In selected experiments, rapamycin 1 or 10 ng/mL or CsA 0.1 or 1.0 mcg/mL was added into cultures containing 100 IU/mL human recombinant IL-2. Multiscreen-IP 96-well microtiter plates (Millipore, Bedford, MA) were coated with a mouse anti-human CD3 mAbs (2 μg/mL) check details and mouse anti-human IFN-γ capture mAbs (4 μg/mL). Freshly isolated T cells (1×105 cells/well in 200 μL) were cultured for 36 h, isolated,

washed and incubated with a biotinylated mouse anti-human IFN-γ mAbs (2 μg/mL). After washing, HRP-labeled streptavidin (DAKO, Carpinteria, CA) was added for 1 h and subsequently the spots were developed with AEC substrate (Sigma-Aldrich, St. Louis, MO) and analyzed in an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH). Cytokine secretion is expressed

as spots/well. CD4+ T cells were stained with up to four directly conjugated fluorescent antibodies or control antibodies for 30 min at 4°C. After extensive washing the cells were fixed and permeabilized using the Fixation & Permeabilization kit (eBioscience), and intracellular staining of FOXP3 and CTLA-4 was performed according to the manufacturer’s recommendations. Data were acquired on a FACsCalibur (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR). For cell sorting experiments, CD4+ cells stained for desired cell surface markers were isolated using a FACSAria or FACSVantage (BD Rucaparib clinical trial Biosciences) apparatus. PCR was performed using the TaqMan Gene Expression Assay Kit (TaqMan, MGC probes, Applied Biosystems,

Foster City, CA) and the 7300 real-time PCR system. Gene-specific primers for the analysis of human Tbet and GAPDH by real-time PCR were obtained from Applied Biosystems. Migration of lymphocyte subpopulations in response to IP-10 (CXCL10) was quantified at single-cell resolution using microfluidic devices and time-lapse microscopy, as described previously 46. Briefly, photoresist (SU8, Microchem, Newton, MA), medroxyprogesterone was patterned within silicon wafers, which were used as a mold to produce a PDMS (Fisher Scientific, Fair Lawn, NJ) device, which was then bonded onto standard 1×3 in. glass slides (Fisher Scientific). The microfluidic network inside each device consisted of an array of up to 450 parallel channels (6×6 μm cross-section and 800 μm long) connected to one main channel, (50 μm tall, 400 μm wide and 10 mm long) with inlets and outlets. The devices were first primed with a solution of IP-10 (100 nM) and fibronectin (250 nM) for 15 min. After priming, sorted populations of either CXCR3+ or CXCR3− CD4+CD25+CD127dim/− Tregs (∼1×105/condition) suspended in 15 μL of media were introduced into the main channel through tubing connected to the main inlet. The cells were flushed through the main channel until media was seen to emerge from the main outlet.

40,41,43 The indirect pathway is supported by observations that in many cases there is no evidence of a specific microbial antigen, and the iNKT cell response involves IFN-γ but not IL-4 production and appears to be completely dependent on costimulation by cytokines such as IL-12p70.41,45 However, because it is difficult to rule out the possibility that microbes for which no iNKT cell antigen has been identified nevertheless do contain cryptic antigens, while microbes

that do contain such antigens will also concurrently provide TLR-mediated stimulation that activates DC cytokine production, it is not clear that these two pathways are actually separate during most physiological infections. Sorafenib cell line For example, it has recently been shown that CD1d-mediated presentation of a lipo-peptido-phosphatidylinositol from Entamoeba histolytica is necessary for secretion of IFN-γ by iNKT cells, but that the response requires simultaneous TLR-induced IL-12 secretion.72 Similarly, in a mouse model of tuberculosis it has recently been shown that iNKT cells have a protective effect through recognition of infected macrophages, and that macrophage production of IL-12 and IL-18 is critical for this effect.73 It is not clear whether recognition of mycobacterial

antigens is required for the iNKT cell-mediated protection; however, a previous study has identified mycobacterial lipids that may serve as iNKT antigens.74 Thus, it seems likely that the two

