Previous studies have shown that aspirin desensitization improves

Previous studies have shown that aspirin desensitization improves olfactory function, reduces the need for topical and systemic corticosteroids and reduces infective sinusitis episodes as well as emergency room visits for asthma exacerbations [110,111]. Oral aspirin desensitization protocol is summarized in Example 6. For a more detailed description of preparation of patients for this procedure and treatment of allergic reactions the reader is directed to recently published practice parameter [108] Begin early

in the morning and establish intravenous access. Carboplatin represents the main drug in the management of ovarian cancer, including treatment of relapses. It is usually well tolerated, but up to 27% of patients treated with seven or this website more cycles with this agent develop type 1 hypersensitivity with cutaneous manifestations in > 90% of patients, and up to 77% show cardiovascular compromise [112,113]. Selleck Cabozantinib The non-irritant concentration for skin test is 1–10 mg/ml [114,115]. Rapid desensitization with carboplatin

has been carried out successfully (Example 7) in these patients, and this is associated with disappearance of skin test reactivity. Step Solution Rate (ml/h) Time Dose (mg) Cumulative dose (mg) Reproduced with permission from Lee CW et al. [96]. Solution A: 0·02 mg/ml [total volume 250 ml; total dose 5 mg]; Solution B: 0·2 mg/ml [total volume 250 ml; total dose 50 mg]; Solution C: 2 mg/ml [total volume 250 ml; total dose 500 mg]. Although

several mechanisms have been delineated, in truth no single mechanism is likely to explain all the observed clinical effects and immunological phenomena; this has been described elegantly in recent reviews [116–120]. Noon’s paper cited the work of William Dunbar, who showed that antibodies to the pollen ‘toxin’ were found in hay fever patients and could be induced in animals by injection of pollen. He reasoned that inducing Olopatadine pollen ‘anti-toxins’ in hay fever patients would neutralize the effect of the pollen. Today, IgG4 antibodies directed against the allergen are still measured as evidence of a response to immunotherapy. The precise role of the antibodies is controversial; they are proposed to bind to the allergen and prevent its causing mast cell degranulation via IgE binding. Levels of allergen-specific IgG (total IgG or IgG4) do not predict or correlate with a clinical response to immunotherapy [74–77]. Alterations of allergen-induced cytokine production profile have been demonstrated in various studies. While the changes seen vary between studies, the overall trend observed is for a switch from a pro-allergenic Th2 profile, including interleukin (IL)-4 and IL-5 production, towards a Th1 profile characterized by increased interferon (IFN)-γ production [119,121,122].

Although comparisons of phenotypic activities among these variant

Although comparisons of phenotypic activities among these variants have been attempted, there are few detailed reports on this. In this study, we examined typical EPEC strains isolated from diarrheal and healthy persons for polymorphism of the bfpA and perA genes, presence or absence Palbociclib purchase of BFP-related genes, and such virulence-associated characteristics as autoaggregation, adherence to HEp-2 cells and contact hemolysis. The nucleotide primer sets eaek1/eaek4 and bfpAks/bfpAkcomas were used for PCR to amplify and identify eae and bfpA genes, respectively (Table 1). A total of 53 typical EPEC strains (eae+ bfpA+) isolated in Japan (27 strains) and Thailand (26 strains) from healthy humans and patients with

diarrhea, and 2 reference EPEC strains, E2348/69 (O127a: H6) (17) and 886L (O111: H2), were used in this study. In addition, the KI1924 and KI1455 strains, neither of which has the eae nor bfpA gene, were used as negative controls. The O and H serotypes were determined with antisera kits (Denka-Seiken, Tokyo, Japan) and H8-antisera (Statens Serum Institut, Copenhagen, Denmark). Detection of eae and BFP-related genes (bfpA, bfpF, perA, Kinase Inhibitor Library perC, and pchA) was performed

by PCR using specific primers for amplification. The specific primers used in this study are shown in Table 1. The DNA template was prepared by suspension of a bacterial culture grown overnight on an antibiotic medium 3 agar plate (Difco, BD, Sparks, MD, USA) with 100 μl of distilled water, followed by boiling for 10 min. PCR assays were performed Sodium butyrate in 25 μl of a reaction mixture consisting of PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl, and 1.5 mM MgCl2), 0.1 mM dNTPs, 0.1 μM of each primer, 1 unit/0.2 μl of Taq polymerase (Promega Corporation, Madison, WI, USA) and 2 μl of template DNA. The reactions were run in a DNA thermal cycler 9600 (Roche Molecular Biochemicals, Indianapolis, IN, USA) for 25 cycles of denaturation (94 C for 30 sec), annealing (50 C or 55 C for 1 min), and extension (72 C for

