A 67-year-old Japanese woman had worsening edema in her right thigh and hip area for 3 years. She had previously undergone extended hysterectomy with lymph node dissection for endometrial cancer 8 years before. Indocyanine green test showed antegrade and retrograde lymph flow. Four LVAs were made in the right medial thigh and right lower abdominal area under local anesthesia. Lymphedema showed rapid improvement within 12 months and compression therapy was not required at 24 months after LVA. Retrograde LVA has a possibility of a more efficacy for secondary lymphedema. © 2012 Wiley Periodicals,

Inc. Microsurgery, 2012. “
“Free tissue transfer has become a popular technique LY2606368 manufacturer for soft tissue defect reconstruction in head

and neck cancer ablation. Although high success rates and good reliability of free flaps are proven, microvascular thrombosis is still the most critical issue for microsurgeons. Pharmacological antithrombotic agents are widely used but their efficacy is still debated. In this study, we analyzed whether prostaglandin-E1 (PGE1) and dextran-40 can improve the outcomes compared to no antithrombotic therapy at all. We retrospectively reviewed 1,351 free flaps performed for head and neck reconstruction after cancer ablation. Three groups defined were 232 flaps received PGE1, 283 flaps received dextran-40, and 836 received no antithrombotic therapy. LBH589 The demographics of these three groups indicated no statistical differences. The results showed that flap survival revealed no significant Flavopiridol (Alvocidib) difference among PGE1, dextran-40, and control group (P = 0.734). There was a tendency to hematomas in PGE1 group (P = 0.056) when compared with other two groups. Dextran-40 significantly increased flap failure rate in high-risk patients with diabetes mellitus (P = 0.006) or hypertension (P = 0.003), when compared with PGE1 and control group. These results revealed antithrombotic therapy with PGE1 and dextran-40 do not determine a significant improvement in flap survival. © 2012 Wiley

Periodicals, Inc. Microsurgery, 2012. “
“Injuries of the common peroneal nerve (CPN) are frequent and associated with poor motor outcomes. So far, the opinion is held, that nerve reconstruction is reasonable and indicated up to 6 months after injury. We describe successful sural nerve interposition grafting in a patient with neuroma-in-continuity formation of the CPN, presenting with foot drop, 13 months after injury. Due to this positive result, we think nerve grafting in neuroma-in-continuity lesions of the CPN should be contemplated in patients with foot drop even more than one year after injury. © 2012 Wiley Periodicals, Inc. Microsurgery, 2013. “
“We developed a biodegradable poly-lactide (PLA) film with a honeycomb-patterned porous structure (honeycomb film). This study investigated the use of this film in neurorrhaphy.

CBA data was analysed using fcap Array software (BD Biosciences). learn more Statistical analyses were performed with GraphPad Prism software (Graphpad Software, Inc., La Jolla, CA, USA). Significance was determined using Kruskal–Wallis analysis with

Dunn’s multiple comparisons post-test and Wilcoxon tests. We analysed NKT cells isolated from fresh human thymus, spleen, cord blood and adult peripheral blood. The mean NKT cell frequency of donor tissues were similar for peripheral blood (0·1 (mean) ± 0·02 [standard error of the mean (s.e.m.)], cord blood (0·06 ± 0·01) and spleen (0·08 ± 0·03), but significantly lower in thymus (0·007 ± 0·001). Most (> 90%) thymus and cord blood NKT cells were CD4+, with CD4− NKT cells seen mainly in peripheral blood and spleen (Fig. 1). In contrast to findings in mice that blood NKT cells provide a poor measure of NKT cell frequency in spleen [18], we found that human spleen and blood had similar mean frequencies of NKT cells and of CD4+ and CD4− NKT cell subsets, although this applies