PLX-4720 solubility dmso pathways of iNKT cell activation are not mutually exclusive, and that they occur simultaneously in many systems. Notably, it is not yet clear whether either the direct or indirect pathways of iNKT cell activation during microbial infection result in the maturation of pro-inflammatory DCs, such RVX-208 as those that are observed after administration of α-GalCer. Induction of a pro-inflammatory DC phenotype was shown in one system to depend on the up-regulation of CD40L expression by iNKT cells as well as their secretion of cytokines such as IFN-γ, both of which are induced by a strong TCR stimulus.65 While self-antigen recognition in the presence of IL-12 and IL-18 is sufficient to induce iNKT cell IFN-γ secretion, the extent to which this form of stimulation also induces cell surface CD40L up-regulation remains unclear. Nevertheless, it is possible that, when combined with a TLR stimulus and IFN-γ, even weak CD40L stimulation from iNKT cells is sufficient to induce the maturation of pro-inflammatory DCs (Fig. 1b). Although mature DCs have the capability to potently activate naïve T cells, it is well established that immature DCs have tolerizing effects.75 Thus, by inducing maturation of immature DCs, iNKT cells may tend to promote pro-inflammatory responses simply by shifting the balance away from the more tolerizing stage of DC differentiation.

[18, 33] It is noteworthy that changes in the severity of colitis caused by IL-33 injection or

ST2 deficiency were not significantly associated with a change in body weight in the mice (Fig. S2A,B). This is consistent with a previous study showing identical Pritelivir order body weight loss in WT C57BL/6 and IL-33−/− mice when fed with DSS.[24] Intriguingly, compared with WT mice, the IL-33−/− mice had a delayed recovery in body weight after withdrawal of DSS.[24] However, this was not the case in ST2−/− mice in the present study and the reason is currently unclear. It may be because of the differences in genetic background of the mice and experimental conditions or the ST2-independent bioactivity of full-length IL-33 as previously suggested.[34] Furthermore, recent evidence suggests that injection of IL-33 may have a beneficial effect on chronic DSS-induced colitis or trinitrobenzene sulphonic acid-induced colitis, a model of Crohn’s disease in mice,[35, 36] suggesting that IL-33 may play a complex role in different types and throughout the duration of colitis. More studies are needed to clarify this issue. Interleukin-33 is clearly expressed in the inflamed mucosa of patients with inflammatory bowel disease, particularly in UC, and is reduced after anti-TNF-α therapy.[20-23] In these cases mucosal expression of IL-33 is also mostly localized to intestinal epithelial see more cells[20, 21, 23]

and in activated sub-epithelial myofibroblasts.[22] However, the clinical relevance of the IL-33/ST2 system in inflammatory bowel disease is unknown. Our results have extended these clinical findings with a putative mechanism and suggest that colon-derived IL-33 may represent an important factor for the development and exacerbation of UC. This study received financial support from the Arthritis Research UK, Medical Research Council UK and the Wellcome Trust, UK. The authors have no financial conflicts Orotidine 5′-phosphate decarboxylase of interest. “
“Trypanosoma congolense strains have been shown to differ in their

virulence both between subgroups and within the Savannah subgroup between strains. This review revisits these findings and complements them with information on the virulence of T. congolense Savannah subgroup strains isolated from cattle (domestic transmission cycle) in different geographical areas and of strains isolated in protected areas where trypanotolerant wildlife species are the reservoir of the trypanosomes (sylvatic transmission cycle). The virulence of a total of 62 T. congolense Savannah subgroup strains (50 domestic and 12 sylvatic), determined using a standard protocol in mice, was compared. Virulence varied substantially between strains with, depending on the strain, the median survival time of infected mice varying from five to more than sixty days. The proportion of highly virulent strains (median survival time <10 days) was significantly (P = 0·005) higher in strains from the sylvatic transmission cycle.

