PD-1 is expressed on activated and exhausted T cells and anti-B7-H1 blockade has been shown to restore T-cell functionality during chronic viral infections 32, 35. Paradoxically, anti-B7-H1 blocking Ab and Fab-fragments also induced inhibition of CD4+ T-cell proliferation. The effect was found to be mediated by IFN-γ-induced production of NO from macrophages 36. This demonstrates that reverse signaling of B7-H1 can further enhance the inhibitory activity of this ligand which may interfere with the potential use of anti-B7-H1-blocking Ab for

therapeutic use. We observed that chitin-mediated upregulation of B7-H1 occurred independently of TLR-mediated signals since the expression was induced in INK 128 clinical trial Fulvestrant BMDM from TLR2-, TLR3-, TLR4-, MyD88- and MyD88/TRIF-deficient mice, although the response was less pronounced in MyD88/TRIF-deficient mice compared with the other strains. At present, it remains unclear how chitin induces B7-H1 expression in macrophages. It could occur by direct activation of signaling pathways that lead to enhanced gene expression or indirectly via induction of IFN or other factors that induce secondary signaling events. We consider it unlikely that the mannose receptor might be involved since inhibition was also observed in cultures where

the mannose receptor was desensitized by soluble mannan. Further analysis of cells from dectin-1-deficient mice should help to clarify whether B7-H1 expression requires signaling via this receptor. Indeed, a recent study showed that dectin-1 alone can be sufficient to mediate chitin-induced expression Phospholipase D1 of TNF-α and IL-10 in macrophages 12. Chitin-mediated inhibition of T-cell proliferation may provide

an explanation for the observed attenuation of the adaptive immune response when chitin was used in murine asthma models 16, 17. However, chitin can at least transiently induce an innate pro-inflammatory immune response in the lung 9, 18. To further define the immunomodulatory functions of chitin in the lung, it would be important to study the outcome of chronic exposure to different chitin concentrations in future experiments. Chitin-derived products are exploited for tissue engineering and as vehicles for vaccine and drug delivery. Due to its biophysical properties, chitin is used to produce complex nanofiber scaffolds that resemble the extracellular matrix and support diverse types of cells to grow into artificial tissues 37. The suppression of T-cell proliferation by chitin-exposed macrophages may help to prevent rejection of these structures. However, there are no studies at present that analyzed the immune response against such chitin-based tissues. Since chitin can induce macrophages to produce pro-inflammatory cytokines including IL-17 and TNF-α but also inhibitory molecules such as IL-10 and B7-H1, it appears that the context of chitin recognition (e.g.

Figure S4. Gating strategy on CD8+ OT-1 T cells after 24 h and 42 h culture. Figure S5. PMN-MDSCs increase IFN-γ secretion levels upon co-culture with OVA-stimulated OT-1 splenocytes. Figure S6. IFN-γR-/- and IRF-1-/- MDSCs enhance IFN-γ production by activated CD8+ T cells on a per cell basis. Figure S7. MO- and PMN-MDSCs do not augment IL-12 levels upon

co-culture with OVA-stimulated OT-1 splenocytes. Figure S8. MO- and especially PMN-MDSCs suppress T-bet expression in activated CD8+ T cells. Figure S9. MDSCs down-modulate IL-2 production by activated CD8+ T cells. Figure S10. MO-MDSCs down-regulate CD25 expression and STAT5 phosphorylation. Figure S11. MDSCs alter the expression levels of cell adhesion molecules on CD8+ T Selleck PS341 cells. Figure S12. MO-MDSCs augment Fas expression on activated CD8+ T cells. RGFP966 order Figure S13. Neither MO- nor PMN-MDSCs are targets for OVA-specific CTLs, nor do they affect the cytotoxic activity of mature CTLs. Figure S14. Unseparated splenic MDSCs affect CD8+ T-cell activation events. Figure S15. RMA-OVA-induced splenic MDSCs affect CD8+ T-cell activation events. Figure S16. MDSCs differentially affect CD8+ T-cell activation events upon polyclonal

