[82] In the uninephrectomised sheep, plasma sodium levels were significantly elevated between week 6 and 10 after birth and blood volume and arterial pressure Napabucasin chemical structure became elevated at a postnatal age of 6 months.[81] Furthermore, urinary excretion of sodium was significantly reduced in the

uninephrectomised animals at the age of 6 months but at 2 years, excretion of sodium was similar to that of the sham animals.[81] This shows that the reduction in excretion of sodium may contribute to the increase in blood pressure at the age of 6 months. Furthermore, the normalization of excretion of sodium at 2 years suggests that a rightward shift in pressure natriuresis had occurred to increase blood pressure chronically, in a manner that allowed maintenance of salt and water homeostasis in the animals with one kidney. In models of developmental programming of low nephron endowment and hypertension an increase in expression of sodium transporters and channels has also been observed in kidneys of offspring[83-85] suggesting that alterations in handling of sodium via the renal GSK1120212 manufacturer tubules may be a common pathway leading to hypertension in models of low nephron endowment. Compensatory renal growth appears to be a contributing factor to the genesis of hypertension, but very little is known

about the actual mediators of compensatory renal growth.

Multiple factors have been identified in the compensatory growth process including, insulin-like growth factors, transforming growth factor beta-1 and glucose transporters.[86] Furthermore, indirect evidence suggests Sitaxentan a role for renal sympathetic nerve activity. Uninephrectomy in the rat has been demonstrated to increase mean renal nerve activity by as much as 80% compared with the control animals by day 3 after nephrectomy.[87] This increase in mean renal nerve activity also correlated with the increase in weight of the remnant kidney.[87] The ontogeny of the renal sympathetic nerves is poorly understood, but developmental increases in sympathetic innervation have been linked to hypertension in adulthood.[88-90] Based on the evidence examined in this review, we propose that factors, which contribute to the compensatory hypertrophy of the kidney, in the long term, contribute to the later elevation in arterial pressure and reduction in GFR. As depicted in Figure 3, following a reduction in renal mass there is an increase in SNGFR. This increase in SNGFR is associated with hypertrophy of glomeruli. One explanation for the increase in SNGFR following nephron loss may be reduced preglomerular vascular resistance as evidenced by increased renal blood blow.

Rather, these data add to emerging evidence suggesting that individual differences in R788 cost face scanning might reliably predict aspects of later development. “
“Infants greatly refine their ability to discriminate language sounds by 12 months, yet 14-month-olds appear to confuse similar-sounding

novel words. Two explanations could account for this phenomenon: infants initially have incomplete phoneme representations, suggesting developmental discontinuity; or word-learning demands interfere with use of established phonetic detail. These hypotheses were tested at 14 months by pairing a novel word with an object preexposed to half the infants and novel to the other half. If demands are key, only preexposed infants should efficiently use phonetic detail; there is no need to concurrently learn object details with the word. If representations lack detail, object familiarity should not matter. Only infants preexposed to the object noticed a change in its label, thus challenging the discontinuity position and demonstrating the impact of object familiarity on early word learning. “
“Pattern perception and

organization are critical functions of the visual cognition system. Many organizational processes are available early in life, such that infants as young 3 months of age are able to readily utilize a variety of cues to organize visual patterns. However, other processes are not readily evident in young infants, and their development involves perceptual PD0325901 concentration learning. We describe a theoretical framework that addresses perceptual learning in infancy and the manner in which it affects visual organization and development. It identifies five kinds of experiences that induce learning, and suggests that they work via attentional and unitization mechanisms to modify visual organization. In addition, the framework proposes Atazanavir that this kind of learning is abstract, domain general, functional at different ages in a qualitatively similar manner, and has a long-term impact on development through a memory reactivation process. Although most models of development

assume that experience is fundamental to development, very little is actually known about the process by which experience affects development. The proposed framework is an attempt to account for this process in the domain of perception. “
“This study employed a new “anticipatory intervening” paradigm to tease apart false belief and ignorance-based interpretations of 18-month-olds’ helpful informing. We investigated in three experiments whether 18-month-old infants inform an adult selectively about one of the two locations depending on the adult’s belief about which of the two locations held her toy. In experiments 1 and 2, the adult falsely believed that one of the locations held her toy. In experiment 3, the adult was ignorant about which of the two locations held her toy.

