Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and

quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. Methods: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained selleck chemicals for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask selleckchem that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy,

intermediate muscular dystrophy and normal control tissue. Results: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin : spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. Conclusion: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies. “
“The brain is vulnerable to a number of acute insults, with traumatic brain injury

being among the commonest. Neuroinflammation is a common response to acute injury and microglial activation is a key component of the inflammatory response. In the acute and Reverse transcriptase subacute phase it is likely that this response is protective and forms an important part of the normal tissue reaction. However, there is considerable literature demonstrating an association between acute traumatic brain injury to the brain and subsequent cognitive decline. This article will review the epidemiological literature relating to both single and repetitive head injury. It will focus on the neuropathological features associated with long-term complications of a single blunt force head injury, repetitive head injury and blast head injury, with particular reference to chronic traumatic encephalopathy, including dementia pugilistica. Neuroinflammation has been postulated as a key mechanism linking acute traumatic brain injury with subsequent neurodegenerative disease, and this review will consider the response to injury in the acute phase and how this may be detrimental in the longer term, and discuss potential genetic factors which may influence this cellular response. Finally, this article will consider future directions for research and potential future therapies. “
“Dentatorubral-pallidoluysian atrophy (DRPLA) is a hereditary spinocerebellar degeneration.

At the same time, the globally sustained hypoxic pulmonary vasoconstriction allows for a limit on the shunt effect and maintains gas exchanges. Such mechanisms may account for the alterations of capillary-alveolar function coexisting with normal

blood gases that was observed in our lungs treated with 30 μM of CsA. A possible limit encountered in our study might be the short ischemic time (135 ± 21 minutes) to which our lungs have been exposed. Indeed, a longer ischemia may provoke a more severe IRI and perhaps give the opportunity for the CsA to emphasize its positive effects. Nevertheless, the duration of ischemia in our model was similar to several other studies performed with CsA [15, 25, 30]. A possible bias may also be related to the induction of anesthesia with Isoflurane CX-4945 in vitro in live animals. Indeed, several works show that halogen gases inhibit the MPTP [10, 23, 31, 34], which could interfere with the CsA action in the prevention

of IRI. This preventive action was expected for Sevoflurane [10, 31], while Isoflurane showed contrasting results [23, 34]. In our protocol, Isoflurane was only used for the induction of general anesthesia before euthanasia and lung procurement surgery. As observed in the exhaled gas analysis we assumed that there was almost no gas left in the alveoli at reperfusion time. Moreover, Isoflurane has been used in every group, thus limiting the effects of possible drug interference in the results analysis. IRI prevention is a major challenge in lung transplantation. In our pig EVLP model, CsA showed a dose-dependent

improvement in PaO2/FiO2 ratio that may be related to a parallel enhancement of hypoxic pulmonary vasoconstriction. Low Galunisertib doses of CsA showed a non-significant trend toward an improvement in capillary-alveolar membrane IKBKE injury. Lungs treated with high doses of CsA (30 μM) presented an aggravation in lung permeability and cytokines concentrations, suggesting a deleterious imbalance between the possible beneficial properties of CsA on IRI cells and their hemodynamic effects in microvascularization. Further studies should focus more on lungs subjected to longer ischemia and treated with low or moderate doses of CsA. We evaluated for the first time the effects of CsA on IRI in ex vivo reperfused pig lungs. Our data suggests a possible deleterious imbalance between the beneficial cell properties of CsA and its hemodynamic effects on microvascularization. For future experiments, it would be interesting to focus more on smaller doses of CsA which might limit hemodynamic drawbacks on lung microcirculation, while keeping their beneficial cellular effect on IRI. Unlike our experiment, in which the length of cold ischemia was limited, other experiments should test CsA in various cold ischemic time situations (i.e., broad spectrum of IRI severity) for highlighting the efficacy of CsA. This study was funded by the French Health Ministry and by the association “Vaincre la mucoviscidose.” We thank Mr.