stimulation. Figure S17. Tumor-infiltrating MO-MDSCs are strongly anti-proliferative and recapitulate only some aspects of their splenic counterparts. “
“In clinical Neratinib in vitro practice it is possible to find patients with clinical signs suggestive of anti-phospholipid syndrome (APS) who are persistently negative for the routinely used anti-phospholipid antibodies (aPL). Therefore, the term proposed for these cases was seronegative APS (SN-APS). We investigated the clinical

usefulness of thin-layer chromatography (TLC) immunostaining in detecting serum aPL in patients presenting clinical features of SN-APS. Sera from 36 patients with SN-APS, 19 patients with APS, 18 patients with systemic lupus erythematosus (SLE), 20 anti-hepatitis C virus (HCV)-positive subjects and 32 healthy controls were examined for aPL using TLC immunostaining. Anti-β2-glycoprotein-I, anti-annexin II, anti-annexin V and anti-prothrombin antibodies were tested by enzyme-linked immunosorbent assays (ELISA). Eahy926, a human-derived endothelial cell line, was incubated with immunoglobulin (Ig)G fraction from SN-APS patients and analysis of phospho-interleukin (IL)-1 receptor-associated kinase (IRAK) and phospho-nuclear factor (NF)-κB was performed by Western blot, vascular cell adhesion molecule 1 (VCAM-1) expression by cytofluorimetric analysis and supernatants tissue factor (TF) levels by ELISA. TLC immunostaining showed aPL in 58·3% of SN-APS patients: anti-cardiolipin in 47·2%, anti-lyso(bis)phosphatidic acid in 41·7% and anti-phosphatidylethanolamine in 30·5%. Six of 36 patients showed anti-annexin II.

5 mL sterile PBS (pH 7.2). GDC-0941 purchase Mice injected with sterile PBS were used as sham controls. Mice were housed at the Department of Immunology animal facilities and fed with sterilized food and acidified water. This work was approved by the Ethical Committee for Animal Research of the Biomedical Sciences Institute of the University of São Paulo, Brazil. At 15 and 120 days of infection, mice were euthanized, and surgical procedures were done according to approved protocol by the Ethical Committee for Animal Research of the University of São Paulo, Brazil. The peripancreatic/perisplenic

omentum, the target organ of ip P. brasiliensis infection, (Xidieh et al., 1999; Nishikaku & Burger, 2003c) was collected and fixed in Methacarn solution (60% methanol, 30% chloroform, and 10% acetic acid) for 3–4 h in a shaker at 4 °C. Tissues were embedded in paraffin, and 5 µm sections were used for histologic and immunohistochemical procedures according to Nishikaku & Burger (2003a). The immunohistochemical reactions were done

according to the protocol described previously (Nishikaku & Burger, 2003a; Nishikaku et al., 2008). In brief, slides with deparaffinized tissue sections were incubated overnight at 4 °C with anti-mouse IFN-γ mAb (hybridoma XMG 1.2, dilution in PBS – 0.3% Tween 20). Biotinylated see more anti-rabbit IgG (Rockland, Gilbertsville, PA) was applied to tissues, followed by incubation with streptavidin-peroxidase (Vector Laboratories, Burlingame, CA). The chromogen 3.3′ diaminobenzidine tetrahydrocloride (Sigma-Aldrich, St. Louis, MO) was used, and sections were then counterstained with Mayer’s Hematoxylin and examined using a light microscope (Hund Wetzlar H500, Germany). Image capture was carried out using a microscope coupled to a video camera (Kodo, Tokyo, Japan) and a microsoft video capture software for Windows. Control slides were made with specimens of uninfected mice and without primary antibody replaced

