Noticeably, BIs are consistently fused in sarcoma (FUS) positive. NIFIs are by definition immuno-positive for class IV IFs including three Akt inhibitor NF triplet subunit proteins and α-internexin but negative for tau, TDP-43, and α-synuclein. In NIFID cases several types of inclusions have been identified. Among them, hyaline conglomerate-like inclusions are the only type that meets the above immunohistochemical features of NIFIs. This type of inclusion appears upon HE staining as multilobulated, faintly eosinophilic or pale amphophilic spherical masses with a glassy appearance. These hyaline conglomerates appear strongly argyrophilic, and robustly and consistently immuno-positive

for IFs. In contrast, this type of inclusion shows no or only occasional dot-like FUS immunoreactivity. Therefore, BIs and NIFIs are distinct from each other in terms of morphological, tinctorial and immunohistochemical features. However, basophilic inclusion body disease (BIBD) and NIFID are difficult check details to differentiate clinically. Moreover, Pick body-like inclusions, the predominant type of inclusions seen in NIFID, are considerably similar to the BIs of BIBD in that this type of inclusion is basophilic, poorly argyrophilic, negative for IFs and intensely immuno-positive for FUS. As BIBD

and NIFID share FUS accumulation as the most prominent molecular pathology, whether these two diseases are discrete entities or represent a pathological continuum remains a question to be answered. “
“An 84–year-old man with rheumatoid arthritis (RA) treated with methotrexate, developed progressive confusion and cerebellar symptoms, and died approximately 2 months later. Neuropathological examination revealed progressive multifocal leukoencephalopathy (PML) involving the cerebellum and brainstem. The affected tissues

4-Aminobutyrate aminotransferase displayed intense infiltrations by CD8+ T-cells and microglia. JC virus was localized in oligodendroglia and cerebellar granule cells. This case illustrates unusual localization of inflammatory PML in a patient with RA treated with methotrexate. Progressive multifocal leukoencephalopathy (PML) is a demyelinating, usually non-inflammatory disorder of the CNS caused by reactivation of a latent JC virus (JCV), in the setting of immunosuppression.[1-4] The most frequent underlying conditions are HIV/AIDS, myelo- and lymphoproliferative disorders, autoimmune and chronic granulomatous diseases, as well as the use of immunomodulatory medications.[1-4] Among autoimmune disorders, the most common is systemic lupus erythematosus.[5-7] PML as a complication of rheumatoid arthritis (RA) treated with immunosuppressive medication is rare.[8-19] We present a patient with RA treated with methotrexate who developed an uncommon form of inflammatory PML limited to the infratentorial compartment.

Switzerland). For FRET analysis, the WT and MUT ζ cDNAs were cloned into the Clontech expression vectors pEYFP-N1 to obtain YFP-tagged ζ proteins, and actin to pECFP-C1 to obtain the CFP-tagged

actin. The actin plasmid was cotransfected into COS-7 cells (Lipofectamin 2000) with either WT or MUT ζ. G-actin was prepared from rabbit muscles and polymerized when required as previously described [36]. For cosedimentation, tested protein was added to prepolymerized F-actin, incubated for 20 min at 25°C and centrifuged at 80 000 rpm for 1 h at 4°C. Supernatants and pellets were separated, resolved on SDS-PAGE, and stained with Coomassie brilliant blue. For EM, samples were fixed on carbon-coated grids and negatively stained with 1% uranyl acetate. The grids were viewed under a Jeol 100cx (Jeol-LTD. Tokyo Japan) scanning SCH727965 chemical structure EM. For cell activation, 5 × 105 cells coated with anti-CD28 Abs were mixed with an equal number of 6-micron diameter polystyrene beads (Polysciences Inc, PA, USA) precoated

with A2B4 Abs. After brief centrifugation, samples DAPT concentration were incubated for various time periods at 37°C, transferred to poly-l-lysine coated slides (Lab-Tek), fixed, washed, and stained for CD3 expression. Confocal analysis was performed using LSM 410 microscope (Carl Zeiss MicroImaging, Inc.). TCR clustering formation was scored as positive if at least one distinct cap was observed at the cell–bead contact area. At least 100 cells in contact with beads were counted, and the percent cap formation was calculated. For specific T-cell activation, APCs (LK B-cells) were labeled with blue cell tracker CMAC (Molecular Probes), washed, and incubated with or without the specific peptide (cytochrome C, 81–104 aa). After washing, a 1:1 ratio of LK cells and WT or MUT T cells were mixed and incubated at 37°C for different time periods. Cells were seeded onto a