On the other hand, unpublished data from our laboratory indicate that αDCs (derived from healthy controls) matured in CellGro medium produce approximately a 10-fold lower level of CXCL9 and CCL3 than in AIM-V, reflecting the chemokine levels found in these two studies. So far, only two clinical trials exploring the role of matured

DCs loaded with tumour cell lysate in patients with CLL have been published [6, 31]. In both studies, in which TNF-α solely was used for final DC maturation, the authors could show that a tumour-associated antigen-specific CTL induction was possible to achieve YAP-TEAD Inhibitor 1 supplier but the clinical effect was relatively modest. Moreover, there is clinical data indicating that also PGE2-matured DCs might be insufficient for cancer treatment: a phase III trial in patients with malignant melanoma failed to show the advantage of PGE2DCs over standard dacarbazine chemotherapy [32]. Instead, it has been shown in vitro that even though αDC1s and PGE2DCs induced similar CD8+ T cell expansion, only

αDC1 could induce cytolytic functional CTLs with tumour-relevant homing capacity [33]. In addition, a most recent phase I/II study could show that αDC1s, loaded with glioma-associated antigens, induced both immunological and clinical responses in patients with different brain PD332991 tumours [34]. Thus, it is tempting to speculate that inadequate maturation conditions of DC vaccines could be one important reason for previous failure of DC-based antitumour vaccination in patients with CLL. Another major challenge in the development of a successful tumour vaccination method is to avoid the recruitment of suppressive Tregs to sites of antigen-specific

DC–T cell interactions within vaccine-draining lymph nodes that could hinder such optimal activation. Notably, we found that PGE2DCs, in contrast to αDC1s, preferentially produced the Th2- and Treg-attracting chemokines CCL17/TARC and CCL22/MDC, data that corroborate Mirabegron with previous in vitro studies on healthy donors [16, 17]. Further, in a clinical study on patients with myeloma, injected PGE2-matured DCs expanded even more FOXP3+ Tregs than immature DCs and they concluded that vaccine-mediated induction of Tregs may be an underappreciated effect in clinical trials of human DC vaccination [35]. Together, our in vitro data and observations by others underline the importance of optimal DC maturation conditions and illustrate the value of also taking the chemokine profile into account when designing and evaluating potential cancer vaccines. Even though this was not the primary focus of our study, an important issue in optimizing DC vaccines is the choice of antigen source for DC loading. DCs and/or macrophages that have endocytosed cells in early apoptosis are known to reduce their ability to secrete proinflammatory mediators, including IL-12p70 [36], CXCR3-ligands [37, 38] and CCL3/MIP-1α [39].

Other confounders in the analysis included type of initial CNI (cyclosporine or tacrolimus) and antimetabolite agent (mycophenolate

mofetil or azathioprine or none), as well click here as transplanting centres and transplant period. Transplant period was divided into four cohorts for analysis (i.e. 1995–1997, 1998–2000, 2001–2003, 2004–2005). Transplanting centres were categorized into the five transplanting states in Australia including Western Australia, New South Wales, Victoria, Queensland and South Australia. The report of comorbid medical conditions was collected at the commencement of renal replacement therapy. The clinical outcomes of this study were acute rejection occurring in the first 6 months post-transplant, overall graft survival (including death-censored graft failure (DCGF) and death with functioning graft (DFG)), patient survival and estimated GFR (eGFR) calculated by Modification of Diet in Renal Disease formula14 at 1 and 5 years post-transplant. Data on acute rejection were collected only from Dabrafenib concentration 1997. For the purpose of this study, outcome data of all patients were censored at December

2006. Results were expressed as frequency (percentage) for categorical data or as mean and standard deviation for continuous data. Comparisons of baseline characteristics between the use of IL-2Ra were made by chi-square test or Fisher’s exact test, as

appropriate. Acute ADAM7 rejection was modelled using log-binomial regression to estimate relative risk (RR). Linear regression was used to examine eGFR at 1 and 5 years by estimating differences in mean. Graft and patient survival were examined using standard survival methodology using Kaplan–Meier methods, including Cox regression for adjusted analyses. Log–rank tests were used to test equality of survival curves. As DFG and DCGF are competing risks, differences in the cumulative incidences of DFG and DCGF were tested using the Pepe and Mori test. All point estimates are presented with 95% confidence interval (95% CI). The covariates included in the adjusted models include donors’ characteristics (age, source and gender), recipients’ characteristics (gender, BMI, age, diabetes mellitus, vascular disease, smoking, time on dialysis), transplant centres and period. Statistical analysis was performed using Stata/IC 10 statistical software program (Stata Corporation, College Station, TX, USA). Two-tailed P-values of less than 0.05 were considered statistically significant. Of the low-risk recipients, 218 of 1220 (18%) received IL-2Ra induction therapy whereas 883 of 3204 (28%) intermediate-risk recipients received IL-2Ra.