by diluent (PBS – 0.3% Tween 20). The quantitation of IFN-γ in the lesions was done using a reticulated eyepiece (×12.5) with square grid and a ×40 objective (total magnification: ×500, total area = 280 μm2). This method was previously standardized by the same authors (Xidieh et al., 1999; Nishikaku et al., 2009b). The number of positive cells was counted Docetaxel nmr in 10 fields randomly chosen for each tissue slides (three mice per group) blindly by two examiners, and the results were expressed as mean ± standard error of the mean (SEM) of IFN-γ-positive cells/μm2. Two observers blindly analyzed the percentage of weakly and strongly IFN-γ-positive cells. Immunohistochemical data were expressed as mean ± SEM. The results were analyzed using the graph instat software version 2.04a. Differences were observed using the analysis of variance (anova) with Tukey–Kramer multiple comparisons test, and considered statistically significant when P < 0.05.

[35]. Subsequently, the sections were rinsed again

in TBS and coverslipped with glycerol/gelatin (Sigma). Alternatively, sections were rinsed with TBS, briefly washed with distilled water, mounted onto glass slides, air-dried and coverslipped with Entellan in toluene (Merck, Darmstadt, Germany). Control experiments were performed by omitting the primary antibodies or switching the fluorophores related to the different markers. All calculations were performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA). Differences between https://www.selleckchem.com/products/gdc-0068.html groups were checked for significance using one-way analysis of variance (anova) with Bonferroni post hoc test, or unpaired t-tests. Data are shown as mean ± SEM. Significance levels were determined as follows: *P < 0.05, **P < 0.01. Prior to immunolesioning experiments with 12-month-old mice, the occurrence of AD-like alterations in this age group had been verified. Concomitant β-amyloidosis and allocated hyperphosphorylated tau were revealed by double fluorescence labelling of hippocampal sections with antibodies recognizing total Aβ and the established marker for phospho-tau, AT8 (Figure 1a). Additionally, the combined staining of APP and 4G8 (raised against Aβ17–24, but with reported cross-reactivity for APP [36]) resulted in strong red fluorescent APP immunosignals and numerous

green fluorescent 4G8-monolabelled deposits, but also a portion of yellowish appearing structures immunopositive for both markers (Figure 1b). The efficacy of immunolesioning in 16-month-old

AZD0530 cell line mice that underwent icv immunotoxin injection 4 months before was routinely analysed by immunofluorescence labelling with affinity-purified goat-anti-ChAT as a marker for cholinergic neurones. Thereby, ChAT immunolabelling revealed the expected cholinergic chemoarchitecture in the forebrain of age-matched untreated control mice, e.g., the basal forebrain projection neurones and the more laterally located striatal interneurones (Figure 2a), which was not distinguishable from the staining Fenbendazole of cholinergic cells in mice 4 months after sham-injection with anti-p75 (Figure 2b). In contrast, 16-month-old immunolesioned mice were nearly devoid of ChAT-immunopositive neurones, whereas the respective striatal staining remained (Figure 2c). Additionally, selected sections containing the MS/DB were applied to p75 immunolabelling; thereby, forebrain sections from naive animals (Figure 2d) and from mice that had underwent sham-injections (Figure 2e) appeared nearly identical, i.e. the CPN neurones displayed the expected staining, whereas the striatum was devoid of p75-immunoreactivity. On the other hand, after successful immunolesion nearly no p75-immunoreactivity of CPN remained (Figure 2f).

