, Foster City, CA, USA). Assumptions and formulation.  The PLN and each islet are assumed to be well-mixed, spatially homogenous compartments. Each islet bin, as described above, contains the same model architecture. Differences in simulated behaviours in islets of different bins result from sequential and progressive lymphocyte infiltration of different islets and islet bins, leading to different

degrees of accumulated infiltrate, see more local inflammation and damage at a given time. Common functions represented in all compartments include mediator synthesis, cellular proliferation, apoptosis and activation. Each of these functions are regulated by cell contact and soluble mediators with the following basic approach: (i) a baseline rate is Decitabine in vitro assigned if data suggest a constitutive activity; (ii) additional stimulatory effects are assumed to be additive; (iii) regulators that synergize or amplify the impact of another are treated as potentiating them and represented as having multiplicative effects; (iv) inhibitory

effects are represented as fractional reductions in baseline and/or stimulated effects as indicated by the data; and (v) an upper limit may be imposed, such as when the rate is proportional to the fraction of cells involved (e.g. proliferation) and saturates at 100% involvement. The likelihood of cell contact within a compartment is a function of the relative numbers of each cell type within the total cellular population. Mediator concentrations in each compartment are a function of the synthesis rate (i.e. ng/1e6 cells/h), the number of mediator-producing cells, mediator half-life and the compartment volume. Because the effect of each regulator

is dependent on its concentration/activity, ADAMTS5 a standard dose–response curve was employed to describe the relationship between the regulator and its effects (Fig. 3). Published data were used to define the effective concentration range and the maximum effect. If the effective concentration range had not been published, the available data were used to define the saturating concentration and a three-log range of dose-sensitivity is assumed. Parameterization.  Parameter values were derived directly from (or calculated to be in agreement with) published data wherever quantitative data were available. Preference was given to NOD mouse data. If unavailable, data from other mouse strains, other animal species or human cells were used. The determination of the rate of tumour necrosis factor (TNF)-α synthesis by activated CD8+ T lymphocytes from Utsugi et al. [79] is a relevant illustration of data usage. They reported TNF-α production by NOD CD8+ T cell clones stimulated with islet cells. In all similar cases where parameters were extracted/calculated from specific literature, the references are cited in the location within the model where the parameter was used. Thus, all directly derived parameters are referenced.

MEK5 induction of KLF4 is mediated by ERK5. MEK5/CA-transduced HDMECs are less responsive

to TNF, an effect partly mediated by KLF4. Conclusions:  MEK5 activation by LSS inhibits inflammatory responses in microvascular ECs, in part through ERK5-dependent induction of KLF4. “
“Please cite this paper as: Su S-W, Catherall M and Payne S. The Influence of Network Structure on the Transport of Blood in the Human Cerebral Microvasculature. Microcirculation 19: 175–187, 2012. In this article, we explore how the structural properties of miniature networks influence the transport of blood through the human cerebral microvasculature. We propose four methods for generating such networks, and investigate both how the resulting network properties match available experimental data from the human cortex and how these properties affect the flow of blood through

the networks. As the nature of such microvascular MG-132 nmr flow patterns is inherently random, we run multiple simulations. We find that the modified spanning tree method produces artificial networks having characteristics closest Selleck Selumetinib to those of the microvasculature in human brain, and also allows for high network flow passage per unit material cost, being statistically significantly better than three other methods considered here. Such results are potentially extremely valuable in interpreting experimental data acquired from humans and in improving our understanding of cerebral blood flow at this very small length scale. This could have a significant impact on improving clinical outcomes for vascular brain diseases, particularly vascular dementia, where localized flow patterns are very important. “
“Please cite this paper as: Mahé G, Durand S, Humeau-Heurtier A, Leftheriotis G, Abraham P. Impact of experimental conditions on noncontact laser recordings in microvascular studies. Microcirculation 19:

669–675, 2012. Microcirculation, especially skin microcirculation, is a window toward systemic vascular function in magnitude and underlying mechanisms. Different techniques have been developed to assess the microcirculation. Among these techniques, laser technology is used to perform noninvasive microvascular check details assessments. In the 1970s, the laser Doppler flowmetry (LDF) technique was proposed to monitor microvascular blood flow. More recently, noncontact technologies including laser Doppler perfusion imaging (LDI) and laser speckle contrast imaging (LSCI) have improved the reproducibility of the microcirculation measurements and facilitated some clinical evaluations such as on wounds and ulcers. However, due to the absence of contact between tissue and sensors, it is likely that different technical and environmental conditions may interfere with microvascular recordings. This review presents major technical and environmental conditions, which may interfere with noncontact laser recordings in microvascular studies.