In our unpublished meta-analysis, we searched PubMed using the key words ‘birthweight’, ‘intrauterine growth retardation’, ‘intrauterine growth restriction’, ‘creatinine clearance’, ‘glomerular filtration rate’ and ‘renal function’; five studies which observed 2733 subjects aged 18 years or older were included, and we found that

GFR of LBW people was approximately 3 mL/min per 1.73 m2 lower than that of normal counterparts (Fig. 1). One study compared the birthweight between 1230 end-stage renal disease (ESRD) patients and 2460 healthy controls and revealed that birthweight less than 2.5 kg or higher than 4.0 kg was associated with the highest ESRD risk selleck chemical and birthweight between 3.5–4.0 kg was associated with the lowest ESRD risk.34

Whereas another matched case–control study did not reveal the association between birthweight and ESRD in a population of 1162 subjects.35 A longitudinal study with a duration of 38 years observed over 2 million people, and results showed that Protein Tyrosine Kinase inhibitor LBW people had 1.5 times higher risk of ESRD. However, in this study, ESRD mainly occurred before the age of 14 years old, which was possibly due to the higher incidence of congenital or inherited renal disease in the LBW population.36 In a study on the familial aggregation of ESRD, LBW was not an influence factor but high birthweight was considered as a protective factor.37 Three meta-analyses showed that birthweight was negatively associated with blood pressure in different age stages, with every 1 kg increase of birthweight resulting in a 1.2–2 mmHg decrease of blood pressure,38–40 possibly old resulting from kidney hyperfiltration caused by glomerulosclerosis and damage of renal sodium excretion capacity. The risk of diabetes and dyslipidaemia was also higher in LBW people.41,42 This could be explained by their susceptibility to obesity and insulin resistance and their special growth process, namely,

malnutrition in uterine, relative over-nutrition after birth and excessive fast growth in the early stage of life.43 LBW also influenced the structure and function of the cardiovascular system,44 such as the damage of vessel dilation function and the turbulence of endo-epithelial function. It is a reasonable speculation that this kind of abnormality could also exist in the capillary of nephrons and the function of glomerular endothelium. LBW also influences sympathetic nerve45 and renin–angiotensin system activity.46 Some researchers owed the higher risk of CKD in certain races such as black people47 and goajiro Indians48 to their higher LBW mortality. However, one study revealed that low nephron number and LBW may play a role in the development of hypertension in white subjects but not in black.49 Another study showed that the more severe hypertension found in black subjects could not be attributed to racial differences in number of glomeruli or birthweight.

However, TDP-43 has since been detected in conditions such as Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB) but is often confined to the limbic region rather than the more widespread pattern seen in FTLD-TDP. Previous work has suggested some relationship between hippocampal sclerosis and TDP-43 expression. A number of AD cases of both moderate and high stage were examined Ivacaftor nmr to determine whether the pattern of TDP-43

immunohistochemical expression differed and whether any relationship to hippocampal sclerosis could be detected. Cases of hippocampal sclerosis from surgical epilepsy specimens were examined to determine whether hippocampal sclerosis alone could cause abnormal TDP-43 expression. To establish whether abnormal TDP-43 expression in other neurodegenerative diseases resembled the pattern and distribution in FTLD-TDP we examined multiple blocks from a variety of neurodegenerative conditions. In 75% of cases of high-stage AD there was abnormal TDP-43 positivity compared to 57% of moderate-stage AD. While the abnormal TDP-43 positivity was confined to the limbic regions in the moderate stages, occasional cases in the high stages showed neocortical positivity. Also amygdala and/or entorhinal positivity appeared to precede positivity in the dentate gyrus. No relationship could be established between abnormal TDP-43

expression and degree of hippocampal sclerosis either in the surgical or autopsy cases. The pattern of distribution of TDP-43 inclusions from cases of dementia pugilistica most closely resembled that in FTLD-TDP. This raises the question as to whether there may be some shared pathogenic GDC-0941 molecular weight mechanisms between the two conditions. “
“F. Junyent, L. de Lemos, E. Verdaguer, M. Pallàs, J. Folch, C. Beas-Zárate, A. Camins and C. Auladell (2012) Neuropathology and Applied Neurobiology38, 311–321 Lack of Jun-N-terminal kinase 3 (JNK3) does not protect