The cells on coverslips were infected with DsRed- and/or EGFP-tagged adenoviruses, at moi of 100, in the presence or absence of 0.5–1 μmol/L MG-132 (Sigma) or 5 mmol/L 3-methyladenine (3MA; Sigma). After 48 h, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with Epigenetics inhibitor 100% methanol, washed with PBS, and immunostained overnight at 4°C with the following primary antibodies at 1:200 dilutions; mouse monoclonal TuJ1 (R&D Systems, Minneapolis, MN, USA), mouse-O4 (R&D), mouse anti-p62 (BD Biosciences, San Jose, CA, USA), rabbit anti-TDP-43 C-terminus (Cosmo Bio), rabbit anti-FUS (Sigma), rabbit anti-GFAP (DAKO,

Glostrup, Denmark), rabbit anti-ubiquitin (DAKO) and rabbit anti-choline acetyltransferase (ChAT;

Millipore, Billerica, MA, USA). The cells were then incubated with Alexa Fluor 350 or 488-conjugated goat anti-rabbit or anti-mouse antibodies (Invitrogen) at 1:400 dilutions for 1 h at room temperature, followed by incubation for 15 min with 2 μg/mL Hoechst 33342 (Invitrogen). After washings, coverslips were mounted on glass slides with Gelvatol (20% glycerol/10% polyvinyl alcohol in 0.1 mol/L Tris selleck chemicals buffer, pH 8.0). Immunostained cells were examined under an Olympus AX80TR microscope equipped with DP70 CCD camera. The experimental protocols were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Medical Science. Adult Fischer 344 male rats (8–12 weeks old, 150–200 g) were anesthetized with intraperitoneal injection of pentobarbital sodium (40 mg/kg). Under a dissecting

microscope, the right facial nerve was exposed and 10 μL solution in total of recombinant adenovirus(es) (1 × 108 plaque-forming units (pfu) each for single and combined injection) was slowly HA-1077 manufacturer injected into three facial nerve branches using a 33G microsyringe (Hamilton, Reno, NV, USA). The virus suspension was mixed with Evans blue (0.01% final; Sigma) to confirm visually that the injection was successfully performed. The wounds were covered with a small piece of gelatin sponge (Gelfoam; Pharmacia Upjohn, Bridgewater, NJ, USA) and suture closed, and the animals were killed at 3–7 days post-operation as described below. Rats were anesthetized with a lethal dose of pentobarbital sodium and transcardially perfused with 0.1 mol/L phosphate buffer, pH 7.4 (PB) followed by 4% paraformaldehyde in 0.1 mol/L PB. The brain stem tissue containing facial nuclei and their intramedullary nerve tracts was dissected and immersion fixed in the same fixative as described.[24] The brain stem tissues were cryoprotected in 30% sucrose in 0.1 mol/L PB and serial transverse sections (15 μm thickness) were made by cryostat.

4, P < 0·05). Triptolide and dexamethasone were equally effective in reducing levels of BALF TGF-β1 (512 ± 54 mTOR inhibitor versus 524 ± 67 pg/ml, Fig. 4, P > 0·05). There was no significant difference between the TRP and DEX groups. We demonstrated that triptolide inhibited airway remodelling and reduced TGF-β1 expression. Recent reports have demonstrated an improved method for investigating the expression of active TGF-β1 signalling in situ,25 which involves examination of the expression of the intracellular effectors, Smads. Therefore, we investigated the expression patterns of phosphor-Smad2/3 (pSmad2/3) and Smad7 in the lung specimens following administration

of dexamethasone to investigate any effect on active TGF-β signalling in airway lesions. Data were normalized to the levels of GAPDH. An increase buy Napabucasin in expression of pSmad2/3 was observed during prolonged allergen challenge, whereas administration of triptolide and dexamethasone both considerably decreased pSmad2/3 expression (0·73 ± 0·07 versus 0·55 ± 0·04 and 0·51 ± 0·07, Fig. 5, Table 2, P < 0·01). In contrast with pSmad2/3, Smad7 was markedly up-regulated in mice treated with triptolide or dexamethasone compared with the OVA-sensitized/challenged group (0·44 ± 0·03 and 0·44 ± 0·04 versus 0·29 ± 0·06, Fig. 5, Table 2, P < 0·01). There was no significant difference of pSmad2/3 and Smad7 in mice treated with triptolide

and dexamethasone (Fig. 5, Table 2, P > 0·05).

