Cells were incubated for 1 h at 37°C with 125 µM substrate. The fluorescence of the cleaved reporter group was measured at

an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Camptothecin (an extract of selleck chemical the Chinese tree Camptotheca acuminata) is a potent inhibitor of topoisomerase I, a molecule required for DNA synthesis. Camptothecin has been shown to induce apoptosis and was therefore used for positive controls. The percentage of apoptotic and nectrotic cells was quantified by performing a cell staining with annexin V and 7-amino-actinomycin D (7-AAD) (PE Annexin V Apoptosis Detection Kit I; BD Biosciences, Franklin Lakes, NJ, USA). Apoptosis was quantified directly with annexin V, measuring the translocation of phospholipids phosphatidylserines from the inner to the outer leaflet of the plasma membrane in apoptotic cells. The loss of membrane integrity in late apoptotic or necrotic NVP-AUY922 molecular weight cells was assessed by 7-AAD staining. 7-AAD intercalates into double-stranded nucleic acids. It is excluded by viable cells, but can penetrate cell membranes of dying or dead cells. For

analysis, a fluorescence activated cell sorter (FACSCalibur) flow cytometer (Becton Dickinson) was used. Results are expressed as median and the error bars are plotted as median with interquartile range for the caspase assays. Values from stimulated cells are shown as percentage compared to control values of 100%. All experiments were conducted at least four times. Analysis of variance (anova) and Kruskal–Wallis multiple comparison tests were performed to assess the statistical significance of differences, using GraphPad Prism version 4·0 software (San Diego, CA, USA). For flow cytometry analysis, box-plots were designed using the spss program. P-values <0·05 were considered significant. To determine a possible effect of hypoxia on apoptosis in

alveolar macrophages and neutrophils caspase-3 as the key enzyme in the final pathway was determined as well as caspase-8 and -9 to distinguish between intrinsic and extrinsic pathways. Interestingly, the two cell types, although belonging to the group of effector cells, did not experience the same changes. While the Edoxaban apoptosis rate did not change under hypoxic conditions in alveolar macrophages at early time-points compared to control cells, caspase-3 activity increased by 80% in the LPS group and caspase-8 activity showed a threefold increase in the same group after 4 h (P < 0·05) (Fig. 1a). After 8 h, caspase-3 activity was enhanced by 240%, caspase-8 activity by 148% and caspase-9 activity by 85% in the LPS group (P < 0·05) (Fig. 1b). Figure 1c shows the 24 h caspase-3, -8 and -9 activities with no significant changes, except again for the LPS group, where caspase-3 level was increased by 277%, caspase-8 level by 41% and caspase-9 by 198% (P < 0·05).

The only site of unique conserved sequence in the Sorafenib clinical trial KIR locus is in the 14-kb intergenic region that separates 3DP1 from 2DL4 and divides the locus into Cen and Tel parts of

similar size [26, 27]. It was recently shown that a certain Cen variant (Cen-B/B) is associated with a lower risk of relapse after unrelated transplantation for acute myelogenous leukaemia [5]. Therefore, we analysed the distributions of KIR Cen and Tel parts between patients with syphilis and controls. Our data showed that there were no significantly different distributions in the Cen part between the two groups (Table 5). Interestingly, a KIR genotype (Tel-B/B) was significantly increased in patients with syphilis (P = 0.024) compared to healthy controls, while another KIR genotype (Tel-A/B) was close to significantly increased in controls (P = 0.049, this needs more work to confirm) compared to patients with syphilis. As there are more activating see more KIR genes in Tel region than those in Cen region, our data showed clearly that Tel-B/B encoding a dominant activating KIR gene repertoire conferred

increased risk for syphilis in Chinese population. Dissimilarly to our results, Dring et al. [28] found that KIR Cen-A/B was significantly increased in patients with hepatitis C virus infection compared to controls, and no significant Edoxaban difference was observed in Tel region between the two groups. These data suggested that different regions of KIR gene cluster might provide different immune responses to non-virus and virus infections. The biologic relevance of dominant activation KIR gene repertoire in syphilis pathogenesis remains unclear because the ligands for activating KIRs are unknown. Certain activating KIRs are predicted to bind to the same HLA class I

ligands in peptide-dependent manner as their structurally related inhibitory KIRs [29, 30]. We speculate from our data that the signals transduced by the activating KIRs binding to their ligands may overcome HLA class I-dependent inhibition. This favours the activation status of the host NK cells and participates in the physiopathological process either by excessively destroying infected cells or by non-specific inflammatory responses such as oxidative DNA damage, which may increase risk of syphilis. Recent studies have demonstrated that KIRs expressed on the surface of NK cells play a key role in the regulation of immune responses via the transduction of inhibitory or activating signals [12, 31]. NK cells can produce IFN-γ in response to microbial stimulation [13, 32]. It was reported that both primary and secondary syphilis lesions contained IFN-γ mRNA [33], and the peak IFN-γ production directly preceded the clearance of treponemes and the beginning of lesion healing [34].