The increased level of IFN-γ resulted from both the CD4+ T and the CD8+ T cells, particularly

click here from CD8+ T cell. Interestingly, the ubiquitination strategy designed to improve MHC I-mediated cellular responses also resulted in improved cytokines and proliferative responses mediated by CD4+ T cells. It could be that the increasing protein degradation by the proteasome also yields peptides that could be taken up by MHC II molecules. That modulation of immune response in our experiment is helpful for the protective immunity of Mycobacterium tuberculosis. The modulated immune response indicated that the expressed Ag85A protein had a higher rate of intracellular degradation in a proteasome pathway because of the addition of UbGR. Our result is consistent with the Dobaño’s report [24], which showed that immunization with DNA vaccine encoding PyHEP17 fused to Ub induced higher IFN-γ, cytotoxic and proliferative T cell responses than those of unmodified vaccines. However, no effect was seen for another antigen PyCSP using the same targeting strategies. Rodriguez’s report [26] demonstrated that the ubiquitinated DNA vaccine targeted to the protein degradation pathway enhanced

cytotoxic T lymphocyte induction and abrogated antibody induction. However, in Vadlin’s study [27], when ub fused with hepatitis C virus (HCV) core antigen, an undetectable antibody response and no increase Neratinib in CTL activity were observed compared with the non-fusion vaccine. In our study, the humoral immune old responses were not completely abrogated. Those different results may correlate with the different antigenicity of protein and the different dependence of antigen on

ub. In conclusion, the data presented above suggested that the fusion of UbGR to DNA vaccine significantly increased the antigen-specific cellular immune response. Infection with M. tuberculosis remains largely confined to an intracellular localization. Thereby, it is greatly accepted that protective immune response against M. tuberculosis infection involved a cell-mediated response rather than humoral response on the part of the host defences, involving both CD4+ and CD8+ T cells and the ability to respond with Th1-type cytokines, particularly IFN-γ. Taken together, our results demonstrated that the fusion of UbGR to Ag85A DNA vaccine could be a new strategy to improve the efficacy of TB DNA vaccines. We thank Dr. Xiao An for providing us the sera from patients infected with Mycobacterium tuberculosis. This research was funded by the fund of Bureau of Public Health, Shanghai (number 2009132) and the National Natural Science Foundation of China (Grant No. 31070121). No competing financial interests exist. “
“Nearly all proteins entering the lumen of the endoplasmic reticulum (ER) become glycosylated en route to a cellular organelle, the plasma membrane, or the extracellular space.

The aim of this study was to investigate whether diabetes and insulin resistance affect B-1 cells and their production of natural IgM. We found that diabetic db/db mice have check details lower levels of peritoneal B-1a cells and a decreased

IgM response to pneumococcal immunization and TLR-4 activation. Furthermore, our in-vitro studies showed that glucose in high concentrations reduces B-1 cell IgM secretion and differentiation into antibody-producing cells concurrent with proliferation arrest and increased apoptosis. Specific pathogen-free C57BL/6 mice were purchased from Taconic (Skensved, Denmark). For isolation of peritoneal B-1 cells, male and female C57BL/6 mice were fed a normal chow diet. As a model for insulin resistance, 8-week-old male C57BL/6 mice were assigned randomly to a low glycaemic control diet or a high-fat diet (Harlan

Laboratories, Madison, WI, USA) for 12 weeks. On a caloric basis, the low glycaemic control diet contained 16·8% fat, 60·9% carbohydrate and 22·3% protein (3·3 Kcal/g), whereas the high-fat diet contained 60·3% fat, 21·3% carbohydrate and 18·4% protein (5·1 Kcal/g). The diets contained comparable amounts of vitamins and minerals. Male db/db mice and control mice (+/+ or +/db) on a C57BL/6 background from Jackson Laboratories (Bar Harbor, ME, USA), and db/db and wild-type controls (+/+) on a BKS background from Taconic, were maintained on a normal chow diet. For in-vivo assessment Vemurafenib price of the effect of TLR-4 agonist, 10–12-week-old db/db mice (on a C57BL/6 background) and controls MRIP were injected intraperitoneally with 0·34 mg/kg of the TLR-4 agonist Kdo2-Lipid A (Avanti Polar Lipids, Inc., Alabaster, AL, USA) or vehicle. For immunization studies, 10–12-week-old db/db mice and controls (on a C57BL/6 or BKS background) and C57BL/6 mice maintained on diets for 3 months were injected intraperitoneally with 11·5 μg of a 23-valent vaccine (Pneumovax; Sanofi Pasteur MSD, Lyon, France), containing 0·5 μg each of 23 types of polysaccharides from S. pneumoniae

