Results:  Proteinuria was reduced after tonsillectomy over 2 years of follow-up

in both early and later groups compared with proteinuria in the 6 months preceding surgery. Complete remission was achieved in 10 patients, most often among those having surgery within 3 years, while patients refusing surgery failed to attain complete remission of urinary findings. Histological activity decreased in both groups, significantly when surgery was early. Complement component C3 deposition selleck inhibitor and activated macrophages in glomeruli decreased after tonsillectomy, especially with early surgery. Conclusion:  Tonsillectomy improved clinicopathological features in relatively severe paediatric IgA nephropathy, especially with the early-surgery group. Therapeutic mechanisms may include inhibition of complement activity in glomeruli and AZD6244 in vitro glomerular infiltration by activated macrophages. “
“MicroRNAs (miRNAs) are short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression via blockade of protein translation or by inducing mRNA degradation. These miRNAs potentially

regulate the expression of thousands of proteins. As a result, miRNAs have emerged rapidly as a major new area of biomedical research with relevance to kidney disease. MiRNA expression has been shown to differ between the kidney and other organs as well as between different kidney regions. Furthermore, miRNAs have been found to be functionally important in models of podocyte development, diabetic

nephropathy and polycystic kidney disease. Of particular interest, podocyte-specific deletion of Dicer, a key enzyme in the biogenesis of miRNA, results in proteinuria and severe renal impairment in mice. One miRNA (miR-192) can also act as an effector of transforming growth factor-β activity in the high-glucose environment of diabetic nephropathy. Differential expression of miRNAs has been reported in kidney allograft rejection. It is anticipated that future studies involving miRNAs will generate new insights into the complex pathophysiology underlying various kidney diseases, generate diagnostic biomarkers and might be of value as therapeutic targets for progressive kidney diseases. The purpose of this review is to highlight key miRNA developments in kidney Ergoloid diseases and how this might influence the diagnosis and management of patients with kidney disease in the future. MicroRNAs (miRNAs) are endogenous non-coding RNA molecules, 20–22 nucleotides in length. The discovery and characterization of miRNA in the last decade is revolutionizing our understanding of gene regulation, cell differentiation, proliferation, apoptosis, metabolism and pathophysiology of many diseases including kidney diseases. The understanding of miRNA biology and its role in various diseases is still in its early stage but is expanding rapidly.

Adult neurogenesis, a dramatic form of adult brain circuitry plasticity, has been implicated in physiological brain function and appears to be of pivotal importance for certain forms of learning and memory. In GSK-3 cancer addition, failing or altered neurogenesis has been associated with a variety of brain diseases such as major depression, epilepsy and age-related cognitive decline. Here we review recent advances in our understanding of the basic

biology underlying the neurogenic process in the adult brain, focusing on mechanisms that regulate quiescence, proliferation and differentiation of NSPCs. In addition, we discuss how neurogenesis influences normal brain function, and in particular its role in memory formation, as well as its contribution to neuropsychiatric diseases. Finally, we evaluate the potential of targeting endogenous NSPCs for brain repair. The brain is challenged

every day by new experiences that have to be integrated into previously acquired knowledge and skills. Changes in neural function and subsequent connectivity are referred to as neural plasticity. It was believed for a long time that experience-induced changes of neural networks could only affect existing neuronal cells (i.e. cells that were generated during embryonic or early postnatal development). This central dogma was based on the idea that the brain is too complex an organ to allow for the generation and subsequent integration

of newborn neurones, especially in the adult. However, initial Maraviroc solubility dmso evidence dating back to the 1960s, which was debated for decades and finally accepted in the mid-1990s, showed that the next adult mammalian brain contains substantial numbers of neurogenic neural stem/progenitor cells (NSPCs) that retain the ability to generate new neurones throughout life [1–4]. Thus, these seminal findings challenged previously held concepts about brain function and added a novel level of complexity to our understanding of adult neural plasticity. However, the process of adding new neurones into the preexisting neural circuitry, called adult neurogenesis, is not widespread throughout the brain but rather limited to two main neurogenic areas: the subventricular zone (SVZ) lining the lateral ventricles where NSPCs divide and give rise to cells that migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into distinct types of olfactory neurones; and the hippocampal dentate gyrus (DG) where NSPCs generate cells that differentiate into newborn granule cells (substantial amounts of neurogenesis have been identified in these two brain regions in adult rodents and non-human primates; the evidence for adult neurogenesis in humans will be discussed below) [5–7].

