© 2011 Wiley-Liss, Inc. Microsurgery, 2011. “
“Introduction The aim of this study

was to compare magnetic resonance angiography (MRA) with digital subtraction angiography (DSA) in the preoperative assessment of crural arteries and their skin perforators prior to free fibular transfer. Patients and methods Fifteen consecutive patients, scheduled for free vascularized fibular flap transfer, were subjected to DSA as well as MRA of the crural arteries of both legs (n = 30). All DSA and MRA images were assessed randomly, blindly, and independently by two radiologists. Each of the assessors scored the degree of stenosis of various segments on a 5 point scale Ibrutinib cell line from 0 (occlusive) to 4 (no stenosis). The Cohen’s Kappa coefficient was used to assess the agreement between DSA and MRA scores. In addition, the number of cutaneous perforators were scored and the assessors were asked if they would advise against fibula harvest and transplantation based on the images. Results A Cohen’s Kappa of 0.64, indicating “substantial agreement of stenosis severity scores” was found between the two imaging techniques. The sensitivity of MRA to detect a stenosis compared with DSA was 79% (CI95%:60–91), and a specificity of 98% (CI95%: 97–99). In 53 selleckchem out

of 60 assessments, advice on suitability for transfer were equal between DSA and MRA. The median number of cutaneous perforators that perfuse the skin overlying the fibula per leg was one for DSA as well as MRA (P = 0.142).Conclusions A substantial agreement in the assessment of stenosis severity was found between DSA and MRA. The results suggest that MRA is a good alternative to DSA in the preoperative planning of free fibula flap transplantation. © 2013 Wiley Periodicals,

Inc. Microsurgery 33:539–544, 2013. “
“Background: The use of pressor drugs after microsurgical free tissue transfer remains controversial because of potential vasoconstrictor effects on the free flap. Noninvasive monitoring of free flaps with laser Doppler flowmetry may provide further information regarding the local regulation of blood flow in the flap tissues during pressor infusions. FER This study evaluated the effects of four commonly used pressor agents. Methods: Twenty four patients (25 data sets) undergoing head and neck cancer resection and free flap reconstruction were recruited. Epinephrine, norepinephrine, dopexamine, and dobutamine were infused in a random order at four infusion rates, after surgery, with free flap and control area (deltoid region) laser Doppler skin blood flow monitoring. Frequency analysis of the Doppler waveform was performed utilizing the time period immediately before the first drug infusion for each patient as baseline. Results: At baseline there was less power at the 0.002–0.6 Hz frequency in the flap compared with control tissue consistent with surgical denervation.

Conflict of interest: The authors declare no financial or commercial conflict

of interest. See accompanying Commentary: http://dx.doi.org/10.1002/eji.201040447 “
“Epigenetic control of gene expression is critical for cellular differentiation and development. Macrophage development, polarization and activation are also controlled by DNA and histone modifications. This Viewpoint summarizes the recent findings on Opaganib supplier the role of histone modifications regulating macrophage polarization toward M1 and M2 subtypes. Macrophages play pleiotropic roles in responding to various stresses such as infection, genotoxic stress and injury 1. Furthermore, macrophages are critical for tissue remodeling and angiogenesis in the late stages of inflammation, tumor progression and metabolic homeostasis. Macrophages develop from hematopoietic stem cells through common myeloid progenitors in the BM, and repopulate in peripheral tissues 2. Currently, macrophages can be classified into several different subtypes, based on their reactions to different stimuli 3–5. Macrophages involved in inflammatory responses to bacterial and viral infection are called M1 macrophages. M1 macrophages produce high

amounts of www.selleckchem.com/products/chir-99021-ct99021-hcl.html proinflammatory cytokines, such as TNF, upon recognition of invading pathogens

by a set of pattern-recognition receptors including TLRs, Quisqualic acid RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) 6–8. M1 macrophages are known to produce nitric oxide (NO) by expressing inducible NO synthase (iNOS) and are critical for clearing bacterial, viral and fungal infections. IFN-γ produced by activated T cells and TLR ligands, induces M1 macrophage generation in vitro. On the other hand, macrophages involved in responses to parasite infection, tissue remodeling, angiogenesis and tumor progression are called “alternatively activated macrophages” or “M2 macrophages” 3. M2 macrophages are characterized by their high expression of markers of alternative activation, including arginase-1 (Arg1), chitinase-like Ym1 (Chi3l3), found in inflammatory zone 1 (Fizz1), mannose receptor (MR), chemokines such as CCL17, CCD24 and so on 9–13. The pattern-recognition receptor system responsible for the recognition of helminth infection and M2 polarization has yet to be identified; however, stimulation of macrophages with the Th2 cytokines IL-4 or IL-13 induces M2-type macrophages 4, 14. In addition, immune complex formation, IL-10 and glucocorticoid or secosteroid hormones are also known to generate M2 macrophages.

