Lower-dose intradermal treatment has been better tolerated and as

Lower-dose intradermal treatment has been better tolerated and associated with improvement in airway hyper-responsiveness, late-phase skin test

response to whole allergen, reduction in NVP-BKM120 manufacturer nasal symptoms together with up-regulation of CD4+ T cells producing IFN-γ cells but not regulatory T cells following cat peptide immunotherapy [126–130]. It is also possible to induce in-vivo production of allergen by vaccinating with DNA encoding the allergen. While this often produces a Th1-biased response, it is highly dependent on the DNA construct and mode of delivery. Clinical studies of these agents have not progressed [131]. Recombinant allergens offer the hope of better standardization, but their biological efficacy has been uncertain. Recombinant BetV1 protein has also been proven to be as effective as native BetV1 or conventional birch pollen extract in birch pollen SCIT [132,133], and in a recent clinical trial recombinant grass pollen vaccine has also been shown to be clinically safe and effective selleck kinase inhibitor [134]. Use of recombinant allergens may not only be safer, but may also allow patient-specific vaccines to be produced based on the individual’s

in vitro IgE reactivity pattern. While current native allergen vaccines modulate the patient’s existing allergen-specific IgE, they can also induce new sensitizations to other epitopes of the allergen, previously not present in the patient’s serum. The clinical consequences of this, if any, are not known, so any clinical advantage of vaccines based on component-resolved diagnostics remains to be demonstrated. Enhancement of the allergen with adjuvants itself is not new. Enzyme-potentiated immunotherapy represented an early attempt to increase the potency of the allergen not by adding a β-glucuronidase, protamine sulphate and cyclohexanediol. It was not widely adopted, and was shown subsequently to be ineffective [135]. Another adjuvant, monophosphoryl lipid A (MPL) has been investigated

in allergy vaccines. MPL is a purified lipopolysaccharide extracted from the cell walls of Salmonella minnesota[136–138] and induces a Th1 response via Toll-like receptor-4. A large recent multi-centre study with pollen allergoids adsorbed on L-tyrosine formulated with MPL has shown good efficacy and tolerability. Other adjuvants that have been investigated for their strong Th1-evoking ability include immunostimulatory DNA sequences [139] (ISS) and heat-killed Mycobacterium vaccae[140]. The latter need further investigation in clinical trials. Many alternative modes of allergen delivery for specific immunotherapy (SIT) aim to induce a T cell response but avoid IgE-binding. Because allergen is presented to T cells in the context of MHC class II, steering allergen towards this pathway is an attractive possibility.

6e) To determine if xeno-GVHD resulted

from a loss of pe

6e). To determine if xeno-GVHD resulted

from a loss of peripheral tolerance, we evaluated the levels of human Treg detectable in the blood of standard NSG–BLT mice (with irradiation) over time (Fig. 6f). The percentage of CD25+/CD127dim/FoxP3+ cells in the blood of NSG–BLT mice did not decrease over time. To determine the contribution of irradiation in the development of xeno-GVHD in BLT mice, we compared the survival of NSG–BLT mice that were either irradiated or non-irradiated (Fig. 6g). Overall, there was an increased survival of non-irradiated NSG–BLT mice; however, these animals TSA HDAC cell line ultimately developed GVHD-like symptoms. The BLT mouse, also referred to as the Thy/Liv mouse, is an ideal model to study human immune and T cell functions, as the implant of human thymic tissues and autologous human HSC enable the efficient development of HLA-restricted human CD4 and CD8 T cells [63]. Following implantation into the subcapsular ABT263 renal space, the human fetal thymus grows significantly, is populated with a normal distribution of human thymocyte subsets and allows high levels of human T cells to repopulate the peripheral lymphoid tissues [21-23]. The BLT model is based on the severe compromised immunodeficient-humanized

(SCID-hu) mouse described by McCune and colleagues [6]. The original SCID-hu model was created using CB17-scid mice and involved the transplant of human fetal thymic tissues in the renal subcapsular space and i.v. injection of autologous or allogeneic HSC derived from the fetal liver. The SCID-hu mouse enabled the development of human T cells, which required both the implant of thymic tissues and injection of HSC. However, in CB17-scid mice the Phloretin persistence of human T cells in the peripheral