chamber slides, fixed, washed, stained, and analyzed as described. In ex vivo experiments, splenocytes Histamine H2 receptor were activated with anti-CD3ε Abs and processed as described. TCR clustering was detected by using anti-TCRβ Abs (Biolegend). FRET was measured by donor-sensitized acceptor fluorescence [37]. CFP (excitation, 458 nm; emission, 465−510 nm) was used as a donor and YFP (excitation, 514 nm; emission, 530 nm) as an acceptor. The results were verified by using the acceptor photobleaching techniques as previously described [38]. Detailed description is provided in the Supporting Information. FRET was corrected and the FRET efficiency was determined. Both WT and MUT cells were activated for 16 h at 37°C with PMA (40 ng/mL) and Ca ionophore (1.5 μm; Sigma) or with LK cells loaded with Pigeon cytochrome C peptide. Following activation, cells were washed, and assessed for CD25 and CD69 expression by FACS analysis.

The durations of gazes at and away from mother’s face, however, were not predicted by one another. This pattern suggests that infants exhibit distinct and temporally stable levels of interest in social and nonsocial features of the environment. In this report, we discuss the implications of these results for parents, for experimental research using looking time measures, and for our understanding of infants’ developing communicative abilities. “
“Infants’ early communicative repertoires include both words and symbolic gestures. The current study examined the extent to which infants

organize words and gestures in a single unified lexicon. As a window into lexical organization, eighteen-month-olds’ (N = 32)

avoidance of word–gesture overlap was examined and compared with avoidance of word–word overlap. The current study revealed that when presented with novel words, infants avoided lexical overlap, Vismodegib in vivo mapping novel words onto novel objects. In contrast, when presented with novel gestures, infants sought overlap, mapping novel gestures onto familiar objects. The results suggest that infants do not treat words and gestures as equivalent MAPK Inhibitor Library datasheet lexical items and that during a period of development when word and symbolic gesture processing share many similarities, important differences also exist between these two symbolic forms. “
“Most words that infants hear occur within fluent speech. To compile a vocabulary, infants therefore need to segment words from speech contexts. This study is the first to investigate whether infants (here: 10-month-olds) can recognize words when both initial exposure and test presentation are in continuous speech. Electrophysiological evidence attests that this indeed occurs: An increased extended Progesterone negativity (word recognition effect) appears for familiarized target words relative to control words. This response proved constant at the individual level: Only infants who showed this negativity at test had shown

such a response, within six repetitions after first occurrence, during familiarization. “
“This paper examines the relative merits of looking time and pupil diameter measures in the study of early cognitive abilities of infants. Ten-month-old infants took part in a modified version of the classic drawbridge experiment used to study object permanence (Baillargeon, Spelke, & Wasserman, 1985). The study involved a factorial design where angle of rotation and presence or absence of an object were crossed. Looking time results are consistent with previous work and could suggest object permanence if one ignored data from all cells of the factorial design. When all cells are considered, the data rather suggest a perceptual interpretation. Dynamic changes in pupil diameter uniquely support this interpretation, illustrating which aspects of events (and when) infants primarily respond to.

Expression of the proteins was observed using a fluorescent microscope (Leica Microscopy system Inc., Buffalo Grove, IL, USA). Immunization with recombinant adenovirus.  Six- to 8-week-old female C57BL-6 mice were purchased from Charles River

Laboratories (Wilmington, MA, USA) and maintained under specific pathogen-free conditions at the animal facility buy Alvelestat at Colorado State University. For intranasal immunization (i.n.) of one dose of recombinant adenovirus, 5 × 107 PFU of recombinant adenovirus or AdLac Z was diluted with phosphate-buffered saline (PBS) to a total volume of 20 μl and delivered into the airway of a mouse with a fine pipette tip [12]. Mycobacterium bovis BCG at a dose of 5 × 105 CFU/mouse was diluted in PBS to a total volume of 100 μl and injected subcutaneously. Lymphocyte isolation and in vitro antigen stimulation.  At 4 weeks post-vaccination, mice were humanely euthanized, and the spleens were removed. Single-cell suspensions were prepared as described previously [12] and cultured. Approximately 1 × 106 cells per well were seeded in 96-well plates in RPMI-1640 medium containing 10% foetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mm of l-glutamine at 37 °C in 5% CO2 with or without antigen stimulation (ESAT-6 at 5 μg/ml). ICG-001 The culture supernatants were collected after 24 h

and stored at −80 °C until used. Analysis of ESAT-6-specific T cells by ELISPOT assay.  Isolated splenocytes were seeded at 1 × 106 per well many in a 96-well PVDF microplate (Millipore, Bedford, MA, USA) that was precoated overnight with a mouse IFN-γ capture antibody (R&D Systems, Minneapolis, MN, USA;