1.5 min), with a final extension at 72 C for 10 min. PCR products were electrophoresed on a 13% polyacrylamide gel electrophoresis system and visualized with ethidium bromide under ultraviolet light. The typing of eae and bfpA was performed by HMA as previously described (34, 35). HMA is a convenient way of determining the similarity of sequences from their heteroduplex mobility in polyacrylamide gel electrophoresis (36). Amplicons obtained from the bfpA-PCR and perA-PCR were subjected to HMA. An appropriate amount of amplicons was mixed with 2 μl of the amplicons from a reference strain, 2 μl of 50 mM EDTA [pH 8.0], and sterile distilled water added to 10 μl. The mixture was denatured at 94 C for 5 min, re-annealed at 72 C for 3 min and at 50 C for 1 hr. The heteroduplexes were electrophoresed on a 10% polyacrylamide gel, containing 5% stacking gel, in Tris-glycine buffer without SDS.

When we try and reach the best coverage of the immunological repe

When we try and reach the best coverage of the immunological repertoire, we actually aim to sequence as many immunoglobulin sequences as possible, out of the whole repertoire. That is, we aim to maximize the ratio between sequenced immunoglobulins (SI) to the total number of immunoglobulins (TI) in

the organism. We aim to reach an SI : TI ratio of 1. When this SI : TI ratio has been reached, an account for the entire repertoire can be obtained. Peptide 17 price Smaller model organisms, therefore, provide a better starting point from which to reach this ratio. Smaller organisms contain significantly fewer cells in total and, obviously, fewer immune cells. Much smaller organisms (e.g. the round worm) are sufficient for some aspects of immunology (see refs 31,32) but not for studying the lymphocyte repertoire. Zebrafish, Danio rario, are an ideal model system for studying the adaptive immune system for two reasons: first, they have the earliest recognizable adaptive immune system whose features match the essential human elements, and second, the zebrafish immune system has only ∼ 300 000 antibody-producing B cells, making it three orders of magnitude simpler than mouse and five

orders of magnitude simpler than human. Recent works study the zebrafish B-cell repertoire via high throughput analysis.33 An important issue in the immune receptor diversity analysis is clone identification, e.g. classification of the obtained reads into clusters, under the assumption that relatively close sequences originate from the same clonally expanded cell. V(D)J segment identification is usually carried out by performing local alignment to germline sequences [available on the International ImMunoGeneTics (IMGT) database34]. However, D segment classification is more complex because of the short length of the sequence, as opposed to V and J genes. Furthermore, nucleotide deletions and P/N additions occur frequently during somatic recombination processes at the V–D and D–J junctions. Much immunological interest is focused on the complementarity determining region 3 (CDR3) of the chains,14,18–20,22,25,33 C-X-C chemokine receptor type 7 (CXCR-7) the most

variable locus of the three CDR regions, and especially the β chain of the TCR.14,18–20,22,25 A recent study focused only on a small portion of the TCR-β repertoire by capturing only sequences generated by a specific gene recombination.22 Read length is a critical parameter in this case, as the entire V(D)J region is ∼ 300 nucleotides in length, including its V and J segments. This has been solved by either using the 454 method (with longer reads), the Douek approach (see above) or special methods of read assembly as in refs 14,25. Once sequences are available, different perspectives portray the repertoire: the size of the repertoire; similarities between repertoires; V(D)J segment use; nucleotide insertions and deletions; CDR lengths; and amino acid distributions along the CDRs.

On the other hand, negative selection in the HY model was slightl

On the other hand, negative selection in the HY model was slightly impaired in KSR1−/− mice. However, a defect in negative selection was not apparent in the AND TCR model system or in an endogenous superantigen-mediated Inhibitor Library manufacturer model of negative selection. These results suggest that, despite a requirement for KSR1 for full ERK activation in thymocytes, full and efficient ERK activation is not essential for the majority of thymocyte selection events.