to group analysis, rather than to each individual donor. A recent publication identified diversity within CD4+, CD4− and CD8+ NKT cell subsets, but these cells had been expanded prior to analysis. We analysed cell surface antigen expression by CD4+ and CD4− NKT cell subsets without in-vitro expansion and compared blood-derived NKT cells to those from selleck chemical cord blood, thymus and spleen (Fig. 2). Many antigens were expressed differentially by the CD4+ and CD4− NKT cell subsets (Fig. 2a–j), including CD56 and CD161 (confirming these as ineffective surrogate markers for human NKT cells), with CD161 expressed more highly in peripheral blood and spleen Olopatadine than cord blood or thymus. This confirms CD161′s status as a marker of NKT cell maturity [19, 22, 23]. Interestingly, CD161 was expressed by more CD4− than CD4+ NKT cells (Fig. 2a), which supports the hypothesis that comparatively immature precursors

of CD4− NKT cells are present within the CD4+ subset [22] [19, 23]. Our analysis did not identify any preferential surface antigen expression by either of the CD4+ or CD4 NKT cell subsets. CD8, CD45RA and CD94 were expressed typically by more CD4− NKT cells (Fig. 2i,j and data not shown), whereas CD62L, CD127 and LAIR-1 (Fig. 2c,d,b) were expressed by a higher proportion of CD4+ NKT cells. CD25, CD56, CD16, CD45RO, CD84, CCR7 and signalling lymphocyte activation molecule (SLAM) were expressed differentially by both CD4+ and CD4− NKT cell subsets, but the pattern of expression was similar for each subset (Fig. 2a–j and data not shown). NKT cells from thymus, cord blood, peripheral blood and spleen expressed similar levels of most antigens, although there were exceptions: CD4 was expressed by more NKT cells in thymus and cord blood, CD161 was higher in peripheral blood, CCR7 expression was lowest in peripheral blood and CD25 was highest in cord blood.

3). As these mice received different wt vaccines (Table 1), the potential bactericidal activity elicited in NMRI mice by the increased OpcA level in the wt 1 vaccine (Fig. 1A) was not apparent with the target strain of low OpcA expression, as noted above. NMRI mice responded to the wt vaccine with similar titres as C57BL/6 mice receiving the Omp85+ vaccine, but they were lower compared with the Omp85+ vaccine in Balb/c mice (P = 0.008). The titres induced by the two wt vaccines in Balb/c mice were not www.selleckchem.com/products/AG-014699.html significantly different (data not shown).

With target strain B1723, all mice strains had significantly lower serum bactericidal titres (P ≤ 0.001) compared with strain 44/76 (Fig. 3). Only a few of the total sera (3/47; 6.4%) had log2 titres > 2 with strain B1723. Six sera from Balb/c mice with high Omp85 antibody levels following the Omp85+ vaccine (Fig. 2A) and six sera from Balb/c mice immunized with the wt vaccine were also tested in SBA with two heterologous meningococcal strains. No titres (i.e. log2 < 3) were observed

with strain B16B6 (B:2a:P1.5,2), whereas low titres (log2 range 3–4) were found with strain B:4:P1.19,15 that were not significantly different for the two vaccines. These results supported those with strain B1723 of PorA being the dominant bactericidal antigen. Pooled sera from Balb/c and C57BL/6 mice, immunized with the Omp85+ or wt vaccines, were tested in OPA with live 44/76 meningococci.

For each mouse strain, distinct opsonic titres were obtained that were similar for the two vaccines (log2 17-DMAG (Alvespimycin) HCl titre of 7 for Balb/c Rapamycin purchase mice and log2 titre of 6 for C57BL/6 mice). Adsorption of the same sera with recombinant Omp85 coupled to magnetic beads, followed by OPA with the PorA-negative strain B1723, gave lower but similar titres for the two vaccines. Thus, the OPA and SBA experiments indicated that the increased Omp85 levels in the Omp85+ vaccine did not induce higher functional antibody activities than the wt vaccine. In this study, the vaccine potential of meningococcal Omp85 was investigated in terms of the functional serum bactericidal and opsonic activities raised in inbred mouse strains (C57BL/6 and Balb/c mice) and in outbred strains (OFI and NMRI mice). Because Omp85 is essential for bacterial viability, knockout mutants of Omp85 are unavailable for such studies [22, 41]. We therefore examined the functional activities in the mice following immunization with a genetically modified OMV vaccine expressing fivefold higher Omp85 levels than a control wt vaccine. The increased expression of Omp85 was found to induce high antibody levels, but these antibodies did not appear to have higher functional activities related to protection against meningococcal disease [14, 15, 42] than the wt vaccine. Specific Omp85 and PorA antibody levels were measured by digital scanning of the same immunoblots with denatured Omp85+ OMV as antigen.

