In this study, we determined the fate and function of Lgr5-expressing cells Hippo pathway inhibitor during thymic development. We show that TECs transiently express Lgr5 during fetal development and specifically marks a subset of TECs at E10.5 and E11.5. However, presence of Lgr5 is not essential for proper thymic development. Finally, lineage tracing confirmed that fetal Lgr5+ TECs do not generate detectable progeny in vivo. The presence of Lgr5 transcripts has been reported at E13.5 of thymic development in mice with a TEC-specific overexpression of β-catenin

[31]. We first set out to determine the temporal regulation of these Lgr5 transcripts in the fetal thymus. Fetal thymi of different ages were evaluated for presence of Lgr5 transcripts. Low levels of Lgr5 message were detected in the fetal thymus at E13.5 and E14.5 by RT-PCR. With increasing gestational age, the levels of Lgr5 transcripts gradually decreased and were undetectable from E19.5 onwards (Fig. 1A). To determine whether the observed Lgr5 transcripts lead to Lgr5 protein expression and to identify the cells expressing Lgr5, individual fetal thymi from Lgr5-EGFP-IRES-CreERT2 reporter mice in which EGFP marks

cells expressing Lgr5, were collected and single cell suspensions were made. First, the hematopoietic (CD45+) fraction was analyzed for the presence of Lgr5+ cells; however, at early and later embryonic age no considerable amount could be detected (Fig. 1B). Next, the epithelial fraction (CD45−EpCAM+) was analyzed by flow cytometry for EGFP expression GSK126 chemical structure (Fig. 1C and D). In agreement with our transcript analysis, we found that the percentage of EGFP+ TECs was highest at E13.5 with a range from 2.17 to 7.37% (3.95 ± 1.51%). At later embryonic ages, Lgr5+ TECs

could still be detected; however, the number decreased with age; 0.02–0.64% (0.36 ± 0.19%) for E14.5, 0.05–0.242% (0.12 ± 0.10%) for E16.5 and 0.00–0.04% C-X-C chemokine receptor type 7 (CXCR-7) (0.05 ± 0.03%) at E19.5. In order to confirm that the Lgr5+ cells are indeed located within the thymus and to determine their in situ localization, fetal thymi of Lgr5-reporter embryos were analyzed by immuno-histochemistry. E10.5 complete embryos were sectioned and analyzed for the presence of Lgr5+ cells in the thymic anlage. The 3rd pharyngeal pouch at E10.5 clearly showed EGFP+ cells within the thymic primordium and these cells coexpressed the epithelial marker epithelial cell adhesion molecule (EpCAM) (Fig. 2A). At the right side of the pharyngeal region the number of EpCAM+EGFP+ cells appeared to be higher, consistent with earlier observations that there is asymmetry in developmental timing between the two sides of the embryo [32]. Next, sections of whole E11.5 embryos were analyzed. Also at E11.5, EpCAM+EGFP+ cells were clearly detectable within the thymic primordium and marked a subpopulation of fetal TECs.

Canine distemper is considered an interesting model of virus encephalitis, which can be associated with

a chronic progressing disease course and can cause symptomatic seizures. Methods: To determine the impact of canine distemper virus (CDV) infection on hippocampal neurogenesis, we compared post-mortem tissue from dogs with infection with and without seizures, from epileptic dogs with non-viral aetiology and from dogs without central nervous system diseases. Results: The majority of animals with infection and with epilepsy of non-viral aetiology exhibited neuronal progenitor PF-562271 manufacturer numbers below the age average in controls. Virus infection with and without seizures significantly decreased the mean number of neuronal progenitor cells by 43% and 76% as compared to age-matched controls. Ki-67 labelling demonstrated that hippocampal cell proliferation was neither affected by infection nor by epilepsy of non-viral aetiology. Analysis of CDV infection in cells expressing caspase-3, doublecortin or Ki-67 indicated that infection of neuronal progenitor cells is extremely

Fluorouracil rare and suggests that infection might damage non-differentiated progenitor cells, hamper neuronal differentiation and promote glial differentiation. A high inter-individual variance in the number of lectin-reactive microglial cells was evident Acesulfame Potassium in dogs with distemper infection. Statistical analyses did not reveal a correlation between the number of lectin-reactive microglia cells and neuronal progenitor cells. Conclusions: Our data demonstrate that virus encephalitis with and without seizures can exert detrimental effects on hippocampal neurogenesis, which might contribute to long-term consequences of the disease. The lack of a significant impact of distemper virus on Ki-67-labelled cells indicates that the infection affected neuronal differentiation and survival of newborn cells rather

than hippocampal cell proliferation. “
“Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases. To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay.