Double- and triple-colour fluorescence images were acquired using a Leica microscope. CXCR3 expression was detected on acetone-fixed tissue sections using a polyclonal rabbit

anti-mouse antibody to CXCR3 (0·5 µg/ml final concentration; Zytomed) followed by the tyramide signal amplification (TSA) system with peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) (5 µg/ml; Jackson Immunoresearch) and FITC-tyramide (PerkinElmer Life Sciences, Boston, MA, USA). CD117+ lin- precursor-enriched lamina propria mononuclear cells (lamina propria MCs) were finally isolated subsequently using lineage-marker [negative depletion with antibodies to CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), 7-4 and Ter-119] and c-kit microbeads (positive selection) and MACS techniques (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s BIBW2992 molecular weight Selleckchem GS 1101 instructions. Total RNA of isolated precursor cells and bone marrow-derived dendritic cells (bmDCs) was isolated

using TRIzol (Sigma-Aldrich, Hamburg, Germany) according to the manufacturer’s recommendations. Reverse transcription into complementary DNA was performed using the Moloney murine leukaemia virus (MMLV) reverse transcriptase (Life Technologies Inc., Carlsbad, CA, USA) method. Chemokine receptor expression was analysed using two multiplex PCR kits (Maxim Biotech, San Francisco, CA, USA) including CCR1-9 and CX3CR1, according to the manufacturer’s instructions. Notch 1–4 expression by Arachidonate 15-lipoxygenase IEL precursors and mature IEL was analysed by RT–PCR as described elsewhere [11]. Notch-ligand expression on bmDC was analysed 24 h after incubation with various concentrations of rmMip3a (R&D Systems) by real-time PCR as described elsewhere [11]. For isolation of bmDC, bone marrow was isolated from femur and tibia and erythrocytes were lysed. The remaining cells were plated at a density of 106 per ml in six-well plates in RPMI-1640 (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) and containing 10 ng/ml of murine granulocyte–macrophage colony-stimulating factor (GM-CSF) and 1 ng/ml of murine IL-4 (Peprotech, Rocky Hill, NJ, USA). The cells were incubated

at 37°C with 5% CO2. After 2 days of culture the cells were washed gently and replaced with RPMI-10 containing the same concentration of GM-CSF and IL-4 for an additional 5 days and semi-adherent cells were harvested for further experiments. For maturation, bmDC were stimulated further with 1 µg/ml LPS for 24 h and incubated with variable concentrations of rmMip3a (R&D Systems). Colitis was induced by addition of 3% DSS (molecular weight 40 000; ICN Biomedicals, Aurora, OH, USA) to drinking water for 7 days. Citrobacter rodentium was grown overnight in Luria–Bertani broth at a concentration of 2·5 × 109/ml. Adult (10-week-old) CCR6 heterozygous mice were infected with 200 µl of the bacterial suspension (5 × 108 bacteria) by oral gavage.

45-μm filter) and stored at room temperature protected from light. Working concentrations of 3M-003 for each experiment were prepared from the stock solution using complete tissue culture medium (CTCM) consisting of RPMI-1640, 10% fetal bovine serum, penicillin 100 U mL−1, and streptomycin 100 μg mL−1. Recombinant murine IFN-γ (0.98 mg mL−1, 3.84 × 107 U mg−1) was supplied by Genentech (S. San Francisco, CA). Unless otherwise