tissues was transient, as CD3+ cells were not detectable in the peripheral blood at 12 weeks post-implant and the ability of these cells to mediate an immune response was limited [64]. The persistence and functionality of human T cells was improved significantly by the use of NOD-scid mice as recipients of human thymic and liver tissues [22, 23]. However, engraftment of fetal thymic and liver tissues into NSG mice enhances human cell chimerism significantly, including reconstitution of a mucosal immune system, compared to other mouse strains [17, 65]. Continued improvement of the NSG mouse by the transgenic expression of human-specific cytokines and growth factors and expression of HLA that will allow matching with the donor tissues will further augment the development of human immune systems in BLT mice [3, 66]. In an effort to provide an analysis of optimal parameters for establishing the NSG–BLT model, we have assessed the requirement for irradiation to attain high-level human cell chimerism, the optimal implantation sites for thymic tissues, the stability of human cell chimerism and the longevity of engrafted mice.

To identify previously unrecognized responses triggered by KIR2DS

To identify previously unrecognized responses triggered by KIR2DS1 or KIR2DL1

binding to HLA-C2, Xiong et al. performed microarray-based MG-132 price genomic profiling of the following four dNK subpopulations: KIR2DS1+, KIR2DL1+, KIR2DS1+KIR2DL1+, and KIR2DS1–KIR2DL1– [49]. KIR2DS1+KIR2DL1+ dNK cells exhibited different responses than the KIR2DL1+ single-positive dNK cells, whereas only HLA-C2-activated KIR2DS1+ dNK cells produced several soluble products, such as GM-CSF, that enhanced the migration of primary trophoblast and JEG-3 trophoblast cells in vitro [49]. These findings provide a possible molecular mechanism for the fact that expression of activating KIR receptors on maternal dNK cells can be beneficial for placentation. The liver is an immunotolerant organ containing a large proportion of innate immune selleck chemicals llc cells such as NK cells, NKT cells, γδT cells, and macrophages [50]. These immune cells play an important role in inhibiting autoimmune diseases as well as in maintaining immunotolerance and homeostasis [51]. In humans, 30–50% of

intrahepatic lymphocytes are NK cells [52]. In mice, NK cells account for approximately 10–15% of intrahepatic lymphocytes and can be divided into two distinct subpopulations: CD49a+DX5– and CD49a–DX5+ NK cells [51, 53]. We performed gene expression microarray analysis of ∼22 000 genes to explore the differences in the transcriptional signatures of hepatic DX5– and DX5+ NK cells in mice [53]. Although nearly half of the tested genes were identically expressed between the DX5– and DX5+ NK-cell subpopulations, these Dichloromethane dehalogenase two subpopulations were distinct from each other in the following ways: among the 1507 genes found to be significantly different between the subpopulations, 566 genes enriched in DX5– NK cells were associated with negative regulation and immune tolerance, while the 941 genes enriched in DX5+ NK cells were instead associated with migration,

proliferation, immune responses, and cell maturation [53]. DX5– NK cells expressed relatively high numbers of genes related to IL-17 production and Th17-cell development (including Il21r, Rora, and Ahr) [54] as well as genes preferentially expressed by Treg cells (including LAG-3, Helios, and Egr-2) [55, 56], raising the possibility that DX5– NK cells might exert negative regulatory control within the liver. Microarray datasets are not only used to find previously unrecognized gene changes under various conditions but also to establish a molecular definition of cell identity. Clustering and other classical techniques, such as principal component analysis (PCA), are useful methods for analysis of gene expression data [41, 57]. The relatedness of NK-cell subpopulations to each other and to other leukocyte populations have been investigated using hierarchical clustering or PCA.

Kidney Disease Outcomes Quality Initiative: No recommendation UK

Kidney Disease Outcomes Quality Initiative: No recommendation. UK Renal Association: No recommendation. Canadian Society of Nephrology: No recommendation. European Best Practice Guidelines: No recommendation. International Guidelines: No recommendation. No recommendations. The evidence related to protein requirements in the early post-transplant period is limited to small studies on patients receiving prednisone

at levels which may be higher than currently used. Multi-centre trials are needed to confirm the dietary protein requirement of kidney transplant recipients in the early post-transplant period receiving lower doses of prednisone. There is also limited research on the effects of a moderate dietary protein restriction, though the evidence to date suggests that such a restriction may improve X-396 clinical trial glomerular perm-selectivity click here in adult kidney transplant recipients with chronic allograft nephropathy. Multi-centre trials are needed to establish the safe level of dietary protein restriction and to assess the long-term efficacy and safety of protein restriction on the progression of allograft nephropathy. All of the authors have no relevant financial affiliations that would cause a conflict of interest according to the conflict of interest statement set down by CARI. These guidelines were developed under a project funded by the Greater Metropolitan Clinical Taskforce, New South Wales. “
“According to