In this study, we Dynein established a mouse model of airway remodelling by repetitive OVA-challenge which replicated many of the features of the human disease asthma with a high degree of fidelity. Therefore, we investigated whether administration of triptolide could inhibit the progress of airway remodelling in mice exposed to repetitive allergen challenge, as well as determining whether triptolide could modulate the expression of signalling molecules of the TGF-β1/Smad pathway, which may in turn modulate airway remodelling. Recent morphological examination of airway tissues with bronchial asthma has revealed that abnormalities in airways, including goblet cell hyperplasia, mucous gland hypertrophy, subepithelial fibrosis and smooth muscle cell hyperplasia or hypertrophy, are in part irreversible.2,3 It is generally accepted that tissue remodelling is a process of wound healing for the maintenance of homeostasis after various injuries. Normally the process means the repair of injured tissues both morphologically and functionally; however, prolonged inflammation may induce remodelling of airways which could differ from wound healing. True to the observed clinical and symptomatic variability, remodelling can be elevated by as much as 50–300% in asthma patients who have died, and from 10 to 100% in subjects who have milder cases.26 Triptolide may offer a much needed therapeutic strategy for asthma airway remodelling.

This, however, is in contrast with previous studies, which reported that eosinophils mainly secrete Th2-type cytokines in response to parasite antigens and allergens.33,34 The GM-CSF is a cytokine expressed by a variety of cells, including activated T cells, Mφ, fibroblasts and epithelial cells. GM-CSF

is required for the recognition of pathogens, the timely development and proper compartmentalization of the immune response and the control of pulmonary growth of C. neoformans.35 Furthermore, GM-CSF stimulates the functional activity of eosinophils and maintains the maximum viability of cells,13 and GM-CSF-activated Nutlin3 eosinophils have been reported to be capable of acting as specific APCs to a T-cell BGJ398 concentration clone derived from mice infected with Mesocestoides corti.27 The results of the present study showed that

GM-CSF only modified the MHC class II expression levels on eosinophil surfaces cultured with C. neoformans. Moreover, C. neoformans-pulsed eosinophils in the presence of GM-CSF expressed threefold more MHC class II than C. neoformans-pulsed eosinophils in the absence of this stimulating factor (Fig. 2b). In contrast, GM-CSF did not modify phagocytosis of the fungus, the expression of MHC class I, CD80 or CD86, cytokine production or the fungicidal molecules released by eosinophils incubated with the fungus. Related to this, Feldmesser et al.19 have demonstrated that short-term incubation with IL-5, GM-CSF and lipopolysaccharide (LPS) did not appear to enhance eosinophil phagocytosis. Phagocyte–microbe contact is accompanied by intracellular signals that trigger cellular processes as diverse as cytoskeletal rearrangement, alterations in membrane trafficking, activation of microbial killing mechanisms, production of pro- and anti-inflammatory cytokines and chemokines, activation of apoptosis and the production of molecules required for efficient antigen presentation to the adaptive immune system.36,37 In this regard, it has been shown that eosinophils are able to produce H2O2 in response to phagocytosis

of heat-killed Staphylococcus aureus38 and excretory–secretory products (ESP) from interacting with Fasciola hepatica.8 In addition, Phipps et al.39 suggests that eosinophil-derived NO contributes to innate protection against the respiratory syncytial virus. In fact, in cryptococosis, the generation of NO is required Methocarbamol for resistance to primary fungal infections. Moreover, mice deficient in inducible nitric oxide synthase (iNOS) did not survive a primary infection.40 Snelgrove et al.41 have shown that NADPH oxidase-deficient mice elicited a heightened Mφ-driven Th1 response with the containment of cryptococci within pulmonary granulomatous lesions. They also observed improved clearance of pathogen in lung and airways, with reduced dissemination to the brain. In the present study, opsonized C. neoformans down-regulated NO and H2O2 synthesis by eosinophils in an FcγRII-dependent manner.

