In a recent study, Warren et al.14 sequenced the TCR repertoire, and successfully obtained more than one billion Hydroxychloroquine manufacturer raw reads from a single blood sample, which is the deepest immune receptor sequencing to date, with a yield of about 200 million TCR-β nucleotide sequences. There are other sequencing machines available, each with its own advantages and disadvantages. We concentrate on the two machines mentioned above, as they are the only machines used so far in sequencing the immunological repertoire.

Other machines include the SOLiD sequencer (Life Technologies, Grand Island, NY), Helicos (Cambridge, MA), PacBio (Menlo Park, CA), and IonTorrent (Life Technologies, Grand Island, NY).11,15,16 The task at hand, for unbiased Rep-Seq protocols, is to isolate the relevant sequences, from the source B and T cells. These sequences are then sequenced by an NGS machine. To determine relative abundance of different sequences within the repertoire, a

proper account for each of the source sequences is made. Any biased amplification of some of the sequences will leave us with a skewed view of the repertoire. If, for example, one of the sequences in the process is favoured for amplification in one of the stages of the protocol, then we are left unable to discriminate such amplification from actual dominance of the clone in the repertoire. Causes for amplification are therefore an extremely sensitive issue in Rep-Seq and different groups provide different solutions (see below). Upon isolation of the appropriate genetic material (RNA/DNA, B cells/T cells), Rep-Seq requires Palbociclib the ‘lifting’ of the relevant immunoglobulin coding region. This is mostly done through a PCR-based amplification step. This amplification involves DNA primers with complementarities to the target regions. The standard technique uses multiple sets of primers, which are usually compatible with germline V

and J segments17–22 (Fig. 2a). It is impossible to design primers for all the numerous gene segments; for this reason Cediranib (AZD2171) primers are designed for families of genes or consensus sequences so that most gene segments are detected.23 A common primer should be designed to recognize the highest consensus region, whereas unique or family primers should recognize the least consensus region within a segment. In addition, specific tags can be added to the primers; for example, to identify from which sample a sequence was amplified.21 However, using a multiplex PCR amplification system, a strong bias is expected towards specific V and J segments, and so observed sequence relative abundances may not accurately reflect real amounts. To deal with these issues, 5′ rapid amplification of cDNA ends (5′-RACE) has been used (see refs 14,24,25; Fig. 1b). The group of Daniel Douek at the National Institutes of Health (Bethesda, MD) have recently established their own 5′ RACE protocol.

07% in a relatively large screen

of HLA-A2 donors without melanoma [14]. Interestingly, tetramer-binding CD8+ T cells are Opaganib supplier also detectable in HLA-A2-negative healthy subjects at frequencies that are barely detectable ex vivo and approximately one order of magnitude lower than those detected in the HLA-A2+ individuals [15]. In both HLA-A2+ and A2– healthy donors, the phenotype and functional profile of these tetramer-binding CD8+ T cells are indistinguishable from that of the naïve CD8+ T-cell pool [13-15]. These findings were surprising and had no precedent in either the human or the mouse immune systems. For most other epitopes of CD8+ check details and also CD4+ T cells, the precursor frequency of naïve cells is far below the limit of detection of tetramers by ex vivo, multiparameter flow cytometry analyses. The estimates of such frequencies after magnetic

bead pull down of tetramer+ T cells have been approximated at one specific T cell per one million T cells [16-18]. In fact, the frequencies of Melan-A/MART-1-specific CD8+T cells in healthy individuals are comparable to those measured of T cells specific for some viral epitopes [19]. In sharp contrast, however, T cells specific for viral epitopes are phenotypically and functionally antigen-experienced memory T cells, corresponding to the previous exposure to the respective antigens [20]. Thus, the question was how such an abundant repertoire of naïve antigen-specific T cells could be generated, at least a hundred times more abundant than most other antigen-specific naïve T-cell precursors measured by tetramer binding

assays (Fig. 1). Two major reasons have emerged upon careful study of these cells in the human thymus and the composition of their TCR repertoire. It became clear, on the one hand, that a significant proportion of human subjects (more than half) contain detectable Melan-A/MART-1 tetramer+ CD8+ T cells in cord blood Liothyronine Sodium lymphocytes [21]. Moreover, these cells are also measurable in single CD8+ thymocytes in thymuses from children. Thus, it appears that a high thymic output is one of the reasons for the high frequency of these cells. This is coupled with a slow in vivo turnover of these cells during adult life, as could be directly estimated by measuring two tell-tale features of proliferative history in human lymphocytes: the length of chromosomal telomeres and the levels of TCR-alpha excision circles [21]. To this day, the remarkable stability of the naïve Melan-A specific T-cell repertoire remains most intriguing. Indeed, the antigen Melan-A is normally expressed by melanocytes and even keratinocytes which receive from melanocytes melanosomes containing the Melan-A/MART-1 polypeptide [22].