the Indian chronic kidney disease registry, in 2010 only 2% of end stage kidney disease patients were managed with kidney transplantation, 37% were managed with dialysis and 61% were treated conservatively without renal replacement therapy. In countries like India, where a well-organized deceased donor kidney transplantation program is not available,

living donor kidney transplantation is the major source of organs for kidney transplantation. The most common reason to decline a donor for directed living donation is ABO incompatibility, which eliminates up to one third of the potential living donor pool. Because access to transplantation with human leukocyte PJ34 HCl antigen (HLA)-desensitization protocols and ABO incompatible transplantation is very limited due to high costs and increased risk of infections from more intense immunosuppression, kidney paired donation (KPD) promises hope to a growing number of end stage kidney disease patients. KPD is a rapidly growing and cost-effective living donor kidney transplantation strategy for patients who are incompatible with their healthy, willing living donor. In principle, KPD is feasible for any centre that performs living donor kidney transplantation. In transplant centres with a large living donor kidney transplantation program KPD does not require extra infrastructure, decreases waiting time, avoids transplant tourism and prevents commercial trafficking. Although KPD is still underutilized in India, it has been performed more frequently in recent times.

Blood lymphocytes were washed once in cold PBS, and following cen

Blood lymphocytes were washed once in cold PBS, and following centrifugation (680 g, 18°C, 10 min) the pellet was resuspended in 2 ml red blood cell lysis buffer [0·15 m NH4Cl, 0·01 m KHCO3 and 10 µm ethylenediamine tetraacetic acid (EDTA) Na2·2H2O] and incubated for 2 min at room temperature. The volume was then adjusted to 30 ml using

sterile PBS and centrifuged. Following two subsequent washes, the cell pellet was resuspended in IMDM (Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco, Mulgrave, Australia) and anti-mycotic solution (10 mg/l; Sigma). PBMCs/CRL-9850 cells were plated in six-well tissue culture plates (Corning, Sigma) at 5 × 106 cells/well and incubated at 5% CO2, 37°C for 24 h prior to stimulation with bacteria, as described by Amrouche et al. [21]. Briefly, 106 freshly prepared viable (live or GIT) or equivalent (∼106 CFU/ml) heat-killed bacteria were added per 106 cells Cobimetinib molecular weight and co-cultured for 72 h at 5% CO2, 37°C. At 6, 12, CP-673451 chemical structure 24, 48 and 72 h, 500 µl samples of the culture medium were collected and analysed for cytokine secretion by

ELISA (Becton Dickinson, San Jose, CA, USA), in accordance with the manufacturer’s instructions. Data are expressed as the mean cytokine response minus background (pg/ml) of each treatment from triplicate wells, plus or minus the standard error of the mean. Treg/Th17 populations were characterized following PBMC/bacteria co-culture. Briefly,

106 PBMC were co-cultured with either live or killed bacteria, lipopolysaccharides (LPS; Sigma) or media alone, in a 24-well plate at 37°C in 5% CO2 for 96 h, then cells were washed twice using FACS buffer (PBS + 2% FCS) and centrifuged at 500 g for 10 min. PBMC were resuspended at 106 cells/ml, and surface marker staining was performed using fluorescein isothiocynate (FITC)-labelled anti-human CD4, allophycocyanin-labelled anti-human CD25/CD3 (Becton-Dickinson), peridinin chlorophyll protein (PerCP)-labelled anti-human CD3 (Biolegend, San Diego, CA, USA) and PerCP cyanine (Cy)5·5-labelled anti-human CCR6 (CD196). Intracellular staining was performed using phycoerythrin (PE)-labelled anti-human FoxP3/RORγt (BD MG-132 ic50 Pharmingen and R&D Systems, Minneapolis, MN, USA, respectively), according to the manufacturer’s instructions. Samples were read using a BD FACSCalibur, data acquired using CellQuest program (Becton Dickinson Biosciences), and analysis performed using Gatelogic version 3·07 software (Inivai, Victoria, Australia). Absolute numbers of Treg cells and Th17 cells were calculated as a percentage of the total lymphocyte number. All co-cultures were carried out in triplicate. Results obtained were analysed as a split plot in time design with three main factors: strains (six levels) and treatments (three levels) as the main plot and time (five levels) as a subplot.