T-cell development is a complex, multistep process that begins with seeding of the thymus by progenitor cells arising from the bone marrow. Progenitor cells progress through three distinct stages before exiting the thymus as mature T cells into the periphery. These developmental stages can be characterized by the expression of the cell-surface markers CD25, CD44, the coreceptors CD4 and CD8, as well as the TCR itself. Early in development, thymocytes that lack expression

of either co-receptor (dominant negative, DN) begin to rearrange and test their TCR α-chains. Once successful generation of the TCR α-chain has been accomplished, thymocytes begin the processes of positive and negative selection. At this stage, Neratinib datasheet both CD4 and CD8 coreceptors are expressed (DP) and interactions with self-peptide and MHC molecules are critical in determining thymocyte fate. Thymocytes must receive the appropriate signal through their TCR to undergo positive selection in order to escape death by neglect and develop into CD4 or CD8 lineage cells 1, 2. Further, the signal delivered through the TCR via MHC/self-peptide

must not be too strong or the programmed Pregnenolone cell death of thymocytes will be induced, a process termed negative selection 3, 4. The critical role of the ERK-MAPK signaling cascade in T-cell development has been well studied but the results have been inconsistent 5–12. Many of these studies used transgenic mice expressing dominant-negative or constitutively active forms of MAPK pathway components. These studies generated conclusions that ERK was implicated in either positive but not negative selection, or in both positive and negative selection 3, 6, 8, 9, 12. A more definitive study used conditional deletion to remove both ERK isoforms at various stages of thymocyte development 7. These studies demonstrated that thymocytes lacking both isoforms of ERK have a partial developmental block at the DN3 stage. If ERK was deleted following the DN3 stage, however, a complete block in positive selection but not negative selection was observed 7, 13. Interestingly, when double ERK knockout mice were analyzed on a TCR transgenic background, some positive selection did occur. This study also suggested that ERK2 plays a more important role in CD4+ T-cell development compared with CD8+ T-cell development 7. A more recent study has suggested that the degree and duration of ERK activation may distinguish positive and negative selection and possibly CD4 versus CD8 lineage decisions 14.

The rise in IFN-γ observed in mice 24 h after infection with the

The rise in IFN-γ observed in mice 24 h after infection with the self-resolving P. yoelii 17XNL or Plasmodium chabaudi parasite [47, 48] resembled findings in vaccinated mice, compared with unvaccinated controls [24]. Epigenetics Compound Library concentration Both T cells and NK cells contributed to IFN-γ production. Tumour necrosis factor alpha (TNF) concentrations also increased 24 h after infection with P. yoelii 17XNL, P. chabaudi or P. berghei ANKA, although with the latter parasite species they continued

to increase, and 5 and 7 days later, the mice developed signs of experimental cerebral malaria (ECM) [49]. By contrast, early IFN-γ production was associated with the absence of ECM in mice that were infected with both ECM-inducing P. berghei ANKA together with non-ECM P. berghei K173 parasites [50]. This was consistent with the observation of raised concentrations of IFN-γ and TNF soon after FK506 ic50 infection with nonlethal P. yoelii 17XNL [47, 48] or with P. chabaudi AS associated with protective immunity in resistant mouse strains [51]. The presence of high concentrations of TNF [49], including CD4+ T-cell-derived TNF [52],

later in P. berghei ANKA infection was associated with the development of ECM. TNF is also likely to be released by macrophages activated directly by parasite-derived exoantigens [53], including glycosylphosphatidylinositol [54, 55] the anchor molecule for some merozoite and sporozoite

surface antigens [56, 57]. Activated macrophages release both IL-12 [58] and IL-18 that stimulate NK cells to release IFN-γ, leading to further activation of macrophages, amplification of TNF release and increased phagocytic activity. The roles of IFN-γ and IL-12 have been much studied in murine malaria infections. Mice depleted of IFN-γ and IL-12 by specific antibodies and also cytokine gene knockout mice failed to control nonlethal P. chabaudi infections [20], and IL-18 knockout mice failed to control nonlethal P. yoelii 17XNL infections [59]. Conversely, administration of recombinant oxyclozanide IL-12 conferred protection against P. chabaudi infection [20]. Similarly, raised concentrations of IFN-γ and IL-12 during early infection were associated with protection in human malaria [60-62]. Early TNF production was associated with rapid control of parasitaemia and faster recovery in patients with uncomplicated malaria while higher levels of TNF, IL-6 and IL-8 were associated with severity of disease [63, 64]. Treatment with antibody against TNF delayed parasite clearance [65]. Although the persistence of proinflammatory cytokines, in particular TNF and IFN-γ, was associated with severe malaria [66, 67], induction of the anti-inflammatory cytokine IL-10 was critical in preventing severity. Young African children with low levels of IL-10 or high TNF:IL-10 ratios were more likely to die [68, 69].