Rep-Seq produces orders of magnitude less data. The issue of allocating storage for Rep-Seq experimentation is therefore easily absorbed into the public storage space currently allocated for sequencing projects. Furthermore, cloud computing is being actively used by different groups worldwide for NGS.47 There are multiple cloud providers, both commercial and open source, such as Amazon, Rackspace, GoGrid, Nimbus and Eucalyptus, this website all provide central processing units,

memory and storage devices.48 Cloud-based data storage and data processing not only provides dynamic and parallel storage services but also enables easy on-demand file sharing and easy access to these data worldwide. In immunology, the International ImMunoGeneTics STI571 nmr database,49 has positioned itself as a highly useful tool. ImMunoGeneTics is a high-quality integrated database specializing in immunoglobulin, TCRs and MHC molecules of all vertebrate species. ImMunoGeneTics is the main and only database that curates all

these data in one place and has actively gathered tools for sequence analysis and alignment. However, the rapid changes and development in the field of repertoire sequencing call for new databases and tools for the analysis of whole repertoires, and for the comparisons between species. Rep-Seq provides a segue to systems immunology approaches that, with the combination of new computational system-based tools, promise to enrich immunology. The complexity that characterizes the immune system and immune response can only be fully understood by a systems-approach to integrate processes, experimental data and high-level computational algorithms. “
“Inflammatory bowel disease is characterized by dysregulated immune responses in inflamed intestine, with dominance of interleukin-17 (IL-17) -producing cells and deficiency of regulatory T (Treg) cells. The aim of this study was to investigate the effect and mechanisms of sirolimus, an inhibitor of the mammalian target of rapamycin, on immune responses in a murine model of Crohn’s disease. Murine colitis was induced by intrarectal

administration of 2,4,6-trinitrobenzene sulphonic acid at day 0. Mice were then treated intraperitoneally with sirolimus daily for 3 days. The gross and histological Carbohydrate appearances of the colon and the numbers, phenotype and cytokine production of lymphocytes were compared with these characteristics in a control group. Sirolimus treatment significantly decreased all macroscopic, microscopic and histopathological parameters of colitis that were analysed. The therapeutic effects of sirolimus were associated with a down-regulation of pro-inflammatory cytokines tumour necrosis factor-α, IL-6 and IL-17A. Intriguingly, sirolimus administration resulted in a prominent up-regulation of the regulatory cytokine transforming growth factor-β.

Secondly, all Gram-positive bacteria, but none of the virus, induced IL-12p40 responses,

but the IL-12p40 responses did not affect Th1 cytokine production (IFN-γ). Instead, Th1 responses were correlated with the capacity to induce IFN-α secretion, which in cord cells were induced by S. aureus and influenza virus alone. These data imply that enveloped virus can deviate Th2 responses in human cord T cells. Allergic diseases among children and youth are one of the most common Smoothened antagonist chronic diseases in the Western world and the prevalence has increased drastically during the last 40 years [1]. The hygiene hypothesis states that a reduced exposure to microbes increases the risk of developing allergies. This hypothesis was originally based on observations showing that children with many siblings, children

attending early day care or children growing up in poverty are less prone to develop allergies [2]. It is, however, not yet clear which microbes that can and cannot affect allergy development. Epidemiological studies show that certain viral and bacterial infections correlate with a reduced incidence of allergic manifestations. click here We have recently shown that infection with human herpes virus type 6 (HHV-6) is associated with reduced allergic sensitization in 18-month-old children [3]. We have confirmed this in an experimental animal model of allergic asthma, where mice that are exposed to HHV-6 are protected against allergic inflammation. Mice exposed to HHV-6 have significantly lower levels of allergen-specific IgE, eosinophils and Th2 cytokines as compared to allergic control mice [4]. In addition, previous infection with EBV [5, 6] and Hepatitis A virus [7, 8] has been associated with a reduced incidence of allergic sensitization and allergic symptoms in human subjects. Infection with orofecal and foodborne