These cells stimulate T helper type 1 (Th1) helper cells that in turn elicit the production of cytotoxic T lymphocytes (CTL) [22]. These cytotoxic effector cells attack infected cells, resulting in resolution of the infection [23]. However, little is known about how to modulate these immune responses. Prophylactic vaccination. 

Vaccination p38 MAPK phosphorylation with VLPs gives rise to virus-neutralizing antibodies in serum. Vaccination by intramuscular injection of L1 VLPs has been shown to be highly immunogenic and well tolerated in Phase I trials [24–27]. Three randomized placebo-controlled Phase II trials with, respectively, a monovalent HPV16 vaccine, a bivalent HV16/18 vaccine and a quadrivalent HPV6/11/16/18 vaccine candidate have consistently demonstrated almost complete protection against persistent infection with the targeted HPV types [28–32]. Moreover, these trials confirmed Seliciclib order the safety of the vaccines and showed strong immunoresponses that were several orders of magnitude higher than those observed after natural infections. Two pharmaceutical companies [Merck Sharp & Dohme (MSD) and GlaxoSmithKline (GSK)] have completed large multi-centre Phase III vaccine trials

in all continents except Africa [33–35]. In addition, the National Cancer Institute (United States) is conducting a population-based trial in Costa Rica using the bivalent vaccine [36]. These Phase III trials demonstrated that vaccines protect against histologically confirmed high-grade cervical intraepithelial neoplasia (CIN) and adenocarcinoma in situ (AIS) associated with the targeted HPV types under the condition that subjects were not infected with one or more vaccine types at baseline [33–35]. Both vaccine formulations have a good safety profile. Tangeritin Neither has noted any therapeutic effect, as women who test positive for HPV DNA prior to vaccination show no protection against disease end-points associated with that type. Modest cross-protection to closely related high-risk types HPV

31, 33, 45 was found with bivalent vaccine [Cervarix(R)][37] and also to some extent with the quadrivalent vaccine [Gardasil(R)][38,39]. Therapeutic HPV vaccines.  Development of cervical precursors, their maintenance and progression to invasive cancer requires the continued intracellular expression of the viral oncoproteins E6 and E7 [40,41]. Therefore, therapeutic vaccines have been directed towards stimulating T cell responses against these viral early oncogenes. The approaches include administration of peptide antigens or recombinant proteins, plasmid DNA vaccines, viral vector vaccines and administration of E7-pulsed dendritic cells, but despite being variably immunogenic have not shown an impact upon invasive cancer but appear to induce some degree of clearance of cancer precursors or anogenital warts [23,42–44].

151 However, investigators have shown that no interaction occurs when the itraconazole capsule is co-administered with the non-buffered enteric-coated ddI formulation that is currently marketed.152 Early studies of antacid co-administration

with posaconazole tablets suggested that elevations in gastric pH did not produce clinically significant changes in selleck chemical posaconazole concentrations or exposure.153 However, a well-designed study using the currently marketed formulation and a proton pump inhibitor clearly demonstrates that posaconazole absorption is significantly impacted by changes in pH and food.45 Co-administration with a proton pump inhibitor reduces posaconazole Cmax and exposure selleck chemicals llc by 46% and 32% respectively.45 Food, irrespective of whether it is a solid or liquid and regardless of fat content, significantly increases the bioavailability of posaconazole.46,47,153 Indeed, the effect of food on posaconazole