Chronic kidney disease (CKD) is a global public health problem involving increased risk of cardiovascular disease (CVD) and premature death. Psychosocial explanations of health involving social, psychological and physiological processes all interact to affect the aetiology and development of CKD.[1] For example, social processes such as social support may lead to psychological changes at the individual level which may influence health directly via physiological processes or modified behaviours.[2] Psychosocial factors are important both because they have an

impact on quality of life and have been shown to influence the progression of various chronic diseases.[3, 4] However, our understanding of the burden and impact of these potentially modifiable risk factors in CKD is limited. Rates of CKD are increasing in Australia this website PS-341 chemical structure with the number of patients commencing renal replacement therapy (RRT; dialysis or transplantation) between 1990 and 2009 escalating by 321%.[5] In addition to those being treated, around 36% of people with advanced CKD are not being dialysed[6] and a similar proportion are dying via withdrawal from dialysis.[7] In light of this increasing social and economic burden, examining the role of potentially modifiable non-biological risk factors on the disease trajectory of CKD should

be a priority. This paper examines the prognostic role of several key psychosocial factors (depression, anxiety and perceived social support) and health-related quality of life (HRQOL) in adults with CKD (i.e. CKD stage 1–5, unless otherwise stated) prior to RRT. We explore current gaps in the literature and examine potential mechanisms through which these factors may affect health outcomes. Potential interventions and suggestions for future research are also outlined. Depression is a chronic and recurrent illness associated with substantial morbidity Ribonucleotide reductase and all-cause mortality. Comorbid depressive disorders in patients with chronic disease reduce quality of life, and increase functional disability and use of healthcare services.[8] Unemployment,

low income, low perceived social support, and changes in familial and occupational roles are recognized risk factors for depression in people with CKD.[9-12] While identifying depression in patients with kidney disease is complicated by the potential misclassification of uraemic symptoms as somatic symptoms of depression, prevalence estimates for clinical depression in dialysis patients (CKD 5D) range from 20% to 30%.[13, 14] Similarly, around 22% of individuals with pre-dialysis CKD fulfil the criteria for major depression[15, 16] while 37–55% report depressive symptoms.[16-18] This is higher than the prevalence of depressive disorders in the general population (7%)[9] and in those with other chronic diseases including cancer (11%).

Transfection of a variety of cell lines with HERV-W env induced cellular fusion that was reduced when the cell

cultures were treated with an antibody against the HERV-W Env protein.21,26 In addition, induction of fusion of BeWo cells (a human trophoblastic choriocarcinoma cell line) by forskolin was associated with increased expression of syncytin.21 Moreover, inhibition of syncytin 1 expression in primary trophoblast cells reduced the number and size of syncytia formed during culture.30 The Env glycoprotein of HERV-FRD, termed syncytin 2, is structurally similar to syncytin 1 (see Fig. 2); however, it entered the primate genome before the split of the New World and the Old World Monkeys more than 40 million years ago, while syncytin

1 entered the primate genome approximately 25 millions DNA Damage inhibitor years ago and is not present in Old World Monkeys.31 Syncytin 2 also elicits cell fusion when transiently transfected into several different cell lines.32 Interestingly, the two syncytins display different properties as both are fusogenic, but syncytin 2 has immunosuppressive properties unlike syncytin 1.33 The Env protein of ERV3 is also present in syncytiotrophoblasts and was the first ERV Env for which a potential physiological function was described.34 Although it has a long open reading frame, the protein is prematurely terminated by the presence of a stop codon in the transmembrane region (Fig. 2),

which truncates the hydrophobic domain that is required for anchoring to the Bortezomib cost cell membrane.35 It also lacks a leader and a fusion peptide and, although it harbors a region with the characteristics of an immunosuppressive domain, its function is likely diminished by the lack of membrane anchorage.36 ERV3 Env does not elicit cell fusion, although its expression increases in BeWo cells treated with forskolin. When ERV3 Env is stably expressed in undifferentiated BeWo cells, it induces changes characteristic of trophoblast differentiation, such as increased levels of chorionic gonadotropin, growth inhibition, and altered morphology.37 Considering that the ERV3 Env is expressed in a variety of normal tissues heptaminol and particularly in hormone-producing organs, including adrenal and sebaceous glands and testis, it may play a general role in hormone production.36 However, 1% of 150 healthy Caucasian individuals were found to be homozygous for a premature stop codon that would theoretically result in a severely truncated non-functional protein;38 thus, it is debatable whether the ERV3 Env has a critical biological function. Two murine ERV env genes, syncytin-A (Gm52) and syncytin-B (D930020E02Rik), were identified and found to be expressed in the syncytiotrophoblast component of the labyrinthine zone of the mouse placenta.20 Both are highly fusogenic in transfection assays.