1:60 dilution). Cells were incubated for 24 h with or without stimulation by ESAT-6. The plate was then developed by a standardized streptavidin-conjugated alkaline phosphatase and chromogen method (R&D Systems). The number of IFN-γ-releasing cells was determined using a Cellular Technology Ltd Series 5 UV Immunospot Analyzer (Shaker Heights, OH, USA). TNF-α measurement.  The level of TNF-α in the supernatants was measured with a mouse-specific enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). Stored samples were thawed, and the manufacturer’s protocol was used to assess the concentration. The sensitivity of detection was 2 pg/ml. Low-dose aerosol infection with Mycobacterium tuberculosis.  Animals were infected with live M. tuberculosis H37Rv via the aerosol route in a Glass-Col Airborne inhalation exposure system that delivered approximately 100 CFU M. tuberculosis bacilli per mouse. At 4 weeks post-infection, the protective efficacy was evaluated by plating serial 10-fold dilutions of lung and spleen homogenates on Middlebrook 7H11 agar plates. Plates were then incubated at 37 °C for 21 days, and colonies were counted. Data analysis.

The temperature programme was a 5-min denaturing step at 94 °C, 35 amplification cycles (94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s), and a final extension step of 72 °C for 10 min. After amplification, 5-μL samples of the PCR products were separated on a 1.5% agarose

gel and stained with ethidium bromide. Images were recorded and analysed using an EDAS 290 system (Kodak, NY), with band density measurements expressed in pixels. The integrated density value (IDV) was determined based on the number MG-132 supplier of registered pixels minus background: IDV=Σ(each pixel value minus background). The IDV of each band expressed in nanograms was obtained by comparison with the 300-bp band (equivalent to 80 ng μL−1) of the GeneRuler molecular weight marker (Fermentas Life Sciences, MD). To compare the values obtained from the different study groups with the basal values, a one-sample t-test was performed using the statistica 8 (2007) software for Windows. P<0.05 was considered significant. Fragments of tissue from one mouse of each group (NI-MG, ISSI-MG, CI-MG, and NbI-MG) were obtained and fixed in phosphate-buffered saline with 10% formaldehyde buy NVP-AUY922 for 24 h. They were then washed in Tris-HCl buffer (0.1 M, pH 7.2), longitudinally cut, and decalcified in a 10% EDTA aqueous solution for 15 days. The tissue was embedded in paraffin, and five sections of 5 μm were hydrated and antigenically reactivated in a citrate buffer (0.01 M citric acid,

0.01 M sodium citrate) according to the method of Pérez-Torres et al. (2009). Endogenous peroxidase was blocked with aqueous 3% H2O2. Nonspecific antigenic

sites were blocked with 4% bovine serum albumin, fraction V, dissolved in Tris-HCl and 0.01% Triton X-100 for 20 min at room temperature. The blocking solution was decanted, and the primary antibody for TLR2 or TLR4 was added (rabbit and goat polyclonal anti-mouse TLR2 and TLR4 antibodies, respectively; Santa Cruz Biotechnology, CA) in a 1 : 50 dilution in Tris-HCl. After an overnight incubation at 4 °C, the secondary antibody (anti-rabbit for TLR2 (Match 4 Kit, Biocare Medical Co. CA) or anti-goat Methane monooxygenase for TLR4 (Goat HRP-Polymer Kit, Biocare Medical Co.) was added, and the tissue was incubated for 60 min in a humid chamber at room temperature. The horseradish peroxidase-coupled complementary polymer (MHR2P for Match4 and Goat HRP-Polymer for Goat Kit, Biocare Medical Co.) for the secondary antibody was added and incubated at room temperature for 30 min. Colour development was assessed after incubation for 5 min with diaminobenzidine (DAB500 Chromogen System, Biocare Medical Co.) at room temperature. Specimens were counterstained with Mayer’s haematoxylin. Finally, the tissue was dehydrated and mounted with resin (Ecomount Mounting Medium, Biocare Medical Co.) for analysis under a light microscope. Negative staining controls were run in parallel for all mouse groups without anti-TLR2 and anti-TLR4 antibodies.