or saline. As indicated for each experiment, body weight, plasma insulin, glucose and antibody titres were followed in longitudinal blood samples. Before blood sampling, mice were fasted for 4 h. Plasma glucose in blood samples from fasted, non-anaesthetized animals was determined with a glucose dehydrogenase method by using HemoCue® B-glucose microcuvettes (HemoCue®, Ängelholm, Sweden) and insulin was determined by a mouse insulin enzyme-linked immunosorbent assay (ELISA) (Mercodia, Uppsala, Sweden). Plasma triglycerides and cholesterol were measured using Konelab 20 Autoanalyzer (Thermo Electron Corporation, Vantaa, Finland). All mice were housed in a controlled environment and all experimental protocols were approved by the animal ethical committee in Gothenburg.

Consequently,

P and V proteins share the same 317 residues at the amino terminus (P/V common region), while the two proteins have unique carboxyl termini. The V protein contains a 67-residue unique KU-60019 price carboxyl terminus (Vu region), which is characterized by highly conserved 15 amino acids in almost all of the members of the subfamily Paramyxovirinae. The conserved residues include seven cysteine residues, forming a zinc-finger motif that binds two zinc ions (4, 5, 6). Phenotypes of  V-deficient viruses provided insights into the role of the V protein in virus infection in mice (reviewed in (7, 8)). V-knockout virus obtained by mutations at the RNA editing site (SeV V[-]) was cleared from mouse lungs at an early stage of infection, although the virus propagated as efficiently as the wild-type virus in cultured cells (9). A similar phenotype was also observed in SeV possessing truncated V protein lacking the Vu region (SeV VΔC) (10). Both the V(-) and VΔC viruses are remarkably attenuated in virulence in mice, indicating a substantial role of the V protein, predominantly the Vu domain, in SeV pathogenicity in vivo. Amino acid substitutions at the conserved residues of the Vu region also resulted

in suppression of virus growth in mouse lungs and attenuation in virulence, Decitabine research buy accompanying a defect of zinc binding to the mutant Vu region (11, 12). We have shown that growth of SeV V(-) was restored in interferon regulatory factor-3 (IRF3) knockout (KO) mice (13). IRF3 is a transcriptional factor that facilitates expression of IFN and IFN-related genes and plays an important role in innate immunity responding to viral infection. Recent progress in research of innate immunity has revealed detailed signaling pathways leading to IRF3-activation and IFN-β production in response to virus infection (reviewed in (14, 15)). Intracellular dsRNA and/or 5’-terminal triphosphate of RNA generated during viral replication are detected by the cytoplasmic proteins RIG-I (16, 17, 18) and MDA5 (19, 20). TBK-1 and IKKɛ kinases, both of which

Cytidine deaminase form a heterotrimeric complex with TANK, are then activated through IPS-1, and IRF3 is further phosphorylated and activated by the activated kinases. Paramyxovirus V proteins including the SeV V protein have been shown to bind MDA5 and to disturb activation of IRF3 and production of β-interferon (19, 20). Thus, it has been hypothesized that V function related to viral pathogenesis can be explained by interaction of V and MDA5. In the present study, we tested this hypothesis by investigating interactions of the mutant V proteins with MDA5. 293T cells (human renal epithelial cells expressing the SV40 large T antigen; Riken Bio Resource Center, Japan) were propagated in DMEM supplemented with 10% fetal calf serum. Wild-type SeV derived from a cDNA of the Z strain (21) and its V mutant viruses were propagated in embryonated chicken eggs.