IFN-β-mediated immunomodulatory functions may differentially operate depending on the responding cell subset acting on T- or B-cell proliferation, Selleckchem Fostamatinib modulation of cytokine production, and regulation of adhesion molecules involved in lymphocyte migration across the blood-brain barrier [18]. For these reasons, investigating the action of IFN-β therapy on B cells might be of great relevance to understand

their pathogenic role in the development and regulation of autoimmune inflammatory response in MS. There is increasing recognition that TLRs and TLR-driven responses can play a key role in the pathogenesis of several autoimmune diseases, including MS. TLR7 and TLR9 are selectively expressed by B cells, and when activated by specific ligands, lead to their proliferation and differentiation into Ig-secreting cells. Given the key importance of B lymphocytes in MS disease, we investigated whether IFN-β therapy would modulate Ig synthesis in MS patients by performing a longitudinal study conducted with unseparated PBMCs isolated from 15 Talazoparib concentration MS patients before (T0) and

1 month after (T1) the beginning of IFN-β therapy. Moreover, PBMCs isolated from 10 healthy donors (HDs) were also included in this study as comparative control. To this end, PBMCs were cultured in vitro with either a specific TLR7 (the synthetic small molecule

3M001) or TLR9 (a type B CpG, 2006) agonist for 7 days and then IgM (Fig. 1A) and IgG production were Rebamipide evaluated by Elispot (Fig. 1B) and Elisa assay (Supporting Information Fig. 1). The TLR9-mediated B-cell stimulation led to a similar frequency of IgM- and IgG-secreting cells in both HD- and MS-affected individuals and this Ab release was not modified in response to IFN-β treatment. On the other hand, it was very interesting to find that the basal level of TLR7-induced Ig production was significantly lower in MS patients as compared with that in HD, showing a specific defect in TLR7 responses in B cells from MS sufferers. Surprisingly, 1 month of IFN-β therapy was able to partially restore this deficiency and selectively increase the production of IgM and IgG upon TLR7 triggering, re-establishing the level of Ab release found in HDs. The analysis of Ig content by Elisa confirmed the results obtained by Elispot assay (Supporting Information Fig. 1). IFN-β-mediated effect was long-lasting since it was still observed after 6 months of IFN-β treatment (data not shown). However, IFN-β did not enhance auto-Ab production as demonstrated by measurement of both homogeneous and speckled patterns of anti-ANA Abs on sera of MS patients before and after therapy (data not shown) [19].

At the completion of the

experiments, blood was harvested by cardiac puncture with a heparinized syringe and the animal was killed. Blood was assessed for lactate concentration, leukocyte count, and hematocrit Selleck JQ1 using standard assays in the clinical hematology laboratory of Hamilton Health Sciences Corporation, McMaster site. Purified human AGP was radiolabeled using 125I by the Iodogen method [12] and injected into C57BL/6 mice either intravenously or intraperitoneally, using a dose of 3.3 × 106 counts per minute in 0.1 mL of normal saline; the acid-precipitable radioactivity in plasma samples obtained by sampling from the tail vein was followed over time, and reported as a percentage of the total injected radiolabeled AGP dose as previously described [39, 2]. All values are reported as the mean ± the SEM. Data were analyzed using GraphPad