The age of patients were between 5 and 64 and all of them were males. The wound sizes in these patients ranged between 31–35 × 10–12 cm and flap dimensions

were between 38–48 × 6–8 cm. Perforator branches of the 10th intercostal vessels were dissected and supercharged to the flaps to reduce the risk of ischemia of the inferior cutaneous extensions. The secondary pedicles were anastomosed to recipient vessels other than the primary pedicles. Recipient areas were consisted of lower extremities. Four patients suffered of early arterial failure in the major pedicle and all revisions were successfully attempted. Neither sign of venous congestion nor arterial insufficiency were observed Fluorouracil at the inferior cutaneous extensions of the flaps, and all defects were reconstructed successfully. All donor Selleck C59 wnt sites were primarily closed, only two patients suffered from a minor area of superficial epidermal loss at the donor site, without suffering any adjunct complications. In conclusion coverage of large defects can be safely performed with extending the skin paddle of latissimus dorsi flap as a bipedicled free flap. © 2009 Wiley-Liss, Inc. Microsurgery, 2010. “
“A 67-year-old man with squamous cell carcinoma underwent reconstruction with a free anterolateral thigh myocutaneous flap. Unroofing the skin perforators found that the skin perforators originated

from the oblique branch Non-specific serine/threonine protein kinase of the lateral circumflex femoral artery with no connections with the descending branch. Thus, the flap was harvested based on the oblique branch, leaving the descending branch in situ. Reconstruction was completed uneventfully and he had an excellent outcome at 1-year follow-up. The anterolateral thigh myocutaneous flap was reputed to be a technically easy flap to harvest. The perforators supplying the

skin were visualized and a block of muscle incorporating the perforators harvested with the descending branch of the lateral circumflex femoral artery as the pedicle of the flap. However, not infrequently with this approach, the flap thus harvested has a well-perfused muscle component, whereas the skin component was not viable. This situation is explained anatomically by the potential occurrence of an alternative pedicle that supplies the anterolateral thigh flap, called the oblique branch of the lateral circumflex femoral artery. Our case presented here was a “classic” intraoperative finding of this potential trap and the importance of defining the anatomy before committing oneself to the harvest by unroofing all the skin perforators was emphasized. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“A 26-year-old man presented with a nonhealing ulcer on the plantar aspect of the left foot of five years duration. Initial investigations were unremarkable. It was only after careful neurological examination that an inherited neuropathy was suspected.

Such an interaction has been shown to promote the activation of microglia in vitro[65] and its genetically engineered or pharmaceutical abrogation results in amelioration of EAE expression.[66] Ponomarev et al. described a two-step process for microglia activation in EAE. They proposed that the CD40-independent first step occurring just before EAE onset is mediated by pro-inflammatory cytokines released by encephalitogenic T cells, such as IFN-γ, and results in selleck chemicals microglial cell proliferation and up-regulation of MHC class II, CD40 and CD86; the second step of activation, which is CD40-dependent, occurs at the peak of disease and is characterized by a further

increase in expression of activation markers and a reduced proliferation.[64] Upon full activation, microglia can act as antigen-presenting cells to present phagocytosed myelin antigen to encephalitogenic T cells leading to their expansion in the CNS and severe disease expression.[64] However, antigen presentation

is unlikely to be the main mechanism of damage mediated by microglia in EAE; rather, release of inflammatory cytokines and reactive oxygen species may be more relevant. A recent study identified Peli1, a family member of E3 ubiquitin ligases implicated in TLR and IL-1 receptor signalling in innate immune cells, as an essential regulator of microglia activation during EAE pathogenesis that is required for mitogen-activated protein kinase (MAPK)-dependent production of pro-inflammatory cytokines and chemokines.[67] Peli1 knockout mice Glycogen branching enzyme were refractory to EAE click here induction and showed reduced numbers of CNS-infiltrating cells and activated resident microglia. Peli1 was abundantly expressed in microglia and absolutely required for microglial activation during EAE, as shown by studies in Peli1-knockout GFP-expressing chimeric mice.[67] In vitro studies showed that Peli1 affects MAPK activation through MyD88-dependent TLR regulation, and acts by promoting

degradation of TNF receptor-associated factor 3, a potent inhibitor of MAPK activation and gene induction.[67] Another molecule that plays an important role in microglia activation leading to neurotoxicity is macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine identified as a marker of clinical worsening in MS patients.[68] A recent study has shown that MIF can drive the activation of microglia both in vitro in primary microglia cell cultures, and in vivo in EAE-affected mice or in the focal EAE model in MIF-deficient mice.[69] Increasing concentrations of MIF induced dose-dependent changes in expression of inflammatory molecules, such as TNF-α, IL-1β, IL-6 and inducible nitric oxide synthase, in primary microglia in vitro, that were accompanied by morphological changes from resting to activated and/or phagocytic phenotype.