pharmacokinetics is much greater than that of pH.45,153 Increases in gastric emptying caused by prokinetic agents such as metoclopramide may result in reductions in Cmax and exposure that are likely not clinically significant.45 In contrast, the co-administration of this azole with loperamide, an antikinetic agent, produces no clinically relevant effects on posaconazole pharmacokinetics.45 In patients who require acid suppression therapy and treatment with either itraconazole or posaconazole, the interactions can be managed. In patients requiring itraconazole therapy, the solution should be employed. For protracted courses of therapy, the solution may be impractical and an appropriate alternative antifungal agent should be considered. To maximise posaconazole absorption in patients requiring acid suppression therapy, the drug should be administered in divided doses with or after a high-fat meal, or at least with any meal, a nutritional supplement, or an acidic beverage.45 Induction of antifungal biotransformation.  Antifungal agents can produce additive toxicities with other

medicines and alter the distribution, metabolism and elimination of many other drugs. However, few drugs can enhance the toxicity, or decrease the Bacterial neuraminidase serum concentrations or systemic exposure of antifungal agents. Medicines that affect the disposition of antifungal agents do so by inducing enzymes involved in oxidative or conjugative metabolism, or transport proteins. Interactions affecting the disposition of antifungal agents typically involve phenytoin, phenobarbital, carbamazepine, rifampin, ritonavir, efavirenz and other well-known inducers of CYP3A4. In addition, as illustrated by the interaction between rifampin and caspofungin, our understanding of the induction of transport proteins will grow as their role in drug disposition continues to evolve. The majority of interactions affecting the disposition of antifungal agents involves the induction of CYP3A4.

Mice were housed and bred in the Biomedical Research Facility at University of North Dakota. All the animal procedures have been approved by the UND IACUC committee. K. pneumoniae (ATCC 43816 serotype II) was provided by Dr. V. Miller (Washington University, St. Louis) [[41]]. Bacteria were grown overnight in LB broth at 37°C with shaking. The bacteria were pelleted by centrifugation at 5000 × g. We then anesthetized mice with 45 mg/kg ketamine and intranasally instilled 2 × 105 colony-forming units (CFUs) of K. pneumoniae in

PBS (50 μl). BAL was performed 5 times with 1.0 mL volumes of lavage fluid, while the first 0.5 mL was saved separately for cytokine detection. A cell smear was made from Ivacaftor ic50 learn more the BAL fluid and stained with HEMA-3 (Fisher, Rockford, IL) for cell differential counting. AMs were collected

from the BAL fluid precipitate after centrifuging at 2000 × g for 5 min at 4°C and cultivated in RPMI 1640 medium supplemented with 10% newborn calf serum and penicillin/streptomycin in a 5% CO2 incubator. After BAL procedures, the lung, liver, and kidneys were aseptically harvested for homogenization or fixed in 10% formalin or OCT [[42]]. For evaluating bacterial burdens in BAL AMs, and lung tissue, BAL was performed to get rid of the free bacteria. Homogenization of lung tissue was done using liquid nitrogen and samples kept on dry ice before dissolving in RIPA buffer for western blotting analysis or in PBS for CFU and superoxide analysis. For western blotting, the samples were sonicated for three times at 10 s each. Histology slides were made after formalin fixation, and stained with the standard hematoxylin-eosin method [[43]]. For immunohistochemistry assays, we performed OCT fixation and cryosection and stained the slides using the methods described previously [[44]]. AMs were resuspended in lysis solution. Lung or other tissues were homogenized by pestle/mortar in liquid nitrogen and followed

by brief sonication. AMs from BAL fluid or homogenized tissues of the lung, liver, and kidneys were spread on LB plates to enumerate the bacteria that have invaded into AMs or tissues. Free bacteria were killed with polymycin B (200 μg/mL) for 1 h and washed away by lavage. Selected unlavaged selleck inhibitor samples were also saved and assessed to evaluate the differences in cell signaling. The plates were cultured in a 37°C incubator for 18 h, and bacterial colonies were counted [[22]]. Triplicates were done for each sample and control. Cytokine concentrations in BAL fluids (the first 0.5 mL lavage solution) or tissues were measured by standard ELISA kits according to the manufacturer’s instructions (eBioscience company, San Diego, CA) [[45]]. To overcome detection limits (5 pg/mL), we have only used the initial 0.5 mL of lavage solution to determining cytokine concentrations.