Health-related quality of life in elderly dialysis patients appears to be decreased compared with elderly persons in the general population[19] although may be better preserved

than in a younger cohort of patients where the perceived reduction in health-related quality of life associated with dialysis is greater.[20] Many factors will impact on a patient’s quality of life and may influence their decision to dialyse or not. An important concept is that of hospital free survival. Dialysis in elderly patients is associated with increased hospitalization with rates of hospitalization in elderly RRT patients of 20–35 days per year[9, 21] compared with 10–16 days per year[9, 17] in those on non-dialysis pathways. One UK study published by Carson et al.[9] concluded that elderly haemodialysis patients spent almost 50% of the time they survived in hospital or attending to dialysis compared with those on non-dialysis CHIR-99021 price pathways who spent just 4.3% of their days. This crucial information is frequently not imparted

to patients or considered by nephrologists when discussing the option of RRT. Evidence Bortezomib supplier also exists that elderly dialysis patients have one of the highest prevalence rates for frailty of any single population and that initiation of dialysis may be associated with considerable functional decline. Jassal et al.[22] showed that in those aged ≥80 who commenced dialysis (80% of whom were living independently at home), 30% had functional

loss 6 months after dialysis initiation (required community/carer support or transfer to a nursing home). Another study by Kurella Tamura et al.[14] showed that the majority of elderly nursing home residents have died (60%) or lost function (27%) 12 months after dialysis initiation. The elderly can have specific medical issues and needs that are best assessed by an Aged Care Physician. This is recommended particularly when assessment of cognitive function is a part of the considerations in determining whether dialysis is appropriate or not. Finally carers of elderly dialysis patients also have impaired quality of life with all components of The Short Form (36) Health Survey (SF36) affected and 32% of carers with signs of depression in one study.[23] We have no information on the impact of carers of elderly patients on non-dialysis pathways and further studies are required. acetylcholine Jennifer Robins and Ivor Katz Documenting five key variables important in determining mortality associated with dialysis: Nephrologist response to the Surprise Question. Age. Comorbidities. Functional status. Nutritional status. Use of the Surprise Question in all patients: on dialysis or those patients on, or being considered for, a non-dialysis pathway. Use of the clinical score by Couchoud et al. (2009) for patients being considered for a non-dialysis pathway. Use of the modified Charlson score (MCS) and the clinical score by Cohen et al.

This specific induction has been demonstrated to be mediated by Ag presentation mechanism — via CD80/86, HLA-DR — and IL-15 pathways [102]. Together, the above findings support a model in which LCs provide important regulatory feedback to the immune system, but also selectively contribute to effector T-cell responses. Resident commensal organisms on the skin are necessary for optimal cutaneous immunity, through the increase of IL-1β signaling and amplifying responses in accordance with the local inflammatory environment [85]. Screening mice deficient in factors known to drive IL-17A production, Hanski et al. showed

that IL-1R1, and its downstream signaling complex MyD88, play a dominant role JNK inhibitor in controlling the production of IL-17A, but not IFN-γ, by cutaneous T cells. selleck IL-1α production by cutaneous cells was significantly reduced in germ-free

mice and monoassociation with S. epidermidis restored the production of this cytokine, showing that resident bacteria are necessary to drive effector T-cell function in the skin [85]. The skin can be a point of entry for fungal infections when the epithelial barrier is breached, or it can be a site for disseminated, systemic fungal diseases. For example, the dryness associated with AD compromises the barrier function of the skin and as a result AD is associated with high susceptibility to viral, bacterial, and fungal skin infections [103]. To determine whether