no. 553142; BD Pharmingen, Becton Dickinson, San Jose, CA, USA). Staining was carried out in 5H buffer to detect H-2Db (expressed on NOD, C57BL/6J and CByB6F1/J lymphocytes) and H-2Kb– (C57BL/6J and CByB6F1/J mice) using the following antibodies: α-H-2Db-phycoerythrin

(PE) (clone KH95, cat. no. 111507; BioLegend, Inc., San Diego, CA, USA), α-H-2Kb-AlexaFluor 647 (cat. no. 116511, clone AF6-88.5; BioLegend), α-CD4-Horizon (cat. no. 48-0042-82, clone RM4-5; eBioscience, Inc., San Diego, CA, USA), α-CD8α-biotin (cat. no. 13-0081-82, clone 53-6.7; eBioscience) in combination with streptavidin–AlexaFluor 488 (cat. no. S32354; Molecular Probes, Invitrogen). 7-Aminoactinomycin D (7AAD) (cat. no. 559925; BD Pharmingen, Becton Dickinson) was BTK inhibitor order used for live/dead cell discrimination. Diabetes-free survivals in the experimental groups were assessed by Kaplan–Meier analysis and comparisons between groups were calculated using the

log-rank test. From groups B1, B2 and C2, the three mice that did not deliver a litter were excluded from the analyses. Multivariate analysis of diabetes outcome was performed using the Cox proportional hazards model, which included the covariates mating group and insulin autoantibody Rucaparib titre at the time of mating. Comparisons of insulin autoantibody titres between group A1 and C1 were made using Student’s t-test. Two-tailed P-values of < 0·05 were considered significant. For all statistical methods, PASW statistics version 18 (SPSS, Chicago, IL, USA) was used. Mating at age 10 weeks did not accelerate diabetes, but resulted in a significant delay of diabetes development in the NOD dams (unmated females, 81% diabetes by age 28 weeks, mated females, 60% by age 28 weeks; P = 0·04; Fig. 1a). Differences were observed between mating partners. Mating at 10 weeks with NOD males had no effect on diabetes incidence (71%

by age 28 weeks, P = 0·38), whereas mating with MHC haploidentical CByB6F1/J male mice had the strongest Tideglusib effect on diabetes development (38% by age 28 weeks, P = 0·01 versus unmated NOD females; P = 0·08 versus NOD male mated females). Mating with fully MHC mismatched C57BL/6J males did not delay diabetes significantly (73% by age 28 weeks, P = 0·22 versus unmated females). Mating at age 13 weeks did not affect diabetes development significantly in NOD females (unmated females, 94% diabetes by age 28 weeks, mated females, 72% by age 28 weeks; P = 0·22; Fig. 1c) although, again, diabetes development was lowest in females mated with CByB6F1/J male mice (64% by age 28 weeks, P = 0·13).

The wztYS-11 was introduced into strain 455, and the changes in the phenotype were observed by SEM. The fragment including the wztYS-11 ORF was amplified by PCR using the primers wzt-EcoF and wzt-PstR (Table 1). An EcoRI site or a PstI site (Table 1, underlined) was introduced into the 5′ end of the PCR product. The reaction

mixture contained 10 ng μL−1 genome DNA of strain YS-11, 1 × PCR buffer, 0.2 mM dNTPs, 0.5 μM each primer, and 25 mU μL−1 KOD dash DNA polymerase (Toyobo, Osaka, Japan), and sterile-distilled water was added to the mixture to a final volume of 50 μL. The reaction conditions were as follows: 94 °C for 6 min, 35 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, and 72 °C for 2 min with a PCR thermal cycler (Takara Bio). The PCR product purified with the QIAquick gel extraction kit (Qiagen) was digested with EcoRI and PstI (Takara Bio), and ligated BAY 80-6946 mw to the plasmid vector pSTV28 (Takara Bio), which was predigested with the same combination of restriction enzymes. Ligation was performed using a DNA ligation kit ver. 2.1 (Takara Bio)