InStat version 3.01 statistical analysis software (GraphPad Software, Inc., San Diego, CA, USA). For multiple comparisons, data were analyzed using ANOVA with Tukey’s post-test, if the data sets met conditions of normal distribution and similarity of standard deviations, and non-parametric ANOVA (Kruskal–Wallis) with Dunn’s post-test if they did not. For comparisons of two groups, a non-paired, two-tailed Student’s t-test was used for parametric analysis if these conditions were met and the Mann–Whitney test was used if they were not. Statistical significance was set at p < 0.05 in all cases. In all experiments, whether involving endotoxemia or CLP, all animals were alive and active four hours post-LPS or CLP, when subjected to anesthesia in CT99021 solubility dmso preparation for intravital microscopy; in addition, none died under Celastrol anesthetic cover prior to the point in the protocol at which euthanasia was planned. As shown in Table 1, there were no significant differences among groups of mice in the endotoxemia experiments in either hematocrit or lactate levels, suggesting that not only did the mice have similar intravascular fluid status but that they were also well resuscitated. A similar

situation was found with respect among groups of mice in the CLP experiments (see Table 2). Administration of LPS significantly reduced circulating leukocyte counts, irrespective of whether saline, AGP, or HAS were employed as the resuscitation fluid (see Figure 1A). Leukocyte counts were reduced to 23 ± 8% of levels seen in sham-treated mice by LPS treatment with saline resuscitation, and to 18 ± 8% and 13 ± 4%, respectively, in LPS-treated mice resuscitated with AGP or HAS, respectively (mean ± SD). These reductions were highly statistically significant with reference to their respective sham values but did not differ significantly among the three resuscitation fluid groups. As shown in Figure 2A, the leukopenia associated with CLP was less marked than that associated with endotoxemia; reductions in leukocyte counts of 50–60% were observed, relative to sham-treated mice, for both saline- and AGP-treated mice.

In conclusion, this study demonstrates that AFP impair the DC ability of activation of NK cells. These findings might provide new insight into understanding the mechanisms underlying the suppression of innate immune responses

in chronic liver disease patients with high serum AFP levels. This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Research on Hepatitis and BSE from the Ministry of Health, Labour and Welfare of Japan. The authors have no conflicts of interest. “
“Antineutrophil cytoplasm autoantibodies (ANCA) directed against bactericidal/permeability-increasing Ibrutinib protein (BPI) are common in patients with cystic fibrosis (CF), and serum levels are correlated with lung colonization by Pseudomonas aeruginosa and the severity of lung damage. The production of BPI-ANCA may be due to the costimulation of BPI when mounting an immune response against P. aeruginosa. The effect of surgery aiming to eradicate bacteria and infected tissue on BPI-ANCA levels is sparsely described. A cohort of patients with CF were included: 53 patients having extensive

image-guided sinus surgery (EIGSS) with topical postoperative antibiotic treatment, 131 non-operated controls and 36 who had double lung transplantation (LTX). In all 219 patients, serum samples before and after surgery or at similar intervals were analysed for IgG and IgA BPI-ANCA. The EIGSS group showed a highly significant decrease selleck in both IgA and IgG BPI-ANCA levels compared with their own preoperative values and control

group values (P < 0.001–0.02). The LTX patients also showed a highly significant decrease in both IgA and IgG BPI-ANCA levels (P < 0.001). EIGSS and LTX decrease IgA and IgG BPI-ANCA levels in patients with CF, indicating that extensive removal of infected tissue influences the pathogenic ifoxetine process of autoantibody production. The results shown herein are in favour of applying EIGSS in selected patients with CF and for using BPI-ANCA as a surrogate marker for guiding further therapeutic interventions. The paranasal sinuses in patients with cystic fibrosis (CF) are often colonized with CF-lung pathogens, especially Pseudomonas aeruginosa [1, 2]. Bacteria from the sinuses can be aspirated to the lower airways and thereby initiate or maintain deleterious lung infections [3]. Antineutrophil cytoplasm autoantibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) are frequently seen in patients with CF [4], especially in those with severe lung damage [5, 6]. IgG BPI-ANCA is common and occur in approximately 70% of patients with CF, whereas IgA BPI-ANCA is found in about 35% [7]. There is a strong association between BPI-ANCA and lung infection by P. aeruginosa, and BPI-ANCA levels are significantly correlated with the severity of lung damage [5, 8].