against neurodegeneration induced by 3-nitropropionic acid Aims: 3-Nitropropionic acid (3-NP) is a toxin that replicates most of the clinical and pathophysiological symptoms of Huntington’s disease, Wnt inhibitor inducing neurodegeneration in the striatum due to the inhibition of mitochondrial succinate dehydrogenase. Different pathways have been implicated in the cell death induced by 3-NP in rodents. One of them is the Jun-N-terminal kinase (JNK) pathway, which may play a role in the neurodegenerative process in different diseases. Moreover, the lack of one isoform of JNK (JNK3) has been associated with neuroprotection in different experimental models of neurodegeneration. Therefore, in the present study the role of JNK3 in the experimental Huntington’s model induced by 3-NP administration was evaluated. Methods: 3-NP was intraperitoneally administered once a day for 3 days to wild-type and Jnk3-null mice. Coronal brain sections were used to determine cell death and astrogliosis in striatum.

In addition, this NFR-like pattern is

never associated with EMA and, in the controls treated with 12 months of gluten withdrawal, it did not disappear, showing the absence of a gluten dependency. On the other hand, as only two of 20 CD patients evaluated in this study show serum ANA-positive results, it is possible to conclude that NFR antibodies are different buy X-396 from the classics ANA. Incidentally, the ANA prevalence observed in our CD patients does not exceed the frequency reported currently for different classes of healthy individuals [35]. In conclusion, this is an early translational study describing a new autoantibody named NFR related to CD. In fact, the presence of NFR antibodies in CD patients’ serum is gluten-dependent and, accordingly, they could be considered to be CD-specific. The identity of NFR-related 65- and 49-kDa autoantigens is yet unknown, and therefore further investigations should be addressed to either obtain new knowledge on the humoral response of CD or to facilitate the development of a novel and promising serological test. In this regard, if our data are confirmed by large clinical trials, serum NFR antibody detection might to become a useful tool to monitor treated CD patients. The present study was supported by research funds assigned to Antonio Picarelli MD from the Sapienza University, Rome, PD-1 antibody inhibitor Italy. Authors declare that there

are no financial or other relationships that might lead to conflicts of interest. “
“This Viewpoint series provides authoritative and detailed outlines of exciting areas of DC research. Some of the subjects that frequently come up include development of DC; distribution of DC in lymphoid and non-lymphoid tissues such as skin, intestine and lung; different

forms or subsets of DC; and the role of DC in initiating tolerance and immunity. In this Preface, I will introduce the Viewpoints and consider some future challenges as well as the medical relevance of DC research. The development of DC, at least in mice, can be described with increasing precision because of discoveries summarized in the Viewpoint by Liu and Cediranib (AZD2171) Nussenzweig 1: (i) in the steady state, DC arise from a bone marrow progenitor that is shared with monocytes and macrophages 2; (ii) this progenitor gives rise to two cell types in the steady-state bone marrow: monocytes and a common DC progenitor 3–5; (iii) the latter gives rise to committed preDC that express some MHC II and CD11c, leave the marrow and circulate briefly in the blood before populating lymphoid and non-lymphoid organs 6, 7; (iv) Flt3 ligand (Flt3L) drives DC development 8, so that Flt3 knockout mice have a DC deficit 9, while administration of Flt3L expands DC numbers at least ten-fold in mice 10 and in humans 11. The discovery of distinct steps in DC development should make it possible to identify the relevant transcription factors and, in turn, new markers to improve the definition and understanding of the DC lineage.

Intracranial localization is very rare and only a few cases have been reported. This report intends to present the clinical, radiological and pathological pictures of a primary central nervous system angiosarcoma along with a review of the literature. A 35-year-old woman presented at our institution with weakness and sensory disturbances of her

right hand. Neuroimaging revealed a roughly round, hemorrhagic and moderately enhancing lesion in the left frontal posterior region. The tumor was totally removed under awake anesthesia and continuous monitoring of motor and language functions. Histopathology revealed CX-5461 molecular weight an epithelioid angiosarcoma. Radical removal, followed by adjuvant radiotherapy and chemotherapy, is able to completely control the disease for a relatively long period. “
“We studied one frontal lobe tumor and multiple spinal cord tumors (one in an extramedullary location) that had been resected from a 24-year-old man. The frontal lobe tumor was well demarcated and non-infiltrating, and consisted of eosinophilic, elongated fibrillary cells arranged in a fascicular pattern. A similar histology was reproduced