according to the manufacturer’s directions. Escherichia coli DH5α (Invitrogen) was transformed with this ligation solution. https://www.selleckchem.com/products/Deforolimus.html The constructed plasmid, named pWZT, was purified from a colony grown on TSAY containing 20 μg mL−1 of chloramphenicol. Ten nanograms of constructed plasmid was added to 50 μL of the competent cell of strain 455, and transformation was carried Casein kinase 1 out as described above. Measurement of the viscosity of spent culture media and SEM observation for the presence of meshwork-like surface structures were carried out on the recombinants grown on the TSAY containing 50 μg mL−1 of kanamycin and 20 μg mL−1 of chloramphenicol. The wztYS-11 on the pWZT was fused with the α-peptidase gene on pSTV28 so that the viscosity and the cell surface-associated phenotype were examined under culture conditions with or without 1 mM isopropyl-β-d(−)-thiogalactopyranoside (IPTG; Wako Pure Chemical Industries, Osaka, Japan). Strain 455 with pSTV28 and E. coli DH5α with pWZT were used as controls. The bacterial strains and plasmids used in this study are listed

in Table 2. Escherichia hermannii strains YS-11 and 455-LM with meshwork-like structures were compared with those of strains 455 and ATCC33650 that lacked this phenotype for the ability to induce abscess formation in mice. Bacterial strains were cultured in TSBY for 12 h (early stationary phase). Five hundred microliters of bacterial suspensions (107–109 CFU mL−1) were injected subcutaneously into the inguen of each BALB/c mouse (male, 4 weeks; three mice per strain). Changes in abscess lesions were recorded photographically using a camera (Nikon FIII, Nikon, Japan) set at a fixed magnification for five consecutive days. Stock cultures of YS-11 were inoculated into TSBY and grown for 48 h. The viscosities of the spent culture media were measured using a rotary viscometer.

The

RYR1 mutations associated with CCD are usually dominant but recessive inheritance has also been reported, whereas cases identified as MmD are exclusively linked to recessive mutations [2–7] and recently in patients with fibre type disproportion as their only pathological feature. [8] Classically in the RYR1 sequence, three hot-spots are considered, two in the large hydrophilic domain of RyR1 and one in the C-terminal hydrophobic domain. Most of the heterozygous dominant CCD mutations are mapped to the C-terminal domain, whereas the recessive CCD and MmD mutations are more extensively distributed along the RYR1 sequence. Additionally, a heterozygous de novo RYR1 mutation in the C-terminal region of the protein has been found in a 16-year-old female patient initially diagnosed with selleck screening library centronuclear myopathy (CNM) with ‘core-like’ lesions and central nuclei in up to 50% of fibres in the muscle biopsy

[9], and a heterozygous de novo RYR1 mutation in the N-terminal domain has been found in a patient presented with King-Denborough syndrome and MHS [10]. In RYR1-related congenital myopathies, the histological phenotype varies widely. It comprises central and eccentric cores, unique and multiple, structured and unstructured, well-delimited cores spanning the entire fibre length or poorly defined cores that spread only a few sarcomeres, and occasionally ATR cancer a variable degree of sarcomeric disorganization [2,11–13]. These structural abnormalities are sometimes associated with an increased number of internal myonuclei (up to 30% of the fibres) and variable degrees of fibrous and adipose tissue replacement [6,14,15]. There also exist biopsies without major alterations showing only a type I fibre predominance or uniformity [16]. Moreover, a histopathological continuum has been suggested linking the diverse RYR1-related core myopathies [17–20]. On the other hand, centronuclear myopathies (CNM; OMIM 310400, 160150 and 255200), comprise X-linked recessive, autosomal dominant and autosomal recessive forms, associated, respectively,

with myotubularin 1 (MTM1), dynamin 2 (DNM2) and amphiphysin 2 Erythromycin (BIN1) genes [21–23]. The histopathological presentation of these distinct forms of CNM has been well established [24]; so far, neither cores nor minicores have been described in such genetically determined CNM forms. Here we report clinical, histological and molecular characterization of seven patients initially diagnosed with CNM due to the significantly high number of fibres with internalized nuclei (up to 51% of the fibres). However, the key histopathological feature that led us to screen RYR1 gene for mutations was the invariable presence of large areas of sarcomeric disorganization in the muscle fibres, despite the number and location of internalized nuclei.

