5A–D); this effect was significantly enhanced by TRIF (Fig 5A an

5A–D); this effect was significantly enhanced by TRIF (Fig. 5A and C). Also, suppression of IRF7 expression impaired poly(I:C)-mediated IFN-β gene induction, confirming that IRF7 is involved in poly(I:C)-mediated induction of IFN-β (data not shown). Interestingly, we demonstrate that although ectopic expression of Mal or the TIR domain of Mal dose-dependently inhibited IRF7:TRIF-induced activation of the IFN-β and PRDI-III reporter genes, the N-terminal region of Mal did not (Fig. 5A and C). Additionally, Mal did not affect TBK1/IKKε-induced activation of the IFN-β and PRDI-III reporter genes nor the IRF3/IRF7 transactivation reporter gene induction (Supporting Information Fig. 3).

We also show that Mal and its variants did not significantly affect IRF3:TRIF-induced activation LY2606368 mw of the IFN-β and PRDI-III reporters (Fig. 5B and D). Given that our data suggest that selleck products the TIR domain of Mal negatively regulates TLR3:TRIF:IRF7-induced IFN-β gene induction, we sought to further explore the mechanism involved. Thus, we examined the ability of Mal to modulate poly(I:C)-mediated IRF7 phosphorylation and nuclear translocation 28. We clearly demonstrate that IRF7 undergoes poly(I:C)-induced phosphorylation

and this effect is blocked by Mal (Fig. 6A). Moreover, poly(I:C) induced the phosphorylation of endogenous IRF7 to a greater extent in BMDM lacking Mal (Fig. 6B) and densitometric analysis revealed that ∼50% greater phosphorylation of IRF7 was evident in Mal-deficient cells when compared with WT cells following poly(I:C) stimulation. On the contrary, equivalent IRF3 phosphorylation is evident in WT and Mal-deficient

BMDM following poly(I:C) stimulation (Fig. 6B, lower). As a further test of the negative role of Mal on IRF7 activation, we examined the effect of Mal Thymidine kinase on the nuclear translocation of IRF7. We demonstrate that over-expression of Mal blocked poly(I:C)-induced nuclear translocation of IRF7 (Fig. 6E). As expected, Mal did not affect the nuclear translocation of IRF3 following ligand stimulation (Fig. 6E). We also show that Mal colocalises with IRF7, not IRF3 within the cytosol of HEK293:TLR3 cells (Supporting Information Fig. 4). Together, these data show that Mal inhibits IRF7, but not IRF3, functionality and concomitant IFN-β gene induction. Given that previous studies show an interaction between IRF and Mal 27, we hypothesised that Mal may be directly binding to IRF7 and thus prevent its phosphorylation and translocation. We found that full-length Mal co-immunoprecipitates with IRF7, but not IRF3 (Fig. 6C and D). Further, co-immunoprecipitation experiments show that the TIR-domain of Mal, but not the N-terminal domain of Mal, co-immunoprecipitates with IRF7, but not with IRF3 (Supporting Information Fig. 5) and supports the hypothesis that Mal impacts on TLR3:IRF7, not TLR3:IRF3-mediated IFN-β induction.

Tetracycline-mediated inhibition of de novo bacterial protein syn

Tetracycline-mediated inhibition of de novo bacterial protein synthesis promotes the loss of ubiquitinated proteins from the AVM. This effect is reversible, as removal of tetracycline restores AVM ubiquitination to pretreatment levels. These results demonstrate a novel mechanism

by which A. phagocytophilum remodels the composition of its host cell-derived vacuolar membrane and present the first example of a Rickettsiales pathogen co-opting ubiquitin during intracellular residence. Anaplasma Crenolanib in vivo phagocytophilum is a tick-transmitted obligate intracellular bacterium that infects neutrophils to cause the emerging and acute febrile infection, human granulocytic anaplasmosis (HGA) (Chen et al., 1994; Rikihisa, 2011). In nature, A. phagocytophilum is maintained in an enzootic cycle between its tick vector and mammalian hosts. Humans are accidental hosts. HGA clinical manifestations range in severity from asymptomatic to severe disease and death. Although often self-limiting, severe complications such as prolonged fever, shock, leucopenia, thrombocytopenia, high levels of C-reactive protein