The concentration of peptide required to generate 50% of the maximal response was used as a measure of avidity. Mice were sacrificed 14 days after a single priming vaccination. Single-cell suspensions from individual spleens were cultured in complete medium in 25 cm2 upright flasks (3×106 cells/mL) supplemented with 10−8 M of the corresponding PSMA HLA-A*0201-binding peptide and 20 IU/mL IL-2 (R&D Systems, Abingdon, UK). Following 6 days stimulation in vitro, the cytolytic activity of the CTL cultures was assessed in Kinase Inhibitor Library price a standard

5-h 51Cr-release assay. Target cells were labeled with 51Cr with or without peptide for 1 h. Target (T) cells (5×103) were then cultured with effector (E) T cells at different E:T ratios.

Specific % lysis was calculated by the formula: (release by CTL−release by targets alone)/(release by 4% NP40−release by targets alone)×100. Splenocytes harvested from naïve HHD mice were pulsed with 1 mM PSMA27, PSMA663, or control HLA-A*0201-binding anti-PD-1 antibody inhibitor peptide (VLHDDLLEA) at a concentration of 2×107/mL in PBS. The cells were then labeled with either 0.5 or 5 μM CFSE (Molecular Probes, Invitrogen) for 8 min at 37°C before adding FCS to a final concentration of 20% to quench the reaction. After washing, the cells were mixed at a 1:1 ratio such that each prevaccinated mouse received 1×107 cells pulsed with PSMA peptide and the same number pulsed with control peptide in 0.1 mL PBS by intravenous injection. Splenocytes were harvested from individual mice 20 h later, lymphocytes were isolated using density gradient centrifugation and CFSE staining was analyzed by FACS Canto (BD Pharmingen). Lymphocytes from the same mice were also used in an ELISpot assay as described. HHD mice were vaccinated with p.DOM-PSMA27, p.DOM-PSMA663, or p.DOM control vaccine 13 days prior to the assay. TRAMP-PSMA+ HHD+, and TRAMP-HHD+ cells were labeled with 10 and 1 μM CFSE, respectively, as described above and then mixed in a 1:1 ratio. The cell suspension was then mixed

with Matrigel® (BD Biosciences) at a ratio of 1:1 so that each mouse received 1×106 of each population in a total volume of 150 μL by subcutaneous injection. Matrigel® cell suspensions were kept on ice 3-mercaptopyruvate sulfurtransferase until the time of injection, according to the manufacturer’s protocol. After 5 days, the Matrigel® plugs were harvested and digested with 1 mg/mL collagenase/dispase and 0.5 mg/mL DNAseI. CFSE staining of the cells released from the plug was analyzed using a FACS Calibur (BD). The spleens of the same mice were used in an ELISpot assay, as described, to identify those responding to vaccination; animals with an IFN-γ response of less than twice the background or <50 SFCs/106 cells were excluded from the analysis. Experimental groups were compared using a Mann–Whitney U-test. In vivo tumor lysis was analyzed using Fisher’s exact test.

The data showed consistency with a recent report suggesting the expression of Il10 mRNA in CD19+ B cells of draining LN of susceptible mice at the first day post-inoculation, and it was shown that B cells play as a source of IL-10 which influences the susceptibility of BALB/c

mice to L. major infection [30]. At the late stage of the infection, augmented expression of this cytokine was documented at W3 and then tended to gradually decrease at W5 and W8 post-infection. DE5 strain showed the highest level of expression at W3 post-infection. It seems that IL-10 along with IL-4 cytokine is responsible for the susceptibility of the BALB/c mice to L. major infection, as suggested before [31]. Hence, our results showed that the contribution of www.selleckchem.com/products/MLN8237.html DA39 strain in eliciting Il10 mRNA expression is lower than most strains at 16 h and during the late stage of infection. Taken together, the results of this study show that different strains of L. major display different virulence and induce different patterns of cytokine expression in BALB/c mice. While DA39 strain induced the lowest parasite load, high-level expression of Th1-related cytokines mRNA MK-2206 ic50 and higher Ifng/Il4 mRNA ratio in LN of BALB/c mice, the SH25 strain elicited the highest number

of viable parasite in LN of the infected mice and a lower level of Ifng/Il4 mRNA ratio than DA39 strain at 40 h and 8 weeks post-infection. Interestingly, DA39 strain has failed to induce higher expressions of both Il4 and Il10 mRNA, especially at the late stage of the infection. It is noteworthy that in our previous study, similar results in the parasite burden and the generation of IFN-γ induced by DA39 strain were reported at 4 weeks post-infection, however at that study, we reported