bacteria, including Toxoplasma gondii and Helicobacter pylori, or exposure to bacterial components, such as endotoxin, have also been demonstrated PRKD3 to be inversely related to atopic allergy [8–11]. Furthermore, the composition of the intestinal commensal flora has been suggested to affect the risk of developing allergic disease, where early colonization with bifidobacteria and lactobacilli is associated with a lower prevalence of allergy in young children (0–2 years of age) [12–14]. The allergic response is driven by Th2 cells, and their secretion of IL-4, IL-5 and IL-13. The initiation of the T cell response and the subsequent maturation of the T cells, including their differentiation into Th1 or Th2 cells, are regulated by dendritic cells (DC) [15]. These cells are generally divided into two major subsets; myeloid CD11c+CD123− DC (mDC) and plasmacytoid CD11c−CD123+ DC (pDC). MDC are the main source of IL-12, which is pivotal in the differentiation of naïve CD4+ T cells into the favoured Th1 phenotype [16–18].

The 1H,13C HSQC and HBMC spectra of the polysaccharide showed minor CH3/CH3 and CH3/CO cross-peaks at δ 2.08/21.9 and δ 2.08/174.2, respectively, which indicated the presence of a minor O-acetyl group. However, its position could not be determined owing to its too low content and, as a result, the lack of NMR signals potentially useful for determination of the site of O-acetylation. The data obtained indicated that the O-antigen of P. alcalifaciens

O40 has the structure 1 shown in Fig. 4, where d-Qui3NFo stands for 3,6-dideoxy-3-formamido-d-glucose. This monosaccharide derivative occurs rarely in bacterial polysaccharides; to the best of our knowledge, Autophagy Compound Library in vivo earlier it has been reported only once as a component of the O-antigen of Hafnia alvei 1204 (Katzenellenbogen et al., 1995). The O-antigen structure and the presence of an O-acetyl group ALK inhibitor were confirmed independently by negative ion high-resolution ESI MS of oligosaccharide

fractions A and B. The mass spectrum of fraction B showed a major ion peak for a Hex4HexA1Hep3Kdo1Ara4N1P1PEtN3 oligosaccharide (where Ara4N indicates 4-amino-4-deoxyarabinose, Hep – heptose, Hex – hexose, HexA – hexuronic acid, Kdo – 2-keto-3-deoxyoctonic acid, PEtN – phosphoethanolamine) (experimental and calculated molecular masses 2200.55 and 2200.54 Da, respectively). The major causes of structural heterogeneity were the presence of compounds having one less or one more PEtN group (∆m 123.01) and the occurrence of incomplete core glycoforms lacking one or two hexose residues

(∆m 162.05 u each). Therefore, fraction B represents a core oligosaccharide with composition typical of Providencia species (Kondakova et al., 2006; Ovchinnikova et al., 2011), which was derived from the R-form LPS devoid of any O-antigen. The mass spectrum of Edoxaban fraction A demonstrated ion peaks for compounds with the molecular masses 3037.78 and 3079.80 Da accompanied by related species with one less and one more PEtN group. The mass differences of 714.23 and 756.24 Da between these compounds and the core oligosaccharide corresponded to the Qui3NFo1Gal1GlcA1GalNAc1 and Qui3NFo1Gal1GlcA1GalNAc1Ac1 tetrasaccharide O-units, respectively. Therefore, the fraction A oligosaccharides were derived from the SR-form LPS and consist of an O-unit, either O-acetylated or not, attached to the core. In addition, fraction A was found to contain the cyclic Fuc4N4ManNA4GlcN4Ac11 (where Fuc4N indicates 4-amino-4-deoxyfucose, ManNA – mannosaminuronic acid) dodecasaccharide enterobacterial common antigen (experimental and calculated molecular mass 2386.88 Da) and two minor dodecasaccharides having one less and one more acetyl group (∆m 42.01 Da). Enzyme-immunosorbent assay with rabbit polyclonal O-antiserum against P. alcalifaciens O40 was used to characterize the O-antigen specificity of this bacterium and to reveal possible relationships of the O40-antigen with those of other Providencia O-serogroups.