stated, all reagents were purchased from Sigma Chem. Co. (St. Louis, MO). Pathogen-free BALB/c mice, 7–8 weeks old, from Simonsen Lab (Gilroy, CA), were used for isolation of monocytes, neutrophils, and macrophages. Mice selleck chemicals llc were housed and maintained in the animal facilities at the California Institute for Medical Research (CIMR, San Jose, CA). In studies in which PBMC supernatants were generated at 3M Co. and shipped in dry ice to CIMR, pathogen-free BALB/c mice 4–6 weeks of age were used. The project was approved by the institutional animal care and use committees at the 3M Co. and the CIMR. Peripheral blood was obtained by axillary bleeding, 10 mice per experiment, and heparinized (30 U mL−1). Heparinized blood was mixed 1 : 1 in saline and 4 mL was layered over 4 mL of Histopaque 1077 per 15-mL conical centrifuge this website tube. After centrifugation at 400 g for 30 min, PBMC layers were

collected, diluted with RPMI-1640, and PBMC pelleted by centrifugation (400 g, 10 min). PBMC were suspended in CTCM and counted in a hemacytometer. PBMC (5 × 106  mL−1 CTCM) were dispensed, 0.2 mL per microtest plate well (Costar 5936, Corning Co., Corning, NY). After incubation at 37 °C in a 5% CO2 incubator for 2 h, nonadherent cells were removed by aspiration. The number of adherent cells was calculated to be 5 × 105 per well by subtracting nonadherent cells from plated cells. The pelleted PBMC (erythrocytes and neutrophils) resulting from the centrifugation of heparinized Decitabine blood over Histopaque 1077 were collected in saline and mixed

1 : 1 in 3% Dextran 500 (w/v saline). After sedimentation for 1 h at 1 g at 37 °C, the white blood cell layer (neutrophils) was collected and cells were pelleted by centrifugation (400 g, 10 min). Pelleted cells were treated with 0.85% NH4Cl to lyse contaminating red blood cells. Treated neutrophils were suspended in CTCM, counted in a hemacytometer, and plated at 105 per well. Peritoneal macrophages were selected for study as representative of tissue macrophages, a cell type C. albicans would encounter in deep infections. Resident peritoneal cells were collected by lavage of peritoneal cavities (10 mL RPMI/mouse) from 10 mice per experiment. Peritoneal cells were pelleted by centrifugation (400 g, 10 min), pooled, suspended in CTCM, and counted. Peritoneal cells (2 × 106 mL−1 CTCM) were plated, 0.2 mL per microtest plate well, incubated for 2 h at 37 °C in 5% CO2 incubator, and then nonadherent cells aspirated.

Cleavage of fB by fD results in formation of the initial AP C3 convertase C3(H2O)Bb, which, like the classical C3 convertase C4bC2a, can cleave C3 into C3b and C3a. The generation of C3b allows the AP to be fully activated via formation of the bona fide AP C3

convertase Maraviroc price C3bBb (Fig. 1). Newly formed C3bBb is stabilized by the plasma protein properdin that binds to the complex and slows its deactivation.4 In fact, it should be noted that while the spontaneously generated C3(H2O)Bb is unique to AP, the C3b fragment generated from any of the pathways can bind to fB and, with the participation of fD, can form the AP C3 convertase C3bBb, which serves as an amplification loop for the entire complement system by rapidly augmenting the conversion of C3 to C3b necessary for full activation of the system and its downstream effects (Fig. 1).4 The cleavage of C3 to C3b is therefore the key step of convergence in the activation of the complement cascade.3

Apart from initiating the AP complement, C3b attaches to cells or immune complexes through covalent bonding; the opsonization of these targets by C3b or its further cleavage fragments facilitates their transportation and disposal through the endoreticular system. Additionally, C3b can associate with either of the C3 convertases to form the C5 convertase that cleaves C5 into C5a and C5b and initiates the terminal complement cascade, ultimately resulting in the formation of the multimeric membrane attack complex (MAC) (Fig. 1). In contrast to the early steps of complement activation,

assembly of the cytolytic MAC on the cell surface R788 in vivo is a tuclazepam nonenzymatic process, initiated by association of C6 and C7 to C5b and subsequent insertion of the C5b-7 complex into the cell membrane through a hydrophobic domain in C7.5 Further attachment of C8 and multiple copies of C9 to the membrane-residing C5b-7 leads to assembly of the MAC, which creates physical pores in the cell membrane and causes lysis.3,5 Although the above scheme of complement activation is well established, two recent findings have provided novel insight into the activation mechanism of the AP. Biochemical and gene-targeting studies have revealed a critical role of properdin in initiating AP complement activation on some, although apparently not all, susceptible surfaces.6–10 Accumulating evidence supports the conclusion that, in addition to serving as a stabilizer of C3bBb, properdin can function as a pattern recognition molecule to trigger AP complement activation and in some instances such an activity of properdin is indispensible for the AP.6,7 The second notable finding of recent studies is the requirement of MASP1/3 for normal AP complement activity.11 It has been shown that MASP-1/3 cleaves inactive fD zymogen into the active form of fD that is normally present in plasma.