higher levels of IFN- produced by DE5 strain than DA39 at W8 post-infection [14]. The reason for this discrepancy may be attributed to the methods used for the cytokine evaluation. It might be considered that the expression of the cytokines mRNA by real-time PCR seems to be a more precise method than assessment of the cytokine in lymphocyte culture. Moreover, the Methocarbamol present study was repeated for three times, and the third experiment results were reported as representative. Therefore, DA39 strain might be considered as an ideal strain for the vaccine studies. In conclusion, our results showed variable parasite loads and different expressions of cytokine mRNA in LN of mice infected with the four strains of L. major. Amongst the four strains isolated from the four endemic areas of Iran and analysed by SSCP, DA39 strain induced lower load of parasites in LN of the inoculated BALB/c mice. Moreover, this strain elicited higher expressions of Ifng and Il12 mRNA and lower expressions of Il4 and Il10 mRNA in draining LN of the infected BALB/c mice at early and late stages post-infection.

It has been shown that recipients with third party anti-HLA Abs (antibodies against HLA antigens that are not donor-specific) have reduced graft survival compared with recipients without any anti-HLA antibodies and furthermore those with DSAbs have worse graft survival than those with third-party anti-HLA Abs.24 Therefore, the presence

of a DSAb suggests inferior graft survival compared with no DSAb even in the presence of a negative CDC crossmatch.23 One advantage Luminex testing has over other forms of crossmatching is the removal of false positives because of antibody binding to non-HLA antigens. In addition, because the antigens present on Luminex can be controlled, confusion regarding the class of HLA they Venetoclax are binding to is eliminated; remembering that in B-cell crossmatching class I and II antigens are present. The presence of a DSAb detected by Luminex in the setting of a negative CDC crossmatch

appears to have prognostic importance in terms of graft survival and acute rejection risk; however, there is insufficient data to determine the meaning of a DSAb with a negative flow crossmatch.19,23,25,29 In each assay negative control beads provide a minimum threshold for a positive result. Positive results can then be graded as weak, moderate find more or strong on the basis of the degree of fluorescence of the positive bead. This result can be scored as a mean fluorescence index or molecules of equivalent soluble fluorescence. The molecules of equivalent soluble fluorescence of a DSAb has been shown to correlate with antibody titre and predict graft failure.30 Recently, it has become evident that while adding sigificantly to the area of crossmatching, Luminex testing has

some limitations including possible interference of the assay by IgM, incomplete antigen representation on bead sets and Farnesyltransferase variation in HLA density on beads.29,31,32 Those interested in more detail regarding Luminex testing should read the recent review paper in this journal covering the topic.26 All the above-mentioned crossmatching techniques attempt to detect a donor-reactive antibody likely to result in acute or chronic antibody-mediated rejection. The presence of sensitization of the cellular arm of the immune system, particularly T cells, can be assessed by cytokine assays such as ELISPOTs. These assays detect the number of recipient T cells producing cytokines such as interferon gamma when encountering donor antigen presenting cells. The assays are conducted in plates coated with a capture antibody for the cytokine of interest. The mixed donor and recipient leucocytes are added to the plate and incubated. After washing to remove the cells the reaction is developed by adding a second antibody for the cytokine of interest and then stained for that antibody.

Over the years, this polysaccharide has been referred to as a PS/A by this research group. Later, Christensen et al. (1990) described a slime-associated antigen (SAA) isolated from the same strain and having a similar function. SAA was claimed to be different from PS/A. However, Baldassari et al. (1996) suggested that SAA and a hexosamine-containing polysaccharide intercellular adhesin (PIA) of S. epidermidis strains RP62A and 1457, Selleckchem RXDX-106 described at that time by Mack et al. (1994), could be the same antigenic molecule. Mack et al. (1996) were the first to elucidate the chemical structure of PNAG (called, according to its biological properties, PIA). Using a combination

of analytical methods and nuclear magnetic resonance (NMR), PIA was identified as a linear β(1,6)-linked N-acetylglucosaminoglycan containing c. 130 N-acetylglucosamine (GlcNAc) residues, partially substituted with O-succinyl groups, partially de-N-acetylated and apparently phosphorylated (Mack et al., 1996). The genes encoding PNAG biosynthesis are