Due to the strong correlation between the induction of an

efficient immune response to late-stage antigens and the control of latent Mtb infection, HspX may be an ideal candidate antigen for vaccines against latent tuberculosis. The addition of late-stage antigens such as HspX to the well-established prophylactic vaccines (Weinrich Olsen et al., 2001; Agger et al., 2006) might convert them into multistage tuberculosis vaccines that not only defend against all stages of Mtb infection, but also prevent reactivation of latent infections. For subunit vaccines, adjuvants are needed to increase the immunogenicity of the antigens. Aluminum hydroxide is widely used as one of two currently approved adjuvants (Gupta et al., 1995). The use of aluminum hydroxide in preclinical and clinical tests and its prevalent use in approved vaccines for millions of individuals show that aluminum hydroxide cancer metabolism inhibitor is safe, well tolerated and capable of enhancing the immune response to a wide range of antigens (Singh et al., 2006). The mechanism of the aluminum reaction is largely

unknown; in addition to the depot effect theory (Gupta et al., 1995), the ability of aluminum salts to promote antigen uptake and presentation by dendritic cells (DCs) (Sokolovska selleckchem et al., 2007; Kool et al., 2008) have also been discussed. More recently, other theories about the mechanism of its adjuvant activity have been suggested. Kool et al. (2008) proposed that the cytotoxicity of aluminum salts leads to the release of uric acid in vivo, which acts as a damage-associated molecular pattern that is required for the adjuvant activity of aluminum. Other research has shown a requirement for caspase 1 activation in vivo, which is mediated by nucleotide-binding domain and leucine-rich repeat-containing gene (NLR) family, pyrin domain-containing 3 (NLRP3) and apoptosis-associated speck-like protein containing a CARD (ASC), collectively known as the nlrp3 inflammasome (Eisenbarth et al., 2008). However, there is still much controversy concerning

these new proposals. CpG DNA is a novel adjuvant that contains unmethylated CpG motifs that are recognized by the innate immune system via TLR9 (Cornelie et al., 2004). The recognition by the innate immune system induces broad adjuvant effects Dipeptidyl peptidase such as the direct activation of B cells, macrophages and DCs as well as the secretion of IL-6 and IL-12 cytokines (Krieg et al., 1995; Askew et al., 2000; Cornelie et al., 2004). Although the immune reaction induced by CpG is nonspecific, it can be used to enhance the immune responses to specific antigens or to switch the immune response from Th2 to Th1. In vaccine trials for bacterial, viral and parasitic infections, CpG increased both the innate immune response and protective immunity (Davis et al., 1998; Decker et al., 2000; Deng et al., 2004).

Here we show that in Th17 cells, the more phenotypically flexible Th lineage, the PcG proteins Mel-18 and Acalabrutinib manufacturer less strikingly Ezh2 are associated differentially with the Il17a promoter. Using the RNAi approach, we found that Mel-18 and Ezh2 positively regulate the expression of Il17a and Il17f. The inducible binding of Mel-18 and Ezh2 at the Il17a promoter was dependent on signaling pathways downstream of the TCR. However, a continuous presence of TGF-β, the cytokine that is necessary to maintain Il17a expression, was required to preserve the binding activity of Mel-18, but not of Ezh2, following restimulation. The binding of Mel-18 at the Il17a promoter

was correlated with the recruitment of the lineage-specifying transcription factor RORγt. Altogether, our results suggest that in Th17 cells the TCR and polarizing cytokines synergize to modulate the binding activity of Mel-18 at the Il17a promoter, and consequently to facilitate Il17a expression. Naive