and hepatic transaminases, pneumonitis, acute renal failure, and hemorrhages can result. Doxycycline is the drug of choice for treating HGA (Thomas et al., 2009). Following entry, A. phagocytophilum facilitates survival by replicating exclusively within a host cell-derived vacuole that exhibits altered www.selleckchem.com/products/Gefitinib.html fusogenicity. The A. phagocytophilum-occupied Sinomenine vacuole (ApV) does not mature along the endosomal pathway, does not acidify, avoids lysosomal fusion, and prevents bacterial exposure to reactive oxygen species by avoiding fusion with secretory vesicles and specific granules harboring

NADPH oxidase (Webster et al., 1998; Gokce et al., 1999; Mott et al., 1999; Carlyon et al., 2004; IJdo & Mueller, 2004; Huang et al., 2010a). The ApV is not an inert compartment that is completely sequestered from interfacing with its host cell. Rather, it co-opts membrane traffic, host cell molecules, and cellular processes to camouflage itself and obtain requisite nutrients. For example, the ApV selectively recruits recycling endosome-associated Rab GTPases while excluding Rabs that would otherwise direct A. phagocytophilum to the lysosome (Huang et al., 2010a, b, c). Also, the ApV membrane (AVM) has been shown to accumulate early autophagosomal markers, caveolae markers, and cholesterol, each of which is important for bacterial survival, as well as multiple signaling molecules (Lin & Rikihisa, 2003a, b; Niu et al., 2008). These phenomena serve as harbingers that the A. phagocytophilum likely hijacks additional host cell molecules as part of its intracellular survival strategy. Post-translational modification by ubiquitin is a highly conserved eukaryotic cell-specific process. Ubiquitin is a 76 amino acid protein that is covalently attached to lysine residues of target proteins.

7,28,30 As BAs are part of the enterohepatic circulation, the ile

7,28,30 As BAs are part of the enterohepatic circulation, the ileum, mesenteric lymph node and liver may be candidates as sites where BAs act to modulate DC differentiation. The authors

thank T. Yajima, M. Uo, H. Naruse, S. Ando and Y. Wada for helpful discussions and critical comments. This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Japan Society for the Promotion of Science, and the Keio University Medical Fund. The authors declare no conflict of interests. RI, TT, KY performed the experiments. RI, TT, KY, NK, MK, HH, SO, MW, TK and HI designed the experiments, collected data and wrote the manuscript. T. Hisamatsu reviewed the manuscript GSK458 molecular weight and T. Hisamatsu and T. Hibi supervised and compiled the final version of the manuscript. Figure S1. Cell viability of peripheral blood monocyte derived DCs. Figure S2. mRNA transcript of proinflammatory cytokines in TGR5-DCs. “
“Benaroya Research Institute, 1201 Ninth Avenue, Seattle, WA 98101, USA A fundamental component of signaling initiated by the BCR and CD19 is the activation of phosphoinositide 3-kinase. Downstream

of phosphoinositide 3-kinase, the protein kinase AKT phosphorylates several substrates, including MLN0128 purchase members of the forkhead box subgroup O (Foxo) transcription factor family. Among the Foxo proteins, Foxo1 has unique functions in bone marrow B-cell development and peripheral B-cell function. Here, we report a previously unrecognized role for Foxo1 in controlling the ratio of mature B-cell subsets in the spleen. Conditional deletion of Foxo1 in B cells resulted in an increased percentage of marginal zone B cells and a decrease in follicular (FO) B cells. In addition, Foxo1 deficiency corrected the absence of marginal zone B cells that occurs in CD19-deficient mice. These findings show that

Foxo1 regulates the balance of mature B-cell subsets and is required for the marginal zone B-cell deficiency phenotype Erastin ic50 of mice lacking CD19. BCR crosslinking activates phosphoinositide 3-kinase (PI3K), the lipid products of which orchestrate the assembly of membrane-associated signaling complexes 1. One group of proteins, termed the BCR signalosome, is responsible for maximal activation of phospholipase Cγ and subsequent phosphoinositide hydrolysis and Ca2+ mobilization. Another outcome of PI3K signaling is the activation of AKT. The AKT serine/threonine kinases have numerous substrates, whose phosphorylation state controls diverse processes including proliferation, survival, metabolism and differentiation. The roles of most AKT substrates in B-cell biology have not been defined. CD19 is a transmembrane protein that enhances BCR signaling by multiple mechanisms 2, 3.