There is a need for additional, improved animal models to study r

There is a need for additional, improved animal models to study roles of mast

cells and/or their products in vivo. Mast cells are present at all sites through which pathogens enter the body, including the lung. Michael Gurish (Boston, MA) described factors controlling the early mast cell progenitor recruitment to inflamed lung. Pulmonary mast cell progenitor numbers increase dramatically in the lung of sensitized and aerosolized Ag-challenged mice. This increase depends on 4 integrins expressed on the circulating mast cell progenitors and on VCAM-1 and CXCR2 expression on the vascular endothelium 23. It further requires memory CD4+ T cells present at the time of Palbociclib price challenge, as no increase in mast cell progenitors occurs in the lungs of sensitized

and challenged T-cell deficient mice or in WT mice after mAb depletion of CD4+ but not CD8+ cells before aerosol Ag challenge. Sensitized and Ag-challenged IL-9-deficient mice and sensitized WT mice given mAb to IL-9 just before Ag challenge show significant reductions in elicited lung mast cell progenitors. CD1d-deficient mice and WT mice receiving anti-CD1d before Ag challenge also show significant reductions in elicited lung mast cell progenitors, revealing an additional requirement for mast cell progenitor recruitment. Surprisingly, anti-CD1d treatment of IL-9-deficient mice or anti-IL-9 treatment of CD1d-deficient mice did not further Selleckchem MAPK Inhibitor Library reduce the significant partial impairment of mast

cell progenitor recruitment occurring with a single deficiency. Dr. Gurish noted that these findings implicate NKT cells and IL-9 as central regulators that function in the same pathway mediating the Ag-induced increase in numbers of pulmonary mast cell progenitors 24. Mast cells can also participate in T-cell activation. Silvia Bulfone-Paus (Borstel, Germany) addressed host defense by mast cell-CD8+ T-cell interactions. Upon stimulation with polyinosinic-polycytidylic acid (pIC) or Newcastle disease virus, mast cells actively respond to TLR-3 engagement 25. Incubation of mast cells with pIC, which mimics the natural TLR-3 ligand dsRNA, or Newcastle Disease Virus is followed by a rapid phosphorylation of TLR-3 resulting in the production of IFN-β, as GPX6 well as upregulation of costimulatory molecules and the release of chemokines regulating T-cell functions. The production of type I interferons is crucial for the induction of antiviral protein synthesis, the upregulation of TLR-3 expression, and the activation of cytotoxic CD8+ T cells. The upregulation of mast cell costimulatory molecules by pIC indicates the potential of mast cells to shape adaptive antiviral CD8+ T-cell responses 26. The cytokines and chemokines produced by mast cells alter the nature and the strength of the adaptive immune response by specifically recruiting CD8+ T cells to sites of challenge. Further, using three-model Ag, Dr.

6, top left and middle left) Tactile and erogenous sensitivity w

6, top left and middle left). Tactile and erogenous sensitivity was also rated as excellent. Urethrography carried out by the urologists showed a stricture at the urethral anastomosis 4 months postoperatively which required an open urethroplasty. Two months later, another urethroplasty was necessary due to recurrent stricture. Twelve months postoperatively, the patient was able to urinate while standing (Fig. 6, bottom left). No donor-site complications were recorded. The patient regained full range of motion of the wrist with unimpaired

strength. No nerve-related complications were encountered. The patient was a 48-year-old female-to-male transsexual with an osteogenesis imperfecta, arterial hypertension, and a heavy smoking find more history with chronic obstructive pulmonary disease. In this case, vaginectomy had been performed in a previous procedure combined with adnexectomy and hysterectomy. We performed the free sensate RFF-phalloplasty

from the right side using the Chang-design. The microsurgical anastomoses were performed in the right groin: the radial artery onto the common femoral artery in an end-to-side fashion, Vismodegib nmr and three venous end-to-end anastomoses of the flap onto branches of the greater saphenous vein. Both antebrachial nerves were coapted to the ilioinguinal and to one of the dorsal clitoral nerves respectively. The same pharmacological and flap monitoring protocol was followed as for case 1. Starting from POD 11, a partial flap necrosis appeared, Glutamate dehydrogenase affecting the areas of the lateral flap borders. The debridement resulted in a complete loss of the neo-urethra. We decided to apply an identical approach to reconstruct the neo-phallus and the neo-urethra. The same modified, shortened Chang-designed RFF was harvested on the contralateral left forearm. The flap dimensions were identical to the ones described in case 1 (Fig. 2). The anastomoses were carried out in the intact left groin: an end-to-side anastomosis of the radial artery onto the common femoral artery,