Our results showed that the percentage of infected monocytes did not change upon treatment with captopril, as the percentages of infection were similar when comparing PI3K inhibitor captopril-treated with untreated cultures. Moreover, these results allowed for the selection of the 3-h time-point to evaluate the extent of parasite internalization in monocyte suspensions, using CFSE-labelled T. cruzi as the read-out. Our flow cytometry results (Fig. 1c and d) showed that intensity of CFSE fluorescence in infected CD14+ cells increased 27% in monocyte suspensions supplemented with captopril compared to untreated monocytes

(1552·3 versus 1128·4; Fig. 1c and d). Collectively, these data indicate that captopril increased markedly the extent of parasite uptake per host cell and, although it did not affect the proportion of infected monocytes, it favoured the penetration of a higher number of parasites per cell. Antigen-presenting cells (APC) play a key role in the induction of immune responses, and it is well established that monocytes are able to present major histocompatibility complex (MHC)-restricted epitopes to T cells [19]. ACE was found in tissue macrophages as well as in cultured monocytes [20]. Due to its dipeptidyl carboxydipeptidase selleck activity, ACE enhances the presentation of endogenously synthesized

peptides to MHC class I by generation of optimally sized class I-binding peptides from a larger protein fragment [21]. In order to determine if ACE 4��8C expression is altered by T. cruzi infection and/or captopril treatment, we stained PBMC with anti-CD14 (monocyte marker) and anti-CD143 (ACE) antibodies after 3 h of incubation with T. cruzi in the presence or absence of captopril. We found that T. cruzi infection led to an increase in the frequency of CD14+CD143+ cells in relation to non-infected

cells (8·05% ± 2 versus 3% ± 1; Fig. 2a). The same results were observed in infected cells upon treatment with captopril: we observed an increase in CD14+CD143+ cells in cultures treated with the ACE inhibitor compared to captopril-treated, but non-infected cultures (7·7% ± 2 versus 3·3% ± 1; Fig. 2a). Thus, our results indicated that captopril by itself was not able to induce alterations in ACE expression either in infected or non-infected monocytes, as the percentage of expression of CD143 was similar in captopril-treated or untreated cultures (Fig. 2a). T. cruzi induction of CD143 expression by CD14+ cells is consistent with the role of ACE in intracellular antigen presentation [21]. In addition to antigen presentation, monocytes participate in immunoregulatory functions via cytokine production. We then evaluated if the expression of IL-10 and IL-12 by monocytes was altered by T. cruzi infection and/or by captopril treatment (Fig. 2b and c). T. cruzi infection increased significantly the expression of IL-10 by monocytes compared to uninfected cells (9·5% versus 4·5%; Fig. 2b). Interestingly, we found that T.

Expression of XBP1 and antioxidant molecules was also detected in surgically excised specimens from 30 patients with glioma, and 10 normal brain control specimens obtained at autopsy. Results: XBP1 knockdown significantly enhanced the cell death fraction, MMP loss and ROS levels in H2O2- or As2O3-treated glioma cells, concomitant with a decrease of several antioxidant molecules including catalase. Moreover, the abundant expression of XBP1 and antioxidant molecules was also observed in human glioma specimens, as compared with normal brain tissues. Conclusions: check details XBP1 confers an important role in protection against oxidative stress in gliomas, potentially

via up-regulation of antioxidant molecules such as catalase. Targeting XBP1 may have synergistic effects with ROS inducers on glioma treatment. “
“R. A. Armstrong and N. J. Cairns (2010) Neuropathology

and Applied Neurobiology36, 248–257 Analysis of β-amyloid (Aβ) deposition in the temporal lobe in Alzheimer’s disease using Fourier (spectral) analysis Aim: To determine the spatial pattern of β-amyloid (Aβ) deposition throughout the temporal lobe in Alzheimer’s disease (AD). Methods: Sections of the complete temporal lobe from six cases of sporadic AD were immunolabelled with antibody against Aβ. Fourier (spectral) analysis was used to identify sinusoidal patterns in the fluctuation of Aβ deposition in a direction parallel to the pia mater or alveus. Results: Significant sinusoidal fluctuations in density were evident in 81/99 (82%) analyses. In 64% of analyses, two frequency components find more were present with density peaks of Aβ deposits repeating every 500–1000 µm and at distances greater than 1000 µm. In 25% of analyses, three or more frequency components were present. The estimated period or wavelength (number of sample units to GNA12 complete one full cycle) of the first and second frequency components did not vary significantly between gyri of the temporal lobe, but there was evidence that the fluctuations of the classic deposits had longer periods than the diffuse and primitive deposits.