We performed a multicentre, observational,

We performed a multicentre, observational, Fer-1 manufacturer retrospective study in 17 renal transplant units from Spain. We collected data from renal recipients

with hypercalcaemic (calcium >10.2 mg/dL) SHPT (intact parathyroid hormone (iPTH) > 120 pg/mL) who initiated cinacalcet in the clinical practice. We included 193 patients with a mean (standard deviation (SD)) age of 52 (12) years, 58% men. Cinacalcet treatment was initiated at a median of 20 months after RT (median dose 30 mg/day). Mean calcium levels decreased from a mean (SD) of 11.1 (0.6) at baseline to 10.1 (0.8) at 6 months (9.0% reduction, P < 0.0001). Median iPTH was reduced by 23.0% at 6 months (P = 0.0005) and mean phosphorus levels increased by 11.1% (P < 0.0001). The effects were maintained up to 3-years. No changes were observed in renal function or anticalcineurin drug levels. Only 4.1% of patients discontinued cinacalcet due to intolerance and 1.0% due to lack of efficacy. Idasanutlin manufacturer In renal transplant patients with hypercalcaemic SHPT,

cinacalcet controlled serum calcium, iPTH and phosphorus levels up to 3 years. Tolerability was good. “
“To determine whether complexity of chromatin structure in kidney macula densa cells (MDC) decreases during postnatal development in mice. The levels of chromatin structural complexity were measured by determining fractal dimension of MDC nuclei. Kidney tissue was obtained from the total of 32 male Swiss albino mice divided into four age groups (n = 8): newborn (0 days), 10 days old, 20 days old and 30 days old. For a total of 640 MDC chromatin structures, fractal dimension, lacunarity, as well as parameters of Grey level co-occurrence matrix STK38 (GLCM) texture were determined. Chromatin fractal dimension in animals aged 10 days, 20 days and 30 days was significantly lower (P < 0.05,

P < 0.01 and P < 0.001, respectively), compared with newborn mice. This complexity reduction of chromatin architecture is in accordance with previously published studies, which detected generalized and sustained loss of both tissue and cell complexity during aging. The loss of complexity was texture-independent, since there was no statistically significant difference (P > 0.05) in both chromatin angular second moment and inverse difference moment between the age groups. Our results indicate that age-related nuclear intrinsic factors which do not influence chromatin texture may have an important role in MDC postnatal development. Macula densa (pars maculata tubuli distalis nephroni) represents a group of epithelial cells in the wall of the nephron distal convoluted tubule, near the vascular pole of the kidney glomerulus. These cells have an important role in regulation of glomerular filtration rate.

While mild, well-controlled asthma is not a contraindication for

While mild, well-controlled asthma is not a contraindication for SCIT with

aero-allergens, injections must not be administered during intercurrent respiratory infection when there is exaggerated bronchial reactivity, which may predispose patients to the development of a systemic reaction. Poor asthma control is suggested by excessive use of short-acting β2 agonist (more than twice a day), nocturnal symptoms, recurrent courses of oral steroids and hospitalization for acute asthma. A more objective evaluation of asthma control can be obtained by reviewing peak flow charts recorded twice daily for 3–4 weeks with documentation of click here short-acting β2 agonist usage, as well as baseline spirometry [forced expiratory volume in 1 s (FEV1) should be ≥ 70% predicted]. VIT injections must also not be administered during intercurrent respiratory infection. It is imperative to optimize the anti-inflammatory