one of the comitant veins and a total of three subcutaneous veins of the flap onto branches of the great saphenous vein in an end-to-end fashion. No nerve reconstruction was performed. The postoperative course was uneventful. No flap-related complications occurred. Due to a filiform stricture at the urethral anastomosis, the patient underwent open urethroplasty 10 months postoperatively. Twelve months postoperatively, the patient was able to urinate while standing. The appearance of the neo-phallus was subjectively rated as good, and the patient reported on an excellent tactile and erogenous sensitivity (Fig. 6, right column). No donor-site complications were recorded. Partial flap necrosis is reported to occur in 7–11% of phalloplasty cases.[1-3] The largest series published by Doornaert et al. showed a rate of 7.2% (23 out of 316 cases) with a higher incidence in smokers, patients who insisted on large-sized neo-phalluses, and after anastomotic revision.

These receptors can be classified into two groups, EphAs and EphB

These receptors can be classified into two groups, EphAs and EphBs, based on their sequence homology. Ligands for Eph receptors, so called ephrins, are also divided into two classes. Some are membrane anchored by a glycosylphosphatidylinositol linkage Daporinad ic50 (ephrin-A) and the others through a transmembrane domain (ephrin-B). In mammals, there are nine EphAs that bind to five ephrin-As, and five EphBs (B1, B2, B3, B4, B6) that bind to three ephrin-Bs (B1, B2, B3). The interactions between Ephs and ephrins are promiscuous; one Eph can bind to multiple ephrins and vice versa, including some exceptional interactions between different

classes [[1, 2]]. The ephrins can also function as reciprocal receptors for Ephs and this axis works as a bidirectional signal transduction system between two cells upon selleck chemical direct contact [[2, 3]]. The functions of Ephs and ephrins have been extensively demonstrated in the control of accurate spatial patterning and cell positioning in the development and repair after injury of the nervous system [[2, 3]]. Recent studies have also elucidated cross-talk with many other signaling pathways [[4]] and the critical roles in a wide variety of fields, such as angiogenesis, glucose homeostasis, bone maintenance and remodeling, intestinal homeostasis, and cancer development

[[2]]. While some members of Ephs/ephrins are also expressed in the lymphoid organs [[2, 5, 6]], their physiological role in immune responses are still not known. Studies have shown that a deficiency of certain Ephs leads to a defect in thymocyte maturation because of abnormal development of the stromal cells [[7-10]]. The effects of Eph receptors expressed on mature T cells have been reported, such as modulation

of chemotaxis by certain ephrin-As and ephrin-Bs [[11-14]]. Eph signaling in thymocytes has been reported to blunt the effects of high T-cell receptor (TCR) signaling [[15-17]], suggesting the possible inhibition of negative selection of self-reactive Temsirolimus in vivo thymocytes. In contrast, Wu and colleagues have proposed promotional TCR costimulatory effects of all ephrin-Bs by using their original ephrin-B-Fc chimeric proteins [[18-20]]. However, the molecular basis for an Eph/ephrin system to inhibit or promote TCR signaling in each cell type remains unknown. In the central nervous system, it is now clear that Eph receptors have functional versatility, namely, both repulsive and attractive signals [[21-25]]. This bifunctional guidance cue may be regulated by developmental time and location, most likely characterized by the concentration and combination of the ligands. Recently, another remarkable feature of ephrins, a concentration-dependent transition from promotion to inhibition in retinal axon growth, has emerged for ephrin-As [[21]].

7 ± 41 9, endocardial 130 2 ± 29 2); 70% baseline-flow (epicardia

7 ± 41.9, endocardial 130.2 ± 29.2); 70% baseline-flow (epicardial 160.4 ± 27.7, endocardial 112.1 ± 15.1); 30% baseline-flow (epicardial 44.3 ± 5.5, endocardial 32.9 ± 9); 20 minutes reperfusion (epicardial 175.8 ± 33.6, endocardial 126.5 ± 30); 120 minutes reperfusion (epicardial 146.3 ± 31.1, endocardial 107.1 ± 29.7); and complete LAD occlusion