While a number of functions are mediated by Abs without additiona

While a number of functions are mediated by Abs without additional mediators or cells, others require interactions between Abs and other components of the immune system, e.g. complement, phagocytic cells, or effector cells (e.g. NK cells). The best-documented direct effect of Abs is neutralization. Ab-mediated neutralization

of bacterial toxins was already reported in the 19th century (pioneered by Adolf Emil Behring and Kitasato Shibasaburo) and is essential for the buy Tigecycline vaccine-mediated resistance against diphtheria, tetanus, and pertussis toxins. Furthermore, neutralization by Abs plays an important role in immune responses against viruses, as the Abs are able to inhibit virus attachment to specific host cell receptors, to block uncoating of the virus and therefore interfere with productive infection, and to inhibit viral assembly and release 1. Very recently, an additional mechanism of Ab-mediated interference of viral replication was described, showing that Abs bound to the capsid of nonenveloped viruses can bind to the cytoplasmic Fc-binding protein TRIM21 and target these cytosolic viruses for proteasomal degradation 2. The ability of Abs to block receptors required for pathogen uptake and thereby to inhibit

infection is not limited to viruses, but has also been reported for intracellular bacteria and for the malaria-causing protozoan parasite Plasmodium falciparum3, 4. Furthermore, Abs specific for effector proteins secreted by bacteria, such as listeriolysin O, the pore-forming toxin of Listeria monocytogenes, can neutralize JAK inhibitor these effectors and thereby protect

the host from productive infection 5. Similarly, Abs directed against pathogen components involved in locomotion, e.g. the flagella of Pseudomonas aeruginosa, mediate their protective effect by interfering with pathogen motility 6. Abs also prevent pathogen Interleukin-2 receptor entry at mucosal sites and play an important role in promoting compartmentalization of bacteria in these tissues 7; however, Abs can not only block infection but, under certain circumstances, also enhance infection as has been documented for Dengue virus and HIV 8. In addition to mediating direct protective effects, Abs can fulfill protective functions via activation of the classical complement pathway, which results in pathogen opsonization, chemoattraction of leukocytes, and the formation of the membrane attack complex 9. Abs also mediate a number of effector functions through the interaction with Fc receptors (FcRs) on innate immune cells, thereby linking the specificity of the humoral immune response to the powerful effector functions of innate immunity. One such effector mechanism is ADCC, an important effector mechanism for the elimination of virus-infected cells, multicellular parasites, and tumor cells. ADCC directs nonspecific cytotoxic cells, such as NK cells, neutrophils, and eosinophils, in an FcR-dependent manner to specific target cells which are marked by Ab bound to surface Ag.

Solomon and colleagues assessed the relationship among the initia

Solomon and colleagues assessed the relationship among the initial haemoglobin response to darbepoetin after two weight-based doses, the haemoglobin level achieved after 4 weeks, the subsequent darbepoetin dose and outcomes in 1872 patients from the TREAT trial who were randomized to darbepoetin.15 The initial dose of darbepoetin was 0.75 µg/kg Selleck Palbociclib of body weight and was repeated after 2 weeks if haemoglobin values did not exceed 140 g/L. Poor initial response to darbepoetin was defined as the lowest quartile of per cent change in haemoglobin level (<2%) after the first two standardized doses of the drug. Patients in the lowest quartile of haemoglobin responsiveness were more likely to have cardiovascular

disease, high CRP levels and low ferritin and transferrin saturation levels. The average haemoglobin level after 12 weeks remained marginally but statistically significantly lower among patients with a poor initial response (122 ± 9 g/L) than among those with a better initial response (124 ± 7 g/L, P < 0.001). The average monthly dose of darbepoetin after 12 weeks and throughout the remainder of the trial was substantially higher among patients a poor initial response (median dose, 232 µg; interquartile range, 126 to 390) than those with a better initial response (167 µg; interquartile

range, 95 to 310; P < 0.001). There was significant difference CB-839 in the use of intravenous iron or blood transfusion throughout the trial. Compared with patients with a better initial response, those with a poor initial response were at increased risk of a cardiovascular composite event (HR 1.31, 95% CI 1.09–1.59) and all-cause death (HR 1.41, 95% CI 1.12–1.78). The event rates for the cardiovascular composite outcome and all-cause death in the better initial response group