This newly developed animal model now includes three major hallmarks BEZ235 solubility dmso of AD: genetically related age-dependent β-amyloidosis and tau hyperphosphorylation, complemented with experimentally induced cholinergic cell loss. Prospectively, such an attempt using 3xTg mice with modifiable cholinergic dysfunction appears promising for studies addressing different aspects of this devastating disease. Currently, acetylcholinesterase

inhibitors are still, despite their limitations, the most widely used drugs for symptomatic AD therapy [81]. Selective α7 nicotinic acetylcholine receptor partial agonists are now in clinical trials and have been demonstrated to be beneficial in preclinical studies by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors [82]. The presented data support the view that drugs targeting the cholinergic neurotransmission remain justified as a potential treatment strategy of AD (for review see [47]). The authors thank Drs Reinhard Schliebs and Thomas Arendt for critical reading of an earlier version from this article. We are

thankful to Dr Peter Davies (Pathology, Albert https://www.selleckchem.com/products/avelestat-azd9668.html Einstein College of Medicine, New York, USA), Dr Sascha Weggen (Neuropathology, University of Düsseldorf, Germany) and Dr Christian Czech (Hoffmann-La-Roche, Basel, Switzerland) for the donation of antibodies and Drs Frank M. LaFerla and Salvatore Oddo (University of California, Irvine, CA, USA) for pairs of triple-transgenic and WT mice. The technical assistance of Dr Anke Hoffmann, Ute Bauer and Marita Heindl is gratefully acknowledged. The biochemical part of the study was supported by the Alzheimer Forschung Initiative e.V. (to O.W.). The study was designed by Wolfgang Härtig who also performed the histological work together with Simone Goldhammer (SG) as part of her MD thesis. Immunolesions were made by Johannes Kacza. All biochemical data were generated by Annika Saul and Oliver Wirths. Histological Rho figures were produced by Jens Grosche, Simone Goldhammer and Dominik Michalski. The manuscript was written

by Wolfgang Härtig and considerably improved by Oliver Wirths and Dominik Michalski. “
“Upon denervation, skeletal muscle fibres initiate complex changes in gene expression. Many of these genes are involved in muscle fibre remodelling and atrophy. Amyotrophic Lateral Sclerosis (ALS) leads to progressive neurodegeneration and neurogenic muscular atrophy. Disturbed calcium homeostasis and misfolded protein aggregation both in motor neurons and muscle fibres are key elements of ALS pathogenesis that are mutually interdependent. Therefore, we hypothesized that the calcium sensor STIM 1 might be abnormally modified and involved in muscle fibre degeneration in ALS and other types of NMA. We examined ALS and NMA patient biopsy and autopsy tissue and tissue from G93A SOD1 mice by immunohistochemistry and immunoblotting.

Mouse IgG subclasses IgG1, IgG2a, IgG2b and IgG3 were examined with strip-immobilized goat anti-mouse antibodies (Serotec, Raleigh, NC, USA) according literature [19, 20]. The intensity of the resulting bands indicated specific antibody concentrations in the tested antisera (n = 5 mice from each group). Evaluation was done by calculated integral optical density (IOD) (software Gel-Pro Analyser 3.1; Media Cybernetics, Santa Barbara, CA, USA). Peripheral blood

leucocytes population was obtained from the heparinized complete peripheral blood of mice as described before [14]. Briefly, polymorphonuclear cells (PMN) were isolated by Ficoll-Urografin gradients following dextran sedimentation of erythrocytes and finally adjusted Lapatinib to 1 × 106 cells/ml in RPMI 1600. C. albicans CCY 29-3-100 (serotype A) cells (100 μl, 5 × 106 cells/ml) were pre-incubated with 100 μl of heat non-inactivated serum samples and heat-inactivated serum samples (n = 5 mice from each