the skin microbiota of patients with AD is different from that of healthy individuals, Zhang and co-workers used an rRNA gene clone library of 3647 Lonafarnib mw clones to identify 58 fungal species and seven unknown phylotypes from AD patients and healthy individuals [104]. As expected, Malassezia species were predominant in AD patient skin, accounting for 63–86% of the clones identified from each subject. Overall, the non-Malassezia yeast microbiota of the patients was more diverse than that of the healthy subjects. Candida albicans, C. diffluens, and C. liquefaciens as well as the filamentous fungi Cladosporiumngi spp. and Toxicocladosporium irritans were detected in AD samples but were seldom detected in healthy samples [104]. Although Malassezia yeasts are a part of the mycobiota of healthy skin, they have also been associated with a number of diseases affecting the human skin, such as pityriasis versicolor, folliculitis, seborrhoeic dermatitis and dandruff, psoriasis, and AD (for a review see [105]). Changes in the fungal microbiota of the scalp that accompany dandruff have been examined [106]. While fungi of the Ascomycota dominated in both healthy individuals and dandruff patients, fungi of the Basidiomycota phyla (which include Malassezia) were significantly increased in dandruff-afflicted scalps [106].

As described below, repeated measures of spleen volume and cell content were made learn more in four inoculated calves whereas change in regional distribution of phenotyped cells was determined by sequential euthanasia of six inoculated calves in comparison with two un-inoculated calves. Magnetic resonance imagery was performed with a 1·0 Tesla machine (Philips Intera, Andover, MA, USA). Sequences were acquired in a dorsal plane. The area imaged was from the spine to the ventral abdominal wall. A 40 cm field-of-view ensured that the entire spleen could be visualized. One-centimetre-thick

slices with a 2 mm gap were acquired using a short tau inversion recovery (STIR) sequence. This sequence resulted in a hyperintense spleen on a low intense background. The volume was calculated by tracing the outline of the spleen for the area on each slice and multiplying by the number of slices plus gap thickness

(3D-DOCTOR; Able Software Corporation, Lexington, MA, USA). Each calf’s spleen volume was calculated on the day prior to infection and then at 11 or 12 dpi, 2 calves each. Immediately following each MRI procedure, a 1 cm3 biopsy of marsupialized spleen was removed under local Erlotinib manufacturer lidocaine anaesthesia for determining differential cell counts. Each biopsy was immediately processed into a single cell suspension using a tissue grinder (Tenbroek; Bellco Glass, Inc., NJ, USA), suspended in 50 mL of PBS and enumerated for differential cell counts by standard methods used for whole blood (28). Six inoculated calves were euthanized by captive bolt and jugular exsanguination enough for collection

of spleen tissue: one calf each on dpi 7, 8, 9 (fever day 1) and 14 (fever day 5), and two calves at 13 dpi (fever days 4 and 5). In this way, the spleens from three calves each were examined from two periods: a period just prior to, or including, the initiation of fever (7, 8 and 9 dpi) and a period several days after fever initiation (13 and 14 dpi). Spleen tissue from two uninfected calves was similarly collected. Multiple 15 × 15 × 5 mm sections of spleen were collected from each calf immediately posteuthanasia. Each section was placed into a cryostat mould containing Tissue-Tek® O.C.T.™ Compound (Sakura Fineteck USA, Inc., Torrance, CA, USA), snap frozen by floating on liquid nitrogen, and stored at −80°C. Cryostat sections (15 μm) were mounted on standard SuperFrost™ Plus slides (Electron Microscopy Services, Hatfield, PA, USA), fixed in 95% EtOH for 10 min and allowed to air dry overnight at room temperature. Formalin-fixed, paraffin-embedded samples of spleen were also collected from each calf and routinely stained in haematoxylin and eosin (H&E). Immunolabelling was carried out at room temperature in a humidified chamber. A Super PAP Pen HT™ (Research Products International Corp., Mt. Prospect, IL, USA) was used to create a hydrophobic margin to retain fluid reagents on slides.