FRET was measured by monitoring excitation at 280 nm and emission at 450 nm, and normalized to total AMCA fluorescence (excitation at 350 nm and emission at 450 nm). Complete digestion of HLA-DR1 during the reaction was verified by SDS-PAGE analysis and silver staining of recovered reaction mixtures. Samples were boiled after the addition of 8 × Laemmli SDS-PAGE sample buffer with 5% v/v 2-mercaptoethanol, run on 12% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Freiburg, Germany). Membranes were blocked for 1 hr in blocking buffer (1× PBS, 0·05% Tween 20 and 1× Rotiblock; Roth, Karlsruhe, Germany) and incubated for an

additional hour with the HLA-DR-specific antiserum CHAMP32 diluted in blocking buffer. After washing in PBS with 0·05% Tween 20, horseradish peroxidase (HRP)-conjugated secondary antibody (donkey Dinaciclib ic50 anti-rabbit immunoglobulin; GE Healthcare) was diluted 1 : 5000 in blocking buffer and Ferroptosis cancer incubated for 1 hr. Following additional washes, HRP activity was revealed using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare) and visualized using Hyperfilm ECL (GE Healthcare). Cathepsin G−/− (CG−/−) mice on a C57BL/6 background were received from the laboratory of C. Pham (Department of Internal Medicine, Washington University School of Medicine, Saint Louis,

MO). C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were bred and maintained at the Stanford University Research Animal Endonuclease Facility. The handling of all mice followed guidelines and requirements established by the National Institutes of Health and Stanford University animal research committee. Mice were killed with compressed CO2 gas and by cervical dislocation, and spleens were removed. Single-cell suspensions were prepared by mechanical disruption of the spleen through a 70-μm filter. Spleens were then treated with 1 × red blood cell (RBC) lysis buffer [1·68 m NH4Cl, 0·10 m potassium bicarbonate and 1 mm ethylenediaminetetraacetic acid (EDTA)], washed twice, and used directly for analysis. The following

antibodies were purchased from BD Biosciences (San Jose, CA): anti-mouse I-Ab phycoerythrin (PE), anti-mouse CD11b PE-Cy7, anti-mouse CD11c allophycocyanin (APC), and anti-mouse CD19 APC-Cy7. Anti-mouse CD3 Pacific Blue, anti-mouse CD45R (B220) fluorescein isothiocyanate (FITC), and anti-mouse F4/80 PerCP-Cy5.5 were purchased from eBiosciences (San Diego, CA). Before staining, cell preparations were blocked with 3·3 μg/ml anti-mouse CD16/CD32 (Fc Block; BD Biosciences) in PBS containing 0·5% bovine serum albumin (BSA) and 0·1% NaN3 for 15 min. For intracellular staining, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min on ice, and washed with 1 × Perm/Wash Buffer (BD Biosciences).

Those authors hypothesized that a state of unresponsiveness to the endogenous microflora may be apparent only after a transient mucosal immune response has occurred [24]. The response to bacteria and bacterial antigens we observed in our experiment might be elevated due in part to a transient unphysiological high load of bacteria in the axenic mice; however, it might mimic a response that occurs on a frequent basis, albeit less pronounced,

whenever a new bacterial strain is introduced in the intestinal lumen. The changes in the intestinal milieu with regard to cytokine and chemokine secretion as well as expression of cell surface antigens may instigate the generation of immune-regulatory cells. A crucial role for the presence of a microflora in the induction of regulatory T cells has been demonstrated in a murine transfer model of colitis [25]. Protective T cells showed reduced efficacy in preventing colitis development and demonstrated Vincristine reduced release of IL-10 and IFN-γ Saracatinib research buy when derived from axenic mice as opposed to those derived from conventionally housed mice. While we did not detect a significant increase in systemic T cells with a common