Anti-autonomic autoantibodies had already been detected previously in serum samples from patients with short-term

CRPS [4]. To ascertain buy EX 527 anti-autonomic autoantibodies in long-standing CRPS patients, a laboratory study was carried out using a novel adult cardiomyocyte model [5]. Although cardiomyocytes are not involved in the CRPS pathophysiology, these cells are useful for detecting autoantibodies directed against autonomic receptors, as any functional receptor effect will be indicated by changes in the pattern of the cardiomyocytes’ beatings. Cardiomyocytes treated with serum-IgG preparations from CRPS patients and controls (29 healthy patients, seven with neuropathic pain, nine with myasthenia and 12 with fibromyalgia) were placed into a pulsating electric field to induce calcium influx and contraction. Selleck Panobinostat In the CRPS cells, both the baseline calcium levels and the calcium transient

were reduced; however, the level of cell contraction was the same as that of the control cells, suggesting calcium-independent myofibril sensitization. The calcium effect was confirmed in patch-clamping experiments where calcium influx was reduced in the CRPS group compared to the control preparations. Eleven of 18 CRPS serum-IgG preparations induced functional or calcium abnormalities, while only one in 57 control preparations induced abnormalities (P < 0·0001). These results suggest that long-term CRPS is associated with specific anti-autonomic autoantibodies. Discussions in the field have traditionally assumed that although there might be an immune involvement in the initial CRPS stages the patients' pain would later be maintained by brain factors but, conversely, our results argue that there is an ongoing, potentially treatable immune abnormality. Additionally,

of the 11 serum-IgG preparations available from CRPS patients who participated in the previous IVIg treatment trial [2], all preparations from subjects who responded to IVIg treatment (n = 4) were active in the cardiomyocyte ID-8 assay, but the majority of preparations from non-responders to IgG (n = 4/7) were also active. This therefore indicates that CRPS-specific autoantibodies are not restricted to IVIg responders. The study group also investigated the effect of CRPS serum-IgG in a novel animal model via passive transfer [6]. Serum-IgG preparations from 12 CRPS patients and 12 controls from the previous trial were administered to mice. Behaviour in the open field, stimulus-evoked pain and motor co-ordination were observed in order to ascertain whether the transfer of IgG antibodies produced signs of CRPS. Rearing behaviour was reduced significantly in the CRPS-IgG-treated group, and motor impairment was also observed; however, these mice were not suffering from CRPS, as assays for hyperalgesia revealed no results.

We next analysed binding of CTLA-4-Ig on DCs and B cells after sensitization with DNFB. Mice were treated with 25 mg/kg of CTLA-4-Ig or control protein 1 day prior to sensitization. As shown in Fig. S1A, significant binding of CTLA-4-Ig to DCs could be detected on day 3. Furthermore, we found a significantly reduced expression of CD86 4 and 5 days after sensitization in CTLA-4-Ig-treated mice (Fig. S1B,C). In contrast, no specific binding of CTLA-4-Ig to B cells could be detected at either time-point examined (Fig. S1D), but expression of CD86 on B cells was strongly suppressed at every time-point after sensitization in the CTLA-4-Ig-treated group compared to treatment with isotype control

(Fig. S1E,F). Together, these data suggest that CTLA-4-Ig binds p38 MAPK cancer preferentially to DCs in the draining lymph node after hapten

sensitization, and that CTLA-4-Ig reduces the level of the maturation marker CD86 on both DCs and B cells. Having demonstrated a reduction of CD4+ and CD8+ T cell activation in draining lymph nodes in the presence of CTLA-4-Ig, we wanted to investigate the consequences for the inflammatory reaction in the tissue after challenge. Thus, infiltrating cells were isolated from the inflamed ear 48 h after challenge, stained for activation markers and analysed by flow cytometry. As shown in Fig. 4, CTLA-4-Ig treatment led to a significant reduction in both number and percentage of CD8+ T cells in the inflamed ear compared to controls (Fig. 4a). In contrast, the Cell Cycle inhibitor number of CD4+ T cells was not significantly different, but the percentage of CD4+ T cells was increased in the CTLA-4-Ig-treated group (Fig. 4b). More importantly, CTLA-4-Ig treatment resulted in a reduction in the number of Janus kinase (JAK) activated CD8+ T cells in the inflamed ear

compared to controls. Thus, we observed a decreased number and percentage of CD44+CD62L−CD8+ T cells and CD69+CD8+ T cells in the CTLA-4-Ig-treated group compared to controls (Fig. 4c,d). In conclusion, these results suggest that CTLA-4-Ig inhibits infiltration of activated CD8+ T cells into the challenged tissue. To correlate the reduced cellular infiltration into the target tissue after CTLA-4-Ig treatment with the local production of cytokines and chemokines, homogenates of inflamed ear tissue from CTLA-4-Ig-treated and isotype control-treated animals were analysed for their content of a number of cytokines and chemokines including IL-4, CXCL10 (IP-10), IL-12 (p40), MIP-2, TNF-α, IFN-γ, IL-1β, IL-10 and IL-6. As shown in Fig. 5, IL-1β and IL-4 were suppressed significantly in the CTLA-4-Ig-treated group compared to the control group both in the DNFB- and in the oxazolone-induced models (Fig. 5a–d). Additionally, the concentrations of the chemokines MIP-2 and CXCL10 (IP-10) were reduced in both models (Fig. 5c,d,g,h) after CTLA-4-Ig treatment.