Th cells (CD4+) can differentiate https://www.selleckchem.com/products/bmn-673.html into effector or regulatory lineages, each characterized by distinct expression pattern of cytokines 1–4. The effector Th1, Th2 and Th17 cells express in a TCR-dependent manner the signature cytokines IFN-γ, IL-4 and both IL-17A and IL-17F, respectively. Th17 cells play a critical role in host protection, mainly in eradication of extracellular pathogens, but are also involved in the pathogenesis of autoimmune diseases 5–9. The differentiation of Th17 cells, as of other Th cells, is most efficiently promoted by the cytokine milieu; a combination of TGF-β and the proinflammatory

cytokine IL-6 strongly potentiates the Th17 pathway 10–16. IL-6 activates STAT3, a crucial transcription factor for Th17 development, which can also be activated following differentiation by IL-21 in an autocrine manner or IL-23 after acquisition of the IL-23R expression. IL-23 appears to expand or Fludarabine cell line maintain the Th17 cell population, and it is required for the maintenance of Th17 function and Th17-mediated autoimmunity in vivo 10, 13–15, 17–23. RORγt and RORα are the Th17 lineage-specifying transcription factors, and similar to T-bet in Th1 cells and GATA3 in Th2 cells, establish the lineage fate 24, 25. However, the phenotypes of differentiated Th cells present a higher degree of plasticity than it was previously appreciated 3, 26–34, especially Th17 cells, which are mainly prone to acquire the Th1 phenotype in vitro and in vivo 35–45. Differentiation of Th cells is accompanied by lineage-specific epigenetic marks at cytokine genes 46–49; Il17a and Il17f are differentially associated with permissive chromatin modifications in Th17 cells 42, 43, 50, 51. However, these histone modifications are unstable in the presence of the opposing polarizing cytokines 42.

In contrast, in anergic Th1 cells, p21Cip1 persists and is available to bind to inhibit the MAPK important in early T-cell activation. In addition to partnering with cdk, p21Cip1 can form a binary complex with PCNA.30 PCNA is induced in activated T cells and when T-cell proliferation ceases, synthesis and accumulation of PCNA also stops.13 In case of genetic damage, p53-dependent up-regulation of p21Cip1 leads to cdk-independent inhibition of PCNA-dependent

DNA replication allowing time for DNA repair.30,31 p21Cip1 interaction with PCNA results in the inhibition of PCNA and thereby causes G1 and G2 block in T cells.14,32 There was some association of p21Cip1 with PCNA in stimulated control Th1 cells, but the functional significance of this low-level interaction was not determined. The interaction between p21Cip1 and PCNA was not increased in anergic Th1 cells, which

suggests that PCNA inhibition APO866 solubility dmso by p21Cip1 is probably not the cause of proliferative unresponsiveness in these Th1 cells. p21Cip1 in anergic Th1 cells instead appears to work via the inhibition of MAPK, specifically p-JNK and p-c-jun. In T cells, productive antigen stimulation triggers the activation of MAPK including extracellular signal-regulated kinase, p38 and JNK.33 The JNK is activated through the dual phosphorylation of its Thr and Tyr residues by mitogen-activated kinase kinase MK0683 research buy 4 (MKK4) and MKK7. Activated JNK in turn phosphorylates c-jun in its N terminus, activating the c-jun-containing AP-1 complexes.34 Activation of AP-1 transcription factor eventually results in increased IL-2 transcription. Others have shown defective expression and function of the AP-1 transcription factor as well as reduced JNK

activity in anergic T cells.18–20,35 MycoClean Mycoplasma Removal Kit In accordance with these earlier studies, c-fos and c-jun activity was decreased in Th1 cells anergized by exposure to n-butyrate. Although an ELISA-based method was used, the readout reflects the activity rather than the binding of the transcription factors because the primary antibody provided with this kit is specific for an epitope on the bound and active form of the transcription factor. p21Cip1 has been shown to interact with JNK and inhibit its activity.15,16 p21Cip1-deficient fibroblasts had higher basal levels of JNK1 than controls, an effect that was reversed if the cells were transfected with p21Cip1.16 In T cells JNK activation following release from G1 arrest correlated with dissociation from p21Cip1.17 In T cells, JNK not only promotes IL-2 gene transcription through the activation of c-jun and AP-1,36 but also directly promotes IL-2 messenger RNA stability.37 Consequently, the finding that p21Cip1 interacts with p-JNK and p-c-jun in Th1 cells anergized by exposure to n-butyrate could explain the lack of IL-2 production and related proliferation in these Th1 cells.