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 BMN 673 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 Dabrafenib on Mehran score for risk of development of CIN and analysed the effect of statins. Results:  Three-hundred and fifty-three patients

met inclusion PAK6 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.

To verify this possibility, the concentration dependence of inflammasome activation in WT and KI cells (in the presence and absence of ATP) check details was determined. It was found that while inflammasome activation increased in both the cell types with increasing LPS concentrations, WT cells required massive amounts of LPS (>1000 ng/mL) to activate the inflammasome in the absence of ATP, whereas KI cells required only minute amounts of LPS. It thus appears that KI cells do not require co-stimulation by ATP because the small amounts of TLR ligand that enter in the absence of ATP are sufficient to activate the altered inflammasome.

Overall, these data BAY 80-6946 clinical trial are consistent with the concept previously suggested from studies of CAPS patients that NLRP3 mutations lead to changes in the conformation of the protein that, in turn, result in a reduced activation threshold and thus an inflammasome capable of responding to reduced amounts of TLR ligand or other activating factors 9, 19. However, NLRP3 may not be able to directly bind to such a wide variety of ligands including PAMP and DAMP, rather an endogenous activator induced by all these upstream stimuli may serve as the direct ligand for NLRP3 (Fig. 1). This concept has also been proposed independently by other researchers 20, 21. NLRP3 KI mice bearing an R258W

mutation raised under pathogen-free facility exhibit spontaneous clinical symptoms similar to those of the counterpart Muckle–Wells syndrome patients. These symptoms consist of poor linear growth, reduced reproductive capacity, impaired hair development and, in many animals, severe dermatitis affecting the PRKACG ears, top of

the head and tail base area occurring at 6–12 wk of age that is associated with a deterioration of health. The skin lesions were clinically more severe than the urticaria-like skin disease seen in human CAPS and characterized by neutrophilic infiltration of the dermis and epidermis. Spleen and draining lymph nodes were enlarged in the KI mice and showed poorly developed follicles along with a diffuse infiltrate, again containing many neutrophils. However, these KI mice were free of lung, kidney or gut inflammation and the level of circulating inflammatory cytokines was normal 9. The clinical features of mice bearing A350V and L351P mutations were qualitatively similar to those described for R258W mice, but were far more severe. These A350V/L351P KI mice had lifespan measured in days rather than weeks, and had more widespread skin inflammation and inflammatory infiltration (mainly neutrophilic) of many organs, including the joints, sinus, bone marrow and tongue. In addition, there was evidence of “necrotic degeneration” in the gut and kidney.

hADSCs may play a key role in nerve regeneration by acting primarily as support for local neurotrophic mediation and modulation of nerve growth rather than that of a primary neuronal differentiation agent. © 2013 Wiley Periodicals, Inc. Microsurgery 34:324–330, 2014. “
“Microsurgical

revascularized fibula graft is a standard for the reconstruction of mandible or maxilla after major resection. Usually, screwed implants are inserted as a second procedure for dental rehabilitation. A lot has been published about the advantages of vascularized bone grafts, but check details until now there is only little information about long-term viability of inserted bone grafts. In this study, previously inserted vascularized fibula bone grafts were examined histologically. Bone biopsies were taken during dental implant insertion procedure in average of 19 months after insertion of bone grafts from 10 patients. All bone biopsies showed partially or totally necrotic bone, although clinical examination and postoperative monitoring of the revascularized bone remained

unremarkable. The results of histological examination are surprising, due to the fact of previous insertion of a vascularized bone graft and pretended osseointegration of inserted dental implants with satisfying primary stability. Therefore, one would expect vital bone. For better understanding how much viability is really necessary for sufficient remodeling of learn more inserted bone grafts for adequate functional load, further studies should be performed. © 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“Background: The Iraq and Afghanistan Wars have presented military reconstructive surgeons with a high volume of challenging extremity injuries. In recent years, a number of upper and