Mainly, the tolerogenic functions of LCs in non-inflamed skin are based on their immature state, low migratory properties and low expression of co-stimulatory molecules, as well as release of proinflammatory soluble mediators [11]. Moreover, data from a murine model system using the receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL),

overexpressing keratinocytes showed that LCs down-regulate co-stimulatory molecule expression and induce regulatory T cells, Hydroxychloroquine ic50 thereby modulating the skin immune response and attenuating overactivation even in an inflamed state [12]. However, under some circumstances LCs might also lose their tolerogenic properties and induce immunogenic immune responses during inflammatory conditions. Several FcεRI-bearing subtypes

have been identified so far in human skin of AD patients. Concerning myeloid DCs, both CD207+/CD1a+, i.e. LCs click here as well as CD207–/CD1a+/FcεRI+ DCs, are located in the epidermis [13]. While low numbers of CD207+/CD1a+/FcεRI+DCs occur in the dermis, CD1c+/FcεRI+ DCs represent the major DC subpopulation of the dermal compartment [14]. DC subtypes expressing FcεRI in the skin and blood of AD patients are IgE receptor-bearing epidermal LCs, which predominate in non-lesional AD (Table 1). Further, a subtype of DC, which in contrast to LCs does not have any Birbeck granules but expresses the mannose receptor (CD206), Cediranib (AZD2171) the so-called inflammatory dendritic epidermal cells (IDEC), invades the skin in the acute phase and persists during the chronic phase of AD [15]. PDCs detectable in the epidermal skin of patients with psoriasis, lupus erythematodes or allergic contact dermatitis are almost absent in patients with AD [16]. We know from atopy patch test models that after allergen application to the skin, an eczematous skin reaction develops within 24–48 h in

sensitized patients. This mechanism is in addition to the induction and release of a plethora of chemokines in the upper part of the skin [17] and recruitment of inflammatory cell subtypes such as IDECs from their dermal and blood precursors [18]. The initial predominance of T helper type 2 (Th2) cytokines during the acute phase is attenuated and the amount of Th1 cytokines, in particular IFN-γ, increases [19]. Other exogenous trigger factors such as microbial antigens might lead to very similar recruitment mechanisms. During the flare-up phase of AD, epidermal LCs up-regulate their FcεRI and co-stimulatory and major histocompatibility complex (MHC) expression [18]. Furthermore, they release chemotactic factors, but prime naive T cells primarily into T cells of the Th2 type.

As expected, FACS analysis showed a clear titration in the percentage of 5C.C7 (Vβ3+,CD4+) T cells seeding the recipients

(Fig. 1A). In order to derive a reliable value for the number of T cells that populate the animal, we combined two such experiments (n = 6–7 mice) and calculated the recovery of 5C.C7 cells as a fraction of injected cell numbers (Supporting Information Fig. 1A). After eliminating the outliers, we calculated the mean seeding efficiency for each dilution (Supporting Information Fig. 1B). As shown in Figure 1B, the recovery is close BGB324 mouse to 20% of the input at all dilutions (linear regression coefficient of 0.9969) except the lowest. In the experiments that follow (Fig. 2B and D), we use this calculated efficiency to normalize T-cell expansion (as a function of the actual initial frequency). So, an injection dose of 103 corresponds to an actual precursor frequency of 129 ± 33 5C.C7 T cells in the recipient while that of 105 amounts to 21,866 ± 1320 cells. The presence of a large frequency of antigen-specific T cells at the beginning

of the response has been shown to blunt the clonal expansion and accelerate the subsequent clonal contraction after an acute antigenic immunization [9]. Similar to those studies, 5C.C7 T cells challenged acutely with PCC (Pigeon Cytochrome C) peptide (with LPS as an adjuvant) attained PF-562271 in vivo an expansion maximum that was inversely proportional to the initial precursor frequency (Fig. 2A). This is most evident in Figure 2B where expansion is represented as the fold increase from the initial seeding frequency on day 1. At the peak of their expansion (day 4), the 105 group increased in number by around 40-fold. However, lower frequencies resulted in a significantly greater burst — 175- to 456-fold for the 104 and 1367- to 3504-fold for the 103, albeit at a later time point (day 8). These data are Dichloromethane dehalogenase consistent with the idea that T cells can clonally compete for antigen [8, 9]. Each