Another important consideration learn more in translating the in vitro murine data to the bedside is the dosing regimen. Serum concentrations of atorvastatin, for lipid lowering, are in the nanomolar range [42], while inhibition of T cell activation and MMP-9 production occurs only at micromolar concentrations in tissue culture. Direct comparisons of human serum concentrations to in vitro experiments are not appropriate, especially for a lipophilic drug such as atorvastatin. Additionally, previous

work has shown that statin treatment inhibits MMP-9 production indirectly in the vessel wall of abdominal aortic aneurysms [43,44], thus serum levels may not reflect accurately local tissue concentrations at work, similar to the disconnect between serum and tissue levels of MMP-9 in KD [45]. An altered lipid profile has been reported in children with KD. During the acute phase of disease a pro-atherogenic lipid profile [46,47], with a decrease AZD1208 clinical trial in total cholesterol, high-density lipoprotein-cholesterol (HDL-C), apoA1 and apoA2 and an increase in triglycerides and apoB, is observed [46,48,49]. Total cholesterol returns quickly to normal, but HDL-C recovery is slow and remains

significantly lower than expected up to years later [46]. In addition to the observed mild dyslipidaemia in patients with KD, arterial function may be abnormal with abnormal measures

of endothelial dysfunction even in those without aneurysms [50–53]. Carotid artery intima-media thickness among KD patients is greater, and endothelial dysfunction has been reported both in children with persistent coronary lesions as well as in those without detectable early coronary artery involvement, Liothyronine Sodium indicated by decreased brachial artery flow-mediated dilatation [40,48,49,53]. Thus, all patients with KD, even in the absence of echocardiographic evidence of coronary artery involvement, may be at risk for premature atherosclerosis even if managed appropriately during the acute phase of the illness. The potential benefits of statin therapy are recognized outside the acute phase of illness. Recognizing the limitations of the in-vitro results reported, confirmation of the immunomodulatory effects of atorvastatin are needed in vivo using the LCWE-induced coronary arteritis animal model of KD. This model, which mimics accurately the histopathological changes seen in the coronary arteries of KD patients [19,20,54], provides a unique opportunity to study treatment protocols and potential side effects of statin therapy in young animals, providing important insight prior to human studies.

Quantitative analysis of regenerated nerves between experimental groups showed that those repaired by direct contact of the stumps with fibrin glue showed significant increase in the myelin and fiber areas. The tubulization groups, repaired by suture or fibrin glue, provided similar results. G-ratio analysis revealed that the regenerating axons of all experimental groups presented values equivalent Osimertinib to control (crushing group). These results suggest that the use of fibrin glue in nerve repair by either direct coaptation or tubulization

is an alternative to conventional suture repair, particularly in case of small-size-nerve reconstruction. © 2013 Wiley Periodicals, Inc. Microsurgery 33:468–477, 2013. “
“Microvascular procedures not only demand precise movements but also usually

require a long operation time. Using a conventional surgical microscope, microvascular surgeons need to keep the neck in a fixed flexion posture, which can lead to physical fatigue. Thus, our aim was to develop a three-dimensional (3D) monitoring system to improve the microsurgery environment. It consists of four main parts: the surgical microscope, the charge-coupled devices, the 3D multiplexer, and the 3D monitor. Two patients with head and neck cancers who underwent tumor resections were reconstructed with free flap microsurgeries. Both artery anastomoses were completed successfully and the postoperative courses of the two patients were smooth. Vascular anastomosis can be performed successfully with the help of the new 3D display system. Although the artery anastomosis procedures took longer than under a surgical Selleckchem Small molecule library microscope, the 3D system offers another option to improve the working environment for surgeons. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“The

goal for treatment of osteonecrosis of the femoral head (ONFH) is to relieve pain, preserve the contour of the femoral head, and delay the need for total hip arthroplasty. The free vascularized fibular grafting (FVFG) has been shown to support the subchondral architecture as well as restore local circulation for the necrotic femoral Clostridium perfringens alpha toxin head in treatment of ONFH. This report aimed to present the clinical results of the use of a modified surgical technique of FVFG for treatment of ONFH. Four hundred and seven patients with 578 hips of ONFH were included. The patients’ average age was 36.7 years old (ranging 19–55 years old). The disease was staged from II to V based on the Steinberg classification system. By the modified procedure, the vascularized fibular graft was harvested via a lateral incision with fibular osteotomy prior to the exposure of the vascular pedicle, and the removal of necrotic tissue and inset of graft were performed through an anterior approach. The operative time averaged 90 min for unilateral ONFH (ranging 75–110 min) and 190 min for simultaneous treatment of bilateral ONFH (ranging 160–230 min).

