxygen species (ROS)-generation in the Afatinib in vivo up-regulation of NKG2D and DNAM-1 expression was reported by A. Santoni (Rome), while C. Watzl (Heidelberg) showed that CD107a, a protein present on the inner leaf of cytotoxic granules, protects NK cells from degranulation-associated damage. C. Romagnani Cell Cycle inhibitor (Berlin) dissected NK-cell differentiation stages according to the CD62L and other markers and showed that studying NK-cell clustering by principal component analysis enables immature and mature NK cells to

be tracked in vivo after NK-cell adoptive transfer and hematopoietic stem cell transplantation. The role of the CX3CR1/CX3CL1 axis was studied by G. Bernardini (Rome) in a modified mouse model in which the CX3CR1 gene was replaced by GFP, showing that CX3CR1 regulates NK-cell accumulation in the bone marrow, likely by affecting NK-cell differentiation into KLRG1+ cells. J. D. Haas (Hannover) studied

the ontogeny of IL-17-producing γδ T cells, and found that IL-17 was not generated after the induction of Rag-1 in an inducible Rag-1 KO mouse model. However, the generation of γδ T17 cells could be restored by thymus transplantation in adult animals. C. Agostini (Padova) reported on the role of common variable immunodeficiency (CVI) in provoking damage in the lung. CVI was also investigated by M. Lima Gomes Ochtrop (Freiburg), who described a number of abnormalities among bone marrow-resident Racecadotril T and B cells, such as the presence of diffuse and nodular CD3+ infiltrates, or a partial block in B-cell development at the B-I to pre-B-II cell stage. H. Eibel (Freiburg) had screened a large cohort of patients that suffer from primary antibody deficiency and found that two of them had a homozygous deletion in the BAFF-R gene causing a severe block of B-cell development at the stage of transitional B cells. O. Pabst (Hannover) demonstrated that oral tolerance requires the sequential interaction of T cells with different populations of APCs in the mesenteric lymph nodes and thereafter in the intestinal lamina propria.

To evaluate the development and time course of cellular immune responses in the presence of MDA, pigs born to immune sows were vaccinated with modified live commercial vaccine based on the Bartha strain. The lymphocyte proliferation assay (LPA) was used to estimate the antigen-specific proliferation of lymphocytes. In addition, we investigated the nature of protective immunity induced by

systemic delivery of glycoprotein E (gE)-deleted attenuated vaccine (the Th1–Th2 polarization of immune response) by examining selleckchem the profile of Th1 and Th2 cytokines produced by PBMC stimulated in vitro with the live ADV. Eight seronegative pregnant sows and their litters were used. gE-deleted, attenuated vaccine against Aujeszky’s disease was used as a model (Akipor 6.3, Merial, France). All pigs were vaccinated with the same dose of vaccine (2.0 mL) by intramuscular injection. Sows were vaccinated twice, at 6 and 2 weeks before parturition. Piglets were assigned to six groups: five were vaccinated and one (group 1, n=13) served as unvaccinated control for evaluation of the duration of maternal antibodies in piglet sera. Control pigs received placebo [phosphate-buffered

saline (PBS)] instead of vaccine. The vaccination schedule of piglets was as follows: group 2 (n=15) was vaccinated following the vaccine manufacturer’s recommendations at 10 and 14 weeks of life, and groups 3 (n=13) and Small molecule library mw 4 (n=14) were vaccinated once at 8 and 12 weeks of life, Isotretinoin respectively. Piglets from groups 5 (n=13) and 6 (n=14) were vaccinated at 7 days of age and the booster doses were administrated at 8 and 12 weeks of life, respectively. The Local Ethical Commission approved all procedures involved

in the study. Specific antibodies to the gB and gE (gp1) antigen were determined using blocking ELISA tests (HerdChek*Anti-PRVgB or HerdChek*Anti-PRVgp1, Idexx Laboratories), as directed by the manufacturer. The presence or absence of antibodies to investigated antigen was determined by calculating the sample to negative (S/N) ratio (OD of test serum/mean OD of negative reference serum). Samples were considered to be positive for gB antigen if the S/N ratio was ≤0.5, and for gE antigen if the S/N ratio was ≤0.6. Blood from piglets was collected from vena cava cranialis in vacuum tubes containing EDTA-K3 as an anticoagulant (Medlab, Poland). The blood was taken 2 weeks after the final vaccination in parallel with unvaccinated animals, and the second time at 20 weeks of life. The PBMC were isolated from blood samples by density gradient centrifugation. The blood (3 mL) was layered on an equal volume of Histopaque 1.077 (Sigma), and centrifuged for 30 min at 1500 g at 20 °C. Buffy coats, which contain the PBMC, were collected and washed in PBS.