(epicardial 10.5 ± 5.8 endocardial 1.4 ± 0.3) (r = 0.986–0.962, p < 0.001). Conclusions:  This new blood pressure waveform-triggered laser Doppler probe is able to measure RMBF at different depths online in the beating heart. "
“G-CSF and EPO have shown a notable capability in neovascularization. However, their use is limited because of untoward leucocytosis, erythrogenesis, see more and short half-life in the plasma. Herein, we examined whether G-CSF and EPO released from fibrin gel injected into ischemic tissues would synergistically promote neovascularization with limited systematic effects in a rat hindlimb

ischemic model. In vivo study, group learn more Gel received an intramuscular injection of fibrin gel; group Gel+G-CSF received fibrin gel containing human G-CSF; group Gel+EPO received fibrin gel containing human EPO; group Gel+G-CSF&EPO received fibrin gel containing G-CSF and EPO; group G-CSF&EPO received G-CSF and EPO. Through promoting the expression of SDF-1, local high concentration of EPO could traffic

CXCR4+ cells mobilized by G-CSF Baricitinib to enhance neovascularization in ischemic muscle. The treatment with Gel+G-CSF&EPO was superior to the other treatments on blood flow reperfusion, capillary density, and α smooth muscle actin-positive vessel density. And this treatment induced a modest WBC count increase in peripheral blood. G-CSF and EPO released from fibrin gel had a combined effect on postischemia neovascularization. This treatment may be a novel therapeutic modality for ischemic peripheral artery disease. “
“Please cite this paper as: Chakraborty, Nepiyushchikh, Davis, Zawieja and Muthuchamy (2011). Substance P Activates Both Contractile and Inflammatory Pathways in Lymphatics Through the Neurokinin Receptors NK1R and NK3R. Microcirculation18(1), 24–35. Objective:  The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. Methods:  A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics. Results:  We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs.

The frequency of circulating CD3+ cells was 23 0 ± 6 4% of periph

The frequency of circulating CD3+ cells was 23.0 ± 6.4% of peripheral blood mononuclear cells (PBMC) for the control group and 27.9 ± 10.8% for the Aire group; the difference was not statistically significant. This indicated significant reconstitution of the T cell compartment

in both recipient groups, as the normal frequency of CD3+ selleck chemicals llc T cells in the blood of WT C57BL/6 mice has been reported to be 21% [35]. At the time of termination, we determined the clinical status of the recipients using a clinical score adapted from Cooper et al. [34]. There was no difference between the groups in weight loss or hunching, symptoms that generally indicate wasting. Overall, the clinical scores were similarly low in both groups Dasatinib manufacturer (data not shown) and in both the Aire and control groups the animals remained clinically healthy. Only two animals in the control group lost over 10%

of their original body weight. The recipients were euthanized 2 months after the transfer and tissues harvested for detailed analysis. In PBMC, the frequency of CD3+ cells was comparable in the Aire and control group (25.6 ± 12.0% and 21.9 ± 21.5%, respectively), but the frequency of CD3+ cells expressing the cell cycle marker Ki-67 was significantly higher in the Aire group (Fig. 1A). Similarly, in spleen the frequency of Ki-67+ cells within the CD3+ population was higher in the Aire group (Fig. 1B), and this also resulted in the accumulation of CD3+ splenocytes at a higher frequency (Fig. 1C). In both blood and spleen of the Aire group recipients, the increased expression of Ki-67 was found particularly in the CD8+ T cells (Fig. 1D,E), and in the spleen the Aire group had a significantly higher frequency of CD8+ cells within the CD3+ population (Fig. 1F). Together, these

data show that cells originating from Aire−/− donors hyperproliferate in response to lymphopenia, and that CD8+ T cells are mostly responsible for this hyperproliferation. At the time of termination, we collected tissues reported GBA3 to be targets of the autoimmune attack in Aire−/− animals [9, 10, 12]. Histological analysis of stomach, adrenals, ovaries, liver, salivary glands and pancreas showed low-level lymphocytic infiltrates in the three last-mentioned tissues, but no significant differences were observed between the recipient groups. To confirm this lack of difference with a more quantitative method, we used qPCR to measure the amount of T cell receptor gene constant alpha (TCR Cα) mRNA, normalized against the house-keeping gene Hprt mRNA levels. Again, we did not see any significant difference between the recipient groups (Fig. 2), confirming that T cells were present at equal numbers in the tissues studied in both groups. It has been reported that liver infiltrates in Aire−/− mice consist mainly of B cells [26], so we also measured the amount of CD19 mRNA in the liver tissue of the recipients.