group, final serum dilution 1:50) and PBS as control for 30 min at 37 °C. Next, C. albicans cells samples were washed with PBS and incubated with isolated PMN (1 × 106 cells/ml), to obtain target cells to effector cells ratio 5:1, for 60 minutes at 37 °C. After incubation, PMN were lysed with sodium deoxycholate [13, 14, NSC 683864 manufacturer 21]. Propidium iodide (PI, 0.02 μg/ml, redistilled water, Sigma) and fluorescein diacetate (FDA, 5 mg/ml stock solution in acetone, 50 μg/ml, redistilled water, Lachema) staining was carried out by incubating 100 μl of the Candida suspension with 50 μl of PI and 50 μl of FDA for 30 min at room

temperature in darkness. Incubations and staining steps were done under static conditions. Spleens aseptically removed from immunized and control mice were placed in ice-cold PBS. Spleens were washed out with PBS (5-ml syringe, 1 ml per spleen) to rinse cells. The cell suspension was centrifuged at 800 × g buy Afatinib for 10 min at 4 °C. The cell pellet was resuspended in 5 ml of ACK lysing buffer (0.15 m NH4Cl, 1 m K2CO3, and 0.01 m EDTA, pH 7.2) and incubated at room temperature for 5 min to lyse the red blood cells. The cell suspension was washed twice with PBS and resuspended in RPMI-1640 containing 10% foetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin sulphate. The cell density was adjusted to 1 × 106 cells per ml with RPMI-1640 after determination of cell viability using trypan blue dye exclusion method. The ELISPOT assay was used to analyse mannan-specific antibody-secreting cells in spleen of immunized mice. C. albicans serotype A or C. albicans serotype B purified mannan was diluted in carbonate – bicarbonate coating buffer (pH 9.6) at a concentration 10 μg/ml and 100 μl of the solution was applied to each well. The plates were incubated at 4 °C overnight. The plates were washed three times with PBS and blocked by incubation with RPMI 1640 medium containing 10% foetal bovine serum for 2 h at room temperature. The plates were washed twice with PBS.

In lymphoid tissues ATP and Idasanutlin molecular weight ADP are primarily hydrolyzed to AMP by NTPDase1/CD39, and further to adenosine by CD73. To trigger signaling cascades in the responding cells ATP and ADP bind to a series of ligand-gated (P2X) and G-protein-coupled (P2Y) receptors, whereas adenosine binds to one of the four adenosine receptors. Intriguingly, ATP and ADP generally evoke proinflammatory signals, whereas adenosine shows opposite effects by acting as an anti-inflammatory mediator.

Along with the “classical” nucleotide-inactivating chain, the counteracting adenylate kinase (AK) and nucleoside diphosphate (NDP) kinase enzymes co-exist on the cell surface. The balance between these opposing nucleotide-scavenging and ATP-regenerating pathways may represent a key element in controlling the duration and magnitude of purinergic signaling 1–3. CD73 is a glycosylphosphatidylinositol-linked surface protein expressed

on subsets of leukocytes, vascular endothelial cells and on certain epithelial cells 4–7. The preferential expression of CD73, together with NTPDase, on CD4+CD25+FoxP3+ immunosuppressive Tregs has recently drawn much attention 8–11. The enzymatic activity of CD73 modulates leukocyte–endothelial Selleckchem LDK378 cell contacts and it improves barrier functions of the vascular lining 12–14. Altered inflammatory reactions have been reported in CD73-deficient mice in multiple selleck chemicals different models, including ischemia-reperfusion injuries and autoimmune diseases 13, 15–19.

CD73 can be expressed on several cancer types such as leukemia, glioblastoma, melanoma, and ovarian, bladder, thyroid, eosophageal, gastric, colon, prostate and breast cancer 3. The ecto-nucleotidase activity on the malignant breast cancer cells is known to enhance the migration, invasion and neovascularization of these cells and to support the growth of tumors 20, 21. CD73 expression has even been suggested to serve as a prognostic marker in certain cancer types, such as breast cancer 21. Although the functions of CD73 in cancer cells have been studied to some extent, the contribution of host CD73 activity to cancer progression has not been addressed. Here, we report that CD73-deficient T cells show up-regulated NTPDase activity, and that tumor progression and intratumoral accumulation of Tregs and mannose receptor (MR)+ macrophages, which are typically considered to be type 2 macrophages 22–24, are attenuated in CD73-deficient mice. Moreover, the composition of intratumoral leukocyte populations and tumor growth can be therapeutically manipulated by targeting CD73 and NTPDase. These data indicate that suppression of the host’s CD73 activity might be a new tool to keep cancer cells under the control of anti-tumor immune responses.