were comparable with oxyclozanide the placebo group. These findings indicate that requirement of high-dose ESA to achieve target haemoglobin rather than achieved haemoglobin may be responsible for the poor outcome. Interestingly, the event rates for stroke were comparable in the two response groups, but higher in both groups than in the placebo group. It still remains unclear whether the use of ESA or high haemoglobin target contributed to increased risk of stroke in patients treated with darbepoetin. A summary of the observational studies is provided in Table 2. In a US Medicare study of 75 283 prevalent patients receiving haemodialysis between July–December 1993, a haematocrit level of 33–36% was associated with a similar risk of mortality (adjusted RR 0.96, 95% CI 0.91–1.01) compared with a reference haematocrit level of 30–33%.16 In contrast, lower haematocrit levels were associated with increased risk of mortality (haematocrit <27% RR 1.33, 95% CI 1.26–1.40; haematocrit 27–30% RR 1.12, 95% CI 1.08–1.17). The pattern of higher mortality with lower haematocrit was similar in diabetic and non-diabetic patients.

In the cultures of lung CD34+ cells (Fig  2a), we detected 2 ± 1 

In the cultures of lung CD34+ cells (Fig. 2a), we detected 2 ± 1 CFU/well in the control culture, and 8 ± 3 versus 6 ± 2 CFU/well in cultures where either IL-5 or rmEotaxin-2 was added alone, respectively. When the combination of rmIL-5 and rmEotaxin-2 was added to the culture of lung CD34+ cells, no further significant increase in CFU/well was observed (10 ± 1 CFU/well; Fig. 2a). Interestingly, previous studies have shown that BM-derived CD34+ cells form CFU when stimulated with rmIL-5.9 Hence, BM CD34+ cells were cultured in parallel as a control for our system. In the cultures of BM CD34+ cells, we detected 1 ± 1

BM CFU/well in the control cultures (no cytokines INCB024360 added), whereas we found no BM CFU in the cultures where rmEotaxin-2 alone was added. In contrast, the cultures where rmIL-5 was added alone, or together with rmEotaxin-2, had 27 ± 3 and 26 ± 2 CFU/well, respectively (Fig. 2b). The optimal time for BM CFU growth was after 8 days of culture, and in lung after 8–14 days of culture. The cells Acalabrutinib were identified as eosinophils on the basis of morphologically homogeneous appearance. A multiparametric cell cycle analysis was used to assess whether the magnetically enriched CD34+ or Sca-1+ newly produced eosinophil-lineage-committed cells proliferate locally within the airways in response to allergen, by analysis of BrdU staining together with 7-AAD staining (total DNA stain). We found

a significant increase in the number of CD34+ CCR3+ BrdU+ and Sca-1+ CCR3+ BrdU+ proliferating cells (i.e. cells within S phase or G2/M phase) Carnitine palmitoyltransferase II in the allergen-exposed animals when compared with the saline exposed animals. This increase was paralleled with an increase in proliferating cells in both SSChigh and SSClow lung cell populations, representing eosinophils

and their progenitors (Fig. 2c,d). We employed double staining of CCR3 together with MBP to further assess whether the CCR3+ cells were committed to the eosinophil lineage. Almost all of the CCR3+ cells gated on both the SSChigh and SSClow cell population co-expressed MBP (ranging between 75 and 99%) (data not shown). Bone marrow, lung and BAL cells were stained for CD34+ CD45+ IL-5Rα+ to evaluate the amount of CD34+ progenitors (CD34+ CD45+ cells) and the classical eosinophil progenitors (CD34+ CD45+ IL-5Rα+ cells) in our model. No differences were found in BM eosinophil progenitors of allergen-exposed animals compared with saline-exposed animals (data not shown). In contrast, lung and BAL CD34+ CD45+ IL-5Rα+ cells were significantly increased in the allergen-exposed animals compared with the saline-exposed animals (Fig. 3a). To further assess whether the IL-5Rα+ newly produced cells proliferate locally within the airways in response to allergen a multiparametric cell cycle analysis for BrdU+ cells together with 7-AAD staining (total DNA analysis) was used.