Membrane-bound TGF-β or other this website contact-dependent factors have been shown to be the main mediators of Foxp3+ Treg action in direct co-culture experiments 34, 35. Previous studies using type I diabetes and chronic colitis models have suggested the possible involvement of contact-dependent mechanisms in NKT-mediated immune suppression 25, 32, although these reports did not evaluate the specific effects on Th17 differentiation. It is known that NKT cells express several inhibitory molecules on their surface, and these molecules are upregulated when NKT cells are activated 18, 19. We are currently attempting to identify the responsible

molecules expressed on the NKT cells and blocking antibodies against CD40L, 4-1BB, 4-1BBL, CTLA-4, Fas, 2B4, NKG2D, GITR, and PD-1 failed to abrogate NKT inhibitory effects on PLX4720 Th17 differentiation. Therefore, additional experiments are needed to find out the target molecules involved in NKT:CD4+ T-cell interaction

and we are also examining the role of APC in the NKT cell-mediated inhibitory process. The regulatory role of NKT cells on TH differentiation was confirmed in vivo using an EAU model. CD1d−/− and Jα18−/− mice displayed a more severe disease phenotype compared with WT mice (Fig. 5A and B). Upon closer examination of the data, the disease severity appears milder in Jα18−/− mice compared with CD1d−/− mice, and thus we cannot completely rule out the effect of type 2 NKT cells present in Jα18−/− mice. However, as the difference between CD1d−/− and Jα18−/− was not statistically significant (p=0.203), we used CD1d−/− mice in the majority of the following experiments. The adoptive transfer of WT NKT cells decreased the degree of uveitis in CD1d−/− mice to that of WT mice (Fig. 5H). Moreover, the profile of disease regulation following adoptive transfer of NKT cells from different cytokine-deficient mice (Fig. 5H) paralleled the inhibitory effects of cytokine-deficient NKT cells on Th17 differentiation in vitro (Fig. RVX-208 2A and B), which is consistent with recent reports demonstrating that experimental uveitis induced following immunization with uveitogenic antigens was predominantly mediated through Th17 effector

pathways 15, 17. Grajewski et al. also reported the regulatory role of invariant NKT cells in experimental uveitis 36. In this report, however, CD1d-deficient mice did not show enhanced susceptibility to uveitis. In contrast to their observation, invariant NKT cell-deficient mice, both CD1d−/− and Jα18−/− mice, revealed great increases in disease severity in our study. Discrepancies might lie on the different antigen types used: we used human IRBP peptide fragments 1–20, which could discriminate increased pathogenesis between IFN-γ−/− and WT B6 mice 37. The relative lack of IL-10 induction with IRBP peptides 1–20 37 compared with the IRBP protein used in a previous study 36 could explain the increased sensitivity in disease pathogenesis of NKT cell-deficient mice in our experiments.

): negative conversion, improved, unchanged, worsened, not assessable Major items: clinical symptoms (fatigue, sputum, sputum cruentum, cough), fever, diagnostic imaging Minor items: nutrition status, inflammation findings improvement, no change, aggravation, indetermination The major drawback of these studies is the heterogenous criteria used for defining overall response with some utilising stabilisation and some using improvement in clinical, radiological and mycological (or serological) findings or their combination thereof. For instance, the response rates vary from 13–14% to 44–61% in CCPA if one

uses improvement or stabilisation, respectively, to define response.[11, 22, 27] Another drawback is that many studies have not differentiated between the different entities of CPA. Chronic pulmonary aspergillosis is a progressive pulmonary syndrome characterised by the presence LY294002 mw of multiple Daporinad concentration cavities and evidence of the presence of Aspergillus (either immunological or microbiological detection). In some patients, the disease can follow a progressively relentless course. The results of this study suggest that there was better outcome with itraconazole, but it may not confer long-term clinical or radiological benefit in patients with CCPA. In fact,