Recent evidence suggests that statins have multiple effects and are able to modulate the immune response independent of their cholesterol attenuating ability [25]. The anti-inflammatory and immunomodulatory selleckchem effects of statins stem from downstream effects of inhibiting the mevalonate pathway leading to decreased activity of the small guanosine triphosphate (GTPases) Rac, Ras and Rho [26], which are crucial for many cellular functions including proliferation and transcriptional regulation [27], key processes in inflammation. We hypothesize a beneficial

therapeutic effect of statins in SAg-mediated diseases through the modulation of T cell activation and MMP-9 production. In this study, we studied

the role of atorvastatin in modulating three critical steps in the pathogenesis of coronary artery inflammation and aneurysm formation in a disease model of KD. These include T cell proliferation, TNF-α cytokine production and TNF-α-mediated MMP-9 production [28,29]. We show that atorvastatin inhibits each one of these critical processes leading to aneurysm formation, suggesting a potential beneficial effect of statins in the treatment of KD. Atorvastatin calcium Paclitaxel datasheet (Pfizer, Kirkland, Quebec, Canada) was dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA). Mevalonic acid (MVA) (Sigma-Aldrich) was also dissolved in DMSO, and Staphylococcal enterotoxin B (SEB) (Toxin Technology Inc, Sarasota, FL, USA) was dissolved in phosphate-buffered saline (PBS). LCWE was prepared as described previously [19]. Briefly, Lactobacillus casei (ATCC 11578) was harvested after ∼18 h and washed in PBS. Bacteria lysis by overnight sodium dodecyl sulphate (SDS) incubation was followed by incubation with DNAase I, RNAse and trypsin (Sigma Chemicals) to remove any adherent material from the cell wall. The cell wall was fragmented through sonication in a dry ice/ethanol bath for 2 h. BCKDHA Phenol-sulphuric colorimetric determination assay was used to determine the measurement of rhamnose concentration,

which was expressed in mg/ml PBS. Total protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Mississauga, ON, Canada) following the manufacturer’s instructions. Wild-type 6–12-week-old C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed under specific pathogen-free conditions at the Hospital for Sick Children under an approved animal use protocol. Splenocytes (5 × 105) from C57BL/6 mice were cultured in medium alone (Iscove’s supplemented with 10% heat-inactivated fetal bovine serum (FBS), sodium pyruvate, non-essential amino acid, 50 µM 2-mercaptoethanol (ME), 2 mM l-glutamine and 10 mM HEPES), medium containing 0·03125 µg/ml highly purified SEB (Toxin Technology Inc.

The UK Expert Consensus Group have developed GPCR Compound Library manufacturer evidence-based guidelines for symptom management in adults who are dying from ESKD.4 These guidelines developed from the Liverpool Care Pathway for the Dying Patient, which was used initially for terminal cancer but subsequently for stroke and heart failure patients. An Expert Consensus Group for patients dying with renal failure found those dying with renal failure had similar symptoms to those dying with terminal cancer hence the Renal Liverpool Care Pathway prescribing guidelines

were developed with the aim of controlling these symptoms.78 The NKF KDOQI guidelines state Nephrologists should be familiar with the principles of palliative care and should not neglect hospice referral for patients with advanced kidney failure.2,5 The CARI guidelines do not address palliative care15 and formulating guidelines in the Australian context should be a high priority. However, the Kidney Health Australia website provides information for patients on conservative approaches both pre-dialysis and withdrawing from dialysis.79 National Kidney Foundation core curriculum in nephrology summarized the relevance of palliative care and https://www.selleckchem.com/products/AG-014699.html its incorporation into

dialysis units.5 It highlights the usefulness of advanced care planning in patients with ESKD and strategies to increase its use. The American Society of Nephrology and the Renal Physicians Association produced a position statement on End of Life Care in 2002.1 This is a comprehensive document that addresses

advanced care planning and directives, hospice care and palliative care. It also makes recommendations, which includes ensuring education of multidisciplinary renal team members in palliative care principles including Methane monooxygenase advanced care planning, supporting the patient requesting dialysis withdrawal with palliative care referral and the development of renal unit policies and protocols to ensure advanced care planning occurs. The Renal Physicians Association and the American Society of Nephrology also provide a clinical practice guideline on dialysis initiation and withdrawal.80 Standards for providing Quality Palliative Care for all Australians were published in 2005.81 Although there is no specific reference to patients with kidney disease the standards provide guidelines that can be applied to all diseases. The standards do emphasize the need to encompass the patient and their family’s wishes and needs in the decision-making process of care planning. In addition, access to palliative care services should be available independent of diagnosis and should be based on clinical need. The only tool in the public domain that we could find was in the National Health Service National End of Life Care Program to enhance end-of-life care in those without cancer. It introduced the tool to support patients with kidney failure.