therapy for asthma prior to commencing VIT and to perform an objective evaluation XAV-939 supplier of asthma as above. VIT is contraindicated in severe or ‘brittle’ asthma, but the approach is somewhat different in moderate asthmatics where a careful ‘risk–benefit’ analysis must be performed for VIT, taking into consideration co-morbid factors, occupation, hobbies and social circumstances as well as patient choice. Allergen immunotherapy in any form must not be initiated during pregnancy [38]. Although allergen immunotherapy is not known to have teratogenic effects, it should ideally be avoided in pregnancy, even in patients established on treatment who are in maintenance phase, in view of the rare but real possibility of anaphylaxis which may cause fetal Proteases inhibitor hypoxia [38]. Beta-blocker therapy is generally

considered an absolute contraindication during allergen-specific immunotherapy due to the risk of refractory anaphylaxis [36–38,80]. This is related to reduced therapeutic efficacy of adrenalin in anaphylaxis due to underlying beta blockade. Therefore, as far as possible, it is better to avoid beta-blockers during immunotherapy, but there are some special circumstances in patients requiring VIT where withdrawal of beta-blockers may put the patient at risk (such as of underlying tachyarrhythmias) [80,81]. In such circumstances, a careful ‘risk–benefit’ analysis must be undertaken, and liaison with the patient’s family physician and cardiologist will be beneficial. Where benefit of continuation of treatment of beta-blocker clearly outweigh the risk of their discontinuation, short-acting beta-blockers may be discontinued temporarily prior to injections or during the induction phase of VIT. Some groups have undertaken VIT successfully alongside treatment with beta-blockers. In such circumstances, glucagon must be readily available to treat refractory anaphylaxis [80].

gov were searched Obesity was defined as a BMI ≥ 30 Comparable

gov were searched. Obesity was defined as a BMI ≥ 30. Comparable data from observational studies find more was combined for pooled analysis and quality assessment of observational studies was performed. Fourteen studies met the inclusion criteria (n = 6,043 patients). Pooled data analysis demonstrated significantly higher prevalences of overall complications, recipient site complications overall, donor site complications overall, donors site wound infection, donor site seroma, abdominal bulge/hernia, mastectomy skin flap necrosis, recipient site delayed wound healing, and partial flap failure, in obese (BMI ≥ 30) compared with nonobese (BMI < 30) patients. A BMI

of 40 was identified as a threshold at which the prevalence of complications became prohibitively high. No randomized-controlled trials were found and all studies had methodological weaknesses. Complications in obese patients following free autologous breast reconstruction were higher than in their nonobese counterparts; however the majority of these NVP-BGJ398 cell line complications were reported in the studies as being minor. Until better evidence is available this information will help when counseling patients. © 2014 Crown Copyright. Microsurgery 34:484–497, 2014. “
“In spinal cord injuries at the C6 level, elbow extension is lost and needs reconstruction. Traditionally, elbow extension

has been reconstructed by muscle transfers, which improve function only moderately. We have hypothesized that outcomes could be ameliorated by nerve transfers rather than muscle transfers. We anatomically investigated nerve branches to the teres minor and posterior deltoid as donors for transfer to triceps motor branches. In eight formalin-fixed cadavers, the axillary

nerve, the teres minor branch, the posterior deltoid branch, the triceps long and upper medial head motor Dimethyl sulfoxide branches, and the thoracodorsal nerve were dissected bilaterally, their diameters measured and their myelinated fibers counted. To simulate surgery, using an axillary approach in two fresh cadavers, we transferred the teres minor or the posterior deltoid branch to the triceps long head and to the thoracodorsal nerve. The posterior division of the axillary nerve gave off the teres minor motor branch and then the branch to the posterior deltoid, terminating as the superior lateral brachial cutaneous nerve. The diameters of the teres minor motor branch, posterior deltoid, triceps long and upper medial head branches, and the thoracodorsal nerve all were ∼2 mm, with minimal variation. The nerves varied little in their numbers of myelinated fibers, being consistently about 1,000. Via an axillary approach, either the teres minor or the posterior deltoid branch could be transferred directly to the thoracodorsal nerve or to triceps branches without any tension. © 2011 Wiley-Liss, Inc. Microsurgery, 2011.