five patients had clinical and/or radiological worsening in the next 6 months after stopping itraconazole. Most of our patients had been treated for pulmonary tuberculosis in the past, which reflected the occurrence of CCPA in the upper lobes similar to previous reports.[31, 32] Itraconazole can penetrate into the walls of the cavity and even inside the fungal balls.[17] Hence, azoles are considered as an important therapeutic option in patients with aspergilloma (and CCPA). The earliest

study on CPA by De Beule et al. showed global improvement in 66% of CNPA and 56% of aspergilloma treated with oral itraconazole.[13] In this study, patients with CNPA demonstrated good radiological response whereas most patients with aspergilloma showed only symptomatic Ketotifen response.[13] Similarly, Dupont et al. showed an overall improvement of 92.8% with itraconazole given at a dose 100–200 mg day−1 in CNPA.[12] Denning et al. have reported the efficacy of oral itraconazole, voriconazole and posaconazole in patients with CPA with majority of the cases being those of CCPA.[2, 10, 19, 22] The efficacy rates of different azoles in various studies ranged from 61% to 71% and the efficacy rates were not different between the azoles.[2, 10, 19, 22] The results of our systematic review suggest a wide variation in efficacy rates with different agents, with the response lower in CCPA and highest in CNPA. Although both CNPA and CCPA are characterised by lung cavities what differentiates them is the course of the disorder which is far more rapid and destructive in the former.

Genes encoding SLAM family receptors are located at 1q23, implicated in systemic lupus erythematosus (SLE). In this study, we have investigated

the expression and alternative splicing of CS1 and 2B4 in immune cells from SLE patients. The surface expression of CS1 and 2B4 on total peripheral find more blood mononuclear cells (PBMCs), T, B, natural killer (NK) cells and monocytes in 45 patients with SLE and 30 healthy individuals was analysed by flow cytometry. CS1-positive B cell population was increased significantly in SLE patients. Because CS1 is a self-ligand and homophilic interaction of CS1 induces B cell proliferation and autocrine cytokine secretion, this could account for autoreactive B cell proliferation in SLE. The proportion of NK cells and monocytes expressing 2B4 on their surface was significantly lower in patients with SLE compared to healthy controls. Our study demonstrated altered expression of splice variants of CS1 and 2B4 that mediate

click here differential signalling in PBMC from patients with SLE. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease, characterized by the improper regulation of B cells that leads to the production of autoantibodies. The incidence of disease is gender-biased, with a female to male ratio of 9 : 1, and the onset of disease is usually during the child-bearing years [1]. Using lupus model mice such as MRL/lpr, NZB/NZW and NZM2410, which develop SLE spontaneously, Etofibrate mouse chromosome 1 has been shown to contain lupus susceptibility genes [2–5]. Genomic characterization of the Sle1b locus, the most potent member of lupus susceptibility region on murine chromosome 1, identified a highly polymorphic cluster of genes coding for the signalling lymphocyte activation molecule (SLAM) family receptors [6]. Similarly, genome-wide linkage analyses of SLE families have shown a strong association of SLE with the 1q23 region of the human genome, which also includes SLAM family receptors [7–9].

SLAM family receptors are expressed broadly on haematopoietic cells, and play an important role in immune regulation. Members of this family are SLAM (SLAMF1, CD150), CD229 (SLAMF3, Ly-9), 2B4 (SLAMF4, CD244), CD84 (SLAMF5), NTB-A (SLAMF6; Ly108 in mouse) and CS1 (SLAMF7, CRACC, CD319). All these receptors have immunoreceptor tyrosine-based switch motifs (ITSMs) in their intracellular domain, which can be bound by small adaptor proteins such as SLAM-associated protein (SAP, SH2D1A), Ewing’s sarcoma (EWS)-activated transcript 2 [Ewing's sarcoma-activated transcript-2 (EAT-2), SH2D1B] and EAT-2-related transducer (ERT, SH2D1C, only in rodents). Mutations in SH2D1A, the gene encoding SAP, are responsible for the primary immunodeficiency X-linked lymphoproliferative disease (XLP) in humans [10–12].