“
“Henoch-Schoenlein nephritis (HSPN) is find more a severe disease in adults and may cause renal insufficiency

in a large portion of patients. But its rarity has led to lack of data. There are few controlled studies on therapy with immunosuppressants in HSPN adults. This study aims to evaluate the effect of leflunomide on HSPN adults with nephrotic proteinuria. We retrospectively studied 65 adult patients who had biopsy-proven HSPN with nephrotic proteinuria. Twenty-seven patients (Group P) only received steroids, and 38 (Group P + L) were treated with leflunomide in addition to steroids. The clinical features, laboratory data and pathological findings of both groups were analyzed. The two groups were well-matched at baseline. After 24 months of treatment, urinary protein excretion of both groups decreased significantly from the baseline, and the estimated glomerular filtration

rate (eGFR) was higher in Group P + L. Four patients in Group P and three in Group P + L developed to end-stage renal disease at the most recent follow-up. Group P + L showed better renal outcome INCB024360 mouse than Group P. The treatment group and the degree of mesangial hypercellularity were significantly related to renal prognosis. Leflunomide combined with steroids is effective for treating adult HSPN with nephrotic proteinuria. “
“Aim:  A more precise understanding of the aetiology and sequelae of muscle wasting in end-stage renal disease (ESRD) is required for the development of effective interventions to target this pathology. Methods:  We investigated 49 patients with ESRD (62.6 ± 14.2 years,

0.3–16.7 years on haemodialysis). next Thigh muscle cross-sectional area (CSA), intramuscular lipid and intermuscular adipose tissue (IMAT) were measured via computed tomography as indices of muscle quantity (i.e. CSA) and quality (i.e. intramuscular lipid and IMAT). Additional health and clinical measures were investigated to determine associations with these variables. Results:  Age, energy intake, disease burden, pro-inflammatory cytokines, nutritional status, strength and functioning were related to muscle quantity and quality. Potential aetiological factors entered into forward stepwise regression models indicated that hypoalbuminaemia and lower body mass index accounted significantly and independently for 32% of the variance in muscle CSA (r = 0.56, P < 0.001), while older age and interleukin-8 accounted for 41% of the variance in intramuscular lipid (r = 0.64, P < 0.001) and body mass index accounted for 45% of the variance in IMAT (r = 0.67, P < 0.001). Stepwise regression models revealed that intramuscular lipid was independently predictive of habitual gait velocity and 6 min walk distance, while CSA was independently predictive of maximal isometric strength (P < 0.05).

2; P = 0.037) and CPSI (26.1 ± 5.0 vs 17.2 ± 8.3; P = 0.0016) scores improved from baseline to end of treatment. Incontinence episodes per day improved slightly (P = 0.042). When only those completing at least 8 weeks

of treatment were RXDX-106 ic50 examined (n = 9), significant changes in CPSI, VAS, and PSQI were still observed. At the final visit, 8/9 (88.9%) men also reported some improvement in pain related to sex. Side-effects were generally mild and well tolerated. Conclusion: These results suggest that apremilast may improve CP/CPPS symptoms with only mild side-effects. However, placebo controlled studies are necessary to determine efficacy. “
“Over the past decade, the use of quality of life (QOL) questionnaires in the evaluation of pelvic organ prolapse (POP) has become a standard part of most clinical studies. Investigators have attempted to correlate QOL scores with objective findings and treatment efficacy and as outcome measures in comparing different treatment modalities. Many of the QOL questionnaires are available in short forms, making them easier to adapt to clinical settings. This article includes an overview of several validated QOL questionnaires and their application in studies whose results provide useful