Interestingly, MoDC that was incubated with PIC-CM prior to coculturing them with allogeneic PBMC generated a highly increased buy FDA-approved Drug Library release of IFN-γ in MLR culture supernatants. Both changes in MoDCs, i.e. upregulation of CD40, CD86, and increased MLR stimulation, were abrogated by blocking IFN-β. Surprisingly, MoDC incubated with PIC-CM did not induce IL-12p70 secretion; however, previous data showed that under certain conditions, IL-12p70 can be dispensable for IFN-γ induction. Indeed, in some virus infections, the lack of IL-12 has little or no effect on the induction

of Th1 immunity and systemic production of IL-12p70 could not be detected after in vivo administration of poly I:C, whereas poly I:C was superior at inducing systemic type I IFNs and Th1 immune response [42-45]. Murine BMDCs also secreted higher levels of IL-12p70 when they were matured in the presence of PAU-B16 CM. Therefore, a novel aspect of the use of dsRNA mimetics in cancer immunotherapy can be assumed: when tumor

cells are activated with dsRNA ligands, they secrete IFN-β at levels that are capable of improving the maturation state and function of DCs, promoting a Th1 response that could be independent of the induction of IL-12. Tumor-derived factors significantly alter the generation of DCs from hematopoietic progenitors, increase the accumulation of JQ1 datasheet myeloid suppressor cells, and inhibit DCs maturation [22, 23]. When MoDCs were matured with different TLR ligands in the presence of tumor CM, expression of co-stimulatory molecules, secretion of IL-12p70, and induction of IFN-γ in MLR were significantly diminished. In contrast, when the maturation was done in the presence of PIC-CM, all Palmatine these parameters were improved. Indeed, TLR-induced IL-12p70 secretion by DC has been

shown to depend on a type I IFN autocrine–paracrine loop [26]. Thus, the simultaneous presence of IFN-β plus the exogenously added TLR ligand, and/or other factors present in PIC-CM such as HMGB1 or other cytokines, could be producing a synergistic effect on maturing MoDCs that can be readily observed in the enhanced values of secreted IL-12p70 and the better capacity of driving an IFN-γ response in the MLR. Similar results were obtained in our previous work, in which murine prostate adenocarcinoma and melanoma cells (TRAMPC2 and B16, respectively) secrete low but reliably detected levels of IFN-β upon TLR4 activation [19]. These low levels of IFN-β were enough to enhance the expression of co-stimulatory molecules on BMDCs as well as to increase the levels of IL-12 secreted. In addition, the frequency of CD11c+ tumor infiltrating cells expressing IL-12 was increased in mice bearing LPS-B16 tumors [19].

They produce high levels of IFN-γ and tumor necrosis factor-α (TNF-α), and can kill infected cells through the release of granzymes and perforin into the immunological synapse [60]. The cytokines IL-2 and IL-12 drive effector CTL differentiation by triggering STAT4 and STAT5 signaling, as well as through the phosphoinositide-3-kinase–Akt–mTOR and the rat sarcoma (RAS)-rat fibrosarcoma (RAF)–mitogen-activated protein kinase (MAPK) pathways [61]. After resolution of infection, the bulk of CD8+ T cells die; however, a small Nutlin3a fraction remains as long-lived memory CD8+ T cells that respond to re-exposure to the cognate pathogen with strong proliferation and rapid conversion into

effector cells. Already at early stages of the response, phenotypic and functional markers help to distinguish between short-lived effector