Autopsy examination, limited to the intracranial tissues, revealed marked infiltration of IgG4-containing plasma cells in the adventitia and media of the vertebral and basilar arteries. Multiple

fibrous nodules forming pseudotumors were also evident on the outer surface of the affected arteries. These histological features were very similar to those of arteriopathy, such as inflammatory aortic aneurysm, which has been described in patients with IgG4-related disease, suggesting that autoimmune mechanisms, known to be involved in the pathogenesis of visceral lesions in the disease, also played a role in the etiology of VBD in the present patient. In conclusion, we consider that the present case may represent VBD as a manifestation of IgG4-related selleck inhibitor disease. “
“C. B. Carroll, M.-L. Zeissler, C. O. Hanemann and J. P. Zajicek (2012) Neuropathology and Applied Neurobiology38, 535–547 Δ9-tetrahydrocannabinol

(Δ9-THC) exerts a direct neuroprotective effect in a human cell culture model of Parkinson’s disease Aims:Δ9-tetrahydrocannabinol (Δ9-THC) is neuroprotective in models of Parkinson’s disease (PD). Although CB1 receptors are increased within the Talazoparib ic50 basal ganglia of PD patients and animal models, current evidence suggests a role for CB1 receptor-independent mechanisms. Here, we utilized a human neuronal cell culture PD model to further investigate the protective properties of Δ9-THC. Methods: Differentiated SH-SY5Y neuroblastoma cells were exposed to PD-relevant Lonafarnib purchase toxins: 1-methyl-4-phenylpyridinium (MPP+), lactacystin and paraquat. Changes in CB1 receptor level were determined by quantitative polymerase chain reaction and Western blotting. Cannabinoids and modulatory compounds

were co-administered with toxins for 48 h and the effects on cell death, viability, apoptosis and oxidative stress assessed. Results: We found CB1 receptor up-regulation in response to MPP+, lactacystin and paraquat and a protective effect of Δ9-THC against all three toxins. This neuroprotective effect was not reproduced by the CB1 receptor agonist WIN55,212-2 or blocked by the CB1 antagonist AM251. Furthermore, the antioxidants α-tocopherol and butylhydroxytoluene as well as the antioxidant cannabinoids, nabilone and cannabidiol were unable to elicit the same neuroprotection as Δ9-THC. However, the peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist T0070907 dose-dependently blocked the neuroprotective, antioxidant and anti-apoptotic effects of Δ9-THC, while the PPARγ agonist pioglitazone resulted in protection from MPP+-induced neurotoxicity. Furthermore, Δ9-THC increased PPARγ expression in MPP+-treated SH-SY5Y cells, another indicator of PPARγ activation.

Our previous studies show that hepatic natural killer T (NKT) cells play a significant role in the pathogenesis of NAFLD. In this study, we explore the mechanism by which modification of gut flora leads to the alteration of hepatic NKT cells and improvement of steatosis. Mice were fed a high-fat (HF) diet to induce NAFLD. Some of them also received different doses of mixed-strain probiotics (VSL#3); single-strain probiotic (Bifidobacterium infantis) or antibiotics. Animal weight, glucose tolerance, liver steatosis and hepatic NKT cells were assessed. Lipid extracts from probiotics were tested for their ability to activate NKT

cells. Toll-like receptor 4 (TLR4) knockout mice Natural Product Library ic50 were also evaluated for their responses to HF diet. High-dose VSL#3 was more effective

than low-dose VSL#3 and B. infantis for the improvement of hepatic NKT cell depletion and steatosis. The lipids extracted from VSL#3 stimulated NKT cells both in vivo and in vitro. In contrast, lipids R428 mouse from B. infantis decreased α-GalCer-mediated NKT cell activation in vitro, but were able to stimulate NKT cells. TLR4 knockout mice have a similar response to HF-diet-induced NKT cell depletion and obesity. These results suggest that alterations in the gut flora have profound effects on hepatic NKT cells and steatosis, which are both strain-specific and dose-dependent, but not through TLR4 signalling. Furthermore, these data suggest that probiotics may contain bacterial glycolipid antigens that directly modulate the effector functions of hepatic NKT cells. “
“Citation Wang B, Koga K, Osuga Y, Hirata T, Saito A, Yoshino O, Hirota Y, Harada M, Takemura Y, Fujii T, Taketani Y. High mobility group Box 1 (HMGB1) levels in the placenta and in serum in preeclampsia. Am J Reprod Immunol 2011; 66: 143–148 Problem Preeclampsia is a pregnancy disorder characterized