guidelines for health care professionals who diagnose and manage women with POP. Pelvic organ prolapse (POP) is a condition that affects millions of women with a prevalence estimated in a clinical population to be 40% of parous women.[1] Age[2, 3] and parity[4] are well known risk factors for the development of POP, parity being the strongest risk factor selleck compound with an adjusted risk ratio of 10.85.[4] Neurologic injury to the pelvic floor[5, 6] and underlying connective tissue disorders[7] have also been implicated. Other suspected predisposing factors include chronic conditions that increase abdominal pressure such as heavy lifting, chronic cough, bowel dysfunction, previous Dolutegravir molecular weight hysterectomy, estrogen deficiency[8-10] as well as obesity in some[11, 12] but not all[13, 14] studies. The development of POP

in nulliparous women combined with its absence in many multiparous women suggests that genetics may also play a role.[15] Though POP and its associated disorders are rarely life threatening, they have a direct and profound impact on quality of life (QOL). Historically, objective evaluation of POP was commonly done by physical examination alone or in combination with instruments that addressed only a single organ, making it difficult to assess multi-organ involvement. Further, the absence of valid and reliable tools to measure QOL issues made the assessment of outcomes to various treatment modalities incomplete. Over the years, researchers and clinicians have recognized the need to develop (i) a comprehensive staging system that involved all pelvic organs and (ii) standardized quality of life assessment tools specifically designed for POP disorders that would better evaluate treatment efficacy.

We show that the two-stage activation process that was previously described only in vitro26 can adequately explain the situation in vivo. However, tumor escape seems to be more complex than might be suggested by definitions in terms of type 1 or type 2 resistance. λ-myc transgenic mice express the myc oncogene under the control of Ig-λ chain regulatory sequences and spontaneously develop tumors of the B-cell lineage that share multiple features of human Burkitt lymphoma 29. Animals with lymphadenopathy were sacrificed and NK cells from spleens and lymph nodes were phenotypically analyzed. The absolute number of NK cells was strongly increased in tumor lymph nodes.

The highest numbers were found in cervical and mandibular lymph nodes, Decitabine cost the primary site of lymphoma growth (Fig. 1A). Inguinal and axillary lymph nodes and other lymphoid organs are infiltrated by tumor cells later during disease progression. Obviously, there is either an active migration of NK cells into the developing lymphomas or an enhanced proliferation in the tumor lymph nodes.

Most activating receptors including NKG2D and the inhibitory receptors tested were diminished, and expression of typical activation markers, such as CD45R and CD69, was enhanced (Fig. 1B). We assume that interaction of NK cells with tumor cells gave rise to NK-cell activation entailing up- or down-regulation of several surface receptors. A correlation between NK-receptor levels and NK/tumor-cell ratios in the different compartments was not seen in mice with visible tumor burdens suggesting strong activating signals as soon as visible tumor AZD6244 in vivo growth has started. To obtain more information on NK-cell activation in vivo, we also analyzed transgenic mice prior to macroscopic

tumor manifestation. NK cells from these animals already showed slight alterations of the surface molecules (data not shown), which might be due to incipient, yet undetectable lymphomas. To investigate effector functions, NK cells were tested for cytotoxicity by chromium release assay and for IFN-γ expression by RT-PCR and protein staining. In contrast to normal NK cells, highly enriched NK cells from tumor-bearing animals did not exert any cytotoxicity against the NK-sensitive STK38 YAC-1 target (Fig. 2A). Lytic activity of NK cells from clinically unapparent λ-myc transgenic mice (before manifestation of visible tumors) was also impaired but its decrease was often less pronounced than in tumor-bearing mice. For IFN-γ mRNA expression, a clear hierarchy was observed in NK cells derived from WT, clinically unapparent λ-myc transgenic and lymphoma-bearing animals, respectively (Fig. 2B). These differences were confirmed at the protein level by IFN-γ capture assays and intracellular IFN-γ staining (Fig. 2C). As in T lymphocytes activation-induced anergy may be overcome by stimulation of TLR, we treated freshly isolated NK cells with CpG-oligonucleotide (CpG-ODN) 1668, a stimulatory TLR9 ligand.