Patient management following microsurgical flap failure includes strategic abandonment of reconstruction in some cases, use of conventional procedures in a majority of cases, and further microsurgical procedures in one-third of cases. The reconstructive surgeon should have this range of possibilities available for these difficult

cases. © 2009 Wiley-Liss, Inc. Microsurgery, 2010. “
“The aim of this study was to investigate the correlation between contractile function recovery and changes of acetylcholine receptors (AChR) in a transferred muscle flap following reinnervation. Orthotopic transfer of the gracilis muscle flap with repair of its nerve was performed bilaterally in 48 rats. The rats were randomly divided into six experimental groups based on the time intervals for assessments (1, 4, 5, 10, 20, and 30 weeks). Sixteen learn more ABT-888 cell line gracilis muscle samples from eight rats without surgery were used as the controls. In each group, muscle contractile force and weight were measured

(n = 16). The AChR numbers (n = 8) and subunits (ϵ and γ) mRNA (n = 8) were examined using [125I]-α-bungarotoxin and fluorescent quantitative-PCR. The results showed the AChR numbers in the muscle flap increased from 4 to 20 weeks after reinnervation and correlated with recovery of the tetanic contraction force. However, correlation between the increase of AChR number with the specific tension (peak contractile force normalized to wet muscle weight) was only found from 4 to 10 weeks postoperatively. The expression of γ-subunit mRNA increased at the early period after flap transfer and then decreased rapidly, whereas the ϵ-subunit mRNA recovered gradually since fourth week postoperatively. A small amount of γ-subunit mRNA could still be detected at 30 weeks

after surgery. In conclusion, following reinnervation of the transferred muscle flap, the contractile functional recovery is partially correlated to increase of the AChRϵ. Our findings may provide evidence for further study of improving muscle function in functional reconstruction Galeterone by targeting the AChR. © 2010 Wiley-Liss, Inc. Microsurgery 2010. “
“We report the case of a 46-year-old patient who suffered from huge tophus masses involving the metatarsal joints of the big toes of both feet, with infection and skin necrosis secondary to chronic tophaceous gout. After conventional curettage and debridement of each lesion, a free anterolateral thigh flap (ALTF) was used to resurface the circumferential wound, protect the underlying structures, and provide a gliding surface for the exposed tendons. The flap was safely raised and debulked during revision surgery, and excellent functional and cosmetic results were apparent at the 2-year follow-up. We consider ALTF to be a valuable option for the coverage of necrotic skin over tophi after adequate debridement. © 2009 Wiley-Liss, Inc.

2006AA02A109. 2006AA02A115); the National Natural Science Foundation of China (no. 30570771); the Beijing Ministry of Science https://www.selleckchem.com/products/DAPT-GSI-IX.html and Technology (no. D07050701350701) and the Cheung Kong Scholars programme. All disclosures were provided in the ‘Acknowledgements’ section. “
“Thomas Jefferson University, Philadelphia, PA, USA Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years

because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular Erlotinib mw CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast,

virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these

observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4+ T-cell responses to viral and tumour antigens. “
“TCR repertoire diversity can influence the efficacy of CD8+ T-cell populations, with greater breadth eliciting better protection. We analyzed TCRβ diversity and functional capacity for influenza-specific CD8+ T cells expressing a single TCRα chain. Mice (A7) transgenic enough for the H2KbOVA257–264-specific Vα2.7 TCR were challenged with influenza to determine how fixing this “irrelevant” TCRα affects the “public” and restricted DbNPCD8+versus the “private” and diverse DbPACD8+ responses. Though both DbNPCD8+ and DbPACD8+ sets are generated in virus-primed A7 mice, the constrained DbNPCD8+ population lacked the characteristic, public TCRVβ8.3, and consequently was reduced in magnitude and pMHC-I avidity. For the more diverse DbPACD8+ T cells, this particular forcing led to a narrowing and higher TCRβ conservation of the dominant Vβ7, though the responses were of comparable magnitude to C57BL/6J controls. Interestingly, although both the TCRβ diversity and the cytokine profiles were reduced for the DbNPCD8+ and DbPACD8+ sets in spleen, the latter measure of polyfunctionality was comparable for T cells recovered from the infected lungs of A7 and control mice.