by systemic inflammation. High mobility group box 1 (HMGB1) check details is a molecule known to act as a ‘danger signal’ by participating in various inflammatory processes, but data in regard to preeclampsia are sparse. The aim of this study was to analyze placental and serum HMGB1 levels in normal pregnancy and preeclampsia. Method of study Sera were collected from women with preeclampsia soon after the manifestation of the disease and before commencing any medication. Placental samples were collected immediately after delivery. Expressed isoforms of HMGB1 (28- and 30-kDa) in the placenta were evaluated by Western blot analysis. Serum HMGB1 concentrations were measured using enzyme-linked immunosorbent assays (ELISA). Results Two isoforms of HMGB1 are expressed by the human placenta. The 28- and 30-kDa HMGB1 isoforms were expressed highly in preeclamptic placental tissue; however, compared with normotensive control tissue, differences in detected expression levels did not reach statistical significance.

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteer learn more donors provided by the “Etablissement Français du Sang” (EFS, Marseilles, France) and isolated by fractionation over a density gradient of Lymphoprep© (Abcys). Human CD4+ T cells were negatively selected from isolated PBMCs by depletion of non-CD4+ T cells with magnetic beads using the T-cell isolation kit II from Miltenyi Biotec®. Isolated CD4+ T cells were used for further experiments when purity was superior than 90%. PBMCs from healthy donors were stained with 5 μL of the following mouse anti-human mAbs per million of cells: ECD-conjugated anti-CD3, PC5-conjugated anti-CD14, PC5-conjugated anti-CD19 (to

select CD3+CD14−CD19− cells) (all from Beckman Coulter), Pacific Blue-conjugated anti-CD4, Alexa700-conjugated anti-CD8 (all from BD Pharmingen, San Diego, CA, USA), APC-Alexa750-conjugated anti-CD27 (Invitrogen), PC7-conjugated anti-CD45RA (BD Biosciences), Alexa647-conjugated anti-CD277 (clone 20.1, IgG1) 1. The CD277 mAb (clone 20.1) was labeled with

Alexa Fluor 647 using a commercial kit (Invitrogen). APC-conjugated IgG1 (Beckman Coulter) was used as a negative control and LIVE/DEAD Fixable Dead Cell Stain Kit was used for viability. MAPK inhibitor Cells were incubated for 20 min at 4°C, then washed twice in PBS fixed with 2% paraformaldehyde, and analyzed by an FACSAria flow cytometer (BD Biosciences). Interleukin-3 receptor Data were analyzed using the FlowJo Software (TreeStar, Ashland, OR, USA). Purified CD4+ T cells (2×105 cells/well) from thawed human PBMCs were cultured during 96 h in RPMI 1640 10% FBS in flat bottom 96-well plates (Microtest™ 96, Becton Dickinson), which have been previously incubated with CD3 mAb (clone OKT3) plus CD28 mAb (clone CD28.2) 23 or isotypic control (IgG1). Anti-CD3 and anti-CD28 mAbs were used at 0.3 μg/mL and 10 μg/mL, respectively. Cells were placed into

an atmosphere of 5% CO2 at 37°C in a humidified incubator. Every 24 h, cells were transferred in a conic bottom 96-well plate (Nunc™, Denmark) and stained for 30 min at 4°C with 3 μL of purified anti-PD-1 (clone PD-1.3.1) 24, washed three times in PBS/FBS 0.2%/NaN3 0.02%, then stained with PE-conjugated goat anti-mouse (1/80, Beckman Coulter), washed and stained with 3 μL of each of PC7-conjugated anti-CD4, FITC-conjugated anti-CD3 (all from BD Biosciences) Alexa647-conjugated anti-CD277 and 6 μL of 7-AAD (BD Biosciences) for 30 min at 4°C. Purified IgG1 and APC-conjugated IgG1 were used as controls. Immunostained cell samples fixed with 2% paraformaldehyde were analyzed on a BD FACS Canto (BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo Software (TreeStar, Ashland, USA). Mononuclear cells were obtained from LNs by crushing fresh tissue samples in RPMI 1640 10% FBS.