lower extremity injuries requiring multiple tissue transfers for multiple limb salvages in the same casualty have been encountered. Our group will discuss the microsurgical challenges, algorithms, and success and complication rates for this cohort of war injured patients. Methods: this website All consecutive limb salvage cases requiring free flaps from 2003 to 2012 were reviewed. Cases involving simultaneous free tissue transfers were identified. Data collected included success rates and complications with comparisons made between the single and multiple free-flap limb salvage cohorts. Results: Seventy-four free flap limb salvage cases were performed over the 10-year period. Of these cases, four patients received two free flaps to separate upper and lower extremity injuries for limb salvage within a single operative setting. The complication rate was 63%, which was significantly higher than those cases in which a single microvascular anastomosis was performed (26%, p = 0.046). However, the higher complication rate did not increase the flap or limb salvage failure rates (p = 0.892 and 0.626).

Progression of immature thymocytes through the DN and DP stages was uninhibited in KSR1−/− thymi, indicating that suboptimal ERK activation is enough for thymocytes to proceed through

developmental checkpoints that require TCR signaling. Consistent with previous studies, we found a more complex role for ERK in negative selection. Of the three model systems examined in this study, attenuated ERK activation diminished the efficiency of negative selection only for the HY TCR. Determining the exact nature of the learn more role of ERK activity in negative selection will help shed light on the signaling mechanisms responsible for distinguishing positive and negative selection. KSR1−/− mice were previously generated on a DBA1/LacJ background 18. For TCR transgenic experiments, these mice were backcrossed more than ten times to C57BL/6 (Jackson Laboratory). KSR1−/− TCR transgenic mice were generated by breeding KSR1−/− C57BL/6 mice with AND 24 (Jackson Crizotinib in vitro Laboratory) or HY 25

TCR (Taconic) transgenic mice. AND mice were crossed with AKR.B6 mice (Jackson Laboratory) to generate AND TCR transgenic mice with the H-2k haplotype. Superantigen deletion experiments were performed in the original DBA1/LacJ KSR−/− mice. All mice were housed under specific pathogen-free condition in the Washington University animal facilities in accordance with the institutional guidelines. Single-cell suspensions were generated from thymi excised from 6- to 8-wk-old mice. Total thymocytes were stimulated

with 40 ng/mL PMA or 5 μM anti-CD3 for various time points, lysed in NP-40 buffer and resolved on a 10% SDS-PAGE gel. Total ERK and ppERK were detected using polyclonal rabbit antibodies from Santa Cruz (anti-ERK2) and Cell Signaling Technology (anti-pERK1/2, (Thr202/Tyr204)), respectively. HRP-conjugated anti-Rabbit secondary antibody (Jackson ImmunoResearch) followed by ECL Western blotting Amino acid substrate (Pierce) was used for detection. Single-cell suspensions were generated from thymi of 4- to 6-wk-old mice. Cells were stimulated with 1 μg biotinylated anti-CD3 (BD Biosciences) followed by 1 μg/mL unconjugated SA (Jackson Immunoresearch) for 3 min followed by fixation with 4% PFA and permeablization with 95% methanol. Cells were first stained with anti-pERK1/2, (Thr202/Tyr204) from Cell Signaling overnight and then stained with CD4 APC and CD8 PE-Cy5 antibodies from BD Biosciences and an anti-rabbit PE-conjugated secondary (Jackson ImmunoResearch). FACS analysis was performed on single-cell suspensions of thymus and spleen. Following passage through a cell strainer (Fisher), cell suspensions were pelleted and resuspended in PBS+2% FBS and counted using trypan blue exclusion. Cells were then stained with various combinations of the following antibodies from BD Biosciences: CD4 FITC, Vβ9 FITC, CD4 PE, Vβ6 PE, Vβ7 PE, Vβ8.1 PE, HY TCR PE, Vα11 PE or eBiosciences: CD8 PECy7 and CD3 APC. Samples were run on a BD FACSCalibur instrument and analyzed using FlowJo software.