Inhibition of CD26 activity results in reduced T cell activity [9]. Interestingly, CD26 can increase T cell activation by ABT-263 increasing the co-stimulator CD86 on antigen-presenting cells in a process that requires enzymatic activity [10]. CD26 associates with other membrane proteins on T cells, including the tyrosine phosphatase CD45 and the ectoenzyme adenosine deaminase (ADA), which might be important

for the co-stimulatory activity of CD26 [8, 11]. However, inhibition of DPP-4 enzymatic activity may not block all these immune activities; the ability of soluble CD26 to bind ADA and enhancement of T cell proliferation can usually occur even when the active site of DPP-4 has been mutated [12, 13]. CD26 is also expressed on myeloid cells, and enzymatic inhibition decreased macrophage activation and migration into

adipose tissue [14]. In addition to GLP-1, DPP-4 also cleaves immune peptides, including neuropeptide Y (NPY) and chemokines such as interferon gamma-induced protein (IP)-10, stromal cell-derived factor (SDF)1-alpha and regulated upon activation normal T cell expressed and secreted (RANTES) [15]. DPP-4 cleavage can affect chemokine activity or receptor specificity; therefore, CHIR-99021 ic50 inhibition of DPP-4 could alter leucocyte chemotaxis [16]. In humanized mice, human haematopoetic stem cells show enhanced engraftment with DPP-4 inhibition, which may be due to altered migration of these cells [17]. Clinical trials are now under way

to test if sitagliptin can improve cord blood transplant outcomes (NCT00862719). In mouse models of T cell-mediated autoimmunity, inhibitors of DPP-4 can reduce disease severity and are associated with increases in transforming growth factor (TGF)-β levels and improvements in immune tolerance induction [18, 19]. Interestingly, in human autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, increased Idoxuridine CD26 levels on leucocytes are observed, yet there is decreased DPP-4-associated peptidase activity [20-22]. The reason for the discrepancy between activity and membrane CD26 levels is unclear, but this could be due to decreased shedding of CD26 from the membrane or decreased levels of other peptidases that cleave the same substrate. Despite evidence that sitagliptin might alter immune activity, few direct measurements of immune function after sitagliptin treatment in humans have been undertaken [23]. Therefore, we set up a double-blind clinical protocol in which healthy individuals were given either sitagliptin or placebo daily for 4 weeks. We chose to enrol healthy volunteers to separate effects of sitagliptin from disease effects on immune readouts (e.g. in type 2 diabetes).

The carotid bifurcation is the primary site for atherosclerotic changes, for which extensive clinical trials and pathological analyses on carotid endarterectomy specimens have been performed. Plaque rupture and erosion give rise to thrombus formation, which leads to brain ischaemic injury. These changes have much in common with atherosclerotic lesions of the subepicardial coronary arteries. Emboli of various types of particles are characteristics of brain ischaemic injury. Thrombi rich in fibrin and red blood cells (red thrombi) that develop in the cardiac chambers are common

sources of cerebral emboli. Small-vessel Selleck INCB018424 disease of the brain induces fibrinoid necrosis, microaneurysm, fibrohyalinosis, lipohyalinosis and microatheroma, changes commonly associated with hypertension. The acute hypertensive small-vessel changes organize to create segmental arterial disorganization and deep Venetoclax price small infarcts when they escape from rupture. Some specific vascular diseases responsible for brain ischaemic injury are briefly reviewed also. “
“Transactivation response (TAR) DNA-binding protein of Mr 43 kDa (TDP-43) is a major component of the tau-negative and ubiquitin-positive inclusions that characterize amyotrophic

lateral sclerosis (ALS) and frontotemporal lobar degeneration which is now referred to as FTLD-TDP. Concurrent TDP-43 pathology has been reported in a variety of other neurodegenerative disorders such as Alzheimer’s disease, forming a group of TDP-43 proteinopathy. Accumulated TDP-43 is characterized by phosphorylation and fragmentation. There is a close Rucaparib chemical structure relationship between the pathological subtypes of FTLD-TDP and the immunoblot pattern of the C-terminal fragments of phosphorylated TDP-43. These results suggest that proteolytic processing of accumulated TDP-43 may play an important role for the pathological process. In cultured cells, transfected C-terminal fragments of TDP-43