However, a few studies have reported that artificially programmed DCs exhibited remarkable changes in phenotype. Immature DCs pre-treated with dexamethasone and subsequently stimulated with tumor necrosis factor-α (TNF-α) exhibited an endocytic

capacity four times higher (at maximum dexamethasone concentration) than iDCs treated with only TNF-α.[34] Clingan et al.,[35] reported that pre-treatment of iDCs with either interleukin-4 (IL-4) or interferon-γ (IFN-γ) inhibited the migration of iDCs in response to CCL3. Coincidentally, they observed that when IL-4 or IFN-γ pre-treated DCs were incubated with FITC-dextran in the presence of CCL3 for 2 min, dextran uptake capacity of the DCs was significantly enhanced by approximately fourfold (IFN-γ) or fivefold (IL-4) versus MG-132 cost without CCL3. Yanagawa and Onoe,[36] found that CCL3 and CCL19 rapidly (in less than an hour) p38 MAPK inhibitors clinical trials and selectively enhanced the internalization ability of iDCs and mDCs, respectively, when dextran and chemokines were added simultaneously to

the cell culture. They also noted that CCL19 induced an actin-reorganization related to the endocytic behaviour of mDCs.[37] Moreover, the synergistic effects of combinations of cytokines have been shown on the expansion of blood progenitors,[38] on the endocytic pathway in insulin-producing cells,[39] and on the migration and development of other phenotypes in endothelial cells.[40] Hence it may be possible, using selected chemokines or their combinations, to artificially program iDCs, thereby controlling their phenotypes and maturation status in order to enhance antigen uptake and presentation. We report here the first study to engineer DC phenotypes with select chemokine application to enhance antigen uptake and processing capacity of DCs, which can directly affect antigen presentation and DC-based vaccine efficiency in future. Dendritic cells were pre-treated with Dichloromethane dehalogenase the individual chemokines CCL3, CCL19, or their combination in various ratios. Then, 24 hr later, DCs were exposed to lipopolysaccharide (LPS), [a Toll-like receptor 4 (TLR4) ligand], to induce maturation. We demonstrate that when DCs are pre-treated with a chemokine combination of CCL3 : CCL19

in a specific ratio then subsequently stimulated with LPS, certain phenotypic changes arise that are significantly different from those of iDCs or DCs stimulated only with LPS. Dendritic cells programmed with a specific chemokine combination (CCL3 : CCL19 = 7 : 3) retained antigen uptake capacity and exhibited antigen-processing capacity, even after subsequent LPS maturation stimulus, at levels higher than iDCs (36%), and iDCs treated only with LPS (27%), respectively. Along with antigen uptake, this chemokine programming of DCs also modulated expression of MHC molecules and various cytokine responses of DCs even after maturation of DCs. Results here suggest chemokine programming may be a new tool for enhancing ex vivo and in vivo immunotherapy vaccine strategies.

However, strong CD8+ T-cell recall responses have also been demonstrated to cause undesired and sometimes learn more lethal immunopathology in certain circumstances [[9, 10, 16, 31]]. Therefore, rational vaccine design needs to take into account the delicate balance between robust immunity

and lethal CD8+ T-cell-mediated immunopathology. Following LCMV-Arm infection, wild-type mice mount vigorous antiviral CD8+ T-cell responses and clear the virus in a perforin-dependent manner [[40]]. PKO mice fail to clear LCMV-Arm and develop chronic infections [[14]]. Moreover, the requirement for perforin-mediated cytolysis in resistance to primary infection with LCMV is well documented [[41]] and PKO mice are models for FHL [[16-19]], a uniformly fatal disease associated with viral infection in human with mutations in perforin gene [[20, 23-25, 42]]. Thus, perforin OSI-906 mouse deficiency represents an immunocompromised state in which defective antiviral CD8+ T-cell response results

in the establishment of chronic infection [[16]]. Previous work in our laboratory demonstrated that vaccination to generate memory CD8+ T cells can overcome perforin deficiency and provide enhanced resistance against intracellular infection with LM [[27, 30]]. In contrast, vaccination of BALB/c-PKO mice results in accelerated mortality following LCMV infection [[16]]. In this case, vaccination of PKO hosts converts a nonlethal persistent infection into a rapidly fatal disease mediated by CD8+ T cells. To understand why vaccination leads to mortality in the absence of perforin, we analyzed multiple parameters that could potentially contribute to the drastic, and ultimately fatal response Etofibrate observed. We have shown that vaccination-induced mortality is mediated by massive expansion of