T cell at lower frequencies can have more access to the antigen, resulting in stronger initial stimulation. The extended expansion could then be a programmed consequence of this initial signal [16]. Alternately, since acute antigen can linger in vivo for over 3 days, the extended proliferation by the lower frequency groups could also be a result of continuing to receive stronger stimulation at these later times [17]. Regardless, after this phase, the expanded cells begin classical clonal contraction. In this model, the contraction is not much influenced by the initial frequency and all groups decay similarly — even over longer time frames (Fig. 2E). In contrast, even the first phase of the response of 5C.C7 T cells to a chronic self-antigen (PCC expressed constitutively from an MHCI promoter) was less dependent on initial frequency (Fig. 2C, D, and F).

In DO11.10 T-cell hybridoma, ERK1/2-RSK pathway was shown to phosphorylate Nur77 at residue 354. An alanine substitution at this site impairs Nur77 nuclear export and apoptosis 26. To see if this might be true in DP cells, we used 16610D9 cells, a CD4+CD8+ thymoma cell line that possesses many characteristics of primary DP thymocytes 48. As shown in Fig. 6A, 16610D9 cells express very little endogenous Nur77 (lanes 1, 4, 7 and 10).

Infection of these cells with Nur77 retrovirus led to expression of Nur77 in the nuclear compartment (lanes 2 and 5). However, very little Nur77 was found in the mitochondria/cytoplasmic fractions unless PMA/ionomycin were added (see lane 11 versus lane 8). Interestingly, expression of Nur77(354A) mutant (mutation verified by sequencing) led to constitutive translocation of Nur77 Kinase Inhibitor Library cell assay to the mitochondria/cytoplasmic fraction (lane 9). These data show that phosphorylation of Nur77 at residue 354 might have a different effect in DP cells from DO11.10 cells and that regulation of Nur77 nuclear transport and its association with Bcl-2 is more complicated than initially thought. Nur77 has been reported as the target of numerous kinases including protein

kinase A, PKC, Akt, JNK, ERK5 and p90 ribosomal S6 kinase 23, 24, 49–51. Phosphorylation of Nur77 by these proteins was demonstrated in various in vitro and in vivo experiments. However, the functional consequence of Nur77 phosphorylation remains controversial. Here, we report that the PKC proteins regulate Nur77 phosphorylation and nuclear/cytoplasmic translocation Sorafenib solubility dmso in thymocytes during apoptosis that mimics negative selection. Chemical inhibition of PKC proteins prevented Nur77 and family member Nor-1 from targeting the mitochondria and their targeting of Bcl-2. In contrast, inhibition of AKT, JNK, ERK1/2 and p38 did not affect the subcellular localization of Nur77 family proteins in thymocytes. These results are different from mitochondria

translocation of Nur77 induced by the retinoid analog CD437, which requires activation of the JNK and inhibition of the AKT pathways 23. Inhibition of ERK1/2 was also Tryptophan synthase recently reported to block Nur77 mitochondria translocation in DO11.10 T-cell hybridoma cells 26. The discrepancy with our results is most likely due to the differences in the cells used. Consistent with this, we found that alanine mutation at Nur77 residue 354, which impairs mitochondrial translocation in DO11.10 cells, causes constitutive translocation of Nur77 in 16610D9 CD4+CD8+ cells. Thus, the involvement of kinase pathway(s) in Nur77 mitochondria translocation is cell type and stimulus specific. Though calcium signals alone were adequate in causing Nur77 to be localized to the mitochondria, these levels may be inadequate for binding Bcl-2, as no Bcl-2/Nur77 interaction could be detected in ionomycin treated thymocytes. In addition, ionomycin could not induce Nor-1 to any appreciable levels.