For example, C57Bl/6 strains differ significantly and the differe

For example, C57Bl/6 strains differ significantly and the difference between various B6 substrains are often larger than the differences when

comparing a specific C57Bl/6 with other inbred strains such as B10. In addition, using strains from other colonies means that the mice also differ in epigenetic- and environmental-caused selection. A recent example is the lack of segmented filamentous bacteria (SFB) in the Jackson Laboratory animal house as compared with some other animal houses that dramatically affected an IL-17-associated phenotype 14. Another example is the induction of inter-male aggressiveness among non-littermate adult males that, in fact, results in severe arthritis in many mouse strains 15. There is one obvious solution to this problem and that is to

use littermates. This will ensure that not only https://www.selleckchem.com/products/epz-6438.html is the genetic background comparable but also the environment. Another advantage is that the mice do not require full backcrossing, as the difference in genes will be neutralized when littermates are compared although less backcrossing might result in a requirement for increased numbers of mice in the experiments as the variability will increase. The exception for not using littermate GDC-0973 supplier controls is to use mouse strains that can be demonstrated to be genetically identical. However, in these cases the experiments still need to be controlled for environmental factors. Thus, the control and test mice need to be balanced in terms

of cages, age, sex, etc. and the experiments need to be blinded as has recently been highlighted by the new guidelines for reporting animal experiments, the ARRIVE guidelines 16. The suggestions to use littermate controls and to control for linked fragments may raise the threshold for reporting new findings and limit the quantity of unreliable results. The drawback is, of course, that it gives an extra Phospholipase D1 burden of labor, in particular when more complicated modifications are to be studied and sometimes it is simply impractical. That is most likely one reason why scientific journals, including EJI, have not yet implemented this requirement. Given the present explosion of the data and publication pool, which we first enjoy swimming in but soon discover that we cannot keep up with and end up drowning in, it is of particular importance for high-quality journals to set quality standards for reporting data. Conflict of interest: The authors declare no financial or commercial conflicts of interest. The authors are members of the Executive Committee of EJI but it should be noted that the views expressed in this Commentary are the personal views of the authors and do not represent EJI policy.

IRF-8 was originally identified as a repressor of IFN-stimulated

IRF-8 was originally identified as a repressor of IFN-stimulated response elements and through its ability to inhibit the transcriptional activation Enzalutamide of other IRFs [50, 51]. Yet, studies of human monocytes and murine cDCs found that IRF-8 promoted type I IFN production [35, 52]. Current findings show that IRF-8 is a strong negative regulator of CpG-driven IFN-β and IL-6 production by human pDCs (Fig. 4B). This is an important observation, as pDCs constitutively express high levels of IRF-8 [13] and IRF-8 KO mice

fail to generate pDCs [36]. Taken together, current findings demonstrate that IRF-8 expression plays a role in negatively regulating pro-inflammatory and IFN responses following TLR9 stimulation of pDCs. We are in the process of examining whether the elevated levels of IRF-8 in the nucleus of unstimulated pDCs (Fig. 2) reflect a constitutive role for IRF-8 in the regulation of gene activation and whether IRF-8 interacts with IRF-5. Several findings support the technical reliability of results from the knockdown experiments upon which these conclusions are largely based. First, no off-target (i.e. nonspecific) Sotrastaurin solubility dmso activity was detected

with any of the siRNAs tested (Fig. 3A and C and 4A, and Supporting Information Fig. 2A–C). Second, cells transfected with siRNA were not stimulated unless CpG ODN was added (in contrast to the report by Hornung et al. [34]) (Supporting Information Fig. 2D and E). Third, siRNA administration significantly reduced the level of expression of both mRNA and protein of the targeted gene (Fig. 3A and C and 4A, Supporting Information Fig. 2A–C). Finally, siRNA knockdown of MyD88 and TRAF6 blocked the induction of IFN-β and IL-6 mRNA by CpG-stimulated

pDCs, consistent with earlier reports (Fig. 3B; [15, 31, 32]). K” ODN triggered the rapid translocation of NF-κB p50 and p65 (RelA) from the cytoplasm to the nucleus in CAL-1 cells and human pDCs (Fig. 2D, 6, and 7). Interestingly, the knockdown of p105/p50 but not p65 significantly reduced IFN-β production (Fig. 3D), whereas both p105/p50 and p65 contributed to the induction of IL-6. Accumulating evidence indicates that IκBξ (also known as MAIL, a nuclear ankyrin repeat protein) is required for TLR-dependent upregulation of IL-6 [53, 54]. As IκBξ associates with both p50 and Fluorometholone Acetate p65 [55], current findings suggest that eliminating either impairs IκBξ-dependent induction of IL-6. K” ODN induced the rapid nuclear translocation of both IRF-5 and NF-κB p50 (Fig. 2, 6, and 7). PLA, a technique used to identify protein–protein interactions under physiologic conditions, was employed to examine whether these transcriptional factors associated upon stimulation [40]. Only proteins in close proximity (<40 nM) are visualized by PLA, yielding results comparable to resonance energy transfer techniques (such as fluorescence resonance energy transfer analysis).