Conclusions: (i) Aβ deposits exhibit complex sinusoidal fluctuations in density in the temporal lobe in AD; (ii) fluctuations in Aβ deposition may reflect the formation of Aβ deposits in relation to the modular and vascular structure of the cortex; and (iii) Fourier analysis may be a useful statistical method for studying the patterns of Aβ deposition both in AD and in transgenic models of disease. “
“Clear cell meningioma (CCM) is an uncommon variant of meningioma, corresponding to WHO grade II. We present a case of CCM with histologically aggressive appearance and clinically aggressive behavior. The tumor demonstrated rapid regrowth and brain metastasis. The histological progression from the ordinal CCM to the atypical area and higher MIB-1 index was observed.

Specifically patients with deferoxamine-therapy, hyperglycaemic with or without ketoacidosis, or other forms of acidosis are uniquely

predisposed to mucormycosis. In this review, we discuss the molecular mechanisms of infection in these patient categories in an attempt to identify novel therapies for a disease with poor prognosis. Emphasis on the effect of glucose and free iron on host–pathogen interactions are also covered. Mucormycoses are buy ABT-888 rare life-threatening fungal infections caused by fungi of the order Mucorales.[1-3] Rhizopus species remain the most common cause of infection, although more mucormycosis cases caused by Mucor, Lichtheimia and Apophysomyces are being reported.[4-7] These infections usually afflict patients with classical immunosuppression due to neutropenia, haematologic malignancies or corticosteroid treatment.[8, 9] Additionally, hyperglycaemia, diabetic ketoacidosis (DKA) and other forms of acidosis predispose patients to mucormycosis.[3, 10] Although burn and trauma patients have long been known to be susceptible to this infection,[9, 11] recent data showed that outbreaks of mucormycosis are also associated with natural

disasters[12, 13] and even in military personnel who are injured in combat operations.[14, 15] Therefore, mucormycosis are becoming more prevalent in the last two decades. Indeed, there has been a considerable rise in the incidence of mucormycosis at Peptide 17 major transplant centres.[16, 17] In fact, in high-risk patients the prevalence of mucormycosis can be up to 8% in autopsied patients with leukaemia.[18] A population-based study carried out in France demonstrated a 70% increase in mucormycosis cases between 1997 and 2006.[19] In addition, data from a tertiary care centre in India demonstrated ≥400% increase in mucormycosis incidence, mainly among DKA patients in a 16-year period.[20, 21] The standard therapy for invasive

mucormycosis includes reversal of the underlying predisposing factors (if possible), emergent, wide-spread surgical debridement of the infected area, and antifungal therapy.[2, 22, 23] Although amphotericin B (AmB) remains the only Fossariinae antifungal agent approved for the treatment of invasive mucormycosis,[2, 23, 24] it is widely accepted that lipid formulation of AmB are the first line therapy for this disease. This is because Mucorales are relatively resistant to AmB, and higher doses (1–1.5 mg/kg/day) are required for effective treatment. Due to the less toxicity of lipid formulations of AmB, it is now possible to administer more effective higher doses of these lipid formulation drugs. However, in the absence of surgical removal of the infected focus (such as excision of the eye in patients with rhinocerebral mucormycosis), antifungal therapy alone is rarely curative.[2, 23] Moreover, even when surgical debridement is combined with high-dose lipid formulation AmB, the overall mortality associated with mucormycosis reaches 50%.

The course of systemic vasculitis differs considerably from one patient to another. For example, a

patient with early Wegener’s granulomatosis in the nose, ear or sinuses may not have detectable lung or renal involvement. Early diagnosis and treatment would aim to reduce upper airway damage and hearing Small Molecule Compound Library loss. If involvement of the lungs or glomeruli were to occur later the clinical situation would alter significantly, as more potent and potentially toxic immunosuppressive therapy would be necessary to rescue vital organ functions. If the clinical onset is manifested mainly by renal disease, the underlying systemic vasculitic condition may take longer to diagnose. The consequences can be detrimental because kidney function is often lost very quickly, and irreversible changes in the glomeruli may have occurred by the time diagnosis is made [5]. Missed or delayed diagnosis influences prognosis strongly if critical organs are involved,

and less so when structurally and functionally less critical organs are affected. Careful management, with long-term follow-up, attempts to preserve health. Economic consequences Belnacasan will depend on the health cost for the patient and society as a result of damage. A systematic approach to diagnosis and follow-up will take into account the relapsing remitting nature of the disease, damage caused by low-grade grumbling disease and side effects of medication. Active inflammation requires an aggressive approach, which is entirely inappropriate in quiescent disease with extensive scarring, although the features of the clinical presentation may overlap. The initial assessment will be to make a diagnosis, categorize disease severity and formulate Fossariinae a management plan. Subsequent assessments review the success of treatment and detect new organ involvement. The Birmingham Vasculitis Activity Score (BVAS) may be used to summarize this information systematically.