regulatory phenotype, such as CD25-positive T cells, we cannot exclude the generation of a specific population of cells with regulatory function in mucosal tissues and/or systemically. The increased CD11b-positive leucocyte population may be involved in the suppression of activated T cell responses. Myeloid-derived suppressor cells with a CD11b-positive, Gr-1-positive phenotype and immunosuppressive function have been described and have been implicated in Chloroambucil the protection of T cell-mediated chronic enterocolitis [26,27]. We have demonstrated previously a similar rapid onset of proinflammatory cytokine and intestinal injury in adult axenic IL-10 gene-deficient mice following colonization with commensal faecal flora [8]. A similar uncontrolled proinflammatory cytokine response to commensal bacterial antigens has also been found to play a crucial role in the human leucocyte antigen-B27 (HLA-B27) transgenic rat

colitis model [28]. Our results demonstrate for the first time that bacterial colonization in wild-type mice initially triggers a similar proinflammatory immune response, causing temporary intestinal inflammation. Endogenous bacterial antigens are treated as ‘foreign’ and stimulate an antigen-specific systemic immune response. However, in contrast to colitis susceptible rodents, wild-type mice are able to down-regulate the initial proinflammatory immune response and establish mucosal as well as systemic tolerance. Acquisition of immunological homeostasis appears to follow a defined inflammatory response pattern when first exposed to faecal bacteria and antigens, which probably plays an important role in the induction of tolerance to the endogenous microflora.

However, we believe it is mechanistically relevant to the BTLA pathway, as Sedy et al. described an ex vivo analysis of these cells using a co-culture system with

CHO cells presenting the OVA antigen ± the BTLA ligand HVEM, and demonstrated inhibition of DO11.10 T cell proliferation when the HVEM molecule was presented appropriately to the T cells [9]. Taken together, the in vitro and in vivo data set we have generated suggests that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation. selleck chemical All authors were employees of Amgen Inc. at the time this work was conducted and the manuscript written. Fig. S1. Anti-B and T lymphocyte attenuator (BTLA) monoclonal antibodies (mAbs) do not inhibit mouse T cell proliferation in the mixed lymphocyte reaction (MLR) in vitro. Mouse T cells and mitomycin C-treated antigen-presenting cells were cultured at a 1:1 ratio in selleckchem the presence of plate coated anti-BTLA antibodies clone 6G3, 6H6 and mouse immunoglobulin isotype control (10 µg/ml in phosphate-buffered saline, 100 µl per well). Mouse CTLA4-Fc was added to the indicated wells as a positive control inhibitor of T cell proliferation. The cells were cultured for 5 days and [3H]-thymidine was

pulsed for the last 18 h. T cell proliferation was measured by scintillation counting as described in the Materials and methods on day 5. Fig. S2. Anti-B and T lymphocyte attenuator (BTLA) monoclonal antibodies (mAbs) do not inhibit antigen-induced mouse DO11.10 T cell

proliferation in vitro. DO11.10 mice CD4+ T cells and mitomycin C-treated antigen-presenting cells were cultured at a 1:1 ratio in the presence of plate-coated anti-BTLA antibodies clone 6G3, 6H6 and mouse immunoglobulin isotype control (10 µg/ml in PBS, 100 µl per well). Mouse CTLA4 Fc was added to the indicated wells as positive control inhibitor of T cell proliferation. The cells were stimulated with ovalbumin peptide at 0·05 µg/ml for 3 days and [3H]-thymidine was pulsed for the last 18 h. T cell proliferation was measured by scintillation counting on day 5. Fig. S3. Inhibitory anti-B and T lymphocyte attenuator (BTLA) monoclonal antibodies (mAbs) bind to a different epitope on muBTLA than do non-inhibitory Molecular motor anti-BTLA mAbs. Anti-BTLA mAb 6F7, which does not inhibit in vitro T cell proliferation, was immobilized on a CM5 sensor chip, and mBTLA-mFc was captured on the antibody surface, followed by injection of inhibitory anti-BTLA antibody. If the immobilized antibody and the injected antibody bind to the same epitope, a second binding event will not be observed; if they bind to distinct epitopes, a second binding event will be seen. Events during the experiment are represented by letters, with ‘A’ corresponding to injection of mBTLA-mFc, ‘B’ corresponding to the end of the mBTLA-mFc injection, ‘C’ corresponding to injection of the second mAb, and ‘D’ corresponding to the end of the second mAb injection and start of the buffer wash.