in the spinal cord tumors, with additional areas showing standard features of ependymoma. Immunohistochemical and ultrastructural observations revealed that all the tumors were ependymal in nature with positivity for GFAP and epithelial membrane antigen and negativity for oligodendrocyte transcription factor 2, showing intra- and intercellular microrosettes, leading us to a diagnosis of tanycytic ependymoma for the frontal lobe tumor and tanycytic ependymoma RAD001 solubility dmso with ordinary ependymomatous component for the spinal cord tumors. The spinal extramedullary tumor was a schwannoma. Importantly, SPTLC1 a heterozygous truncating mutation in the NF2 gene was identified in the blood lymphocytes from the patient. It is known that multiple nervous system tumors can occur in neurofibromatosis type 2 (NF2), which is caused by mutation in the NF2 gene, and that

occurrence of ependymoma, including the tanycytic variant, can be associated with this genetic condition. The present case provides further information about the clinicopathology of tanycytic ependymoma with details of the immunohistochemical, ultrastructural and genetic features. “
“Chordoid glioma is a rare, slowly growing tumor of the CNS, which is always located in the third ventricle of adults. Chordoid glioma has classic histological features consisting of clusters and cords of epithelioid tumor cells embedded within a mucinous stroma with rich lymphoplasmacytic infiltrate. The important distinctive immunohistochemical feature of this neoplasm is strong and diffuse reactivity for GFAP. Here, we report four cases of chordoid glioma that occupied the anterior portion of the third ventricle or suprasellar region. These four cases were all adult females with almost typical clinical, radiological, histologic and immunohistochemical characteristics of chordoid glioma.

Consequently, we are today limited to the default postulate that the regulation of class is determined solely by germline-selected processes [6, 8]. This can be rationalized in evolutionary terms as the origin of the effector mechanism that rids a pathogen is an outcome of the same interactive germline-selection pressures operating between pathogen and host that gave rise to the innate system. Using the Matzinger and Kamala [30] suggestion as a base, an effector class is defined here as the collection of compatible EPZ-6438 datasheet elements (cell

types, interleukins, chemokines, immunoglobulins, etc.) that synergize or cooperate to rid a given category of pathogen. This will be referred to as an ‘effector ecosystem’. The elements of an ecosystem act in concert and will eventually have to be detailed. In the end, the detritus produced by the biodestrucive effector activities is ridded by macrophage phagocytosis, requiring that all effector ecosystems feed into that mechanism. Therefore, each ecosystem must include a humoral component that arms phagocytosis. The cell-mediated system might stop

selleck the development of a pathogen, but cannot rid it. As a dead reckoning estimate to simplify the discussion, there are four effector ecosystems, an initially expressed or naive effector system and three systems to which the naive effector system can switch or differentiate in response to Eliminon-driven additional signalling. Adopting a simplified nomenclature based on that used for the humoral system, these four ecosystems would be M, G, A and E. Admittedly, this nomenclature might become misleading. One should

be cautious as there may not be a totally faithful concordance between the Ig-subtype and membership in a given ecosystem. The four effector ecosystems are, at least in part, incompatible with each other because they express activities that are mutually inhibitory. For example, IgA that does not activate C’-lysis can inhibit the activation of C’-lysis by IgM or IgG2 and eTh1 can inhibit the induction of eTh2 and vice versa. Therefore, keeping the ecosystems functionally separated when responding to multiple Eliminons interacting with or derived from a given tissue is a problem that must eventually be faced [6]. The antigen-responsive cells, iT/B, are born as part of the crotamiton M-ecosystem. It consists of virgin iTh0, iTc0, Bμ/δ and the eTh0 that are required to prime the response. Included, of course, in this ecosystem are the APCs, macrophages and several other cell types, as well as the interleukins and other factors required for induction to effectors and their functioning. As a minimum, no trauma signals need be postulated for the induction of the M-ecosystem to effectors. The M-ecosystem is the virgin or initial state. The virgin M-ecosystem has the potential to either respond as such or to differentiate to any one of the three other ecosystems, G, A or E.