NP118-specific memory CD8+ T cells and the associated aberrant cytokine production in PKO mice. Different vaccine strategies did not alter the outcome as long as the number of NP118-specific memory CD8+ T cells exceeds a certain threshold number. In our adoptive transfer experiments (Fig. 3), we observed that the majority of PKO mice succumbed to LCMV infection if they received at least 8 × 104 NP118-specific CD8+ T cells. Assuming 10% “take” of the transferred number, this result indicated that as few as 8000 NP118-specific CD8+ T cells in the spleen at the time of LCMV infection would be sufficient to cause mortality in these PKO mice. Although we did not observe any mortality in mice that received 8 × 103 NP118-specific memory CD8+ T cells (i.e. 800 memory cells in the spleen, assuming 10% take), we documented severe morbidity as significant weight loss in these mice following LCMV infection (Fig. 3C). Thus, even a small number of NP118-specific memory CD8+ T cells is sufficient to cause immunopathology after LCMV infection of PKO mice.

Migration assay.  Migration of NK and NKT cells towards chemokines produced by DCs matured with the indicated stimuli was GSK2126458 evaluated by using a transwell assay. Briefly,

lower chambers of 24-(trans)well plates, 8.0-μm-pore-size filters covered with matrigel matrix, (BD Biosciences) were filled with 600 μL culture supernatants collected from previously washed and tumour-pulsed, mature or immature DCs. Six hundred microlitres of medium only was used as a control to determine spontaneous fallout. PBMCs isolated from patients with CLL were partly depleted of B cells with CD19+ magnetic beads (Miltenyi Biotec). After CD19 depletion, the percentage of B cells was 5–30% (median 10%). A total of 1 × 106 PBMCs were added in 400 μl CellGro medium in the upper chamber, and the plate was incubated at 37 °C for 24 h. Cells that migrated to the lower chamber were RG-7388 mouse harvested and stained with anti-CD5 PerCP, anti-CD56 PE-Cy7, anti-HLA-DR APC, anti-CD3 APC-Cy7 and anti-CD45 AmCyan (all from BD Biosciences) in TrueCounts tubes (BD Biosciences). Absolute numbers

of cells were calculated according to the manufacturer’s instructions. CD3− CD56+ NK cells, CD3+ CD56+ NKT cells and CD3+ total T cells were subsequently defined and counted using flow cytometry. Migration was presented as absolute numbers of migrated cells, or when indicated, as a quota of control (spontaneously migrated cells, migrated to medium only). CCL3/MIP-1α, CCL4/MIP-1β and IL-12p70 production after CD40 ligation.  To evaluate the ability of the differentially matured DCs to secrete CCL3/MIP-1α, CCL4/MIP-1β and IL-12p70 upon the follow-up activation in draining lymph nodes, DCs were stimulated with soluble, histidine-tagged, CD40L protein (R&D systems; 200 ng/ml) followed by the addition of an antipolyhistidine MoAb (R&D Systems; 4 μg/ml) 20 min later. DCs used in this experiment were pulsed with heat-stressed,

necrotic CLL cells and stimulated with either the gold standard or the αDC1 maturation cocktail for 24 h, washed twice and replaced in the well and cultured in fresh medium for a further 24 h followed by CD40 ligation. CD40-stimulated immature DCs were Dynein used as a control. Supernatants were collected after 24 h and tested for the presence CCL3/MIP-1α, CCL4/MIP-1β and IL-12p70 by ELISA (R&D Systems). The lower limits of detection were 7.8 pg/ml for CCL3/MIP-1α and CCL4/MIP-1β while 31.3 pg/ml for IL-12p70. Statistical analysis.  The statistical significance of differences between experimental samples was determined using the Wilcoxon signed-rank test and IBM SPSS statistics 19 software (IBM, Stockholm, Sweden). To efficiently recruit NK and probably also NKT cells within the draining lymph node, immigrating DC should produce CXCR3 ligands.