Assessment of damage provides clinical and prognostic information on organ scarring caused by the disease and its treatment but does not represent ongoing active inflammation. Suitable tools for this include the Vasculitis Damage Index (VDI) and Disease Extent Index (DEI). Finally, assessment of function considers the overall impact of the disease on the physical, social and psychological function, including quality of life and employment. Tools include the Short Form 36 (SF36) and Health Assessment Questionnaire (HAQ), which are questionnaire-based. Clinical assessment of patients with giant cell arteritis and Takayasu’s arteritis includes palpation of peripheral pulses for asymmetry, bilateral blood pressure assessment, auscultation for bruits and laboratory tests for evidence of systemic inflammation. Further diagnostic information is provided by temporal artery biopsy (TAB) in giant cell arteritis and imaging of the arterial tree by conventional angiography, magnetic resonance imaging (MRI) or positron emission tomography (PET) [17].

The empty vector was used to generate CAL-1-EV cells. Lentiviral particles were produced in 293T cells by calcium phosphate transfection. Spin transduction of CAL-1 cells with 8 μg/mL Polybrene was performed at 1800 selleck chemical rpm for 90 min. GFP-positive CAL-1 cells were sorted under low-pressure conditions on the

FACSAria. For RNA interference, CAL-1 cells were transfected with 75 nM siRNA directed against NAB2 (siRNA ID: s9248; Ambion/Applied Biosystems) or the Silencer Selected Negative Control siRNA #1; Ambion/Applied Biosystems) together with 25 nM siGLO Transfection Indicator (Dharmacon) with transfection reagent DharmaFECT 4 (Dharmacon) according to the manufacturer’s protocol. Transfection efficiency was determined by flow cytometry (Supporting Information Fig. 3A), and silencing was confirmed at protein levels by western

blot (Supporting Information Fig. 3B). A total of 105 primary human pDCs were stimulated with 12.5 μg/mL CpG A (Invivogen) or left untreated for 4 h or overnight in complete medium in a 96-well plate for RT-PCR and flow cytometry or western blot analysis, respectively. CAL-1 cells (7 × 105) were seeded overnight in a 24-well plate in 2 mL medium. A total of 1.1 mL medium was replaced with 100 μL FBS-free RPMI medium containing 12.5 μg/mL CpG B or Ctrl CpG B, 5 μg/mL Imiquimod (Invivogen), or 100–200 ng/mL IFN-β (PBL Medical Laboratories) to prevent FBS-mediated NAB2 induction see more ([14], data not shown). A total of 50 μM SB203580, 2.5 μM BAY11–7082, 5 μM PI-103 (Tocris Bioscience), 200 mM Rapamycin (Calbiochem) or DMSO alone, or 0.1 μg/mL B18R (eBioscience) were added to cells 30 min prior to CpG stimulation. After stimulation, supernatant was harvested for cytokine analysis and cells were washed once with PBS before further analysis. pDC cell sorting was performed with anti-CD45RA-FITC (BD Biosciences) and anti-CD123-PE (Miltenyi Biotec). Cell surface staining was performed Liothyronine Sodium with Anti-CD40-PE (Beckman Coulter) or isotype control

IgG1-PE (BD Biosciences), and anti-TRAIL (2E5; Enzo Life Sciences), or control mouse IgG1 (BD Biosciences), followed by anti-mouse IgG1-Biotin (Enzo Life Sciences) and Steptavidin-allophycocyanin (BD Pharmingen). Dead cell exclusion was performed with propidium iodide. Intracellular IRF-7 staining was performed by fixation and permeabilization with Cytofix/cytoperm Solution (BD Biosciences) and PBS containing 0.5% saponin and 2% FCS, followed by staining with IRF-7 (H-246; Santa Cruz Biotechnologies) or isotype control (Imgenex) and anti-rabbit IgG Alexa 568 (Invitrogen). Flow cytometry was performed with FACS Calibur or LSRII (BD Biosciences). Analysis was performed with FlowJo software (Tristar).