organized in the icaADBC (intercellular adhesion) operon, which consists of four ORFs (Heilmann et al., 1996; Gerke et al., 1998) with a transcriptional repressor gene, icaR, located upstream and transcribed in the opposite orientation (Conlon et al., 2002). The ica locus was later found in a number of S. aureus strains, and its presence was related to the ability to form a biofilm in vitro (Cramton et al., 1999). A recombinant Saracatinib strain of Staphylococcus carnosus (pCN27), containing icaABC of S. epidermidis RP62A, unlike the parent S. carnosus strain, which is biofilm-negative, was adherent to glass and revealed the ability to form intercellular aggregates as well as to produce PNAG (Heilmann et al., 1996). McKenney et al. (1998) subsequently demonstrated that the recombinant S. carnosus (pCN27) antigen was identical to PS/A and ‘chemically related’, but distinct from PIA in molecular size, solubility, and substitution of the majority of the amino groups of the glucosamine residues with succinate. This polymer,

named poly-N-succinyl-β-(1,6)-glucosamine (PNSG), was suggested as a potential vaccine candidate against staphylococcal infections (McKenney et al., 1999, 2000). However, Meloxicam subsequent studies carried out by the same group showed that the presence of the N-succinyl substitution was an analytical artefact (Joyce et al., 2003). These authors used a ‘PS/A overproducing strain’S. aureus MN8m, and the corresponding polysaccharide was named SAE (S. aureus exopolysaccharide). Detailed NMR studies, in combination with the chemical modifications, allowed a complete assignment of NMR spectra of SAE. According to Joyce et al. (2003), the main differences between SAE and PIA were phosphorylation (absence of a phosphate substitution in SAE) and molecular mass [>300 kDa for SAE and ∼30 kDa for PIA (Mack et al.

The results we present here for purified memory-phenotype CD4+ T cells and for effector-memory Th17 cells derived from obstructed kidney indicate suppression of IL-17A secretion comparable to that of naïve CD4+ T cells. In the case of memory-phenotype CD4+ T cells activated in vitro under Th17-skewing conditions, MSC contact was also associated with inhibition of proliferation and of CD25 up-regulation. These results RG7204 concentration are in-line with the in vitro and in vivo findings of Rafei et al. for MSC effects on MOG-specific Th17 cells in mouse EAE 14. In addition, MSC-mediated suppression

of Th17 responses has been reported for antigen-specific Th17 cells in rat EAE and autoimmune myasthenia gravis and in established autoimmune diabetes mellitus in NOD mice 32, 33. Interestingly, however, evidence for enhancement of Th17 differentiation and IL-17A production

by MSCs and fibroblasts has also been presented in a small number of studies 34, 35. The reported results suggested that MSC production of IL-6 as well as stimulation of IL-1 and/or IL-23 secretion by APCs were responsible for the observations 34, 35. In our own experiments, we have observed that administration of a non-selective COX inhibitor in MSC/Th17 co-cultures is associated with enhancement of IL-17A secretion compared with control Th17 cultures (Fig. 5A and our unpublished observation). We have also confirmed production of IL-6 and TGF-β1 by MSCs co-cultured with activated T cells (our unpublished observation). Thus, it is important to consider that MSC selleck Sulfite dehydrogenase inhibition of Th17 cell differentiation and activation, while potent, is conditional, being dependent upon opportune MSC/T-cell contact and upon inducible mechanisms which, when absent or subject to blockade, may unmask a paradoxical

capacity for enhancement of Th17 activity. Furthermore, in the case of naturally occurring Th17 cells from obstructed kidney (or other sites of inflammation and autoimmunity), additional experimental work will be required to distinguish between direct and indirect MSC effects on this T-cell effector phenotype. From a mechanistic perspective, we provide compelling evidence that the induced production of PGE2 by MSCs in direct contact with CD4+ T cells undergoing activation was primarily responsible for suppressive effects on naïve- and memory-phenotype Th17 cells in vitro as well as on in vivo-derived effector-memory Th17 cells. This is consistent with the report of Ghannam et al. in which indomethacin reversed MSC-mediated suppression of Th17 differentiation from human naïve, cord-blood CD4+ T cells as well as IL-17A production by Th17 clones 9. By utilizing FACS to re-purify MSCs, we convincingly demonstrate significant up-regulation of COX-2 and production of PGE2 by these cells within 12–24 h of placement in Th17-skewing cultures.