Third, bounded by the progress of renal function, the area under

Third, bounded by the progress of renal function, the area under the curve of serum NGAL was 0.872 (95% confidence interval, 0.786–0.933), which suggests a blood NGAL cut-off level of 246 ng/mL (sensitivity 85.19%, specificity 81.54%). Fourth, Kaplan–Meier survival curve analysis showed that the serum NGAL level was closely related to the

end-point of renal function in patients with CKD. Fifth, Cox multivariate regression analysis showed that the estimated glomerular filtration rate and blood NGAL are associated with progression of CKD. Serum NGAL is an effective biomarker for detecting early-stage renal damage in CKD patients. Serum NGAL was significantly correlated with the severity of renal damage and the progression of renal Selleck RO4929097 function deterioration. “
“Aim:  Oxidative stress and ischaemia are suggested as possible mechanisms of contrast-induced nephropathy (CIN). Statins may offer renoprotection in both acute and chronic kidney diseases because of their antioxidant and anti-inflammatory properties. We investigated whether use of statins before non-emergent percutaneous coronary intervention

(PCI) reduces the incidence of CIN. Methods:  We retrospectively evaluated 540 consecutive adult patients who underwent non-emergent PCI over a 3 year period at a tertiary care centre. CIN was defined as 25% or 44 mmol/L increase from baseline creatinine at 48–72 h. In addition, we classified patients based find more on Mehran score for risk of development of CIN and analysed the effect of statins. Results:  Three-hundred and fifty-three patients

met inclusion Ergoloid criteria. Two-hundred and thirty-nine patients were taking statins before PCI and 114 were not. Baseline characteristics were similar for both groups. CIN occurred in 75 patients (21.2%). There was a higher incidence of CIN in patients on statins as compared with patients not on statins (24.7% vs 14%; 95% CI: 1.09–3.67; P = 0.02). However, propensity-based adjustment for receipt of statins revealed no significant differences in CIN between both groups (OR: 1.6; 95% CI: 0.87–3.22; P = 0.12). Multivariate logistic regression revealed Mehran score to be independently predictive of CIN. None of the patients who developed CIN required dialysis. Conclusions:  Statin use before non-emergent PCI is not associated with reduction in CIN. Further randomized controlled trials based on proper risk adjustment for development of CIN are needed. “
“Atheromatous renovascular disease is an increasingly common diagnosis in our ageing population. Although there is a wide spread of experience in its treatment, the evidence base is heterogeneous and inconclusive. The Angioplasty and Stenting for Renal Artery Lesions trial has provided some, but not all, answers regarding the place of renal revascularization therapy and has also raised more questions and generated further debate.

IFN-β-mediated immunomodulatory functions may differentially oper

IFN-β-mediated immunomodulatory functions may differentially operate depending on the responding cell subset acting on T- or B-cell proliferation, find more 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 Lenvatinib molecular weight 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 Terminal deoxynucleotidyl transferase 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].

1c) These

1c). These Decitabine results suggest that the C-terminal transactivation domain and the phosphotyrosine-mediated dimerization, are

not important for the regulation of constitutive GILT expression. The remaining portion of STAT1 includes the DNA-binding domain,27,28 which may be responsible for constitutive binding of STAT1 to the GILT promoter. Previously, several groups have shown that the mutation of specific amino acids within the DNA-binding and linker regions in Stat1 can affect Stat1 binding and nuclear retention.29–31 Thus, we generated three Stat1 constructs mutated at DNA-binding sites and tested them in the luciferase reporter gene assay. The first mutant, Stat1-V426D/T427D, is defective in IFN-γ-induced Stat1 DNA binding to specific GAS sites and also shows weakened, non-specific protein–DNA interactions.29 The DNA-binding-deficient Stat1 mutant, E428A/E429S, has been shown to be tyrosine phosphorylated in response to IFN-γ and can be translocated to the nucleus, but cannot induce activation of the reporter gene.30 The third DNA-binding

mutant, Stat1-K544A/E545A, previously characterized by Darnell et al.,31 has been shown to have increased off-rates from GAS sites. Hence, this mutant is present at the GAS sites for much shorter times than the WT protein but has this website been found to accumulate within the nucleus upon IFN-γ stimulation.29Stat1−/− and WT MEFs were co-transfected with a firefly luciferase reporter gene under the control of GILT promoter and either WT Stat1α or one of the three described DNA-binding mutants. Expression of either Stat1α (Fig. 2a) Exoribonuclease or two of the DNA-binding mutants (E428A/E429S and K544A/E545A) (data not shown) in Stat1−/−