are more prone to form aggregates than full-length TDP-43. Transfecting the C-terminal fragment of TDP-43 harboring pathogenic mutations of TDP-43 gene identified in familial and sporadic ALS cases into cells enhanced the aggregate formation. Furthermore, we found that methylene blue and dimebon inhibit aggregation of TDP-43 in these cellular models. Understanding the mechanism of phosphorylation and truncation of TDP-43 and aggregate formation may be crucial for clarifying the pathogenesis of TDP-43 proteinopathy and for developing useful therapeutics. “
“E.-L. von Rüden, J. Avemary, C. Zellinger, D. Algermissen, P. Bock, A. Beineke, W. Baumgärtner, V. M. Stein, A. Tipold and H.

The coating buffer was removed from the plates, optimal concentrations of 2 × 105 responder cells in IMDM with 5% FCS were put into each well and 5 × 104 tumour target cells or lysates (equivalent of 2 × 104 cells), optimal concentrations of αGalCer (100 ng/ml, obtained from Dr H. Ovaa, the Netherlands Cancer Institute, Amsterdam, the Netherlands) or phorbol myristate acetate (PMA) (50 ng/ml) plus ionomycin (1 µg/ml) were HSP targets added and the plates were incubated overnight at 37°C. In experiments with NK T cell lines, optimal concentrations of responders were used at 5 × 102/well, targets at 2 × 103 cells/well and external antigen-presenting cells C1R-huCD1d or C1R

at 2 × 103 cells/well. After removal of the cell suspension, the plates were washed with PBS, developed according to the manufacturer’s instructions and read using the Bioreader 4000 pro-X ELISPOT reader (Bio-sys, Karben, Germany). Plasma samples were obtained at various time-points during IFN-α therapy,

preserved at −70°C and tested using ELISA according to the manufacturer’s instructions (human IL-7 Quantikine ELISA kit HS750, human IL-12 Quantikine ELISA kit D1200 and human IL-15 Quantikine ELISA kit D1500; R&D Systems, Abington, UK). PBMC subset analysis was performed as described previously [23]. Briefly, cells or cell lines were stained for MG-132 molecular weight 20 min at room temperature followed by washing steps in PBS containing 0·5% BSA with the following conjugated antibodies directed at: CD3-fluorescein isothiocyanate [fluoroscein isothiocyanate (FITC)/CD(16+56]-phycoerythrin (PE) (B&D Biosciences), CD8-FITC, CD56-PE cyanine5 (PC5),

CD19-PC5, CD69-PE Texas Red [electrochemical detection (ECD)], CD8-PC5, CD3-PE cyanine7 (PC7), CD45-FITC/CD14-PE, CD45RO-ECD and CD4-ECD (all from Beckman Coulter, Woerden, the Netherlands). For detection of NK T cells, staining with anti-TCR Vα24-FITC and Vβ11-PE in combination with anti-CD3-PC7 was used; in some experiments, NK T cells were measured using anti-TCR Vβ11-PE in combination with 6B11-FITC [24] (BD Biosciences Pharmingen, San Diego, CA, USA). Further NK T subset typing was performed using antibodies Bcl-w to CD4-ECD, CD8-PC5, CD56-PC5, CD69-ECD, CD45RO-ECD (all Beckman Coulter) and CD161-biotin (Ancell, Bayport, MN, USA). For enumeration of regulatory T cells (Treg), antibodies were used directed at: CD4-FITC, CD8-PE, CD45-ECD, CD25-PC5, CD3-PC7 (Beckman Coulter) and forkhead box P3 (FoxP3) (eBioscience kit; eBioscience, Inc. San Diego, CA, USA). In all experiments gates were set on viable [propidium iodide (PI)-negative] cells and fluorochrome-labelled isotype control antibodies were included in each assay to determine background staining. FACS analysis was performed with a Beckman Coulter flow cytometer FC500 and computer software Beckman Coulter program CXP.