To counter this, codes such as the HONcode (Health on the Net code) have been developed, and can be used

to assess the reliability and validity of information on the Internet. Clinicians and health workers are often asked by patients and their carers for direction to reliable websites containing information on nephrology-related issues. Equally, many nephrologists have been confronted by patients who have found unreliable, erroneous or misleading health information on the Internet. Table 3 RAD001 contains a list of reliable Internet sites that may be of interest to the Nephrologist and to patients and their carers (but this is by no means exhaustive), as well as a link to the HONcode. While general news is easy to access through traditional broadcasting and print services, general health and discipline specific news is a bit harder to come by and even harder to keep pace with. There are a number of services that you can use to keep up to date, ranging from Google News through to specialist services: Medical News Today (http://www.medicalnewstoday.com/sections/urology-nephrology/) MK-1775 purchase offers subject specific news, albeit with a US/UK focus. Google

News (http://news.google.com.au/news?pz=1&ned=au) can be searched using a search string such as kidney or renal site: au to retrieve news from Australian sources. Sciencedaily (http://www.sciencedaily.com/news/health_medicine/kidney_disease/) provides general nephrology news, as well as articles, video, images, as well as book reviews. Click on the RSS icon (see boxed text and Fig. 1) on the page of each of these sites to subscribe to the feed. Web 2 and its associated technologies offer many

opportunities for the Nephrologist to keep up to date with the latest news and research within the discipline. By exploring and exploiting the Oxalosuccinic acid various nephrology resources, after a small investment of time to set up automated systems, a clinician can easily establish a personalized system whereby they are regularly updated with news about their profession, as well as developments in their area of practice. “
“Aim:  It has been well described that large residual urine volumes (≥300 mL) affect renal function in advanced benign prostatic hyperplasia (BPH). However, it is not clear whether small residual urine volumes (<100 mL) are related to renal function. The present study was performed to examine the association between chronic kidney disease (CKD) and the post-void residual urine volume (PVR) in BPH patients. Methods:  A cross-sectional study was performed in 160 consecutive BPH patients with PVR of less than 100 mL. We first determined the stage of CKD and compared the PVR in subjects with/without CKD.

Furthermore, practical and predictive humanized animal models would be beneficial to evaluate the induction of human immune responses, at both cellular and humoral levels by candidate dengue vaccines in development.12 Our group and several others have shown that humanized mice provide a tractable animal model that permits in vivo infection of human cells with

DENV and elicits human DENV-specific immune responses.13–16 Using cord blood haematopoietic stem cell (HSC)-engrafted NOD-scid IL2rγnull (NSG) mice we previously showed that the engrafted mice support DENV infection. Human T cells from infected NSG mice expressing the HLA-A2 Selleck INCB018424 transgene produced interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) upon stimulation with DENV peptides. These mice also developed moderate levels of IgM antibodies directed against the DENV envelope protein.14 We speculated that suboptimal positive selection of HLA-restricted human T cells on murine thymus in NSG mice may have led to reduced human T-cell and B-cell responses. Humanized fetal liver/thymus (BLT-NSG) mice were developed to provide a microenvironment for human T-cell development.17 In these mice, human

fetal liver and thymus tissue are implanted under the kidney capsule to produce a thymic organoid that allows the education of human T cells on autologous thymus. Then, HSC from the same liver and selleck thymus donor are injected intravenously into the transplanted mice. Engrafted BLT-NSG mice develop robust populations of functional human T lymphocytes within mouse lymphoid tissues. Following infection of BLT-NSG mice with Epstein–Barr virus and HIV, antigen-specific cellular and humoral

immune responses have been detected.17–20 In this manuscript we tested the hypothesis that the education and maturation of human T cells on autologous human thymic tissue in the BLT model and subsequent infection of BLT-NSG mice with DENV would lead to heightened Rucaparib clinical trial DENV-specific cellular and humoral immune responses. The NOD.Cg-PrkdcscidIl2rgtm1Wjll/SzJ mice (NSG) were bred at The Jackson Laboratory and subsequently maintained in the animal facilities at the University of Massachusetts Medical School. All experiments were performed in accordance with guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School and the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996). NSG mice at 6–8 weeks of age were irradiated (200 cGy) and received surgical implants under the kidney capsule of 1-mm3 fragments of HLA-A2-positive or negative human fetal thymus and liver on the same day as the tissues were received. Tissues were purchased from Advanced Bioscience Resources (Alameda, CA).