cells, decreased the luciferase activity. However, the cells transfected with the DNA-binding mutant V426D/T427D behaved like Stat1−/− cells, suggesting that this particular site is important for constitutive binding of STAT1 to GILT promoter in MEFs. Promoter regions of IFN-γ-inducible genes usually have a conserved nucleotide sequence, TTNCNNAA, known as the GAS, which directs rapid transcriptional activation upon Stat1 binding.28 Therefore, the mouse GILT promoter was analyzed for transcription of GAS sites using the Matinspector program.32 Two putative GAS sites were identified (Fig. 3a). Biotinylated oligonucleotides corresponding to these two sequences – STAT1 GAS Site Probe 1 (GCGGAGCCTTCAGGAAAGGAGTCCCAGG) and STAT1 GAS Site Probe 2 (CACACTCAGTTGCTGGAAGCAAGTACCTCA) – were tested for their ability to bind Stat1 in DAPA.33 These oligonucleotides were incubated with whole-cell lysates from WT or Stat1−/− MEFs (Fig. 3b). In order to confirm the specificity of binding, lysates from Stat1−/− and WT MEFs were also tested for binding in the presence of excess non-biotinylated competitors: either with excess Stat1 consensus sequence or with excess of a non-specific p53 oligonucleotide (Fig. 3c).

Mast cells are activated by antigen crosslinking of IgE-bound hig

Mast cells are activated by antigen crosslinking of IgE-bound high-affinity receptor for IgE (FcεRI) receptors, and aggregation of these receptors results in rapid phosphorylation of tyrosine residues in the ITAMs of β and γ chains by lyn kinase, which leads to recruitment and activation of spleen tyrosine kinase (syk) and fyn. Selleck MLN0128 Both fyn and syk phosphorylate downstream targets, leading to calcium mobilization,

degranulation, arachidonic acid metabolization, and cytokine and chemokine gene transcription 9, 10. As opposed to activation, desensitization is a process in which mast cells are rendered hypo-responsive to an activating challenge, either by exposure to low antigen doses in calcium-depleted conditions 11 or by exposure to incremental

doses of antigen, in the presence of calcium 12, 13. Calcium-depleted conditions cannot be applied to human desensitizations, and few studies have addressed physiological desensitizations, since events occurring in the absence of extracellular calcium may not reflect the same pathways DAPT in vivo as those occurring in the presence of calcium 14. Internalization of FcεRI through progressive crosslinking at low levels of antigen has been postulated as the likely mechanism for cell-surface depletion of IgE and cellular unresponsiveness to specific activating doses of allergen 12. Depletion of molecular targets of activation such as syk has been shown in prolonged antigen desensitization, indicating a universal rather than specific desensitization 15. Based on our previous study 16, we report here a model of mouse BM-derived mast cell (BMMCs) specific rapid desensitization to DNP and OVA antigens in the presence of physiologic levels of calcium. Increasing doses of antigen delivered at fixed time intervals induced a highly specific and prolonged hypo-responsiveness to triggering doses of the desensitizing antigen.

Mast cells desensitized to DNP or OVA demonstrated almost complete inhibition of β-hexosaminidase and pre-formed TNF-α release, calcium flux and arachidonic acid metabolization. They did not release significant amounts of newly generated IL-6 or TNF-α and failed to phosphorylate STAT6 and p38 MAPK. When sensitized to both DNP and OVA antigens, DNP-desensitized cells responded fully to OVA and vice versa. Most importantly, Lepirudin specific rapid desensitization targeted the internalization of antigen/IgE/FcεRI complexes since antigen-specific IgE bound to the α chain of the FcεRI remained at the membrane level. This model may provide support for the specificity and effectiveness of human desensitizations. In order to compare single-dose antigen delivery (activation) with sequential cumulative doses (rapid desensitization), we first assessed the dose response curve to DNP-human serum albumin conjugated (DNP-HSA) antigen, by β-hexosaminidase release, with cells sensitized with anti-DNP IgE (see Fig. 1A).