CTLs and T cells that can give rise to long-lived memory cells. The CD44hiCD62Llokiller cell lectin-like receptor 1(KLRG1)hiIL7-Rαlo phenotype is characteristic for effector CTLs, whereas the memory precursors can be defined as CD44hiKLRG1loIL7-Rαhi. The differentiation of naïve CD8+ T cells into effector and memory CTLs is regulated by balanced expression of several transcription factors. Whereas BCL-6, Ibrutinib eomesodermin (EOMES), inhibitor of DNA-binding (ID) 3 and T-cell factor 1 (TCF1) are associated with memory cell differentiation and longevity, T-BET, ID2, and BLIMP-1 promote effector cell development [60]. Like in Th17 cells, TGF-β Silibinin acts in combination with IL-6 or IL-21 to promote differentiation of IL-17-producing and ROR-γt-expressing Tc17 cells, which are detectable during viral infections, autoimmunity, and in tumor environments. Tc9-cell development parallels that of Th9 cells and is also induced by TGF-β and IL-4. These cells are detectable in the lamina propria of mice and in the periphery of mice and humans with atopy [62, 63]. In contrast

to CTLs, Tc9 and Tc17 cells display low cytotoxic activity [63-68]. Three recent studies demonstrated essential roles for IRF4 in effector CTL development. Although dispensable for initial activation and proliferation, IRF4 was required for CTL expansion, sustained expression of the effector CTL phenotype, and function. This was shown in three experimental models of infection with intracellular pathogens, namely in mice infected with lymphocytic choriomeningitis virus (LCMV), influenza virus, and L. monocytogenes [22, 23, 25]. Although WT mice can clear infection with L. monocytogenes within 10 days, Irf4–/– mice failed to clear the bacteria. This was caused by defective CD8+ T-cell function that was T-cell intrinsic, as transfer of WT CD8+ T cells into Irf4–/– mice rescued bacterial clearance [23]. Furthermore, mice with conditional deletion of IRF4 in CD8+ T cells failed to control influenza infection [25]. Similarly, defective CTL development in the absence of IRF4 was shown in response to infection with LCMV [22, 69].

193%) whereas the background staining among TCRβ-positive cells was much lower (0.06%, data not shown).

Last, consistent with iNKT cells being the major PLZF-expressing T-cell population, most PLZF+ αβ T cells expressed NKR-P1A/B at intermediate levels (Fig. 2F). Apart from F344 inbred rats, we also examined the widely used LEW inbred rat strain. The LEW strain is well known for its susceptibility to induced organ-specific autoimmunity, which is not to be found in F344 rats [24-26]. As shown in Figure 2F LEW rats lack the PLZF+ NKR-P1A/B-intermediate T-cell Belnacasan population found in F344 and show no specific binding of α-GalCer-CD1d dimers (Fig. 2B). Nevertheless, the few cells stained with α-GalCer-CD1d dimers in the liver of LEW rats showed some increase of the DN fraction in comparison with the cells stained with vehicle-CD1d dimers (Fig. 2B). Therefore, it is conceivable that these DN cells are iNKT cells, which may Tanespimycin be missed due to nonspecific staining of the vehicle control. However, even if it is postulated that all the DN α-GalCer-CD1d-stained cells would be bona fide iNKT cells, their frequency would be a maximum of 0.003% in IHLs (i.e., about 2% of the iNKT cells found in F344 liver). Next, we examined the presence

of iNKT cells in the thymus of both inbred rat strains by flow cytometry and compared it with that of C57BL/6 mice (Fig. 2G). We used both rat and mouse CD1d dimers, but none of them revealed a distinct iNKT-cell population among F344 or LEW thymocytes. In contrast, C57BL/6 thymocytes contained a distinct fraction of α-GalCer-CD1d dimer-stained cells. The analysis of iNKT cells in mouse thymi is commonly carried out after exclusion of HSAhigh (CD24) immature thymocytes. The commercially available anti-rat HSA isothipendyl mAb does not stain rat thymocytes. Therefore, we analyzed CD8− cells (CD8αβ− in case of rat and CD8αα−/CD8αβ− in case of mouse), stained with anti-TCRβ mAb and CD1d dimers. This approach has been chosen to specifically enrich

the populations among which rat (CD4+, DN, and CD8αα+) or mouse (CD4+, DN) iNKT cells are expected and found to result in an eightfold increase of the relative iNKT-cell frequency among C57BL/6 thymocytes. However, we were still not able to detect a distinct iNKT-cell population among F344 or LEW thymocytes (Fig. 2G). In addition to flow cytometry experiments, we also examined the expression of AV14-containing TCRs by RT-PCR (Supporting Information Fig. 1F). First, we analyzed the expression of TCRα chains comprised by AV14 and AJ18 gene segments. The highest expression levels were found among F344 IHLs, followed by F344 splenocytes, and thymocytes. In contrast, analysis of LEW-derived RNA gave only very weak or no signals. Importantly, the differences between LEW and F344 were already found in thymocytes. AV14-AJ18 rearrangements were also analyzed by sequencing the RT-PCR products.